Document 6427072
Transcription
Document 6427072
EDITORIAL BOARD DR K M L PATHAK Deputy Director-General (Animal Sciences) Indian Council of Agricultural Research, Krishi Bhavan, New Delhi 110 114 DR M K BHOWMIK Foremerly Director (Research), P-26, Udyan Park, 3, Chanditala Branch Road, P.O. New Alipur, Kolkata 700 053 DR P K JOSHI Director, National Academy of Agricultural Research Management, Hyderabad, Andhra Pradesh 500 030 DR N KONDAIAH Director, National Research Centre on Meat, Hyderabad, Andhra Pradesh 500 030 DR LAL KRISHNA Assistant Director-General (Animal Health), Indian Council of Agricultural Research, Krishi Bhavan, New Delhi 110 014 SHRI R R LOKESHWAR Former Chief Editor (Eng.), 5712 Vedavathy Block, Nandi Enclave, 2nd Cross, 3rd Phase, 5th Block, Banashanker Stage-3, Bangalore, Karnataka 560 085 DR (MRS) A A SHERIKAR Formerly Professor and Head, Atlanta Co-op. Housing Society, 6-Sherikar Niwas, Plot 29, Sector 40, Nerula (Sea woods), Navi Mumbai 400 706 DR K T SAMPAT Director, National Institute for Animal Nutrition and Physiology, Adugodi, Bangalore, Karnataka 560 030 DR B C SARKHEL Director, Biotechnology Centre, Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur, Madhya Pradesh 482 104 DR M C SHARMA Director, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh 243 122 DR A K SRIVASTAVA Director, National Dairy Research Institute, Karnal, Haryana 132 001 DR T P TRIVEDI Project Director, Directorate of Information and Publications of Agriculture, KAB I, Pusa, New Delhi 110 012 DR A K MISHRA Project Director, Project Directorate on Cattle, Meerut Cantt, Uttar Pradesh 250 001 Assistant Editor : ARUNA T KUMAR Incharge (English Editorial Unit) : DR R P SHARMA Chief Production Officer Technical Officer (Production) : : VIRENDER KUMAR BHARTI ANIL KUMAR SETH Senior Artist : NARENDRA BAHADUR Annual Subscription Individual Institutional Single copy Inland Rs 500 Rs 1500 Rs 125 Foreign US $ 125 US $ 375 US $ 32 For any editorial enquiry please contact: ARUNA T KUMAR, Ph: 011–25841004, 011–25842221 EXTN 612 E-mail: [email protected] [email protected] For subscription and advertisement please contact: S K JOSHI Business Manager DIRECTORATE OF INFORMATION AND PUBLICATIONS OF AGRICULTURE INDIAN COUNCIL OF AGRICULTURAL RESEARCH KRISHI ANUSANDHAN BHAVAN, NEW DELHI 110 012 Telefax: 011–25843657, E-mail: [email protected] • All disputes are subject to the exclusive jurisdiction of competent courts and forums in Delhi/New Delhi only • The Council does not assume any responsibility for opinions offered by the authors in the articles and no material in any form can be reproduced without permission of the Council • The Council is not responsible for any delay, whatsoever, in publication/delivery of the periodicals to the subscribers due to unforeseen circumstances or postal delay • Readers are recommended to make appropriate enquiries before sending money, incurring expenses or entering into commitments in relation to any advertisement appearing in this publication. The Council does not vouch for any claims made by the advertisers of products and services. The publisher and the editor of the publication shall not be held liable for any consequences in the event of such claims not being honoured by the advertisers. THE INDIAN JOURNAL OF ANIMAL SCIENCES Previous Issue : Vol. 80, No. 3, pp. 189–272 Vol. 80, No.4 April 2010 CONTENTS ANIMAL HEALTH Molecular detection of Staphylococcus aureus mastitis in crossbred cows based on genus specific gap gene and species specific aroA gene PCR assay Neelesh Sindhu, Anshu Sharma and V K Jain Detection and differentiation of leptospires using real-time polymerase chain reaction G Malathi, K G Tirumurugaan, K Vijayarani and K Kumanan Activity of extracts of Synzium aromaticum against microbes of veterinary importance Meenakshi Virmani, S L Garg, Nitin Virmani and S K Batra Uterine torsion in bovines: a review S P S Ghuman Insulin-like growth factor-I and -II in buffalo ovary: mRNA expressions and partial sequences R Dev, M K Sharma and Dheer Singh Effect of Bt cotton plants on oxidative stress in sheep B Anilkumar, A Gopala Reddy, B Kalakumar, K Jyothi and K S Gopi Micro-minerals status in goats of different age in semi arid region of India Neeru Bhooshan, Puneet Kumar and M C Yadav Effect of age and reproductive state on phosphatase enzymes and steroid hormones profile in Indian goats Neeru Bhooshan, Puneet Kumar and M C Yadav 275 279 284 289 306 313 317 321 Short Communications Comparison of PCR with conventional techniques for the diagnosis of brucellosis in cattle P Kaushik, D K Singh and A K Tiwari Therapeutic and residual efficacy analysis of some anti-tick compounds against natural Boophilus microplus infestation in crossbred cattle Hira Ram and A K Sharma Differentiation of the tubular components and collecting duct system of nephron in buffalo kidney during prenatal life Monika Suman, Neelam Bansal and Varinder Uppal Histogenesis of lingual epithelium during prenatal life in buffalo Deepanjali Verma, Varinder Uppal and Neelam Bansal Ultrastructure of gut associated lymphoid tissues in Kadaknath fowl P C Kalita, G K Singh and B S Dhote Biochemical and enzymatic changes in downer cow syndrome Subhash Kachhawaha and R K Tanwar Alteration in surface body temperature and physiological responses in Sirohi goats during day time in summer season S K Phulia, R C Upadhyay, S K Jindal and R P Misra 326 329 331 333 336 338 340 ANIMAL PRODUCTION Inventorization of Gaushala resources and their use in breed improvement and conservation programmes Dinesh Kumar Yadav and Pradeep Kumar Vij Environmental and genetic effects on growth traits of Chokla sheep B P Kushwaha, Ajoy Mandal, Ravindra Kumar and Sushil Kumar 343 346 Growth rate and wool production of Marwari lambs under arid region of Rajasthan H K Narula, Ajay Kumar, M Ayub and Vimal Mehrotra Effect of supplementing bypass fat prepared from soybean acid oil on milk yield and nutrient utilization in Murrah buffaloes S S Thakur and S K Shelke On-farm evaluation of urea molasses multi-nutrient blocks enriched with minerals in goats R Singh, S Kumar and R Bhardwaj Carcass and meat quality characteristics of designated indigenous sheep breeds of India A R Sen and S A Karim Carcass and meat characteristics of Soviet Chinchilla rabbits as influenced by age A R Sen and S A Karim 350 354 358 362 366 Short Communications Identification of single nucleotide variations in the genes related to reproduction in riverine buffalo (Bubalus bubalis) J Thanislass, R Sumathy, S Venkatesa Perumal and K V Subba Reddy Effect of farms on growth pattern of crossbred cattle Surendra Singh, A K Vasisht, A K Paul and L M Bhar Influence of genetic and non genetic factors on growth profile of Bharat Merino sheep in semi arid region of Rajasthan Ashish Chopra, L L L Prince, G R Gowane and A L Arora Impact of breed improvement programme on goat production under farmers’ flocks M K Singh, A K Goel, B Rai, Ashok Kumar and M C Sharma Evaluation of growth, feed conservation efficiency and carcass traits of Jamunapari goats under intensive feeding system M K Singh, T K Dutta, R B Sharma, A K Das and N P Singh Housing and feeding managemental practices for goats followed in South Gujarat G P Sabapara, S B Deshpande, V B Kharadi and P K Malik 370 373 376 379 382 385 Indian Journal of Animal Sciences 80 (4): 275–278, April 2010 Molecular detection of Staphylococcus aureus mastitis in crossbred cows based on genus specific gap gene and species specific aroA gene PCR assay NEELESH SINDHU1, ANSHU SHARMA2 and V K JAIN3 CCS Haryana Agricultural University, Hisar, Haryana 125 004 India Received: 5 August 2009; Accepted: 4 October 2009 ABSTRACT The present study aimed to diagnose Staphylococcus aureus from mastitic milk samples of crossbred cows using genus specific PCR assay based on gap gene (encoding glyceraldehyde –3–phosphate dehydrogenase) and species specific PCR assay based on aroA gene (encoding 5–enolpyruvylshikimate–3–phosphate synthase). For internal control, a set of universal primers were included. Out of 770 milk samples tested, 50.74 and 52.21% samples were diagnosed to be positive for Staphylococcus spp. by cultural examination and gap gene based genus specific PCR, whereas as many as 41.91% and 43.38% samples were found positive for Staphylococcus aureus by bacteriological examination and aroA gene based species specific PCR respectively. The assay could also detect the “no-growth” milk samples. Using this rapid, sensitive and specific method, staphylococcal isolates can be differentiated at the subspecies or strain level within 6–8 h and it can contribute to an increased understanding of mastitis epidemiology and control options for overall increase in lifetime productivity of crossbred cattle Key words: aroA, Crossbred cows, gap, Mastitis, PCR, Staphylococcus aureus from functional quarters of lactating crossbred cows (Hariana × Holstein Friesian with mixed crosses of Jersey, Sahiwal and Brown Swiss) from organized farms and individual farmers brought to Veterinary College Central Laboratory, COVS, CCS Haryana Agricultural University, Hisar, were included in the present investigation. Bacteriological examination: Milk (10 microlitres) from each sample was streaked on 5% sheep blood agar plates and MacConkey’s lactose agar plates separately. All Gram positive, catalase positive and oxidase negative isolates were confirmed as belonging to genus Staphylococcus. Staphylococcal isolates were further classified as coagulase positive and coagulase negative by standard citrate and rabbit plasma coagulase test. Coagulase positive isolates were further characterized biochemically by standard thermostable nuclease test, latex agglutination test (Staph latex test kit) and utilization of mannitol. DNA extraction from milk: Mastitic milk (1.5 ml) was centrifuged at 5 000 rpm for 2 min. Pellet was resuspended in 600 μl NTE buffer [0.1 M NaCl, 20 mM Tris-HCl and 1 mM EDTA], then the suspension was treated with 100μl of 24% sodium dodecyl sulphate and digested with proteinase K (20 mg/ml) and ribonuclease A for 2 h. 100 μl of 5 M NaCl and 80 μl of CTAB-NaCl [10% CTAB, 0.7 M NaCl] was added and incubated in water-bath at 65°C for 10 min. Then phenol: chloroform: isoamyl alcohol [25: 24: 1] and chloroform: India stands first in milk production in the world (Lahoti and Chole 2009). Infection of the cow’s udder (bovine mastitis) has remained one of the major constraints in growth of dairy industry in India and abroad (Sasidhar et al. 2002, Osteras and Solverod 2009). In India, among various pathogens responsible for mastitis, Staphylococcus aureus presents growing and formidable challenges for animal health concerns and remains predominant organism in crossbred cows (Singh and Kataria 2009, Peer et al. 2009). Several nucleic acid amplification assays based on specific gene targets and universal sequences were reported for diagnosis of Staphylococcus aureus from bovine mastitis in India (Sindhu et al. 2008, Dubey et al. 2009, Sharma et al. 2009). The present investigation describes a suitable and specific method for molecular diagnosis of Staphylococcus aureus from milk samples from crossbred cows infected with mastitis using gap gene and aroA gene as target for amplification. MATERIALS AND METHODS Collection of milk samples: Milk samples (770) collected Present address: 1 Ph.D. Scholar, 3 Associate Professor, Department of Veterinary Clinical Medicine, Ethics and Jurisprudence (E mail: [email protected]); 2 Scientist cum-Incharge, Veterinary College Central Laboratory, College of Veterinary Sciences. 3 276 SINDHU ET AL. [Indian Journal of Animal Sciences 80 (4) Table 1. 5’–3’ primer sequences for gene amplification Name Primer sequence 5’–3’ Reference Universal F: AGTGGAATTCCATGTGTAGC R: GAGTGCTTAATGCGTTAGCT F: ATGGTTTTGGTAGAATTGGTCGTTTA R: GACATTTCGTTATCATACCAAGCTG F: AAGGGCGAAATAGAAGTGCCGGGC R: CACAAGCAACTGCAAGCAT Riffon et al. (2001) 210 Yugeros et al. (2001) 933 Marcos et al. (1999) 1 153 gap aroA Expected product size (bp) encoding glyceraldehydes–3–phosphate dehydrogenase is a part of glycolytic operon in Staphylococcus aureus and was used to characterize Staphylococcus spp. (Yugeros et al. 2000, Ghebremedhin et al. 2008). 5–enol pyruvlshikimate –3– phosphate synthase is a key enzyme of aromatic amino acids and the folate universal biosynthetic pathway and aroA gene, which encodes this enzyme was described as a tool for identification of Staphylococcus aureus in milk of cows and ewes (Marcos et al. 1999, Saei et al. 2009) but on extensive search of literature, no study was found to be reported from India regarding diagnosis of mastitis using gap and aroA gene in crossbred cows. On bacteriological examination of 770 milk samples, 272 samples were found culturally positive revealing 138 staphylococci, 87 streptococci and 47 E. coli isolates. Staphylococcal culture isolates were further differentiated into 114 coagulase positive and 24 coagulase negative staphylococci. Based on biochemical tests, all coagulase isoamyl alcohol [24: 1] extraction was done until the interface was clear. Precipitation of DNA was done by sodium acetate (pH 5.2) and chilled 100% ethanol. Finally DNA was dissolved in 50 μl of in TE buffer (pH 8.0) (10 mM Tris HCl, 5 mM EDTA) and stored at –20°C till further use. DNA extraction from bacterial culture isolates: In brief, single colony from overnight grown culture was inoculated in 25μl of TE buffer (pH 8.0) (10 mM Tris HCl, 5 mM EDTA) and heated at 99°C for 15 min and cooled immediately by putting on ice. The resultant template DNA was stored at – 20°C and 5μl of each sample was used for PCR analysis. Amplification and analysis: PCR reactions were standardized using different MgCl2 concentrations, Taq DNA polymerase concentrations, primer concentrations, annealing temperature and number of cycles in thermocycler. The PCR reaction mixture optimized was 200 μm deoxynucleotide triphosphate (dNTP) mix, 1X PCR buffer (with 10 mM TrisHCl, pH 8.8, 50 mM KCl and 0.8% Nonidet P 40), 1.5 mM MgCl2, 20 pmol of each primers, 2.5 U Taq DNA polymerase, 200 ng DNA as template and nuclease free water. Sequences of oligonucleotide primers taken are indicated in Table 1. The optimized PCR conditions are indicated in Table 2. DNA isolated from pure bacterial culture of Staphylococcus aureus ATCC 25923 was taken as positive control, nuclease free water was taken as negative control and PCR mixture without template was taken as PCR control to check the possibility of contamination. After amplification, amplified products were subjected to 2% agarose gel prepared using 0.5X TBE buffer [pH 8.0, 0.045 M Tris borate, 0.001 M EDTA] containing ethidium bromide (0.2 μg/ml) with 100 bp gene ruler as marker and visualized using UV transilluminator. Sensitivity of PCR primers was evaluated by using different dilutions (CFU/ml) of bacteria. Specificity of PCR primers was checked with milk samples inoculated with Streptococcus dysgalactiae, Streptococcus agalactiae, Streptococcus uberis and E.coli. L 1 2 3 4 5 6 7 1153 bp 953 bp 210 bp Fig 1. PCR amplification using universal primers, gap gene and aroA gene Lane 1: Sample with Staphylococcus aureus with universal primers Lane 2: Sample with Streptococcus spp. with universal primers Lane 3: Sample with E.coli. with universal primers Lane 4: Sample with Staphylococcus spp. with gap gene primers Lane 5: Sample with Staphylococcus aureus with aroA gene primers Lane 6: Negative control without DNA Lane 7: Negative control with nuclease free water RESULTS AND DISCUSSION Glyceraldehydes–3–phosphate dehydrogenase catalyses conversion of glyceraldehyde–3–phosphate to 1, 3 diphospho glycerate and so enhances binding of transferring and serine protease plasmin for causing virulence activity. gap gene 4 April 2010] MOLECULAR DETECTION OF STAPHYLOCOCCUS AUREUS 277 Table 2. Optimized PCR amplification conditions Action Universal primers gap gene aroA gene Temperature (°C) Duration Cycles Temperature (°C) Duration Cycles Temperature (°C) Duration Cycles 94 94 56 72 72 4 5 min 45 sec 1 min 1 min 10 min infinite 1 35 35 35 1 – 95 95 55 72 72 4 3 min 30 sec 30 sec 30 sec 7 min infinite 1 40 40 40 1 – 94 94 58 72 72 4 5 min 1 min 1 min 1 min 10 min infinite 1 36 36 36 1 – Initial denat-uration Denat-uration Annealing Extension Final extension Storage Table 3. Comparison of details of bacterial isolates and their correlation with PCR assay Number of samples 770 Culturally positive for Staphylococcus spp. (both coagulase positive and coagulase negative) Coagulase positive (All S.aureus) gap gene PCR from Culture gap gene PCR from milk directly aroA gene PCR fromCulture aroA gene PCR from milk directly 138 114 138 142* 114 118* *4 samples showing negative results for bacterial isolation were positive for gap gene and aroA gene by PCR directly from milk. positive isolates were identified as Staphylococcus aureus and no isolate of other coagulase positive staphylococci (viz. Staphylococcus intermedius and Staphylococcus hyicus) was found. When staphylococcal isolates (both coagulase positive and coagulase negative) were subjected to gap gene based genus specific PCR, all the isolates were found positive for PCR with amplicons of size 933 bp (Fig. 1). Similar results were observed when culture isolates positive for Staphylococcus aureus were screened by aroA gene based species specific PCR with amplified products of size 1153 bp (Fig. 1), indicating 100% sensitivity of PCR assay on cultural isolates. The results obtained are in direct accordance to the studies reported by others (Marcos et al. 1999, Yugeros et al. 2001, Ghebremedhin et al. 2008, Saei et al. 2009) from cultural isolates obtained from milk samples of cows and ewes. None of the isolates were found positive for Streptococcus dysgalactiae, Streptococcus agalactiae, Streptococcus uberis and E.coli with PCR when amplified with primers encoding gap and aroA gene, although all these samples amplified 210 bp product with universal primers showing 100% specificity of primers. When samples were subjected to PCR directly on milk based on universal primers, gap and aroA genes, 276, 142 and 118 samples were found positive respectively. Comparison of details of bacterial isolates and their correlation with PCR assay is shown in Table 3. In our study, 4 culturally negative samples were found positive by PCR directly on milk. These samples had history of prior administration with antibiotics for treatment which may have inhibited growth of bacteria. As reported by Pheuktes et al. (2001), another reason for negative culture results might be due to the presence of leucocytes in milk samples with high somatic cell count. The “no-growth” samples have remained problematic for mastitis laboratories, veterinarians, and dairy producers and studies reported failure of growth of bacteria in up to 30% of milk samples from clinical and subclinical bovine mastitis even after 48 h of conventional culture (Sharma et al. 2009, Taponen et al. 2009). A reduction in morbidity due to preventable and prevalent endemic diseases like contagious mastitis caused by Staphylococcus aureus is needed to maximise overall lifetime productivity of cattle without compromising animal welfare. The application of molecular or DNA-based methods in mastitis research and diagnostics has contributed to an increased understanding of mastitis epidemiology and control options. Using these methods, staphylococcal isolates can be differentiated at the subspecies or strain level within 6– 8 h allowing for improved recognition of sources and transmission routes of pathogens. In conclusion, this assay based on detection of gap and aroA genes can be adopted for screening of large organized herd and for direct detection of Staphylococcus spp. and Staphylococcus aureus from mastitis samples respectively. ACKNOWLEDGEMENT The authors gratefully acknowledge technical help rendered by Mr Bhupender Singh and Mr Randhir Singh of Veterinary College Central Laboratory, College of Veterinary Sciences. 5 278 SINDHU ET AL. REFERENCES [Indian Journal of Animal Sciences 80 (4) J. 2001. Development of a rapid and sensitive test for identification of major pathogens in bovine mastitis by PCR. Journal of Clinical Microbiology 39: 2 584–89. Saei H D, Ahmadi M, Mardani K, Batavani R A. 2009. Molecular typing of Staphylococcus aureus isolated from bovine mastitis based on polymorphism of the coagulase gene in the north west of Iran. Veterinary Microbiology 137: 202–06. Sasidhar P V K, Reddy Y R and Rao B S. 2002. Economics of mastitis. Indian Journal of Animal Sciences 72(6): 439–40. Sharma A, Sindhu N and Jain V K. 2009. 16S–23S rRNA intergenic spacer based molecular detection of Staphylococcus aureus directly from mastitic milk of crossbred cows. Indian Journal of Animal Sciences 79: 350–52. Sindhu N, Sharma A and Jain V K. 2008. Phenotypic and genotypic characterization of Staphylococcus aureus isolated from mastitis cases of cows and buffaloes. Journal of Immunology and Immunopathology 10(2): 119–23. Singh J and Kataria A K. 2009. Relationships between bacterial species and total somatic cell counts in sub-clinical and latent mastitis in cattle. Indian Journal of Animal Sciences 79(1): 38– 40. Taponen S, Salmikivi L, Simojoki H, Koskinen M T and Pyorala S. 2009. Real-time polymerase chain reaction-based identification of bacteria in milk samples from bovine clinical mastitis with no growth in conventional culturing. Journal of Dairy Science 92(6): 2 610–17. Yugueros J, Temprano A, Sanchez M, Luengo J M and Naharro G. 2001. Identification of Staphylococcus spp. by PCR-restriction fragment length polymorphism of gap gene. Journal of Clinical Microbiology 39: 3 693–95. Dubey A, Ghorui S K and Kashyap S K. 2009. Differentiation of Staphylococcus aureus strains based on 16S–23S ribosomal RNA intergenic space polymorphism. Indian Journal of Biotechnology 8: 276–79. Ghebremedhin B, Layer F, Konig W and Konig B. 2008. Genetic classification and distinguishing of Staphylococcus species based on different partial gap, 16S rRNA, hsp60, rpoB, sodA, and tuf gene sequences. Journal of Clinical Microbiology 46: 1 019–25. Lahoti S R and Chole R R. 2009. Dairymen’s preferences for mode of training. Indian Dairyman 61(5): 53–55. Marcos J Y, Cascon A, Sanchez M, Hernanz C, Suarez S, Smeltzer M S and Naharro G. 1999. Rapid identification and typing of Staphylococcus aureus by PCR-restriction fragment length polymorphism analysis of the aroA gene. Journal of Clinical Microbiology 37: 570–74. Osteras O and Solverod L. 2009. Norwegian mastitis control programme. Irish Veterinary Journal 62 (supplement): 26–33. Peer F U, Bhattacharyya H K and Ansari M M. 2009. Studies on the epidemiology and therapeutic management of bovine mastitis. Indian Journal of Dairy Science 62 (1): 39–42. Phuektes P, Mansell P D, and Browning G F. 2001. Multiplex polymerase chain reaction assay for simultaneous detection of Staphylococcus aureus and streptococcal causes of bovine mastitis. Journal of Dairy Science 84: 1 140–48. Riffon R, Sayasith K, Hayssam K, Dubreuil P, Drolet M and Lagace 6 Indian Journal of Animal Sciences 80 (4): 279–283, April 2010 Detection and differentiation of leptospires using real-time polymerase chain reaction G MALATHI1, K G TIRUMURUGAAN2, K VIJAYARANI3 and K KUMANAN4 Tamil Nadu Veterinary and Animal Sciences University,Chennai, Tamil Nadu 600 007 India Received: 4 March 2009; Accepted: 22 October 2009 ABSTRACT Leptospirosis, an important emerging infectious disease of man and animals world-wide, is caused by helical motile spirochetes of the genus Leptospira necessity. In the present study, primer’s targeting the locus LA0322 of L. interrogans was used to compare the efficiency of conventional and SYBR green based real-time PCR assay. In serum and urine samples spiked with known amount of leptospires the conventional PCR had a average detection limit of 3.2–4.6 × 103 while real-time PCR was sensitive enough to detect as minimum as 32 to 41 leptospires per milliliter. Melting curve analysis indicated an average Tm of 79.5°C for L. interrogans, while it was for 85.6°C L. borgpetersenii. Our results indicate the ability of real-time PCR to differentiate leptospires in a single tube reaction. Further studies are required with the inclusion of other serovars. Key words: Conventional PCR, Melting curve, SYBR green real-time PCR, Typing Leptospirosis, an important emerging infectious disease of man and animals world-wide, is caused by helical motile spirochetes of the genus Leptospira (Ko et al. 1999, Levett 2001). Molecular tools such as conventional PCR and realtime PCR (Merien et al. 2005, Palaniappan et al. 2005, Fearnley et al. 2008) have made a great impact due to their rapidity, greater sensitivity and specificity in diagnosis during early stages of the infection. These techniques also have the ability to differentiate between the pathogenic and nonpathogenic serovars. However, the design of primers to differentiate non-pathogenic from pathogenic serovars in the above assays had always remained a challenge. In the present study, we used the primers targeting the locus LA0322 of L. interrogans and attempted for a SYBR green based real-time PCR assay. We compared the efficiency of the real-time PCR assay for the detection of leptospires with the conventional PCR, as well as its ability to help in typing. Table 1. Pathogenic and non-pathogenic Leptospira used in the study Serogroup Serovar Strain Pathogenic leptospires (Leptospira interrogans) Icterohaemorrhagiae Icterohaemorrhagiae RGA Hebdomadis Hebdomadis Hebdomadis Pyrogenes Pyrogenes Salinem Canicola Canicola Hond Utrecht IV Javanica Javanica Poi Pomona Pomona Pomona Pathogenic leptospires (Leptospira borgpetersenii) Sejroe Sejroe M84 Ballum Ballum Mus 127 Non-pathogenic leptospires (Leptospira biflexa) Andamana Andamana CH11 Semeranga Patoc Patoc I MATERIALS AND METHODS EMJH liquid medium and maintained in semi-solid media. Dark field microscopy was used to monitor the growth of the cultures before using for subsequent procedures. The cultures diluted in 10 folds were counted using Petroff Hausser chamber and spiked into serum and urine samples for use in conventional and real-time PCR assay. Isolation of genomic DNA from the leptospires: DNA was isolated from the culture of leptospires as well as the spiked samples according to Fischer and Lerman (1979). The precipitated DNA pellet was finally resuspended in TE buffer Bacterial strains: Six different pathogenic leptospires of Leptospira interrogans, 2 pathogenic leptospires of L. borgpetersenii and 2 non-pathogenic leptospires of L. biflexa belonging different serogroup and serovars used in the study were maintained at the Leptospira unit of Veterinary College are listed in the Table 1. The leptospires were cultured in Present address: 1, 2, 3, 4Department of Biotechnology, Madras Veterinary College, Chennai 600 007 (e-mail: kumananrani @hotmail.com) 7 280 MALATHI ET AL. [Indian Journal of Animal Sciences 80 (4) to 95°C with a linear temperature transition rate of 0.1°C/. The first derivate (dF/dT) of the melting curve (performed automatically by the software) were plotted against temperature for determination of the melting temperature (T m). Since all real-time PCR runs were performed in duplicates, the coefficient of variation for each dilution of the leptospires was calculated to show the repeatability. Fig. 1. Agarose gel electrophoresis of 331 bp amplicon of LA03222 gene sequence amplified only in pathogenic leptospira species. Lane M, 100 bp ladder; Lane 1 and 2, negative control; Lane 3, L. interrogans serovar Pyrogenes; Lane 4, L. interrogans serovar Hebdomadis; Lane 5, L. interrogans serovar Javanica; Lane 6, L. interrogans serovar Pomona; Lane 7, L. interrogans serovar Canicola; Lane 8, L. Borgpetersenii serovar Sejroe; Lane 9, L. Borgpetersenii serovar Ballum; Lane 10, L. interrogans serovar Icterohaemorrhagiae; Lane 11 and 12, Non-pathogenic L. biflexa serovar Patoc and L. Biflexa serovar Andamana. RESULTS AND DISCUSSION Conventional PCR assay sensitivity and specificity: The DNA of the reference leptospira serovars (Table 1) belonging to 8 different pathogenic species and 2 non-pathogenic species were included to determine the specificity of the conventional PCR. All the pathogenic leptospires DNA were tested positive that resulted in a 331-bp amplicon which was visualized by agarose gel electrophoresis (Fig. 1). The amplicons were also sequenced and blast results confirmed the sequence to be that of LA0322 gene. All the nonpathogenic samples resulted in no amplification (Fig. 1). The detection limit of the conventional PCR with the spiked samples corresponded to an average of 3.2–4.6 × 10 3 leptospires. Sensitivity of the real-time PCR assay: The sensitivity of the real-time PCR assay to detect leptospires were done by testing spiked samples with different dilutions of L. interrogans serovars Canicola and L. borgpetersenii serovar Sejroe. The obtained Ct values were plotted against the different dilution of leptospires and the linear regression line resulted in a correlation coefficient value of more than 0.9. Thus based on the standard curve, the minimum detection limit of the real-time PCR assay with the LA0322 primers corresponded to an average of 32 to 41 leptospires per ml (Figs 2a and 2b). Typing of leptospira by melting curve analysis: The melting curve analysis was performed on the DNA of different pathogenic L. interrogans and L. borgpetersenii species. The melting temperature of L.interrogans serovar Pyrogenes was 79.8°C, L.interrogans serovar Hebdomadis 79.3°C, L. interrogans serovar Javanica 79.4°C, L.interrogans serovar Pomona 79.1°C, L. interrogans serovar Canicola 79.1°C and L. interrogans serovar Icterohaemorrhagiae 79.6°C (Fig. 3a). The mean melting temperature of L. interrogans serovars was found to be 79.3°C. The L.borgpetersenii serovars Sejroe had a melting temperature of 85.7°C, while L.borgpetersenii serovars Ballum had 86.0°C with an average temperature of 85.5°C (Fig. 3b). Compared with the conventional methods for detecting leptospires, the real-time PCR assay using SYBR Green mix offers many advantages in that it is simple and in-expensive. Real-time PCR assays can also detect as little as 2-fold change. The conventional PCR in spiked samples had a detection limit that corresponded to an average of 3.2–4.6 × 103 leptospires. However, real-time PCR targeting LA0322 locus following spiking of known quantities of various and used as template for standardization of the real-time PCR assay. Conventional PCR: PCR was performed in the study using 20 pmoles of the following primers LFB1F (5’CATTCATGTTTCGAATCATTTCAAA-3’) and LFB1R (5’GGCCCAAGTTCCTTCT AAAAG-3’) that targeted a conserved hypothetical protein of LA0322 gene from pathogenic leptospira. A 25 ml reaction was performed using 2x Red dye master mix in an Eppendorf gradient cycler under the following conditions: 94°C for 5 min denaturing, 35 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 30 sec, and a final extension at 72°C for 7 min. The PCR amplicons were checked in a 2% TAE agarose gel and visualized by staining with ethidium bromide. Real-time PCR assay for quantitation of leptospires: The counted cultures of leptospires were spiked into serum or urine samples in a total volume of 10 ml. The samples were denatured at 95°C for 10 min and snap cooled. To the denatured mixture 100 mM of primers (LFB1F and LFB1R) and the SYBR green master mix were added and the reactions were performed under the following conditions in an ABI 7 500 machine: 50°C for 2 min, 95°C for 10 min followed by 50 cycles at 95°C for 15s, 59°C for 32 s, and 60°C for 32s. The ROX dye in the master mix was used as a passive reference dye. All reactions were carried out in duplicate with negative and positive controls. The data were accepted only in the absence of amplification in no template control (NTC). The data were analyzed using the biosystem sequence detection software (V.1.3.1.22). The obtained Ct (cycle at threshold) values were plotted against the log quantity of the leptospires to determine the standard curve. The curves with correlation coefficient values of greater than 0.9 were only accepted. Real-time PCR assay for typing of leptospires: For typing the leptospires using real-time PCR, based on melting curves, the reaction mix and the conditions remained the same as above except for the inclusion of a single dissociation step at the end of the 50 cycles. The melting curve analysis was done by continuous acquisition of the fluorescence from 65 8 April 2010] DETECTION AND DIFFERENTIATION OF LEPTOSPIRES 281 Delta Rn vs Cycle Positive control 6 3.2 × 10 3.2 × 104 3 3.2 × 10 2 3.2 × 10 1 3.2 × 10 1.0e+001 1.0e+000 Delta Rn 1.0e-001 1.0e-002 1.0e-003 1.0e-004 y=2.408x + 20.449 R2=0.9979 1.0e-005 1.0e-006 1 3 5 7 9 11 13 15 17 19 21 23 25 27 Cycle number 29 31 33 35 37 39 41 43 45 47 49 Fig. 2a. Representative results of the leptospira amplicon (L. interrogans serovar Javanica) detected by real-time PCR. Amplification plots of serially diluted leptospira cultures with 3.2 × 105, 3.2 × 104, 3.2 × 103, 3.2 × 102, 3.2 × 101 and 3.2 × 10° organisms. DNA was replaced with PCR-grade water in no template control. Insert shows the establishment of standard curve with the above dilutions of the organisms (correlation coefficient= 0.996). Each point represents the average values from 3 different experiments. Delta Rn vs Cycle Positive control 5 4.12 × 10 4.12 × 104 3 4.12 × 10 2 4.12 × 10 1 4.12 × 10 1.0e+001 1.0e+000 Delta Rn 1.0e-001 1.0e-002 1.0e-003 1.0e-004 y=3.413x + 18.384 R2=0.9954 1.0e-005 1.0e-006 1 3 5 7 9 11 13 15 17 19 21 23 25 27 Cycle number 29 31 33 35 37 39 41 43 45 47 49 Fig. 2b. Representative results of the leptospira amplicon (L. borgpetersenii serovar Sejroe) detected by real-time PCR. Amplification plots of serially diluted leptospira cultures with 4.12×105, 4.12×104, 4.12×103, 4.12×102, 4.12×101 and 4.12×10° organisms. DNA was replaced with PCR-grade water in no template control. Insert shows the establishment of standard curve with the above dilutions of the organisms (correlation coefficient = 0.996). Each point represents the average values from 3 different experiments. leptospira serovars was sensitive enough to detect as minimum as 32 to 41 leptospires per milliliter. The detection limit of any PCR assay depends on the copy number of the gene that is being targeted (Palaniappan et al. 2005, Slack et al. 2007). Slack et al. (2007) had reported an analytical sensitivity of 10 copies of the gene per reaction in a Taqman based real-time PCR assay. Targeting of the 16S gene of leptospira (Smythe et al. 2002) in the real-time PCR showed the ability to detect as low as 2 cells of leptospira due to the high copy number of the gene. The slightly lower sensitivity observed in our study could be because of the low copy number of the gene that had been targeted for the real-time PCR assay. In addition, the aim of the study was not only to detect pathogenic leptospires but also to assess the applicability of real-time PCR to simultaneously type the leptospires in a single tube reaction. Typing of leptospira is routinely being done by serological methods based on the antigenic difference in the lipopolysaccharide envelope that surround the cell wall (Mathew 2001). However, recently several molecular techniques have been tested for typing of leptospira with varied sensitivity. These molecular tools also avoid the tedious procedure of maintaining a panel of reference cultures and sera. Targeting genes that differ in their GC content in PCR would result in amplicons that differ in their Tm. These differences in the Tm would result in changes in their melting curves that can be appreciated in a SYBR based real-time PCR with a final dissociation step. Such a methodology was successfully applied to differentiate bacterial species (Jeffery et al. 2007,Portoni et al. 2006). Levett et al.(2005) attempted melting curve analysis for typing the leptospires. Interestingly in their study, the melting temperatures ranged between 82.5°C and 86°C depending on the species tested. Merien et al. (2005) were also able to assign a specific Tm to a single species or a set of species within a range of 3°C. Reports also indicated that L. alexanderi and L. borgpetersenii showed a mean highest Tm of 86.5°C, while the mean lowest Tm of 83.4°C was observed for L. interrogans s.s. and L. 9 282 MALATHI ET AL. Coefficient of variance = 0.001949 0.24 86°C 0.20 Derivative borgpetersenii had 85.6°C as mean melting temperature. The specific single peaks obtained in the melting curve analysis indicate amplification with no evidence of primer-dimer or other non-specific products. Our results indicated lower melting temperature for L. interrogans and a higher Tm L. borgpetersenii. Hence, in diagnostic laboratories, specific detection of pathogenic leptospires with the simultaneous ability to type the leptospires will be of real advantage. This avoids the need to maintain additional reagents that are required for conventional serotyping and also had a short turn around time. The preliminary results obtained in our study are encouraging and indicate that real-time PCR can be effectively used differentiate between L. interrogans and L. borgpetersenii indicating the usefulness of this technique. However, further studies are required with the inclusion of various other serovars to assess out the usefulness of this technique Dissociation curve 0.28 85.7°C 0.16 0.12 0.08 0.04 0.00 –0.04 –0.08 60 65 70 75 80 85 Temperature (°C) 90 95 Fig. 3a. Melting curve analysis of pathogenic Leptospira interrogans s.s after real-time PCR following amplification with primers targeting the LA03222 gene sequence and SYBR Green dye. The first derivative of the initial melting curve is plotted against the temperature for improved determination of the melting temperature (Tm). Note the duplicates for each sample giving the same Tm. The Tm of L.interrogans serovar Pyrogenes was 79.8°C, L.interrogans serovar Hebdomadis 79.3°C, L. interrogans serovar Javanica 79.4°C, L.interrogans serovar Pomona 79.1°C, L. interrogans serovar Canicola was 79.1°C, and L. interrogans serovar Icterohaemorrhagiae was 79.6°C. The mean melting temperature of L. interrogans s.s is 79.3°C with a co-efficient of variance of 0.00419. Dissociation curve Coefficient of variance= 0.00419 0.50 Average Tm –79.3°C ACKNOWLEDGEMENTS We thank the DBT, Government of India, New Delhi, for the funding and the TANUVAS, Chennai, for providing the necessary facilities to carryout this research. REFERENCES Fearnley C, Wakeley P R, Gallego-Beltran J, Dalley C, Williamson S, Gaudie C and Woodward M J. 2008. The development of a real-time PCR to detect pathogenic Leptospira species in kidney tissue. Research in Veterinary Science 85: 8–16. Fischer S G and Lerman L S. 1979. Lenth-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis. Cell 16: 191. Jeffery N, Gasser R B, Steer P A and Noormohammadi A H. 2007. Classification of Mycoplasma synoviae strains using singlestrand conformation polymorphism and high resolution meltingcurve analysis of the vlhA gene single-copy region. Microbiology 153: 2 679–88. Ko A I, Galvao Reis M, Ribeiro Dourado C M, Johnson Jr W D and Riley L W. 1999. Urban epidemic of severe leptospirosis in Brazil. Salvador Leptospirosis Study Group. Lancet 354: 820– 25. Levett P N. 2001. Leptospirosis. Clinical Microbiology Review 14: 296–326. Mathew J R. 2001. Leptospirosis- Medicine. Website www.emedicine.com/med/topic 1283.htm. Merien F, Portnoi D, Bourhy P, Charavay F, Berlioz-Arthaud A and Baranton G. 2005 A rapid and quantitative method for the detection of Leptospira species in human leptospirosis. FEMS Microbiology Letter 249: 139–47. Palaniappan R U, Chang Y F, Chang C F, Pan M J, Yang C W, Harpending P, McDonough S P, Dubovi E, Divers T, Qu J and Roe B. 2005. Evaluation of lig-based conventional and real time PCR for the detection of pathogenic leptospires. Molecular Cell Probes 19: 111–17. Portnoi D, Sertour N, Ferquel E, Garnier M, Baranton G and Postic D. 2006. A single run, real-time PCR for detection and identification of Borrelia burgdorferi susu lato species, based 0.60 Delta Rn 0.40 0.30 79.8°C 79.3°C 79.4°C 79.1°C 79.1°C 79.6°C 0.20 0.10 0.00 –0.10 60 65 70 80 75 Temperature (°C) 85 90 [Indian Journal of Animal Sciences 80 (4) 95 Fig. 3b. Melting curve analysis of pathogenic Leptospira borgpetersenii after real-time PCR following amplification with primers targeting the LA03222 gene sequence and SYBR Green dye. The first derivative of the initial melting curve is plotted against the temperature for improved determination of the melting temperature (Tm). Note the duplicates for the each giving the same Tm. The L.borgpetersenii serovars Sejroe had a melting temperature of 85.7°C while L.borgpetersenii serovars Ballum had 86.0°C with an average temperature of 85.5°C for L.borgpetersenii serovars with a coefficient of variance of 0.001949. santarosai. In our study to assess the ability of real-time PCR to differentiate pathogenic leptospires the mean melting temperature for L. interrogans was 79.5°C, whereas L. 10 April 2010] DETECTION AND DIFFERENTIATION OF LEPTOSPIRES on the hbb gene sequence. FEMS Microbiology Letter 259: 35– 40. Slack A, Symonds M, Dohnt M, Harris C, Brookes D and Smythe L. 2007. Evaluation of a modified Taqman assay detecting pathogenic Leptospira spp. against culture and Leptospiraspecific IgM enzyme-linked immunosorbent assay in a clinical 283 environment. Diagnostic and Microbiology of Infectious Diseases 57: 361–66. Smythe L D, Smith I L, Smith G A, Dohnt M F, Symonds M L, Barnett L J and McKay D B. 2002. A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp. BMC Infectitous Diseases 2: 13. 11 Indian Journal of Animal Sciences 80 (4): 284–288, April 2010 Activity of extracts of Synzium aromaticum against microbes of veterinary importance MEENAKSHI VIRMANI1, S L GARG2, NITIN VIRMANI3 and S K BATRA4 CCS Haryana Agricultural University, Hisar, Haryana 125004 India Received: 3 March 2009; Accepted: 20 November 2009 ABSTRACT The study was conducted with the objective to evaluate the antibacterial as well as antiviral activity of the aqueous and alcoholic extracts and oil of the flower buds of clove against the microbes responsible for causing diseases in poultry and livestock. The aqueous and alcoholic extracts of dried flower buds of clove were obtained by extraction in soxhlet apparatus using water and ethanol (95%) as solvents respectively. Oil was obtained from the clove flower buds by steam distillation. Both the extracts and oil were assessed for their antibacterial activity against Streptococcus zooepidemicus, Salmonella Gallinarum, Escherichia coli, Staphylococcus aureus and Pasteurella multocida while antiviral activity was tested against equine herpes virus – I (EHV I) and infectious bursal disease (IBD) virus. The extracts as well as oil were effective against the bacteria tested with zone of inhibition ranging from 11.67±0.33 to 24.00±0.00 mm. The Minimum inhibitory concentration (MIC) values for the extracts ranged from 0.75 to 4.00 mg/ml. Clove was also found to have potent antiviral activity against EHV–1 up to 104TCID50/ml, while IBD virus was found to be resistant to the clove extracts. Key words: Antimicrobial activity, Escherichia coli, Pasteurella multocida, Salmonella gallinarum, Staphylococcus aureus, Streptococcus zooepidemicus and Synzium aromaticum there is a need of developing some plant based derivatives which protect the poultry and livestock against the diseases as well as maintain or enhance their production performance. With this aim, we selected Synzium aromaticum to assess its antibacterial and antiviral activity against pathogens of animal health importance. The pathogens selected for the present study included bacteria, viz. Streptococcus zooepidemicus, Salmonella Gallinarum, Escherichia coli, Staphylococcus aureus and Pasteurella multocida and virus (Equine Herpes Virus 1 and infectious bursal disease virus) which are of veterinary importance as they cause huge economic losses to the country due to morbidity and mortality in livestock as well as poultry and thereby leading to decrease in productivity. Indians have been traditional users of plant derived medicines both directly and as an integral constituent of plethora of packages and practices of indigenous medicine. These plants and their extracts are being used in the pharmaceutical preparations of modern medicine, veterinary and in agriculture (Iyengar 1985, Chopra et al. 1992, Zafar 1999). The antimicrobials obtained from plants are of much therapeutic potential and are effective in treatment of infectious diseases while simultaneously mitigating many of the side effects that are often associated with synthetic antimicrobials (Kokoska et al. 2002). Synzium aromaticum (clove, family Myrtaceae) is an aromatic plant, grown extensively in India and is known for its antiseptic, carminative, stimulant, analgesic, antispasmodic properties (Prajapati et al. 2003). Clove is being used by the Indian Ayurvedic healers since ancient times to treat various ailments. To have suitable therapeutic control of the diseases of known importance of the livestock, MATERIALS AND METHODS Plant material and preparation of extracts The dried flower buds of clove were purchased from the local market.The dried flower buds were ground to powder form, soaked for 48 hr in distilled water and 95% (v/v) ethanol for aqueous extraction and alcoholic extraction, respectively and extracted as per procedure of Virmani et al. (2008). Clove oil was obtained from the clove buds by steam distillation. Per cent yield of various extracts from flower buds of clove Present address: 1Assistant Professor, Department of Veterinary Physiology, GADVASU, Ludhiana 141 009 Punjab. 3National Research Centre on Equines, Sirsa Road, Hisar 125 001, Haryana, India. 2Department of Veterinary Biochemistry and 4Department of Veterinary Microbiology, College of Veterinary Sciences. 12 April 2010] ACTIVITY OF EXTRACTS OF SYNZIUM AROMATICUM AGAINST MICROBES 14 1: 1000 multiplicity of infection (m.o.i) and maintained in MEM containing 2% foetal calf serum and incubated at 37°C with 5% CO2 tension in an incubator (Virmani et al. 2004) Cytotoxicity assay: Each extract was separately diluted 1: 10 with the maintenance medium and filter sterilized through 0.22μ millipore filter. Further dilutions (1: 20, 1: 40, 1: 60, 1: 80 and 1: 160) were made from the stock. The cytotoxicity assays were carried out using 100μl of cell suspension containing 104 cells in each well of the microtitre plate. The highest concentration of each extract found to be non-toxic to the cells was recorded and used further for antiviral assay. Antiviral assay: The non-toxic concentrations of the extracts were checked for antiviral property by cytopathic effect inhibition assay in cell monolayers infected with serial dilutions of virus (101 to 104 TCID50/ml). The cultures were overlaid with maintenance medium containing plant extracts at dilutions mentioned in Table 2 and incubated at 37°C for 5 days. Every 24 hr, the observations were made and cytopathic effects recorded. The antiviral activity was determined by the inhibition of CPE when compared with controls. For IBD virus, Chicken Embryo fibroblast (CEF) cells were freshly prepared and used for propagation of the virus, cytotoxicity assay and antiviral assay. The assays were performed as per the protocol mentioned for Equine Herpes Virus 1. However, the medium used for CEF was M 199 and the extracts were diluted in ratio of 1: 50, 1: 100, 1: 150 and 1: 200. Rest of the procedure was similar as for EHV 1. Phytochemical analysis: The qualitative phytochemical analysis of the alcoholic extract of flower bud of clove as well as oil of clove bud was undertaken using standard qualitative methods as described by other workers (Vogel 1958, Kapoor et al. 1969, Scalbert 1991 and Chukwurah 1997). The plant extracts were screened for the presence of biologically active compounds including glycosides, phenolics, alkaloids, tannins, flavonoids and saponins.` 12 % yield 10 8 6 4 2 0 Aq. extr. Alc. Extr. 285 Oil Fig. 1 Per cent yield of extractive from Synzium aromaticum flower bud. is given in Fig. 1. Antibacterial sensitivity assay and determination of minimum inhibitory concentration (MIC) An inoculum of each of the bacterial strains (single colony) was suspended in 5 ml of broth (tryptose soy broth for S. zooepidemicus) and incubated at 37°C for 18 hr. The antibacterial activity was tested by the disc diffusion assay (Bauer et al. 1966). 0.1 ml of inoculum (105 CFU/ml) was spread on sterile Mueller Hinton plates (blood agar for S. zooepidemicus and P. multocida) and sterile paper discs were placed on the inoculated surface. The discs were impregnated with 15μl of each of the extract at 10% concentration, kept at room temperature for absorption of extract in the medium and then incubated at 37°C in the incubator for 24 hr. The antimicrobial activity was evaluated by measuring the diameter of inhibition zone as per the procedure described by Kim et al., 1995. The experiment was carried out in triplicate and the mean of the diameter of inhibition zones was calculated. Four commercially available antibiotic drugs, viz. gentamycin, ciprofloxacin, cephotaxime and cefixime were used simultaneously as control. For determination of MIC, 1 ml of broth medium was taken into 10 test tubes for each bacteria. Different concentrations of plant extracts ranging from 0.25 to 10.00 mg/ml concentration were incorporated into the broth and the tubes were then inoculated with 0.1 ml of inoculum of respective bacteria (105 CFU/ml) and kept at 37°C for 24 hr. The test tube containing the lowest concentration of extract which showed reduction in turbidity, when compared with control was regarded as MIC of that extract. The optical density of each tube was measured in the spectrophotometer (at 620 nm). RESULTS AND DISCUSSION The extracts of Synzium aromaticum (clove) were found to be active against the bacteria tested (Table 1). The aqueous and alcoholic extracts of dried flower buds gave the inhibition zones of diameter ranging from 11.67±0.33 mm to 21.00±0.57 mm, while clove oil showed inhibition zones up to 24.00±0.00 mm for S. zooepidemicus. Clove oil was observed to have most potent antibacterial activity with MIC of 0.75 to 2.00 mg/ml against different bacteria (Fig. 2). Our findings corroborate with the findings of other workers who also reported clove to have high antimicrobial activity against different bacteria (Deans et al. 1995, Ahmad et al. 1999 and Nascimento Gislene et al. 2000). Arora and Kaur (1999) tested the spices against human pathogenic bacteria and yeasts and found that aqueous extract of clove possesses antimicrobial activity against Shigella flexnerii and Candida Antiviral activity Propagation of equine herpes virus 1 in RK–13 monolayer: RK–13 cells, grown as monolayer in MEM (minimum essential medium) containing 10% foetal calf serum, were infected with the virus (104.2 TCID50/ml) at 13 286 VIRMANI ET AL. 4 Aqueous extract Alcoholic extract of all the 3 extracts (aqueous, alcoholic and oil) was assayed and their cytotoxic levels were recorded. Alcoholic and aqueous extracts of Synzium aromaticum were found to be cytotoxic for both RK–13 cells and Chicken embryo fibroblasts at the concentration of 5 mg/ml while the oil extract was found to be cytotoxic at 1250 mg/ml (Table 2). The extracts of clove (both aqueous and alcoholic) as well as clove oil showed potent antiviral activity against EHV 1 up to the level of 104TCID50/ml, while none of the extracts had any antiviral activity against any of the dilutions of IBD virus. Benecia and Courreges (2000) reported Eugenol, an active component of clove oil to possess antiviral activity against Herpes Simplex virus 1 (HSV 1) and Herpes Simplex virus 2 (HSV 2) viruses and showed inhibitory concentration 50% values for the anti HSV effect to be 25.6 μg/ml and 16.2 μg/ml respectively for HSV 1 and HSV 2. The higher dose requirement in present study might be due to the crude preparations used as against the purified eugenol used by Benecia and Courreges (2000). Vijayan et al. (2004) observed different parts of medicinal plants of Nilgiris and found Hypericums myorense, Hypericums. hookerianum and Usnea complanta to be having antiviral activity against HSV 1 which is a virus belonging to Alpha herpes virinae similar to Equine Herpes Virus 1. Phytochemical analysis of the extracts depicted that the alcoholic extract of flower bud of clove was having glycosides, flavonoids, phenolics and tannins while only flavonoids and phenolics were present in oil of the clove bud. The results are in agreement with the reports of other workers (Chopra et al. 1992, Bruneton 1995 and Ahmad and Beg 2001). Sakagami et al. (1995) have suggested that a major part of antiviral activity in polyphenols probably derives from their direct inactivation of the virus and or from the inhibition of the virus binding to the cells. They also noted the inhibitory effect of polyphenols on the viral replication enzymes. Kurokawa et al. (1998) extracted the phenolic compound eugeniin (ellagitannin) from Genum japonicum and Synzium aromaticum and found it to be having anti-HSV activity. Kurokawa et al. (1998) and Liu et al. (1999) reported that one of the major target sites of inhibitory Oil 3.5 3 MIC (mg/ml) [Indian Journal of Animal Sciences 80 (4) 2.5 2 1.5 1 0.5 0 P.m. S.a. E.c. S.g. S.z. Bacteria S.z. Streptococcus zooepidemicus S.a. Staphylococcus aureus, S.g. Salmonella Gallinarum P.m. Pasteurella multocida E.c. Escherichia coli Fig. 2. Minimum inhibitory concentration (MIC) of the extracts (mg/ml) against bacteria. species resistant to the antibiotics. Dormans and Deans (2000) observed that the volatile oils of clove exhibited inhibitory effects against all the organisms under test mainly Escherichia coli, Salmonella pullorum, Staphylococcus aureus, Bacillus subtilis, Clostridium sporogenes, etc. and were reported to be equally effective against Gram + and Gram – organisms. Kim et al. (1995) studied the antibacterial activity of some essential oil components including eugenol from clove oil against the food borne pathogens at 5, 10, 15 and 20% levels using paper disc method and observed MIC for eugenol to be 1000 μg/ml for all the bacteria tested. Eugenol showed dose related increase in the zone of inhibition against all pathogens. Suresh et al. (1992) found eugenol to be sensitive at a concentration of 5 g/assay against E. coli, Enterobacter sakazaki and Klebsiella pneumoniae which were resistant to antibiotics like ampicillin, erythromycin and sulfamethizole. The antibacterial activities of the standard antibiotics, viz. gentamycin, ciprofloxacin, cephotaxime and cefixime are mentioned in Table 1. The extracts were screened for antiviral activity against EHV 1 and IBD virus as well. The cytotoxic concentration Table 1. Inhibition zone diameter (mm) of extracts against bacteria (mean ± SE) Plant extract/antibiotics Aqueous extract Alcoholic extract Oil Gentamycin Cephotaxime Ciprofloxacin Cefixime - means no activity S.z. S.g. E.c. S.a. P.m. 19.00±0.57 15.00±0.00 24.00±0.00 12 26 21 16 21.00±0.57 15.67±0.33 23.33±0.33 24 22 36 24 – 14.33±0.33 16.33±0.33 30 32 30 26 11.67±0.33 17.33±0.67 17.33±0.67 22 31 32 12 20.67±0.67 16.33±0.88 21.83±1.05 24 26 30 24 S.z., Streptococcus zooepidemicus; S.g., Salmonella Gallinarum; E.c., Escherichia coli; S.a. Staphylococcus aureus; P.m., Pasteurella multocida 14 April 2010] ACTIVITY OF EXTRACTS OF SYNZIUM AROMATICUM AGAINST MICROBES 287 Table 2. Cytotoxicity assay and antiviral activity of plant extracts against EHV-1 virus Plant extract Aqueous extract Alcoholic extract Oil Cytotoxicity (μg/ml) Concentration of extract used for antiviral activity (μg/ml) 10 TCID50* 102 TCID50 103 TCID50 104 TCID50 4000 4000 1000 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 2/3 3/3 5000 5000 1250 CPE inhibition assay *Each dilution of virus was used in triplicate.Numerator, number of wells showing no CPE, Denominator, total number of wells per dilution of virus. action of eugeniin is viral DNA synthesis. Meerbach et al. (2001) while working with 17 polyhydroxycarboxylates, a phenolic compound, against herpes simplex virus type 1 (HSV 1), HSV 2, thymidine kinase deficient HSV 1, human cytomegalovirus (HCMV) and HIV 1 and HIV 2 have attributed the antiviral properties of phenolic compounds to the presence of the carboxylic groups which cause inhibition of the virus adsorption. Phenolic compounds which possess C3 side chain at a lower level of oxidation and containing no oxygen are classified as essential oils such as Eugenol and often cited as antimicrobials (Cowan 1999). This study is an evaluation of the antibacterial and antiviral activity of Synzium aromaticum against some important selected pathogens (bacteria and viruses) of veterinary importance, which are known to cause huge losses to livestock and poultry industry. Synzium aromaticum was found to be effective against all the selected bacteria while antiviral properties were found against EHV 1 virus only. Active phytochemicals from Synzium aromaticum in alone and in synergism with drugs especially such as Acyclovir against Herpes virus need to be studied further along with the in vivo trials of the extracts of the plant. Bauer A W, Kirby W M M, Sherris J C and Turch M. 1966. Antibiotic susceptibility testing by a standardized single disc method, American Journal of Clinical Pathology 45: 493–96. Benecia F and Courreges M C. 2000. In vitro and in vivo activity of eugenol on human herpes virus. Phytotherapy Research 14: 495–500. Bruneton J. 1995. Pharmacognosy, Phytochemistry of Medicinal plants. (Lavoisler, France), 265. Chopra R N, Nayer S L and Chopra I C. 1962. Glossary of Indian Medicinal Plants. 3rd edn. Council of Scientific and Industrial Research, New Delhi, India, 7. Chukwurah B K C. 1997. Antimicrobial activity of Holarrhena floribunds stem bark ethanol extracts. Fitoterapia 68: 180–81. Cowan M M. 1999. Plant products as antimicrobial agents. Clinical Microbiological Reviews 12: 564–82. Deans S G, Noble R C, Hiltunen R, Wuryani W and Penzes L G. 1995. Antimicrobial and antioxidant properties of Synzium aromaticum (L.) Merr. And Perry: impact upon bacteria, fungi and fatty acid levels in ageing mice. Flavours and Fragrance Journal 10: 323–28. Dorman H J D and Deans S G. 2000. Antimicrobial agents from plants: antibacterial activity of plant volatile oils. Journal of Applied Microbiology 88: 308–16. Field A K and Biron K K. 1994. The end of Innocerice revisited: resistance of herpes virus to antiviral drugs. Clinical Microbiological Reviews 7: 1–8. Iyenger M A. 1985. Study of Crude Drugs. 2 nd edn. College of Pharmaceutical Sciences, Kasturba Medical College, Manipal, India, 13. Kapoor L D, Singh A, Kapoor S L, Srivastava S N. 1969. Survey of Indian medicinal plants for saponins, alkaloids and flavonoids. Lloyds 32: 297–302. Kim J, Marshall M R and Wei C. 1995. Antibacterial activity of some essential oil components against five food borne pathogens. Journal of Agricultural Food Chemistry 43: 2839– 45. Kokoska L, Polesny Z, Rada V, Nepovim A and Vanek T. 2002. Screening of some Siberian Medicinal Plants for antimicrobial activity. Journal of Ethnopharmacology 82: 51–53. Kurokawa M, Hozumi T, Basnet P, Nakano M, Kadota S, Namba T, Kawana T and Shiraki K. 1998a. Purification and characterization of eugeniin as an anti-herpesvirus compound from Geum japonicum and Synzium aromaticum. Journal of Pharmacology and Experimental Therapeutics 284: 728–35. Liu K C, Lin M T, Lee S S, Chiou J F, Ren S and Lien E J. 1999. Antiviral tannins from two Phyllanthus species. Planta Medica 65: 43–46. Meerbach A, Neyts J, Balzarini J, Helbig B, De Clercq E and ACKNOWLEDGEMENTS The investigation was financially supported by Scientist’s Pool Scheme to the first author from the Council of Scientific and Industrial Research, New Delhi. The authors are thankful to Dean, College of Veterinary Sciences, CCSHAU, Hisar for providing the facilities to conduct the present work. REFERENCES Ahmad I and Beg A Z. 2001. Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multi-drug resistant human pathogens. Journal of Ethnopharmacology 74: 113–23. Ahmad I, Zaiba, Beg A Z and Mehmood Z. 1999. Antimicrobial potency of selected medicinal plants with special interest in activity against phytopathogenic fungi. Indian Veterinary Medical Journal 23: 299–306. Anne Xavier S R, Sivsankari V and Mary D P A. 2001. Antibacterial activity of the essential oils of Anisomelous malabarica R. Br. and Ocimum gratissimum Hook. Indian Journal of Microbiology 41: 231–32. Arora D S and Kaur J. 1999. Antimicrobial activity of spices. International Journal of Antimicrobial Agents 12: 257–62. 15 288 VIRMANI ET AL. Wutzler P. 2001. In vitro activity of polyhydroxycarboxylates against herpes viruses and HIV. Antiviral Chem. Chemotherapy 12: 337–45. Nascimento-Gislene G F, Locatelli-Juliana, Freitas-Paulo C and Silva-Giuliana L. 2000. Antibacterial activity of plant extracts and phytochemicals on antibiotic resistant bacteria. Brazilian Journal of Microbiology 31: 247–56. Prajapati N D, Purohit S S, Sharma A K and Kumar T. 2003. A Handbook of Medicinal Plants – A complete sourcebook. (Agrobios, India). p 226. Sakagami H, Sakagami T and Takeda M. 1995. Antiviral properties of polyphenols. Polyphenol Actualites 12: 30. Scalbert A.1991. Antimicrobial properties of tannins. Phytochemistry 30: 3875–77. Somchit M N, Reezal I, Nur I E and Mutalib A R. 2003. In vitro antimicrobial activity of ethanol and water extracts of Cassia alata. Journal of Ethnopharmacology 84: 1–4. Suresh P, Ingle V K and Vijayalakshmi V. 1992. Antibacterial [Indian Journal of Animal Sciences 80 (4) activity of Eugenol in comparison with other antibiotics. Journal of Food Science and Technology 29: 254–56. Vijayan P, Rsghu C, Ashok G, Dhanaraj S A and Suresh B. 2004. Antiviral activity of medicinal plants of Nilgiris. Indian Journal of Medical Research 120: 24–29. Virmani, Meenakshi, Garg, S.L. and Sharma Anshu 2008. In vitro studies on antimicrobial activity of Allium sativum against bacterial and fungal pathogens of animal health importance. Journal of Immunology and Immunopathology 10(1): 46–49. Virmani N, Verma P C, Panisup A S, Singh B K and Batra Munish. 2004. Studies on neurotropic properties of indigenous strains on EHV–1 in murine model. Indian Journal of Animal Sciences 75(4): 393–96. Vogel A I. 1958. A Textbook of Practical Organic Chemistry. pp 90–92. Longman, Green and Co. Ltd. London). Zafar M. 1999. ‘Studies on antimicrobial properties of some Indian medicinal plants.’ PhD thesis submitted to Aligarh Muslim University, Aligarh (India). 16 Indian Journal of Animal Sciences 80 (4): 289–305, April 2010 Uterine torsion in bovines: a review S P S GHUMAN1 Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab 141004 India Received: 7 August 2009; Accepted: 30 October 2009 ABSTRACT The bovines are at a higher risk of uterine torsion around the start of parturition process. Various existing suppositions concerning the maternal and the fetal destabilizing factors liable for the occurrence of uterine torsion in bovines are unrealistic, however some of these have been justified by logical interpretations. Considerable correlation exists between the constriction of uterine blood vessels and the degree of uterine torsion. This is illustrated by the decrease in blood circulation to uterus with an increase in the degree of torsion. Pathological changes of uterus and cervix are severe with an increase in severity (higher degree of torsion persisting for a prolonged duration) of uterine torsion. The occurrence of uterine torsion increases adrenocortical activity and influences blood vascular cellular components as well as the metabolism of liver, kidney and muscular system. The recorded alterations in blood parameters are suggestive of deteriorating condition of the dam and thus help to decide about the institution of various therapies, viz. anti-stress, liver protection and electrolyte therapy. For considering the prognosis of a uterine torsion case at the time of presentation, the patients can be categorized into either of the 3 stages, viz. stage of positive prognosis, less positive prognosis or poor prognosis. Duration and degree of torsion is taken into account while deciding about the survival prognosis of unborn calf and dam as well as the future reproductive health of dam. Prognosis is best when duration of torsion is <36 h and worsens with the further elapse of time. Ovariohysterectomy is considered to increase the chances of survival of bovines with severe uterine torsion and uterine tissue compromise. At the end, acute nature of this emergency warrants timely diagnosis and treatment. Key words: Bovine, Dystocia, Physiopathology, Survival, Uterine torsion of great importance due to its incidence. Uterine torsion cases in buffaloes are 67–83% of the dystocia presented at the referral hospitals (Vasishta 1983, Malhotra 1990, Singh 1991a, Prabhakar et al. 1994, Srinivas et al. 2007). The cattle dystocia handled under field conditions or presented at the referral hospitals report uterine torsion incidence as 3–11% and 4–28%, respectively (Morten and Cox 1968, Pearson 1971, Schultz et al. 1975, Manning et al. 1982, Singla et al. 1992a, Menard 1994, Frazer et al. 1996, Laven and Howe 2005). In bovines, the striking feature of uterine torsion is its association with advanced pregnancy and process of parturition. Usually, uterine torsion occurs before the onset or during the late first stage of parturition as the cervix is partially or completely dilated prior to or immediately after the correction of torsion (Wright 1958, Pearson 1971, Nanda and Sharma 1986), and this condition is rarely encountered during the early second stage of parturition (Noakes et al. 2001). Among the referred cases of torsion, pregnancy period is generally complete in 83–85% buffaloes (Prabhakar et al. 1994, Srinivas et al. 2007) and 77–100% cattle (Pearson 1971, Manning et al. 1982, Prabhakar et al. 1994, Frazer et Uterine torsion, first reported in 1766 by Boutrolle, is rotation of the pregnant uterus on its longitudinal axis (Fleming 1930). Since then, occurrence of uterine torsion has been diagnosed in various domesticated species like cattle (Cergolj et al. 1999), buffalo (Prabhakar et al. 1994), doe (Dhaliwal et al. 1986), ewe (Ijaz and Talafha 1999), llama (Hopkins et al. 1991), camel (Cebra et al. 1997), mare (Jung et al. 2008), bitch (Brown 1974) and queen (Thilagar et al. 2005), and even in laboratory species like rabbit (Hobbs and Parker 1990) and guinea pig (Kunstyr 1981). Out of the domestic species mentioned, torsion is limited to a uterine horn or a part of the horn in bitch and queen, whereas in the remaining, torsion is of the uterine body (Berchtold and Rüsch 1993, Prabhakar et al. 1995b, Barrand 2009). In bovines, torsion of the pregnant horn in front of intercornual ligament without the involvement of uterine body or nonpregnant horn is rarely encountered (Matthijsen and Putker 1989, Dhaliwal et al. 1993). As far as bovine species is concerned, torsion of uterus is Present address: 1 Associate Professor (e-mail: ghuman_s @yahoo.co.in), Department of Veterinary Gynaecology and Obstetrics. 17 290 GHUMAN al. 1996, Srinivas et al. 2007). Occasionally, uterine torsion can occur around 2 months (Roberts 1986) or between 5–8th month of pregnancy (Singh et al. 1979, Ruegg 1988, Penny 1999, Biggs and Osborne 2003) as well as when parturition date is overdue by 1–3 weeks (Manning et al. 1982, Frazer et al. 1996) or even in post-parturient bovines (Matthijsen and Putker 1989, Willetto et al. 1996). The incidence of uterine torsion as well as the time of its occurrence in bovines emphasizes its impact on dam’s health and thus the dairy herd profitability (Schönfelder and Hasenclever 2005). Cost-utility analysis of a bovine with uterine torsion indicated that total loss of untreated or euthanized animal could be around Rs 30,000–50,000, mainly due to expenses for the replacement of animal, whereas the loss of a treated animal could be around Rs 5,000–10,000. This includes losses due to calf, reduced milk yield and handling of subsequent conditions, viz. delayed uterine involution, endometritis and infertility (Schönfelder et al. 2003, 2005a). Moreover, torsion of uterus may accompany ovarian vein rupture (Blanchard 1981), rotation of urinary bladder (Kochhar et al. 1994), intestinal obstruction (Dhaliwal et al. 1992), haemoperitoneum (Jadhao et al. 1993), uterine perforation (Pickel et al. 1990) and formation of adhesions of uterus with surrounding viscera (Siddiquee 1988). Thus, looking into the magnitude of adverse impact of uterine torsion in bovines, the purpose of this review is to understand: (a) the reasons underlying the predisposition of bovine uterus to torsion, (b) physiopathological consequences, (c) diagnosis, (d) treatment, (e) events during post-detorsion period, and (f) survival and fertility prognosis of bovines in the event of uterine torsion. [Indian Journal of Animal Sciences 80 (4) horns is minimized (Berchtold and Rüsch 1993, Chaney et al. 2007). However, bovine uterus is conducive to torsion due to the facts that: (a) bovines have sub-ilial attachment of broad ligaments, (b) broad ligaments are attached along the lesser (ventral) curvature of uterus, thus leaving the greater (dorsal) curvature free, (c) uterine horns are not fixed by the broad ligaments but are lying free, and (d) as the pregnancy advances in bovines, there is a relatively small increase in the length of the broad ligaments but the pregnant horn extends massively beyond the area of attachment (Wright 1958, Pearson 1971, Schulz et al. 1975, Sloss and Dufty 1980, Manning et al. 1982, Baker 1988). This relatively unstable anatomical arrangement predisposes bovines to uterine torsion during the last trimester of pregnancy (Roberts 1986, Noakes et al. 2001). This concept is supported by the fact that uterine torsion is extremely rare in cases of bicornual pregnancy and feto-pelvic disproportion, both conditions tend to stabilize the broader base of pregnant uterus, and thus prevent uterine torsion (Manning et al. 1982, Roberts 1986, Noakes et al. 2001). Nevertheless, a rare case of uterine torsion was reported during twin pregnancy (Wardrope and Boyes 2002). Between bovine species, anatomical variability in the attachment of broad ligaments to uterus seems to have a role in predisposition of pregnant uterus to torsion. The likelihood of torsion is less in Bos indicus cattle compared to Bos taurus cattle, crossbred cattle and buffaloes (Prabhakar et al. 1994, Frazer et al. 1996, Srinivas et al. 2007, Aubry et al. 2008). In Bos indicus cattle, pregnant uterus is more stable due to attachment of broad ligaments on the dorsal side in the anterior two-thirds of uterus and on the ventrolateral side in the posterior one-third of uterus (Sloss and Dufty 1980, Roberts 1986). In Bos taurus cattle, crossbred cattle and buffaloes, pregnant uterus is unstable due to attachment of broad ligaments on the ventrolateral side of uterus (Wright 1958, Sloss and Dufty 1980, Singh 1991b, Noakes et al. 2001). Additionally, higher incidence of uterine torsion in buffaloes than cattle is partly due to the big length of broad ligaments in buffaloes which makes the pregnant uterus less stable in buffaloes as these ligaments position the uterus at time of parturition to facilitate the expulsion of fetus (Singh 1991b). Musculature of the broad ligaments: Role of broad ligament musculature in the occurrence of uterine torsion is highlighted from the observation that broad ligaments of bovines suffering from uterine torsion are thin and have less muscles compared to their counterparts with other types of dystocia (Singh 1991b). At least 25% females born to uterine torsion affected dams and 11% non-pregnant buffaloes have poorly developed muscles in broad ligaments (Brar et al. 2008). Additionally, broad ligament musculature is better arranged in cattle compared to buffaloes, thus providing better stability to the pregnant uterus of cattle (Prabhakar et al. 1994, Brar et al. 2008). Predisposition of bovine uterus to torsion A number of concepts exist for explaining the predisposition of bovine uterus to torsion; however, the maternal and the fetal destabilizing factors behind occurrence of uterine torsion are not well understood (Schönfelder and Sobiraj 2005). The proposed concepts confront with reality as only some and not all the bovines develop torsion of uterus. In fact, uterine torsion is hypothesized to be of fetal and not maternal origin as the affected animal is not predisposed to torsion in the future pregnancies (DeBruin 1910, Taylor 1942). Maternal destabilizing factors Attachment of the broad ligaments: The uterus is held in its position mainly by the mesometrium which consists of 2 folds of peritoneum called the broad ligaments. The importance of attachment of broad ligaments with regard to occurrence of uterine torsion can be observed from the fact that in mares, torsion of uterus is less frequent because the attachment of broad ligaments is sub-lumbar and the ovaries are fixed in the lumbar region, thus the mobility of uterine 18 April 2010] UTERINE TORSION IN BOVINES Enlargement of the pregnant uterine horn: A hypothesis was proposed that pregnant uterine horn due to its larger mass and imbalanced suspension protrudes ventrally and leads to rotation of uterus in the direction of pregnant horn (Desliens 1967, Pearson 1971). However, this hypothesis appears doubtful as in 80% uterine torsions, the direction of torsion is towards non-pregnant horn (Gloor 1973). Moreover, in cattle with didelphic uterus, in which nonpregnant horn is absent, torsion of the pregnant horn without the involvement of rudimentary accessory horn is reported during consecutive pregnancies (Dhaliwal et al. 1988, 1989). Location of the pregnant uterine horn: For stability, the pregnant uterine horn is usually located inside bursa supraomentalis (Schönfelder and Sobiraj 2005). However, in 80% cattle with uterine torsion, pregnant horn is usually present outside bursa supraomentalis as observed at the time of caesarean section (Gloor 1973). This may lead to instability of pregnant horn, and thus dam is predisposed to the torsion of uterus (Schönfelder and Sobiraj 2005). Unfilled rumen: The role of rumen in preventing torsion of uterus is evident from the fact that presence of rumen on left side increases the incidence of right side uterine torsion (Siddquee 1988). If rumen is unfilled, space in the abdominal cavity is increased and the relatively unstable pregnant uterus gets predisposed to torsion, especially in case of sudden fall and the way bovines normally get up and lie down (Moore and Richardson 1995, Drost 2007). Furthermore, the type of diet and hence the rumen volume is expected to be the reason behind variation in incidence of uterine torsion (1–7% of dystocia) between Australian, European, North American and British literature. In some regions, bovines that are reared on concentrate diet have small rumen volume compared to bovines reared on pasture, and thus there is more space in the abdominal cavity for the unstable pregnant uterus to rotate (Morten and Cox 1968, Sloss and Dufty 1980, Manning et al. 1982). Body frame of the dam: It was hypothesized that large framed Brown Swiss cattle is likely to have torsion of uterus compared to other cattle breeds due to their roomy abdomen (Desliens 1967, Elmore 1993). Moreover, capacious and pendulous abdomen seems to facilitate easy rotation of pregnant uterus in buffaloes compared to cattle and in pluriparous buffaloes compared to primiparous buffaloes (Sloss and Dufty 1980, Siddiquee 1988, Singh 1991b). This concept can be ruled out as the body frame (scapula to tuber sacral distance as well as depth of the abdomen at fifth lumbar vertebra) of cattle suffering from uterine torsion had no relation with torsion of uterus (Berger-Pusterla 1995). Type of housing: Stall fed, and not the pastured, bovines were proposed to be more prone to uterine torsion (Williams 1948, Agarwal 1987). Confinement of animals in stables for long periods may lead to weakness of the abdominal muscles due to lack of exercise and thus may support the occurrence of uterine torsion (Sloss and Dufty 1980). This concept is 291 not supported by all studies in which either pastured cattle are at higher risk or the incidence is similar between stall fed and pastured cattle (Wright 1958, Desliens 1967). Nevertheless, a stall fed pregnant cattle housed in a group is at the risk of torsion of uterus due to the chance of being bumped on its side by the accompanying cattle (Aubry et al. 2008). Sudden movements of the dam: Advanced pregnant bovines may get predisposed to uterine torsion following sudden fall, sudden push from other animal and bumpy movements during transportation (Schulz et al. 1975, Elmore 1993, Berger-Pusterla 1995, Moore and Richardson 1995). In these cases, the fetus may respond with violent movements, and thereafter the heavy uterus may take time to return to its original position, while the dam may change their position quickly to expose unstable pregnant uterus to torsion (Rüsse 1963, Schönfelder and Sobiraj 2005). In addition, while lying down, bovines go down on forelegs first and while getting up, the hindquarters are elevated first, thus each time, the pregnant uterus is temporarily suspended in the abdominal cavity and is prone to torsion (Drost 2007). However, other contributory factors must be present in addition to instability that occurs during sudden movements of the dam, otherwise uterine torsion would have been frequent in advanced pregnant animals compared to during the first stage of parturition (Frazer et al. 1996, Amer and Hashem 2008). In addition, history of transportation and fight between animals is absent in uterine torsion cases presented at a referral hospital (Nanda and Sharma 1986). Unsteady walk: None of the uterine torsion affected buffaloes presented at a referral hospital had the history of wallowing or grazing on hills (Nanda and Sharma 1986). Unsteady walk of buffaloes in ponds at the time of wallowing was ruled out as a predisposing factor for uterine torsion when attempts to induce torsion in advanced pregnant buffaloes by forced wallowing, for an hour daily for about a month, were not successful (Agarwal 1987). Moreover, minor differences in the incidence of uterine torsion in bovines reared either in the hilly tracts or the plains rules out the concept of predisposition to uterine torsion during unsteady walk (Ishaque et al. 1960, Singh 1991a). Age of the dam: The influence of age on occurrence of uterine torsion remains controversial, as there is no age predisposition in torsion-affected buffaloes and cattle of 2– 18 years age (Manning et al. 1982, Agarwal 1987, Tamm 1997). Others propose that 70–77% torsions occur in the pluriparous and 23–30% in the primiparous cattle and buffaloes (Pearson 1971, Roberts 1986, Singla 1988, Srinivas et al. 2007). The proposed reasons include larger abdominal cavity, stretching of pelvic ligaments, loose and long broad ligaments together with loosening of uterine tissue and decreased uterine tone in old aged bovines (Roberts 1986, Berger-Pusterla 1995, Drost 2007, Aubry et al. 2008). On the contrary, cattle with minimum 5 parities were at less risk 19 292 GHUMAN of uterine torsion, as 74% uterine torsions occur in cattle during first to third parity (Pearson 1971, Frazer et al. 1996). In addition, increased thickness of uterine muscles in multiparous bovines rejects the concept of loosening and destabilization of uterine tissue (Mochow and Olds 1966). Plasma hormonal profiles: Low progesterone and high estradiol are essential for the onset of uterine contractions at the onset of parturition, whereas high progesterone and low estradiol during immediate pre-partum period may make the uterus flaccid, and hence increases risk of its torsion (Bugalia et al. 1995, Nanda and Sharma 1986, Abdel-Ghaffar and Abou-El-Roos 2002, Prabhakar et al. 2007, Amer and Hashem 2008). The reason for hormonal imbalance could be non-conversion of progesterone to estrogen by nonfunctional placenta (Agarwal 1987, Siddique 1988) and partial degeneration of corpus luteum of pregnancy (Nanda and Sharma 1986). [Indian Journal of Animal Sciences 80 (4) cause the rotation of uterus (Schönfelder and Sobiraj 2005). In addition, reduced amount of amniotic fluid decreases size of uterus which allows free intra-abdominal movement of uterus followed by the occurrence of uterine torsion (Schönfelder and Sobiraj 2005). Fetal movements and uterine tone: About 90% uterine torsions are encountered during the late first stage of parturition process (Noakes et al. 2001). At this stage, conditions favourable for torsion are created because cervix has started to dilate, uterus has begun to contract and gets molded on fetus (Taylor 1942). However, some active force must be present during the late first stage of parturition that should have a role in the development of uterine torsion, otherwise torsion would have occurred frequently during the late pregnancy (Frazer et al. 1996). It is hypothesized that during first stage of parturition, the forces impulsive for the rotation of unstable uterus are strong intrauterine movements of fetus that are invoked by myometrial contractions, changes in intra-uterine pressure as well as changes in fetal blood flow (Dufty 1973, Jose et al. 1983, Duncanson 1985, Baker 1988). In addition, at this stage, uterine muscles are not in much tone, thus uterus is not able to restrict the movement of upper portion of fetus and the relaxed and unstable uterus may be a cause for the occurrence of uterine torsion (Rüsse 1963, Schönfelder and Sobiraj 2005). In fact, uterine instability may induce torsion only up to 180º, whereas torsions of ≥360º require active fetal movements (Noakes et al. 2001). Fetal destabilizing factors Calf birth weight and sex: The concept that excessive calf birth weight is a predisposing factor for the development of uterine torsion gets support from the observation that 90% cattle with uterine torsion deliver calves which have birth weight above the breed average (Wright 1958, Pearson 1971, Frazer et al. 1996). During normal parturition, average size fetus is able to rotate and flex its limbs within the boundaries of uterine wall, however, when fetus is oversized, fetal limbs may get entangled in the uterine wall and the continued vigorous movements of fetus may lead to rotation of uterus (Frazer et al. 1996). On the contrary, uterine torsion is rare in cases of feto-pelvic disproportion (Aubry et al. 2008). Other studies indicate that neither calf birth weight nor sex have influence on the predisposition of dam to torsion of uterus (Potjans 1988, Prabhakar et al. 1994, Berger-Pusterla 1995, Matharu and Prabhakar 2001). Pattabiraman et al. (1979) and Frazer et al. (1996) recorded that 63–69% calves from torsion-affected dam are male, whereas others record 55–66% as female (Vasishta 1983). Fetal presentation: Abnormal presentation may not be a factor in the development of uterine torsion (Wright 1958, Sloss and Dufty 1980). In fact, 80–100% calves from uterine torsion-affected bovines usually deliver in anterior presentation with majority in dorso-ilial (17%) or dorso-pubic (43%) position (Vasishta 1983, Prabhakar et al. 1994, Frazer et al. 1996, Drost 2007, Aubry et al. 2008). This is in concurrence with expected proportion of calvings in posterior presentation (5–10%) among all normal calvings in bovines (Noakes et al. 2001). Reduced amount of the amniotic fluid: This is generally observed in cattle with uterine torsion and leads to decrease in distance between the fetus and the uterine wall (Zimmermann 1950, Uray 1956). Hence, the fetus feels abrupt movements of dam as a painful stimulus and in response, performs strong reflexive movements which may Physiopathological alterations following uterine torsion Uterine blood flow: Rotation of uterus compresses middle uterine vein which results in disturbances in venous circulation and increases carbon dioxide tension in the fetal blood. Consequently, uncomfortable fetus makes vigorous movements that may further increase the degree of uterine torsion. With the increase in degree of torsion, there is compression of middle uterine artery and oxygen going to the fetus is decreased (Schultz et al. 1975, Schönfelder et al. 2005b). Doppler ultrasound of uterine arteries in 360° uterine torsion reveals that blood flow is almost reduced to zero and there is a good correlation of the degree of torsion with the constriction of uterine arteries and blood flow resistance as well as velocity (Schönfelder et al. 2005b). Electrocardiogram alterations following experimental induction of uterine torsion in buffaloes also reveal cardiac abnormalities (Siddiquee et al. 1992). Limited arterial perfusion and venous outflow in the twisted uterus leads to ischemia, hypoxia and cell death causing irreversible damage to the endometrium, myometrium and ultimately death of the fetus. Continued failure of blood supply results in loss of uterine wall elasticity and viability, and hence the uterine wall becomes necrosed, brittle, fragile and prone to rupture (Baker 1988, Pearson and Denny 1975, Tamm 1997, Noakes et al. 2001). With 20 April 2010] UTERINE TORSION IN BOVINES prolonged obstruction, inflammation progresses, and bacterial infection can spread to fetus, amniotic fluid, placenta and uterine wall (Frazer et al. 1996, Schönfelder et al. 2005b). Inflammatory changes can cause adhesions of uterus with surrounding abdominal tissues (McEntee 1990, Sell et al. 1990). Ultimately, delay in correction of uterine torsion causes death of the dam due to generalized bacteremia, endotoxemia or cardiovascular failure (Roberts 1986). Pathological changes in the uterus: Macroscopically, following rotation of uterus, the colour of uterine wall changes from rose-pink to blue-purple and grey indicating the progressive metabolic deterioration of uterus (Schönfelder et al. 2007b). Uterine wall may become inelastic, jammed and in extreme cases can reach a thickness of 8–12 cm from an initial value of <2 mm (Kuller 1960, Rüsse and Grunert 1993). The surface of uterus may reveal congested vessels and hematoma. Amniotic fluid is usually blood-coloured with strong edema of fetal membranes and circular hematoma around placentomes. Congestion, edema and hematoma is present in the uterine ligaments, mesoovarium and ovaries (Schönfelder et al. 2007b). Histological examination of endometrium reveals haemorrhage, edema, thrombosis, coagulative necrosis and inflammation with varying degree of endometrial gland and stroma degeneration (Malik 1986, Matharu 1997). The damage to uterine tissue and its regenerative potential following the rotation of uterus can be accessed from plasma indicators, viz. haptoglobin and creatine kinase. Haptoglobin, an acute phase protein, is a good indicator for the degree of uterine tissue impairment following spontaneous or assisted delivery in bovines (Eckersall and Conner 1988, Horadagoda et al. 1999, Schönfelder et al. 2005c). Uterine-torsion affected bovines exhibit an increase in plasma haptoglobin, which increases further following correction of torsion by the rolling of dam (Schönfelder et al. 2006, Ghuman et al. 2009). During post-delivery period, plasma haptoglobin remains high in detorted animals compared to their counterparts in which fetal delivery was achieved by fetal mutations (Ghuman et al. 2009). Surgically corrected cases of uterine torsion in cattle revealed correlation of plasma haptoglobin with the severity of disease, wound healing and secondary diseases. In addition, an increase of plasma haptoglobin in about 1 week after surgical treatment of torsion can help in predicting the regenerative potential and future fertility of uterus (Schönfelder et al. 2005c). Moreover, the plasma picture of creatine kinase, a muscle-specific enzyme, can reveal time required by injured uterine muscles for regeneration in torsion-affected animals (Schönfelder et al. 2007a). Clinical consequences of the pathological changes in uterus appear more severe with increasing degree and duration of the torsion. With >360° torsion, there is massive edema, muscle fibre degeneration and necrosis. In long standing cases, uterine wall becomes brittle and disintegrating, and is prone to rupture either spontaneously 293 or at the time of detorsion. In rescued cases of torsion, hypoxemia and regenerative inflammation have a decisive influence on prospective fertility (Pearson and Denny 1975, Dzuba 2000). Cervical damage: The viscoelastic properties of cervix responsible for the dilatation of cervix are disturbed following the torsion of uterus (Breeveld-Dwarkasing et al. 2003). Depending upon the degree and duration of torsion, there is variable amount of cervical ischemia leading to hypoxic degeneration of cervical epithelium, marked fragmentation of elastic fibres and irreparable coagulative necrosis of smooth cells in the cervical tissue (Singla et al. 1989). A recent study has categorized the cervical condition of uterine torsion-affected animals on the basis of per-vaginally palpable cervical texture and its correlation with histopathological alterations of the cervix (Honparkhe et al. 2009). Class-A cervix has soft and smooth cervical texture without any lobulations. Histopathology of this type of cervix reveals hemorrhage, congestion, edema, occasional patches of necrosis and intact cervical wall. Class-B cervix is moderately soft and partially lobulated with marked necrosis, fibrosis and tearing of cervical wall. Class-C cervix is described as very hard and completely lobulated (Honparkhe et al. 2009). Necrotic changes in cervical epithelium and musculature of class-B and class-C cervix are responsible for their failure to dilate following successful detorsion of uterus (Singla et al. 1989, Honparkhe et al. 2009). In fact, early correction of torsion may prevent cervical fibrosis (Barber 1995). Following vaginal delivery in successfully detorted buffaloes, the basal cell layer of epithelium proliferates, replaces the necrosed epithelium, smooth cells become hypertrophied and elastic and collagen fibres start organizing into denser connective tissue (Singla et al. 1989). Blood cellular components: Uterine-torsion affected buffaloes suffer from normocytic normochromic anaemia (decrease in the RBCs, Hb and PCV) due to accumulation of metabolic waste products or relatively large loss of blood during abnormal parturition (Amer and Hashem 2008). The leukogram of these buffaloes reveals lymphocytopaenia, neutrophilia and monocytosis in association with eosinopenia (Pattabiraman and Pandit 1980, Kaur et al. 1993, Malhotra et al. 1993, Amer and Hashem 2008), which continues till day third postpartum in surgically corrected cases of uterine torsion (Phogat et al. 1991). Erythrocytes are relatively fragile in torsion-affected buffaloes and remain so in animals that fails to survive during post-detorsion period. However, in the survivors, erythrocytes get stabilized by 18 h postdetorsion due to alleviation of stress of torsion (Ghuman et al. 1997b). Liver functions: Normal parturition in bovines has negligible influence on the plasma enzymes (Schönfelder et al. 2007a, Hussein and Abd Ellah 2008), however, following uterine torsion and after its correction by detorsion or surgical treatment, the activities of aspartate amino transferase (AST), 21 294 GHUMAN alanine amino transferase (ALT), glutamate dehydrogenase (GLDH), creatine phosphokinase (CK) and gamma glutamyl transferase (GGT) are increased (Pattabiraman and Pandit 1980, Frazer 1988, Phogat et al. 1991, Singla et al. 1992b, Kaur et al. 1993, Kuhad et al. 1996, Amer and Hashem 2008), which usually gets stabilized within 10 days after surgical treatment of uterine torsion (Schönfelder et al. 2007a). The increase in plasma AST and muscle specific CK is attributed to great muscular exhaustion produced by strong abdominal contractions following uterine torsion (Kraft and Durr 2005, Hussein and Abd Ellah 2008). The increase in liver specific plasma GGT and GLDH in torsion-affected buffaloes that are subjected to caesarean sections indicates hepatic dysfunction (Pearson 1990, Hussein and Abd Ellah 2008). During hepatic dysfunctions, the esterification of cholesterol is disturbed in the liver, however, no consensus can be drawn from the results of plasma cholesterol in torsion-affected bovines, as there is either no alteration (Ghuman et al. 1996, Hussein and Abd Ellah 2008) or concentrations are high (Phogat et al. 1991, Singla et al. 1992b). The latter condition can be due to stress-induced increase in adrenal activity and thyroid hypofunction. Renal functions: At the time of presentation of a uterine torsion case, a substantial increase in plasma urea and creatinine indicates poor prognosis (Frazer 1988, Schönfelder et al. 2007a, Amer and Hashem 2008). In uterine torsion, ureters lying in the broad uterine ligaments are constricted thus the urine output reduces and renal functions may get affected (Schönfelder et al. 2007a). Moreover, the presence of stress-induced decrease in blood flow to kidneys, shock, dehydration and nephropathy resulting from toxic substances liberated by dead fetus may cause acute or chronic renal insufficiency, leading to decrease in the urea and creatinine elimination (Noakes et al. 2001, Amer and Hashem 2008). Plasma proteins: Huge decrease in the total plasma proteins and albumin is consistently observed in torsion affected buffaloes (Pattabiraman and Pandit 1980, Manju et al. 1985, Khatri et al. 1986, Singla et al. 1992b, Amer and Hashem 2008). This hypoproteinemia is associated with liver malfunction and negative nitrogen balance because of reduced protein intake (Rowlands et al. 1980, Schoenfelder et al. 2003). Plasma cortisol: Occurrence of torsion of uterus is a highly stressful event as revealed by the huge increase in plasma cortisol which increases further by 15–30% following detorsion of uterus through the rolling of dam (Ghuman et al. 1996, 1997a, Amer and Hashem 2008). The buffaloes that die subsequent to detorsion of uterus have persistently elevated plasma cortisol during post-detorsion period suggesting that continued presence of stress is detrimental for the dam survivability (Ghuman et al. 1998a). Plasma glucose: Hyperglycemia in torsion-affected buffaloes is related to activation of stress axis and increased cortisol release which leads to gluconeogenesis (Ghuman et [Indian Journal of Animal Sciences 80 (4) al. 1996, Amer and Hashem 2008). In fact, plasma ketones are elevated in these buffaloes due to improper utilization of glucose (Ghuman et al. 1996). Plasma electrolytes: In torsion affected cattle, minor changes can be noticed in plasma calcium, inorganic phosphorus, sodium and potassium; however the presence of low plasma magnesium during post-detorsion period indicates poor prognosis (Schönfelder et al. 2007c). Hypomagnesaemia leads to decrease in activity of various tissues like heart and skeletal muscle, nerve tissue, brain and spinal fluid and liver, which explains muscle tremor, decreased movement, pain and recumbency following obstetrical interventions (Schönfelder et al. 2007c). The importance of plasma magnesium during the initial postoperative period can be further emphasized from the findings that the uterine torsion affected cattle that are subjected to caesarean and ultimately fail to survive have marked hypomagnesaemia and hypomagnesaemia-induced renal potassium loss by day 4 post-caesarean (Thomas 2005, Schönfelder et al. 2007c). During this period, regeneration of liver and all the reactions involving ATP is hampered due to huge requirement of magnesium for its role in activity of around 300 enzymes (Kolb 1985, Rükgauer 2005). The factors responsible for hypomagnesaemia are—stress of uterine torsion and surgery, lowering down of renal threshold for the excretion of magnesium due to high-dose glucose infusion, and reduced food intake during post-caesarean period (Durlach and Rayssignier 1980, Durlach 1992, Rükgauer 2005). Diagnosis History and external signs: Diagnosis is easy when the abnormal symptoms appear at time of parturition. Typical history of a case of uterine torsion will indicate that animal was about to calve, as exhibited by letdown of milk and relaxation of pelvic ligaments, but adequate time has passed and still there is neither the rupture of fetal water bags nor the appearance of fetus from vulvar lips (Wright 1958, Prabhakar et al. 1995a). On the contrary, dam is suffering from tachycardia, tachypnoea, restlessness (frequently gets up and down), and severe abdominal pain (due to stretching of the broad ligament) as manifested by kicking of the abdomen with her hind legs (Wright 1958, Sloss and Dufty 1980, Frazer et al. 1996, Noakes et al. 2001). With the increase in degree of torsion (>270°), the stretch receptors present in the vagina are stimulated and lead to severe abdominal straining (Frazer et al. 1996). If the uterus is not detorted during this period, then the history will indicate that the continuous straining initially exhibited by the animal to deliver the fetus has ceased followed by the tightening of pelvic ligaments and reabsorption of milk. If the condition remains undetected for several days, then appetite diminishes, rumination ceases and faeces become hard (Pearson 1971, Sloss and Dufty 1980, 22 April 2010] UTERINE TORSION IN BOVINES Manning et al. 1982, Ruegg 1988, Singla et al. 1992a, Frazer et al. 1996, Srinivas et al. 2007). At the referral hospitals, history will also suggest that based upon the symptoms of abdominal pain and discomfort, the farmer was misguided by the unqualified practitioners and the animal was said to be suffering from a simple case of digestive disorder and was treated for the same. Later on, when there were no signs of improvement, animal has been diagnosed with uterine torsion by other practitioners and is referred (Personal observations). External signs of uterine torsion like displacement of upper commissure of vulva towards inward, left or right, vulvar edema due to compression of the vaginal veins and lymphatic drainage, and a slight depression of lumbo-sacral vertebrae are not the consistent features (Pearson 1971, Frazer et al. 1996, Schönfelder et al. 2003). Determining time elapsed since the occurrence of uterine torsion: Estimate of duration of torsion based upon the behavioral history provided by the farmer is necessary to have idea about the prognosis of a case. It is not unusual for a torsion to have occurred between 6 and >72 h before the case is presented (Roberts 1986, Prabhakar et al. 1995a). The estimated duration of labor in 63% torsions is between 8–12 h and >12 h in 21% torsions (Manning et al. 1982). In another study, the percentage of torsions presented in <6 h, 6–24 h, 24–48 h, 72–96 h and 96–168 h after onset of torsion is 5, 55, 16, 17 and 7, respectively (Frazer et al. 1996). From a practical point of view, the duration of occurrence of torsion at the time of case presentation to a veterinarian can be judged from the status of pelvic ligament relaxation and mammary gland engorgement. In the buffaloes with uterine torsion of <36 h and 36–72 h, both these signs are usually evident in 90 and 37% cases respectively. Beyond 72 h, milk usually gets reabsorbed and pelvic ligaments are tightened in 80% torsions (Prabhakar et al. 1995a). 295 palpable and cervix is not accessible. In these vaginal folds, if the fingers go to the left side and the hand to right side then torsion is of right side; however the side of torsion needs confirmation by rectal examination (Noakes et al. 2001, Aubry et al. 2008). Pre-cervical uterine torsion: During pre-cervical torsion, the twist of rotated uterus lies on the body of uterus and does not extend beyond the cervix, thus folds on vaginal wall are absent and cervix is approachable during vaginal examination (Noakes et al. 2001). Pre-cervical torsions are more likely to occur during the last trimester (Sloss and Dufty 1980). Bos indicus cattle is more prone to pre-cervical torsion (57%; Roberts and Hillman 1973, Prabhakar et al. 1994, Prasad et al. 2000), whereas the preponderance of torsion is postcervical in Bos taurus cattle (Pearson 1971), cross-bred cattle (56–99%; Singla et al. 1992a, Sharma et al. 1995) and Bubalus bubalis buffaloes (87–99%; Vasishta 1983, Malhotra 1990, Prabhakar et al. 1994, 1997, Sharma et al. 1995, Srinivas et al. 2007). Rectal examination Pre-cervical uterine torsion: During rectal examination, attention should be paid to the course of broad ligaments to rule out pre-cervical torsion. In normal pregnant animal, the broad ligaments can be palpated on the sides of uterus, whereas in pre-cervical (and post-cervical) torsion, the orientation of broad ligaments is altered and these can be felt by crossed and twisted uterus (Noakes et al. 2001). Direction of uterine torsion: Accurate determination of the direction of torsion through rectal examination is necessary prior to making attempts at correction, as detorsion in the wrong direction will worsen the problem (Noakes et al. 2001). The direction of post- or pre-cervical torsion is clockwise (right) or counter-clockwise (left) (Sloss and Dufty 1980, Noakes et al. 2001). The broad ligament ipsilateral to the side of torsion is pulled vertically downward beneath the uterus, whereas the contralateral broad ligament is tightly stretched diagonally above the uterus, thus the examiner’s hand will move in a pouch formed at either right or left side of uterus (Pearson 1971, Manning et al. 1982, Berchtold and Rüsch 1993, Noakes et al. 2001, Drost 2007). Differences exist between bovine species regarding the predominance of right or left uterine torsion. It is proposed that pregnant horn usually rotates over the non-pregnant horn, thus, left torsions are associated with right horn pregnancies, and vice versa (Sloss and Dufty 1980). Moreover, it is presumed that rumen checks the rotation of right horn over to left (Wright 1958, Sloss and Dufty 1980, Noakes et al. 2001). However, both these concepts do not hold good in Bos taurus cattle as 59–75% torsions in this species are towards left (Pearson 1971, Manning et al. 1982, Baker 1988, Frazer et al. 1996, Aubry et al. 2008). In fact, Bos taurus cattle has additional muscular fold on the right broad ligament which prevents the right horn to rotate downward (Singh Vaginal examination Post-cervical uterine torsion: Post-cervical torsions can be easily diagnosed by vaginal examination. About 66–96% torsions are post-cervical in which the twist of rotated uterus extends caudal to the cervix and involves the anterior vagina in rotation (Frazer et al. 1996, Noakes et al. 2001, Aubry et al. 2008). Anterior vagina is the weakest point of the bovine genital tract due to the absence of muscles in the cervical area of broad ligaments (Singh 1991b). In 34% torsions, vaginal involvement is not obvious during vaginal examination as the cervix is approachable. This is due to post-cervical torsion of less degree or if there is pre-cervical torsion (Pearson 1971, Frazer et al. 1996). During vaginal examination, if post-cervical torsion is <180°, then the spiral folds or twists are present in the vaginal wall along an accessible cervix (Pearson 1971, Noakes et al. 2001, Drost 2007). When post-cervical torsion reaches more than one revolution (>180°), then only vaginal folds are 23 296 GHUMAN 1991b). On the contrary, rotation of uterus is predominantly towards right in Bos indicus cattle (83%; Roberts and Hillman 1973, Prabhakar et al. 1994, Prasad et al. 2000), crossbred cattle (79%; Singla et al. 1992a) and Bubalus bubalis buffaloes (95–98%; Vasishta 1983, Malhotra 1990, Prabhakar et al. 1994, Srinivas et al. 2007). In buffaloes, the absence of a muscular fold on right broad ligament increases the possibility of right torsion (Singh 1991b). Degree of uterine torsion: Degree is determined by the number of twists present on the body of uterus. During rectal examination, these are palpable like the twists on a screw. Torsion of >45° may result in dystocia (Sloss and Dufty 1980). Sometimes, dystocia due to fetal positional abnormalities (dorso-ilial and dorso-pubic) is actually a uterine torsion of low magnitude (Morten and Cox 1968, Roberts 1986). In general, torsion of 90°–180° is common, but a marked difference exists between referral and field cases (Wright 1958, Pearson 1971, Manning et al. 1982, Frazer et al. 1996). Torsions of <180° account for 75% torsions handled in the field and 6–15% torsions handled at the referral hospitals (Pearson 1971, Sloss and Dufty 1980, Manning et al. 1982, Aubry et al. 2008). About 57–80% torsions at referral hospitals are of 180°–270° (Manning et al. 1982, Frazer et al. 1996, Aubry et al. 2008). At a referral hospital, where mainly buffaloes are presented, the percentage of torsions of <90°, 90°–180°, 180°–360° and >360° is 12–21, 57–59, 12–28 and 3% respectively (Malhotra 1990, Singla et al. 1992a, Srinivas et al. 2007). Interestingly, in one referral population, 66% torsions are of 360°, although in general, only 1–9% torsions are >360° and torsions of 720°–1080° seldom occur (Pearson 1971, Manning et al. 1982, Ruegg 1988, Frazer et al. 1996, Noakes et al. 2001, Aubry et al. 2008). Peri-uterine adhesions and uterine texture: In delayed cases, rectal examination is necessary to rule out uterine adhesions with the other abdominal structures. If adhesions are present, the examiner will not be able to move his hand on the either side of uterus during rectal examination (Noakes et al. 2001). The palpation of uterine texture is important for determining the prognosis of a case and will be discussed later in this review. [Indian Journal of Animal Sciences 80 (4) and Rudloff 1983). In torsion-affected bovines, clenbuterol administration helps in better assessment of the direction of torsion, easier passage of hand through the vaginal folds, easier rotation of fetus through the vagina and easy detorsion of the uterus. Moreover, the number of surgical deliveries decreases with tocolytics as the uterus is maintained in a relaxed state after detorsion, thus allowing better relaxation and dilation of the birth canal soft tissues (Menard 1994). The likelihood of delivery by surgical procedures is much high if tocolytics are not routinely used in torsion-affected cattle (64–82% versus 23%; Pearson 1971, Manning et al. 1982, Menard 1994). Nevertheless, use of tocolytics is not effective in long-standing torsions and in >360° torsion. In these cases, hampered circulation prevents tocolytics from reaching and activating sufficient number of receptor sites thus leads to poor uterine relaxation (Menard and Diaz 1987, Menard 1994). Per-vaginal rotation of the fetus: Degree of torsion and the amount of cervical dilatation are critical factors for the success of this method (Pearson 1971, Sloss and Dufty 1980, Noakes et al. 2001, Kruse 2004). With rotations of ≤90°, the fetus is easily rocked manually into a normal dorso-sacral position (Drost 2007). Success rate is high if dam is standing, cervix is sufficiently dilated to grasp the fetus and the fetus is live. However, increased fetal size and the weight of reproductive tract can make the correction of torsion difficult (Morten and Cox 1968, Noakes et al. 2001). This method is effective in 96% torsions when correction per-vaginum is attempted as a first choice in the field, however at the referral hospitals, the success rate of this method is 20–62% due to more difficult cases of torsion being referred and delay in handling of torsions (Pearson 1971, Sloss and Dufty 1980, Manning et al. 1982, Frazer et al. 1996). In fact, a field survey reported that manipulation per-vaginum is the treatment of choice, with rolling being the alternate approach (Morten and Cox 1968). Rolling of the dam: Rolling is indicated if the dam is recumbent, the fetus is not approachable due to the severity of torsion, or if the torsion has occurred before the expected time of parturition (Roberts 1986, Noakes et al. 2001). Following the rolling of dam without a plank, detorsion of uterus is successful in 18–100% cases (Roberts and Hilman 1973, Sloss and Dufty 1980, Frazer et al. 1996, Kruse 2004). However, success rate is 84–90% using Schaffer’s method (Schaffer 1946) of rolling in which a plank (12 feet long and 10 inch wide) is placed on the upper paralumbar fossa at the time of rolling (Arthur 1966, Roberts and Hilman 1973, Noakes et al. 2001, Aubry et al. 2008). In Schaffer’s method, theory is to rotate the dam to the same degree and direction to which the uterus has rotated, keeping the fetus fixed by fixing uterus with a plank (Schaffer 1946). In brief, after ascertaining the side of torsion, animal is casted carefully in lateral recumbency on the side of direction of torsion and the front and hind legs are secured Treatment The technique to be selected for detorsion of uterus in bovines varies with expertise of veterinarian, stage of pregnancy, severity of torsion as well as condition of dam, uterus and fetus. The most commonly used techniques are per-vaginal rotation of fetus, rolling of dam and caesarean section. Use of tocolytics prior to uterine detorsion: Sympathomimetic compounds like clenbuterol selectively block smooth muscle contraction, can induce uterine relaxation and their effect on uterus is termed as tocolysis (Ballarini et al. 1978, Zerobin and Kundig 1980, Bostedt 24 April 2010] (A) UTERINE TORSION IN BOVINES (B) 297 (C) Fig. 1. Diagrammatic representation of Sharma’s modified Schaffer’s method for uterine detorsion of buffaloes. (A) Three assistants are standing on the lower end of plank and another assistant is ready to press the upper end of plank, B) and C) while the buffalo is being rolled, two assistants fix the lower end of plank, one assistant moves upon the plank and other assistant modulates the pressure on the plank by pressing the upper end of plank. separately. The plank is placed on the upper paralumbar fossa of dam in an inclined manner with lower end on ground. Next step is to slowly roll over the dam on to its back. For this, the front and hind legs are pulled up and over the recumbent dam. At the same time, an assistant stands on the plank to modulate pressure first on left side (when animal is casted on right side), followed by ventral abdomen and lastly on right side. After each roll, effectiveness of roll is judged by vaginal or rectal examination. If the roll is successful, disappearance of the vaginal spirals or rectal pouch can be immediately palpated by the examiner. If the roll is not successful, then the dam is returned slowly to her original position and the whole procedure needs to be repeated (Roberts and Hillman 1973, Singla et al. 1992a, Noakes et al. 2001). In Egyptian buffaloes, Schaffer’s method was described as the best method for rolling (El-Naggar 1978, Samad et al. 1981). However, thick skin of Indian buffaloes causes skidding of the plank at the time of rolling. Moreover, pendulous abdomen of Indian buffalo warrants greater pressure for the fixation of pregnant uterus. Therefore, modifications were made in Schaffer’s method and the method is termed as Sharma’s modified Schaffer’s method (Fig 1; Singh and Nanda 1996). The modifications made were: (a) alteration in the dimensions of plank (length: 11.9 feet, width: 9 inch and thickness: 2 inch) to suite the buffaloes, (b) while rolling, plank is anchored by 1–2 medium weight assistants who stand still upon the lower end of plank and another assistant moves on the plank, (c) an additional assistant modulates the pressure on the plank by pressing the upper end of plank, and (d) buffalo is rolled quickly. In fact, Sharma’s modified Schaffer’s method of detorsion was developed based upon the principle of lever (fulcrum, load and effort). In this, fulcrum is lower end of plank that does not move, load is the weight of assistants standing and moving on the plank and effort is the force used by the assistant on the upper end of plank (Fig 1). Using this method, the detorsion rate in Indian buffaloes was 90% in comparison to 40% success rate achieved by Schaffer’s method (Singh and Nanda 1996, Srinivas et al. 2007). The success rate of uterine detorsion after rolling of the dam depends upon the location, degree and the duration of torsion. When torsion is pre-cervical and duration is <36 h, 36–72 h or >72 h, the animals successfully detorted are 82, 100 or 67%, respectively (Prabhakar et al. 1997). In buffaloes, detorsion followed by vaginal delivery is easily accomplished when torsion is <180°. With an increase in degree of torsion, more number of rolls is required to detort the uterus as well as the likelihood of vaginal delivery is decreased (Amer and Hashem 2008). It is suggested that if the torsion is not relieved after 3 rolls then failure should be admitted and surgery is indicated (Nanda et al. 1991). Buffaloes subjected to injudicious rolling (>3 rolls) has least survival rate as compared to those where rolling was well planned (Dhaliwal et al. 1991). Depending upon the prior handling of a case of torsion, buffaloes subjected to 1–3 rolls had 44– 78% survival rate, whereas those subjected to more than 3 rolls had 35–56% survival rate (Dhaliwal et al. 1991). In another study, survival rate of buffaloes requiring 1–2 rolls and 3–4 rolls for complete detorsion of uterus was 85 and 43%, respectively (Ghuman et al. 1997a). Excessive adrenal stimulation due to 1 to 2 extra rolls causes slower decline in plasma cortisol during post-detorsion period (Ghuman et al. 1997a). In long standing cases of torsion (>72 h), with apparent reabsorption of milk and tightened pelvic ligaments, attempts to achieve detorsion of uterus are usually unsuccessful due to development of adhesions between the uterus and the adjoining abdominal organs (Dhaliwal et al. 1991). Detorsion of the uterus in these cases is not possible even after detachment of adhesions (Sharma et al. 1995, Luthra and Khar 1999). Both tissue anoxia and serosal injury following the torsion of uterus are important factors in promoting adhesion formation in the abdominal cavity (Henderson 1982). Caesarean section: Most uterine torsions do not warrant surgical intervention and cesarean section is never performed as the first choice. In a study, where 62% uterine torsion affected cattle were subjected to caesarean, only in 35% cases caesarean was the first choice (Frazer et al. 1996). At referral 25 298 GHUMAN hospitals, caesarean is usually attempted in 11–26% torsions, in which all other methods of detorsion had failed or there is failure of complete cervical dilatation subsequent to successful detorsion (Arthur 1966, Manning et al. 1982, Baker 1988, Cergolj et al. 1999, Murty et al. 1999, Amer and Hashem 2008). In these cases, the main hope to deliver the fetus is caesarean and the uterus is untwisted manually after suturing (Drost 2007). Delayed (>72 h) uterine torsions should be directly subjected to caesarean in order to avoid the undue stress of rolling (Prabhakar et al. 1995a). Animal with a friable, septic uterus containing an emphysematous fetus is a poor candidate for the abdominal surgery (Amer and Hashem 2008). While attempting caesarean, the cost of operation and value of the animal should be judiciously considered. In rare instances, flank laparotomy is indicated when a preterm torsion is diagnosed and attempts at correction by rolling the dam are unsuccessful (Roberts 1986). In these cases, if the uterus has not ruptured and the fetus is still alive, the pregnancy may be continued under observation (Morten and Cox 1968). Reducing the stress of uterine torsion: Uterine torsion affected buffaloes are under acute stress as suggested by elevated plasma cortisol and suppressed ruminal, liver and kidney functions (Ghuman et al. 1996, 1997a, 1998a). In addition, the process of detorsion further enhances adrenocortical activity and potentiates the existing stress on the dam. Attempts made to alleviate stress of uterine detorsion by administration of tranquilizers, viz. acepromazine, chlorpromazine or diazepam before subjecting the dam to detorsion process have failed to achieve desired success as revealed by the regular monitoring of plasma cortisol (Ghuman et al. 1997c). However, dexamethasone administration during immediate post-partum period of successfully detorted animals decreases stress and thus increases the chances of survival of dam (Sathya et al. 2005). Moreover, stress-induced oxidative damage is reduced and post-dystocia convalescence is improved when antioxidants like Vitamin E and Selenium are administered to uterine torsion affected bovines (Sathya et al. 2007). As revealed by plasma concentrations of liver enzymes in uterine torsion affected bovines, the first post-operative week should be critically observed and a liver protection therapy should be instituted during this period (Singh 1991a). Uterine torsion cases suffer from respiratory alkalosis without much metabolic alterations which stabilizes following detorsion (Ghuman et al. 1998b). Determination of blood-gas and acid-base parameters followed by appropriate fluid and electrolyte therapy in torsion affected bovines shall prognosticate the chances of survival of dam as well as the calf. As a matter of critical care, monitoring blood-gas and acid-base status as well as electrolytes should be a mandatory procedure in torsion affected bovines (Ghuman et al. 1998b). [Indian Journal of Animal Sciences 80 (4) With the increase in duration of uterine torsion, plasma and blood volume decreases and animal progresses towards dehydration and toxaemia, this suggests the requirement of immediate fluid therapy (Dhindsa et al. 2005, 2007). Torsion cases can be resuscitated successfully using intravenous administration of small amount of hypertonic saline and dextran–40 followed by oral administration of large amount of fresh water. This is a quicker, practical, easy and effective method compared to intravenous administration of huge amounts of normal saline (Kumar et al. 2009a). Dextran–40 can maintain elevated plasma and blood volume for longer durations and thus decreases the degree of dehydration (Kumar et al. 2009b). In fact, alterations in plasma cortisol, albumin and glucose following administration of hypertonic saline, dextran–40 and oral fluids lead to alleviation of stress (Kumar et al. 2009a). Events during immediate post-detorsion period Uterine rupture: Uterine rupture usually occurs when torsion is >270°. This is due to vascular compromise and resulting edematous changes which weakens myometrial fibers (Frazer et al. 1996). In 9–27% torsions, rupture of uterus can occur either at the time of uterine detorsion or during obstetrical manipulations in the field or at the referral hospital (Pearson 1971, Pearson and Denny 1975, Frazer et al. 1996). Duration of occurrence of uterine rupture can be judged from the gross appearance of wound edges and the severity of peritonitis (Pearson 1971). Following uterine rupture, animals can be euthanized or subjected to corrective surgery depending upon the viability of uterus (Pearson and Denny 1975, Frazer et al. 1996). Cervical dilatation subsequent to the detorsion of uterus: Following successful detorsion of uterus, 18–50% animals fail to exhibit complete dilatation of cervix, which warrants cesarean section for fetal delivery (Manning et al. 1982, Malhotra 1990, Frazer et al. 1996, Aubry et al. 2008). Duration of torsion has a significant role in the likelihood that cervix will completely dilate subsequent to detorsion. When buffaloes are subjected to detorsion process in <36 h, 36–72 h and >72 h of occurrence of torsion, the possibility that cervix will dilate and there will be vaginal delivery is 83, 52 and 9%, respectively (Dhaliwal et al. 1991, Prabhakar et al.1995a, Honparkhe et al. 2009). Location of uterine torsion also has impact on the probability of cervical dilatation. Pre-cervical torsion is more detrimental to cervix due to severe ischemia of cervical tissue compared to post-cervical torsion. In bovines with precervical torsion of <36 h, the fetuses were delivered through vaginal route in 67% cases, whereas the cervix failed to dilate in all the animals which had pre-cervical torsion of >36 h (Prabhakar et al. 1997). Uterine contractions induced by live fetus help to achieve complete dilatation of cervix (Breeveld-Dwarkasing et al. 2003). Thus, viability of fetus at the time of uterine detorsion 26 April 2010] UTERINE TORSION IN BOVINES has a major impact on the post-detorsion likelihood of complete cervical dilatation (Honparkhe et al. 2009). In fact, cervix seldom dilates if the fetus is found dead immediately after detorsion (Frazer et al. 1996). It is strongly recommended that following detorsion, if the fetus is dead, the dam should be immediately subjected to cervical dilatation approaches, otherwise leaving even soft (class-A) or moderately soft (class-B) cervix to dilate on its own will lead to complete hardening of cervix (class-C) within 24 h, followed by its failure to dilate (Honparkhe et al. 2009). Otherwise, if the fetus is alive, one may elect to wait a few hours to assess if parturition proceeds to second stage. However, this occurs only in a few cases and waiting period of >3 h is dangerous, especially if the fetal membranes have already ruptured (Pearson 1971). In successfully detorted bovines, non-hormonal and hormonal approaches can be applied to achieve complete cervical dilatation followed by fetal delivery. According to some workers, an indurated cervix is unlikely to dilate and manual stretching of a partially dilated cervix is seldom successful (Pearson 1971, Sloss and Dufty 1980). Others reveal that cervical manipulations through non-hormonal approaches can effectively dilate cervix, in particular, if unborn fetus is dead. In this technique, immediately after detorsion, a soft (class-A) or moderately soft (class-B) cervix should be subjected to cervical massage (three times, 15 min each, at 1 h interval) with warm sodium carboxy methyl cellulose (SCMC) gel (Honparkhe et al. 2009). If the vaginal canal is relaxed and the fetus is not excessively large, sectioning of the cervical rim can be attempted. This approach is contra-indicated if the cervix is thick and indurated (Pearson 1971). Thus, it is sensible that a successfully detorted bovine diagnosed with hard cervix and dead fetus should be immediately subjected to cesarean as non-hormonal approaches are also unable to dilate cervix of hard texture (Honparkhe et al. 2009). Hormones like oxytocin can be used for inducing cervical dilatation in cattle; however literature has not consistently proven its efficiency (Sloss and Dufty 1980, Roberts 1986). Multiple intra-cervical injections of prostaglandin-F2α do not elicit cervical dilatation (Malhotra et al. 1994). Furthermore, hyaluronidase reduces the adhesions of cervical cells by neutralizing hyluronic acid and thus helps in softening of the cervix and cervical dilatation (Sharma and Singh 1984). Hyaluronidase injected into the cervix (through vagina) for cervical dilatation was successful in 83% detorted animals, and thereafter vaginal delivery occurred between 17–27 h, although this needs further validation as the sample size (n=6) of the study is insufficient (Malhotra et al. 1994). Survival of the calf: Survival rate of calves delivered from torsion affected bovines presented at the referral hospitals varied between 4–56% (Pearson 1971, Pattabiraman et al. 1979, Manning et al. 1982, Vasishta 1983, Prabhakar et al. 1994, Sharma et al. 1995, Tamm 1997, Schönfelder et al. 299 2005a). Both the duration and the degree of torsion appear to influence the calf survival rate (Frazer et al. 1996, Amer and Hashem 2008). Calf survival can be as high as 44–58% if there is timely diagnosis and correction of torsion (Sloss and Dufty 1980, Manning et al. 1982). Delay in diagnosis can cause fetal hypoxia due to placental separation even in the presence of intact water bags (Sloss and Dufty 1980, Noakes et al. 2001). Once uterine torsion is corrected, the delay in achieving complete cervical dilation may further compromise calf viability (Sloss and Dufty 1980). Delay of even 2–3 h results in the death of calf (Pearson 1971). Increasing degree of torsion has impact on uterine vascular compromise and hence calf survival (Frazer et al. 1996, Amer and Hashem 2008). Chance of rupture of water bags and blood colored uterine discharge is less with torsion of <90° and increases with the degree of torsion (Amer and Hashem 2008). Condition of the dead calf may be fresh or even autolytic, with 14% being emphysematous, and is influenced by the duration rather than the degree of torsion (Frazer et al. 1996). Survival of the dam: Survival rate of torsion affected bovines varies between 78–95% depending upon whether torsion is handled in the field or at the referral hospital (Pearson 1971, Sloss and Dufty 1980, Frazer et al. 1996). Main factor playing a role in the survivability of dam is time elapsed between the onset of parturition and the correction of torsion. Actually, survival rate in the torsion affected bovine declines linearly (from 87 to 43%) with an increase in the duration of torsion (Prabhakar et al. 1995a). When the buffaloes are subjected to detorsion in <36 h, 36–72 h and >72 h of exhibition of clinical signs of uterine torsion, there is a survival rate of 96, 61 and 35%, respectively (Srinivas et al. 2007). Furthermore, in a study on pre-cervical torsion in buffaloes, dam survival rate at similar time points is 54, 50 and 47%, respectively (Prabhakar et al. 1997). Survival rate of buffaloes which have completely dilated, incompletely dilated or closed cervix at the time of detorsion is 95, 67 and 50%, respectively (Nanda et al. 1991). In these cases, when more time is taken by the dam for complete cervical dilatation, she remains under stress for prolonged period which is fatal for the dam. Moreover, the duration of the torsion and the time taken for complete dilatation of cervix determine the severity of uterine necrosis, fetal putrefaction, maternal toxemia, dehydration, shock and peritonitis which is fatal to dam (Singh and Dhaliwal 1998, Luthra and Khar 1999, Murty et al. 1999, Matharu and Prabhakar 2001, Noakes et al. 2001). Correction method also influences dam survival rate. Torsions in which detorsion is achieved through the manual correction or rolling of the dam are usually discharged on the same day, while most cases subjected to caesarean are hospitalized for 2–7 days (Frazer et al. 1996). Dam survival rate following delivery of calf through vagina or caesarean section is 88–100% and 25–95%, respectively (Pattabiraman 27 300 GHUMAN et al. 1979, Malhotra 1990, Prabhakar et al. 1995a, Sharma et al. 1995, Frazer et al. 1996). The survival rate of torsion affected bovines that are subjected to caesarean for delivery of fetus is also dependent upon the duration of torsion. When caesarean section is conducted in <36 h, 36–72 h and >72 h of occurrence of torsion, the dam survival rate is 100, 57 and 33%, respectively (Srinivas et al. 2007). Also, survival rate of the dams that are first rolled and thereafter subjected to cesarean in <36 h and 36–72 h of occurrence of torsion is 100 and 50%, respectively (Srinivas et al. 2007). In studies where torsion affected bovines are subjected to caesarean, 31–47% animals are euthanized during caesarean due to compromised state of the uterus (Frazer et al. 1996, Amer and Hashem 2008). Torsion affected bovines that usually fail to survive or are euthanized exhibit thrombosed uterine vessels, intra-mural hemorrhage and necrosis, and fibrinous peritonitis (Sloss and Dufty 1980, Roberts 1986). Depending upon injudicious handling of the case, survival rates of torsion affected animals presented in <36 h, 36–72 h and >72 h of occurrence of torsion are 52–86, 29–74 and 32–62%, respectively. Cases around lower range of the survival rate in each time point are those which are first handled in the field, then subjected to unsuccessful detorsion at the referral hospital followed by caesarean. Cases around higher range of the survival rate in each time point have no history of handling in the field and are delivered through vaginal route (Dhaliwal et al. 1991). Attempted detorsion by rolling before caesarean section reduces dam survival rate as the stress of rolling together with delay in removal of dead fetus decreases post-operative chances of survival (Nanda et al. 1991, Dhaliwal et al. 1991). In a study where dam survival was 69%, the main factor responsible for dam mortality was an increase in the degree of torsion (Amer and Hashem 2008). Degree of torsion and thus the vascular compromise influence the development of uterine edema and ischemic necrosis which leads to endotoxic shock (Sloss and Dufty 1980, Manning et al. 1982). [Indian Journal of Animal Sciences 80 (4) examination is a reliable prognostic indicator of congestion and damage to the uterine wall and thus subsequent survival and fertility. Bovines with uterine elasticity have better prognosis and bovines with bulging tense and inelastic uterus have a greater casualty rate and lower fertility (Schönfelder et al. 2003). Thus, with the help of case history, external clinical signs such as mammary engorgement and relaxation of sacrosciatic ligaments and rectal examination, the owner can be informed about the prognosis of the presented case (Pearson 1971, Pattabiraman et al. 1979, Schönfelder et al. 2003). With these parameters, and by taking into account the degree and duration of torsion as well as the condition of fetus, a three-stage based classification is provided for assessing the survival and fertility prognosis of a uterine torsion affected bovine. Stage of positive prognosis: Animals have variable degree (90º–360º) of uterine torsion of short duration (<12–36 h) and exhibit relaxation of pelvic ligaments and complete milk letdown (Prabhakar et al. 1995a, Schönfelder et al. 2005a). During vaginal examination, fetal viability can be checked through partially dilated cervix if the degree of torsion is less. Rectal examination will reveal elastic uterine wall with easily palpable fetal reflexes. Depending upon the degree of torsion, torsion can be easily corrected by per-vaginal detorsion of fetus or by the rolling of dam (Noakes et al. 2001, Hantschmann 2004). Chances of calf survival after correction of torsion are very high if degree is towards lesser side. Survival and subsequent fertility of bovines is >90 and 70%, respectively (Schönfelder et al. 2005a, 2007b). Stage of less positive prognosis: Animals have variable degree (180º–360º) of torsion of long duration (>36–72 h) and only half of them exhibit relaxation of pelvic ligaments and complete milk letdown. In the remaining, milk is reabsorbed and pelvic ligament are tightened (Prabhakar et al. 1995a, Schönfelder et al. 2005a). Rectal examination will reveal elasticity in the uterine wall. Subsequent to detorsion, usually by rolling of the dam, per-vaginal delivery of dead fetus is possible only after cervical massage and manual dilatation of cervix (Honparkhe et al. 2009). About 87% cases will survive when torsion is corrected around 36 h after the occurrence of torsion (Nanda et al. 1991). Subsequent fertility of bovines is 40% (Schönfelder et al. 2005a, 2007b). Stage of poor prognosis: Prognosis for survival is unfavorable as the presented case will have long standing (>72 h) torsion of >180º (Schönfelder et al. 2003, Schönfelder et al. 2005a). In majority of the bovines, milk is reabsorbed and pelvic ligament are tightened (Prabhakar et al. 1995a). Congestion resulting from long standing torsion of uterine vessels causes fetal death and subsequent autolytic and bacterial changes in fetus which lead to a bulged, tense, inelastic and fragile uterine wall with an immovable uterus (Schönfelder et al. 2005a). As the uterine wall is highly damaged, spontaneous uterine rupture can occur if rolling of dam is attempted by the plank method. Prognosis Survival of a torsion affected bovine depends upon the severity of vascular compromise that makes uterus friable, duration of uterine torsion and correct diagnosis followed by judicious manipulation (Sloss and Dufty 1980, Noakes et al. 2001, Drost 2007). Subsequent fertility is negatively correlated with both the degree and duration of torsion (Schönfelder et al. 2005a). After general examination of a uterine torsion affected bovine, the owner is usually impatient to know about the likelihood of survival of dam and fetus, treatment costs, and post-delivery duration of illness and livestock use or future fertility. To respond, a veterinarian requires immediately identifiable parameters that may help to identify the prognosis of case. Elasticity of the uterus that is palpable during rectal 28 April 2010] UTERINE TORSION IN BOVINES Caesarean is usually advised for detorsion of uterus. During post-operative period, there is delayed involution due to overexpansion of uterine structures, perfusion obstruction, inflammation, accumulation of toxins, and myometrial damage (Randt 1993, Frazer et al. 1996, Schönfelder et al. 2005a). During caesarean, if uterine wall is found highly damaged and uterine closure is no longer useful, an immediate ovariohysterectomy could be an alternative treatment, especially because of prospective infertility (Schönfelder and Soboraj 2006a, 2006b, Schönfelder et al. 2005a, 2007b). After the removal of dead fetus, partial (10– 25%) or complete ovariohysterectomy of ischemic or necrosed uterus is a standardized procedure in cattle and buffaloes although this procedure requires expertise to overcome some technical difficulties (Mannari et al. 1982, Cochran and Cochran 1983, Matthijsen and Putker 1989, Singh et al. 2006). Ovariohysterectomy can be lifesaving and should be considered if salvage is desirable to the owner (Schönfelder and Sobiraj 2006a, Singh et al. 2006). At this stage of torsion, dam survival rate is <43% (Nanda et al. 1991). Nevertheless, this stage usually leads to infertility (Schönfelder et al. 2005a, 2007b). 301 appropriate therapies during post-detorsion period. Uterine torsion cases can be categorized into three stages namely stage of positive prognosis, less positive prognosis or poor prognosis. Categorization is based upon the degree and duration of torsion at the time of admission of animal and may provide an appropriate survival and fertility prognosis. Impact of uterine torsion on calf as well as dam mortality and morbidity can be significantly reduced if abnormal signs at the time of parturition are treated as an emergency. REFERENCES Abdel-Ghaffar A E and Abou-El-Roos M E A. 2002. New trends regarding the diagnosis, prognosis and treatment of uterine torsion in buffaloes. Assiut Veterinary Medical Journal 46: 249– 61. Agrawal R G. 1987. ‘Some studies in uterine torsion with special reference to its etiology and treatment in buffaloes.’ Dissertation, Punjab Agriculture University Ludhiana. Amer H A and Hashem M A. 2008. Relationship between clinical and biochemical picture of uterine torsion in Egyptian buffaloes (Bubalus bubalis). The Internet Journal of Veterinary Medicine 4: 1. Arthur G H. 1966. Recent advances in bovine obstetrics. Veterinary Record 79: 630–44. Aubry P, Warnick L D, DesCôteaux L and Bouchard E. 2008. A study of 55 field cases of uterine torsion in dairy cattle. Canadian Veterinary Journal 49: 366–72. Baker I. 1988. Torsion of the uterus in the cow. In Practice 10: 26. Ballarini G, Belluzi G, Beltrami F, Brisighella G, Fanini G B and Signorini G C. 1978. Indications for induction of uterine relaxation in cattle, using clenbuterol. Clinica Veterinaria 101: 766–71. Barber S M. 1995. Complications of chronic uterine torsion in a mare. Canadian Veterinary Journal 36: 102–3. Barrand K R. 2009. Unilateral uterine torsion associated with haematometra and cystic endometrial hyperplasia in a bitch. Veterinary Record 164: 19–20. Berchtold M and Rüsch P. 1993. Birth interference from the mother. Animal obstetrics. pp. 222–44. (Eds) Richter J, Götze R, Grunert E and Arbeiter K. Parey Buchverlag Berlin. Berger-Pusterla J. 1995. ‘Investigations on the aetiology of intrapartum uteri torsio in cattle.’ Dissertation, University of Zurich. Biggs A and Osborne R. 2003. Uterine prolapse and mid-pregnancy uterine torsion in cows. Veterinary Record 152: 91–2. Blanchard T L. 1981 Uterine torsion with ovarian vein rupture in a ewe. Journal of American Veterinary Medical Association 179: 1402–03. Bostedt H and Rudloff P R. 1983. Prophylactic administration of the beta-blocker carazolol to influence the duration of parturition in sows. Theriogenology 20: 191–96. Brar P S, Saigal R P, Sharma R D and Nanda A S. 2008. Histology and histochemistry of broad ligaments in buffaloes. Indian Journal of Animal Sciences 78: 464–67. Breeveld-Dwarkasing V N, Struijk P C, Lotgering F K, Eijskoot F, Kindahl H, van der Weijden G C and Taverne M A. 2003. Cervical dilatation related to uterine electromyographic activity and endocrinological changes during prostaglandin F(2alpha)induced parturition in cows. Biology of Reproduction 68: 536– Future thrust areas Whether genetic reasons underlie the predisposition of buffalo uterus to torsion can be explored. Predicting the chances of cervical dilatation subsequent to detorsion of uterus needs further research. Still there is lack of an appropriate treatment that can improve the chances of cervical dilatation especially in cases where fetus is found dead following detorsion of uterus. Further attempts should be made to reduce the amount of stress inflicted upon the bovines following occurrence of uterine torsion and during detorsion procedures. Studies on evaluation of blood-gas and acid-base parameters in bovines with different duration of torsion have not been carried out. Based upon the results of these studies, the institution of an appropriate fluid therapy can improve the likelihood of dam survival during postdetorsion period. CONCLUSIONS Reassessment of various speculations made for justifying the higher incidence of uterine torsion in bovine in comparison to other species has produced some realistic explanations. Small quantity of fetal fluids and associated decrease in size of uterus at the end of pregnancy seems to be a realistic justification for the occurrence of uterine torsion in bovines. Destabilizing factors such as weak broad ligament musculature, lower tone of uterine muscles, uterus outside bursa supraomentalis along with sudden movements of the dam and the fetus can further add up to probability of occurrence of uterine torsion. Physiopathological alterations suggest that torsion affected bovines are under huge stress and require timely correction of torsion along with 29 302 GHUMAN [Indian Journal of Animal Sciences 80 (4) Durlach J. 1992. Magnesium in clinical practice. pp. 130–67. Fischer-Verlag, Jena. Durlach J and Rayssignier Y. 1980. New data on magnesium and chronic alcoholism. Reviews in Alcohol 26: 1–26. Dzuba L. 2000. Case report—a complicated bovine uterine torsion. Bovine Practice 34: 58–59. Eckersall P and Conner J. 1988. Bovine and canine acute phase proteins. Veterinary Research Communications 12: 169–78. Elmore R. 1993. Focus on bovine reproductive disorders: Diagnosing and treating uterine torsion. Veterinary Medicine 88: 170–73. El-Naggar M. 1978. Evaluation of field oriented treatment of uterine torsion in buffaloes in Egypt. Indian Veterinary Journal 55: 61–67. Fleming G. 1930. Fleming’s Veterinary Obstetrics. pp. 235–50. Baillière, Tindall and Cox, London. Frazer G S. 1988. Uterine torsion followed by jejunal incarceration in a partially everted urinary bladder of a cow. Australian Veterinary Journal 65: 24–25. Frazer G, Perkins N and Constable P. 1996. Bovine uterine torsion: 164 hospital referral cases. Theriogenology 46: 739–58. Ghuman S P S, Nanda A S, Prabhakar S and Sharma R D. 1996. Stress related endocrine and metabolic changes in normally calving and torsion affected buffaloes. Indian Veterinary Journal 73: 1142–46. Ghuman S P S, Sharma R D, Prabhakar S and Nanda A S. 1997a. Plasma cortisol and blood glucose milieu as an index of stress in buffaloes with uterine torsion. Indian Journal of Animal Reproduction 18: 83–84. Ghuman S P S, Nanda A S, Sharma R D and Prabhakar S. 1997b. Osmotic resistance of erythrocytes in uterine torsion affected buffaloes. Indian Journal of Animal Science 67: 139–40. Ghuman S P S, Nanda A S, Sharma R D and Prabhakar S. 1997c. The effect of tranquilizers on stress response in buffaloes affected with uterine torsion. Indian Veterinary Journal 74: 1034–36. Ghuman S P S, Sharma R D, Nanda A S and Prabhakar S. 1998a. Effect of certain clinical procedures and stressors on plasma cortisol milieu in dystocia affected buffaloes. Buffalo Journal 1: 71–79. Ghuman S P S, Sharma R D, Mirakhur K K, Prabhakar S and Nanda A S. 1998b. Acid base status and blood gas tensions of normally calving and uterine torsion affected buffaloes. Buffalo Journal 14: 355–60. Ghuman S P S, Ajeet Kumar and Honparkhe M. 2009. A preliminary study on plasma haptoglobin concentrations in buffaloes following spontaneous or assisted delivery. Buffalo Bulletin 28: 127–30. Gloor H. 1973. Etiology of uterine torsion in the cow. Schweizer Archiv für Tierheilkunde 115: 74–80. Hantschmann B. 2004. Uterine torsion corrected by conservative method in cattle from the perspective of the practicing veterinarian. Tierärztliche Umschau 59: 350–52. Henderson R A. 1982. Controlling peritoneal adhesions. Veterinary Surgery 11: 30–33. Hobbs B A and Parker R F. 1990. Uterine torsion associated with either hydrometra or endometritis in two rabbits. Laboratory Animal Science 40: 535–36 Honparkhe M, Ghuman S P S, Ajeet Kumar, Sood N K, Gupta K and Ahuja C S. 2009. Cervical massage with sodium carboxy 42. Brown A J. 1974. Torsion of the gravid uterus in a bitch. Veterinary Record 94: 202. Bugalia N S, Phogat J B and Umed Singh. 1995. Peripheral plasma progesterone and oestradiol–17ß profiles in buffaloes affected with uterine torsion. Buffalo Journal 12: 353–56. Cebra C K, Cebra M L, Garry F B and Johnson L W. 1997. Surgical and nonsurgical correction of uterine torsion in new world camelids: 20 cases (1990–1996). Journal of American Veterinary Medical Association 211: 600–02. Cergolj M, Tomaskovic A and Makek Z. 1999. Diagnosis and treatment of uterine torsion during pregnancy in cattle. Tierärztliche Umschau 54: 79–83. Chaney K P, Holcombe S J, LeBlanc M L, Hauptman J G, Embertson R M, Eric Mueller P O and Beard W L. 2007. The effect of uterine torsion on mare and foal survival: A retrospective study 1985–2005. Equine Veterinary Journal 39: 33–36. Cochran M L and Cochran J. 1983 Ovariohysterectomy in complicated bovine cesarean sections. Journal of American Veterinary Medical Association 183: 120–21. DeBruin M G. 1910. Abnormal parturition—dystokia. Bovine Obstetrics. pp 144–70. (Ed.) Wyman W E A. W R Jenkins Co, New York. Desliens L. 1967. Torsion of the uterus in the cow—etiology and practical considerations. Bulletin de l’Académie vétérinaire de France 40: 147–56. Dhaliwal G S, Vashista N K and Sharma R D. 1986. Uterine torsion in a goat—a case report. Indian Journal of Animal Reproduction 7: 90–91. Dhaliwal G S, Sharma R D, Singla V K and Prabhakar S. 1988. Torsion of didelphic uterus in a cow. Indian Journal of Animal Reproduction 9: 71–72. Dhaliwal G S, Prabhakar S and Sharma R D. 1989. A note on recurrence of uterine torsion in a cow with didelphic uterus. Indian Journal of Animal Reproduction 10: 171–72. Dhaliwal G S, Prabhakar S, Singh P and Sharma R D. 1991. Effects of injudicious handling of uterine torsion on survival rate of dam in buffaloes (Bubalis bubalis). Pakistan Veterinary Journal 11: 117–19. Dhaliwal G S, Prabhakar S and Sharma R D. 1992. Intestinal obstruction in association with torsion of uterus in a buffalo. Pakistan Veterinary Journal 12: 42–43. Dhaliwal G S, Prabhakar S and Sharma R D. 1993. Torsion of pregnant horn in a cow—A case report. Indian Journal of Animal Reproduction 14: 129. Dhindsa S S, Gandotra V K, Nanda A S, Singh S P S, Jindal R and Brar P S. 2005. Effect of duration of dystocia on haematobiochemical alterations in buffaloes. Indian Journal of Animal Reproduction 26: 117–19. Dhindsa S S, Gandotra V K and Prabhakar S. 2007. Plasma and blood volume in dystocia affected buffaloes. Indian Journal of Animal Sciences 77: 837–39. Drost M. 2007. Complications during gestation in the cow. Theriogenology 68: 487–91 Dufty J H. 1973. Clinical studies on bovine parturition—foetal aspects. Australian Veterinary Journal 49: 177–82. Duncanson G. 1985. Uterine torsion in cattle—a review of 18 severe cases. British cattle Veterinary Association Proceedings. pp. 133–35. 30 April 2010] UTERINE TORSION IN BOVINES 303 1021–23. Kumar A, Ghuman S P S and Honparkhe M 2009b. Effect of oral fluid therapy in combination with intravenous dextran–40 and hypertonic saline solution on plasma and blood volume in dystocia affected buffaloes. Indian Journal of Animal Science 79: 479–82. Kunstýr I. 1981. Torsion of the uterus and the stomach in guinea pigs. Z Versuchstierkd 23: 67–69. Laven R and Howe M. 2005. Uterine torsion in cattle in the UK. Veterinary Record 157: 96. Luthra R A and Khar S K. 1999. Survival rate following different regimes of treatment in cases of uterine torsion in buffaloes. Indian Veterinary Journal 76: 399–402. Malhotra P. 1990. ‘Some studies on the cervical changes in the uterine torsion cases with special reference to cervical filtration in buffaloes.’ M.V.Sc. Thesis, Punjab Agriculture University, Ludhiana, India. Malhotra P, Sharma R D and Singla V K. 1993. Haematological studies in periparturient and with uterine torsion buffaloes. Indian Veterinary Medical Journal 17: 111–14. Malhotra P, Sharma R D, Singla V K and Pangawkar G R. 1994. Effect of hyaluronidase and PGF2a on the dilatation of cervix in buffaloes suffering from uterine torsion. Indian Veterinary Medical Journal 18: 46–47. Malik J S. 1986. ‘Histochemical and histopathological studies on uterine torsin in buffaloes (Bubalus bubalis).’ M.V.Sc. Thesis, Haryana Agriculture University, Hisar, India Manju T S, Verma S K, Gupta R C, Manadskhot V M and Krishnaswamy A. 1985. Profiles of some plasma biochemical constituents associated with uterine torsion and following its correction by laprohysterotomy in buffaloes. Indian Journal of Animal Reproduction 6: 57–61. Mannari M N, Tadkod D M and Parsania R R. 1982. Partial resection of the uterus in she-buffalo. Indian Journal of Veterinary Surgery 3: 103–05. Manning J, Marsh P, Marshall F and McCorkell R 1982. Bovine uterine torsion: a review illustrated by cases from the Western College of Veterinary Medicine, Large animal clinic. Bovine Practice 17: 94–98. Matharu S S. 1997. ‘Endocrinological and histopathological investigations on corpus luteum and uterus of buffaloes affected with uterine torsion.’ M.V.Sc. Thesis, Punjab Agriculture University, Ludhiana, India. Matharu S S and Prabhakar S. 2001. Clinical observations and success of treatment of uterine torsion in buffaloes. Indian Journal of Animal Reproduction 22: 45–48. Matthijsen H F and Putker P H. 1989. Postpartum torsion of the right uterus horn in a cow. Tijdschrift voor diergeneeskunde 114: 17–19. McEntee K. 1990. The uterus: degenerative and inflammatory lesions. In: Reproductive Pathology of Domestic Animals. pp. 143–66. (Ed.) McEntee K. NY Academic Press, New York. Menard L. 1994. The use of clenbuterol in large animal obstetrics: Manual correction of bovine dystocias. Canadian Veterinary Journal 35: 289–92. Menard L and Diaz C S. 1987. The use of clenbuterol for the management of large animal dystocias: Surgical corrections in the cow and ewe. Canadian Veterinary Journal 28: 585–90. Mochow R and Olds D. 1966. Effect of age and number of calvings on histological characteristics of the bovine uterus. Journal of methyl cellulose for achieving complete cervical dilatation in successfully detorted uterine torsion affected buffaloes. Indian Journal of Animal Science 79: 26–29. Hopkins S M, Althouse G C, Jackson L L and Evans L E. 1991 Surgical treatment of uterine torsion in a llama (Lama glama). Cornell Veterinarian 81: 425–28. Horadagoda N, Knox K, Gibbs H, Reid W, Horadagoda A, Edwards S and Eckersall P. 1999. Acute phase proteins in cattle: discrimination between acute and chronic inflammation. Veterinary Record 144: 437–41. Hussein H and Abd Ellah M R. 2008. Effects of dystocia, fetotomy and caesarian sections on the liver enzymes activities and concentrations of some serum biochemical parameters in dairy cattle. Animal Reproduction Science 105: 384–91. Ijaz A and Talafha A Q. 1999. Torsion of the uterus in an awassi ewe. Australian Veterinary Journal 77: 652–53. Ishaque S M, Khan A A, Praced M M, Singh J K and Narayan K G. 1960. Successful caesarean section in a cow with torsion of uterus. Indian Veterinary Journal 37: 321–25. Jadhao P T. Markandeya N M and Rautmare S S. 1993. Uterine torsion along with haemoperitoneum in a buffalo. Indian Journal of Animal Reproduction 14: 59. Jose V M, Neelakanta Iyer C P, Madhavan E. 1983. Positional and postural changes of the foetus prior to and during parturition in cattle. Aust ralianVeterinary Journal 60: 148–49. Jung C, Hospes R, Bostedt H and Litzke L F. 2008. Surgical treatment of uterine torsion using a ventral midline laparotomy in 19 mares. Australian Veterinary Journal 86: 272–76. Kaur T, Singh B, Kaur T and Singh B. 1993. Reproductive disorders in buffaloes: haemato-biochemical studies. Indian Journal of Veterinary pathology 17: 118–21. Khatri C K, Khar D K, Singh J and Luthra R A. 1986. Changes in biochemical blood constituents of buffaloes with uterine torsion and the effect of caesarean section and certain post-operative therapeutic measures. Archiv für experimentelle Veterinärmedizin 40: 461–68. Kochhar H P S, Prabhakar S, Singh Prakash, Nanda A S and Singh P. 1994. Rotation of urinary bladder with torsion of uterus in a buffalo. Indian Veterinary Journal 71: 388–89. Kolb E. 1985. Recent insights on the biochemical metabolism of the magnesium in ruminants as well as the emergence and treatment of hypomagnesemia with particular attention to the preparation for parturition. Mh Veterinary Medicine 40: 615– 19. Kraft W and Dürr U. 2005. Clinical laboratory diagnostics in veterinary medicine, pp. 6. Aufl. Schattauer, Stuttgart, New York. Kruse M. 2004. ‘Genetic and environmental influences on the occurrence of torsio uteri in dairy cows.’ Thesis, Tierärztliche Hochschule, Hannover. Kuhad K S, Sharma D K, Singh Prem, Nagi R S, Bugalia N S, Khanna B M and Singh P. 1996. Serum transaminases in prolonged cases of uterine torsion in buffaloes. Indian Journal of Veterinary Surgery 17: 115. Kuller H. 1960. Casuistic contributions to the surgical treatment of torsio uteri in cattle and horses. Mh Veterinary Medicine 2: 32–5. Kumar A, Ghuman S P S, Ahuja C S and Honparkhe M. 2009a. Effect of oral fluid therapy on endocrine and biochemical status of dystocia affected buffaloes. Indian Veterinary Journal 86: 31 304 GHUMAN [Indian Journal of Animal Sciences 80 (4) torsion. Indian Journal of Animal Sciences 77: 956–59. Prasad S, Rohit K and Maurya S N. 2000. Efficacy of laparohysterotomy and rolling of dam to treat uterine torsion in buffaloes. Indian Veterinary Journal 77: 784–86. Randt A. 1993. ‘Twins in cattle from obstetric perspective.’ Dissertation, University of Leipzig. Roberts S J. 1986. Diagnosis and treatment of the various types of dystocia. Veterinary Obstetrics and Genital Diseases (Theriogenology). pp. 213–17, 230–33, 337–43, 357–59. Woodstock, Edwards Brothers Inc. Roberts S J and Hillman R B. 1973. An improved technique for the relief of bovine uterine torsion. Cornell Veterinarian 63: 111– 16. Rowlands G J, Manston R, Sturk A J, Bussell A M, Collis K A and Collis S C. 1980. Studies on some blood constituents in pregnant buffaloes. Journal of Agriculture Science Cambridge 94: 517– 27. Ruegg P L.1988. Uterine torsion of 720 degrees in a midgestation cow. Journal of American Veterinary Medical Association 192: 207–08. Rükgauer M. 2005. Magnesium. Laboratory and diagnostics, indication and evaluation of laboratory results for medical diagnosis. 6th edn, pp. 492–94. TH-Books Verlagsgesellschaft mbH, Frankfurt, Germany. Rüsse M. 1963. ‘The birth sequence in cattle-an examination of the functional activity of the normal birth.’ Dissertation, University of Munich. Rüsse I and Grunert E. 1993. Gravid genitalia. Animal obstetrics. pp. 58–61. (Eds) Richter J, Götze R, Grunert E and Arbeiter K. Parey, Berlin. Samad H A, Ali C S and Ahmad K M. 1981. Schaffer’s method for the correction of uterine torsion in the buffalo. Pakistan Veterinary Journal 1: 25–27. Sathya A, Prabhakar S and Ghuman S P S. 2005. Effects of dexamethasone administration on cortisol concentration and biochemical profile in buffaloes suffering from dystocia. Animal Reproduction 2: 233–39. Sathya A, Prabhakar S, Sangha S P S and Ghuman S P S. 2007. Vitamin E and selenium supplementation reduces plasma cortisol and oxidative stress in dystocia-affected buffaloes. Veterinary Research Communications 31: 809–18. Schaffer W. 1946. Schweizer Arch. Tierheilk 88: 44. Schönfelder A, Richter A and Sobiraj A. 2003. Prognostic indicators for conservatively incorrectable uterine torsion in the cow. Tierärztliche Umschau 58: 512–17. Schönfelder A and Hasenclever D. 2005. Cost-utility analysis of surgical obstetrics in bovines with torsio uteri intra partum. Berliner und MünchenerTierärztliche Wochenschrift 118: 490– 94. Schönfelder A and Sobiraj A. 2005. Etiology of torsio uteri in cattle—a review. Schweizer Archiv für Tierheilkunde 147: 397– 402. Schönfelder A, Richter A and Sobiraj A. 2005a. Stages of surgically incorrectable uterine torsion of cows: associations with clinical progress. Tierärztliche Umschau 60: 199–205. Schönfelder A, Richter A and Sobiraj A. 2005b. Doppler ultrasound studies of the uterine arteries in cows with spontaneous parturition and with uterine torsion intra partum. Wiener Tierärztliche Monatsschrift 92: 74–80. Schönfelder A, Schrödl W, Krüger M, Richter A and Sobiraj A. Dairy Science 642–46. Moore A A and Richardson G F. 1995. Uterine torsion and fetal mummification in a cow. Canadian Veterinary Journal 36: 705– 06. Morten D H and Cox J E. 1968. Bovine dystocia. A survey of 200 cases met within general practice. Veterinary Record 83: 530– 37. Murty K K, Prasad V and Murty P R. 1999. Clinical observations on uterine torsion in buffaloes. Indian Veterinary Journal 76: 643–45. Nanda A S and Sharma R D. 1986. Studies on serum progesterone levels in relation to occurrence of uterine torsion in buffaloes (Bubalus bubalis). Theriogenology 26: 383–89. Nanda A S, Sharma R D and Nowshehri M A. 1991. Outcome of various regimes of treatment of uterine torsion in buffaloes. Indian Journal of Animal Reproduction 12: 197–200. Noakes D E, Parkinson D J and England G C W. 2001. Maternal dystocias. Arthurs veterinary reproduction and obstetrics, (Ed.) Noakes D E. Saunders Harcourt, India. Pattabiraman S R and Pandit R K. 1980. Studies on haematological and biochemical constituents in blood of buffaloes with uterine torsion. Cherion 9: 338–40. Pattabiraman S R, Singh J, Rathor S S and Dhablania D C. 1979. Non-surgical method of correction of bovine uterine torsion— a clinical analysis. Indian Veterinary Journal 56: 424–28. Pearson H. 1971. Uterine Torsion in Cattle: a review of 168 cases. Veterinary Record 89: 597–603. Pearson H and Denny H R. 1975. Spontaneous uterine rupture in cattle—a review of 26 cases. Veterinary Record 97: 240–44. Pearson E G. 1990. Diseases of the hepatobiliary system. Large Animal Internal Medicine. pp. 837. (Ed.) Smith B P. The C V Mosby Co., St. Louis. Penny C D.1999. Uterine torsion of 540° in a mid-gestation cow. Veterinary Record 145: 230. Phogat J B, Bugalia N S, Verma S K and Singh Inderjeet. 1991. Plasma cholesterol and haemogram in buffaloes affected with uterine torsion. Indian Veterinary Journal 68: 1048–52. Pickel M, Dirksen G and Grunert E.1990. Uterine perforation following an experiment in the correction of a uterine torsion in the 5th month of pregnancy. Tierarztliche Praxis 18: 469– 72. Potjans J. 1988. ‘Contribution to the etiology of torsio uteri in cattle.’ Dissertation, University of Munich. Prabhakar S, Singh P, Nanda A S, Sharma R D and Singh P. 1994. Clinico-obstetrical observations on uterine torsion in bovines. Indian Veterinary Journal 71: 822–24. Prabhakar S, Dhaliwal G S, Sharma R D and Nanda A S. 1995a. Success of treatment in relation to milk letdown and pelvic relaxation in bovines with torsion of uterus. Indian Journal of Dairy Science 48: 323–25. Prabhakar S, Kochhar H P S and Nanda A S. 1995b. Torsion of one uterine horn in a primigravid Great Dane Bitch. Indian Veterinary Journal 72: 416–17. Prabhakar S, Dhaliwal G S, Sharma R D and Nanda A S. 1997. Success of treatment and dam survival in bovines with precervical uterine torsion. Indian Journal of Animal Reproduction 18: 121–23. Prabhakar S, Matharu S S, Gandotra V K and Brar R S. 2007. Corpus luteum does not contribute to higher progesterone concentration around parturition in buffaloes with uterine 32 April 2010] UTERINE TORSION IN BOVINES 305 buffaloes—Modification of Schaffer’s method. Indian Journal of Animal Reproduction 17: 33–34. Singh M P, Prasad J K, Das A K and Prasad S. 2006. Subtotal hysterectomy of ischemic uterus during caesarean section in buffaloes. Indian Journal of Animal Reproduction 27: 89–90. Singla V K. 1988. ‘Some studies on uterine torsion in buffaloes.’ M.V.Sc. Thesis, Punjab Agriculture University, Ludhiana, India. Singla V K, Roy K S, Sharma R D, Gandotra V K and Dhaliwal G S. 1989. Histopathological studies of cervix and vagina in buffaloes suffering from uterine torsion. Proceedings of VIIIth National symposium of Indian Society for the Study of Animal Reproduction, Anand, Gujrat, India. Singla V K, Sharma R D, Dhaliwal G S, Gandotora V K and Prabhakar S. 1992a. Uterine torsion in cows—an analysis of 34 cases. Indian Veterinary Journal 69: 281–82. Singla V K, Sharma R D, Gandotora V K and Prabhakar S. 1992b. Changes in certain serum biochemical constituents in buffaloes with uterine torsion. Indian Veterinary Journal 69: 805–07. Sloss V and Dufty J H. 1980. Obstetrical physiology. Obstetrical pathology. Obstetrical procedures. Handbook of Bovine Obstetrics. pp. 39, 105, 108–11, 180–83. (Eds) Sloss V and Dufiy J H. Williams and Wilkins, Baltimore. Srinivas M, Sreenu M, Lakshmi Rani N, Subramanyam Naidu K and Devi Prasad V. 2007. Studies on dystocia in graded murrah buffaloes: a retrospective study. Buffalo Bulletin 26: 40–45. Tamm T. 1997. ‘Studies on bovine uterine torsion.’ Dissertation, Tierärztliche Hochschule Hannover. Taylor W A. 1942. Torsion of the uterus: with special reference to epidural anaesthesia. Canadian Journal of Comparative Medicine: Veterinary Science 6: 102–06. Thilagar S, Yew Y C, Dhaliwal G K, Toh I and Tong L L.2005. Uterine horn torsion in a pregnant cat. Veterinary Record 157: 558–60. Thomas L. 2005. Labor and diagnosis: indication and evaluation of laboratory results for medical diagnosis. 6th edn, pp. 450. TH-Books Verlagsgesellschaft mbH, Frankfurt. Uray H. 1956. The bimanual correction of torsio uteri. Wiener Tierärztliche Monatsschrift 43: 610–12. Vasishta N K. 1983. ‘Torsion of uterus in buffaloes in relation to incidence, etiology and treatment.’ M.V.Sc. Thesis, Punjab Agriculture University, Ludhiana, India. Wardrope D D and Boyes G W. 2002. Uterine torsion in twin pregnancies in dairy cattle. Veterinary Record 150: 56. Willetto C E, Morin D E and Constable P D. 1996. Uterine torsion in three post parturient dairy cows. Agriculture Practice 17: 23–27. Williams W L. 1948. The basic causes of dystocia. Torsion of the uterus (Uterine volvulus). Veterinary Obstetrics. pp. 196–199, 295–307. (Ed.) Williams W L, Mass, Ethel Williams Plimpton. Worcester. Wright J G. 1958. Bovine dystocia. Veterinary Record 90: 347–56. Zerobin K and Kundig H. 1980. The control of myometrial functions during parturition with a beta–2 mimetic compound, Planipart®. Theriogenology 14: 21–35. Zimmermann K. 1950. About uterine torsion. Wiener Tierärztliche Monatsschrift 37: 130–34. 2005c. The change of acute phase protein haptoglobin during regular parturition and caesarean section with or without torsio uteri intra partum in cattle. Berliner und Münchener Tierärztliche Wochenschrift 118: 240–46. Schönfelder A and Sobiraj A. 2006a. Cesarean section and overiohysterectomy after severe uterine torsion in four cows. Veterinary Surgery 35: 206–10. Schönfelder A and Sobiraj A. 2006b. Complications during surgical treatment of torsio uteri intra partum in cattle and their handling. Wiener Tierärztliche Monatsschrift 93: 86–89. Schönfelder A, Schrödl W, Krüger M, Richter A and Sobiraj A. 2006. Plasma haptoglobin concentration in bovine with surgically corrected torsio uteri intra partum. Berliner und MünchenerTierärztliche Wochenschrift 119: 81–85. Schönfelder A, Fürll M, Richter A and Sobiraj A. 2007a. Enzyme activities and substrate concentrations in blood plasma of bovines with surgically treated uterine torsion intra partum. Tierarztliche Praxis 35(G): 101–10. Schönfelder A, Schoon H-A and Sobiraj A. 2007b. Macroscopical and histological findings on the uterus in cattle with surgically treated uterine torsion. Tierarztliche Praxis 35(G): 325–34 Schönfelder A, Fürll M, Richter A and Sobiraj A. 2007c. Dynamic of the electrolyte concentration in blood plasma of bovines with surgical treated uterine torsion during parturition. Tierarztliche Praxis 35(G): 414–21. Schulz J, Beuche W and Elze K. 1975. Torsio uteri in cattle. Mh Veterinary Medicine 30: 659–65. Sell F, Eulenberger K and Schulz J. 1990. Use of clenbuterol to control dystocia of cattle. Mh Veterinary Medicine 45: 413–17. Sharma M and Singh V K. 1984. Cervix-hyalse alone and in combination with other drugs. Journal of Obstetrics and Gynaecology of India 34: 1023–31. Sharma S P, Agrawal K B P and Singh D P. 1995. Torsion of gravid uterus and laparohysterektomy in bovine—a report on 72 clinical cases. Indian Veterinary Journal 72: 1180–82. Siddiquee G M. 1988. ‘Studies on etiopathology and therapeutics of uterine torsion in buffaloes.’ Thesis, Punjab Agriculture University, Ludhiana, India. Siddiquee G M, Sharma R D and Sobti V K. 1992. Changes in the electrocardiogram and cardiac vectors following induction uterine torsion in buffaloes (Bubalus bubalis). Proceedings of National Symposium and Xth Annual Convention of Indian Society for the Study of Animal Reproduction, Madras. Singh J and Dhaliwal G S. 1998. A retrospective study on survivability and fertility following caesarean section in bovines. Indian Journal of Animal Reproduction 19: 21–23. Singh J, Nauriyal D C, Pattabiraman S R and Sharma R D. 1979. Uterine torsion at mid-gestation in a buffalo. Indian Veterinary Journal 56: 700. Singh M. 1991a. ‘Studies on changes in blood and ruminal functions in buffaloes with dystocia.’ Thesis, Punjab Agriculture University, Ludhiana, India. Singh P. 1991b. ‘Studies on broad ligament in relation to uterine torsion in buffaloes.’ Thesis, Punjab Agriculture University, Ludhiana, India. Singh P and Nanda A S. 1996. Treatment of uterine torsion in 33 Indian Journal of Animal Sciences 80 (4): 306–312, April 2010 Insulin-like growth factor-I and -II in buffalo ovary: mRNA expressions and partial sequences R DEV1, M K SHARMA2 and DHEER SINGH3 National Dairy Research Institute, Karnal, Haryana 132 001 India Received: 5 September 2008; Accepted: 17 October 2009 ABSTRACT In the present investigation, expressions of mRNAs encoding IGF-I and IGF-II were detected in intact follicles of different sizes, granulosa cells, corpus luteum (CL) and corpus albicans (CA) from buffalo ovary, using semi-quantitative RT-PCR technique. The effect of FSH and LH on expression of mRNAs encoding IGFs and sequencing of partial IGFI and IGF-II cDNAs were studied. The results for expression of mRNAs encoding IGF-I and IGF-II in small (< 5 mm), medium (6–9 mm) and large (> 10 mm) intact follicles and granulosa cells revealed significant increase in mRNAs expression with increase in follicle size. During luteal phase, the presence of expression of mRNAs encoding IGFs in corpus luteum (CL) and corpus albicans (CA) indicated their role in growth, differentiation and regression of luteal tissues. The treatment of granulosa cells culture with varying doses of FSH, did not show any stimulatory effect on expression of mRNAs encoding IGF-I and IGF-II. However, LH induced significantly IGF-I mRNA expression. The partial cDNA products of IGF-I and IGF-II, were sequenced. The blasting of IGF-I sequence (196 nucleotides) with that of other species exhibited the homology in the range of 93–99% whereas it was in the range of 88–100% for IGF-II sequence (154 nucleotides). The results showed that IGF-I and IGF-II play important role during follicular and luteal phases of buffalo ovary. Key words: IGFs, Insulin like growth factor, mRNA Expression, Ovary, Partial cDNA sequences et al. 2000). The expression of mRNAs encoding IGF-I and -II in ovarian follicles is developmentally regulated in a species specific manner (Armsrong et al. 1998). Zhou et al. (1997), suggested that expression of IGF-I mRNA is FSH independent. However, bovine (Spicer and Chamberlain 2000) and ovine (Khalid et al. 2000) granulosa cells during culture, exhibited increased production of IGF-I in response to FSH. Hirakawa et al. (1999) reported that LH enhances IGF-I mRNA expression. In view of the importance of these growth factors and no information on their localization and expression in buffalo ovary, the present investigation was envisaged. In mammalian ovary, the insulin-like growth factor-I and –II (IGF-I and -II) regulate proliferation and differentiation of granulosa cells in an autocrine and/or paracrine fashion synergizing with gonadotropins. The IGFs are mitogenic polypeptides that stimulate cellular proliferation and differentiation (Jones and Clemmons 1995). The IGFs exert their physiological actions by interacting with specific cell surface membrane receptors, called type I (IGF-IR) and type II (IGF-IIR) receptors (Nissley and Lopaczynski 1991). Although IGF-IIR is important for IGF-II internalization and degradation, it is not much clear whether this receptor actively mediates IGF-II signaling. In most instances, the metabolic and growth promoting effects of IGF-II appear to be mediated by IGF-IR (Willis et al. 1998) or insulin receptor. Whether IGF-I or -II is primary stimulus to the ovary, may depend on the species since granulosa cells of human follicles contain IGF-II mRNA and not IGF-I mRNA (Zhou and Bondy 1993) whereas bovine granulosa cells contained both (Armsrong MATERIALS AND METHODS Eagle’s minimum essential medium (MEM) with HEPES, fetal bovine serum (FBS), diethylpyrocarbonate (DEPC), TRI reagent, agarose, chloroform, Trypan blue and ethidium bromide were procured commercially. Collection of buffalo ovaries: Buffalo ovaries were collected in sterile normal saline containing antibiotics (penicillin 1000 U/ml; streptomycin, 100 μg/ml), from New Delhi abattoir. These were further washed thoroughly with normal saline and extra tissues were removed. Present address: 1House No.: 795, First Floor, Sector-5, Gurgaon 122 001 (e-mail: [email protected]) 2, 3Molecular Endocrinology Laboratory, Animal Biochemistry Division. 34 April 2010] INSULIN-LIKE GROWTH FACTORS IN BUFFALO OVARY Isolation of follicles, luteal tissues and granulosa cells: The non-atretic follicles, based on their morphological appearances, were isolated mechanically (Moore et al. 1978) with forcep and scissor asceptically and were pooled based on the size as small (≥ 5 mm), medium (6–9 mm) and large (≥ 10 mm), representing different stages of development. Post-ovulatory structures such as corpus luteum and corpus albicans were isolated based on morphological appearance. The granulosa cells were aspirated from small, medium and large follicles, separately with sterile syringes. The follicular fluid containing granulosa cells was spinned at 2000 rpm for 5 min to get pellet of cells for RNA isolation and cell culture. Isolation of total RNA: Total RNA was isolated using TRI reagent by modification of original method of Chomczynski and Sacchi (1987). TRI reagent (1 ml) was added to granulosa cell pellet obtained from 2 ml of follicular fluid. Granulosa cells grown in monolayer culture were lysed directly in culture dish with 1 ml of TRI reagent/10 cm2 of culture dish after removal of spent media. Luteal tissues (100 mg) were immersed in 1 ml of TRI reagent and homogenized. Similarly, intact 7–8 small, 3–4 medium and 1–2 large size follicles were homogenized, separately in 1 ml of TRI reagent. After 5 min, the homogenate was centrifuged at 12879 g for 5 min/ 4°C to remove debris. The supernatant was transferred to fresh Eppendorf tube and 0.2 ml chloroform/ml of TRI reagent was added and shaken vigorously for 15 sec. The content was kept at room temperature for 15 min and spinned at 12879 g/15 min/4°C. The aqueous phase was transferred carefully in fresh Eppendorf tube. RNA was precipitated by adding isopropanol (0.5 ml/1 ml TRI reagent). After 10 min at room temperature, the content was centrifuged at 12879 g/15 min/4°C. The pellet so obtained was washed with 1 ml of 75% ethanol and RNA was collected at 5031 g/5 min/ 4°C. The purity of RNA was determined by finding A260/ A280 ratio which was in the range of 1.80–2.00. The integrity of RNA was evaluated (result not shown) by agarose (1%) gel electrophoresis, after its denaturation with glyoxal and dimethylsulfoxide (McMaster and Carmichael 1977). Reverse transcription-polymerase chain reaction (RTPCR): The RT-PCR protocol was followed according to MMuLV RT-PCR kit with some modifications. First strand DNA synthesis was done in sterile RNase-free PCR tube in a 20 μl volume. RNA (1μg) was diluted to 9.5 μl with nuclease-free water. After adding 0.5 μl of random hexamer (1μg/μl), a brief spin was given and the content was incubated at 65°C for 30 min in thermocycler and then kept at room temperature for 2 min. The RT reaction was carried out by adding nuclease-free water, 1.5 μl; RT buffer (5X), 4 μl; dNTP mix (30 mM), 2 μl ; DTT (0.1 M), 1 μl; RNase inhibitor (10 U/μl), 1 μl and M-MuLV RT enzyme (50 U/μl), 0.5 μl. The control (RT-) contained 0.5 μl nuclease-free water in place of enzyme. After brief spin of content, the tubes were placed in thermocycler and reaction was carried out at 37°C 307 for 1 hr.; 95°C for 5 min and 4°C with pause. The amplification of RT-product was done in PCR tubes by adding PCR assay buffer (10X), 5 μl; dNTP mix (30 mM), 1 μl; gene specific primers (Table 1), 1 μl (final concentration 0.2 μM); cDNA product, 2 μl and Taq DNA polymerase (1 U/ μl), 1 μl. Total volume was made to 50 μl with nuclease-free water. After brief spin, the PCR cycles were followed as 95°C for 3 min (inhibition of RT enzyme); 95°C for 1 min (denaturation of RT product); 55°C (annealing); 72°C for 1 min (extension); 72°C for 4 min (final extension) and 4°C pause. During PCR with primers for receptors, the annealing temperature was 60°C for 1 min. The performance of PCR reaction largely depends on concentrations of primers and MgCl2 and cycle numbers. Therefore, these parameters were optimized for each target and control gene. Analysis of PCR products: The PCR product (7 μl) mixed with 1.4 μl of 6X gel loading dye was loaded on agaroge (2%) gel. The samples were run in mini-submarine gel electrophoresis system in approximately 250 ml of TBE buffer (1X) at 100 V for 1 hr. The gel was stained with ethidium bromide (0.5 μg/ml). The amplified products were visualized on UV-transilluminator. The intensity of individual bands was measured by densitometry using an image analysis programme (Quantitation 1). The results were expressed as ratio obtained by dividing the band intensity of target gene with intensity of control gene. The statistical analyses for triplicate observations were carried out by ANOVA followed by least significance difference (LSD). Granulosa cell culture: The granulosa cells were cultured as per Blasubramanian et al.(1997). Approximately 8×106 viable granulosa cells were plated in 6–well culture plate using bicarbonate buffered MEM (2 ml/well) supplemented with antibiotics (penicillin, 1000 U/ml and streptomycin, 100 μg/ml) and fetal bovine serum (3%) to permit cell anchorage. The cells were incubated at 37 °C in CO2 incubator. After 24 h, the cells were observed under microscope for attachment and viability. The spent media was aspirated and cells were washed with plain media. The cells were treated with different concentrations of FSH and LH contained in plain MEM. For each concentration (0, 5, 25, 50, 100 ng/ well), 3 wells were used for treatment, and similar number of wells was used as control having plain MEM. After 24 h of further culturing of cells, spent media was removed and RNA was isolated from these monolayer cells as described earlier. Purification of PCR products, cloning and sequencing: The standard protocols for purification and cloning were used as provided with kits. The PCR products were purified using kit. The purified PCR product for IGF-I was cloned using TA cloning kit dual promoter. The INVαF’ cells having pCRII vector containing insert were further grown overnight and plasmid was isolated. Restriction digestion was done to confirm the presence of insert in the vector. The amplification with gene specific primers was also done, using plasmid as 35 308 2 3 4 5 6 7 8 9 10 11 12 1.05 1 0.95 bc ab 0.9 0.85 c b a b LF CL 5 6 7 8 IGF-I IGF-II b a 0.75 MF 4 IGF-I IGF-II a SF 3 G3PDH 0.8 0.7 2 G3PDH IGF-I IGF-II c [Indian Journal of Animal Sciences 80 (4) 1 Ratio of target mRNA/G3PDH rnRNA Ratio of target mRNA/G3PDH rnRNA 1 DEV ET AL. CA 1.2 IGF-I IGF-II 1.1 b ab 1 0.9 a a c b 0.8 0.7 0.6 GC small GC medium GC large Fig. 1. Expression of mRNAs encoding IGF-I and -II in follicles and luteal tissues from buffalo ovary. Lanes 2 and 7, small follicles; lanes 3 and 8, medium follicles; lanes 4 and 9, large follicles; lanes 5 and 10, CL; lanes 6 and 11, CA; lanes 1 and 12, 100 bp DNA ladder and control, respectively. Values within a column lacking a common superscript differ significantly (P< 0.05). Fig. 2. Expression of IGF-I and -II mRNAs in granulosa cells isolated from different sizes of follicles from buffalo ovary. Lanes 2 and 5, GC small; lanes 3 and 6, GC medium; lanes 4 and 7, GC large; lanes 1 and 8, 100 bp DNA ladder and control, respectively. Values within a column lacking a common superscript differ significantly (P< 0.05). source of template to reconfirm presence of the insert. The sequencing of IGF-I cloned PCR product and direct sequencing of purified IGF-II PCR product was got done from commercial agency. differentiated states of follicles. There was significant increase (P<0.05) in expression of IGF-I mRNA with increase in size of follicles (Fig.1), being highest in large follicles, however, its expression was significantly lower (P< 0.05) in post-ovulatory structures as compared to that in large follicles. The expression of IGF-II mRNA also showed similar pattern in follicles, CL and CA. The expression of mRNAs encoding IGF-I and -II, were detected in relation to G3PDH as house keeping (control) gene in granulosa cells isolated from small, medium and large healthy follicles. Evidently, there was significant increase (P< 0.05) in expression of mRNAs encoding IGF-I and -II (Fig. 2) with increase in follicle size. The expression of IGFII mRNA was more than that of IGF-I in granulosa cells from all sizes of follicles. Effect of gonadotropins (FSH and LH) on mRNAs encoding IGF-I and -II in cultured granulosa cells: The granulosa cells were isolated from large healthy follicles and cultured as described. The FSH treatment did not exhibit (Fig. 3) any significant effect in expression of mRNAs RESULTS AND DISCUSSION Purity and integrity of isolated RNA: The RNA was quantified spectrophotometrically and A260/A280 ratio was found in the range of 1.80–2.00, an acceptable purity index, for all RNA preparations. The integrity of RNA was evaluated by agarose (1%) gel electrophoresis, after its denaturation with glyoxal and dimethylsulfoxide. Expression of mRNAs encoding insulin-like growth factors in follicles, luteal tissues and granulosa cells from buffalo ovary: The expressions of mRNAs encoding insulin-like growth factors (IGF-I and IGF-II) were detected in relation to G3PDH (control) gene, in developing healthy follicles. The small, medium and large follicles represented the different stages of growth and post-ovulatory structures, such as corpus luteum (CL) and corpus albicans (CA) were Table 1. Primers used for amplification during PCR Name of gene (Accession No.) IGF-I (X15726) IGF-II (X53553) G–3–PDH (M17701) Forward primer (5″–3″) Reverse primer (5″–3″) DNA product size (bp) CTGCGGGGCTGAGTTGGTCCT TCTGTGCGGCGGGGAGCTGGT AAACCCATCACCATCTTCCAG CGACTTGGCGGGCTTGAGAGGC AGTCTCCAGCAGGGCCAGGTCG AGGGGCCATCCACAGTCTTCT 196 154 360 36 April 2010] 1 2 INSULIN-LIKE GROWTH FACTORS IN BUFFALO OVARY 3 4 5 6 7 8 1 9 10 11 12 2 3 4 5 6 309 7 8 9 10 11 12 0.95 IGF-I IGF-I G3PDH IGF-II IGF-I IGF-II Ratio of target mRNA/G3PDH rnRNA Ratio of target mRNA/G3PDH rnRNA G3PDH IGF-II 0.9 0.85 0.8 075 0.7 Control 5 25 FSH (ng/ml) 50 100 Fig. 3. Effect of FSH treatment on expression of mRNAs encoding IGF-I and -II in granulosa cells during culture. Lanes 2 and 7, control; lanes 3 and 8, 5 ng/ml; lanes 4 and 9, 25 ng/ml; lanes 5 and 10, 50 ng/ml; lanes 6 and 11, 100 ng/ml; lane 1, 100 bp DNA ladder; lane 12, RT (control). 1 IGF-I IGF-II 0.95 0.9 0.85 0.8 0.75 ab a c c bc 0.7 0.65 0.6 Control 5 25 LH (ng/ml) 50 100 Fig. 4. Effect of LH treatment on expression of mRNAs encoding IGF-I and -II in granulosa cells during culture. Lanes 2 and 7, control; lanes 3 and 8, 5 ng/ml; lanes 4 and 9, 25 ng/ml; lanes 5 and 10, 50 ng/ml; lanes 6 and 11, 100 ng/ml; lane 1, 100 bp DNA ladder; lane 12 RT (control). encoding IGF-I and -II in cultured granulosa cells. The relative expression of IGF-I mRNA in cultured granulosa cells was significantly (P< 0.05) increased with increased dose of LH treatment (Fig. 4). There was no significant change in expression of IGF-II mRNA. Sequencing: The presence of 212 bp band (IGF-I) and 170 bp band (IGF-II) indicated (Fig. 5A, B and Fig. 6A, B) successful insertion of target genes in the vector. However, little higher size of inserts resulted due to inclusion of additional base pairs of EcoR1 restriction sites in IGF-I and IGF-II, respectively. The presence of desired insert was also confirmed by PCR using vector containing insert as source of template for IGF-I and IGF-II genes. The presence of bands at 196 bp (IGF-I, Fig. 5B) and 154 bp (IGF-II, Fig. 6B) positions ascertained the insertion of partial genes in the vector. The sequence of cloned partial IGF-I and -II gene and amplified IGF-I and -II cDNA product was similar (Fig.7A, B). The insulin-like growth factors (IGF-I and IGF-II) and their receptors (IGF-IR and IGF-IIR) are important components of IGF system and play significant role in the regulation of ovarian processes such as follicular growth, development, differentiation, ovulation and steroidogenesis. The localization and expression of mRNAs encoding these molecules were found to be species-specific (Armstrong et al. 2000). The present investigation deals with the detection of expression of mRNAs encoding these molecules in granulosa cells of different sizes of follicles and luteal tissues from buffalo ovary. The relative abundance of mRNAs for 2 1 1 2 3 plasmid fragment IGF-I IGF-I (212 bp) A B Fig. 5. Cloning of partial IGF-I cDNA in pCRII vector. (A) agarose (1%) gel electrophoresis of plasmid after restriction digestion with EcoR1. Plasmid sample was digested with EcoR1 to confirm the insertion of IGF-I cDNA (lane 2) which is of little larger size than the normal amplified (196 bp) since it contains part of plasmid being sites for EcoR1. (B) PCR derived confirmation for insertion of IGF-I cDNA in plasmid. Plasmid containing insert was used as source of template for amplification of insert. A band corresponding to 196 bp position (lanes 2, 3) confirms the insertion of IGF-I cDNA in plasmid. Lane 1, 100 bp DNA ladder. 37 310 DEV ET AL. 1 2 3 [Indian Journal of Animal Sciences 80 (4) A CCTCTGCGGGGCTGAGTTGGTGGATGCTCTCCAGTTCGTGTGCGGAGACAGGGGCT TTTATTTCAACAAGCCCACGGGGTACGGCTCGAGCAGTCGGAGGGCGCCCCAGACA GGAATCGTGGATGAGTGCTGCTTCCGGAGCTGTGATCTGAGGAGGCTGGAGATGTA CTGCGCGCCTCTCAAGCCCGCCAAGTCG B TCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTTGTCTGTGGGGACCGCGGCT TCTACTTCAGCCGACCATCCAGCCGCATAAACCGACGCAGCCGTGGCATCGTGGAA GAGTGTTGCTTCCGAAGCTGCGACCTGGCCCTGCTGGAGACT 6 Plasmid fragment Fig. 7. Sequence of partial (A) IGF-I cDNA (196 bp) and (B) IGF-II cDNA (154 bp) from buffalo ovary. et al. 1992) and sheep (Khan-Dawood et al. 1994). There was higher expression of mRNAs for IGFs during early luteal phase followed by a decrease during mid- and late-luteal phase (Berisha and Schams 2005). The localization of mRNAs encoding IGFs in ovary was reported to exhibit marked species specificity. The presence of IGF-I mRNA expression in buffalo granulosa cells agrees with previous reports from pig (Yuan et al. 1996) and mouse (Wandji et al. 1998). The IGF-I mRNA could not be detected in sheep follicles by in situ hybridization (Perks et al. 1995), however, its expression was shown in bovine granulosa cells (Badinga et al. 1992). The expression of IGF-I mRNA could not be detected in bovine granulosa cells (Armstrong et al. 2000) with RT-PCR but the expression was found in buffalo granulosa cells using same gene-specific primers at 35 amplification cycles. The expression increased as the size of follicle increased, a pattern also reported by Yuan et al.(1998). The expression of IGF-II mRNA was found in buffalo granulosa cells isolated from all 3 sizes of follicles and was similar to that in human (Zhou et al. 1993) but in contrast to sheep granulosa cells (Perks et al. 1995). The expression of IGF-II mRNA was detected in bovine granulosa cells, using in situ hybridization (Yuan et al. 1998) but was not detected in these cells by RT-PCR (Armstrong et al. 2000), instead it was found in theca cells. The treatment of buffalo granulosa cells with increasing concentrations of FSH showed non-significant effect on expression of mRNAs encoding IGF-I and IGF-II. The IGFI mRNA levels in granulosa cells were not reduced in hypophysectomized or FSH knockout mice (Zhou et al. 1997) suggesting that the expression of IGF-I mRNA is independent of FSH. On the other hand, culturing of bovine (Spicer et al. 2000) and ovine (Khalid et al. 2000) granulosa cells exhibited increased production of IGF-I in response to FSH. The expression of pregnancy associated plasma protein-A (PAPPA) is induced by FSH in rat granulosa cells (Resnick et al. 1998), which causes a decrease in IGF binding proteins, releasing free IGF-I. Recently, FSH was found to increase PAPP-A mRNA expression in cultured buffalo granulosa cells (unpublished). Thus, the higher free IGF-I production in response to FSH might be due to induced PAPP-A making more free IGF-I available. The LH treatment of buffalo granulosa cells in vitro resulted in higher IGF-I mRNA expression whereas increase in IGF-II mRNA was nonsignificant. There was increased IGF-I mRNA expression in cultured and LH-treated porcine granulosa cells (Hatey et IGF-II (170 bp) A B Fig. 6. Cloning of partial IGF-II cDNA in pCRII vector. (A) agarose (1%) gel electrophoresis of plasmid after restriction digestion with EcoR1. Plasmid sample was digested with EcoR1 to confirm the insertion of IGF-II cDNA (lanes 2, 3) which is of little larger size than the normal amplified (154 bp) since it contains part of plasmid being sites for EcoR1. (B) PCR derived confirmation for insertion of IGF-II cDNA in plasmid. Plasmid containing insert was used as source of template for amplification of insert. A band corresponding to 154 bp position (lanes 2–6) confirms the insertion of IGF-II cDNA in plasmid. Lane 1, 100 bp DNA ladder. target genes was determined by semi-quantitative RT-PCR, which is a widely used technique (Carding et al. 1992, Ali et al. 1997). The conditions of RT-PCR in terms of concentrations of gene specific primers and MgCl2 and amplification cycle number were optimized in presence of G3PDH as housekeeping (control) gene since these parameters influence the PCR performance. The intact follicles exhibited increased expression of mRNAs encoding IGF-I and IGF-II as follicular size increased. The significantly higher expression of IGFs mRNAs in large follicles (preovulatory) may be related with the characteristics of dominant follicles. The kind of shift was demonstrated (Yuan et al. 1998) in expression of IGF-I, IGF-II and IGF binding protein–2 among dominant (greater IGFs, near absence of IGFBP–2) and subordinate (less IGFs, abundant IGFBP–2) follicles in bovine. The low expression of IGF-I mRNA in bovine small follicles was revealed by in situ study (Perks et al. 1999), whereas follicular thecal layer in small healthy follicles exhibited intense IGF-II mRNA expression. The significant expression of IGFs mRNAs in buffalo follicles also supports their important role in follicular development (Badinga et al.1992, Spicer et al. 1994). The different growth factors, especially IGFs have definite role in development and completion of dense network of capillaries (angiogenesis) during follicular growth and corpus luteum (CL) formation (Schams et al. 2001). In buffalo ovary, the presence of expression of IGFs mRNA in CL and corpus albicans (CA) indicated their role in growth, differentiation and regression of corpus luteum. These results are in agreement with studies in ovine (Perks et al. 1995) and bovine (Woad et al. 2000). These IGFs have stimulatory effect on progesterone secretion by luteal tissues of cattle (Sauerwein 38 April 2010] INSULIN-LIKE GROWTH FACTORS IN BUFFALO OVARY al. 1992) when detected using Northern hybridization. LH increases IGF-I mRNA and IGF-I enhances the half-life of LHR mRNA (Hirakawa et al. 1999). The partial IGF-I (196 bp) and -II (154 bp) cDNAs from buffalo ovary were cloned and were custom sequenced. The blasting of IGF-I sequence with that of other species revealed the homology with goat (97%), cattle (99%), sheep (98%), and pig (93%). The blasting of buffalo IGF-II sequence with that of other species, showed the homology of 100%, 100%, 92% and 88% with that of cattle, sheep, and pig, respectively. It is concluded that a significant amount of mRNAs encoding IGF-I and -II are significantly expressed in follicles, granulosa cells and post-ovulatory structures from buffalo ovary representing different stages of growth, development and differentiation. These growth factors are major intraovarian IGF ligands and have functional importance during follicular and luteal phases of buffalo ovary. Further attempts are needed to find out the molecular signaling pathway induced by IGFs, mediating ovarian steroidogenesis and antiapoptotic processes. 311 Carding SR, Lu D and Botomly K A. 1992. A polymerase chain reaction assay for detection and quantification of cytokine gene expression in small number of cells. Journal of Immunology Methods 159: 277–87. Chomczynski P and Sacchi N. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry 162: 156–59. Hatey F, Langlois I, Mulsant P, Bonnet A, Benne F and Gasses F. 1992. Gonadotropins induced accumulation of insulin-like growth factor-I mRNA in pig granulosa cells in vitro. Molecular Cellular Endrocrinology 86: 205–11. Hirakawa T, Minegishi T, Abe K, Kishi H, Ibuki Y and Miyamoto K. 1999. A role of insulin-like growth factor-I in luteinizing hormone receptor expression in granulosa cells. Endocrinology 140: 4965–71. Jones J I and Clemmons D R. 1995. Insulin-like growth factors and their binding proteins: biological actions. Endocrinology Review 16: 3–34. Khalid M, Haresign W and Luck M R. 2000. Secretion of IGF–1 by ovine granulosa cells: effects of growth hormone and follicle stimulating hormone. Animal Reproduction Sciences 58: 261– 272. Khan-Dawood F S, Gargiulo A R and Dawood M Y. 1994. In vitro microdialysis of the ovine corpus luteum of pregnancy: effects of IGF on progesterone secretion. Biology of Reproduction 51: 1299–1306. McMaster G and Carmichael G G. 1977. Analysis of single and double stranded nucleic acids on polyacrylamide and agarose gel using glyoxal and acridine orange. Proceedings of National Acadamy of Science USA 74: 4835–38. Moore R M, Hay M F and Cran D G. 1978. Macroscopic identification and steroidogenic function of atretic follicles in sheep. Journal of Endocrinology 77: 309–18. Nissley P and Lopaczynski W. 1991. Insulin-like growth factor receptor. Growth Factors 5: 29–43. Perks C M, Denning-Kendall P A, Gilmour R S and Wathes D C. 1995. Localization of messenger ribonucleic acid for insulinlike growth factor I (IGF I), IGF II and type I IGF receptor in ovine ovary throughout the estrous cycle. Endocrinology 136: 5266–73. Perks C M, Peters A R and Wathes D C. 1999. Follicular and luteal expression of insulin-like growth factors-I and-II and type-I IGF receptor in the bovine ovary. Journal of Reproduction and Fertility 116: 157–65. Resnick C E, Fielder P J, Rosenfeld R G and Adashi E Y. 1998. Characterization and hormonal regulation of a rat ovarian insulin-like growth factor binding protein–5 endopeptidase: an FSH inducible granulosa cell-derived metalloprotease. Endocrinology 139: 1249–57. Sauerwein H, Miyamoto A, Gunther J, Meyer H H and Schams D. 1992. Binding and action of insulin-like growth factors and insulin in bovine luteal tissue during the oestrous cycle. Journal of Reproduction and Fertility 96: 103–15. Schams D, Kosmann M, Berisha B, Amselgruber W M and Miyamoto A. 2001. Stimulatory and synergistic effects of luteinising hormone and insulin-like growth factor 1 on the secretion of vascular endothelial growth factor and progesterone of cultured bovine granulosa cells. Experimental Clinical Endocrinology and Diabetes 109: 155–62. Spicer L J, Alpizar E and Vernon R K. 1994. Insulin-like growth ACKNOWLEDGEMENT The authors are thankful to the Director, National Dairy Research Institute, Karnal, for providing the necessary facilities for carrying out this work. The financial assistance as NDRI (ICAR) Senior Fellowships to Mr Rishabh Dev is gratefully acknowledged. FSH and LH were generous gift from Dr A.F. Parlow, National Hormone and Pituitary Program, Harbour-UCLA Medical Centre, Torrance, California. REFERENCES Ali S A, Sarto I and Steinkasserer A. 1997. Production for PCR mimics for any semi- quantitative PCR application. Biotechniques 6: 1060–62. Armstrong D G, Baxter G, Guiterrez C G, Hogg C O, Glazyrin A L, Campbell B K, Bramley T A and Webb R. 1998. Insulin-like growth factor binding protein–2 and –4 messenger ribonucleic acid expressions in bovine ovarian follicles: effect of gonadotropins and developmental status. Endocrinology 139: 2146–54. Armstrong D G, Gutierrez C G, Baxter G, Glazyrin A L, Mann G E, Wood K J, Hogg C O and Webb R. 2000. Expression of mRNA encoding IGF I, IGF II and type I IGF receptor in bovine ovarian follicles. Journal of Endocrinology 165: 101–13. Badinga L, Driancourt M A, Savino J D, Wolfenson D, Drost M, delaSota R L and Thatcher W W. 1992. Endocrine and ovarian responses associated with first wave dominant follicle in cattle. Biology of Reproduction 47: 871–83. Berisha B and Schams D. 2005. Ovarian function in ruminants. Domestic Animal Endocrinology 29: 305–17. Balasubramanian K, Lavoie H A, Garney J C, Stocco D M and Veldhius J D. 1997. Regulation of porcine granulosa cell steroidogenic acute regulatory protein (StAR) by insulin-like growth factor-I: Synergism with follicle stimulating hormone or protein kinase A agonist. Endocrinology 138: 433–39. 39 312 DEV ET AL. [Indian Journal of Animal Sciences 80 (4) mediated via the type-I IGF receptor. Journal of Clinical Endocrinology and Metabolism 83: 1256–59. Yuan W, Lucy M C and Smith M F. 1996. Messenger RNA for insulin-like growth factor-I and –II, insulin-like growth factor binding protein–2, gonadotropin receptors and steroidogenic enzymes in porcine follicles. Biology of Reproduction 55: 1045– 54. Yuan W, Bao B, Garverick H A, Youngquist R S and Lucy M C. 1998. Follicular dominance in cattle is associated with divergent pattern of ovarian gene expression for insulin-like growth factor (IGF-I, IGF-II) and IGF binding protein–2 in dominant and subordinate follicles. Domestic Animal Endocrinology 15: 55– 63. Zhou J and Bondy C. 1993. Anatomy of the human ovarian insulinlike growth factor system. Biology of Reproduction 48: 467– 82. Zhou J, Kumar T R, Matzuk M M and Bondy C. 1997. Insulin-like growth factor-I regulates gonadotropin responsiveness in the murine ovary. Molecular Endocrinology 11: 1924–33. factor–I receptors in ovarian granulosa cells: effect of follicle size and hormones. Molecular Cellular Endocrinology 102: 69– 76. Spicer L J and Chamberlain C S. 2000. Production of insulin-like growth factor-I by granulosa cells but not theca cells is hormonally responsive in cattle. Journal of Animal Sciences 78: 2919–26. Woad K J, Baxter G, Hogg C O, Bramley T A, Webb R and Armstrong D G. 2000. Expression of mRNA encoding insulinlike growth factors I and II and the type 1 IGF receptor in the bovine corpus luteum at defined stages of the oestrous cycle. Journal of Reproduction and Fertility 120: 293–302. Wandji S A, Wood T L, Crawford J, Levison S W and Hammond J M. 1998. Expression of mouse ovarian insulin growth factor system components during follicular development and atresia. Endocrinology 139: 5205–14. Willis D S, Mason H D, Watson H and Franks S. 1998. Developmentally regulated responses of human granulosa cells to insulin-like growth factors (IGFs): IGF-I and IGF-II action 40 Indian Journal of Animal Sciences 80 (4): 313–316, April 2010 Effect of Bt cotton plants on oxidative stress in sheep B ANILKUMAR1, A GOPALA REDDY2, B KALAKUMAR3, K JYOTHI4 and K S GOPI5 Sri Venkateswara Veterinary University, Tirupati, Andhra Pradesh 500 030 India Received: 6 April 2009; Accepted: 15 November 2009 ABSTRACT Sheep (32) of 1–year age belonging to Deccani breed were randomly divided into 4 groups consisting of 8 sheep in each group. Group 1 was maintained on basal diet (green fodder @ 3 kg + concentrate feed @ 300 gm/sheep/day) and 2 on conventional (non-Bt) cotton plants @1.5 kg + green fodder @1.5 kg + concentrate feed @ 300 g/sheep/day, 3 on Bt cotton plants @1.5 kg + green fodder @1.5 kg + concentrate feed @ 300 g/sheep/day and 4 on Bt cotton plants ad lib. + concentrate feed @ 300 g/sheep/day. All the groups were fed for 3 months. Parameters related to oxidative stress in organs of all groups and the concentration of gossypol in sera samples of groups 2, 3 and 4, and Bt (Cry1Ac) protein in rumen liquor in groups 3 and 4 were evaluated at the end of third month of feeding, while the concentration of Bt (Cry1Ac) protein in sera samples of groups 3 and 4 was quantified at monthly intervals for 3 months. The mean concentration of GSH and TBARS, and the activity of SOD and catalase in liver, kidney and heart showed statistically nonsignificant difference among the groups. The Bt (Cry1Ac) protein was not detectable in sera samples of groups 3 and 4 at different time intervals, while rumen liquor had detectable concentrations at the end of third month of feeding. Gossypol was not found in the sera samples of sheep fed on Bt and non-Bt cotton plants. In conclusion, the study revealed that feeding of Bt cotton plants did not induce oxidative stress in sheep and sera samples were negative for Cry1 Ac protein and gossypol. Key words: Bt cotton, Gossypol, Oxidative stress, Sheep showed tolerance to gossypol toxicity (Reiser and Fu 1962). In Andhra Pradesh, due to decline of grazing land, the farmers and shepherds allow their animals into cotton fields after harvesting the cotton. There were reports in the print media that sheep died in Telangana region of Andhra Pradesh after grazing on leaves and pods of harvested Bt cotton plant residue in fields (Anonymous 2007). Cultivation of Bt cotton has raised fears among the shepherds and other livestock owners over the safety of Bt cotton plants and its by-products. Therefore, there was a need to carry out a study on the toxic potential, if any, of Bt protein and gossypol that are present in Bt cotton plants by incorporating in the feed of sheep. Hence, the present study was undertaken in sheep to estimate Cry1 Ac protein and gossypol in sera samples and to assess the biomarkers of oxidative stress in liver, heart and kidney. A genetically modified (GM) cotton variety, known as Bt cotton, has been developed to control the menace of pests. By introducing the genetic information encoding the insecticidal protein of Bt (Bacillus thuringiensis) (Cry 1Ac) into plants, the plants would produce their own insecticides (bio-insecticides) that reduce the application of pesticides, improve insect control and reduce environmental problems. Several studies showed that the amount of expression of Bt protein (Cry 1Ac) in plant is very minute in quantity and its action is limited to certain larval species and may not cause toxicity to mammals (Betz et al. 2000). However, it is desirable to correlate the concentration of Cry 1Ac protein in ruminal fluid and blood with the effects of feeding on digestive and other systems. Gossypol is a toxic component that is produced for defense by the plant and causes deleterious effects to non-ruminants, although ruminants 1Ph.D. MATERIALS AND METHODS 2Professor Present address: Scholar, and University Head, 3 Assistant Professor, Department of Pharmacology. C.V.Sc. Rajendranagar, Hyderabad 500 030 India (email: gopalareddy123 @rediffmail.com) 4H. No. # 5-4-17/1, Bhavani Colony, Rajendranagar. Hyderabad 500 030 India. 5Veterinary Assistant Surgeon, Veterinary Dispensary, Thaiyur, Gingee (Taluk), Villupuram (Dt), Tamil nadu 604 205. Deccani sheep (32),1- year-old, were randomly divided into 4 groups, consisting of 8 sheep in each group, after an acclimatization period of 2 weeks. Sheep were dewormed by administering albendazole (@ 7.5 mg/kg body wt orally) and vaccinated for FMD, HS, PPR and sheep pox, and provided with water ad lib. throughout the experiment. Bt and conventional (non-Bt) cotton crops were cultivated at 41 314 ANILKUMAR ET AL. Student Farm, College of Agriculture, Acharya N.G Ranga Agricultural University (ANGRAU), Hyderabad. The groups were fed as per the following schedule for 3 months: Group 1: Basal diet (green fodder; APPN1 hybrid Napier grass) @ 3 kg + concentrate feed* @ 300 g/ sheep/day) Group 2: Non-Bt cotton plant (50% of greens) @1.5 kg+ green fodder @1.5 kg + concentrate feed @ 300 g/sheep/day Group 3: Bt cotton plants (50% of greens) @1.5 kg+ green fodder @1.5 kg + concentrate feed @ 300 g/sheep/day Group 4: Bt cotton plants ad libitum (3 kg) (without other green fodder) + concentrate feed @ 300 g/ sheep/day. *Composition of concentrate feed (ingredients in kg/100 kg concentrate feed) Maize 25 Ground nut cake 20 Wheat bran 15 Red gram 15 Deoiled rice bran 15 Sorghum straw 07 Mineral mixture 02 Salt 01 The blood samples (n = 8) were drawn from jugular vein at monthly intervals for 3 months for estimation of Cry1Ac protein (groups 3 and 4) and at the end of third month for quantification of gossypol (groups 2, 3 and 4). The rumen liquor was collected at the end of third month for quantification of Cry1Ac protein in groups 3 and 4 (n = 8). The sheep were sacrificed after third month of feeding and the liver, kidney and heart tissues were collected (n = 8 for each organ in each group) and homogenized for the assay of reduced glutathione (GSH) (Moron et al. 1979), thiobarbituric acid reacting substances (TBARS) (Subramanian et al. 1988), super oxide dismutase (SOD) (Madesh and Balasubramanian 1998) and catalase (Sinha 1971). The protein estimation in the organs was done as per Lowry et al. (1951). Sera samples were analyzed for gossypol by HPLC (Nomier and Abou-Donia 1982). The expression of Bt Cry1Ac protein in biological samples (serum and rumen [Indian Journal of Animal Sciences 80 (4) liquor) was done by using ELISA kit for Cry1Ab/Cry Ac. The experimental protocol was approved by the Institutional Animal Ethics Committee. The data were subjected to statistical analysis by applying one-way ANOVA using statistical package for social sciences (SPSS) version 10. Differences between means of different groups were tested using Duncan’s multiple comparison test and significance was set at P<0.05. RESULTS AND DISCUSSION The concentration of TBARS (nM of MDA/g tissue) at the end of third month in the homogenized tissues of liver, heart and kidney in basal diet control (group 1) was 371.26±22.53, 349.35±11.22 and 358.44±15.71, respectively, which did not differ significantly when compared to the remaining groups. The concentration of GSH (mg/g protein) of liver, heart and kidney tissue homogenate was 17.73±2.62, 13.30±1.81 and 8.55±0.41, respectively in group 1, which did not differ significantly when compared to the remaining groups (Table 1). The activity of SOD (U/mg protein) in the liver, heart and kidney homogenate of group 1 was 22.33±1.55, 40.42±8.91 and 26.89±5.26, respectively, and the values did not differ significantly when compared to other groups. Similarly, the activity of catalase (μg of H2O 2 decomposed/min/mg protein) of liver, heart and kidney homogenate of group 1 was 5.72±0.69, 2.24±0.98 and 4.25±0.86, respectively, which did not differ significantly when compared to other groups (Table 2). The anti-oxidant defense profile was studied to assess the possibility, if any, of oxidative stress and free radical damage in the biological system by estimating the concentration of GSH in the liver, kidney and heart as oxidative stress is the central mechanism for the occurrence of a number of diseases and toxicities (Kumar et al. 2002).The concentration of GSH (non-enzymatic antioxidant) was assessed in liver as it is the major organ involved in xenobiotic metabolism and in kidney, which is susceptible to the free radical damage owing to its richness in peroxidizable fatty acids. The concentration of TBARS in liver, kidney and heart was analyzed to determine the extent of lipid peroxidation, and the activity of catalase and SOD was assessed as they form the major components of enzymatic antioxidant defense system in vivo. Lipid Table 1. Concentration of TBARS and GSH in liver, heart and kidney of different groups of sheep at the end of third month of feeding Group Basal diet (green fodder + concentrate) Non Bt cotton + Basal diet Bt cotton 50% + Basal diet Bt cotton ad lib. + concentrate TBARS (nM of MDA/g protein) GSH (mg/g protein) Liver Heart Kidney Liver Heart Kidney 371.26±22.53 349.35±11.22 358.44±15.71 17.73±2.62 8.55±0.41 13.30±1.81 374.46±16.41 373.39±12.78 370.72±9.74 356.30±40.57 353.63±9.84 365.38±17.92 360.57± 14.82 355.76± 14.80 355.23± 16.86 14.11±1.84 14.66±0.81 14.76±3.23 9.04±1.46 9.04±1.46 10.93±3.44 12.46±1.89 12.46±1.89 12.65±1.24 Values are mean±SE (n = 8); one-way ANOVA (SPSS). 42 April 2010] TOXICITY OF BT COTTON IN SHEEP 315 Table 2. Activity of super oxide dismutase (SOD) and catalase in liver, heart and kidney of different groups of sheep at the end of third month of feeding Group Basal diet (green fodder + concentrate) Non Bt cotton + Basal diet Bt cotton 50% +Basal diet Bt cotton ad lib + Concentrate SOD (U/mg protein) Catalase (μg of H2O2 decomposed/ min/mg protein) Liver Heart Kidney Liver Heart Kidney 22.33±1.55 40.42±8.91 26.89±5.26 5.72±0.69 2.24±0.98 4.25±0.86 29.46±2.71 28.70±2.67 26.10±1.94 34.51±3.51 27.82±2.45 21.36±3.21 26.73±2.31 20.26±2.03 19.62±3.93 5.81±0.88 4.31±0.71 4.19±0.67 2.87±0.72 2.01±0.21 2.46±0.37 4.30±1.20 4.08±0.83 5.13±0.64 Values are mean±SE (n = 8); one-way ANOVA (SPSS). peroxidation biomarker and enzymatic and non-enzymatic antioxidant defenses did not differ significantly among the groups in study. Concentration of Bt (Cry1Ac) protein (μg/g) in rumen liquor was analyzed at post-prandial period and it was 1.27±0.06 in sheep fed exclusively on Bt cotton plant (group 4) and 0.25±0.03 in sheep fed on diet containing 50% Bt cotton plants (group 3). Cry1Ac protein was not detected in sera samples of sheep (groups 3 and 4). The Bt. protein may be eliminated in faecal material following degradation without getting concentrated in blood leading to nondetectable levels as observed in this study. Similar estimations were done earlier by Yonemochi et al. (2002), who investigated the transgenic Cry9C protein in tissues from chickens fed maize (event CBH 351) and could not detect this novel protein in blood, liver or muscle samples. Ash et al. (2000) also did not detect transgenic CP4 EPSPS protein in whole egg, egg white, liver and faeces of laying hens fed a diet containing genetically modified (GM) soybean meal. The evidence of possible transfer of plant DNA to tissues and edible products of cattle was studied in animals fed conventional (non-GM) and genetically modified (GM) Btmaize by Einspanier et al. (2004) and found DNA of plant chloroplast in duodenal juice and blood lymphocytes in both the groups, but the DNA was not found in blood, milk and any other tissues of the cows fed with Bt-maize. In this study, sera samples were found negative for the presence of gossypol. In ruminants, gossypol seems to have little effect when whole cottonseed and by-products are fed within the safety limits (Calhoun and Holmberg 1991). Even though toxic effects are expected when high concentrations of free gossypol are fed, ruminants have the capacity to detoxify large amounts by binding it to soluble proteins in the rumen (Reiser and Fu 1962). Gossypol affects liver functions, erythrocyte oxygen-carrying or releasing capacity, respiration rate, feed intake, and production and reproduction efficiency (Gray et al. 1990, Brocas et al. 1997). In conclusion, the study revealed that feeding of Bt cotton plants did not induce oxidative stress as evident from biomarkers of oxidative stress in sheep and further, Bt (Cry1Ac) protein and gossypol were not detectable in sera samples of sheep. ACKNOWLEDGEMENTS The authors thank Sri Venkateswara Veterinary University, Tirupati (Andhra Pradesh), for providing necessary financial assistance, Associate Dean, College of Agriculture, Hyderabad, for sparing land and facilities for cultivation of cotton crops and Head, Livestock Research Station, College of Veterinary Science, Hyderabad, for extending necessary facilities during the experimentation. REFERENCES Anonymous. 2007. News paper reports: Deccan Herald, February 7, 2007; The Hindu, March 2, 2007; GM Watch, March 4, 2007; Hindustan Times, June 17, 2007; GM Watch, June 18, 2007; Hindustan Times, June 18, 2007 Ash J A, Scheideler E and Novak C L. 2000. The fate of genetically modified protein from Roundup Ready soybeans in the laying hen. Poultry Science 79 (Suppl. 1): 26. Betz F S, Hammond B G and Fuchs R L. 2000. Safety and advantages of Bacillus thuringiensis-protected plants to control insect pests. Regulatory Toxicology and Pharmacology 32: 156– 73. Brocas C R M, Rivera F F, Paula-Lopez L R, McDowell M C, Calhoun C R, Staples N S, Wilkinson A J, Boning P J, Chenoweth and Hansen P J. 1997. Deleterious actions of gossypol on bovine spermatozoa, oocytes and embryos. Biology of Reproduction 57: 901. Calhoun M and Holmberg C. 1991. Safe use of cotton by-products as feed ingredients for ruminants: A review. Cattle Research with Gossypol Containing Feeds. pp. 97–129. (Eds) Jones L A, Kinard D H and Mills J S. National Cottonseed Products Association, Memphis, TN. Einspanier R, Lutz B, Rief S, Berezina O, Zverlov V, Schwarz W and Mayer J. 2004. Tracing residual recombinant feed molecules during digestion and rumen bacterial diversity in cattle fed transgene maize. European Food Research Technology 218: 269–73. Gray M L, Randel R D, Greene L W and Williams G L. 1990. 43 316 ANILKUMAR ET AL. Metabolic homeostasis and reproductive endocrine function in post-pubertal beef heifers fed varying levels of dietary free gossypol. Journal of Animal Science 68 (Suppl. 1): 465 (Abstract). Kumar D, Lou H and Singal P K. 2002. Oxidative stress and apoptosis in heart dysfunction. Herz 27: 662–68. Lowry O H, Rosenbrough M J, Farr A L and Rawdell R A. 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193: 265–75. Madesh M and Balasubramanian K A. 1998. Micro titre plate assay for super oxide dismutase using MTT reduction by super oxide. Indian Journal of Biochemistry and Biophysics 35: 185–88. Moron M S, Depierre J W and Mannervik B. 1979. Levels of glutathione, glutathione reductase and glutathione S transferase in rat lung and liver. Biochemica et Biophysica Acta 582: 67– 68. [Indian Journal of Animal Sciences 80 (4) Nomier A A and Abou-Donia 1982. Gossypol: High performance liquid chromatogaphic analysis and stability in various solvents. Journal of Association of official Analytical chemists 59: 556– 59. Reiser R and Fu H C. 1962. The mechanism of gossypol detoxification by ruminant animals. Journal of Nutrition 76: 215–18. Sinha A K. 1971. Colorimetric assay of catalase. Analytical Biochemistry 47: 389–94. Subramanian K A, Manohar M and Mathan V I. 1988. An unidentified inhibitor of lipid peroxidation in intestinal mucosa. Biochimica et Biophysica Acta 962: 51–58. Yonemochi C, Fujisaki H, Harada C, Kusama T and Hanazumi M. 2002. Evaluation of transgenic event CBH 351 (Star Link) corn in broiler chicks. Journal of Animal Science 73: 221– 28. 44 Indian Journal of Animal Sciences 80 (4): 317–320, April 2010 Micro- minerals status in goats of different ages in semi-arid region of India* NEERU BHOOSHAN1, PUNEET KUMAR2 and M C YADAV3 Central Institute for Research on Goats, Makhdoom, Uttar Pradesh 281 122 India Received: 10 June 2008; Accepted: 30 October 2009 ABSTRACT The present study was conducted to evaluate and compare blood zinc (Zn), copper (Cu) and cobalt (Co) status of healthy female goats (210: 105 of Barbari breed and 105 of Jamunapari breed) of different ages, managed under semiintensive system at institute farm. Blood Zn and Cu concentrations were significantly influenced by age of goats, while blood Co concentration was significantly affected by the breed of goats. In Barbari and Jamunapari goats, Zn levels further increased during one month of age. Thereafter it decreased significantly with the advancement of age up to 9–10 months of age in Barbari goats. Zn level was significantly low at pubertal age than pre-pubertal age in Barbari goats. While in Jamunapari goats, Zn level was not different in pre-pubertal, pubertal and post-pubertal age. In these goats, Cu concentration was low at birth which increased with the advancement of age and attained the highest level at 11 to 12 months of age, while, blood Cu concentration was not different at pre-pubertal, pubertal and post pubertal age. Blood Co concentration did not change with the advancement of age. Barbari goats have significantly higher blood Co concentration than Jamunapari goats. Key words: Age, Cobalt, Copper, Goat, Zinc Zinc (Zn), copper (Cu) and cobalt (Co) are micro-minerals essential in multiple enzyme systems. Early deficiency of zinc reduces feed intake, growth rate and feed efficiency (McDowell 2003); and cobalt deficiency impairs energy and protein metabolism and then growth and development of deficient animal (Kadim et al. 2006). However, goats are considered as being more resistant to low levels of dietary cobalt (Mburu et al. 1993). Mineral concentrations in goat blood are different from those of other ruminants such as cattle and sheep (Haenlein 1980), there is a need to fully understand its micro-mineral requirements. Breed, age, productivity, physiological state of the animal, mineral intake, chemical form of elements and interrelationships with other nutrients, affect mineral requirements and status (NRC 1985, Khan et al. 2007). Young animals absorb minerals more efficiently than older animals (McDowell 2003). The objective of the present work was to evaluate and compare micro-minerals status of two breeds of goats of different age groups on the basis of mineral concentrations in blood, so as to form the basis for their optimum growth and fertility. MATERIALS AND METHODS Animals and feeding: Healthy indigenous female goats (210: 105 Barbari and 105 Jamunapari goats) of different age groups (Table 1) maintained under semi-intensive system of management at the institute farm were used in this study. Feeding was done according to NRC (1981). Newly born kids were fed with mother milk for first 15 days with bottle. Weaning was done at 3 months of age. After weaning, experimental goats were allowed 4-6 grazing and were stall fed with dry roughage ad lib. For Barbari goats, 200 g to 350 g/animal/day pelleted concentrate mixture with 13 and 69% digestible crude protein (DCP) and total digestible nutrients (TDN) respectively was given from 2 to 3 months of age to 9 to10 months. An additional 50 g feed per animal was given to Jamunapari goats. Adult goats above 1 year of age were given 400 g concentrate mixture daily. Drinking water was given ad lib. The animals were dewormed regularly as per standard health practices. Analysis of micro-minerals: Blood (5 ml) was collected in nitric acid washed heparinized vials from jugular vein at days 0 (birth), 30, 90, 180, 270–300, 330–360 besides prepubertal, pubertal and post pubertal (one week after estrus) * Part of Ph.D. thesis, Submitted to Dr B.R. Ambedkar University, Agra (Uttar Pradesh). Present address: 1Scientific Officer, U.P. Council of Agricultural Research, 8th Floor, Kisan Mandi Bhawan, Vibhuti Khand, Gomti Nagar, Lucknow, Uttar Pradesh 226 010 (e-mail address: [email protected]). 2Principal Scientist,Division of Goat Physiology. 3Principal, Narain College, Shikohabad, Firozabad, Uttar Pradesh 45 318 BHOOSHAN AND KUMAR ages for estimation of Zn, Cu and Co. Blood samples were digested as per the AOAC (1984). Blood Zn, Cu and Co were estimated in digested samples using flame atomic absorption spectrophotometer. Element specific hollow cathode lamps were used and analytical quality was maintained by repeated analysis of reference samples. Eight working standards were prepared freshly from stock (Naresh 1997). Analysis of data: Data obtained was analyzed using mixed model (MIXMDL PC-2) program with a least square technique for fitting non-orthogonal data and maximum likelihood computer program developed by Harvey (1990). Duncan’s multiple range test (DMRT) modified by Kramer (1957) was used for pair-wise comparison among least squares means for age within breed effect to find out any significant difference among them. Correlation coefficient (r) was carried out in pooled manner by using standard method described by Snedecor and Cochran (1994). [Indian Journal of Animal Sciences 80 (4) calves (Kincacid and Hodgson 1989). In Barbari goats, 11– 12 months of age the Zn level increased significantly (P<0.05). Similarly, in cattle, calves did not carry higher concentration of total body Zn than did mature animals (Akan et al. 1991). In Jamunapari goats, Zn level increased at 11– 12 months of age though not significantly. In Barbari goats, the Zn level was significantly (P<0.05) low at pubertal age than pre-pubertal age. While in Jamunapari goats, the Zn level was not different in pre-pubertal, pubertal and postpubertal age. Similarly, no significant difference between the age groups was found in Assami goats (Bhattacharyya et al. 1995) and in Kivircik lambs (Akdogan et al. 2000). In this study, blood Cu concentration was affected significantly by age (P < 0.01) but no obvious affect of breed and interaction between breed and age on Cu concentration was found. Blood copper concentration increased with the advancement of age which may be due to increasing physiological demands of growth. In Barbari and Jamunapari goats, blood copper concentration was low (Table 1) at birth, which increased with the advancement of age and attained the highest level at 11 to 12 months of age. Copper concentrations are related to age in sheep (Church 1993), in beef and dairy calves (Puschner et al. 2004) and in Sudanese camels (Camelus dromedarius) (Mohamed 2004). Ahmed et al. (2001) showed that an association exists between age and the physiological status of dairy Nubian goats, pregnancy, lactation and concentration of copper and zinc. Plasma copper levels increased significantly in adult compared to young animals. The increase in Cu level with age could be associated with higher concentrations of circulating oestrogens in the mature animals as a consequence of oestrous cycle (Desai et al. 1978) and probably for normal functioning of endocrine glands during puberty (Pathak et al. 1986). In these goats, blood copper concentration was not different at pre-pubertal, pubertal and post pubertal age. Contrary to this, blood copper concentration was significantly higher on the day of oestrus than during the other stages of reproduction in Assami goats RESULTS AND DISCUSSION In this study, the overall means of blood zinc (ppm), copper (ppm) and cobalt (ppm) for Barbari and Jamunapari goats irrespective of age are given in Table 1. In sheep, the blood plasma concentration of Zn, Cu and Co was 8–12 ppm, 0.7–1.3 ppm and 0.1–0.3 ppm respectively (Radostits et al. 2000). Similarly, in adult lactating healthy cow, the Zn, Cu and Co concentration was 8.46 ± 1.10 ppm, 0.62 ppm and 0.40 ± 0.03 ppm respectively (Naresh 1997). Least square analysis of variance indicated that goat blood Zn concentration was significantly (P<0.01) affected by age but not with breed. Concentrations of Zn fluctuate with age, stress, infections and feed restriction (Kincaid 1999). In Barbari and Jamunapari goats at birth, the Zn levels further increased (Table 1) during 1 month of age. Thereafter it decreased significantly (P<0.05) with the advancement of age up to 9–10 months of age in Barbari goats. Similarly, plasma Zn concentration decreased significantly with increase in age in Nubian goats (Ahmed et al. 2001) and in Table 1. Least square mean ± SE of zinc (ppm), copper (ppm) and cobalt (ppm) at various ages for Barbari and Jamunapari goats Age (days) Birth 30 90 180 270–300 330–360 Pre-pubertal Pubertal Post-pubertal Overall n 8 8 15 6 21 9 14 12 12 105 Zinc (ppm) Copper (ppm) Cobalt (ppm) Barbari Jamunapari Barbari Jamunapari Barbari Jamunapari 5.74abc±0.73 6.03abc±0.73 4.51c±0.53 4.29 c ±0.84 4.82 c ±0.45 5.18abc±0.68 6.65ab±0.55 4.84bc±0.59 6.88a±0.59 5.44±0.21 4.26 ±0.69 4.94 ±0.69 4.67 ±0.50 4.89 ±0.79 4.91 ±0.42 5.93 ±0.65 5.40 ±0.65 5.63 ±0.56 5.64±0.56 5.14 ±0.20 0.76 ± 0.10 0.87 ± 0.10 0.91 ± 0.07 0.77 ± 0.12 0.96 ± 0.06 1.08 ± 0.09 0.97 ± 0.07 1.02 ± 0.08 0.96 ± 0.08 0.92 ± 0.03 0.52c ± 0.08 1.01ab ± 0.08 1.02ab ± 0.06 0.90ab ± 0.10 1.03ab ± 0.05 1.08a ± 0.08 0.85b ± 0.06 0.92ab ± 0.07 0.93ab ± 0.07 0.92 ± 0.02 0.41ab ± 0.06 0.34 ab ± 0.06 0.39 ab ± 0.04 0.39 ab ± 0.07 0.24 b ± 0.05 0.25 ab ± 0.06 0.22 b ± 0.05 0.38 ab ± 0.05 0.44 a ± 0.05 0.34 ± 0.02 0.27 ± 0.07 0.27 ± 0.07 0.21 ± 0.05 0.26 ± 0.09 0.27 ± 0.05 0.26 ± 0.07 0.23 ± 0.06 0.29 ± 0.06 0.28 ± 0.06 0.26 ± 0.02 Means marked with different a, b, c superscripts in a column between ages indicates DMRT significance (P<0.05) n= denotes the no. of observations for each age group of each breed. 46 April 2010] MICRO-MINERALS AND BIOCHEMICAL PROFILE OF MARWARI GOATS (Bhattacharyya et al. 1995) and in nulliparous heifers (Small et al. 1997). In the present study, blood Co concentration was significantly (P<0.01) affected by breed but not with age. Barbari goats have significantly (P<0.01) higher blood Co concentration than Jamunapari goats. Zadjali et al. (2004) also reported that there are likely genetic differences between breeds of the same species. Co plays a more important role in early growth and development (Kadim et al. 2006). In Barbari and Jamunapari goats, Co concentration did not change with the advancement of age. Contrary to this, kids in the age group of 1–3 months showed significantly (P< 0.05) lower levels of serum vitamin B 12 than the older animals (Zadjali et al. 2004). Robertson (1971) suggested that need of the young animal for serum vitamin B12 is greater than that of adults because of their higher metabolic rate. Although in Barbari goats, cobalt level slightly decreased at 9 to 10 months of age and remained low up to pre-pubertal age. At puberty, it again increased (though not significantly). While in Jamunapari goats, it did not show any change with the advancement of age. In young lambs (up to 2 months of age) if weaned early, likewise have a need for dietary vitamin B12 (NRC 1985). Cobalt deficiency reduced lamb survival and increased susceptibility to parasitic infection in cattle and sheep (Ferguson et al. 1998). Balanced feeding of Zn, Cu and Co in the diet of indigenous goats is very essential for optimum growth, production and reproduction to achieve maximum output in terms of economic returns as healthy, growing and performing kid which survives, reproduces and produces milk and mutton economically. 319 Church C D. 1993. El ruminates: fisiologia digestive ynutricionpp. 397–408.(Ed.) Acribia S A. Zaragoza, Espana. Desai M C, Thakkar T P, Ami D R and Janakiraman K. 1978. A note on serum copper levels in relation to reproductive performance in Surti buffaloes. Indian Journal of Animal Science 47: 398–409. Ferguson E G W, Mitchell G B and Mac Pherson A. 1988. Cobalt deficiency and Ostertagia circumcincta infection in the lambs. Veterinary Record 124: 20. Haenlein G F W. 1980. Mineral nutrition of goats. Journal of Dairy Science 30: 1729–42. Harvey W R. 1990. User’s Guide for MIXMDL PC-2 version Mixed Model Least –Squares and Maximum Likelihood Computer Program (Mimeo). Ohio State University, Columbus, OH. Kadim I T, Mahgoub O, Al-Ajmi D, Al-Habsi K R and Johnson E H. 2006. Comparative effects of low levels of dietary cobalt and parenteral injections of Vitamin B12 on body dimensions in different breeds of Omani goats. Small Ruminant Research 66: 244–52. Khan Z I, Hussain A, Ashraf M,Ashraf M Y and McDowell L R, 2007. Macromineral status of grazing sheep in a semi-arid region of Pakistan. Small Ruminant Research 68: 279–84. Kincacid R L. 1999. Assessment of trace mineral status of ruminants: A review. Proceedings of American Society of Animal Science. www.asas.org/JAS/symposia/proceedings/ 0930.pd. Kincaid R L and Hodgson A S. 1989. Relationship of selenium concentrations in blood of calves to blood selenium of the dam and supplemental selenium. Journal of Dairy Science 72: 259– 263. Kramer C Y. 1957. Extension of multiple range tests to group correlated adjusted means. Biometrics 13: 13–18. Mburu J N, Kamau J M Z and Badamana M S. 1993. Changes in serum levels of vitamin B12, feed, live weight and haematological parameters in cobalt deficient small East African goats. International Journal of Vitamin Nutrition and Research 63: 135–139. McDowell L R. 2003. Minerals in Animal and Human Nutrition. 2nd edn, 644 pp. Elsevier Science, Amsterdam. Mohamed H E. 2004. The zinc and copper content of the plasma of Sudanese camels (Camelus dromedarius). Veterinary Research Communications 28: 359–63. Naresh R. 1997. ‘Studies on profile of trace and toxic heavy metals in mastitis and ameliorative effects of ascorbic acid therapy.’ M.V.Sc. Thesis, I.V.R.I., Izatnagar. National Research Council (NRC). 1981. Nutrient Requirement of Domestic Animal. No. 15 Nutrient requirement of goats. Angora, Dairy and meat goats in temperate and tropical countries. National Academic of Sciences, Washington, D.C. Osman N I, Johnson E H, Al-Busaidi R M and Suttle N F. 2003. The effects of breed, neonatal age and pregnancy on the plasma copper status of goats in Oman. Veterinary Research Communications 27: 219–29. Pathak M M, Patel A V and Jankiraman K. 1986. Blood serum copper at different stages of pregnancy in Surti buffalo. Indian Journal of Animal Sciences 56: 1202–04. Puschner B, Thurmond M C and Choi Y K. 2004. Influence of age and production type on liver copper concentrations in calves. Journal of Veterinary Diagnostic and Investigations 16: 382– 87. ACKNOWLEDGEMENT The authors are thankful to the Director, Central Institute for Research on Goats, Makhdoom, and Dr D Swarup, Head, Division of Medicine, Indian Veterinary Research Institute, Izatnagar, for providing necessary facilities. REFERENCES Ahmed M M M, Hamed T F M and Barri M E S. 2001. Variation of Zinc and Copper concentrations in the plasma of Nubian goats according to physiological state. Small Ruminant Research 39: 189–93. Akan V D, De Bont J, Holm V and Ramawana S S E. 1991. A study of mineral status of cattle in a dairy farm in Sri Lanka. Indian Veterinary Journal 68: 371–74. Akdogan-Kaymaz A, Bakirel U, Cagtay P and Jan H. 2000. The effects of serum IgG and traceelements Copper and Zinc on the developments of Kivircik lambs following colostrums intake. Veteriner-Fakultesi Dergisi Istanbul 26: 475–78. AOAC.1984. Association of Official Analytic Chemists. Virginia. pp. 444–476. Bhattacharyya B N, Talukdar S C, Baruah R N, Baruah K K Sr, Baruah K K Jr and Baruah A. 1995. Studies on circulatory levels of trace mineral at different reproductive status in goat. Indian Journal of Animal Reproduction 16: 96–98. 47 320 BHOOSHAN AND KUMAR Radostits O M, Goy C C, Blood D C and Hinchclig R W. 2000. Veterinary Medicine: A Textbook the Diseases of Cattle, Sheep, Pigs, Goats and Horses. W.B. Saunders, London. Robertson W W.1971. Cobalt deficiency in ruminants. Veterinary Record 89: 5–12. Small J A, Charmely E, Rodd A V and Fredeen A H. 1997. Serum mineral concentration in relation to estrus and conception in [Indian Journal of Animal Sciences 80 (4) beef heifers and cows fed conserved forage. Canadian Journal of Animal Science 77: 63–68. Snedecor G W and Cochran W G. 1994. Statistical Methods. 8th edn. Iowa State University Press Ames- Iowa. Zadjali A Al, Johnson E H and Srikandakuma A. 2004. Serum vitamin B12 level in Omani goats. Tropical Animal Health and Production 36: 437–82. 48 Indian Journal of Animal Sciences 80 (4): 321–325, April 2010 Effect of age and reproductive state on phosphatase enzymes and steroid hormones profile in Indian goats* NEERU BHOOSHAN1, PUNEET KUMAR2 and M C YADAV3 Central Institute for Research on Goats, Makhdoom, Uttar Pradesh 281 122 India Received: 19 July 2008;Accepted 20 October 2009 ABSTRACT Healthy female goats (290; 145 of Barbari breed and 145 of Jamunapari breed) of different ages, viz. 0 (birth), 30, 90, 180, 270–300, 330–360 days, pre-pubertal, pubertal and post-pubertal (1 week after oestrus), managed under semiintensive system of production were used to evaluate and compare the alkaline and acid phosphatases enzyme activity and estrogen and progesterone steroid hormones profile. The activity of alkaline phosphatase was the highest at birth in both the breeds. The alkaline phosphatase activity decreased as age of goats increased. While the activity of alkaline phosphatase increased at pre-pubertal age in Barbari breed, it did not increase in Jamunapari breed. The activity of alkaline phosphatase in Jamunapari breed at pre-pubertal age was lowest. Low level of alkaline phosphatase activity might be the cause of delayed maturity in Jamunapari goats. Acid phosphatase activity was highest at birth in Barbari goats and at 3 months of age in Jamunapari goats. The lowest values of acid phosphatase were observed at pre-pubertal age in both the breeds. The values of alkaline phosphatase activity increased at pubertal and post-pubertal ages in both the breeds. In Barbari and Jamunapari goat breeds, the estrogen level was high at birth. Thereafter, it decreased abruptly and remained low up to pre-pubertal age. At puberty, the estrogen level was maximum in both the breeds. The progesterone level was highest at birth in Barbari and Jamunapari goat breeds. Thereafter it decreased and remained low till pubertal age. It increased and obtained the highest level at post-pubertal age or one week after oestrus. Estrogen (+0.13) and progesterone (+0.30) showed significant positive correlation with alkaline phosphatase enzyme activity. A nonsignificant association of acid phosphatase was observed with estrogen (–0.04). A significant positive correlation of acid phosphatase with progesterone (+0.19) was observed. Key words: Acid phosphatase, Age, Alkaline phosphatase, Estrogen, Goat, Progesterone MATERIALS AND METHODS Alkaline phosphatase enzyme is involved in energy transfer reactions (Freeland and Szepesi 1971). Phosphatases are thought to be influenced by steroid hormones. The activity and location of these enzymes change during growth and puberty. The aim of this study was to evaluate and compare the steroid hormone and phosphatases profile of two breeds of goat at different age groups and reproductive states to facilitate use of modern technologies of hormonal improvement of fertility in Barbari and Jamunapari goats. For this study, 290 experimental female goats (145 Barbari and 145 Jamunapari goats) of 0 (birth), 30, 90, 180, 270 to 300, 330 to 360 days, pre-pubertal, pubertal and post pubertal age (1 week after showing estrus) maintained under semiintensive system of management at Central Institute for Research on Goats, Makhdoom (latitude 27° 10’ N and longitude 78° 02’ E) were used. Feeding was done according to NRC (1981). For the first 15 days, newly born kids were nourished with mother’s milk with the help of bottle. Weaning was done at 3 months of age. After weaning, experimental goats were allowed 4-6 h grazing. Pelleted concentrate mixture with 13% DCP and 69% TDN was offered @ 200 g to 350 g/animal/day to Barbari goats from 2–3 months of age to 9 –10 months. An additional 50 g per animal to Jamunapari goats was offered. The adult goats were provided with 400 g concentrate feed. Drinking water was given ad lib. The animals were dewormed regularly as per the standard health practices. *Part of Ph.D. thesis, Submitted to Dr B.R. Ambedkar University, Agra, Uttar Pradesh Present address: 1Scientific Officer, U.P. Council of Agricultural Research, 8th Floor, Kisan Mandi Bhawan, Vibhuti Khand, Gomti Nagar, Lucknow, Uttar Pradesh 226 010(e-mail address: [email protected]). 2Principal Scientist,Division of Goat Physiology. 3Principal, Narain College, Shikohabad, Firozabad, Uttar Pradesh 49 322 BHOOSHAN ET AL. Blood samples were collected by jugular venipuncture from each animal into nitric acid washed heparinized vials; 10 ml heparinized blood was centrifuged at 2 500 rpm for 15 min, separated plasma was stored at –20°C till analysis. Alkaline and acid phospatases were estimated in plasma by using commercial kits. In plasma, steroid hormones (progesterone and estrogen) were quantitatively analyzed by an enzyme immuno assay (EIA) method with commercially available diagnostic kits (Bhooshan and Kumar 2007). Standards were provided in the kits. For progesterone, standard ranged from 0 to 50 ng/ml and for estrogen, from 0 to 1000 pg/ml. The minimum detectable concentration of progesterone and estrogen 0.05 ng/ml and 1 pg/ml, respectively. Average intra assay variation for progesterone and estrogen was 10.92 and 7.92% respectively. Inter assay variation for progesterone and estrogen was 17.01 and 11.75% respectively. Results were treated statistically by using mixed model (MIXMDL PC-2) program with a least square technique for fitting non-orthogonal data and maximum likelihood computer program developed by Harvey (1990). To find out any significant difference among least squares means for age within breed, pair wise comparison was carried out by Duncan’s multiple range test (DMRT) modified by Kramer (1957). By using standard method, correlation coefficient (r) was carried out in pooled manner (Snedecor and Cochran 1994). [Indian Journal of Animal Sciences 80 (4) transfer reactions its utility in growth has been established. The plasma alkaline phosphatase activity in healthy goats was reported as 432.78 ± 20.00 IU/L (Sandhu et al. 2001). A wide range of alkaline phosphatase values in lower age group of goats (2.80 to 16.73 BU/100 ml) as compared to in higher age group (1.15 to 7.85 BU/100ml) were reported by Adaval et al. (1969). In Barbari and Jamunapari goats, activity of alkaline phosphatase was the highest (72.95 ± 4.25 and 60.61 ± 4.98 KA units, respectively) at birth. Higher alkaline phosphatase found in early age might be due to higher osteoblastic activity for bone formation and to higher metabolic rate (Freeland and Szepesi 1971). Contrary to these findings, Daramola et al. (2005) reported that in West Arfican Dwarf goats, serum alkaline phosphatase levels were however higher in adult animals compared to young animals (P<0.05). In young animals, alkaline phosphatase comes from osteoblasts and chondroblasts because bone development is active. In older animals, alkaline phosphatase comes from hepatobiliary system (Brar et al. 2000). Higher level of alkaline phosphatase was observed in first 2 months of age in male and female kids of Marwari and Surti breeds (Patel et al. 1992). Similarly, in local Israeli goats (Bogin et al. 1981) and in Black Bengal goats (Kalita and Mahapatra 1998), alkaline phosphatase decreased with increase in age. Alkaline phosphatase plays an important role in certain events of reproductive physiology. In Barbari goats, at prepubertal age, the activity of alkaline phosphatase increased up to the level of 52.36 ± 4.52 KA units. It decreased to the level of 39.82 ± 3.55 KA units, at pubertal age. The alkaline phosphatase activity was maximum during luteal phase of oestrous cycle than in the follicular phase of oestrous cycle in cows and goats (Rama Chandran et al. 1980,Singh and Rajya 1982). Similarly, in Alpine Malabari crossbred goats, serum alkaline phosphatase activity was however high, though not significant on 14th day of the cycle, when the luteal function was more (Mathai and Nirmalan 1992). However, in Jamunapari goats, at pre-pubertal age, the activity of alkaline phosphatase was lowest (12.68 ± 4.84 KA Units). Low level of alkaline phosphatase activity may be the cause of delayed maturity in Jamunapari goats. RESULTS AND DISCUSSION Alkaline phosphatases Phosphatase enzymes: The overall means of alkaline phosphatase in Barbari and Jamunapari goats irrespective of age and season of birth were 44.62±1.30 and 35.49±1.53 KA units respectively (Table 1). In Barbari breed, the activity of alkaline phosphatase was significantly higher (72.95±4.25 KA units) at birth. After that it decreased up to 1 year of age. At pre-pubertal age, the activity of alkaline phosphatase again increased significantly up to the level of 52.64±4.60 KA units. Then it decreased significantly at pubertal and post-pubertal age than that of pre-pubertal age. In Jamunapari goats, the activity of alkaline phosphatase was significantly higher (60.61±4.98 KA units) at birth. After that it decreased up to pre-pubertal age. At pre-pubertal age, the activity of alkaline phosphatase decreased significantly up to the level of 12.38±4.92 KA units. It increased significantly at pubertal and post-pubertal age than that of pre-pubertal age. In Barbari goats, the alkaline phosphatase activity during February–March and September–October season of birth were 42.97±1.90 and 46.27±2.14 KA units, respectively. In Jamunapari goats, the alkaline phosphatase means during two seasons of birth were 39.32±2.23 and 31.66±2.60 KA units, respectively. Alkaline phosphatase enzyme is involved in energy Acid phosphatase (KA units) The least square means ±SE of acid phosphatase in Barbari and Jamunapari breeds from birth to post-pubertal age are given in Table 1. Least square analysis of variance indicated that enzyme activity was significantly different between season (P<0.05) and between breeds and in various ages (P<0.01). The interaction between breed × season and breed × age was also significantly different (P<0.01). A nonsignificant association of acid phosphatase was observed with estrogen (–0.04). A significant positive correlation of acid phosphatase with progesterone (+0.19*) was observed. In Barbari breed, the activity of acid phosphatase was 50 April 2010] PHOSPHATASE ENZYMES AND STEROID HORMONES IN GOATS 323 Table 1. Least square mean ± SE of alkaline phosphatase (KA units) and acid phosphatase (KA units) at various ages for Barbari and Jamunapari goats Age (Days) Birth 30 90 180 270–300 330–360 Pre-pubertal Pubertal Post-pubertal Season of birth Feb–March Sep–Oct Overall n Alkaline phosphatase (KA units) Acid phosphatase (KA units) Barbari Jamunapari Barbari Jamunapari 12 16 15 21 21 14 14 16 16 72.95a ± 4.25 63.74ab ± 3.85 32.11d ± 4.38 37.38d ± 3.53 34.03d ± 3.53 34.77d ± 4.67 52.36bc ± 4.52 39.82cd ± 3.55 34.41d ± 3.55 60.61a ± 4.98 51.19ab ± 4.62 35.84bcd ± 4.84 43.87bc ± 3.85 30.63d ± 3.85 21.76de ± 4.98 12.68e ± 4.84 33.09cd ± 4.98 29.84d ± 4.98 2.85a ± 0.29 2.66a ± 0.25 1.16cd ± 0.28 2.04ab ± 0.23 1.60bcd ± 0.23 1.93abc ± 0.29 0.83d ± 0.29 2.05ab ± 0.25 2.60a ± 0.25 2.82abc ± 0.28 2.60bc ± 0.24 3.63a ± 0.27 2.09c± 0.21 2.64bc ± 0.21 3.35ab ± 0.27 0.99d ± 0.27 2.56b ± 0.24 3.16ab ± 0.24 79 66 145 42.97 ± 1.90 46.27 ± 2.14 44.62 ± 1.30 39.32α ± 2.23 31.66ß ± 2.60 35.49 ± 1.53 1.64α ± 0.13 2.30ß ± 0.15 1.97 ± 0.09 2.69 ± 0.12 2.61± 0.14 2.65 ± 0.08 n= no. of observations for each breed;means marked with different a, b, c, d, e (superscript) in a column between ages indicates DMRT significance (P<0.05). Means marked with different α, ß (superscript) in a column between season of birth indicates DMRT significance (P<0.05). significantly higher (2.85±0.29 KA units) at birth and remained high up to 1 month of age. At 3 month of age, it decreased significantly and showed fluctuating trend up to one year of age. At pre-pubertal age, it attained significant low value (0.83±0.30 KA units). At pubertal and at postpubertal age, it again increased significantly and attained value of 2.05±0.25 and 2.60±0.25 KA units respectively. In Jamunapari goats, activity of acid phosphatase was significantly higher (3.63±0.27KA units) at 3 months of age. The activity decreased and remained lower from 6 months to 1 year of age. The activity further decreased significantly and attained the lowest level (0.99±0.27 KA units) at prepubertal age. At pubertal and post pubertal ages, it again increased significantly and attained values of 2.56±0.24 and 3.16±0.24 KA units respectively. The activity of acid phosphatase during February–March and September–October seasons of birth in Barbari goats were 1.64±0.13 and 2.30±0.15 KA units, respectively, which were significantly different. In Jamunapari goats, the means during 2 seasons of birth were 2.69±0.12 and 2.61±0.14 KA units; respectively, which were not significantly different from each other. In Black Bengal kids, the acid phosphatase showed change with increase in age (Kalita and Mahapatra 1998). Both the phosphatases are intracellular enzymes associated with energy transformation during cellular activity. However, in plasma they represent the enzyme leaked out from the cells. The higher levels of these enzymes in new born kids appear to be due to the higher metabolic rate in cells of the new-born due to higher enzymatic activity required for energy supply thus resulting in greater leakage. The higher levels recorded during sexually active period can be due to higher metabolic activity resulting in passage of enzyme through cell membrane. Steroid hormones Estrogen (pg/ml):Least square analysis of variance (Table 2) indicated that the estrogen level was significantly different (P<0.01) at different ages of goats. Estrogen showed a significant positive association with alkaline phosphatase (+0.13*). There was a low association of estrogen with acid phosphatase. In Barbari breed, the estrogen level at birth was significantly higher (61.05±3.06 pg/ml). Thereafter, it decreased and remained significantly low up to pre-pubertal age. At puberty, estrogen level increased significantly and attained the highest level of 103.83±3.25 pg/ml at puberty. Again, it attained significantly low level after one week of estrus. In Jamunapari goats, the estrogen level at birth was significantly higher (58.77±2.29 pg/ml). Thereafter, it decreased and remained significantly low up to pre-pubertal age. At puberty, estrogen level increased significantly and attained highest level of 102.41±2.43 pg/ml. Again it attained significantly low level after 1 week of estrus. Estrogen stimulates bone growth in young ruminants and exhibits other anabolic effects. In Barbari and Jamunapari goats, estrogen level was higher at birth. Thereafter, it decreased abruptly and remained low up to 3 months of age. There is a small increase in estrogen level from 6 months of age to pre-pubertal age. Hormonal levels begin to rise at the age of 4–5 months. A small rise up to 2 pg/ml was measured 51 324 BHOOSHAN ET AL. [Indian Journal of Animal Sciences 80 (4) Table 2. Least square mean ± SE of estrogen (ng/ml) and progesterone (pg/ml) at various ages for Barbari and Jamunapari goats Age (Days) Birth 30 90 180 270–300 330–360 Pre-pubertal Pubertal Post-pubertal Overall n 9 12 9 8 8 8 8 8 8 78 Estrogen (pg/ml) Progesterone (ng/ml) Barbari Jamunapari Barbari Jamunapari 61.05b ± 3.06 22.72c ± 2.65 22.65c ± 3.06 27.93c ± 3.25 29.02c ± 3.25 30.79c ± 3.25 32.05c ± 3.25 103.83a ± 3.25 31.00c ± 3.25 40.12 ± 1.05 58.77b ± 2.29 24.89cd ±1.99 22.25d ± 2.29 29.89c ± 2.43 25.92cd ± 2.43 28.99cd ± 2.43 30.50c ± 2.43 102.41a ± 2.43 29.74cd ± 2.43 39.46 ± 0.78 12.43a ± 0.43 0.74c ± 0.38 0.88c ± 0.43 0.70c ± 0.45 0.68c ± 0.45 0.76c ± 0.45 0.81c ± 0.45 0.91c ± 0.45 9.28b ± 0.45 3.02± 0.15 13.36a ± 0.54 0.74b ± 0.47 0.73b ± 0.54 0.79b ± 0.58 0.80b ± 0.58 0.81b ± 0.58 0.81 b ± 0.58 1.40 b± 0.58 11.90a ± 0.58 3.48 ± 0.19 n= no. of observations for each breed; means marked with different a, b, c, d (superscript) in a column between ages indicate DMRT significance (P<0.05). in oestradiol at the transition from the pre-pubertal to the pubertal period (Khanum et al. 2000). Estrogen hormones also control development of all female secondary sex characteristics and play a major role in mammary gland development. At puberty, the estrogen level was maximum (103.83±3.25 and 102.41±2.43 pg/ml, respectively) in Barbari and Jamunapari breeds. In goats of mixed breed also, there was change in the plasma estradiol17α during estrous cycle. The concentration did not fluctuate much during the day of estrus (Leyva- Ocariz et al. 1995). On the day of fertile heat the estradiol concentration was 22.25±9.07 and 7.56±1.04 pg/ml for Surti and Marwari goats, respectively (Patel et al. 1992). The average concentration of estradiol-17â on the day of oestrus (Surti, 15.54 pg/ml, Marwari, 15.39 pg/ml) was significantly higher for both the breeds of goat, as compared to other stages of oestrous cycle (Pathak et al. 1990 and Pathak et al. 1992). One week after puberty, the estrogen level decreased. Similarly in Surti and Marwari goats the level decreased gradually as the luteal phase advanced, i.e. up to day 13 of the oestrous cycle (Pathak et al. 1992). Progesterone (ng/ml): The means of progesterone in Barbari and Jamunapari goats from birth to post-pubertal ages have been given in Table 2. Least square analysis of variance indicated that there was significant difference (P<0.01) at different ages. A significant positive trend of association of progesterone was noted with acid phosphatase (+0.19*) and alkaline phosphatase (+0.30**). The overall means of progesterone (ng/ml) for Barbari and Jamunapari goats irrespective of age were 3.02±0.15 and 3.48±0.19 ng/ml respectively. In Barbari breed, the progesterone level at birth was significantly higher. Thereafter, it decreased and remained significantly low up to the pubertal age. It attained the highest level at post pubertal age. In Jamunapari goats, the progesterone level was significantly higher at birth. It decreased significantly up to pubertal age. Thereafter it increased and attained significantly high level of 11.90±0.58 ng/ml at post pubertal age. In Toggenburg and Alpine goats, neither serum estradiol and progesterone nor faeces estrogen and progesterone concentrations were affected by breed (Capezzuto et al. 2008). At puberty, the estrogen level was maximum in both the breeds. Similarly in Damascus does, plasma estradiol levels increased significantly (P< 0.01) during the estrous cycle (Gaafar et al. 2005). In Ethiopian Menz ewe lambs, a progesterone concentration was basal (<1.0 ng/ml) before puberty (Mukasa –Mugerwa and Mutiga 1993). It increased slightly at prepubertal age. In temperate and tropical ewe lambs also, there was transient increase in progesterone before puberty (Quirke et al. 1985 and Oyedipe et al. 1986). In Hebsi and Zomri goats, plasma progesterone concentration was <0.5 ng/ml during the pre-pubertal period and >2 ng/ml at first oestrus (Al-Hozab and Basiquni 1999). During the pre-pubertal period, progesterone remained at basal level of 0.1 to 0.5 ng/ ml (Khanum et al.2000). In the present study, the progesterone level was 0.91 ± 0.45 and 1.40 ± 0.58 ng/ml, respectively on the day of oestus in Barbari and Jamunapari goats. Similarly, the progesterone level was low on the day of oestrus in Surti (0.5 ng/ml) and Marwari (0.76 ng/ml) (Pathak et al. 1990), in German Dairy × Boer goat does (0.10 ng/ml) (Bauernfield and Holtz 1991) and in Damascus does (0.69 ± 0.85 nmol/l) (Zarkawi and Soukouti 2001). In the present study, the progesterone level reached the highest level at post-pubertal age or 1 week after oestrus. Similarly, it increased and reached to a peak by day 9 in Surti and by day 13 in Marwari goats (Pathak et al. 1992), by day 12 to 15 in Alpine × Nubian crossbred goats (10–12 ng/ml) (Leyva–Ocariz et al. 1995) and by day 12 in Damascus does (4.2 ± 0.1 ng/ml) (Gaafar et al. 2005) of oestrous cycle. These transient elevations may be useful for normal corpus luteum function (Mcleod and Haresign 1984). 52 April 2010] PHOSPHATASE ENZYMES AND STEROID HORMONES IN GOATS ACKNOWLEDGEMENT 325 Mathai E and Nirmalan G. 1992. Enzyme profile during growth and oestrous cycle in goats. Recent Advances in Goat Production. Proceedings of 5th International Conference on Goat. New Delhi. pp. 1341–47. McLeod B J and Haresign W. 1984. Evidence that progesterone may influence subsequent luteal function in the ewe by modulating preovulatory follicle development. Journal of Reproduction and Fertility 71: 381–86. Mukasa-Mugerwa E. and Mutiga ER. 1993. Prepubertal plasma progesterone patterns in Ethiopian Menz ewe lambs. Journal of Applied Animal Research 3: 31–38. National Research Council (NRC). 1981. Nutrient Requirement of Domestic Animal. No. 15. Nutrient requirement of goats. Angora, Dairy and meat goats in temperate and tropical countries. National Academy of Sciences, National Research Council, Washington, D.C. Bhooshan N and Kumar P. 2007. Profile of ovarian and thyroid hormones, alkaline phosphatase and cholesterol at oestrous cycle, gestation and early lactation in blood plasma of Marwari goats. Indian Journal of Animal Sciences 77(12): 1233–37. Oyedipe E O, Pathiraja N, Edqvist L E and Bewanendran V. 1986. Onset of puberty and oestrous cycle phenomenon in Yankasa ewes as monitored by plasma progesterone concentration. Animal Reproduction Science 12: 195–99. Patel A V, Pathak M M, Mehta V M and Janakiraman K. 1992. Circulating levels of hormones in pregnant goats. Recent Advances in Goats Production. Proceedings of 5th International Conference on Goats. pp. 1371–772. 8th March, New Delhi. Pathak M M, Patel A V, Jaiswal R S and Mehta V M. 1990. Circulating levels of progesterone and oestrogen in cyclic goats. Indian Journal of Animal Sciences 60(7): 836–37. Pathak M M, Patel A V, Jaiswal R S, Mehta V M and Janakiraman K. 1992. Circulating hormone levels in cyclic goats. Proceedings of 5th International Conference on Goats, pp. 1383–872. 8th March, New Delhi. Quirke J F, Stabenfeldt G H and Bradford G E. 1985. Onset of puberty and duration of the breeding season in Suffolk, Rambouillet, Finish Landrace, Dorset and Finn-Dorset ewe lambs. Journal of Animal Sciences 60: 1463–71. Ramachandran S V, Rao P N, Rao P R and Rao A R. 1980. Histological, histometrical and histochemical changes in the uterine and oviductal epithelium of ewes during oestrous cycle. Indian Journal of Animal Sciences 50: 41–45. Sandhu A K, Saini A and Randhawa R S. 2001. Haematological studies in healthy goats. Indian Veterinary Journal 78(7): 590– 93. Singh N and Rajya B S. 1982. Histochemical studies on the ovary uterus and vagina of goats. Indian Journal of Animal Health 21: 1–3. Snedecor G W and Cochran W G. 1994. Statistical Methods. 8th edn. The Iowa State Univ. Press, USA. Zarkawi M and Soukouti A. 2001. Serum progesterone levels using radio immunoassay during oestrous cycle of indigenous Damascus does. New Zealand Journal of Agricultural Research 44(2/3): 165–69. Sincere thanks are due to Director, CIRG, for providing all necessary facilities to carry out the present investigation. REFERENCES Adaval S G, Morris I D and Khare S G. 1969. Serum alkaline and acid phosphatase activity and inorganic phosphorus in goats. Indian Veterinary Journal 46: 485–90. Al-Hozab A and Basiquni G. 1999. Onset of puberty in Hebsi and Zomri goats as monitored by plasma progesterone concentrations. Journal of Applied Animal Research 15(1): 69– 74. Bauernfield M and Holtz W. 1991. Progesterone and estrogen levels in serum of cycling goats measured by enzyme immunoassay. Small Ruminant Research 6: 95–102. Bogin E, Shimshony A, Avidar Y and Israli B. 1981. Enzymes, metabolites and electrolytes levels in the blood of local Israeli goats. Zentralblatt-fur-Veterinarmedizin-A 28(2): 135–40. Brar R S, Sandhu H S and Singh A. 2000. Veterinary Clinical Diagnosis by Laboratory Methods. Kalyani Publishers, New Delhi. Capezzuto A, Chelini M O M, Flippe E C G and Oveira C A. 2008. Correlation between serum and fecal concentrations of reproductive steroids throughout gestation in goats. Animal Reproduction Science 103: 78–86. Daramola J O, Adeloye A A, Fatoba T A and Soladoye A O. 2005. Haematological and biochemical parameters of West African Dwarf goats. Livestock Research and Rural Development 17(8): 90–95. Freeland R A and Szepesi A. 1971. Control of enzyme activity nutrition factors. Enzyme Synthesis and Degradation in Mammalian System. (Ed.) Recheigi M. Univ., Parl Press, Baltimore. Gaafar K M, Gabr M K and Teleb D F. 2005. The hormonal profile during the estrous cycle and gestation in Damascus goat. Small Ruminant Research 57: 85–93. Harvey W R. 1990. User’s Guide for MIXMDL PC-2 version Mixed Model Least –Squares and Maximum Likelihood Computer Program (Mimeo). Ohio State University, Columbus, OH. Kalita D J and Mahapatra M. 1998. Serum constituents and serum enzyme activities of Black Bengal kids. Indian Journal of Animal Research 32: 38–40. Khanum S A, Hussain M, Ali M, Kausar R and Cheema A M. 2000. Age at puberty in female dwarf goat kids and estrous cycle length on the basis of hormones. Pakistan Veterinary Journal 20: 71– 74. Kramer C Y. 1957. Extension of multiple range tests to group correlated adjusted means. Biometrics 13: 13–18. Leyva-Ocariz H, Munro C and Stabenfeldt G H. 1995. Serum LH, FSH, estradiol-17 β and progesterone profiles of native and crossbred goats in a tropical semiarid zone of Venezuela during the estrous cycle. Animal Reproduction Science 39: 49–58. 53 Short Communications Indian Journal of Animal Sciences 80 (4): 326–328, April 2010 Comparison of PCR with conventional techniques for the diagnosis of brucellosis in cattle P KAUSHIK1, D K SINGH 2 and A K TIWARI 3 Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh 243 122 India Received: 10 April 2009; Accepted: 28 November 2009 Key words: Brucella, Cattle, Polymerase chain reaction (PCR) Brucellosis is a major zoonotic disease endemic in many parts of the world. It is characterized by chronic infection in animals leading to abortion and infertility. The disease is caused by Brucella which is gram negative, nonsporeforming, facultative intracellular organism (Cardoso et al. 2006). It consist of 7 species (Ko and Splitter 2003) based on antigen variation and primary host: B. melitensis (sheep and goat), B. abortus (cattle), B. suis (swine), B. ovis (rams), B. canis (dogs), B. neotomae (wooden rats) and recently recognized B. maris (marine mammals). Besides, restriction in the international trade of animal and animal products, the disease can be transmitted to humans following contact with infected animals or through consumption of contaminated milk and milk products (Young et al. 1985). Serological assays used for the diagnosis of Brucella infection include Rose Bengal plate agglutination test (RBPT), serum tube agglutination test (SAT), compliment fixation test (CFT) and enzyme linked immunosorbant assay (ELISA); however, bacteriological isolation remains the definitive method (Chand et al. 2005). But the tedious isolation process and risk to the laboratory personnel to infection limits regular use of bacteriological isolation in diagnosis. Apart from this, brucellae could not be isolated many a times even from known positive cases (Probert et al. 2004). Therefore, a rapid, sensitive and specific tool like polymerase chain reaction (PCR) for precise detection and identification of such a fastidious organism(s) from a variety of samples (Amin et al. 2001, Leyla et al. 2003 and Probert et al. 2004) is being exploited. Several PCR based methods have been described for the detection of Brucella species using bacterial isolates and field samples (Leal-Klevezas et al. 2000). The present study compares PCR with isolation of Brucella from different samples vis a vis conventional test, viz. RBPT and SAT. A total of 45 samples including blood (15), vaginal swabs (15) and milk (15) were collected simultaneously from cattle population from the dairy farm of the Indian Veterinary Research Institute, Izatnagar. The samples were collected aseptically and immediately transported to laboratory under cold condition. The milk samples were processed for bacteriological isolation as described by Alton et al. (1995) and incubated at 37°C under 5% CO2 for several days and inspected regularly for the development of Brucella, like colonies. The blood containing sodium citrate was inoculated onto Ruiz Castaneda biphasic medium (Leal-Klevezas et al. 2000). The vaginal swabs were washed properly in 2 ml of sterilized phosphate buffer saline (PBS; pH 7.4) and were inoculated onto Ruiz Castaneda biphasic medium as for the blood. The serum were tested by RBPT and SAT as per the standard method (Alton et al. 1975). Serum samples, exhibiting any degree of clumping of coloured antigen in RBPT and 50% agglutination reaction at a dilution of 1: 40 or above in SAT were considered positive. The DNA was extracted from whole blood as described by Leal- Klevezas et al. (2000) whereas DNA extraction kit (Biogene) was used for extracting DNA from fatty top layer of raw milk and vaginal swab washing (each 400 μl) as per the manufacturers guidelines. PCR was performed in 50 μl containing 5 μl of 10X PCR buffer (500 mM KCl, 100 mM Tris- HCl, pH 9.0), 200 μM each of the 4 deoxynucleotide triphosphates, 1U Taq DNA polymerase, 8 pmol each of the primers derived from the BCSP–31 sequence (Serpe et al. 1999) of B. abortus (Forward 5’ GGG CAA GGT GGA AGA TTT 3’ and Reverse 5’ CGG CAA GGG TCG GTG TTT 3’) and 5 μl of template DNA. The amplification was carried out in mastercycler gradient thermocycler with a preheated lid at a denaturation temperature of 94°C for 3 min followed by 35 cycles at 94°C for 45s, 53°C for 45s and 72°C for 5 min. Electrophoresis of PCR product was carried out on 1.5% (w/v) agarose gel in tris-borate EDTA (TBE; 0.5X) to visualize 443bp product in positive samples after staining with ethidium bromide. Out of 15 blood samples, 13 were positive in RBPT and Present address: 1Assistant Professor, Department of Veterinary Public Health, Bihar Veterinary College, Patna, Bihar, India. 2, 3Division of Veterinary Biotechnology. 54 April 2010] COMPARISON OF DIAGNOSTIC TECHNIQUES FOR BRUCELLOSIS of these positive samples, 6 were also positive in SAT with a cut off titre of 80. The PCR was positive in 2 blood samples of animals positive in SAT and RBPT. The result of RBPT and SAT, where 7 samples positive by RBPT turned out as negative in SAT, corroborates the findings of Morgan (1974). Moreover, the RBPT recommended by WHO/FAO Expert committee as a screening test (Anonymous 1986), has been reported to be an oversensitive test (Alton et al. 1975a). The other 6 animals were positive to both RBPT and SAT. No isolation of Brucella could be made from any of the blood, milk and vaginal swabs taken from these animals. But the blood and vaginal swabs from 2 of these 6 animals, which tested positive by both serological tests, were also positive by PCR. Although cultural isolation is the gold standard in the microbial diagnosis, it may sometimes be abolished by factors inherent to the microbes besides it is tedious and not possible to isolate Brucella every time even from the known positive cases (Ray 1979). The observation in the present study that two culture negative but seropositive animals tested positive by PCR supports this contention. In case of vaginal swab, this could even be due to massive contamination of the samples or viability loss of the organism before culturing (Ray 1979) and in all these circumstances DNA can still be detected by PCR. However, the intracellular nature of bacteria may affect the proper release of Brucella from leukocyte and results in the negative isolation of Brucella from blood samples. Brucellae are excreted in milk intermittently which may explain the negative PCR result on milk samples from these animals. Alternatively, this could be because of the presence of the PCR inhibitors or number of organisms below the detection limit, in the milk. The sensitivity of PCR to detect brucellae from milk was also determined by serially diluting B. abortus S19 in uncontaminated milk, followed by DNA isolation as above, which detected as low as 40 cells/ml of milk, which is higher than the previous report (Romero et al. 1995). This detection limit can be further increased by reducing sampling error, since random distribution of the cells in a sample may not have the exact number of cells or gene copy as has been calculated to contain (Wilson 1997). The sensitivity of the test in spiked blood was not performed due to the intracellular nature of the organism in leukocytes. The specificity of the PCR was confirmed by amplification of DNA extracted from different strains of Brucella and the lack of amplification of DNA extracted from closely related organisms, viz. Escherichia coli, Vibrio cholerae, Campylobacter coli, Listeria monocytogenes, L. ivanovi and Salmonella Typhimurium. The failure to detect amplified fragments in the DNA template of the related bacteria supported the specificity of this PCR as a genus specific assay in the detection of brucellae. The results indicated the superiority of the PCR for detecting small amount of the pathogen in various samples over isolation proving the PCR as a highly sensitive, reliable and specific method for detection of 327 Brucella species from various clinical samples and can be used as an adjunct to serological tests in diagnosis of brucellosis. However, there is a need to improve the sensitivity of the PCR. SUMMARY Brucellosis is still a wide-spread zoonosis of international importance. In the present study, a PCR has been compared with conventional methods for detection of brucellosis, viz. RBPT, SAT and bacteriological isolation of the agent. The PCR was found highly specific for identification of Brucella. No isolations could be made even from the animals which were positive to RBPT, STAT and PCR. The study indicated that the PCR could be used as an adjunct in the diagnosis of brucellosis. ACKNOWLEDGEMENT The authors are thankful to the Head, Division of Veterinary Public Health and the Director, Indian Veterinary Research Institute, Izatnagar, for providing necessary facilities to carry out the present work. Financial grant received by the first author in the form of fellowship is also acknowledged. REFERENCES Alton G G, Johnes L M and Pietz D E. 1975. Laboratory Techniques in Brucellosis. TRS No.55. WHO, Geneva, Switzerland. Alton G G, Maw J, Rogerson B A, Mc Pherson G G. 1975a. The serological diagnosis of ovine brucellosis: an evaluation of the complement fixation, serum agglutination and rose Bengal test. Australian Veterinary Journal 51: 57–63. Amin A S, Hamdy M E R and Ibrahim A K. 2001. Detection of Brucella meletensis in semen using the polymerase chain reaction assay. Veterinary Microbiology 83: 37–44. Anonymus. 1986. Joint FAO/WHO Expert Committee on Brucellosis. TRS No. 740. WHO, Geneva. Cardoso P G, Macedo G C, Azevedo V and Oliveira S C. 2006. Brucella spp noncanonical LPS: structure, biosynthesis, and interaction with host immune system. Microbial Cell Factories 5: 1–11. Chand P, Rajpurohit B S, Malhotra A K and Poonia J S. 2005. Comparison of milk- ELISA and serum- ELISA for the diagnosis of Brucella melitensis infection in sheep. Veterinary Microbiology 108: 305–11. Ko J and Splitter G A. 2003. Molecular host pathogen interaction in brucellosis: current understanding and future approaches to vaccine development for mice and humans. Clinical Microbiology Review 108: 65–78. Leal-Klevezas D S, Martinez-Vazque I O, Garciacantu J, LopezMerino A and Martinez-Soriano J P. 2000. Use of polymerase chain reaction to detect Brucella abortus biovar 1 in infected goats. Veterinary Microbiology 75: 91–97. Leyla G, Kadri G and Umran O K. 2003. Comparison of polymerase chain reaction and bacteriological culture for the diagnosis of sheep brucellosis using aborted fetus sample. Veterinary Microbiology 93: 53–61. 55 328 KAUSHIK ET AL. Morgan W J B. 1974. the diagnosis, control and eradication of bovine brucellosis in Great Britain. Veterinary Record 94: 510– 17. Probert W S, Schrader K N, Khuong N Y, Bystrom S L and Graves M H. 2004. Real-time multiplex PCR assay for detection of Brucella spp., B. abortus and B. melitensis. Journal of Clinical Microbiology 42: 1290–93. Ray W C. 1979. Brucellosis due to Brucella abortus and B. suis, Hand Book Series in Zoonosis. Vol. 1, CRC Press Inc., Boca Raton, Florida. pp. 99–185. Romero C, Pardo M, Grillo M J, Diaz R, Blasco J M and LopezGoni I. 1995. Evaluation of PCR and indirect enzyme-linked immunosorbent assay on milk samples for diagnosis of [Indian Journal of Animal Sciences 80 (4) brucellosis in dairy cattle. Journal of Clinical Microbiology 33: 3198–3200. Serpe L, Gallo P, Fidenza N, Scaramuzzo A and Fenizia D. 1999. Single-step method for rapid detection of Brucella spp. in soft cheese by gene specific polymerase chain reaction. Journal of Dairy Research 66: 313–17. Wilson I G. 1997. Inhibition and facilitation of nucleic acid amplification. Applied Environmental Microbiology 63: 3741– 51. Young E J, Borchert M M, Kretzer F L and Musher D M. 1985. Phagocytosis and killing of Brucella by human polymorphonuclear leukocytes. Journal of Infectious Diseases 151: 682–90. 56 Indian Journal of Animal Sciences 80 (4): 329–330, April 2010 Therapeutic and residual efficacy analysis of some anti-tick compounds against natural Boophilus microplus infestation in crossbred cattle HIRA RAM1 and A K SHARMA2 Indian Veterinary Research Institute, Campus Mukteswar, Uttarakhand, 263138 India Received : 15 March 2009;Accepted: 4 October 2009 Key words: Boophilus microplus, Cattle, Residual efficacy, Therapeutic efficacy Effective control of tick infestation on animal is mainly carried out by the use of acaricides. Reports on therapeutic and residual drug efficacies of the dips or spray formulations of generally used acaricides are also available (Srivastava et al. 2001, Vatsya et al. 2007). However, such information is missing for recently available anti-tick compounds having different modes of application (injectable and pour on preparations). Present study was performed with the objective to record efficacy and duration of protection of avermectins (ivermectin and doramectin injections) and flumethrin (pour on) preparations against natural B. microplus infestation in crossbred cattle. Study was conducted at the experimental cattle herd of Indian Veterinary Research Institute, Mukteswar, located 29° 25N, 79° 35E, and 7500′ above the mean sea level (msl) during the monsoon period in 2004. In the study area total annual rainfall recorded was 1067.1 mm (maximum 318 mm in August) whereas annual average humidity was 51.5% (maximum 94% in August and minimum 40% in December). Mean maximum temperature in area ranges between 25.7°C in May (highest) and 11.7°C in January (lowest). Animals (crossbred cattle) of this farm graze daily (partial grazing) in the surrounding forest in addition to the standard concentrate, hay, silage or oak leaves supplementation and ad lib. water supply in the shed under scientific supervision. Previous tick management practices at this farm indicate 6 to 8 applications of cypermethrin compounds (body spray) in all animals in one calendar year because of severe tick infestation. For this study naturally infested 18 crossbred cattle, having more than 500 visible adult ticks on body surface (male, female both) were selected and divided into 3 groups of 6 animal each (group A, B and C). Representative tick samples for identification were collected from other animals of the farm and they were also treated along with this trial using either avermectins (injectable) or with flumethrin (pour on) to minimize over all tick load at the farm. Three drugs having different mode of application (ivermectin, doramectin, and flumethrin) rather than dip or body spray (cypermethrin) were selected as a part of strategy to break resistance power of ticks, gained if any in the past. Pre-treatment tick counts of individual animals were performed by counting the number of ticks on one side of animal and than by multiplying with a factor of 2 to get the total number. All animals of group-A received ivermectin injection (1% w/v) @ 0.2-mg/kg body weight (b.wt.) by subcutaneous route whereas; animals of group-B were treated with doramectin injection (1% w/v) at dose and route as mentioned above for ivermectin. Further, group C animals were treated with flumethrin (1% w/v, pour on) @ 1 ml/10 kg b.wt by applying the drug on the dorsum of animals. Tick survival on the animal body was recorded at weekly interval till the appearance of re-infestation of ticks and comparative efficacy was calculated by indirect assessment of per cent tick survival (group average) on seventh day post treatment (DPT), with reference to the average zero day tick count in a group. Existing severe tick infestation on animals at this farm seems to be due to previous continuous applications of cypermethrin (body spray) alone, leading to resistance progenies of B. microplus in the area of study. Zero day observation on tick counts revealed an average 650, 716 and 742 ticks on the body of groups A, B and C animals, respectively. Therapeutic efficacy calculated on seventh DPT revealed 95, 96 and 97% tick reduction from the animal’s body in ivermectin, doramectin and flumethrin treatment groups respectively (Table 1). Male and fully engorged female ticks were not found over the animal’s body, but partially engorged females were found to survive in all treatment groups on seventh DPT. The number of ticks survived on fourteenth DPT remains almost constant as like seventh DPT. Persistent efficacy or residual effect of ivermectin and doramectin was recorded less than 21 days Present address: 1,2 Senior Scientist,Temperate Animal Husbandry Division (e–mail address: [email protected]). 57 330 RAM AND SHARMA [Indian Journal of Animal Sciences 80 (4) Table 1. Observations on therapeutic and residual efficacies of anti-tick compounds against B. microplus infestation in cattle Group Pre and post treatment tick survival (group average) 0 DPT A (IVM) B (DOM) C (FLM) 650 716 742 7th DPT 14th DPT 21st DPT 32(95% reduction) 28(96% reduction) 22(97% reduction) Constant Constant Constant re-infestation re-infestation Constant Protection period (days) <21 days <21 days <28 days* * re-infestation observed at 28 DPT in flumethrin treated group. IVM, Ivermectin, DOM,Doramectin; FLM, Flumethrin. in the study but the same was one week higher in flumethrin treatment group (<28 days). Further, in spite of high level of drug efficacy in all the treatment groups (Table 1), 100% tick mortality was not observed in any treatment group at prescribed dose rate. However, hundred per cent reported efficacies of ivermectin (Kumar et al. 2005) and doramectin (Gonzales et al. 1993) against B. microplus and flumethrin pour on against B. decoloratus infestation in cattle (Meconnen 2000) supports observation of this study to a great extent. Emergence of tick re-infestation before 21 days of treatment in ivermectin and doramectin groups indicated that a second follow up treatment at or after third week is required for proper tick control. However, in flumethrin treated animal’s repetition needed at or about fourth week of first treatment in case of heavy tick infestations especially in crossbred cattle in similar kind of climatic and managemental conditions. Use of acaricides is considered backbone of tick control programme now a days (Fuente and Kocan 2006), but their use in animals needs to be done with great precautions. For an effective control of B. microplus infestation on an organized farm in Uttarakhand state of India, 5 applications of different groups of acaricides (once in summer, thrice in rainy season and once in winter) on animals as well as in premises were suggested (Vatsya et al. 2007). In this context it is also to mention that time to time monitoring of anti-tick efficacy of drugs to be used against common tick genera and discontinuation of low efficacious agents are also required for effective tick control in a geographical area. efficacy of ivermectin, doramectin and flumethrin, respectively. However, residual drug effect (duration of protection) for ivermectin and doramectin injections was recorded less than 21 days in comparison to flumethrin pour on (<28 days). Further, it is concluded from the study that a second follow up treatment on or after third week in case of ivermectin and doramectin (S/C injections) and on or after fourth week in flumethrin (pour on) medication is required for proper control of ticks in endemic areas. ACKNOWLEDGEMENTS Authors are thankful to the Director, Indian Veterinary Research Institute, for providing necessary research facility. REFERENCES Fuente J D L and Kocan K M. 2006. Strategies for development of vaccines for control of ixodid tick species. Parasite Immunology 28: 275–83. Gonzales J C, Muniz R A, Farias A, Goncalves L C and Rew R S. 1993.Therapeutic and persistent efficacy of doramectin against Boophilus microplus in cattle. Veterinary Parasitology 49: 107– 19. Kumar S, Prasad K D, Dev A R and Kumar A. 2005. Efficacy of ivermectin and neem with karanj oil against natural Boophilus microplus infestation in cattle. Journal of Veterinary Parasitology 19: 59–60. Mekonnen S. 2000. Efficacy of flumethrin 1% pour-on against ticks on cattle under field conditions in Ethiopia. Onderstepoort Journal of Veterinary Research 67: 235–37. Srivastava P K, Raizda A and Agrawal P. 2001. A comparative trial on the efficacy of deltamethrin, cypermethrin and fenvalerate as acaricides in livestock animals. Indian Veterinary Medical Journal 25: 293–94. Vatsya S, Yaday C L, Kumar R R and Garg R. 2007. Seasonal activity of Boophilus microplus on large ruminants at an organized livestock farm. Journal of Veterinary Parasitology 21: 125–28. SUMMARY Therapeutic efficacy of ivermectin and doramectin (S/C injections) and flumethrin (pour on preparation) was evaluated against natural Boophilus microplus infestation in crossbred cattle. Tick mortality observed on seventh day post treatment in different groups indicated 95, 96 and 97% 58 Indian Journal of Animal Sciences 80 (4): 331–332, April 2010 Differentiation of the tubular components and collecting duct system of nephron in buffalo kidney during prenatal life MONIKA SUMAN1, NEELAM BANSAL2 and VARINDER UPPAL3 Guru Angad Dev Veterinary and Animal Science University, Ludhiana, Punjab 141 004 India Received: 20 April 2009; Accepted: 30 September 2009 Key words: Buffalo, Foetus, Histogenesis, Nephron In literature differentiation of the duct system was reported in goat (Gopinath 1985, Chaudhary et al. 2002)) but no work has been reported in buffaloes so the present study was conducted. The present study was made on kidneys of 17 Indian buffalo foetii, collected from pregnant buffaloes slaughtered at abattoir, Saharanpur and from Veterinary Clinics, GADVASU, Ludhiana. The curved crown rump length (CVRL) of the foetal body was measured in centimeters and age of the foetus was calculated by using the formulae given by Soliman (1975). On the basis of CVRL, the foetii were divided into 3 groups i.e. group 1 (3.0 cm to 20.0 cm), group 2 (20.1 cm to 40.0 cm) and group 3 (above 40.1 cm). The tissue of kidneys were fixed in Bouin’s and 10% neutral buffered formalin (NBF) fixatives. After fixation the tissues were processed for paraffin blocks preparation by acetone - benzene schedule and sections of 5–7 μm were cut with rotary microtome. The paraffin sections were stained with hematoxylin and eosin and Masson’s trichrome (Luna 1968). The formation of S shaped vesicles along with metanephric tubules was seen at 3.0 cm CVRL. The tubular segments of nephron were differentiated into proximal convoluted tubules, loop of Henle, distal convoluted tubules and collecting ducts from buffalo foetii of 4.1 cm CVRL onwards. Proximal convoluted tubules (PCT): The PCT was first observed in the 4.1 cm CVRL foetii. These were first formed in to the juxtamedullary area and then appeared in the intermediate and sub capsular areas of the metanephros in the buffalo foetii at the age of 10.3 cm CVRL onwards. These tubules were lined by simple columnar epithelium in the foetii of 4.1 cm and 5.7 cm. The formation of microvilli (future brush border) at the luminal end of the epithelial cells was first formed at the age of 11.2 cm CVRL. The coiling of PCT also appeared at this age. In the foetii of 23.0 cm CVRL these tubules were lined by simple cuboidal to pyramidal type of cells along with microvilli, which indicated its reabsorptive and secretory activities as reported in goat embryos by Gopinath (1985) and Chaudhary et al. (2002). Loop of Henle: The PCT continue into the medullary ray to become the proximal straight tubule (PST) also referred as the descending thick limb of loop of Henle. This proximal straight part of the tubule differentiated in the kidneys of buffalo foetii at 15.0 cm CVRL in the medullary rays (Figs 1, 2). It was lined by simple columnar epithelium. The epithelium of PST abruptly changed from simple columnar to simple squamous. The thick ascending limb of loop of Henle was demonstrated at the age of 23.0 cm CVRL. Similar findings were reported by Gopinath (1985) and Chaudhary et al. (2002) in goat embryos. In present study, loop of Henle was observed to be having thin and thick segment in the medulla and medullary rays of metanephos but occasionally it was also recorded in cortical area. This might be due to the location of subcapsular glomeruli in which the loop did not reach up to the medulla (Banks 1993). The thin segment of the loop was lined by simple squamous epithelium (Fig.4). It is similar to the earlier reports of Gopinath (1985) in goat embryos. Distal convoluted tubules: The distal convoluted tubules appeared in the buffalo foetii of 4.1 cm CVRL. Some of these tubules were observed nearer to the vascular pole of juxtamedullary glomeruli, which may be responsible for the formation of future macula densa cells. From 23.0 cm CVRL, these tubules were lined by simple cuboidal epithelium with relatively less eosinophilic cytoplasm and had darkly stained nuclei. In group 3, the DCT had nuclear condensation, which is referred to be as the future macula densa forming the juxtaglomemlar apparatus as reported earlier in goat embryos at 50 days of gestation (Chaudhary et al. 2002). Leeson (1957) and Gopinath (1985) also reported the similar type of epithelium of distal convoluted tubules in the embryos of rabbit and goat, respectively. Some of the tubules had more than one layers of epithelium at 21.5 cm CVRL, which Present address: 1Formerly PG student, 2Associate Professor, Professor cum Head, Department of Veterinary Anatomy. 3 Associate 59 332 SUMAN ET AL. [Indian Journal of Animal Sciences 80 (4) Figs 1–4. 1. The formation of loop of Henle and collecting ducts (CD) in the medulla of 15.0 cm CRL buffalo foetus. Masson’s Trichrome stain×87.5. 2. The continuation of medulla as medullary rays in between cortex to form renal pyramid in 18.0 cm CRL buffalo foetus. Two adjoining juxtamedullary (JG) glomeruli could also be observed. Masson’ s Trichrome stain × 87.5. 3. The flattened epithelial cells lining the loop of Henle (LH), light (L) and dark (D) epithelial cells of collecting tubules (CT) in 44.0 cm CRL buffalo feotus. Masson’ s Trichrome stain×175. 4. Formation of a bend with bulging epithelial cells into the lumen of loop of Henle (LH) in 75.0 cm CRL buffalo feotus. Hematoxylin and Eosin stain×350. indicated the process of canalization at this stage that completed afterwards. Similar type of canalization of DCT was also reported in metanephros of goat embryos by Chaudhary et al. (2002). Collecting ducts: After the formation of cortex and medulla, the intra-renal collecting duct from its dorso-lateral surface continued into juxtamedullary areas in the form of medullary rays. The lining epithelium of these collecting ducts was simple cuboidal to simple columnar type with the chromatic nuclei in the apical region. The relative occurrence of these tubules was more in the juxtamedullary area of the cortex and in the medulla. The shape of collecting ducts varied from round to ovoid. The collecting ducts were lined by two types of cells, viz. light cells and dark cells (Fig. 3 as reported earlier by Dellmann (1993). The light cells were more in number as compared to the dark cells. The light cells showed pale cytoplasm which might be due to the lack of many organelles in these cells as reported by Dellmann (1993). These ducts ultimately formed the large papillary ducts which terminated at the renal papilla. of 17 Indian buffaloes foetii were used. The tubular segments of nephron were differentiated into proximal convoluted tubules, loop of Henle, distal convoulted tubules and collecting ducts from buffaloes foetii of 4.1 cm CVRL onwards. REFERENCES Banks W J. 1993. Applied Veterinary Histology. 4th edn, pp 374– 89. Mosby Year book, St. Louis, USA. Chaudhary A R, Farooqui M M and Chandra P. 2002. Histological and certain histochemical studies on the metanephros of goat (Capra hircus) in prenatal period-secretory part. Indian Journal of Veterinary Anatomy 14: 8–15. Dellmann H D. 1993. Textbook of Veterinary Histology. pp 213– 21. Lea and Febiger, Philadelphia, USA. Gopinath S. 1985. ‘Morphogenesis of the kidney in goat.’ Ph. D. dissertation. Haryana Agricultural University, Hisar. India. Leeson T S. 1957. The fine structure of the mesonephros of the 17–day rabbit embryo. Experimental Cell Research 2: 670–72. Luna L G. 1968. Manual of histologic staining: Methods of Armed forces Institute of Pathology. 3rd edn, pp 38–196. McGraw Hill Book Co, New York, USA. Soliman M K. 1975. Studies on the physiological chemistry of the allantoic and amniotic fluid of buffaloes at various periods of pregnancy. Indian Veterinary Journal 52: 111–17. SUMMARY Differentiation of the tubular components and collecting system of nephron in buffalo kidney during prenatal, was observed and reported in this study. For this study kidneys 60 Indian Journal of Animal Sciences 80 (4): 333–335, April 2010 Histogenesis of lingual epithelium during prenatal life in buffalo DEEPANJALI VERMA1, VARINDER UPPAL2 and NEELAM BANSAL3 Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab 141 004 India Received: 20 April 2009; Accepted: 30 September 2009 Key words: Buffalo foetii, Lingual epithelium In literature the histogenesis of the tongue has been reported in rat (Farbman 1965), mouse (Cameron 1966), goat (Parmar et al. 1999) and crossbred pig (Sarma et al. 2003) but very scanty literature is available on buffaloes so the present study was conducted. The present study was conducted on tongues of 36 buffalo foetii (age ranging from 34 to 298 days). Curved crown rump length (CVR length) of the foetii was measured along the vertebral column between the most anterior part of frontal bone to the rump at ischiatic tuberosity and age of the foetii was estimated by using the formula given by Soliman (1975) in buffalo. Based on the CVR length the foetii were divided into 3 groups, foetii between 0–20 cm CVR; 2: foetii between 20–40 cm CVR, and group 3: foetii above 40 cm CVR. The tissues were taken from different parts of the tongue and fixed in Neutral buffered formalin and Bouin’s fixatives immediately after collection. After the fixation, the tissues were processed for paraffin blocks preparation by acetone benzene schedule (Luna 1968). The blocks were prepared and the sections of 5–6 μm were cut with the help of rotary microtome. These paraffin sections were stained with hematoxylin and eosin and Masson’s trichrome stains. The micrometrical observations of the epithelium thickness were recorded on the hematoxylin and eosin stained sections. In group 1 at 1.2 cm CVR length (34 days), the tongue was lined by a single layer of cells. Further at 2.5 cm CVR length (40 days), the tongue was lined by 2 layers of epithelium; a deep layer of cuboidal cells and a superficial thin sheet of cells. This thin sheet of cells was observed from 2.5 cm CVR length (40 days) to 6.0 cm CVR length (55 days) and had been described as periderm cell layer (Jung et al 2004). At 3.4 cm CVR length (44 days), the dorsal surface of the tongue was usually lined by 3-layered epithelium; a basal layer of cuboidal cells, an intermediate layer and a superficial layer of squamous cells (Figs a, b) as observed in rabbit at 15th day of prenatal life (Kulawik 2005) and in goat foetuses at 10.0 cm CRL (Parmar et al. 1999). With further advancement of age the basal layer of the epithelium comprised high cylindrical cells along with well defined basement membrane. Cells in the stages of mitosis were frequently observed in epithelium. As compared to dorsal surface the ventral surface was lined by a single layer of cuboidal or low columnar type of cells. At 10.7 cm CVR length (77 days), the lamina epithelialis was distinctly stratified and was divided into dark basal and light superficial zones (Fig. c) as reported earlier in buffalo at 79th day (Uppal et al. 2006) and at 30–35 cm CVR length foetii (Scala et al. 2005) but no signs of keratinization were observed. The basal layer was made up of cuboidal or columnar type of cells with rounded nuclei as observed by Kulawik (2006) at 22nd day of prenatal life in rabbit whereas the other layers were having round or polygonal cells. Most of the cells were having eccentric nucleus as reported in the differentiation of ruminal epithelium (Singh 2002). The nuclei of the surface layer were almost flattened as observed by Kulawik (2005) in rabbit at 18th day of prenatal life. The ventral surface was lined by basal layer of cuboidal cells and 3–4 superficial layers of polygonal cells with a high nucleus to cytoplasm ratio (Fig. d) while Farbman (1971) observed 5–8 layer thick epithelium in rat foetus by E14. Further, this stage of development was characterized by the appearance of regularly spaced dermal papillae formed by evagination and invagination of epithelium on the dorsal surface however such type of papillae were not observed on the ventral surface as reported in rat (Farbman 1971). During this period the interpapillary epithelium increased in thickness and dermal papillae increased in length, which was due to the arrested mitosis in the basal epithelial layer at the base of the dermal papillae and absent in overlying apex but interpapillary epithelium continues to grow by mitosis (Baratz and Farbman 1975, Farbman 1971). In group 2 at 21.4 cm CVR length (122 days), the stratified squamous epithelium was better differentiated at both the dorsal (Fig. e) and ventral surfaces. All the layers of epithelium could be well recognized at 29.0 cm CVR length (139 days). The first indication of presence of keratohyaline granules was observed at 29.0 cm CVR length (139 days) on Present address: 1Ph.D student, 2Associate Professor, 3Associate Professor-cum-Head, Department of Anatomy. 61 334 VERMA ET AL. [Indian Journal of Animal Sciences 80 (4) of keratinization between different species may be different due to difference in the duration of the gestation period (Iwasaki et al. 1999). In the present study the first evidence of keratinization was observed in group 3. The epithelium covering the dorsal surface was thicker while that of the ventral surface was thinner as reported by Dellmann (1993). The mean epithelium thickness on the dorsal surface of apex was 81.49±2.88 μm in group 1, 106.36±7.44 μm in group 2 and 437.24±20.06 μm in group 3. Singh (2001) reported epithelium thickness to be 468.00±45.20 μm in neonate buffaloes. The mean epithelium thickness on the ventral surface of apex was 59.85±4.03 μm in group 1, 74.21±4.12 μm in group 2 and 118.05±5.28 μm in group 3, while Singh (2001) observed the 280.00±43.8 μm in neonatal buffalo. The mean epithelium thickness at body in group 1, 2 and 3 was 72.97±1.91 μm, 97.89±7.21μm and 402.28±27.68 μm, respectively, whereas Singh (2001) observed 416.30±56.20 μm in neonatal buffalo.Epithelium thickness at the level of torus was 77.06±3.71 μm in group 1, 199.47±18.85 μm in group 2 and 417.16±23.93 μm in group 3. Our values in group 3 collaborates well with the values of Singh (2001) in buffalo neonates who reported 412.3±90.6 μm thickness at the levels of torus. Epithelium thickness at the root of the tongue in group 1, 2 and 3 was 68.86±2.47 μm, 180.98±17.82 μm and 317.71±8.41 μm respectively. A statistically significant difference (P<0.05) was observed between epithelium thickness at all the parts among group 1, 2 and 3. Figs a–f. (a) 3.4 cm CVR length (44 days) foetus showing superficial periderm layer (P), dark basal layer (Bl) of epithelium and mesenchymal cell (Ms) in the anterior part of tongue. Hematoxylin and Eosin × 200. (b) 3.4 cm CVR length (44 days) foetus showing superficial periderm layer (P), dark basal layer (Bl) and mesenchymal cells (Ms). Hematoxylin and Eosin × 1000. (c) 10.7 cm CVR length (77 days) foetus showing dome shaped primitive papillae (Pp), dark basal layer (Bl) and propria submucosa (Ps). Hematoxylin and Eosin × 200. (d) 10.7 cm CVR length (77 days) foetus showing basal layer of cuboidal cells (Bl) and superficial layers of polygonal cells (Sf) in the ventral part of tongue. Hematoxylin and Eosin × 200. (e) 21.4 cm CVR length (122 days) foetus showing basal layer (Bl) and superficial layers (Sf) in torus region. Hematoxylin and Eosin × 100. (f) 29.0 cm CVR length (139 days) foetus showing keratohyaline granules (kg) in the epithelium. Hematoxylin and Eosin × 200. SUMMARY Histogenesis of lingual epithelium during prenatal life in buffalo was studied in tongues of 36 buffalo foetii. REFERENCES Baratz R S and Farbman A I. 1975. Morphogenesis of rat lingual filiform papillae. American Journal of Anatomy 143: 283–302. Cameron I L. 1966. Cell proliferation, migration and specialization in the epithelium of the mouse tongue. Journal of Experimental Zoology 163: 271–84. Dellmann H D. 1993. Textbook of Veterinary Histology. 4th edn. Lea and Febiger, Philadelphia Farbman A I. 1965. Electron Microscope study of the developing taste bud in rat fungiform papilla. Developmental Biology 11: 110–35. Iwasaki S, Yoshizawa H and Kawahara I. 1999. Ultrastructural study of the relationship between the morphogenesis of filiform papillae and the keratinisation of the lingual epithelium in the rat. Journal of Anatomy 195: 27–38. Jung H S, Akita K and Kim J Y. 2004. Spacing pattern on tongue surface gustatory papillae. International Journal of Developmental Biology 48: 157–61. Kulawik M. 2005. The development of the mucous membrane of the tongue with special emphasis on the development of fungiform papillae in the prenatal life of the rabbit. Electronic Journal of Polish Agricultural Universities Veterinary Medicine the posterior side or root of the tongue (Fig. f) as reported at P0 (postnatal day 0) in rat (Iwasaki et al. 1999). These keratohyalin granules were the characterstics of stratum granulosum or granular layer (Dellmann 1993). The cells became keratinized before it reaches the surface and usually one or more layers of keratinized cells were formed beneath the superficial non-keratinized cells. So the cells in the intermediate layers released the keratohyaline granules and undergo keratinization as it reached the surface. In the present study it was observed that the lingual epithelium was better differentiated on the posterior side as compared to anterior side at any given stage of development. The keratinization of lingual epithelium of rat occurs just before birth (Farbman 1971). The discrepancy in the timing 62 April 2010] HISTOGENESIS OF LINGUAL EPITHELIUM IN BUFFALOES 335 Pelagalli G V. 2005. The innervation of the fetal buffalo tongue. Veterinary Research Communication 29: 203–06. Singh K. 2001. ‘Age correlated histomorphological and histochemical studies on the tongue of Indian Buffalo (Bubalus bubalis).’ M.V.Sc. thesis, Punjab Agricultural University, Ludhiana, India. Singh O. 2002. ‘Anatomical and histomorphological changes in buffalo stomach during prenatal life.’ Ph. D. dissertation. Punjab Agricultural University, Ludhiana, India. Soliman M K. 1975. Studies on the physiological chemistry of the allantoic and amniotic fluid of buffaloes at various periods of pregnancy. Indian Veterinary Journal 52: 111–17. Uppal V, Roy K S, Bansal N, Bawa B S and Singh O. 2006. Histogenesis of tongue during early prenatal life in Murrah buffalo. Indian Journal of Animal Sciences 76: 894– 96. 8: 4. Kulawik M. 2006. Development of the mucosa on lateral surfaces of the lingual body in the period from day 15 of prenatal life to 6th month of postnatal life in the rabbit Electronic Journal of Polish Agricultural Universities Vet Med 9: 2. Luna L G. 1968. Manual of Histologic Staining: Methods of Armed forces Institute of Pathology. 3rd edn. pp 38–196. McGraw Hill Book Co, New York, USA. Parmar M L Malik M R and Taluja J S. 1999. Morphometry of tongue in goat foetuses. Indian Journal of Animal Sciences 69: 79–81. Sarma K, Goswami R N, Kalita S N, Baishya G and Devi J. 2003. Morphogenesis of tongue in crossbred pig foetuses. Indian Journal of Animal Sciences 73: 1029–30. Scala G, Corona M, Persella A, Girolamo P, Vittoria A, Scala F and 63 Indian Journal of Animal Sciences 80 (4): 336–337, April 2010 Ultrastructure of gut associated lymphoid tissues in Kadaknath fowl P C KALITA1, G K SINGH 2 and B S DHOTE 3 Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, Uttarakhand 263 145 India Received: 6 April 2009; Accepted:1 November 2009 Key words: Kadaknath fowl, GALT, Ultrastructure Lymphoid organs constitute the main structural category of the avian immune system. The secondary lymphoid organs, characterized by aggregates of lymphocytes and antigen presenting cells, are scattered throughout the body. Concomitant with the development of digestive structures and functions, a rapid development of the GALT occurs. The development of the bursa and other GALT starts during late embryogenesis (Kajiwara et al. 2003). Bar Shira et al. (2005) showed that development of GALT in the foregut was only slightly and temporarily impeded by feed withholding (for the first 72 h after hatch) in newly hatched broiler chicks. The present study was aimed to investigate the ultrastructural observations on GALT in the Kadaknath fowl. Birds were obtained from Instructional Poultry Farm (IPF) of the University. The birds were sacrificed by decapitation and tissue samples, viz. segments from duodenum, jejunum and ileum, were collected for transmission electron microscopy and fixed in 2.5% gluteraldehyde and processed for TEM as per the manual of AIIMS, New Delhi. Ultrathin (70-80 nm) sections were stained with uranyl acetate and lead citrate, according to Reynolds (1963) and examined in an electron microscope. Lymphocytes were the main cell type seen aggregated beneath the epithelium of GALT either as germinal centres or as diffuse lymphoid tissue. The nuclear diameter profiles of these cells ranged from 1.5 to 5.2 mm. The small lymphocytes had a thin rim of relatively electron dense cytoplasm and more densely clumped chromatin than the large lymphocytes (Fig. 1). Chromatin was often seen along the inner membrane of the nucleus. A nucleolus was sometimes present. Few cytoplasmic organelles were seen in the small lymphocytes whereas the large lymphocytes had a few mitochondria, granular endoplasmic reticulum and occasionally a moderately developed Golgi Complex. Lymphoblasts were common and could be distinguished by their large size, relatively large amount of cytoplasm, small amount of nuclear chromatin and by the presence of more cytoplasmic organelles than were seen in mature lymphocytes (Fig. 2). Vacuoles were observed sometimes in the cytoplasm of the lymphoblasts. Plasma cells with dilated endoplasmic reticulum were seen among the lymphocytes and lymphoblasts (Fig. 3). It had the typical “clockface” arrangement of nuclear chromatin and was larger than other lymphoid cells. The division of the lymphoid tissue into germinal centres and diffuse lymphoid tissue (Befus et al. 1980, Burns 1982) was not always apparent at the ultrastructural level in fowl and nor was the presence of a dome area of lymphoid tissue (Abe and Ito 1978). However, the principle features of a well developed secondary lymphoid organ–small and large lymphocytes, plasma cells–macrophages and mast cells, were all present in Peyer’s patches from fowls and turkeys (Burns and Maxwell1986). Macrophages in moderate numbers were present throughout the lymphoid tissue. They often contained phagocytosed material; dead and dying cells could be seen within vacuoles in the cytoplasm. The nuclear membrane was often indented by phagocytosed material. A similar location for macrophages was reported in mammalian gutassociated lymphoid tissue (Crabb and Kelsall 1940, Abe and Ito1978, Lause and Backman 1981). Cunningham (1978) stated that macrophages are the main antigen-presenting cells in immune responses. Macrophages are considered to be the professional antigen presenting cells in generation of immune response (Kindt et al. 2007). Mast cells were located in the lamina propria just beneath the epithelium, but never populated intestinal epithelium (Fig. 4). The granules were more varied in size and number than the globules of the globule leucocytes. The granules were moderately electron-dense and were bound by a single membrane (Fig. 4). The nuclei were usually regular in shape being either round or oval. Globule leucocytes and lymphocytes were sandwiched between the epithelial cells throughout the intestine. Almost Present address: 1Associate Professor and Head, Department of Veterinary Anatomy and Histology, College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl, Mizoram. 2 Dean, 3 Associate Professor, Department of Veterinary Anatomy, College of Veterinary and Animal Sciences. 64 April 2010] ULTRASTRUCTURE OF GUT ASSOCIATED LYMPHOID TISSUES IN KADAKNATH FOWL 1 2 3 4 337 5 Figs 1–5. Transmission electron microgrpah of GALT of the Kadaknath fowl showing : 1. small (SL) and large lymphocyte (LL) × 10600; 2. lymphoblast (LB) × 7960; 3. plasma cell with dilated endoplasmic reticulum (Pc) × 10800; 4. mast cell (MC), the granules of which are moderately electron dense (Gn) and are bound by a single membrane × 19900; 5. globule leucocyte (GL). The granules (Gn) are generally homogenous and densely stained × 10600. all the globule leucocytes (GL) were spherical and possessed thick cytoplasmic protrusions prolonged between the epithelial cells. The pattern of the chromatin and the size of the nucleus were similar to small or medium sized lymphocytes in the same area, but the cytoplasm of GL had a slightly lower electron density. Therefore, the cytoplasm was easily distinguished from that of the neighbouring epithelial cells. The globules varied in size and appearance within different leucocytes. They were generally homogenous and densely stained (Fig. 5) though sometimes vacuoles were seen within them. Gregory and Nolan (1981) recorded a direct association between globule leucocytes and lymphoid tissue (in Peyer’s patches from lambs infected with coccidia) and also reported mast cells and globule leucocytes in Peyer’s patches from uninfected control lambs. responses in broiler hatching hindgut following delayed access to feed. Veterinary Immunology and Immunopathology 105: 33– 45. Befus A D, Johnston N, Leslie G A and Bienestock L. 1980. Gut associated lymphoid tissue in the chicken. I. Morphology, ontogeny and some functional characteristics of Peyer’s patches. Journal of Immunology 125: 2626–32. Burns R B. 1982. Histology and immunology of Peyer’s patches in the domestic fowl (Gallus domesticus). Research in Veterinary Science 32: 359–67. Burns R B and Maxwll M H. 1986. Ultrastructure of Peyer’s patches in the domestic fowl and turkey. Journal of Anatomy 147: 235– 43. Crabb E D and Kelsall M A. 1940. Organisation of the mucosa and lymphatic structure in the rabbit appendix. Journal of Morphology 67: 351–67. Cunningham A J. 1978. Understanding Immunology. Academic Press Ltd., London Gregory M W and Nolan A. 1981. Globule leucocytes and Peyer’s patches in lambs infected with coccidia. Research in Veterinary Science 30: 385–87. Kajiwara E, Shigeta A, Horiuchi H, Matsuda H and Furusawa S. 2003. Development of Peyer’s patch and cecal tonsil in gutassociated lymphoid tissues in the chicken embryo. Journal of Veterinary Medicinal Science 65: 607–14. Kindt T J, Goldsby R A and Osborne B A. 2007. Kuby Immunology. 6th edn, pp 209.W. H. Freeman and Company, New Work. Lause D B and Bockman D E. 1981. Heterogeneity, position and functional capability of macrophages in Peyer’s patches. Cell and Tissue Research 218: 557–66. Qureshi M A Husain I and Heggen C L. 1998. Understanding immunology in disease development and control. Poultry Science 77: 1126–29. Reynolds E S. 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. Journal of Cell Biology 17: 208–12. SUMMARY The present study conducted on 112 day-old Kadaknath fowl of both sexes revealed that the GALT were similar in many respects to those found in mammals. They were overlaid by a lymphoepithelium containing undifferentiated intestinal epithelial cells with a well developed microvillous border, lymphocytes and plasma cells. The organ was fully developed and the lymphoid aggregates of GALT comprised of small and large lymphocytes, lymphoblasts and plasma cells. Macrophages and globule leucocytes were seen among the lymphoid cells. REFERENCES Abe K and Ito T. 1978. Fine structure of the dome in Peyer’s patches of mice. Archivum histologium Japonicum 54: 195–204. Bar Shira E D, Sklan D and Friedman A. 2005. Impaired immune 65 Indian Journal of Animal Sciences 80 (4): 338–339, April 2010 Biochemical and enzymatic changes in downer cow syndrome SUBHASH KACHHAWAHA1 and R K TANWAR2 Animal Help Line, Veterinary Hospital, Panna Lal Goshala, Mandore, Jodhpur, Rajasthan 342 023 India Received: 25 May 2009; Accepted: 23 November 2009 Key words: Biochemical, Downer cow, Enzymes Downer cow syndrome is a complication of recumbency associated with milk fever (Radostits et al. 2007). Downer syndrome is characterized by inability of an animal to stand from recumbency voluntarily. Occurrence of this syndrome, has been reported in cows (Wadhwa and Prasad, 2002). The present paper describes biochemical and enzymatic changes in clinical cases of downer syndrome in cows. Sixteen cows of 4–8 years of age with history of inability to rise voluntarily for the varied period of time, were brought to the veterinary hospital, Panna Lal Goshala, Jodhpur, between March 2007 to February 2008. Twelve cows were post parturient and four cows were in advance pregnancy. Clinical manifestations were recorded. Six healthy cows were included to serve as control. About ten millilitre of blood was collected from each downer cow and healthy cows and serum was separated and stored at –20°C. The concentration of calcium, phosphorus, magnesium, sodium, potassium, total protein AST, ALT and CPK were measured in serum using kits. Data were analysed statistically as per Snedecor and Cochran (1994). Clinically the downer cows appeared bright and alert but off-feed. The body temperature was within normal range. Animals tried to stand-up repeatedly but could not rise to their feet. They could raise their fore quarters, but failed to put up weight on hind quarters. The mean ± SE value of minerals, electrolytes, enzymes and total proteins are given in Table 1. The values of calcium, phosphorus, potassium were significantly low (P< 0.05) in downer cows incomparison to healthy control cows (Table 1). Low plasma calcium, phosphorus and potassium concentration have been reported by Wadhwa and Prasad (2007). Radostits et al. (2007) stated that calcium, phosphorus and magnesium levels of blood remained within normal range in downer cow. However, a persistent hypocalcemia following treatment for milk fever may exist in a downer cow. The exact explanation for low calcium and phosphorus in downer cows in present study is not known. Hypokalaemia has been reported in downer cows by Allen and Davies (1981). The persistent hypocalcemia and hypophosphatemia has been regarded as cause of downer cow syndrome associated with milk fever (Radostits 2007). Hypokalaemia in recumbent cows occurs due to the fact that muscle ischaemia as a result of prolonged recumbency increases the cell membrane permeability of muscle fibres and allow loss of potassium from the cell causing myotonia which appears to be the basis of downer cow syndrome (Andrew et al. 1992). The magnesium concentration was slightly high in present study. Hypomagnesemia (Baumgartner and Gattinger 1982, as well as normal magnesium level (Narayana et al. 1977) have been ) recorded in downer cows. The sodium concentration in downer cows was within normal range. Hypokalemia could also occurs due to rapid urinary excretion and diminished alimentary absorption of potassium associated with reduced feed intake. Diminished excitability of nerve and muscle cells, weakness and flaccid paralysis are the consequences of hypokalaemia (Kowalezyk and Mayer 1972). The values of total protein Table 1. Mean ± SE value of minerals, electrolytes, enzymes and total protein in downer cows (n = 16) and healthy control cows (n = 6) Parameter Ca (mg/dl) Pi (mg/dl) Mg (mg/dl) Na (mEq/l) K (mEq/l) Total protein (g/dl) AST (units/l) ALT (units/l) CPK (units/l) Present address: 1SMS (Vety. Sc.), CAZRI, KVK, Pali-Marwar, Rajasthan. 2 Head, Department of Epidemiology and Preventive Veterinary Medicine, College of Veterinary and Animal Science, Bikaner, Rajasthan. *P≤0.05. 66 Downer cows (n = 16) Control (n = 6) 7.62±0.21* 3.94±0.05* 3.33±0.11 119.56±2.83 1.90±0.05* 7.13±0.13 117.88±6.33* 84.75±3.42* 128.27±2.31* 10.90±0.25 5.23±0.24 2.30±0.11 122.17±2.80 4.37±0.20 7.58±0.52 118.00±7.23 54.83±3.67 28.44±0.54 April 2010] BIOCHEMICAL AND ENZYMATIC CHANGES IN DOWNER COW SYNDROME fluctuated within normal range in downer cows. The mean values of AST, ALT and CPK in downer cows were significantly high (P<0.05) in comparison to healthy control cows (Table 1). Prolonged recumbency causes ischaemic necrosis of muscles (Cox et al. 1982) resulting in increased permeability of cell membrane allowing seepage of AST, ALT and CPK enzymes into circulation. Increased levels of AST, ALT and CPK enzymes had been observed in cows by El-Sayed et al. (1994) and Wadhwa and Prasad (2007) in buffaloes. Creatine phosphokinase is considered as a specific marker of muscle damage (Kaneko 1989) and increase in CPK testified ischemic damage to the muscle causing its seepage into the circulation. The CPK levels need to be interpreted in relation to the days of recumbency when the sample is taken. Critical levels may be highest initially up to 50 times and may reduce to 10 times normal range at 7 days of recumbency (Radostits 2007). 339 REFERENCES Allen W M and Dames D C. 1981. Milk fever, hypomagnesemia and the downer cow syndrome. British Veterinary Journal. 137: 435–41. Andrews A H, Blowery R W and Eddy R S. 1992. Bovine Medicine. Diseases and Husbandry of Cattle. A Blackwell Scientific Publications, London. Baumgartner W and Gattinger G. 1982. Parturient paresis in dairy cow due to hypophosphataemia. Wiener Tierarztliche Monatsschrift 69: 315–18. Cox V S, McGrawth C J and Jorgensen S E. 1982. The role of pressure damage in pathogenesis of downer cow syndrome. American Journal of Veterinary Research 43: 26–31 El-Sa ed R E, Asma O A and Fetaih H. 1994. Downer cow syndrome in cows and buffaloes. Assiut Veterinary Medicine Journal 31: 155–63 Kaneko J J. 1989. Clinical Biochemistry of Domestic Animals. IV edn., Academic Press, New York. Kowalczyk D F and Mayer G R. 1972. Cation concentration in skeletal muscles of paretic and non paretic cows. American Journal of Veterinary Research 33: 751–56. Narayana K, Sethy D R L, Sastry K N V and Thandavesh Kar M G. 1977. Downer cow syndrome. Indian Veterinary Journal 54: 148–51. Radostits O M, Gay C C, Hinchcliff K W and Constable P D. 2007. Veterinary Medicine. A Textbook of the Diseases of Cattle, Horses, Sheep, Pigs and Goats. Saunders Elsevier. Snedecor G W and Cochran W G. 1994. Statistical Methods. 8th Edn., New Delhi, Oxford and IBH. Wadhwa D R and Prasad B. 2002. Clinico-therapeutic observations of downer cow syndrome. Indian Veterinary Journal 79: 484–86. Wadhwa D R and Prasad B. 2007. Mineral proifle in downer cow syndrome. Indian Journal of Veterinary Medicine 27(1): 22–24. SUMMARY The biochemical and enzymatic changes were observed in sixteen downer cows. Significantly low concentration of calcium, phosphorus and potassium and significantly higher activities of serum enzymes of creatinine phosphokinase, aspartate and alanine amino transferase were observed in downer cows. Downer cows should be treated with potassium in addition to calcium and phosphorus. ACKNOWLEDGMENTS The author is highly thankful to Head, Department of Veterinary Medicine, College of Veterinary and Animal Science, Bikaner, for providing facilities to carry out the present research work. 67 Indian Journal of Animal Sciences 80 (4): 340–342, April 2010 Alteration in surface body temperature and physiological responses in Sirohi goats during day time in summer season S K PHULIA1, R C UPADHYAY2, S K JINDAL3 and R P MISRA4 Central Institute for Research on Goats, Makhdoom, Farah, Uttar Pradesh 281 122 India Received: 2 June 2009; Accepted: 20 November 2009 Key words: Goats, Kids, Physiological responses, Surface body temperature temperature (37.0, 38.0, 41.0, 39.0 ºC) and the soil temperature (41.8, 46.0, 48.9, 38.0 ºC) in the experimental pen fluctuated at different hours of observations (9.00 AM, 12.00 Noon, 3.00 PM and 6.00 PM). Data were analysed by one way analysis of variance and the differences were tested using Duncan’s multiple range test (SPSS 11.5 Version, 2002). Surface body temperature at different sites differed significantly (P<0.01) between adults and kids during day time. The skin surface temperature in adult goats increased significantly (P<0.01) with increase in the intensity of solar radiation at noon (Table 1). Changes in body surface temperature at different sites were low (P<0.01) in the morning (9.00 AM) and about 5–10 ºC higher in the afternoon (3.00 PM). Fore head, back and rump showed significantly (P<0.01) higher temperature than other sites, but these differences were more pronounced in the adults as compared to kids (Table 1). The kids have higher body surface area per unit of body weight and therefore heat dissipation is faster. As a consequence, small animals require a greater heat production per unit of weight than large animals, if the same body temperature is to be maintained. With declining body size, the increase in metabolic rate will eventually reach a limit because the requirements for ‘Fuel’ became prohibitively high (Bianca 1968). The mean rectal temperature of kids (Table 1) was similar as reported by Singh et al. (2004). The changes in rectal temperature were minimum, but it differed significantly (P<0.05) between day time and again significantly (P<0.05) declined in the evening at 6.00 PM in both kids and adult goats. Surface body temperature of forehead, back and rump increased significantly (P<0.01) with increase in the intensity of solar radiation in both the groups. However, it was more pronounced (P<0.01) in adults, probably due to larger body surface area exposed to solar radiation. The surface temperature of extremities was significantly (P<0.01) lower than other body parts; the extremities were cooler by 4–10ºC as compared to body trunk. The ear and legs have more Goats are well adapted to tropical and subtropical conditions (Finch et al. 1980, Goyal and Ghosh 1987), due to their ability to withstand high ambient temperatures (Finch et al. 1980). Goats maintain homoeothermy through a balance in heat loss and heat production through behavioural mechanisms and physiological changes (Ogebe et al. 1996, Al-Tamimi 2007). Singh et al. (2004) reported on adaptability of goats mainly based on rectal temperature, respiration rate and pulse rate, without considering surface body temperature. Particularly, there is lack of information on summer surface body temperature in Indian breed of Sirohi goats, which could be helpful in improving production through proper management during hot period. The present study was carried out to investigate physiological responses and surface body temperature during day time in semi-arid conditions. Six adults (24–30 months weighing 25–30 kg) and 6 kids (5–6 months weighing 8–12 kg) were selected from institute herd. All these animals were maintained under standard management practice followed at the animal farm. The experiment was conducted during summer and all the animals were exposed to the solar radiations in open goat pen during day time. Ten points (forehead, ear, neck, back, rump, abdomen, shoulder, hip, fore leg and hind leg) were earmarked on the body of animal to record surface body temperature using an Infra-red tela-temp thermometer at 3 h interval during day time (9.00AM to 6.00 PM) in summer. The temperature recorded from ear, fore legs and hind legs was grouped under extremities and rest was grouped under body proper. Rectal temperature was recorded using a clinical thermometer. Respiration rate was recorded on the basis of the flank movement. The average relative humidity, wind speed and minimum temperature were 62±8.3%, 69.3±26.8 m/min and 27.5ºC, respectively. The environmental Present address: 1Senior Scientist, Central Institute for Research on Buffaloes, Hisar 125 001 (e mail: [email protected]), 2Principal Scientist, Dairy Cattle Physiology, National Dairy Research Institute, Karnal 132 001. 3,4Principal Scientist 68 April 2010] ALTERATION IN PHYSIOLOGICAL RESPONSES IN GOATS IN SUMMER 341 Table 1. Surface body temperature (°C) and physiological responses (rectal temperature and respiration rate) of Sirohi adult goats and kids during summer condition (mean±SE) Hours Sites Adults 9.00 AM Surface body temperature 42.4±0.94b Forehead** ** Ear 37.30±0.45a Neck** 38.45±0.47a Back** 48.06±1.10b Rump** 42.35±0.71b Abdomen** 38.08±0.27a Shoulder** 38.11±0.38a ** Hip 39.1±0.97ab ** Fore leg 37.86±0.60a Hindleg** 37.08±0.53a Physiological responses Rectal temp.* (ºC) 38.97±0.15ab Resp. rate/min. 43.66±5.0a Kids 12.00 Noon 3.00 PM 6.00 PM 9.00 AM 12.00 Noon 3.00 PM 6.00 PM 46.80±0.83c 40.25±0.30b 42.2±0.73b 51.5±1.10b 46.28±0.66c 40.75±0.40b 40.28±0.24b 41.60±0.31bc 39.50±3.80b 39.78±0.39b 46.36±1.30c 46.00±1.60b 42.55±0.55b 48.26±2.10b 46.30±1.30c 40.35±0.98b 41.80±1.10b 42.36±1.60bc 40.20±0.71b 39.96±0.84b 35.93±0.22a 36.63±0.85a 36.10±0.56a 36.10±0.33a 36.25±0.12a 36.70±0.08a 36.80±0.15a 36.43±0.11a 36.31±0.13a 36.00±0.19a 40.15±0.84b 37.53±0.14c 38.65±0.44a 46.23±1.63c 44.36±1.20c 37.70±0.37b 37.26±0.37 37.05±0.63bc 36.48±0.48bc 36.20±043bc 35.33±0.12a 36.55±0.11b 36.0±0.11b 35.5±0.26a 35.32±0.32a 35.8±0.18a 35.04±.26 34.83±0.15a 34.51±0.20a 34.26±0.35a 36.45±1.10a 35.2± 0.49a 36.50±1.00b 40.45±1.00b 40.30±0.42b 35.85±0.95a 35.65±1.17 38.60±0.28c 37.50±0.92c 37.55±0.3c 35.85±0.15a 36.93±0.49bc 36.08±0.95b 35.85±0.33a 35.73±0.26a 36.33±0.28a 36.41±0.67 35.41±0.59ab 34.98±0.39ab 34.86±0.43ab 38.90±0.17ab 39.35±0.08b 38.76±0.14a 57.00±5.80a 77.33±8.10b 45.60±02.33a 39.4±0.04b 38.95±0.09a 39.70±0.06c 39.15±0.09ab 51.00±4.10a 48.00±1.60a 56.00±1.84b 41.30±3.12a ** Significant difference (P<0.001) between adults and kids; * Significant difference (P<0.005) between adults and kids;abc Values with a different superscript in the row under adults and kids column differ significantly (P<0.005). result from both central stimuli such as blood temperature supplied to hypothalamus or from peripheral stimuli to the skin (Terill 1968). This was due to commissioning of physiological and behavioral process for maintaining homeothermy in both adults and kids. In adult animals, evaporation through respiratory passage is more than the kids. Rise in surface body temperature, rectal temperature and respiration rate was observed in relation to rise in solar intensity and orientation. At higher environmental temperature and increase solar radiations intensity, goats start panting. Hence under extreme environmental conditions goats must be protected from direct solar radiations. surface area to weight and the volume ratio, hence ear and legs permit greater evaporative cooling. The extremities like ears and legs being highly vascularised result in circulation of larger volume of blood per unit area. In fact long and thin legs and long ears are an adaptation character in the hot environment breeds of the tropics, which facilitate their survival in hot environment. The respiration rate of both adults and kids at 3.00 PM was significantly (P<0.01) higher as compared to those in morning and evening. Present study also showed as increase in respiration rate with increase in environmental/soil temperature (Phulia et al. 2000, Ogebe et al. 1996). The rise in respiration rate probably demonstrated that goats used heat regulating mechanisms by increasing respiratory exchange. Rectal temperature is an important indicator of thermal balance and might be used to evaluate the impact of heat stress (Spiers et al. 2004). Rectal temperature was significantly (P<0.05) different between kids and adults (Table 1). The rectal temperature was highest at 3.00 PM, that declined significantly (P<0.05) during evening (6.00 PM) in both groups. Avendano-Rayes et al. (2006) reported that increased body temperature is a normal mechanism by which animals diffuse heat from their bodies to maintain thermoregulation in hot ambient conditions. In the present experiment, the rectal temperatures of both groups exceed over 39°C. However, Devendra (1987) reported that goats maintain their rectal temperatures below 38.5°C, which is considered normal. Darcan et al. (2008) also reported that there was a diurnal fluctuation in body temperature, that was lowest in early morning and highest in late afternoon. In the present study, with the rise in surface body temperature goats started panting. Thermoregulatory responses of panting may SUMMARY The study was undertaken to evaluate physiological responses and surface body temperature during summer. Adult goats and kids of Sirohi breed were exposed to solar radiations. Body sites, viz fore head, back/middle and rump, which were facing sun radiation directly, showed significantly higher skin surface temperature than the other parts of body. Extremities showed significantly lower surface temperatures than the other parts. In adults, respiration rate was significantly higher at 3.00 PM when ambient temperature was high. Body temperature of adults and kids differed significantly among different hours of the observations. The finding suggests that under extreme environmental conditions goat must be protected from direct solar radiations. ACKNOWLEDGEMENT The authors are highly thankful to the director CIRG, Makhdoom, for providing necessary funds and facilities for this study. 69 342 PHULIA ET AL. [Indian Journal of Animal Sciences 80 (4) heat exchange by radio-telemetry in black desert goats during winter. Journal of Agriculture Science 108: 509–10. Ogebe P O, Ogunmodede B K, Mc Dowell L R. 1996. Behavioral and physiological responses of Nigerian dwarf goats to seasonal changes of the humid tropics. Small Ruminant Research 22: 213–17. Phulia S K, Upadhyay R C, Jindal S K and Misra R P. 2000. Changes in skin surface temperature and physiological reactions in Marwari goats. Proceedings of 7th International Conference on Goats. pp 779–0.France, 15–21 May, 2000. Prasetyo S. 1983. ‘Heat stress on birth weight and vigour of kids.’ Ph.D. Thesis, Animal Production, University of Melbourne. Singh G, Phulia S K, Misra R P, Puneet Kumar and Upadhyay R. C. 2004. Seasonal variation in physiological reactions of Jamunapari Kids. Indian Journal of Small Ruminant 10 (1): 21– 24. Spiers D E, Spain J N, Sampson R P, Rhoads R P. 2004. Use of physiological parameters to predict milk yield and feed intake in heat stressed dairy cows. Journal of Thermal Biology 29: 759–764. Terril C E. 1968. Adaptation of Sheep and Goats. Adaptation of Domestic Animals. (Ed.) Hafez E S E. Lea & Febiger. Washington. REFERENCES Al-Tamimi H. 2007. Thermoregulatory response of goat kids subjected to heat stress. Small Ruminant Research 71 (1–3): 280–85. Avendano-Reyes, Alvarez-Valenzuela L, Correa-Clederon F D, Saucedo-Quintero A, Robinson S and Fadel J G. 2006. Effect of cooling Holstein cows during the dry period on postpartum performance under heat stress conditions. Livestock Science 105: 198–206. Bianca W. 1968. Thermoregulation. Adaptation of Domestic Animals. (Ed.) Hafez E S E. Lea & Febiger. Philadelphia. Darcan Nazan, Cedden Fatin, Cankaya Soner 2008. Spraying effects on some physiological and behavioural traits of goats in a subtropical climate. Italian Journal of Animal Sciences 7: 77– 85. Devendra C. 1987. Goats. Bioclimatology and the Adaptation of Livestock. pp 157–167. (Ed.) Johnson H D. Elsevier, Amsterdam, Netherlands. Finch V A, Dmi’el R, Boxman R, Shkolnic A and Taylor C R. 1980. Why black goat in hot desrets? Effects of coat colour on heat exchanges of wild and domestic goats. Physiology Zoology 53: 19–25. Goyal S P and Ghosh P K. 1987. A note on the measurement of 70 Indian Journal of Animal Sciences 80 (4): 343–345, April 2010 Inventorization of Gaushala resources and their use in breed improvement and conservation programmes DINESH KUMAR YADAV1 and PRADEEP KUMAR VIJ2 National Bureau of Animal Genetic Resources, Karnal, Haryana 132 001 India Received: 4 May 2009; Accepted: 5 November 2009 ABSTARCT A comprehensive digitized inventory on Gaushalas of Haryana State with specific focus on their cattle genetic resources, and material resources was developed to integrate the data on Gaushalas on different aspects at one place. A uniform approach was followed to standardize the data using database normalization techniques. This system is menu driven and is designed using MS-Access at the back end and Visual Basic 6.0 at the front end. A user friendly graphical user interface has been developed for storage and retrieval of information from the database. A model for the use of the database information in cattle breed improvement and conservation programmes has been suggested. Key words: Breed, Cattle, Database, Gaushala, Improvement and Conservation, Relational Database Management System central agencies like Animal Welfare Board of India. In view of this, a computerized inventory is a necessity which could help the Gaushala Managers and other potential users for better management of Gaushala resources. Gupta and Yadav (2005) delineated ten goals for ‘Gaushala Development’ visà-vis role of district administration and research and development agencies. They emphasized on inventorization of Gaushala resources for the purpose of Gaushala development. To fulfill this vital need, we have developed a database with the purpose of establishing an information system to support the day-to-day program and management needs of the Gaushalas. Gaushalas (around 4500 in India) constitute a strong network of indigenous cattle maintained throughout the country and are playing a yeoman service to the welfare of the cow. Most of these are primarily looking after nonlactating, weak, unproductive, and stray cattle. But existing infrastructure, committed private management and leadership of devoted persons coupled with flow of resources are some of the important factors which render Gaushalas as ideal centres for cattle improvement and conservation. Added advantages of Gaushalas are their locations in the nook and corner of the country; a very close association and influence with local livestock keepers/people, and local administration of the area. Nevertheless, Gaushala management in the present context is a challenging task. Lack of appropriate financial aid from governments and constant dependency upon the donations for running day-to-day affairs is one of the chronic problems faced by them. Besides this, lack of authentic database of Gaushala inventory adds much to the vowes of the Gaushala Managers, policy planners, researchers, development agencies and farmers/livestock keepers as they need data on Gaushala resources for evolving realistic strategies for improvement and rearing of cattle. Frequent reports about the population status and other resources are required by developmental agencies like state animal husbandry departments for the purpose of giving maintenance grants as well as planning annual resource requirements for veterinary services to Gaushalas and other MATERIALS AND METHODS Primary data were collected from 105 Gaushalas of Haryana using a pre-tested structured questionnaire. Visits were undertaken to every Gaushala and data were collected from Gaushala records, interviews of management personnel and actual observations. Data included information on various aspects of Gaushalas, viz. its history including name and contact numbers of president, secretary and manager; breed-wise and age-wise cattle population; assets inventory; annual income, budget and expenditure; and miscellaneous information (like entrepreneurial efforts, breed improvement programmes, linkages with other agencies, performance recording, common diseases, flow of animals, problems faced by Gaushala management, manpower employed in the Gaushala and suggestions for making Gaushala selfsufficient etc.). System design and development: A uniform approach was Present address: 1 Senior Scientist (email: dkyadav66 @gmail.cm), 2 Principal Scientist. 71 344 YADAV AND VIJ Identification of Gaushalas with adequate infrastructure, resources and herd size [Indian Journal of Animal Sciences 80 (4) Associated herd progeny testing programme Inventorization of Gaushala resources Elite animals Proven bulls Test bulls Progeny of proven bulls General herd Selection Future test bulls Progeny of initially selected test bulls Proven bulls Overall genetic improvement Improvement in organized herds, Gaushalas and Farmers’ herds CATTLE BREED IMPROVEMENT AND CONSERVATION Fig. 1. Cattle breed improvement and conservation model based on the use of inventorization of Gaushala resources. followed to standardize the data using database normalization techniques (Date 2001) and the same was converted into database format using back-end tool MS-Access relational database management system (RDBMS). A user friendly graphical user interface (GUI) was developed for storage and retrieval of information from the database by using front end programming tool Visual Basic 6.0 (Jennings 2000) and Seagate Crystal Reports Professional Version 7.0. Income sources: This module holds information on data on different income sources of a Gaushala for the financial years 2001–02, 2002–03, 2003–04, 2004–05 and 2005–06. Information on other financial years can also be added. Total budget and expenditure: Budget and expenditure statements of the Gaushalas were compiled for the financial years 2001–02, 2002–03, 2003–04, 2004–05 and 2005–06 under this module of the database. Miscellaneous information: Information provided under this module can prove very handy in judging the farsightedness and forward-looking approach of the management committee of the Gaushala. It provides information-like whether entrepreneurial efforts were made by Gaushala management; breed improvement programmes initiated; linkages with other agencies established; records related to milk recording and pedigree of animals maintained; common diseases prevalent in the Gaushala; flow of animals from the Gaushala; problems faced by Gaushala management; manpower employed in the Gaushala; and suggestions of the management for making Gaushala selfsufficient etc. Various electronic forms for data entry, editing, updating of data (equipped with automatic validation checks to safeguard the database against duplicate and/or unnecessary data entry) were incorporated in the software to facilitate easy data storage. Online querying and report generation modules are available in the system for information retrieval. The whole database system was recorded on the CD-ROM medium in auto installation form. The cattle population in the country is declining and some of the cattle breeds like, Krishna Valley, Vechur and Punganur RESULTS AND DISCUSSION A complete database integrating data of Gaushalas on different aspects at one place was designed and tested. The system is menu driven, designed using MS-Access at the back end and Visual Basic 6.0 at the front end. The salient features of the database system are briefly described here. General details: This module holds information about the year of establishment; registration number and code number allotted by Animal Welfare Board of India; address and telephone numbers of Gaushala, its president and secretary. History, objectives and future plans: Brief information on the history of Gaushala, its current objectives and future plans are provided under this module. Breed-wise cattle population: Breed- wise and sex-wise cattle population of each Gaushala in different age groups (<1 year, 1–3 years and >3 years) are provided under this module. Assets: Details of assets inventory like total land under Gaushala (cultivable, uncultivable, constructed area and grazing area); water sources; transport facilities; gobar gas plants, panchgavya medicines production unit; vermicompost production unit etc. are given under this module 72 April 2010] INVENTORIZATION OF GAUSHALA RESOURCES AND THEIR USE IN BREED IMPROVEMENT 345 their infrastructure for breed improvement and conservation programmes. This database can be utilized by the potential users including Gaushala managers, planners, administrators, policy makers, farmers, dairy industry and the scientific community at large to use these resources in breed improvement and conservation programmes in a sustainable and affordable manner. The database would be helpful in performing roles like- functional support, decision support, communication decision support system and performance monitoring etc. are endangered breeds. Time has come to see the writing on the wall and check this suicidal drift to which the nation is heading. The Gaushalas can prove effective cattle improvement and conservation centres. The digitized inventory of the Gaushalas can play as a catalytic role in redefining their utility as improvement and conservation centres. The model (Fig.1) suggested for achieving this objective. Lack of scientific record keeping and exchange of information among Gaushalas, and between Gaushalas and development/research agencies has limited their utility in the past in the breed improvement programmes of the State and Central Governments. There is a great possibility of improving the livestock wealth of Gaushalas if suitable interventions are assessed and provided at appropriate time. If planned properly, these Gaushalas can become important sources for in-situ conservation of indigenous breeds and for progeny testing of large number of bulls. The database developed for the Gaushalas will help in realizing the objectives of—better utilization of Gaushala resources, making them self reliant, ideal, and most importantly utilizing REFERENCES Date C J. 2001. An Introduction to Database System. 7th edn. Addition Wesley Longman Pvt. Ltd. Indian Branch, Delhi. Gupta R and Yadav D K. 2005. District Administration and Gaushala Development. National Conference on Utility of Gaushalas for Improvement and Conservation of Indigenous Cattle. December 27–28, 2005, National Bureau of Animal Genetic Resources, Karnal, India: 53–57. Jennings. 2000. Database Developer Guide with Visual Basic 6.0 BPB Publications, New Delhi. 73 Indian Journal of Animal Sciences 80 (4): 346–349, April 2010 Environmental and genetic effects on growth traits of Chokla sheep B P KUSHWAHA1, AJOY MANDAL2, RAVINDRA KUMAR3 and SUSHIL KUMAR4 Central Sheep and Wool Research Institute, Avikanagar, Rajasthan 304 501 India Received: 3 April 2009;Accepted: 12 November 2009 ABSTRACT Data on 1810 Chokla sheep, maintained at the Central Sheep and Wool Research Institute (CSWRI), Avikanagar, Rajasthan, collected for a period of 21 years (1980 through 2000) were used to study growth traits and genetic control. The average weights at birth, 3, 6, 9 and 12 months of age were 2.73±0.04, 12.74±0.22, 16.71±0.26, 18.20±0.26 and 21.81±0.35 kg, respectively. Significant effects associated with the period of birth, parity of dam, season of birth and sex of lambs were observed on body weight at different stages of growth. Sire of the lamb contributed significantly on all body weights under study. There were marked periodic differences in the different growth traits of lambs. The lambs born in the dam’s third and later parities were significantly heavier to those born in first or second parities. Season of birth had significant effect on body weights at 3, 9 and 12 months of age. Males were heavier than females at almost all stages of growth and the sex differences tended to increase with age. The heritabilities of body weights at birth and at 3, 6, 9 and 12 months of age were high in magnitude, ranging from 0.32 to 0.48, which indicate the ample scope of improvement of these traits by selection. The medium (0.17±0.16) to high (0.89±0.05) genetic correlation among the body weights at different stages in this study suggest that selection for increased early growth traits will lead to genetic improvement in the subsequent development of body weights. Key words: Genetic analysis, Growth traits, Genetic parameters, Sheep Several non-genetic factors affect growth traits and directly obscure recognition of the genetic potential of animals. The effect of various factors like year of birth, season of birth, type of management and sex of animal on lamb growth of various wool breeds of sheep, has been studied (Mandal et al. 2003, Reddy et al. 2009). The present study was undertaken to identify various factors (viz. period of birth, parity of dam, season of birth and sex of lamb) influencing growth traits and to estimate the genetic and phenotypic parameters of these traits in Chokla sheep. for the region where migration is common practice. The wool of this breed is heterogeneous in quality and is finer than the wool of any other carpet wool producing sheep breeds in the country. Generally the animals were maintained under semiintensive system of feeding management where they are allowed to graze for 10–12 h on natural pasture with supplementation of some amount of concentrate depending upon the status and age category of the animals. Normally, rotational grazing system was followed. Controlled mating was practised in the flock. Majority of the ewes (more than 80%) were bred during autumn (August-September), the main breeding season and some ewes were bred in spring breeding season (March-April), off breeding season. Heat detection of ewes was done with teaser ram in the morning and evening during the breeding season. The ewes in heat were mated in the morning with selected sires. Generally, one breeding ram was allowed to mate 25 to 30 ewes and used for 2 years. At birth, each lamb was weighed and identified by metal ear tag. Weaning of lambs was generally done at 90 days. Shearing of animal was done twice in a year, i.e. in March–April and September–October. Animals were vaccinated against PPR, enterotoxaemia, FMD etc. The detailed descriptions of this breed along with distribution and production performance were described by Kushwaha et al. (1997). MATERIALS AND METHODS Breeding flock and its management: The Chokla sheep, comparatively fine carpet wool producing type sheep among the Indian sheep breeds, is hardy and well adapted to the arid and semi-arid regions of Rajasthan and also best suited Present address: 1Senior Scientist, Animal Breeding, Network project on Bhadawari Buffaloes, Indian Grassland and Fodder Research Institute, Jhansi 284 003 (e-mail: [email protected]). 2Senior Scientist, Genetics and Breeding Division, Central Institute for Research on Goats, Makhdoom, Mathura 281 122. 3Senior Scientist, Animal Breeding, National Bureau of Fish Genetic Resources, Dilkusha, Lucknow 226 002. 4Senior Scientist, Animal Breeding, Central Sheep and Wool Research Institute, Avikanagar 304 501. 74 April 2010] FACTORS AFFECTING GROWTH OF CHOKLA SHEEP Data: Data on 1810 lambs of Chokla sheep, maintained at the Central Sheep and Wool Research Institute (CSWRI), Avikanagar, Rajasthan, India, under the All India Coordinated Research Project (AICRP) on Sheep Improvement (later on, AICRP was renamed as Network Project on sheep improvement since 1990) for a period of 21 years (1980 to 2000) were used for the present study. The body weights at different ages (i.e. at birth, 3, 6, 9 and 12 month) were recorded and classified according to period of birth, season of birth, parity of dam and sex of lamb. The years of lambing were divided into 11 periods, each comprising 2 years except last period, based on the use of a different ram for breeding during each period. Each year of lambing was also divided into 2 seasons, i.e. S–1 (January-February) and S–2 (AugustSept.). Statistical analyses: Data were analyzed using a mixed model least-squares analysis for fitting constants (Harvey 1990) including all main effects and interactions. In the final model, all non-significant interactions were ignored, which is as follows: Yijklmn = μ + Si + Pj + Ak + Sl + Em + eijklmn, where, Yijklmn is the record for the nth lamb, Si is the effect of the ith sire, Pj is the effect of the jth period of birth, Ak is the effect of the kth parity of dam, Sl is the effect of the lth 347 season of birth, Em is the effect of the m th sex of lamb and eijklmn is the residual error element. The genetic parameters of various growth traits were estimated by the paternal half-sib method. The comparison of different sub-groups mean was made by Duncan’s Multiple Range Test (DMRT) as described by Kramer (1957). RESULTS AND DISCUSSION Body weights at different ages: The average weight of Chokla lamb at birth, 3, 6, 9 and 12 month of age were 2.73±0.04, 12.74±0.22, 16.71±0.26, 18.20±0.26 and 21.81±0.35 kg, respectively (Table 1). The overall leastsquares means for lamb weights observed at the different ages in this breed were similar with the findings of Nehra and Singh (2006) but lower than the results of Reddy et al. (2009). The sires of lambs had significant (P<0.01) influence on all the body weights in the present study. Similar significant sire effects on body weights were observed by Mandal et al. (2003) in various sheep breeds. The significant effect of sire on all these traits studied indicated that superior rams could be used effectively for improvement of these traits. Period of birth was also highly significant (P<0.01) in respect of lamb weights at all developmental stages in this study. The present findings corroborated with the results of Table 1. Least- squares means along with standard errors of different body weights in Chokla sheep Effects No. of obs. Birth weight (kg) No. of obs. 3 m weight (kg) No. of obs. 6 m weight (kg) No. of obs. 9 m weight (kg) No. of obs. 12 m weight (kg) Overall Period of birth P–1 P–2 P–3 P–4 P–5 P–6 P–7 P–8 P–9 P–10 P–11 Parity of dam 1 2 3 4 5 6 or > 6 Sex of lamb Male Female Season of birth Jan-Feb Aug-Sep 1810 2.73±0.04 1501 12.74±0.22 1304 16.71±0.26 1132 18.20±0.26 920 21.81±0.35 69 48 84 95 149 188 175 207 323 336 136 2.10±0.28a 2.32±0.24a 2.79±0.16bc 2.59±0.14ab 2.87±0.13cd 2.96±0.12cd 3.20±0.12d 2.79±0.14bcd 2.84±0.15bcd 2.91±0.15cd 2.63±0.17ab 64 43 74 84 113 171 147 158 235 303 109 14.54±1.53de 13.99±1.33cde 13.36±0.93cde 12.77±0.82bcd 14.54±0.76de 15.53±0.66e 12.41±0.69bc 10.65±0.82ab 10.17±0.86a 11.48±0.89abc 10.74±0.97ab 55 36 62 68 76 148 117 141 206 294 101 15.00±1.79 a 14.99±1.49 a 14.71±1.23 a 16.84±1.10 ab 21.82±1.01 d 20.88±0.85 cd 19.02±0.88 bc 16.76±1.02 ab 16.17±1.07 a 16.57±1.11 ab 17.11±1.21 ab 47 33 50 60 64 122 100 110 187 263 96 565 465 334 225 140 81 2.38±0.04a 2.65±0.04b 2.79±0.04c 2.87±0.05c 2.86±0.05c 2.81±0.06c 420 387 297 196 128 73 11.75±0.24a 12.62±0.24b 12.88±0.25bc 12.94±0.28bc 13.58±0.31c 12.71±0.37bc 334 347 261 178 116 68 15.86±0.29a 16.80±0.28b 16.83±0.31b 16.75±0.33b 17.19±0.37b 16.86±0.45b 279 292 232 163 104 62 17.49±0.29a 18.07±0.29ab 18.58±0.30b 18.15±0.33ab 18.25±0.37ab 17.97±0.44c 219 235 187 135 88 56 21.39±0.39 a 21.83±0.38 a 22.01±0.40 a 21.64±0.43 a 21.90±0.47 a 22.10±0.54 a 897 913 2.78±0.04 a 2.67±0.04 b 736 765 13.17±0.23 a 12.32±0.23 b 637 667 17.74±0.27 a 15.69±0.27 b 545 587 19.65±0.27 a 16.75±0.27 b 410 510 23.92±0.37 a 19.70±0.36 b 562 248 2.72±0.03 a 2.74±0.04 a 1317 184 11.97±0.21 a 13.52±0.28 b 1149 155 16.48±0.24 a 16.94±0.36 a 1020 112 18.69±0.23 a 17.70±0.35 b 1835 85 22.31±0.32 a 21.31±0.46 b Means with different superscripts differed significantly (P<0.05) from each other. 75 17.96±2.02 abcd 37 16.92±1.80 abc 31 17.08±1.26 abc 43 18.15±1.13 bcd 56 21.53±1.04 d 60 20.93±0.87 d 63 19.92±0.91 cd 83 16.26±1.04 ab 92 14.91±1.10 a 157 17.07±1.14 abc 207 19.44±1.23 cd 91 20.41±2.50 ab 21.58±2.27 abc 18.71±1.61 a 21.63±1.49 abc 26.24±1.39 d 26.21±1.66 cd 25.05±1.21 bcd 19.68±1.42 a 17.77±1.47 a 19.57±1.51 a 21.07±1.60 ab 348 KUSHWAHA ET AL. [Indian Journal of Animal Sciences 80 (4) Table 2. Estimates of heritability (diagonal), genetic correlations (below diagonal) and phenotypic correlations (above diagonal) of growth traits of Chokla sheep Traits/parameter Birth wt. 3 M BW 6 M BW 9 M BW 12 M BW Birth wt. 3 M BW 6 M BW 9 M BW 12 M BW 0.36±0.07(1810) 0.66±0.12 0.26±0.18 0.28±0.18 0.17±0.16 0.44 0.43±0.09(1501) 0.68±0.10 0.37±0.16 0.23±0.17 0.28 0.69 0.37±0.09(1304) 0.51±0.15 0.46±0.15 0.32 0.55 0.68 0.32±0.09(1132) 0.89±0.05 0.30 0.51 0.61 0.80 0.48±0.11(920) Figures in parentheses indicate number of observations. Sivakumar et al. (2006) and Reddy et al. (2009) who obtained significant effect of period/year of birth on body weights of sheep. The body weight differences among lambs born in different periods in our study may be attributed to differences in management, selection of rams and environmental conditions etc. The parity of dam contributed significantly (P<0.01) for all growth traits of lamb except weight at 12 months of age (Table 1). The lambs born in third or later parities were heavier except at birth than the lambs of younger ewes. Similarly, Mandal et al. (2003) found significant effect of parity of dam on different body weights of Muzaffarnagari lamb. The low birth weight of lambs in younger ewes may be due to the existence of relative competition between the still growing ewes and the developing foetus for nutrients/ food. Season of birth had significant effect on weights of lambs at 3, 9 and 12 months of age. The significant effect of season of birth on body weights was also reported in different breeds of sheep (Kumar et al. 2007, Reddy et al. 2009). The significant effect of the season of birth on 9 and 12 months body weight may be due to those lambs born in AugustSeptember passing through a period with a favorable climate during January to April when the grazing of good quality was available. Male lambs achieved significantly (P<0.01) higher body weights at all stages of growth than their female counterparts (Table 1). The increase in the ratio of male to female body weight as the lambs became older and their body weights increased, probably arises from the increasing differences in the endocrine system between males and females (Swenson and Reece 1993). These sex differences are consistent with results from other investigations (Waghmode et al. 2008, Reddy et al. 2009). selection. Both the phenotypic (0.44) and genetic (0.67) correlation (0.67) of birth weight with weaning weight was moderately high in magnitude, but the correlation of birth weight with body weights at subsequent ages ranged from low to medium and positive (0.17 to 0.32). The phenotypic correlations of weaning weight with 6, 9 and 12 months body weight were also significant and high (0.51 to 0.69) but declined steadily at later stages. The genetic correlations of weaning weight with the body weights at subsequent ages showed a similar trend. The 6-and 9-month body weights had significant, high positive phenotypic and genetic correlations with their subsequent body weights and these correlations were statistically significant (P<0.01). The moderately medium to high genetic correlations between birth weight and weaning weight, and of weaning weight with 6, 9 and 12 months body weight in this study indicates that selection for increased birth weight in the sheep will also result in genetic improvement in the subsequent development of body weights. The present study revealed that different environments affect significantly the growth traits of Chokla sheep. The high heritabilities of body weights and high genetic correlation among the body weights at different stages suggest that selection for increased early growth traits will lead to genetic improvement in the subsequent development of body weights. ACKNOWLEDGEMENTS The author is grateful to the Director, Central Sheep and Wool Research Institute (CSWRI), Avikanagar, Rajasthan, for providing all facilities to conduct this study. Help rendered from the staffs of Chokla sheep unit for data management for this flock is also duly acknowledged. Genetic and phenotypic parameters The genetic and phenotypic correlations and heritability estimates for growth traits are presented in Table 2. The heritability of body weights tended to increase with increasing age from birth to weaning and subsequent weights except for that at 9 months of age. The heritabilities of body weights at birth and at 3, 6, 9 and 12 months of age observed in this study were high (0.32–0.48) in magnitude, as compared to the estimates of Nehra and Singh (2006), which indicate ample scope of improvement of these traits by REFERENCES Harvey W R. 1990. User’s guide for LSMLMW PC–2 Version mixed model least-squares maximum likelihood computer program. Minneograph Columbus, Ohio, U.S.A. Kushwaha B P, Kumar S, Kumar R and Mehta B S. 1997. The Chokla sheep in India. Animal Genetic Resources Information, Rome, Food and Agricultural Organization 22: 19–27. Kramer C Y. 1957. Extension of multiple range tests to group 76 April 2010] FACTORS AFFECTING GROWTH OF CHOKLA SHEEP correlated adjusted means. Biometrics 13: 13. Kumar V Ramesh Saravana, Sivakumar K, Singh D, Anandha Prakash, Ramesh V, Muralidharan J and Devendran P. 2007. Non genetic factors affecting birth weight of Mecheri lambs. Indian Journal of Small Ruminants 13(2): 228–30. Mandal A, Pant K P, Nandy D K, Rout P K and Roy R. 2003. Genetic analysis of growth traits in Muzaffarnagari sheep. Tropical Animal Health and Production 35: 271–84. Nehra K S and Singh V K. 2006. Genetic evaluation of Marwari sheep in arid zone: growth. Indian Journal of Small Ruminants 12(1): 91–94. Reddy Y Ravindra, Naidu P Thyagaraja and Rao S T Viroji. 2009. 349 Growth performance of Nellore breed of sheep in India. Indian Journal of Small Ruminants 15(1): 118–20. Sivakumar T, Soundararajan C, Palanidorai R, Ganeshkumar G, Mahendrans M and Malathi G. 2006. Factors affecting birth weight in Madras Red lambs. Indian Journal of Small Ruminants 12(1): 115–16. Swenson M J and Reece W O. 1995. Dukes’ Physiology of Domestic Animals. 11th edn. Cornell University Press, Ithaca, NY, 962 pp. Waghmode P S, Sawane M P, Pawar V D and Ingawale M V. 2008. Effect of non-genetic factors on growth performance of Madgyal sheep. Indian Journal of Small Ruminants 14(1): 127–30. 77 Indian Journal of Animal Sciences 80 (4): 350–353, April 2010 Growth rate and wool production of Marwari lambs under arid region of Rajasthan H K NARULA1, AJAY KUMAR2, M AYUB3 and VIMAL MEHROTRA4 Central Sheep and Wool Research Institute, Arid Region Campus, Bikaner, Rajasthan 334 006 India Received: 23 April 2009;Accepted: 20 November 2009 ABSTRACT Marwari breed, an important carpet wool producing sheep breed of India, is well adapted to harsh and erratic climatic conditions of hot arid region, and has capacity to cover large area during migration. The present study was conducted to evaluate growth and wool production of Marwari lambs in an organized farm under hot arid climate of Rajasthan. The data on 981 lambs born during 2004–07 were utilized for assessing production potential of Marwari sheep maintained at the Central Sheep and Wool Research Institute, Arid Region Campus, Bikaner. The overall least squares means for birth, 3, 6, 9 and 12 months weight of lambs were 2.93±0.01, 15.88±0.09, 22.59±0.12, 28.22±0.11 and 30.44± 0.12 kg, respectively. The effects of sex, type of birth and year of lambing were highly significant on all the body weights. The improvement in the body weights at all stages was observed during the period under study. The overall least squares means for first and second six monthly greasy fleece weights were 596.93±5.48 and 675.43±7.46 g, respectively. The greasy fleece yield was significantly affected by year, type of birth and sex of lamb. The overall least squares means of fibre diameter, hetro fibres, hairy fibres, medullation, staple length and crimps were 33.56±0.26 μ, 36.00±0.70%, 17.66±0.43%, 53.66±0.85%, 5.15±0.05 cm and 0.66±0.01 per cm, respectively. It was concluded that there was an improvement in growth and wool production in the Marwari sheep due to selection programme being applied in the Network Project on sheep. Key words: Average daily gain, Growth, Marwari sheep, Sheep, Wool yield, Wool quality Marwari sheep at an organized farm under arid conditions of Rajasthan. Marwari, an important carpet wool producing sheep breed having medium and coarse carpet wool, is widely distributed in Jodhpur, Jalore, Nagaur, Pali and Barmer districts of Rajasthan. This breed is well adapted to harsh and erratic climatic conditions of hot arid regions and has capacity to cover large areas during migration. Early expressed growth traits are very important economic traits and could be used for genetic improvement of traits. Better growth is essential for appropriate reproduction, production and survivability in sheep. Better growth rate and wool yield has direct relevance to sheep farmers because heavier lambs with high growth rate and high wool yield will lead to more economic returns to sheep farmers. Besides genetic merit, these traits are largely determined by non-genetic factors. Before planning and implementing a sheep breeding plan through selective breeding, it is necessary to evaluate factors affecting body weights and greasy fleece yields. Therefore, in the present study an attempt was made to evaluate the growth, wool yield, wool quality and factors affecting these traits in MATERIALS AND METHODS The data collected on 972 Marwari lambs born during 2004 to 2007 at the Central Sheep and Wool Research Institute, Arid Region Campus, Bikaner,Rajasthan, under the project entitled “Improvement of Marwari sheep for carpet wool production through selection (Network Project on Sheep Improvement)” were utilized for the present study. The climate of Bikaner is hot arid with normal rainfall of 200–240 mm. The lambs were maintained in groups under semi-intensive system of management with 8 h grazing per day. The lambs were allowed to suckle their dams up to 3 months of age. The suckling lambs were also fed concentrate mixture up to weaning and allowed to graze separately from their dams. The lambs were supplemented with 250– 300 g concentrate ration after weaning during various growth stages. These were offered green as well as dry fodder during lean period. The data were recorded for sex of lamb, date of lambing, dams’ weight at lambing and live body weights at birth, 3, 6, 9 and 12 months of age along with greasy fleece weights in first and second 6 monthly clips. Average daily weight gains of individual lamb during 0–3, 3-6 and 6–12 Present address: 1 Senior Scientist, (E mail hknarula @yahoo.co.in), 2 Scientist. 3,4 Technical Officers, Central Sheep and Wool Research Institute, Arid Region Campus, Bikaner. 78 April 2010] GROWTH RATE AND WOOL PRODUCTION OF MARWARI LAMBS months were calculated. The animals were washed properly 48 h before shearing to remove dirt and dust from wool and for smoothening shearing of animals. All the animals were shorn twice a year, i.e. spring and autumn and shearing was carried out using scissors in March and August, respectively. The wool samples (588) were collected from the mid side region of the body of animals and analyzed for various fibre quality traits, viz. fibre diameter, hetro fibre, hairy fire, medullation, staple length and crimps. The breeding rams were selected on the basis of an index incorporating 6 months live body weight and first six monthly greasy fleece yields. The ewes were mated at random to the selected males. The data on growth traits and greasy fleece yields were classified according to year of lambing, sex of lambs and type of birth. The statistical analysis was carried out (Harvey 1990) to assess the impact of sex of lamb and year of lambing on body weights and average daily weight gains by taking year of lambing, sex of lamb and type of birth as fixed effect. 351 predominant across different months of year, growth of pasture, incidence of certain diseases etc. In addition to this, continuous selection and culling process also created differences in performance. On account of genetic/other factors such as selection of rams for breeding, culling of old and inferior stock and flock strength across different months of year and age composition of flock at different intervals due to overlapping generations, there could be variations in growth and other performance traits. The weight gain/day from birth to 3 months, 3 to 6 months, 6 to 12 months was observed to be 143, 75.14 and 40.77 g, respectively. Comparatively, lower body weights at birth, 3, 6, 9 and 12 months were reported by Nehra and Singh (2006). The significant influence of year of birth on all the body weights at different stages was reported by Narula et al. (2007) and Dass et al. (2008). The average daily gains during 0–3, 3-6 and 6–12 months ranged from 128.67 to 156.13 g, 66.94 to 89.90 gm and 28.71 to 50.67 g, respectively. The growth performance showed an increasing trend with slight fluctuations. The 6 and 12 months body weights improved from 21.07 kg 24.84 kg and 28.2 kg to 30.63 kg, respectively. Sex of lambs had significant effect on all the body weights at different ages and various average daily gains (Table 2). Males were significantly heavier than females at all the stages of growth and it may be due to differences in physiology of two sexes. The findings are in agreement with those reported by Nehra and Singh (2006), Narula et al. (2007), Dass et al. (2008) and Narula et al. (2009).The birth, weaning, 6, 9 and 12 months weights in males were 4.18, 12.44, 19.10, 17.53 and 20.02%, respectively, higher than females. Besides the effects of sex hormones and other physiological factors, differences in body weights of male and female lambs/ hoggets could be due to differential culling rates as more number of males was culled after the hogget stage while RESULTS AND DISCUSSION Growth performance: The overall least squares means for live body weights at birth, 3, 6, 9 and 12 months of age are given in Table 1. The birth weight ranged between 2.98 and 3.07 kg. The 6 and 12 months body weights ranged between 20.58 to 24.84 and 28.20 to 33.08 kg, respectively. All the body weights were influenced significantly (P<0.01) by year of lambing. The year differences could be due to varying availability of feeds/fodders, physical environment and other management factors prevailing in different years. Though, efforts were made to ensure uniform feeding and management of the flock over the years, there were many such factors which were beyond the control of farm managers. To list a few such factors are wind velocity, rainfall, sunshine hours, minimum and maximum temperatures, types of grasses Table1. Least squares means for live body weights (kg) at different ages in Marwari sheep Trait/Effect Birth wt Overall mean 2.93±0.01 (981) Sex ** Male 2.99±0.02 (511) Female 2.87±0.02 (470) Year ** 2004 2.98±0.03 (271) 2005 2.75±0.04 (102) 2006 2.92±0.02 (307) 2007 3.07±0.02 (301) Type of birth ** Single 3.28±0.02 (765) Twin 2.59±0.023(216) Regression coefficient of **0.041±.003 DWts at 32.24±3.97 lambing average 3 M wt 6 M wt 9 M wt 12 M wt 15.88±0.09 (972) ** 16.81±0.11 (504) 14.95±0.11 (468) ** 15.38±0.15 (271) 17.18±0.23 (99) 14.36±0.13 (303) 16.61±0.13 (299) ** 17.05±0.09 (761) 14.71±0.16 (211) 22.59±0.12 (946) ** 24.56±0.15 (486) 20.62±0.15 (460) ** 21.07±0.20 (269) 23.87±0.29 (97) 20.58±0.16 (295) 24.84±0.18 (285) ** 23.68±0.11 (740) 21.51±0.21 (206) 28.22±0.11 (743) ** 30.50±0.14 (375) 25.95±0.15 (368) ** 26.43±0.18 (222) 31.74±0.31 (77) 25.70±0.18 (217) 29.02±0.18 (227) ** 28.53±0.12 (591) 27.05±0.23 (152) 30.44±0.12 (643) ** 33.21±0.17 (298) 27.67±0.16 (345) ** 28.20±0.21 (192) 33.08±0.33 (77) 29.83±0.20 (214) 30.63±0.23 (160) ** 30.73±0.13 (509) 29.27±0.26 (134) **0.281±0.020 32.25±3.96 DWt,Dams’ weight at lambing, ** significant (P<0.01),figures in parentheses are number of observations. 79 352 NARULA ET AL. [Indian Journal of Animal Sciences 80 (4) Table 2. Least squares means for average daily gains (g) in different periods Particulars Overall mean Sex Male Female Year 2004 2005 2006 2007 Type of birth Single Twin ADG 1 (0–3 M) ADG 2 (3-6 M) ADG 3 (6–12 M) Number Mean Number Mean Number Mean 972 143.41±1.01 ** 153.06±1.26 133.68±1.27 ** 130.82±2.06 156.13±2.44 128.67±1.39 150.29±1.46 ** 152.14±2.06 134.69±1.73 940 75.14±1.04 ** 86.36±1.30 63.93±1.31 ** 64.06±1.73 79.68±2.51 66.94±1.43 89.90±1.51 NS 74.17±0.97 76.12±1.79 637 40.77±0.64 ** 44.01±0.83 37.53±0.77 ** 35.80±1.06 48.52±1.44 50.67±0.85 28.71±1.03 ** 38.63±0.58 42.91±1.12 504 468 271 99 303 299 761 211 484 456 263 97 295 285 735 205 females were retained to replenish the fertility of stock through replacement of older ewes with younger and genetically superior ones. Selection intensity in males is much higher than females. Type of birth had significant effect (P<0.01) on all the body weights. Single born animals were heavier than twin born animals at all the stages of growth. The single born animals were heavier by 26.66, 15.90, 10 and 4.98% at birth, 3, 6 and 12 months, respectively. The differences in the body weights were higher at initial stage than later stages. Regression of dam’s weight at lambing was found highly significant on birth and 3 months weight. The results indicated that if dam is heavier at lambing then its lamb will also be heavier at birth and subsequent ages. The higher birth weight of heavier lambs may be due to body conditions of dam during pregnancy and lactation, which in turn resulted into healthy and heavier lambs. Sharma et al. (2003), Narula et al. (2007) and Dass et al. (2008) also found similar trend of regression of dams weight at lambing on birth and weaning weights of lambs. Wool yield: The overall least squares means for first greasy fleece yield and second 6 monthly greasy fleece yields were 596.93 and 675.43 g, respectively (Table 3). In contrast to body weights, the female lambs produced more fleece than males. This may be due to higher primary and secondary follicles in females and also because of higher staple length and crimps in females. The lower values of clips yields were reported by Dass et al. (2008) in Marwari sheep. The influence of year and type of birth was significant (P<0.01) on first and second 6 monthly greasy fleece yield (GFY). The significant influence of year and sex was also reported by Dass and Singh (2002) and Dass et al. (2008). As far as type of birth is concerned, the single born lambs produced more clip yield than twin born lambs which was upto 4.7%. The wool yield of both clips improved over the years with slight fluctuations. The first clip and second six monthly greasy fleece yield improved by 20.17 and 18.14%, 293 344 186 77 214 160 503 134 Table 3. Least squares for different wool (g) Particulars First clip Number Overall mean Sex Male Female Year 2004 2005 2006 2007 Type of birth Single Twin 951 493 458 271 96 295 289 744 207 Second clip Mean Number Mean 596.93±5.48 ** 597.47±6.98 596.39±7.32 ** 521.89±9.24 622.68±13.3 615.96±7.93 627.19±8.15 ** 603.50±5.16 590.36±9.51 590 675.43±7.46 ** 635.59±10.58 715.27±8.90 ** 569.85±12.20 699.87±16.53 758.77±10.83 673.25±11.69 ** 690.94±6.79 659.92±13.17 241 349 184 76 172 158 472 118 First clip, first six monthly greasy fleece yield;second clip, second six monthly greasy fleece yield. respectively, in 2007 (as compared to 2004). Still, there is scope of improvement in greasy fleece yield by introducing more genetic variability and introducing new sire lines in the flock. Wool quality: The overall least squares means for fibre diameter, hetro fibres, hairy fibres, medullation, staple length and crimp were 33.56±0.26 μ, 36.00±0.70%, 17.66±0.43%, 53.66±0.85%, 5.15±0.05 cm and 0.66±0.01 per cm, respectively (Table 4). Comparatively lower fibre diameter and medullation% were reported by Dass et al. (2008) in Marwari breed. All the wool quality traits were significantly affected by year of lambing except medullation%. The significant effect of year on these traits was also reported by Dass and Singh (2002) and Dass et al. (2008). The staple length varied between 4.92 and 5.42 cm and this fulfills the requirement of carpet industry for making good quality carpets. The difference in wool quality parameters in different years may be due to availability of varying levels of feeds 80 April 2010] GROWTH RATE AND WOOL PRODUCTION OF MARWARI LAMBS 353 Table 4. Least squares means for wool quality attributes of Marwari sheep Effect/trait Fibre diameter (μ) Hetro fibre(%) Hairy fibre(%) Medullation (%) Staple length (cm) Crimp(/cm) Overall Mean Sex Male Female Year 2004 2005 2006 2007 33.56±0.26 (588) ** 34.67±0.28 (385) 32.44±0.43 (203) ** 34.56±.49 (141) 34.86±0.46 (121) 31.80±0.37 (183) 34.01±0.44 (143) 36.00±0.70 (588) NS 37.29±0.77 (385) 34.70±1.16 (203) ** 37.56±1.30(141) 31.24±1.25(121) 37.79±1.01(183) 38.95±1.19(143) 17.66±0.43 (588) NS 18.24±0.48 (385) 17.08±0.72 (203) ** 18.31±.79 (141) 22.89±0.77 (121) 14.72±0.62 (183) 15.37±0.74 (143) 53.66±0.85 (588) * 55.54±0.94 (385) 51.79±1.40 (203) NS 55.87±1.64 (141) 54.14±1.52 (121) 52.52±1.22 (183) 54.33±1.44 (143) 5.15±0.05 (558) ** 4.85±0.05 (385) 5.46±0.08 (203) ** 5.19±.07 (141) 5.42±0.08 (121) 5.12±0.07 (183) 4.92±0.09 (143) 0.66±0.01 (558) ** 0.59±0.02 (385) 0.73±0.03 (203) * 0.39±.02 (141) 0.62±0.03(121) 0.72±0.02(183) 0.65±0.03(143) and fodders in terms of quality and quantity during different years. The influence of sex was significant on fibre diameter, medullation%, staple length and crimp per cm. The males were coarser in wool quality as compared to females as indicated by higher fibre diameter. The wool quality parameters indicated suitability of fibres produced by Marwari sheep for use in carpet industry. The percentage of hairy fibres should be between 10 and 15% for quality and life of carpets. So, there is need to further reduce medullation percentage and hairy fibres to establish this breed as best carpet wool breed of arid region. It is concluded from the present study that despite high temperature, poor humidity and low rainfall resulting in reduction of vegetation in pasture land and causing physiological stress to the animals this breed performs well. At present Marwari breed is superior to majority of the indigenous carpet wool producing breeds in body weights and wool quantity. The quality of wool is also suitable for medium quality carpets. Hence it can be used for genetic improvement programmes in the farmers flocks located in the breeding tract of Marwari sheep. REFERENCES Dass G and Singh V K. 2002. Improvement of Marwari sheep in hot arid climate. Indian Journal of Animal Sciences 72: 253– 56. Dass G, Sharma P R and Mehrotra V. 2008. Production performance of Marwari lambs under hot arid region of Rajasthan. Indian Journal of Animal Sciences 78: 117–20. Harvey W R. 1990. Mixed Model Least Squares’ and Maximum Likelihood Computer Programme PC–2 version. Ohio State University, USA. Narula H K, Dass G, Sharma, P R, and Mehrotra, V. 2007. Impact of selection on growth and reproductive performance of Marwari sheep in arid region of Rajasthan. Abstracts. National Symposium on Recent trends in technological interventions for rural prosperity in small holder livestock production systems.College of Veterinary Science, Tirupati from 20–22 June, 2007, pp 49. Narula H K, Dass G, Sharma P R and Mehrotra, V. 2009.Growth performance and survivability of Marwari lambs in an organized farm. National Symposium on Livestock Biodiversity Conservation and Utilization: Lessons from Past and Future Perspectives.NBAGR, Karnal from 12–13, February, 2009, pp 167. Nehra K S and Singh V K. 2006. Genetic evaluation of Marwari sheep in arid zone: Growth. Indian Journal of Small Ruminants 12: 91–94. Sharma M K, Sharma N K, Singh V K and Beniwal B K. 2003. Genetic evaluation of Nali and Marwari sheep in arid zone of Rajasthan. Indian Journal of Small ruminants 9: 65–68. ACKNOWLEDGEMENTS Thanks are due to the Director of CSWRI and Head, ARC Bikaner, for providing necessary facilities and cooperation during the course of this study. 81 Indian Journal of Animal Sciences 80 (4): 354–357, April 2010 Effect of supplementing bypass fat prepared from soybean acid oil on milk yield and nutrient utilization in Murrah buffaloes S S THAKUR1 and S K SHELKE2 National Dairy Research Institute, Karnal, Haryana 132 001 India Received: 6 April 2009;Accepted: 20 November 2009 ABSTRACT This experiment was conducted to study the effect of supplementing Ca salts of soya acid oil fatty acids on milk production, its composition and nutrient utilization in lactating buffaloes. Lactating Murrah buffaloes (16) were divided in 2 groups (milk yield 7.08, 6.92 kg/d; 30 and 25 days in milk, 2–4 lactation) of 6 animals in each group, and the animals in control group were fed with wheat straw, green maize fodder and concentrate mixture as per requirements and the animals in experimental group were fed with the same ration as that of control group along with 4% Ca salts of soya acid oil fatty acids of total DMI for 90 days. The average milk yield in experimental group was higher by 12.43% over that of control group. Similarly, the 4% FCM yield was higher by 13.40% in experimental group over that of control group. There was no difference in milk protein, total solids and solid not fat in both groups except milk fat content which was higher in experimental group than that of control group. The total LCFA and MUFA content in milk fat was higher in experimental group compared to control group. The DMI and CPI were similar in both groups, whereas TDNI was higher in experimental group than that of control group. The digestibility coefficient of DM, CP, CF, NDF and ADF were similar in both groups except EE which was higher in experimental group than that of control group. It could be concluded that supplementation of Ca salts of soya acid oil fatty acids at 4% of DMI improved milk yield and proportion of unsaturated FA and LCFA in milk fat in Murrah buffaloes. Key words: Buffalo, Bypass fat, Ca salts of soya fatty acids, Milk yield, Nutrient utilization (milk yield 7.08, 6.92 kg/d; 30 and 25 days in milk, 2–4 lactation) of 6 animals in each group. The animals in control group were fed with wheat straw, green maize fodder and concentrate mixture as per requirements (Kearl 1982) and the animals in experimental group were fed with the same ration as that of control group along with 4% Ca salts of soya acid oil fatty acids of total DMI for 90 days. The concentrate mixture contained maize 33, groundnut cake 21, mustard cake 12, wheat bran 20, deoiled rice bran 11, mineral mixture 2 and common salt 1 part. The lactating buffaloes were housed in a well ventilated paddock having individual feeding mangers and space for separate tying of individual animal and provided with fresh and clean tap water three times daily at 8.00 h, 12.00 h and 18.30 h. These buffaloes were hand milked twice a day, in the morning and in the evening. Digestibility trial was conducted under the same housing management conditions at the end of trial. Calcium salts of soya acid oil fatty acids were prepared using double decomposition method (Deuel 1951). Soybean acid oil was heated in a metal container; an aqueous solution of sodium hydroxide was added and again heated to cause saponification, sodium salts so formed were dissolved in Feeding Ca soaps of fatty acids, which are inert in rumen, to high producing lactating cows, can enhance dietary energy density and therefore, energy intake in early lactation without compromising the activity of rumen microflora (Jenkins and Palmquist1984). Thus, the deleterious effect of acute negative energy balance on lactation performance in animals can be alleviated. Rumen inert fat provides partial resistance from ruminal biohydrogenation and hence enhances beneficial fatty acid profile of milk and body tissues from human health point of view (Mishra et al. 2004, Tyagi et al. 2009). Such quality milk can be used as a nutraceutical for prevention of various life style diseases. Considering the above points, a feeding trial was conducted to investigate the effect of bypass fat supplementation on milk production, fatty acid profile of milk and nutrient utilization in Murrah buffaloes. MATERIALS AND METHODS Lactating Murrah buffaloes (12) were divided in 2 groups Present address: 1 Principal Scientist (email: sst_ndri @yahoo.co.in), 2Ph. D. Scholar, Dairy Cattle Nutrition Division. 82 April 2010] BYPASS FAT FEEDING TO LACTATING MURRAH BUFFALOES excess boiling water. Calcium chloride dissolved in water was then added slowly to the water soluble sodium soaps with stirring causing immediate precipitation of calcium salts. Excess water was removed by squeezing soaps through cheese cloth. The soap was allowed to air dry and then lumps were broken before being mixed with other concentrate ingredients (Jenkins and Palmquist 1984). The level of protection of the rumen inert fat was judged by estimating the degree of saponification of the Ca soaps (Garg and Mehta 1998). From the pooled milk samples of individual animal, the fatty acid analysis of milk and feed samples, viz. green fodder, concentrate and rumen protected fat was done using saponification method (Gulati and Ashes 2000). The analysis was carried out on GLC fitted with flame ionization detector and 50 m length of capillary column. Initial temperature of the column was 140ºC. The RAMP rate was 2 ºC/min. Identification of peaks was made through retention time of the reference standards. DM, CP, EE, CF and total ash of feed and faecal samples were analyzed (AOAC 2005), and NDF and ADF were estimated (Goering and Van Soest 1970). Milk samples were analyzed for total solids, protein and SNF as per AOAC (2005), whereas, milk fat by REIL, electronic milk fat tester. Statistical analyses of the data were carried out using Students‘t’ test as per Snedecor and Cochran (1980). experimental group. Average 4% FCM yield was 11.86 kg/d in control group, and it was enhanced (P<0.05) by 13.40% in experimental group (13.45 kg) over that of experimental group. Control group reached the peak yield in second fortnight and experimental group at third fortnight but the average peak production was also better (P<0.05) in experimental group. Higher peak yield and milk production observed in experimental group may be attributed to enrichment of ration with Ca salts of soya acid oil fatty acids that increased the energy density of the ration. The present results are in line with the findings of Fahey et al. (2002); McNamara et al. (2003); Mishra et al. (2004) and Tyagi et al. (2009) who observed improvement in milk yield on supplementation of bypass fat to cows. Milk fat ranged from 5.29 to 6.00% in control group and 5.15 to 6.20% in experimental group during different fortnights. The overall average milk fat% was higher (P<0.05) in experimental group (6.16) than that of control (5.94). There was no effect on protein, total solids and SNF contents of milk in the 2 groups (Table 3). Our results are in agreement with those of previous researchers who reported improvement in milk fat but no effect on protein, Total solids and SNF contents on addition of bypass fat (Sklan et al. 1994, McNamara et al. 2003). The contents of myristoleic acid (C14: 1), palmitoleic acid (C16: 1), elaidic acid (C18: 1t9), oleic acid (C18: 1c9), linoleic acid (C18: 2), linolenic acid (C18: 3) and arachidic acid (C20: 0) in the milk fat of experimental group were higher as compared to that of control group (Table 2). Total unsaturated fatty acids increased by 21.76% in experimental group over control group, saturated fatty acid content showed a decrease of 14.14 whereas, that of monounsaturated fatty acids (MUFA) also elevated in experimental group by 9.39% than control group, respectively. The total long chain fatty acids (LCFA) content was also higher in experimental group. Dietary bypass fat alters fatty acid profile of the milk towards the fatty acid content of the supplemental fat. The results obtained with polyunsaturated fatty acids (PUFA) rich protected fat feeding (Sampelayo et al. 2004), with Ca salts of FA (Fahey et al. 2002) and with dietary supplementation of progressively more unsaturated fatty acids of Canola, Soybean, and Linseed oil (Chouinard et al. 1998) to cows are substantiated by the present findings, wherein the PUFA and unsaturated fatty acid contents were elevated on feeding of bypass fat made from soya acid oil. Tyagi et al. (2009) also reported increased MUFA and unsaturated fatty acids content in milk fat in crossbred cows on supplementing dietary bypass fat The DMI ranged from 10.59 to 11.95 kg/d in control group and 11.28 to 12.72 kg/d in experimental group during six fortnights. The overall DMI recorded was 11.38 kg and 12.05 kg/d (Table 4) in control and experimental groups, respectively. The overall DMI/100 kg BW was 2.34 and 2.29 kg/d in control and experimental group, respectively. The RESULTS AND DISCUSSION The total fat content (Table 1) in the supplemented rumen protected fat was 85.35% and the protection level from rumen hydrolysis (saponifiable portion) was 58.5%. The predominant fatty acids in protected fat supplement (Table 2) were palmitic, stearic, elaidic, oleic, linoleic and linolenic acids. Average daily milk production (Table 3) ranged from 7.08 to 10.43 kg/d in control group and 6.92 to 11.17 kg/d in experimental group in different fortnights. Average milk was 9.49 kg/d in control group and it was 12.43% higher (P<0.05) in experimental group (10.67 kg/d) than that of control group. Fortnightly average 4% FCM yield (Table 3) ranged from 8.35 to 12.71 in control group and 8.08 to 14.24 kg/d in Table 1. Chemical composition of feed ingredients offered (% DM basis) Particulars Green fodder Wheat straw Concentrate Ca salts of soya acid oil DM OM CP EE CF NFE NDF ADF Total Ash 22.00 89.00 7.77 1.72 35.75 43.97 70.72 30.72 10.79 92.00 89.82 3.05 1.26 38.87 46.64 77.81 35.55 10.18 92.00 90.99 20.18 3.38 7.99 59.46 38.12 25.14 9.01 96.64 355 85.35 14.58 83 356 THAKUR AND SHELKE [Indian Journal of Animal Sciences 80 (4) Table 2. Fatty acid profile (% of total fatty acids) of feed ingredients and milk Fatty acids (% of total fatty acids) Caprylic acid (C8: 0) Capric acid (C10: 0) Lauric acid (C12: 0) Myristic acid (C14: 0) Myristoleic acid (C14: 1) Palmitic acid (C16: 0) Palmitoleic acid (C16: 1) Margaric acid (C17: 0) Stearic acid (C18: 0) Elaidic acid (C18: 1t9) Oleic acid (C18: 1c9) Linoleic acid (C18: 2) Linolenic acid (C18: 3) Arachidic acid (C20: 0) Total Total unsaturated Total saturated Total LCFA Total MUFA Feeds Milk Green fodder Wheat straw Concentrate feed 0.51 0.78 3.4 2 0.48 20.77 0.63 ND 3.32 2.45 4.31 16.25 40.59 0.69 95.67 ND ND 7.03 1.01 ND 20.25 ND 4.32 42.00 ND 13.62 ND ND 5.63 93.86 0.32 0.55 2.48 1.41 ND 12.83 1.48 ND 34.83 2.65 20.93 2.73 16.26 0.33 96.48 results of the present study indicated that there was no adverse effect of bypass fat supplementation on DMI of lactating buffaloes. Similar observations were made by Salfer et al. (1995) and Elliot et al. (1995) on feeding Ca salts of rapeseed fatty acids at the rate of 500 g/d in lactating cows, Garg et al. (2002) on supplementing bypass nutrients (fats encapsulated in a formaldehyde treated protein) @ 1000 g/d to lactating cows and buffaloes. There was no difference in the CP intake between two groups (Table 4). Average TDNI was 9.13 kg/d in control group and it was higher (P<0.05) by 8.43% in experimental group (9.90 kg/d) over that of control group (Table 4). Average TDN intake/100 kg BW was 2.01 kg/d in experimental group which was higher (P<0.05) by 5.78% over that of control group (1.90 kg/d). In the present Milk yield (kg/d) FCM yield (kg/d) Milk composition (%) Fat Total solid (TS) Solids not fat (SNF) Protein Control group Treatment group 9.49a±0.27 11.86a±0.79 10.68b±0.16 13.45b±0.63 5.94a±0.07 15.26±0.04 9.32±0.061 5.59±0.048 6.16b±0.21 15.40±0.07 9.35±0.175 3.72±0.075 0.51 0.42 0.78 0.27 0.54 8.96 0.78 ND 3.81 1.45 21.31 51.57 5.90 0.56 96.86 Control group Experimental group 1.06 3.04 3.42 11.85 0.85 28.65 1.47 0.51 14.65 2.30 25.21 1.41 0.79 0.41 95.62 32.03 63.59 75.40 29.83 0.92 2.46 3.08 8.50 1.18 25.96 1.62 0.24 12.93 2.77 28.06 4.45 0.92 0.51 93.6 39.00 54.60 76.46 32.63 Table 4. Nutrient intake, digestibility coefficient (%) and efficiency of utilization of nutrients in lactating buffaloes fed experimental diets Attributes Control group DMI (kg/day) 11.38±0.65 CPI (kg/day) 1.59±0.09 TDNI (kg/day d) 9.13a±0.16 DMI (kg/100 kg BW) 2.34±0.051 CPI (kg/100 kg BW) 0.32 ±.007 TDNI (kg/100 kg BW) 1.90a±0.29 Digestibility of nutrients DM 65.83±1.34 CP 60.80±2.69 EE 70.68a±2.29 CF 52.87±1.79 NFE 59.07±3.45 NDF 50.07±1.65 ADF 42.40±2.97 Efficiency of utilization of nutrients DMI (kg/kg) milk yield 1.22±0.019 DMI (kg/kg) FCM yield 0.97±0.017 CPI (g/kg) milk yield 170.09±3.59 CPI (g/kg) FCM yield 137.81± 4.35 TDNI (g/kg) milk yield 960.56±35.56 TDNI (g/kg) FCM yield 766.54±22.34 Table 3. Milk yield and its composition in lactating buffaloes fed experimental diets Particular Bypass fat ab Treatment group 12.05±0.69 1.54±0.08 9.90b±0.22 2.29±0.049 0.30 ±.005 2.01b ±0.21 66.59±1.80 61.56±1.96 82.05b±1.32 53.60±2.58 60.87±2.47 51.32±2.62 43.49±3.20 1.15±0.038 0.90±0.036 157.85±6.09 126.98±8.93 926.65± 15.87 737.86±19.55 - Means having different superscripts in the same row differ significantly (P<0.05). abMeans having different superscripts in the same row differ significantly (P<0.05). 84 April 2010] BYPASS FAT FEEDING TO LACTATING MURRAH BUFFALOES experiment, since the DMI was not adversely affected by supplementing protected fat, it led to significantly higher TDNI in experimental group. There was no effect on the digestibility of DM, CP, CF, NFE, ADF and NDF (Table 4). However, EE digestibility was higher (P<0.05) in experimental group than the control group. In the present study, higher EE digestibility in experimental group supplemented with Ca salts of soya acid oil fatty acids is in agreement with the findings of Chouinard et al. (1998) and Tyagi et al. (2009). Calcium soaps of fatty acids remain in intact form in the rumen and unlike direct fat supplementation, do not lead to any toxic effect on rumen microflora thus not altering the fibre digestion (Schauff and Clark 1989, Harrison et al. 1995). In the present investigation, digestibility of fibre fractions was not adversely affected on supplementation of Ca salts of soya acid oil fatty acids. There was no effect on the efficiency of conversion of DMI, CPI and TDNI to milk and 4% FCM in the 2 groups (Table 4). Our results indicated that bypass fat prepared from soybean acid oil supplemented at 4% of DMI, increased milk production, FCM yield and proportion of unsaturated FA and LCFA in milk fat of Murrah buffaloes. 357 2002. Effect of feeding rumen protected nutrients on milk production in cows and buffaloes. Indian Journal of Dairy Science 56 (4): 218–22. Goering H K and Van Soest P J. 1970. Forage Fiber Analysis. Agriculture Handbook No. 379, US Department of Agriculture, Washington DC. Gulati S K and Ashes J. 2000. Methods specifically developed or modified for the feed technology project. Practical Manual. NDDB, Anand. Harrison J H, Kincaid R L, McNamara J P, Waltner S, Loney K A, Riley R E and Cronrath J D. 1995. Effect of whole cottonseed and calcium salts of long chain fatty acids on performance of lactating dairy cows. Journal of Dairy Science 78: 181–93. Jenkins T C and Palmquist D L. 1984. Effect of fatty acids of calcium soaps on rumen and total nutrient digestibility of dairy rations. Journal of Dairy Science 67: 978–86. Kearl C Leonard. 1982. Nutrient requirement of ruminants in developing countries. International Feedstuff. Utah Agriculture Experiment Station, Utah State University, Logan USA. McNamara S, Butler T, Ryan D P, Mee J F, Dillon P, O’mara F P, Butler S T, Anglesey D, Rath M and Murphy J J. 2003. Effect of offering rumen-protected fat supplements on fertility and performance in spring-calving Holstein-Friesian cows. Animal Reproduction Science 79: 45–46. Mishra S, Thakur S S and Rakesh K. 2004. Milk production and composition in crossbred cows fed calcium salts of mustard oil fatty acids. Indian Journal of Animal Nutrition 21(1): 22–25. Salfer J A, Linn J G, Otterby D E, Hansen W P and Johnson D G. 1995. Early lactation responses of Holstein cows fed a rumeninert fat prepartum, postpartum, or both. Journal of Dairy Science 78: 368–77. Sampelayo M R S, Martín Alonso J J, Pérez L, Gil Extremera F and Boza J. 2004. Dietary supplements for lactating goats by polyunsaturated fatty acid-rich protected fat. effects after supplement withdrawal. Journal of Dairy Science 87: 1796– 1802. Schauff D J and Clark J H. 1989. Effects of prilled fatty acids and calcium salts of fatty acids on fermentation nutrient digestibilities, milk production and milk composition. Journal of Dairy Science 72: 917–27. Sklan D, Kaim M, Moallem U and Folman Y. 1994. Effect of dietary calcium soaps on milk yield, body weight, reproductive hormones, and fertility in first parity and older cows. Journal of Dairy Science 77: 1652–60. Snedecor G W and Cochran W G. 1986. Statistical Methods. 7th edn. Oxford and IBH publishing Co. Pvt. Ltd., New Delhi. Tyagi N, Thakur S S and Shelke S K. 2009. Effect of feeding bypass fat supplement on milk yield, composition and nutrient utilization in crossbred cows. Indian Journal of Animal Nutrition 26: 1–8. REFERENCES AOAC. 2005. Official Methods of Analysis. Association of Official Analytical Chemists. Washington D C. Chouinard P Y, Girad V and Brisson G J. 1998. Fatty acids profile and physical properties of milk fat from cows fed calcium salts of fatty acids with varying unsaturation. Journal of Dairy Science 81: 471–481. Deuel Jr H J. 1951. The Lipids.Vol.1. Chemistry. Interscience Publication Inc, New York. Elliot J P, Drackley J K, Fahey Jr G C and Shanks R D. 1995. Utilization of supplemental fat by dairy cows fed diets varying in content of non-structural carbohydrates. Journal of Dairy Science 78: 1512–25. Fahey J, Mee J F, Murphy J J and Callaghan D O. 2002. Effects of calcium salts of fatty acids and calcium salt of methionine hydroxyl analogue on plasma prostaglandin F2 metabolite and milk fatty acid profile in late lactation Holstein-Friesian cows. Theriogenology 58: 1471–82. Garg M R and Mehta A K. 1998. Effect of feeding bypass fat on feed intake, milk production and body condition of Holstein Friesian cows. Indian Journal of Animal Nutrition 15: 242–45. Garg M R, Sheresia P L, Bhanderi B M, Gulati S K and Scott T W. 85 Indian Journal of Animal Sciences 80 (4): 358–361, April 2010 On-farm evaluation of urea molasses multinutrient blocks enriched with minerals in goats R SINGH1, S KUMAR2 and R BHARDWAJ3 Sher-e-Kashmir University of Agricultural Sciences and Technology, R S Pura, Jammu, Jammu and Kashmir 181 102 India Received: 6 May 2009; Accepted: 25 November 2009 ABSTRACT To assess the effects of supplementary feeding of urea-molasses multinutrient block (UMMB) enriched with minerals on productive traits of goats, a study was undertaken in the Kandi belt of Shivalik Hills of North West Himalayas in Jammu region. Beetal cross goats (25), 1–6 year-old were selected from a private farm of which 20 goats were allowed to lick a UMMB @ 200 g daily for 30 days during June-July and 5 goats were kept as control. Blood samples were analyzed for hemato-biochemical parameters, macro and trace elements and hormonal status (T3 and T4) at the beginning and after completion of trial. Glucose, phosphorus, copper and manganese level increased significantly from 44.62 mg/ dl, 4.16 mg/dl, 18.09 μmol/l and 0.56 μmol/l to 68.50 mg/dl, 5.92 mg/dl, 31.31 μmol/l and 1.40 μmol/l, respectively. No significant effect of UMMB feeding on Hb, PCV, TPP, albumin, globulin, ALP, Ca, Mg, iron, Zn, plasma inorganic iodine (PII) and T3 and T4 was observed. An average increase of 15.30% in body weight, 30% in milk yield and 11.50% in milk fat following UMMB supplementation was observed. It was concluded that UMMB being a good source of energy, protein and minerals enhanced body weight and milk yield in goats. Key words: Goats, Hemato-biochemical, Hormones, Milk yield, Mineral, Urea molasses multinutrient blocks apparently improved the live weight gain in lambs. Thus, the successful treatment and control of mineral deficiencies lies in effective and practical methods of supplementation. The present study was undertaken to evaluate the effect of UMMB containing area specific mineral mixture as a supplementary feeding on the general health condition, milk yield and reproductive performance of goats. Goats in the Kandi areas of subtropical and intermediate zones of Shivalik hills are dependent mainly on grazing poor quality feed resources like grasses, tree leaves, which are characteristically low in fermentable nitrogen, mineral, and readily available carbohydrate. These deficiencies result in poor animal growth rate, poor reproduction and unthrifty condition. Developing alternate feeding strategies for ruminant production based on agro-industrial wastes is, therefore, of prime importance. A UMMB prepared from locally available agro-industrial by-products has been adoptable feed supplement, which improves nutritional status of animals (Kang et al. 2007). Baseline survey to identify the commonly prevalent deficiencies in the goats was carried earlier and on that basis plasma analysis area specific mineral mixture was framed. Rodriguez et al. (1985) observed that feeding of merely the deficient minerals does not improve the general health status, production and reproduction of animals in these areas. However, limited amounts of either a good quality green forage or rumen undegradable protein MATERIALS AND METHODS Beetal cross goats (25), 1 to 6-year-old, reared through grazing in the Parmandal block of Jammu district in Kandi belt of Shivalik hills by a farmer were treated with anthelminthic (fenbendazole) prior to trial. Fifteen goats were in lactation. Trial was conducted from June-July. Goats were divided into 2 groups—group 1: 20 goats supplemented with UMMB, group 2: 5 animals were kept as control. The UMMB were prepared by cold method by mixing molasses (35%), urea (10%), deoiled rice bran (10%), oiled rice bran (10%), groundnut meal (10%), cement (10%), area specific mineral mixture 14% as per Singh (2009);it has DCP- 70%, MgSO4– 29%, CuSO4–0.5%, MnSO4 –0.5%, K. iodate–0.09%, and common salt (1%). Close observations were made on selected goats on changes, if any, in feed intake, milk yield and milk fat production and body weight. The trial lasted for 30 days Present address: 1 Associate Professor, 2 JRF, 3 Assistant Professor, Division of Veterinary Clinical Medicine and Jurisprudence, Faculty of Veterinary Sciences and Animal Husbandry. 86 April 2010] EVALUATION OF UREA MOLASSES MULTI NUTRIENT BLOCKS IN GOATS and all animals were weighed at the beginning and at the end of the trial. Sample collection and analysis: Blood samples of goats were collected on day 0 and 30th. Haemoglobin and PCV were analysed by standard methods. Plasma concentration of glucose, total proteins, albumin, alkaline phosphatase and urea nitrogen were estimated using autopak kits. Levels of triiodothyronine (T3) and thyroxine (T4) were assayed by radioimmunoassay technique using RIA kits procured from BARC, India. Plasma sample (3 ml) was analysed for mineral analysis by digesting in distilled concentrated nitric acid AR (15 ml) and perchloric acid, Merck (3 ml) followed by one cycle of hydrogen peroxide AR (2.0 ml of 30%). Digestates (approximately 1–2 ml) were diluted to 15 ml with double glass distilled water. The concentrations of micro-elements, viz. Cu, Fe, Zn, Mn and Co were measured by atomic absorption spectrophotometer. Calcium was estimated by cresolphthalein complex one method using autopak kit, and Mg was also analyzed using kit. Inorganic fraction of plasma phosphorus was determined by the method of Taussky and Shorr (1953). Plasma inorganic iodine (PII) was determined as per Aumont and Tressol (1987). Statistical comparison of data was done as per Snedecor and Cochran (1989). 359 the improvement in productive performance of the animals on treatment was encouraging. The animals offered UMMB had better body condition and looked healthier than did control group. Liu (1995) reported significantly higher live weight gains (95 g/d) in goats with access to urea-mineral lick block without molasses (ULB) than in those without block (73 g/d). Hadjipanayiotou et al. (1993) observed that effects of urea-containing blocks on live weight gains in cattle and sheep were more pronounced than the effects on feed intake. Haematological parameters: The mean values of hematological indices Hb and PCV in goats before the start of trial were 6.37 g/dl and 22.95% which were below the normal range of 8–12 g/dl and 22–38 per cent, respectively (Kahn 2005). No significant (P<0.05) effect of UMMB supplementation on the level of hemoglobin and packed cell volume was observed in goats after 30 days (Table 1). At the beginning of trial, 5% (1/20) goats had normal Hb (>8.0 g/ dl) and 35% goats had very low (<6.0 g/dl) Hb concentration. After 30 days of UMMB supplementation all the animals were anemic, of which 45% (9/20) had Hb very low (<6.0 g/ dl). On PCV basis also the prevalence of anemia (PCV ≤ 22%) was 25% in goats at the beginning as well as at the end of trial. Thus, present study indicates that supplementation of nutrients through UMMB was either not adequate or the duration of trial was short to improve haematological parameters. Biochemical changes in blood: The average values of total plasma protein, albumin and globulin varied nonsignificantly (P<0.05) from 6.60 g/dl, 2.87 g/dl and 3.80 g/dl) at the beginning of trial to 6.76 g/dl, 2.69 g/dl and 4.18 g/dl, respectively, after 30 days of supplementation (Table 1). Moreover, the average values of total plasma protein, albumin and globulin were within normal range of 6.1– 7.5 g/dl, 2.4– 4.6 g/dl and 2.7– 4.4 g/dl, respectively, quoted by Kahn (2005). Considering the critical level of 6.1 g/dl and 2.4 g/dl for plasma protein and albumin, hypoproteinemia and hypoalbuminemia were observed in 15 and 5% goats, respectively, at 0 day as well after 30 days of UMMB supplementation. Findings of the present study corroborate RESULTS AND DISCUSSION The UMMB was palatable to goats and to avoid an excessive intake, goats were offered UMMB @ 200 g/day individually after grazing in the evening. Observations on all closely monitored goats revealed an average increase of 15.30% (range 7–30%) in body weight, 30% in milk yield and 11.50% in milk fat following UMMB supplementation. The average body weight of goats of groups 1 and 2 at the beginning of trial was 30.15 kg and 27.6 kg which increased to 34.76 kg and 28.1 kg after 30 days of UMMB supplementation, respectively. According to the owner of flock marked improvement in the feed intake was observed however, it could not be estimated as the animals were grazed and trial was “on-farm”. The average milk yield was 0.50 kg and milk fat percentage was 3.12%, which, increased to 0.65 kg and 3.48%, respectively, at the end of the trial. Overall, Table 1. Effect of UMMB supplementation on haemato-biochemical parameters in goats Animals Sampling day Hb (g/dl) PCV (%) Glucose (mg/dl) TPP (g/dl) Albumin (g/dl) Globulin (g/dl) ALP (I.U.l-l) BUN (mg/dl) ALT (I.U.l-l) Group- 1 0 6.37 ± 0.22 5.80 ± 0.19 6.22 0.28 5.36 ± 0.49 22.95 ± 0.58 22.40 ± 0.68 22.20 ± 0.24 19.0 ± 2.30 44.62a ± 2.81 68.50b ± 2.84 56.43ab ± 3.72 66.9ab ± 7.46 6.60 ± 0.16 6.76 ± 0.21 6.31 ± 041 6.44 ± 0.32 2.87abc ± 0.05 2.69ab ± 0.06 2.97 ± ac 0.03 2.62 ± abc0.27 3.80 ± 0.15 4.18 ± 0.25 3.58 ± 0.07 3.82 ± 0.48 98.43a ± 18.54 154.96a ± 23.97 72.60ab ± 23.92 40.71b ± 8.79 14.69a ± 0.57 11.20b ± 0.74 15.09ab ± 1.72 10.80b ± 1.26 11.34a ± 0.64 10.2ab ± 0.93 7.20b ± 1.35 7.66ab ± 1.66 30th Group–2 0 30th •Means marked with different superscripts a, b differ significantly (P<0.05) in a column. 87 360 SINGH ET AL. magnesium, iron, zinc and plasma inorganic iodine showed nonsignificant (P<0.05) variation (Table 3). The average values of calcium and magnesium were within the normal range of 9.0 – 11.6 mg/dl and 2.10 – 2.90 mg/dl quoted by Kahn (2005). Considering the critical level of 9.0 mg/dl, 45% goats were having hypocalcaemia whereas, after 30 days of supplementation 65% goats were having hypocalcaemia. The average value of inorganic phosphorus before the start of trial was 4.16 mg/dl which was within the normal range, i.e. 3.7 – 9.7 mg/dl (Kahn 2005). However, at the end of trial Table 2. Effect of UMMB supplementation on hormonal status in goats Animals Sampling day T3 T4 Group 1 0 30th 0 30th 0.67±0.02a 0.61± 0.06a 0.63±0.04a 0.45± 0.01b 55.42±3.05 55.57± 4.58 56.30±4.16 55.0± 7.63 Group 2 [Indian Journal of Animal Sciences 80 (4) •Means marked with different superscripts a, b differ significantly (P<0.05) in a column. Table 3. Effect of UMMB supplementation on mineral status in goats Animals Sampling day Group 1 Group 2 0 30th 0 30th Ca (mg/dl) Pi (mg/dl) 8.46 ± 0.36 4.16 ± 0.37a 8.89 ± 0.40 5.92 ± 0.37b 8.66 ± 0.42 4.22 ± 0.37a 8.15 ± 0.76 4.73 ± 0.78 Mg (mg/dl) Cu (μmol/l) Fe (μmol/l) Zn (μmol/l) Mn (μmol/l) PII (ng/ml) 2.96 ± 0.08 2.85 ± 0.13 2.82 ± 0.06 2.67 ± 0.09 18.09 ± 1.81a 31.31 ± 2.02b 21.12 ± 2.14ac 30.94 ± 4.29bc 38.99 ± 3.72a 34.64 ± 2.66a 44.27 ± 2.46a 22.08 ± 2.44b 30.0 ± 6.62abc 38.30 ± 5.33abc 26.43 ± 4.82abc 13.50 ± 2.43ac 0.56 ± 0.13a 1.40 ± 0.16b 0.68 ± 0.17a 1.07 ± 0.16ab 72.44 ± 10.42 92.39 ± 10.71 78. 64 ± 12.68 98. 10 ± 20.56 •Means marked with different superscript a, b, c differ significantly (P<0.05) in a column. with Brar and Nanda (2008) who also reported nonsignificant (P<0.05) effect of UMMB supplementation on total plasma protein and albumin level in anoestrus buffaloes after 4 weeks. As the rumenally degradable fraction of protein increases in diet, blood urea nitrogen level increases. However, in present study average value of urea nitrogen showed significant (P<0.05) decrease from 14.69 mg/dl at 0 day to 11.20 mg/dl at 30th day. Contrary to present finding Qreshi et al. (2002) have reported that excessive levels of crude protein in the diet elevated BUN levels. The glucose level of goats increased significantly (P<0.05) from 44.62 mg/dl to 68.50 after 30th day of supplementation of UMMB. The average value at the beginning of trial was below the normal range of 48–76 mg/dl quoted by Kahn (2005). Considering the critical level of 48 mg/dl hypoglycemia was observed in 61.53 and 5.88% goats at 0 and 30th day of trial, respectively. Nonsignificant effect on alkaline phosphatase and alanine aminotransferase levels showed that UMMB supplementation had no harmful effect on the liver and bones. Triiodothyronine (T3) and thyroxin (T4): The average value of T3 in plasma samples of goats at the beginning of trial was 0.67 ng/ml, which showed nonsignificant decrease (Table 2). Likewise, the mean value of T4 in plasma samples of goats at the beginning and on 30th day were 55.42 ng/ml and 55.57ng/ml, respectively. Thus, it can be concluded as per the suggestion of Underwood (1981) that serum thyroxine and triiodo-thyronine were poorly related to thyroidal activity as numerous factors (season, ambient temperature and intestinal parasitism) modified the activity of thyroid gland (Pritchard et al. 1974, Andrewartha et al. 1980). Mineral changes in blood: The average values of calcium, the goats showed significant (P<0.05) increase in inorganic phosphorus level to 5.92 mg/dl (Table 3). Considering the critical level of 3.7 mg/dl, 35% goats were having hypophosphataemia whereas, after 30 days of supplementation 5% animals were having hypophosphataemia. The average values of iron and zinc observed in present study were higher than the normal range of 17.9–35.8 μmol/ l and 12.24–18.36μmol/l, respectively, quoted by Radostits et al. (2000) in sheep. The average values of copper in postsupplementation group were above the normal range, i.e. 11.0–20.46μmol/l (Radostits et al. 2000) for sheep. Manganese level of the plasma samples from group 1 goats increased significantly (P<0.05) from 0.56 to 1.40μmol/l. Compared with present finding, Underwood and Suttle (1999) had cited considerably lower normal range of Mn as 0.03276 to 0.0364 μmol/l in sheep. Thus, present study indicates that supplementation of minerals through UMMB improved phosphorus and copper plasma levels; however calcium and iodine levels did not showed much improvement which could be due to increased demand for higher growth rate and the milk production observed following UMMB supplementation or it could be due to short duration of the supplementation. ACKNOWLEDGEMENT Authors are thankful to Department of Science and Technology, Government of Science and Technology, India, for funding the research project under which the work was carried out. REFERENCES Andrewartha K A, Caple I W, Davies W D and Mc Donald J W. 88 April 2010] EVALUATION OF UREA MOLASSES MULTI NUTRIENT BLOCKS IN GOATS 361 responses of sheep to infection with Trichostrongylus colubriformis. Research in Veterinary Science 17: 182–87. Qreshi M S, Habib G, Samad H A, Siddiqui M M, Ahmad N, Syed M. 2002. Reproduction-Nutrition relationship in dairy buffaloes, 1. Effect of intake of Protein, energy and blood metabolites levels. Asian-Australasian Journal of Animal Sciences 15: 330– 39. Radostits O M, Gay C C, Blood D C and Hinchcliff K W. 2000. Veterinary Medicine.9th edn. W B Saunders Harcourt Publishers Ltd. Rodriguez A, Riley J and Thorpe W. 1985. Animal performance and physiological disturbances in sheep fed diets based on ensiled sisal pulp (Agave fourcroydes). 1. The effect of supplementation with protein, forage and minerals. Tropical Animal Production 10 (1): 23–31. Singh, R. 2009. Project report Mineral Imbalances in Livestock with reference to soil-plant-animal relationship. Department of Science and Technology, Government of India, New Delhi. Snedecor G W and Cochran W G. 1989. Statistical Methods. 8th edn. Iowa State University Press, London. Taussky H H and Shorr E. 1953. A micro-calorimetric method for the determination of inorganic phosphorus. Journal of Biological Chemistry 202: 675–85. Underwood E J. 1981. The Mineral Nutrition of Livestock. Commonwealth Agricultural Bureaux, Farnham Royal, Slough, England. Underwood E J and Suttle N F. 1999. The Mineral Nutrition of Livestock. pp. 397–420. CAB International, Wallingford, Oxon OX10 8DE UK. 1980. Observations on serum thyroxine concentrations in lambs and ewes to assess iodine nutrition. Australian Veterinary Journal 56: 18–21. Aumont G and Tressol J C. 1987. Rapid method for the direct determination of inorganic iodine in plasma using ion-exchange chromatography and the Sandell and Kolthoff reaction. Analyst 112: 875–77. Brar P S and Nanda A S. 2008. Improving performance of anoestrus buffaloes through supplementary feeding of urea molasses multi-nutrient block. Indian Journal of Animal Sciences 78: 606– 08. Hadjipanayiotou M, Verhaeghe L, Kronfoleh A R, Labban L M, Amin M, Al-Wadi M Badran A, Dawa K, Shurbaji A, Houssein M, Malki G, Naigm T, Merawi A R and Harres A K. 1993. Urea blocks. 2. Performance of cattle and sheep offered urea blocks in Syria. Livestock Research for Rural Development 5 (3): 16– 23. Kahn C M. 2005. The Merck Veterinary Manual. 9th edn. Merck & Co., Inc. Whitehouse Station, N.J., USA. Kang R S, Nanda, A S, Brar P S, Honparkhe M, Gandotra V K and Jindal R. 2007. Plasma biochemical alterations in relation to induction of oestrus through supplementary feeding and hormonal treatment in summer anoestrus buffaloes. Indian Journal of Animal Sciences 77: 359–62. Liu, J X, Wu Y M, Dai X M, Jun Y, Zhou Y Y and Chen Y J. 1995. The effects of urea-mineral lick blocks on the liveweight gain of local yellow cattle and goats in grazing conditions. Livestock Research and Rural Development 7(2): 9–13. Pritchard R K, Hennessy D R and Griffiths D A. 1974. Endocrine 89 Indian Journal of Animal Sciences 80 (4): 362–365, April 2010 Carcass and meat quality characteristics of designated indigenous sheep breeds of India A R SEN1 and S A KARIM2 Central Sheep and Wool Research Institute, Avikanagar, Rajasthan 304501 India Received: 20 May 2009; Accepted: 23 October 2009 ABSTRACT Carcass and meat quality traits of 18 lambs of indigenous breeds, viz. Malpura, Garole and Malpura × Garole were analyzed. The pre-slaughter weight was significantly lower in Garole as compared to Malpura and their crosses. The small size Garole had significantly higher dressing yield as compared to large size Malpura. The muscular development as indicated by loin eye area was higher in the Malpura and crossbred lambs as compared to Garole. Differences among the breeds for the proportion of wholesale cuts as a percentage in half carcass tended to be small and mostly nonsignificant. In all the breeds, the leg cut had highest lean content. In the neck region, a significant higher bone content was observed in the large size Malpura as compared to smaller size Garole. Cooking loss percentage was significantly lower in Garole sheep as compared to Malpura and their crosses. The water holding capacity (WHC) is significantly more in Garole rams. Tenderness and juiciness was rated better in Garole and their crossbreds than Malpura. In organoleptic study overall score was ranked in the order of Malpura × Garole>Garole>Malpura. The current study showed that carcass and meat quality of Garole was similar to Malpura or their crossbreds. It was also evident that the overall meat quality was better in Garole compared to Malpura rams. Key words: Carcass, Lambs, Meat quality, mutton (Prasad et al. 1981) while comparative study on carcass and meat quality of different indigenous sheep breeds and their crosses with variable adult weight was limited. This study was designed mainly to investigate the efficiency of Garole sheep as a meat producing breed and to characterize the effect of breed on the carcass and chemical composition as well as sensory characteristics of meat obtained from designated indigenous sheep breed. The evaluation of carcass traits of indigenous sheep is essential both for developing appropriate breeding strategy as well as for recommending breed of sheep for meat production. Malpura is a large size dominant breed of sheep in Rajasthan while Garole sheep is indigenous to Sunderban area of West Bengal, India, and is known for higher prolificacy. The two breeds differ markedly in mature body size. Many studies have analyzed the effect of breed and slaughter weight on the physicochemical characteristics of lamb meat (Perez et al. 2002, Macit et al. 2002, Hoffman et al. 2003, Martinez-Cerezo et al. 2005, Muthukumar et al. 2006). Amount and site of fat in the carcass influences its quality and such relationship between carcass weight and fat content was recognized by Okeudo and Moss (2005). Meat tenderness and flavour appear to be the most important sensory characteristics that determine meat quality (Shahidi et al. 1986, Sanudo et al. 1996). Some reports have appeared on carcass study of Indian indigenous breed Malpura (Basuthakur et al. 1980), Avikalin MATERIALS AND METHODS The experiment was conducted at the Central Sheep and Wool Research Institute, Avikanagar, Rajasthan, India, located at 75°28/E latitude and 26°17/N longitude and 320 m above mean sea level. The climate of the location is classified as hot and semi-arid. Male lambs (6) from each breed (Malpura, Garole and Malpura×Garole) were used in this study. Crossbreeding of Malpura ewes with Garole rams was attempted with the view to infuse the genes responsible for prolificacy into crossbreds. The lambs were let out in the pasture at 0800 hours and grazed for 8 h under supervision of a grazer followed by herding in a side open animal shed till next day morning. The grazing area was dominated by Cenchrus ciliaris (70%) with seasonal shrubs and forbs constituting the other vegetation. Besides Present address: 1 Principal Scientist, NRC on Meat, Chengicherla, P.B.No.19, Uppal P.O. Hyderabad, Andhra Pradesh 500 039.(e mail: [email protected]). 2Director. 90 April 2010] CARCASS AND MEAT QUALITY CHARACTERISTICS OF INDIGENOUS SHEEP grazing, all the animals were offered 250 g concentrate/head/ day. All the animals were slaughtered at 10 months of age. The lambs were fasted over night with free access to water and slaughtered in the experimental abattoir by Halal method. After slaughter, the head was removed at the atlanto-occipital joint and fore and hind feet removed at the carpal and tarsal joints, respectively. The animals were partially skinned lying on their back on the floor. Then the animals were suspended by the hind legs for further skinning. Carcass and non-carcass components were separated immediately after slaughter and recorded and were chilled at 4°C. Lungs, trachea and heart were weighed as one piece and designated as the pluck. Non-carcass components included head, skin, feet, digestive tract, liver, spleen, pancreas, lungs plus trachea. Weight of digestive contents was computed as the difference between full and empty digestive tracts. The empty live weight (ELW) was computed as the difference between slaughter weight and weight of digestive content. Carcass was split along the middle and the left half was cut into leg, loin, rack, neck and shoulder and breast and foreshank as per ISI (1963) specifications. The cuts were dissected into lean, bone and fat (subcutaneous and intermuscular) after overnight chilling. The per cent composition of the cuts was calculated on the basis of chilled carcass weight. Loin eye area (cm2) was recorded on the cut surface of Longissimus dorsi muscle at the interface of 12th and 13th rib on both side of the carcass. Longissimus dorsi was collected and analyzed for meat quality evaluation. Cooking loss% was determined by weight loss after cooking of meat for 1 h in water bath at 80°C (Babiker et al. 1990). Shear force value of cooked meat samples was determined using Warner Bratzler shear press and Water holding capacity (WHC) was estimated by filter paper pressing technique in screw plates (Trout 1988). The sensory evaluation for color, odour, tenderness, juiciness and overall palatability was performed by a semi-trained panel using 6 point scale (6 = excellent, 1= very poor). Statistical analysis of results were done using SPSS statistical software 12.0 version (SPSS 2003). Data were analysed using one way analysis of variance (ANOVA) to test for the effect of breed. Significant group differences were compared by Duncan’s Multiple Range Test (Duncan 1955). 363 Table 1. Carcass traits of Malpura, Garole and their crosses Breed Traits Malpura Garole Pre-slaughter 21.18a±0.42 9.10b±0.90 weight** (kg) Forequarter 4.38a±0.12 1.99b±0.21 weight** (kg) Hindquarter 3.71a±0.14 1.62b±0.22 weight** (kg) Dressing% (LW) 37.71b±0.60 39.59a±081 Dressing% (ELW) 47.44bc±0.59 46.31b±1.00 Blood% 4.47±0.24 4.19±0.69 Skin% 9.69±0.43 7.65±1.55 Alimentary canal% 30.07 a±1.00 25.31b±0.30 Ingesta% 20.49a±1.05 14.50c±0.10 Kidney fat% 0.25 b±0.02 0.29a±0.08 0.32a±0.08 Caul fat% 0.31a±0.04 Head & cannon% 9.39 b±0.14 12.00a±0.20 Pluck% 2.58±0.17 2.84±0.33 Liver% 1.90±0.08 2.37±0.37 Spleen% 0.32±0.03 0.45±0.04 Loin eye area (cm2) 7.90b±0.49 4.98c±0.47 Malpura × Garole 20.68a±0.53 4.62a±0.14 3.94a±0.09 41.41a±0.78 50.24a±0.65 3.98±0.15 8.55±0.45 26.70b±0.64 17.58b±0.68 010c±0.02 0.18b±0.04 9.52 b±0.31 2.55±0.09 1.80±0.08 0.35±0.01 9.22a±0.27 ** P<0.01, means with different superscripts in a row differ significantly (P<0.05); n=6 for each breed. of non-carcass component mainly alimentary canal and ingesta content in Garole rams contributed to the higher dressing out percentage of the breed. The carcass studies showed high empty body weight percentages and commercial dressing percentages and these characteristics varied only for slaughter weight (Perez et al. 2002). The skin yield of Malpura is higher than that of Garole and their crosses. The smaller sheep Garole had proportionally larger head and cannons than Malpura. In general non- carcass fat depot was higher in Garole than Malpura and crossbred, which would indicate that Garole breed can sustain larger period of feed deprivation (Farid 1991). As more fat deposited in the kidney of Garole rams, here we can infer one thing that it is the hardiest among the breeds in the study and is able to survive the long periods of feed shortage during summer. High body fat provides the sheep with the ability to cope with long periods of energy shortage, while low subcutaneous fat cover helps the animals to easily regulate their body temperature in hot environments. Further crossbred lambs (Malpura × Garole) had lower visceral fat (kidney and caul). From the strictly applied viewpoint, any attempt to reduce body fat through crossbreeding or selection may have a negative effect on adaptability of these native breeds to its natural habitat and thus should be accompanied by corresponding improvements in its nutrition and management. The muscular development as indicated by loin eye area was higher in the Malpura and crossbred lambs as compared to Garole. RESULTS AND DISCUSSION Carcass characteristics The pre-slaughter weight was significantly (P<0.01) lower in Garole as compared to Malpura and their crosses (Table 1). In Malpura male lambs the dressing yield was approximately 38% of the live weight while Basuthakur et al. (1980) reported higher dressing yield, which could be ascribed to variations in fasting protocol. Interestingly, the small sized Garole rams had significantly (P<0.05) higher dressing yield as compared to Malpura. The lower proportion 91 364 SEN AND KARIM fat diet and prefer lean meat. Breed-wise composition of different cuts is presented in Table 3. Loin ranked the highest among the cuts for percentage of fat. The highest fat content in the loin region was obtained in Malpura × Garole than Malupra and Garole lambs. In all the breeds, the leg cut had highest lean content. In the neck region, a significant (P<0.05) higher bone content was observed in the large sized Malpura as compared to smaller sized Garole rams, while the lean content followed the reverse order. Hogg et al. (1992) explained that the difference in fat content and consequently of bone and meat of different joints is a reflection of the pattern of fat deposition and stage of maturity of the animal. Table 2. Primal and retail cut yields of Malpura, Garole and their crosses Breed Traits Leg% Loin% Rack% Neck and shoulder% Breast and fores hank% Total separable Lean% Fat% Bone% Malpura Garole Malpura × Garole 33.0±0.43 11.29±0.41 12.97±0.37 24.59±0.29 18.15±0.31 32.66±0.35 12.21±0.41 12.83±0.21 25.56±1.77 16.99±1.46 32.32±0.31 12.18±0.35 13.0±0.52 24.22±0.92 18.23±0.69 69.91±1.08 71.39±2.11 69.12±0.10 6.55b±0.90 23.37±1.81 5.73b±0.98 23.02±.1.26 8.95a±0.60 22.08±1.15 Meat quality attributes Cooking loss% was significantly (P<0.05) lower in Garole sheep as compared to Malpura and their crosses. The water holding capacity (WHC) was significantly (P<0.05) more in Garole rams. The lower cooking loss% in Garole may be attributed to the higher water holding capacity. Meat with lower WHC, water will be expelled in higher quantity and higher cooking loss% (Vergara et al. 1999). It is interesting to note that shear force value was higher in Malpura rams compared to Garole and Malpura × Garole. Generally, shear force values that exceed 5.5 kg would be considered as objectionably tough both by a trained sensory panel and by consumers (Shackelford et al. 1991). Hence, meat from Malpura rams was mostly tougher. Warner et al. (1990) reported that samples of meat from aged wether sheep with Warner Bratzler shear force values of >6.6 are perceived by taste panel to be tough. Sensory characteristics of meat from different breeds are presented in Table 4. Colour-wise meat from Malpura × Garole was more attractive than that from Malpura and Garole. Tenderness and juiciness was rated better in Garole and their crossbreds than Malpura. In organoleptic study overall score was ranked in the order of Malpura × Garole>Garole>Malpura. The current study showed that carcass and meat quality Means with different superscripts in a row differ significantly (P<0.05); n=6 for each breed. The data on primal cuts and total separable lean, fat and bone content of different breeds is presented in Table 2. The proportions of various primal cuts observed in this study are in close agreement with the observation of Sen et al. (2000). Differences among the breed for the proportion of wholesale cuts as a percentage in half carcass tended to be small and mostly nonsignificant. Total separable carcass fat content was lower in Garole as compared to Malpura and crossbred. Accordingly the meat production from Garole sheep is economical due to the higher feed conversion efficiency. Further, modern consumers are more concerned with low Table 3. Lean, fat, bone content in different primal cuts of Malpura, Garole and their crosses Traits Leg Lean Fat Bone Loin Lean Fat Bone Rack Lean Fat Bone Neck and Lean shoulder Fat Bone Breast and Lean fores Fat hank Bone Breed Malpura Garole Malpura × Garole 75.39±2.11 3.39±0.48 20.04±2.43 74.79a±3.70 10.54b±0.67 14.99±3.29 66.53±3.26 4.11±1.20 27.64±3.73 64.60b±14.79 6.84±1.17 28.05a±4.33 61.12±3.02 11.09±2.61 25.45±2.18 73.50±1.50 4.40±0.66 19.85±0.15 66.90b±1.10 13.90b±1.89 15.90±0.10 59.42±4.41 4.63±0.37 34.90±5.10 72.09a±0.83 4.39±0.21 21.91b±1.07 63.17±4.83 5.55±2.78 27.25±1.92 75.63±0.89 5.58±1.24 18.07±2.15 61.97b±4.07 19.85a±0.90 16.19±4.86 68.32±0.55 7.96±3.52 21.24±3.20 69.49b±1.16 7.15±0.63 21.98b±0.24 62.28±4.39 10.22±2.94 26.54±1.09 [Indian Journal of Animal Sciences 80 (4) Table 4. Organoleptic characteristics of meat from Malpura, Garole and their crosses. Breed Traits Colour Odour Tenderness Juiciness Overall palatability Means with different superscripts in a row differ significantly (P<0.05); n=6 for each breed. Malpura Garole Malpura × Garole 3.57b±0.20 3.28±0.18 3.42b±0.20 3.42b±0.20 3.14b±0.14 3.14b±0.34 3.57±0.29 4.14a±0.26 4.00a±0.31 3.57b±0.20 4.28a±0.18 4.42±0.20 4.42a±0.02 4.42a±0.20 4.43a±0.20 Means with different superscripts in a row differ significantly (P<0.05); n=8 for each sensory traits. 92 April 2010] CARCASS AND MEAT QUALITY CHARACTERISTICS OF INDIGENOUS SHEEP of Garole was similar to Malpura or their crossbreds. It was also evident that the overall meat quality was better in Garole compared to Malpura rams. Therefore, Garole which is a very prolific breed, can be crossbred with Malpura to get better returns for the farmers and at the same time consumers will get better quality meat. 365 weight and ageing time effects on physicochemical characteristics of lamb meat. Meat Science 69: 325–33. Muthukumar M, Naveena B M, Babji Y and Sen A R. 2006. Effect of slaughter weight and sex on carcass composition and mutton quality of Nellore sheep. Indian Journal of Animal Sciences 76: 413–15. Okeudo N J and Moss B W. 2005. Interaction amongst carcass and meat quality characteristics of sheep. Meat Science 69: 1–8. Perez P, Maino M, Tomic G, Mardones E and Pokniak J. 2002. Carcass characteristics and meat quality of Suffolk Down suckling lambs. Small Ruminant Research 44: 233–40. Prasad V S S, Bohra S D J and Kamal K. 1981. Note on mutton production potentialities of the new cross-breed wool strains. Indian Journal of Animal Sciences 51: 118–20. Sanudo C, Santolaria M P, Maria G, Osorio M and Sierra I. 1996. Influence of carcass weight on instrumental and sensory lamb meat quality in intensive production systems. Meat Science 42: 195–02. Sen A R, Karim S A and Santra A. 2000. Carcass characteristics of finisher lambs maintained on grazing with concentrate supplementation. Indian Journal of Animal Sciences 70: 988– 90. Shackelford S D, Morgan J B, Cross H R and Savell J W. 1991. Identification of threshold levels for Warner-Bratzler shear force in beef top loin Steaks. Journal of Muscle Foods 2: 289– 96. Shahidi F, Rubin L J and D’Souza L A. 1986. Meat flavour volatiles: a review of the composition, techniques of analysis and sensory evaluation. Critical Reviews in Food Science and Nutrition 24: 141–43. SPSS 12.0. 2003. SPSS Manual, Chicago, USA, SPSS Inc. Trout G R. 1988. Techniques for measuring water binding capacity in muscle foods- A review of methodology. Meat Science 23: 235–52. Vergara H, Molina A and Gallego L. 1999. Influence of sex and slaughter weight on carcass and meat quality in light and medium weight lambs produced in intensive systems. Meat Science 52: 221–26. Warner R D, Butler K M, Bogdanovic B and Hodge R W. 1990. Meat quality of aged wethers. Proceedings of the Australian Society of Animal Production. 18 No. 4200. ACKNOWLEDGEMENTS Authors are grateful to the Director, CSWRI, Avikanagar, for providing necessary research facilities. Thanks are also due to Mr M. Nasimuddin for technical assistance. REFERENCES Babiker S A, Elkhider I A and Shafie S A. 1990. Chemical composition and quality attributes of goat meat and lamb. Meat Science 28: 273–77. Basuthakur A K, Nivsarkar A E and Singh R N. 1980. Studies on some pre and post slaughter parameters in Malpura lambs. 1. Phenotypic biometry and dressing percentage. Indian Veterinary Journal 57: 473–78. Duncan D B. 1955. Multiple range and multiple F test. Biometrics 11: 1–42. Farid A. 1991. Carcass physical and chemical composition of three fat –tailed breeds of sheep. Meat Science 29: 109–20. Hoffman L C, Muller M, Cloete S W P and Schmidt D. 2003. Comparison of six crossbred lamb types: sensory, physical and nutritional meat quality characteristics. Meat Science 65: 1265– 74. Hogg B W, Mercer G T K, Kirton A H and Duganzich D M. 1992. Carcass and meat quality attributes of commercial goats in New Zealand. Small Ruminant Research 8: 243–56. ISI. 1963. Indian standard specification for mutton and goat flesh. Fresh, chilled and frozen. IS 2536. Bureau of Indian Standard Institution, New Delhi. Macit, M., Esenbuga, N., Karaoglu, M. 2002. Growth performance and carcass characteristics of Awassi, Morkaraman and Tushin lambs grazed on pasture and supported with concentrate. Small Ruminant Research 44: 241–46. Martinez-Cerezo S, Sanudo C, Panea B, Medel I, Delfa K, Sierra I, Beltran J A, Cepero R and Olleta J L. 2005. Breed, slaughter 93 Indian Journal of Animal Sciences 80 (4): 366–369, April 2010 Carcass and meat characteristics of Soviet Chinchilla rabbits as influenced by age A R SEN1 and S A KARIM2 Central Sheep and Wool Research Institute, Avikanagar, Rajasthan 304 501 India Received: 20 March 2009; Accepted: 25 November 2010 ABSTRACT Carcass and meat characteristics of Soviet Chinchilla rabbits at an average age of 12(G1), 16 (G2), 20 (G3) and 24(G4) weeks was evaluated in the slaughter study. The pre-slaughter weight (kg) was 1.71 in G1 whereas it was higher in G2, G3 and G4. The dressing yield% ranged from 52.8 to 57.1 and was higher in G3 and G4 than G1 and G2. The standard cuts, viz. hind quarter, loin and forequarter expressed as percentage of carcass weight ranged from 38.6 to 41.0, 19.3 to 20.6 and 39.7 to 41.0%, respectively, and were similar in the 4 groups. The abdominal fat content was similar in G1 and G2 and higher in G3 and more so in G4. Percentage of meat in forequarter, loin and hind quarter, meat to bone ratio and total meat yield of the carcass were more in high (G3 and G4) than low (G1 and G2) age group animals. The shear force value increased with increase in slaughter age of the rabbits. It is concluded from the study that dressing yield was more in 20 weeks old rabbits than other age groups. Further, lean content of the carcass increased while its bone content decreased with the advancement of age. Key words: Age, Carcass, Meat quality, Rabbit greens harvested (after 24 h wilting) till slaughter. A total of 48 Soviet Chinchilla rabbits were used in this slaughter investigation. The weaner kits were slaughtered at the age of 12 (G1), 16 (G2), 20 (G3) and 24 (G4) weeks. The rabbits were slaughtered without fasting by cervical dislocation. Traits evaluated on animals and carcasses were pre slaughter characteristics, carcass weight, abdominal fat, giblets (heart, kidneys and liver), forequarter, loin and hindquarter (Lukefahr et al. 1983). Separation of the forequarter from the loin and separation of the loin from the hindquarter were achieved by making transverse cuts at the last rib and at the posterior lumbar-anterior pelvic region respectively. Dressing percentage was calculated as hot carcass weight plus abdominal fat weight plus giblet weight, divided by pre slaughter weight. Body and carcass loin width were measured as the lateral distance from the right to the left transverse processes of the lumbar vertebrae over the top of the mid loin. Lean yield traits include percentages of bone and meat of the forequarter, loin and hindquarter cuts, overall meat to bone ratio, total meat and bone percentage of the carcass were assessed. For separation of meat and bone, the carcasses were stored overnight at 5° to 6°C and the retail cuts were de-boned manually. The manually separated bone was boiled with 2% KOH and the residue meat was scrapped off in the cuts and weighed. Body and carcass lengths were measured from the first thoracic vertebra to the tuber ischiadicum (pin bones). Longissimus dorsi was collected and analysed for meat Commercial broiler rabbit production is a profitable enterprise in many countries throughout the world. However, rabbit meat consumption is substantially lower in India due to consumer’s preference for sheep and goat meat, marginal economic feasibility of rabbit meat production, minimal attention by the scientific community, pet appearance of rabbits and above all limited supply of rabbit meat. Further, information on carcass composition and quality of rabbit meat is limited in contrast to that available for other meat animals. Reported information indicates differences in carcass characteristics among rabbit breeds and crossbreds (Gupta et al. 2002) and age of slaughter (Bernardini et al. 1994) and sexes (Sen and Bhagwan 1999, Poornima et al. 2003). Line origin has also influence on some sensory traits determining rabbit meat tenderness (Arino et al. 2007). However, such information is lacking for finisher rabbits raised under hot semiarid environment requiring organized slaughter studies to optimize the age at slaughter. The study was therefore conducted to determine the effect of age on carcass and meat quality traits of Soviet Chinchilla rabbits. MATERIALS AND METHODS The kits weaned at 4 weeks of age were maintained in individual cages and fed ad lib. on concentrate pellet and Present address: 1 Principal Scientist, NRC on Meat, Chengicherla, P.B. No.19, P.O. Uppal, Hyderabad 500 039 (email: [email protected]), 2 Director. 94 April 2010] EFFECT OF AGE ON CARCASS AND MEAT CHARACTERISTICS OF RABBITS quality evaluation. Cooking loss was determined by measuring the difference in the weight loss after cooking of meat for 1 h in water bath at 80°C and the values were expressed in percentage (Babiker et al. 1990). Muscle tenderness was assessed by Warner-Bratzler shear press method and the amount of force required to cut through the muscle sample was recorded and expressed as kg/cm2. The data on all the carcass and meat quality traits were subjected to analysis of variance (Snedecor and Cochran 1968) and significant differences were compared by Duncan’s multiple range test (Duncan 1955). 367 weight was similar in G2, G3 and G4. Abdominal fat% significantly (P<0.05) increased with the advancement of age. In general, a heavier carcass (G4) had higher (P<0.05) deposition of abdominal fat. The results are in agreement with the findings of Lukefahr et al. (1983). The average dressing percentage ranged from 52.85 to 57.10% of live weight. Dressing percentage was significantly influenced by slaughter age whereas the rabbits slaughtered at 20 weeks had higher (P<0.05) dressing percentage than those slaughtered at 12, 16 and 24 weeks of age. Dressing percentage values observed in this study are higher than earlier reports (Rao et al. 1978; Nofal et al. 1995). The higher dressing percentage with advancement of age may be due to lower (P<0.05) alimentary canal percentage with increasing age. Slaughter age influenced absolute weights of each physical cuts, however, on their expression as the per cent of carcass, the cuts were found similar in these age groups ranging from 38.6–40.9, 19.3–20.6 and 39.7–41.0%, respectively for hind quarter, loin and forequarter. The results reported here are in agreement with the work of Rao et al. (1978) who reported that dressed carcass weight had nonsignificant effect on the proportion of retail cuts. The proportions of carcass in hindquarter, loin and forequarter cuts, as observed in our study are in close agreement with the observation in New Zealand White and Flemish Giant rabbits by Lukefahr et al. (1983). Body and carcass length and loin width are also presented in Table 1. The higher age group had significantly (P<0.05) wider loin as measured on the live rabbit. Carcass length RESULTS AND DISCUSSION Weaning weight (g) was significantly higher (P<0.01) in G1 than in G2, G3 and G4 whereas the finishing weights (kg) were significantly higher (P<0.01) in G3 and G4 than G1 and G2. The higher finishing weight of G3 and G4 was reflection of their age. The average daily gain (g) was however higher (P<0.01) in G1 and progressively decreased with advancement of age in G2, G3 and G4. Similar trend was also observed in feed conversion efficiency (%), which was 41.5 in G1 and decreased to 21.2, 18.3, and 13.0 respectively in G2, G3 and G4 with increasing slaughter age. Similar types of findings were also observed by Larzul et al. (2005). Age at slaughter significantly (P<0.05) influenced pre slaughter and carcass weights (Table 1). The pre slaughter and carcass weight was lower (P<0.05) at 12 weeks of age compared to other age groups. However, the pre slaughter Table 1. Carcass traits of soviet chinchilla rabbits slaughtered at variable age Age in weeks Traits Pre slaughter wt. (kg) Carcass wt. (kg) Abdominal fat% Giblet (%) Alimentary canal (%) Dressing (%) Hindquarter (%) Loin (%) Forequarter (%) Skin (%) Blood (%) Head (%) Lungs (%) Tail and feet (%) Body length (cm) Loin width (cm) Carcass length (cm) Carcass loin width (cm) G1 12 G2 16 G3 20 G4 24 1.71b±0.11 0.82b±0.08 1.21c±0.09 8.17±0.24 20.46a±0.62 52.85b±0.36 40.99±0.49 19.34±0.59 39.66±0.56 8.26b±0.31 3.80±0.40 8.77±0.34 1.02±0.21 2.92±0.21 21.75±0.46 4.75bc±0.27 19.37b±0.34 4.62±0.25 2.26a±0.14 1.11a±0.11 0.90c±0.26 6.36±0.33 18.47b± 0.56 52.99b±0.37 39.25±0.50 19.77±0.69 40.97±0.60 9.61b±0.33 3.18±0.29 8.65±0.20 0.62±0.19 2.55±0.26 23.37±0.34 5.87b±0.39 23.12a±0.25 5.12±0.25 2.42a±0.12 1.27a±0.12 2.45b±0.34 6.75±0.48 16.68b±0.73 57.10a±0.66 39.74±0.41 19.34±0.66 40.82±0.59 9.99b±0.32 3.12±0.35 8.83±0.23 1.07±0.15 2.59±0.29 22.75±0.40 6.25ab±0.46 21.75a±0.35 5.65±0.28 2.39a±0.18 1.17a±0.17 3.92a±0.37 7.88±0.54 17.29b±0.75 54.25b±0.82 38.64±0.58 20.64±0.65 40.58±0.73 11.27a±0.64 3.78±0.39 8.53±0.15 1.05±0.12 2.87±0.19 23.75±0.49 7.25a±0.27 23.47a±0.46 5.47±0.34 Means bearing different superscripts differ significantly (P<0.05). 95 368 SEN AND KARIM [Indian Journal of Animal Sciences 80 (4) Table 2. Lean yield traits and meat quality of soviet chinchilla rabbits slaughtered at variable age Age in weeks Traits Total separable Lean% Bone% Lean: bone Hind quarter Lean% Bone% Loin Lean% Bone% Fore quarter Lean% Bone% G1 12 G2 16 G3 20 G4 24 77.01c±1.08 16.03a±0.98 4.80 83.0b±0.68 12.61b±0.77 6.58 86.84a±1.33 13.07 b ±0.96 5.40 87.65a±0.32 11.55 b ±0.34 7.59 77.03c±1.45 15.92a±1.35 87.17ab±0.48 10.33 b ±1.41 89.05a±1.60 10.09 b ±0.48 85.58 b ±0.57 12.52 b ±1.11 85.00±1.08 7.50±0.76 82.31±1.28 10.77±0.73 85.59±0.53 10.40±0.99 82.83±1.73 10.37±1.66 73.33c±1.10 20.00a±0.58 79.28 b ±0.71 15.83 b±0.77 85.50a±0.95 11.80c±0.58 79.21 b ±0.17 18.81a±0.20 Means bearing different superscripts differ significantly (P<0.05). increased significantly (P<0.05) up to 16 weeks and remained constant thereafter while the loin width gradually increased with the age. Results indicated that after reaching the particular body conformation, the animal attained a more compact body frame (wider loin and shorter carcass). It is difficult to compare these results objectively with other reports because of breed differences (Lukefahr et al. 1982). Lean yield and meat quality characteristics are presented in Table 2. The lean content significantly (P<0.05) increased with age in G2 and G3 whereas the bone content was higher in G1 than other groups. Meat bone ratio ranged from 4.80 to 7.59 and it was lower (P<0.05) in G1 than other groups. These values are comparatively higher than those reported by Rao et al. (1978) whereas, the increasing meat or bone with age compared favorably with their results. The maximum lean content was noticed in the fore quarter and hindquarter cuts than in the loin. Per cent bone was lowest in the loin cut and highest in the forequarter. The results are in agreement with the findings of Lukefahr et al. (1983). Cooking loss percentage decreased from 25.09 (G1) to 25.06 (G2), 24.56 (G3) and 24.49 (G4) with increasing age however, the differences were statistically non- significant. Shear force value of meat increased in higher age group rabbits and found to be 3.59 kg/cm2 in 20 weeks group and 3.72 kg/cm2 in 24 weeks group as compared to 12 weeks age (3.29 kg/cm2). This could possibly be due to collagen content, its solubility and differences in fatness. Valin et al. (1984) also reported increased cross linking in muscles with advancement of age. It is concluded from the study that average daily gain significantly reduced with the advancement of age and dressing yield was more in 20 weeks old rabbits than other age groups. Further, lean content of the carcass increased while its bone content decreased with the advancement of age. However, no significant differences were observed in meat quality parameters at variable age. ACKNOWLEDGEMENTS The authors are grateful to the Director, CSWRI for providing necessary facilities and technical assistance by Mr M. Nasimuddin is also acknowledged. REFERENCES Arino B, Hernandez P, Pla M and Blasco A. 2007. Comparison between rabbit lines for sensory meat quality. Meat Science 75: 494–98. Babiker S A, Elkhider I A and Shafie S A. 1990. Chemical composition and quality attributes of goat meat and lamb. Meat Science 28: 273–77. Bernardini B M, Castellini C and Lattaioli P. 1994. Rabbit carcass and meat quality: effect of strain, rabbitry and age. Italian Journal of Food Science 6: 157–66. Duncan D B. 1955. Multiple range and multiple ‘F’ tests. Biometrics 11: 1–42. Gupta R B, Rao V P, Reddy C E, Satyanarayana A and Reddy P P. 2002. Effect of genetic and non genetic factors on post weaning body weights and carcass traits of broiler rabbits. Indian Journal of Animal Sciences 72: 70–74. Kawinska J, Niedzwiadek S and Tuczynska J. 1980. Slaughter yield and meat quality of White Angora rabbits. Roczniki Naukowe Zootechniki 7: 147–55. Larzul C, Combes, S and Rochambeau H. 2005. Carcass composition, bone mechanical properties and meat quality traits in relation to growth rate in rabbits. Journal of Animal Science 83: 1526–35. Lukefahr S, Hohenboken W D, Cheeke P R and Patton N M.1982. Carcass and meat characteristics of Flemish Giant and New Zealand White purebred and terminal cross rabbits. Journal of Animal Science 54: 1169–74 Lukefahr S, Hohenboken W D, Cheeke P R and Patton N M.1983. 96 April 2010] EFFECT OF AGE ON CARCASS AND MEAT CHARACTERISTICS OF RABBITS Appraisal of nine genetic groups of rabbits for carcass and lean yield traits. Journal of Animal Science 57: 899–907. Poornima K, Gupta B R, Rao G N and Satyanarayana A. 2003. Evaluation of California white rabbits for carcass traits. Indian Journal of Animal Sciences 73: 564–66. Nofal R Y, Toth S and Virag G Y. 1995. Carcass traits of purebred and crossbred rabbits. World Rabbit Science 3: 167–70. Rao D R, Chen C P, Sunki G R and Johnson W M. 1978. Effect of weaning and slaughter ages on rabbit meat production. 2. Carcass quality and composition. Journal of Animal Science 46: 578–83. Rudolph W, Gauss H and Fischer W. 1980. Meat quality characteristics of broiler rabbits as influenced by age, sex and 369 slaughter weight. Archiv fuer tier Zucht 23: 387–91. Saleh K, El-Hakim A M, Abd-elnani F M and Elqen R Y N. 1988. Efficacy of crossbreeding in improving the productivity of rabbits. 3–slaughter traits. Journal of Agricultural Research Tanta University 14: 1590–1602. Sen A R and Bhagwan P S K. 1999. Appraisal of two genetic groups of rabbits for carcass and meat quality traits in males and females. Indian Journal of Animal Sciences 69: 631–33. Snedecor G W and Cochran W G. 1968. Statistical Methods. 6th edn, pp 299–338.Oxford and IBH Publishing Co Calcutta, India. Valin C, Pinkas A, Dragnev H, Boikovski S and Polikronov D. 1984. Comparative study of buffalo meat and beef. Meat Science 10: 69–84. 97 Short Communications Indian Journal of Animal Sciences 80 (4): 370–372, April 2010 Identification of single nucleotide variations in the genes related to reproduction in riverine buffalo J THANISLASS1, R SUMATHY2, S VENKATESA PERUMAL3 and KV SUBBA REDDY4 Rajiv Gandhi College of Veterinary and Animal Sciences, Kurumbapet, Puducherry 605 009 India Received: 6 June 2009; Accepted: 20 November 2009 Key words: Buffalo, Gene, Genetic marker, Single nucleotide variation Microsatellite and single nucleotide polymorphism (SNP) two types of DNA based genetic markers. Genetic markers are defined as identifiable DNA segments that differ in nucleotide sequence from one individual to the other. Genetic markers or DNA based markers are powerful tools for molecular dissection of traits of economic importance and for their potential application in breeding for more productive and efficient livestock. SNP markers are just a single base changes in a DNA sequence which are extremely stable (Sachidandam et al. 2001), abundant (Heaton et al. 2001) and amenable to high-throughput automated analysis (Lindblad-Toh et al. 2000). Due to these advantages SNPs form a preferred DNA marker for genotyping studies. The livestock industry depends on proper reproduction resulting into optimum production. Reproductive hormones play a very important role in fertilization and maintenance of pregnancy. These hormones trigger broad array of tissue and organ specific physiological responses by binding to their respective receptors. Thus the genes for receptors of the reproductive hormones can be candidate for the production traits. Therefore the identification of DNA based markers in these genes will be economically beneficial. Moreover, in buffaloes, systematic studies to develop DNA based markers are scanty. Hence present study was planned to identify single nucleotide variations in the genes related to the reproduction using PCR-SSCP followed by sequencing. Genomic DNA was isolated from blood of 25 unrelated animals representing five different buffalo breeds, viz. Murrah, Mehsana, Surti, Jaffrabadi and Toda (5 animals in each breed). Polymerase hain reaction was carried out using gene specific primers – 5’AATCCATCCTACCCCTGGAG3’ and 5’GCAATGGA TGGCTAAAGGAG3’ for Estrogen receptor (ER), 5’TCTTGGA GGCCGAAAGTTTA3’ and 5’TCGGAACTTACATATTGATGACCA3’ for progesterone Receptor (PR), 5’GATCCTGATCACCAGCCAGT3’ and 5’AGATGGGAAAAAGGGCAACT3’ for FSH receptor (FR), 5’TTGGGTAAA ATTCAAATGCAGA3’ and 5’ATGATGGTGTGGAGGGGTAA3’ for leptin receptor (LR). PCR was conducted in a 20.0 l reaction mix containing 100 ng of genomic DNA for 30 cycles. The annealing temperature was 59°C for ER and 55°C for PR, FR and LR. The sizes of the PCR products obtained were 212 bp, 316 bp, 362bp and 440 bp for ER, PR, FR and LR, respectively. The PCR products thus obtained were denatured in the SSCP dye (containing formamide and EDTA) at 94°C for 10 min, immediately cooled in an ice bath and used for SSCP analysis. The SSCP analysis was carried out using 15% polyacrylamide gel (49: 1) containing 5% glycerol. Electrophoresis was carried at 25°C, 250 volts for 3 h in 0.5% TBE buffer. The gel was subsequently silver stained (Baseem et al. 1991) and difference in SSCP banding pattern was identified. The samples which had produced varied PCRSSCP pattern were re-amplified, gel extracted and subjected for custom sequencing. The sequences (Accession no. EU662273 to EU662291) obtained were subjected for ‘megaBLAST’(Altschul et al. 1990) analysis to know the specificity of the amplification. The sequences were further subjected for multiple sequence alignment (ClustalW) (Higgins et al. 1994) to identify the single nucleotide variation. The effect of single nucleotide variation on the amino acid sequence and restriction sites was analyzed using ‘BLASTx’ and ‘NEBcutter’. PCR-SSCP is one of the methods of identification of single nucleotide variation which is based on the conformational change of DNA fragments due to single base change. The change in conformation can be detected by non-denaturing polyacrylamide gel electrophoresis. This is one of the simplest and most widely used methods to identify nucleotide variations (Orita et al. 1989). PCR-SSCP can detect 70 to 95% of potential base variations in short 200 or less base Present address: 1 Associate Professor (Email: jthanislass @gmail.com); 2Junior Research Fellow, Department of Veterinary Biochemistry, 3Assistant Professor, Department of Veterinary Biochemistry, 4Professor and Head, Department of Veterinary Biochemistry. 98 April 2010] SNPs IN THE GENES RELATED TO REPORDUCTION IN BUFFALO 371 Table 1. Variations identified and their effect on amino acid sequence and restriction site Gene Nucleotide variation identified Estrogen receptor (ER) – Coding region Progesterone receptor (PR) - Coding region FSH receptor (FR) – Coding region Leptin receptor (LR) – Promoter region Breeds of buffalo A 2 3 1 B Restriction site A→G Toda Nil Nil A→G T→C C→T A→G T→C A→G T →A T→C T→C T →A A→G T→G T →A Surti Murrah and Mehsana Jaffrabadi and Mehsana Toda Surti Surti Mehsana Toda Surti, Jaffrabadi and Mehsana Mehsana and Murrah Jaffrabadi Toda Murrah T→A V→A P→L Nil I→T I→G L→Q C→R Nil — — — — Creation of site for AciI Creation of site for AciI Nil Nil Nil Nil Nil Nil Nil Creation of site for SfaNI Creation of site for HinfI Nil Nil ER gene sequence of five different breeds of buffalo resulted in the identification of one nucleotide variation (A→G) in the Toda breed of buffalo which had correlated with the altered SSCP pattern (Fig. A). However, this nucleotide variation identified neither resulted in amino acid change nor restriction site. PCR-SSCP pattern of PR (Fig. B) was different for each breed of buffaloes. Sequence analysis had also revealed 4 different variations; 2 different nucleotide variations were identified in Mehsana breed of buffaloes. The variations identified are resulted in amino acid change as well as change in restriction site as detailed in the table 2. Analysis of FR and LR sequence had also revealed nucleotide variations which were correlated with PCR-SSCP pattern (Figs C and D). Some of the variations are resulted in amino acid change as well as change in restriction site. Overall, of the total 14 variations identified, 10 of them pair products under optimum conditions (Gross et al. 1999). However the method can be improved on its efficiency to detect nucleotide variations in the DNA fragments size from 400 to 450 bp by using low cross linker percentage and 5% glycerol. This helps in the successful determination of nucleotide variation in large PCR products (Hamzeiy et al. 2002). Therefore, in the present study PCR products of size 212 bp, 316 bp, 362bp and 440 bp of ER, PR, FR and LR, respectively were used for PCR-SSCP analysis and the different SSCP pattern obtained is shown in figure A, B, C, and D, respectively. The PCR products were obtained using the primers designed based on the cattle sequence but they were found to amplify the corresponding gene of buffalo which was confirmed by sequence analysis. The respective PCR products were sequenced and the nucleotide sequences were subjected for ‘BLAST” and “clustalW”. Alignment of 1 Amino acid change 2 3 4 5 1 2 3 4 C 5 1 2 3 4 5 6 7 8 9 10 D Figs A–D: A. PCR-SSCP pattern of estrogen receptor (Template DNA used Lane 1.Jaffrabadi, Lane 2: Murrah and Lane 3: Toda), B. PCR-SSCP pattern of progesterone receptor (Template DNA used Lane 1: Jaffrabadi, Lane 2: Mehsana, Lane 3: Murrah, Lane 4: Surti and Lane 5: Toda), C. PCR-SSCP pattern of FSH receptor (Template DNA used Lane 1: Jaffrabadi, Lane 2: Mehsana, Lane 3: Murrah, Lane 4: Surti and Lane 5: Toda), D. PCR-SSCP pattern of Leptin receptor (Template DNA used Lane 1, 2: Toda, Lane 3, 4: Surti, Lane 5, 6: Murrah, Lane 7, 8: Mehsana, Lane 9, 10: Jaffrabadi). 99 372 THANISLASS ET AL. are transitional in nature. The study on human SNPs from EST traces data bases given transition to transversion ratio of 1.7 (Picoult et al. 1999). The variations identified in chicken EST sequence were found 2.3 (Smith et al. 2001) and 4 (Kim et al. 2002) are higher than mammals. Out of 14 variations identified, 7 variations affected the amino acid sequence and 4 variations resulted in change in restriction sites. The variations which have resulted in amino acid change can be functionally important as these can affect the structure of the protein. The variations which had changed the RE site can be used to develop PCR-RFLP marker. All the nucleotide variations identified in this study are associated with difference in PCR-SSCP pattern, which demonstrate the potential usefulness of PCR-SSCP analysis for the detection of nucleotide variation. The nucleotide variations identified are found in coding region and promoter region of the genes studied, therefore these variations can be utilized as DNA markers of functional importance. The pregnancy rate, outcome of in vivo fertilization, fertility, liter size were shown to be associated with single nucleotide variations identified in the above genes. But the efficacy of single nucleotide variations identified in this study depends on the information content of these variations shown under association studies in the population at large. [Indian Journal of Animal Sciences 80 (4) Biology 218: 403–10. Bassem B J, Caetano-Anollesb G, and Gresshoff P M. 1991. Fast and sensitive silver staining of DNA in polyacrylamide gels. Analytical Biochemistry 196: 80–83. Gross E, Arnold N, Goette J, Schwartz–Boeger U and Kiechle M. 1999. A comparison of BRCA1 mutation analysis by direct sequencing, SSCP and DHPLC. Human genetics 105: 72–78. Hamzeiy U, Mashhadian N V, Edwards H J, and Goldfarb P S. 2002. Mutation analysis of the human CYP3A4 gene 5’ regulatory region: population screening using non-radioactive SSCP. Mutation Research 500: 103–10. Heaton M P, Grosse W M, Kappes S M, Keele J W, Chitko-McKown C G, Cundiff L V, Braun A, Little D P and Laegreid W W. 2001. Estimation of DNA sequence diversity in bovine cytokine genes. Mammalian Genome 12: 32–37 Higgins D, Thompson J, Gibson T, Thompson J D, Higgins D G, Gibson T J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22: 4673–80. Kim H, Schmidt C J, Decker K S and Emara M G. 2002. Chicken SNP discovery by EST data mining, in: Plant, Animal and Microbe. Genome 10: 12–16 Lindblad-Toh K, Winchester E, Daly M J, Wang D G, Hirschhorn J N, et al. 2000. Large-scale discovery and genotyping of singlenucleotide polymorphisms in the mouse. Nature Genetics 24: 381–86 Orita M, Suzuki Y, Sekiya T and Hayashi K. 1989. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5: 874–79. Picoult-Newberg L, Ideker T E, Pohl M G, Taylor S L, Donaldson M A, Nickerson D A and Boyce-Jacino M. 1999. Mining SNPs from EST database. Genome Research 9: 167–74. Sachidanandam R, weissman D, Schmidt S C, et al. 2001. A map of human genome sequence variation containing 1.42 million single nucleotide polymorphism. Nature 409: 928–33 Smith E J, Shi L, Drummond P, Rodriguez L, Hamilton L, Ramalal S, Smith G, Pierce K, and Foster J. 2001. Expressed sequences tags for the chicken genome from a normalized 10–day-old white leghorn whole embryo cDNA library: 1. DNA sequence characterization and linkage analysis. Journal of Heredity 92: 1–8. ACKNOWLEDGEMENT The authors gratefully acknowledge the financial support provided by the Department of Biotechnology (DBT), Government of India in the form of research grant (Ref. BT/ PR4729/AAQ/01/180/2004). The authors also thank Dr.G.Butchaiah, Dean, RAGACOVAS for his support and encouragement in developing this research project. Our grateful acknowledgements are to the authorities of livestock farms, Katuupakkam, Tamil Nadu, Mehsana and Dantiwada, Gujarat. REFERENCES Alstchul S P, Gish W, Miller W, Mayers E W and Lipman D J. 1990. Basic local alignment search tool. Journal of Molecular 100 Indian Journal of Animal Sciences 80 (4): 373–375, April 2010 Effect of farms on growth pattern of crossbred cattle SURENDRA SINGH1, A K VASISHT2, A K PAUL3 and L M BHAR4 Indian Agricultural Statistics Research Institute, New Delhi 110012 India Received: 6 July 2009; Accepted: 5 December 2009 Key words: Growth models, Double and tripple cross, Root mean square error Where Xt is weight of cattle at time t, 1-Asymptotic weight, 2-Scaling parameter, ?3 -Rate of maturity, 4Inflection parameter. For testing for homogeneity of variances at different farm the Bartlett’s test has been used, and it was found with the test result that there is a variability between the farms. Researchers in the field of behavioural and life sciences often come across with the studies on growth. Growth studies are very important for the livestock production because growth is the foundation on which the other forms of production such as milk, meat, wool etc rests. Growth models are used to predict growth rates and change in the shape of the organism. Comparison of nonlinear models for weight age data in cattle has been done under homoscedasticity Brown et al. (1972), Brown et al. (1976), Alessandra et al. (2002), Kolluru et al. (2003). Lambe et al. (2006), also studied different growth models in lambs. Various nonlinear models are available for comparing the growth pattern of cattle, but comparison of growth pattern is needed to find most appropriate model In this paper, comparison of performance of different breeds maintained at different farms has been done on the basis of non-linear models under homoscedastic error variance condition and heteroscedastic error condition on the data of same breed for different farms. Data used in the study were collected from Dehradun, Agra and Bareilly farms for Friesian × Sahiwal × Hariana breed, from birth to 36 months of age. The data for cattle in case of Friesian × Sahiwal breeds was collected for Agra and Dehradun farms for comparing the growth pattern of cattle among farms. Following models are fitted for comparing the performance of double cross and triple cross cattle. 1. Logistic model X = β/(1+β t 2e –β t 3) 3. Richards model X = β exp(1+β t 1 2e –β t 3 1/β 4 ) Measure of model adequacy To determine the adequacy of the models, statistical measure, RMSE which is MSE , is considered for judging the goodness of fit of the model. It is given by 1/2 ⎡ n Y1 –Y$ i ⎤ ⎥ Root mean squared error (RMSE) = ⎢ ∑ ⎢⎣i=1 n–p ⎥⎦ n = number of observations, p = number of parameters in the model. RESULTS AND DISCUSSION From Table 1, it is evident that RMSE (23.9349) is least for Von Bertalanffy model, but RMSE (23.9597) for Gompertz model is very close to this value and Gompertz model gives good prediction for birth and maturity weight, so Gompertz model is the best fitted model for F × S × H breed at Dehradun farm. Asymptotic weight (349.2) is maximum for Gompertz model followed by Richards model (343.2). Growth rate (0.1949) is maximum for logistic model. RMSE (4.4825) values is least in Richards model observed from Table 2 and it also shows that prediction for birth weight and maturity weight is good. Therefore Richard is the best fitted model for F×S breed at Dehradun Station followed by Gompertz model as this model over estimates the weight at birth and gives good prediction of weight at maturity. Table 1 also indicates high RMSE for Von Bertalanffy model than Gompertz model. Asymptotic weight (619.80) is maximum for Richards model followed by Gompertz (382.5) and Von Bertalanffy models. Growth rate (0.1534) is the highest for Brody and Logistic models and the least (0.0207) for Richards model. 2. Gompertz model X = β exp(1β t 1 2e –β t 3) 4. Von Bertalanffy Model X = β /(1+β t 1 2e –β t 3 )3 5. Brody Model X = β /(1+β e t 1 2 ; –β t 3) Present address: 1, 2, 3, 4Indian Institute of Agricultural Statistics Research Institute (email: [email protected]) 101 374 SINGH ET AL. [Indian Journal of Animal Sciences 80 (4) Table 1. Station-wise parameter estimates by different models under homoscedastic error structure model Logistic Parameter ß1 ß2 ß3 Gompertz RMSE ß1 ß2 ß3 Richards RMSE ß1 ß2 ß3 ß4 Brody RMSE ß1 ß2 ß3 Von-Bertalanffy RMSE ß1 ß2 ß3 RMSE Bareilly Agra Dehradun F×S×H F×S×H F×S F×S×H F×S 340.3000 (16.5271) 6.6046 (0.9968) 0.0404 (0.0043) 19.2179 368.0000 (17.2467) 2.3180 (0.1215) 0.0974 (.00905) 13.5646 619.9000 (171.0000) –0.9849 (0.0171) 0.0195 (0.0118) –1.2270 (0.1840) 7.19420 340.3000 (16.5270) –6.6046 (0.9968) 0.1614 (0.0175) 19.2179 355.4000 (16.7750) –1.0792 (0.0864) 0.1185 (.1185) 15.6515 339.5000 (14.1131) 6.9597 (0.9061) 0.1614 (0.0147) 16.1711 367.3000 (13.6603) 2.3697 (0.0978) 0.0967 (0.0070) 10.4996 536.3000 (59.7119) 619.8000 (0.0165) 0.0275 (0.0072) –1.0838 (0.1103) 4.2206 339.5000 (14.1131) –6.9597 (0.9061) 0.1614 (0.0147) 16.1710 354.6000 (13.7857) –1.1132 (0.0733) 0.1180 (0.0093) 12.6185 410.5000 (13.6002) 6.9597 x(0.8657 0.1623 0.0114) 15.0360 446.1000 (12.8978) 2.5350 (0.0807) 0.0951 0.0051) 9.3217 526.3000 (41.1868) –0.8693 (0.0772) 0.0503 (0.0112) –0.6752 (0.1589) 6.8825 410.5000 (13.6002) –8.0829 (0.8657) 0.1623 (0.0114) 15.0360 429.4000 (12.9894) –1.2214 (0.0634) 0.1173 (0.0069) 11.3303 327.8000 (17.2990) 8.5121 (1.9712) 0.1949 (0.0262) 24.3640 349.2000 (23.9764) 2.5636 (0.2814) 0.1156 (0.0176) 23.9571 343.2000 (32.9774) 0.6171 (3.2792) 0.1304 (0.0680 0.1937 (0.8430) 24.8196 327.8000 (17.29900 –8.5121 (1.9712) 0.1949 (0.0262) 24.3640 339.5000 (20.5903) –1.2482 (0.1825) 0.1412 (0.0200) 23.9349 354.4000 (15.6193) 6.6555 (0.8399) 0.1534 (0.0142) 16.7320 382.5000 (15.7104) 2.3190 (0.0966) 0.0927 (0.0072) 11.3109 619.8000 (101.6000) –0.9811 (0.0127) 0.0207 (0.0074) –1.1862 (0.1165) 4.4825 354.4000 (15.6193) –6.6555 (0.8399) 0.1534 (0.0142) 16.7320 369.5000 (15.5772) –1.0815 (0.0703) 0.1128 (0.0093) 13.3384 Figures in brackets indicate standard errors. From the above results it is found that maturity weight is more for F × S breed than F × S×H breed, whereas the growth rate of F × S×H breed is better than F × S breed. It is observed from Table 1 that F×S×H breed growth is maximum at Dehradun farm and minimum at Bareilly farm under homoscedastic error condition using nonlinear models. Maturity weight is found maximum at Dehradun farm and minimum at Bareilly farm. Table 1 revealed that growth rate is found better at Agra farm than Dehradun farm for F×S breed under homoscedastic error condition using nonlinear models and maturity weight is also found to be better at Agra farm than at Dehradun farm. RMSE is least for Dehradun farm for F×S×H breed for logistic model Table 2 and Growth rate is found better for Bareilly farm. The maturity weight is found to be maximum at Dehradun farm and minimum at Agra farm when models 102 April 2010] EFFECT OF FARMS ON GROWTH PATTERN OF CROSSBRED CATTLE 375 Table 2. Station-wise parameter estimates by different model under heteroscedastic error structure Model Logistic Parameter ß1 ß2 ß3 Gompertz RMSE ß1 ß2 ß3 RMSE Bareilly F×S×H Agra F×S×H F×S F×S×H F×S 287.9250 (2.3784) 8.9420 (0.1056) 0.2643 (0.0028) 0.1320 318.6515 (1.1803) 2.4832 (0.0042) 0.1361 (0.0006) 0.0778 285.6676 (2.7842) 9.2417 (0.1272) 0.2618 (0.0032) 0.1427 315.6594 (1.4062) 2.5214 (0.0051) 0.1354 (0.0008) 0.0851 343.9066 (4.0048) 10.4364 (0.1656) 0.2531 (0.0034) 0.1534 315.6594 (1.4062) 2.5214 (0.0510) 0.1354 (0.0008) 0.0851 303.6285 (2.7840) 9.7401 (0.1208) 0.2409 (0.0025) 0.1346 337.3123 (2.4365) 2.5720 (0.0079) 0.1236 (0.0011) 0.1040 289.2903 (3.3954) 8.8038 (0.1473) 0.2659 (0.0040) 0.1571 318.0417 (1.8383) 2.4744 (0.0067) 0.1394 (0.0011) 0.0981 are fitted under heteroscedastic error condition. F×S breed growth rate is found (Table 2) to be better at Dehradun farm than at Agra farm. The maturity weight is also found to be better at Agra farm than at Dehradun farm under heteroscedastic error condition. Dehradun REFERENCES Brown J E, Brown C J and Butts W T. 1972. A discussion of the aspects of weight, mature weight and rate of maturing in Hereford and Angus cattle. Journal of Animal Sciences 34: 525. Brown J E, Fitzhugh H A and Cartwright T C. 1976. A comparison of nonlinear models for describing weight-age relationships in Cattle. Journal of Animal Science 43: 810–18 Draper N R and Smith H.1966. Applied Regression Analysis. New York: Wiley. Lambe N R, Navajas E A, Simm G and Bunger L. 2006. A genetic investigation of various growth models to describe growth of lambs of two contrasting breeds. Journal of Animal Science 84: 2642–54 Ramesh Kolluru, Rana P S and Paul A K. 2003. Modelling for growth pattern in crossbred cattle. Journal of Animal Science 73(10): 1174–79 Richards F J.1959. A flexible growth function for empirical use Journal of Experimental Botany 10: 290–300. SAS. 1990. SAS Users’ Guide Version6. Rdn. 4. SAS Institute Incorporation. USA. SUMMARY Different sigmoidal nonlinear growth models are fitted in growth data of double cross Friesian×Sahiwal and triple cross Friesian × Sahiwal × Hariana breed at different farms. It is found that growth rate of Friesian×Sahiwal× Hariana breed under homoscedastic and heteroscedastic error condition is found maximum at Bareilly farm and minimum at Dehradun farm. Maturity weight is maximum at Dehradun farm and minimum at Agra farm. For Friesian × Sahiwal breed maturity weight is better at Agra farm than Dehradun farm. Growth rate is better at Dehradun farm than at Agra farm. 103 Indian Journal of Animal Sciences 80 (4): 376–378, April 2010 Influence of genetic and non-genetic factors on growth profile of Bharat Merino sheep in semi-arid region of Rajasthan ASHISH CHOPRA1, L L L PRINCE1, G R GOWANE1 and A L ARORA2 Central Sheep and Wool Research Institute, Avikanagar, via- Jaipur, Rajasthan 304 501 India Received: 25 March 2009; Accepted: 23 November 2009 Key words: Bharat Merino sheep, Genetic factors, Growth traits, Non-genetic factors, Sheep Bharat Merino is a synthetic dual type fine wool and mutton sheep with 75% exotic inheritance, developed and maintained at Central Sheep and Wool Research Institute Avikanagar, Rajasthan. The purpose of this study was to quantify the effect of non-genetic factors such as year of birth, season and status of ewe at birth, in order to construct an operational model for the accurate estimation of genetic parameters and prediction of breeding values. Data, collected over a period of 28 years (1980–2007) were classified in 4 classes. The traits included in the analysis were birth weight (BWT), 3 month weight (3WT), 6 month weight (6WT), average daily gain from birth to 3 months (ADG1) and average daily gain from 3 to 6 months (ADG2) of 4,388 lambs pertaining to both sexes. Likewise data were also classified in 4 groups according to dam weight. All the animals in this flock were kept under semiintensive management system. The flock was a closed type where about 250 breedable females were maintained in the flock over the years. These animals were subject to selection mainly for weight at 6 months and first greasy fleece yield. Male to female ratio for breeding was around 1: 25. Regarding feeding, concentrate mixture was offered ad-lib. to suckling lambs from 15-day age till weaning (90 days). After about 3 weeks of age, lambs were sent for grazing in morning and evening. During post-weaning period in addition to 8–10 h grazing and dry fodder supplementation, 300 g concentrate mixture per head to weaner lambs was provided. Since the subclass numbers were unequal and disproportionate the data were analyzed by the least squares procedure of fitting constants and using LSMLMW programme (Harvey 1990). The statistical model included period (four classes), season, sex, dam weight (four classes), interaction between period and sex, interaction between season and sex, interaction between period and season and interaction between period, season and sex as fixed effects and regression of ewe’s age at lambing was taken as covariate. Duncan’s multiple range tests was used for comparing subgroup means. Least squares means for BWT, 3WT, 6WT, ADG1 and ADG2 were 3.31±0.01 kg, 15.67±0.07 kg, 22.39±0.10 kg, 138±01 g and 77±01 g, respectively. Lambing period Data spread over 28 years was included in this study and classified in 4 periods. Effect of period was highly significant (P<0.001) for all the traits (Table1). Body weights and ADGs were significantly higher through out the period of study except during third period, i.e. from 1996 to 2001. Change in the feeding practices where zero supplementation was practised from 1996 onwards for few years might have resulted in down fall in the body weights of the animals. Decreasing trend in the rainfall and higher frequency of draught in last decades faced by this region might have also contributed toward low production performance. Variation in physical environmental conditions, feed and forage availability prevailing in different years could lead to significant period differences. Similar significant effect of year of lambing was also reported by Dixit et al. (2001) in Bharat Merino sheep, Sharma et al. (2003) in Marwari sheep, Nehra and Singh (2006) in Marwari sheep and Rao et al. (2004) in Nellore sheep. Season of lambing Spring born lambs were heavier than the autumn born lambs (Table 1). Spring season is being practised as a major season for lambing in the institute flock. Results indicate that practice of getting major crop in spring season should be continued further for getting heavier lambs. Due to higher exotic inheritance of this strain, extra care was taken to avoid external stress. Pregnant and lactating ewes of this period were compensated for dry matter requirement by supplementary feeding to protect animals from metabolic stress. Lambs were also provided green feed mainly Lucerne with stall feeding. This effect is continued in 3WT and 6WT also as a carry over maternal permanent environmental effect. Pre-weaning daily gain (ADG1) has non-significant effect Present address: 1Scientists, 2PS and Head, Division of Animal Genetics and Breeding (e mail: [email protected]). 104 April 2010] EVALUATION OF GROWTH PROFILE OF BHARAT MERINO SHEEP 377 Table 1. Least squares mean along with standard error (SE) for growth traits of Bharat Merino lambs Traits/factors LSμ±SE Period Up to 1989 1990 to 1995 1996 to 2001 2002 to 2007 SSN 1 2 Sex Male Female Ewe weight at lambing ≤ 33 kg 33.01 to 37.0 kg 37.01 to 41.0 kg ≥ 41.01 kg Period*Sex Season*Sex Period*SSN Period*SSN*Sex Regression of Damage BWT (kg) 3WT (kg) 6WT (kg) ADG1 (g) ADG2 (g) 3.31±0.01 (4388) ** 3.44±0.03a(759) 3.38±0.02a(1082) 3.31±0.02b(1278) 3.12±0.03c(1269) ** 3.46±0.01(3654) 3.16±0.02(734) ** 3.40±0.02(2165) 3.22±0.02(2223) ** 3.01±0.02a(1551) 3.29±0.02b(1256) 3.43±0.02c(818) 3.50±0.02d(763) NS NS * NS NS 15.67±0.07(3875) ** 16.64±0.17a(680) 16.64±0.12a(984) 13.36±0.13 b(1040) 16.02±0.13c(1171) ** 15.82±0.05(3214) 15.51±0.13(661) ** 16.16±0.10(1871) 15.17±0.09(2004) ** 13.94±0.10a (1307) 15.18±0.10 b(1123) 16.29±0.12c(746) 17.26±0.13 d (699) NS NS ** NS ** 22.39±0.10(3468) ** 22.80±0.24a(591) 23.80 ±0.17 b(915) 20.01±0.19c(963) 22.97±0.18a(999) ** 23.00±0.08(2878) 21.78±0.18(590) ** 23.53±0.14(1605) 21.26±0.13(1863) ** 20.52±0.15a (1176) 21.94±0.15 b(996) 22.91±0.17c(678) 24.21±0.18 d (618) NS NS ** NS ** 138±01(3875) ** 147±02a b(680) 148±01 b(984) 112±01c(1040) 144±01a(1171) NS 138±01(3214) 138±01(661) ** 143±01(1871) 133±01 (2004) ** 122±01a (1307) 133±01 b (1123) 143±01c (746) 154±01d (699) NS NS ** NS ** 77±01(3421) ** 72±02a(589) 85±02c (895) 75±02ab (950) 78±02b (987) ** 82±02(2845) 72±01(576) ** 86±01(1586) 68±01(1835) * 74±01a (1166) 76±01a(980) 78±02a b(666) 81±02b (609) NS NS ** NS NS Numbers in parentheses indicates the number of animals, **(P≤0.01), *(P≤0.05), NS (nonsignificant). source of variation in all the body weights (Table 1). The effect of dam weight was more on ADG1 than ADG 2. A clear cut increasing trend towards an increase in the body weights of the lambs with increase in the weight of the ewe at lambing was seen. Heavier dams gave birth to heavier lambs because of better nutrition and more uterine space provided by them for developing foetus. Weaning weight and ADG1 of these lambs were also observed to be significantly high. After weaning, these heavier lambs maintained their superiority in advanced age whereas their ADG 2 was significant only at P<0.05. Similar results were found by Dixit et al. (2001); Dey and Poonia (2005) and Nehra and Singh (2006). of season of lambing. ADG1 is non-significant owing to intensive type feeding management up to 3 weeks of age during pre-weaning period (hence getting the similar environment) is being followed here in Avikanagar. These results corroborated with the observations of Nehra and Singh (2006), Dixit et al. (2001) and Dey and Poonia (2005) whereas Singh et al. (1987) found autumn lambs were heavier than the spring born lambs and Sivakumar et al. (2005) found no effect of season of lambing. The lower body weight of autumn born lambs emphasized the need to provide supplementary feed and adequate management of these lambs, to protect them from the variance inducing factor. Sex The sex of the lamb was a significant (<0.01) source of variation in all the traits (Table1). The male lambs were significantly heavier than the female lambs at birth and this superiority of the male lambs increased with the advancement of age. Higher pre-weaning and post-weaning daily gain was also found in male lambs than females. Better pre-natal and post-natal growth of male lambs may be due to differences in their endocrine profile and in their culling level practised at different ages. These results were in agreement with Sharma et al. (2003), Rao et al. (2004), Dey and Poonia (2005) and Nehra and Singh (2006). Dam’s age at lambing Dam’s age at lambing was a non-significant source of variation at the birth (Table 1). This indicates the avoidance of extreme ends in birth weight by nature to maintain the genetic variance in a trait under selection. Similar results were also reported by Negi et al. (1987) in Gaddi sheep and its crosses. Dam’s age was highly significant (P<0.01) at 3WT and 6WT, may be because older dams have more milk production and good mothering ability. Dixit et al. (2001) reported effect of age of ewe at birth and post-weaning ADG less significant, whereas it was found to be highly significant on all other traits in Bharat Merino sheep which is similar finding to our study. Dam weight at lambing Weight of the dams at lambing was a highly significant 105 378 CHOPRA ET AL. [Indian Journal of Animal Sciences 80 (4) ACKNOWLEDGEMENTS Interaction effects Among the interaction of fixed effect studied, only interaction between period and season had significant effect on birth weight (P<0.05) (Table 1). This indicated the constancy of seasonal variation under the study period which was always high. Interaction between period and season, interaction between season and sex, and interaction between period, season and sex were non-significant. Contrary to our study, the interaction between sex and year of birth was observed to be highly significant by Sharma et al. (2003). Results clearly indicate that all the effects taken in study were either highly or moderately significant sources of variation on the growth traits. These findings demonstrate the need of giving proper attention to the significant factors and requirement of efficient flock management to obtain the optimum productivity. To obtain the more accurate estimates of genetic parameters and breeding values it is necessary to adjust the growth records for significant factors prior to further analysis. Authors thank the Director of this Institute for providing facilities. Assistance in data management by Shri N.C. Gupta, T-5 is acknowledged. REFERENCES Dey B and Poonia J S. 2005. Factors affecting growth traits in Nali sheep. Indian Journal of Small Ruminants 11(1): 77–79. Dixit S P, Dhillon J S and Singh G. 2001. Genetic and non-genetic parameter estimates for growth traits of Bharat Merino lambs. Small Ruminant Research 42: 101–04. Harvey R W. 1990. User’s Guide for LSMLMW. Mixed Model Least squares and Maximum Likelihood computer programme, PC– 2 Version. Ohio State University, Columbus, (Mimeograph) USA. Negi P R, Bhat P P and Garg R C. 1987. Factors affecting preweaning weights in Gaddi sheep and its crosses. Indian Journal of Animal Sciences 57(5): 489–92. Nehra K S and Singh V K. 2006. Genetic evaluation of Marwari sheep in arid zone: Growth. Indian Journal of Small Ruminants 12(1): 91–94. Rao V S T, Reddy R V, Veerabrhmalah K and Suresh J. 2004. Nongenetic factors effecting pre-and post-weaning body weights in two strains of Nellore sheep. Indian Journal of Small Ruminants 10(1): 86–87. Sharma M K, Sharma N K, Singh V K and Beniwal B K. 2003. Genetic evaluation of Nali and Marwari sheep in arid zone of Rajasthan: Body weights. Indian Journal of Small Ruminants 9(1): 65–68. Singh G, Mehta B S, Sethi I C and Arora C L. 1987. Genetic and non-genetic factors affecting growth traits of Nali and its crossbred lambs under semi-arid conditions. Indian Journal of Animal Sciences 57(7): 728–34. Sivakumar T, Soundararajan C, Palanidorai R, Ganeshkumar K, Mahendrans M and Malathi G. 2005. Factors affecting birth weight in Madras Red lambs. Indian Journal of Small Ruminants 12(1): 115–16. SUMMARY Present study was conducted to assess the effect of nongenetic factors on growth traits of Bharat Merino sheep. Effect of period was significant for all the traits. Season was significant for all the traits except ADG1 indicating uniform management during pre-weaning stage. Heavier dams gave birth to heavier lambs because of better nutrition and more uterine space provided by them for developing foetus. Dam’s age at lambing was a non-significant source of variation at the birth, indicating the avoidance of very high and very low weight at birth by nature. Interactions were mostly nonsignificant except the interaction between period and season. 106 Indian Journal of Animal Sciences 80 (4): 379–381, April 2010 Impact of breed improvement programme on goat production under farmers’ flocks M K SINGH1, A K GOEL2, B RAI3, ASHOK KUMA4 and M C SHARMA5 Central Institute for Research on Goats, Makhdoom, Uttar Pradesh 281 122 India Received: 10 September 2009;Accepted: 24 November 2009 Key words: Barbari, Breeding, Goat improvement, Conservation Population of Barbari goats is continuously declining in its home tract which has put this breed in endangered category (Singh and Rai 2006). Important reasons for decline in population are indiscriminate breeding due to lack of pure Barbari bucks and dilution with Sirohi breed of goats as Sirohi breeding bucks are available with large flocks and Sirohi goats are hardy for climatic, feeding and housing stress and perform better under grazing/browsing (Rai et al. 2009). Besides population, performance of these goats is also declining due to breeding with low potential bucks since males with good growth are sold without their replacement. Poor adoption of technology due to lack of knowledge is another reason of low performance. Therefore, to improve the goat productivity under farmers’ conditions, it is necessary to provide quality germ plasm especially breeding male, support services (prophylactic measures, market for their goat and goat produces) and awareness on improved management practices. The Central Institute for Research on Goats, Makhdoom (Uttar Pradesh) launched a multidisciplinary transfer of technology (TOT) programme in the home tract of Barbari goat ie Farah block of Mathura district of Uttar Pradesh in the year 2005. Villages Jalal, Bar Ka Nagla, Popa Burj and Pauri Sahjadpur were adopted under TOT programme on the basis of sizable goat population. Data on socio-economic attributes of goat keeper families, breed and flock composition, housing, disease status and management practices etc. were collected on structured schedule through conducting surveys. Later on 6 breeding bucks and 41 females of Barbari breed were provided to goat keepers of selected villages. Performance data on body weight, reproductive traits and service record of bucks were recorded by performing fortnightly visits and with the help of resource person. Body weights of kids were recorded at an interval of 3 months with respect to breed/type, type of birth and sex by a dial balance. Goat keepers were motivated to adopt breeding package of practices such as advantage of keeping purebred buck, awareness on criteria’s and bases of males and females selection, disadvantage of inbreeding (goat keepers used a buck for 3–5 years and later on its progeny), proper age of first service, proper time of service (mid oestrus), care before and during late pregnancy and at the time of parturition, kidding in favourable seasons (climate and feeding resources), optimum interval for re-breeding etc. Besides personal visits; awareness camps, health camps, farmer’s day etc were also arranged. Data on various traits were analyzed using appropriate statistical tools. Socio-economic attributes: Number of Households were 127, 54, 102 and 202 in the village Jalal, Bar ka Nagla, Popa burj and Pauri Sahjadpur. Out of these households, 33, 28, 30 and 34 goat keepers in the corresponding villages were goat rearing (Table 1). Maximum goat rearing households belong to backward (74%) followed by schedule caste (22%) communities. Maximum goat keepers were marginal (59%), followed by landless (38) and small (3%) landholding category. Goat rearing was mostly (84%) practised as secondary source of income and contributes substantially (20–40%) of their total income. Present results were in agreement with the findings of Gokhale et al. 2002, Kumar et al. 2006 and Singh and Rai 2006. About 20–40% goat keepers also kept buffaloes. There were about 5–7% families in each village whose major source of income was from goats and their secondary sources of incomes were from crop farming and factory labour. Most of the (>55%) goat keepers were illiterate. Male kids were sold through middleman (butchers) between 5 and 9 months of age whereas females Table 1. Goat population profile in adopted villages Present addresses: 1Senior Scientist (e mail: [email protected]), Scientists, Division of Physiology Dynamics. 5Director, IVRI, Izatnagar, Uttar Pradesh 243 122. 2,3,4Principal 107 Villages profile Jalal Bar Ka Nagla Popaburj Pauri Sahjadpur No. of households No. of goat rearing families Average flock size Range 127 33 54 27 102 30 202 31 4.2 1–16 8.6 1–12 5.4 1–12 5.9 1–14 380 SINGH ET AL. [Indian Journal of Animal Sciences 80 (4) Table 2.Status of type of birth and service rendered by the institute buck Village Jalal Bar ka Nagla Popa Burj Pauri Sahjadpur Year Institutes buck (%) Village bucks (%) Single (%) Twins (%) Triplets(%) 2006–07 2007–08 2008–09 2006–07 2007–08 2008–09 2006–07 2007–08 2008–09 2006–07 2007–08 2008–09 82.6 84.3 84.6 52.0 70.0 68.0 57.7 56.0 74.3 64.0 76.8 71.6 17.4 15.6 15.4 48.0 30.0 32.0 42.4 46.0 25.7 36.0 23.2 27.4 50.0 32.5 36.0 35.2 47.2 30.0 32.3 37.0 32.0 35.7 23.3 25.0 50.0 67.5 59.0 54.0 45.5 58.0 67.7 53.0 57 64.3 66.7 66.0 – 0.0 5.0 10.8 8.3 12.0 – 9.0 11.0 – 10.0 9.0 normal requirements besides inadequate protection against inclement weather. The breed composition in terms of Barbari, Barbari type and non-descript goats was 10.8, 41.9 and 47.2%, respectively, in Jalal, 0, 7.6 and 92.4% in Bar Ka Nagla, 0, 22.7 and 72.3% in Popa Burj and 4, 27.6 and 68.4%, respectively in Pauri Sahjadpur, respectively in 2006 (Table 3). There was no buck in the village Jalal and Popa Burj and farmers were dependent on non-descript bucks of low potential of adjoining villages. PPR, ET, diarrhoea, pneumonia and internal parasitic infestation were major cause of morbidity and mortality. Diarrhoea and pneumonia together accounted for 82% mortality in kids in adopted villages. Impact of breeding interventions: The bucks from Institute were provided to the goat keepers to extend services to 85, 68, 75.0 and 72% goat keepers in Jalal, Bar Ka Nagla, Popa Burj and Pauri Sahjadpur villages, respectively (Table 2). The impact of buck distribution over the years (from 2005– 06 to 2008–09) resulted in an increase of Barbari type young kids from 42 to 92.3% in Jalal, from 0 to 30.82% in Bar Ka Nagla, from 23 to 68.18% in Popa Burj and from 31.6 to were mostly sold among goat keepers. Goat management practices: The average flock size (including followers) was 4.2, 8.6, 5.4 and 5.8, respectively, in village Jalal, Bar Ka Nagla, Popa Burj and Pauri Sahjadpur, respectively (Table 1). Goats were maintained mainly on grazing however, grains were also provided in the form of sani consisted of wheat straw mixed with fermented crushed grains of barley or wheat. However, sani is provided to lactating or quality goats for a limited period in a year (40– 100 days). Amount of concentrate varies from 50–250 g/head/ day. The percentage of farmers rearing goats on stall-fed, low level of semi-intensive, moderate level of semi intensive and extensive feeding systems were 0, 7.7, 69.2, and 23%, respectively, in Jalal. Corresponding values were 4.1, 12.5, 29.1 and 45.8%, respectively in Bar Ka Nagla; 7.1, 17.9, 67.8 and 10.7%, respectively in Popa Burj and 7.1, 32.1, 50.0 and 17.9%, respectively in Pauri Sahjadpur. Weaning of kids continued till dam allowed the kids to suckle usually up to 4–6 months of age. Most of the flocks (75%) were provided inadequate housing space. The average open and covered housing space was 3 and 2 times lesser than the Table 3. Breed composition of adult goats and in kids after buck distribution in the year 2005 in adopted villages Village (s) No of goat keepers Year Jalal 26 Bar Ka Nagla 26 Popa –Burj 29 Pauri –Sahjadpur 29 2006–07 2007–08 2008–09 2006–07 2007–08 2008–09 2006–07 2007–08 2008–09 2006–07 2007–08 2008–09 Breed composition in adultgoats (%) Breed composition in kids (%) Barbari Barbari type Non descript Barbari Barbari type Non descript 10.8 25.0 24.4 0.0 2.7 4.2 0.0 3.6 7.8 4.0 5.0 11.4 41.9 43.7 44.2 7.6 6.8 12.5 22.7 25.4 29.2 17.6 22.5 27.5 47.2 31.5 31.2 92.4 90.0 83.3 72.3 70.9 63.0 78.4 72.5 61.1 0.0 40.0 38.0 0.0 20.0 18.0 4.5 6.8 11.6 12.0 10.5 18.2 92.3 47.5 50.0 30.8 55.0 58.0 68.2 62.0 62.6 60.3 68.4 63.8 7.7 12.5 12.0 69.2 25.0 24.0 27.3 31.0 26 27.6 21.0 18.0 108 April 2010] IMPACT OF BREED IMPROVEMENT PROGRAMME ON GOAT PRODUCTION 72.4% in Pauri- Sahjadpur. The overall body weight of goats belonging to Jalal village was 2.63±0.05, 8.03±0.15, 14.90±0.36, 16.89±0.81, 19.43±1.09 and 24.12±0.51 kg at birth and 3, 6, 9, 12 and >18 months, respectively. The corresponding estimates in village Bar Ka Nagla were 2.90±1.04, 8.56±0.79, 13.83±0.99, 17.75±2.03, 23.33±180 and 28.45±0.89 kg, respectively; in Popa-Burj were 3.55±0.09, 8.86±0.24, 14.14±0.35, 17.95±0.91, 22.08±0.73 and 30.11±0.91 kg, respectively and in Pauri-Sahjadpur were 3.34±1.54, 8.73±0.92, 15.73±0.95, 22.51±1.27, 27.66±1.88 and 29.64±1.01 kg, respectively. Body weights of kids at different ages however, were less than the kids maintained at Institute flock under semi-intensive management system (CIRG, 2007). A significant increase was recorded in incidences of multiple births in every adopted village as a result of breeding with Institute buck. The incidences of overall multiple births increased from to 42.8 (2005–06) to 64.7% (2008–09). Barbari bucks distribution in its home tract was quite effective, quick and easily manageable intervention by goat keepers. Such intervention is also useful in mobilizing goats for breed oriented market and ultimately technological improvisation for maximization of production. Distribution of 1–2 Barbari bucks in each village depending upon goat population is very sustainable approach for the improvement and conservation of Barbari goats. Since Barbari goats are found in a very large area, more breeding farms should be established to cater to the need of elite bucks for field. 381 with occasional supplementation of concentrate diet without any prophylactic measures. One or two Barbari bucks were provided in each adopted village which increased Barbari/ Barbari type of adult goats from 52.2 to 61.2% in Jalal, 7.6 to 10% in Bar Ka Nagla, 22.7 to 29.1% in Popa Burj and from 21.6 to 27.5% in Pauri Sahjadpur. The incidences of birth of Barbari/Barbari type kids in above corresponding villages were 87.5, 70.0, 69.0 and 78.9%, respectively. Buck supply also increased incidences of multiple births in adopted villages. ACKNOWLEDGEMENT Authors are thankful to Incharge Barbari Unit of AICRP on Goats for providing bucks and females to goat keepers and Director for help. REFERENCES CIRG. 2007. Annual Progress Report. Central Institute for Research on Goats, Makhdoom, Mathura (UP). Gokhale S B, Gokhale R B, Phadlke N L and Desale R J. 2002. Status in village goat management practices in Maharashtra. Indian Journal of Animal Sciences 72: 810–14. Kumar Shalander, Vaid R K and Sagar R L. 2006. Contribution of goats to livelihood security of small ruminant farmers in semiarid region. Indian Journal of Small Ruminants 12: 61–66. Rai B and Singh M K. 2004. Rearing practices of Jakhrana goats in their native tract. Indian Journal of Small Ruminants 10 (1): 33–35. Rai B, Singh M K, Sharma R B and Sharma M C.2009. Caprine biodiversity and Sustainable Management for their Conservation in National Seminar on Goat Biodiversity Conservation: Challenges and Opportunities. 20–21 March, page. 1–9 held at MPKV, Rahuri, Singh M K, Rai B, Kumar Ashok, Simaria M B and Singh N P 2009. Performance of Zalawadi goats under range conditions. Indian Journal of Animal Sciences 79 (1): 68–73. Singh M K and Rai B. 2006. Barbari breed of goat: Reasons of dilution in its home tract. Indian Journal of Animal Sciences 76: 716–19. SUMMARY Barbari, a highly prolific, dual purpose goat and highly suitable for commercial goat farming has come in endangered category of goat breed. Four villages of Farah block in Mathura district of Uttar Pradesh were selected for motivating farmers to adopt technologies in the form of germplasm and package of breeding practices. None of the village has purebred Barbari buck. Goats were maintained on grazing 109 Indian Journal of Animal Sciences 80 (4): 382–384, April 2010 Evaluation of growth, feed conservation efficiency and carcass traits of Jamunapari goats under intensive feeding system M K SINGH1, T K DUTTA2, R B SHARMA3, A K DAS4 and N P SINGH5 Central Institute for Research on Goats, Makhdoom, Mathura, Uttar Pradesh 281 122 India Received: 20 April 2009; Accepted: 28 November 2009 Key words: Carcass traits, Complete feed, DM intake, Feed Conservation Energy (FCE), Jamunapari goat Jamunapari is one of the important large sized dualpurpose goat breeds of India. These goats have been extensively used across the country and aboard for upgradation of non-descript and poor performing goats. However, presently Jamunapari breed has come under endangered category on account of reduction in population in its home tract mainly due to lack of feeding resources (browsing material), improper marketing policy and inadequate development programmes etc. (Singh et al. 2008). Therefore, it is essential to find out alternate feeds to maintain the production potential of these goats so that sustainable livelihood of goat keepers and conservation of this valuable germplasm could be ensured. Balanced feeding in terms of energy and protein optimizes growth and this interrelationship is additive (Chowdhury and Ørskov 1997). The complete feed pellet (CP12% and TDN 60%) containing Cajanus cajan straw as basal roughage gave 51.11 g ADG in weaned (5–10 months) Barbari kids (Dutta et al. 2003). Development of suitable feeding system for this endangered goat breed is one of the targeted areas for its conservation. Commercial goat farming is coming up with fast pace nowa-day. Therefore, it is high time to develop alternate feedings schedule suitable for pace of changes occurring in land utilization pattern particularly shrinkage of grazing land. Therefore, present study was conducted to evaluate complete feed based legume straw (Cajanus cajan) in 2 physical forms (pellet and total mixed ration) in Jamunapari kids. The experiment was carried out in Jamunapari goat unit of All India Coordinated Research Programme located at the Central Institute for Research on Goats (CIRG), Makhdoom, Farah (P.O.), Mathura, Uttar Pradesh in 2006–07. Eighteen weaned Jamunapari males at 3 months of age were divided into two equal groups based on their body weight. Animals were kept individually during the experimental period i.e. 3 to 9 months of age. Kids under group 1 (T1) were fed ad lib. complete pelleted feed and in group 2 (T2) ad lib. total mixed ration (TMR). The rations were prepared using the ingredients as mentioned in Table 1. Samples of feeds and residues were analyzed for Proximate Principles (AOAC 1984) and cell wall constituents (Goering and Van Soest 1970). Observations on body weights were recorded at weekly interval up to 9 months of age whereas nutrients intake was recorded daily. Freshwater was freely available to kids. These kids at the age of 9 months were slaughtered for carcass evaluation. The animals were weighed prior to slaughter. Bleeding, skinning and evisceration were done as per the standard procedures. After dressing the carcass measurements were taken as per Prasad and Agnihotri (1992). Weight of edible and non-edible offal’s Table 1. Formulation and chemical composition (%) of complete feeds Parameters Ration formulation (%) Maize Barley Groundnut cake Wheat bran Mineral mixture Common salt Cajanus cajan straw Total Chemical composition (%) CP EE Ash NDF ADF (T1) complete pellet feed 10 15 15 8.5 1 0.5 50 100 14.52 2.26 11.04 52.78 28.75 (T2) total mixed ration 10 15 15 8.5 1 0.5 50 100 14.38 2.42 11.63 54.10 31.42 Vitamins supplemented per quintal of feed mixture: -vitaminA 500000 IU, vitamin D3 100000 IU, vitamin B2 0.2 g, vitamin E 75 units, vitamin K 0.1 g, calcium pantothenate 0.25 g, nicotinamide 1.0 g, vitamin B12 0.6 mg. Present address: 1,2,3Senior Scientists, 4Scientist, 5Formerly Director (1email: [email protected]) 110 April 2010] EVALUATION OF GROWTH, FCE AND CARCASS TRAITS OF JAMUNAPARI GOATS 383 Table 2. Nutrient intake and growth in weaned Jamunapari kids under complete feeds Parameters Initial body weight (kg) DM intake (g) DMI (kg)/100 kg BW DMI (kg)/W0.75 kg BW CP intake (g) CPI (g)/W0.75 kg BW NDF intake (g) NDF (g)/W0.75 kg BW Final body weight (kg) Weight gain (kg) ADG (g) Feed conversion efficiency (%) T1 (complete pellet feed) T2 (total mixed ration) Significance 11.33±0.57 891.86±72.84 4.82±0.18 99.41±3.65 129.50±10.58 14.43±0.53 470.72±38.45 52.46±1.93 29.80±2.13 16.42±2.15 90.23±8.92 10.47±0.60 10.61±0.50 663.05±55.90 4.67±0.51 90.64±9.22 95.35±8.04 13.03±1.33 358.71±30.24 49.04±4.99 24.93±2.36 11.81±0.52 64.89±2.89 10.44±1.06 NS NS NS NS NS P<0.05 NS P<0.05 NS NS P<0.05 NS Table 3. Carcass traits, variety meat and different cut weights of Jamunapari kids fed with different pelleted feeds and separated fat to the nearest of 1 g, empty body weight to the nearest of 5 g and dressing percentage were recorded as described by Dhanda et al. (1999). Immediately after dressing carcass measurements were recorded using a measuring tape to the nearest of 1 mm. Different cut weights were taken following standard procedure. Statistical analysis of data was done using student t-Test as per Snedecor and Cochran (1980). The CP, EE and NDF were 14.52, 2.26 and 52.78% in complete feed pellet, corresponding estimates were 14.38, 2.42 and 54.10% in total mixed ration (Table 1). Higher DM intake in T1 than T2 was recorded, though the difference was nonsignificant (Table 2). The DM intake/kg W0.75 was 99.41 in T1 and 90.64 g in T2. Whereas, CP and NDF intakes/kg W0.75 were (P<0.05) higher in T1 (14.43 and 52.46 g) as compared to T2 (13.03 and 49.04 g). Therefore, palatability of complete pelleted feed was better than total mixed ration in these kids. The results corroborated the findings of Dutta et al. (2003) in Barbari kids. Weight gain and final body weight (slaughter) were higher in T1 by 4.61 and 5.33 kg over T2, but the difference was nonsignificant, which might be due to high individual variations within group. However, average daily gain (ADG) was significantly (P<0.05) higher in T1 (90.23 g) than T2 (64.89 g), which was mainly due to higher nutrients intake in pellet fed group. Feed conversion efficiency (%) was statistically similar in both the groups (10.47 vs 10.44). Prakash et al. (2006) in Barbari kids reported similar finding. The complete feed pellet (CP12% and TDN 60%) containing Cajanus cajan straw as basal roughage gave 51.11 g ADG in weaned (5–10 months) Barbari kids (Dutta et al. 2003). Higher growth rate in T1 due to higher intake was almost similar with the results of earlier study in Mawari kids reared under feed lot system (Shinde et al. 2000). The slaughter weight was 4.87 kg higher in T1 than T2 although difference was non-significant (Table 3). Body measurements and carcass measurements were higher in T1 than T2, however significant (P<0.05) effect was observed Traits T1 T2 Significance Slaughter weight (kg) 29.80±2.13 24.93±2.36 Carcass measurements Carcass loin width (cm) 12.94±0.47 11.67±0.34 Chest circumference (cm)66.44±1.77 59.78±2.19 Leg circumference (cm) 29.22±1.26 26.97±1.69 1.08±0.03 GR measurement (mm)# 1.16±0.07 Loin eye area (cm2) 10.68±0.71 9.88±0.54 Fat thickness## Back fat (mm) 1.18±0.07 1.10±0.02 Breast fat (cm) 2.26±0.26 1.72±0.29 Carcass traits Hot carcass weight (kg) 14.29±0.29 11.27±1.25 47.56±1.29 44.71±0.88 Dressing (%)b 55.55±1.20 55.65±0.97 Dressing (%)a Variety meatb Testes (g) 170.00±13.87 125.56±11.68 Pancreas (g) 39.44±2.42 32.22±4.01 Spleen (g) 56.11±8.93 40.00±5.27 Kidney (g) 98.33±6.24 86.11±9.20 Liver (g) 483.33±30.26 401.11±35.68 Heart (g) 117.22±9.39 87.22±9.32 Depot fat (kg) Cod fat (kg) 77.22±8.68 64.44±9.23 Kidney fat (kg) 149.44±20.92 131.11±40.36 Omental fat (kg) 250.00±24.49 194.44±30.76 Cut weights Leg cut weight (kg) 2.19±0.20 1.93±0.13 Loin cut weight (kg) 0.88±0.07 0.82±0.10 Rack cut weight (kg) 0.96±0.09 0.90±0.06 Neck and shoulder 1.66±0.20 1.58±0.16 weight (kg) Breast and shank 1.41±0.12 1.25±0.12 cut weight (kg) Saleable meat yield as 44.03±1.18 40.76±1.08 % of SW NS NS NS NS P<0.05 NS P<0.05 NS NS NS NS NS P<0.05 NS NS NS NS NS NS P<0.05 NS NS NS NS NS NS *P<0.05; # GR measurement is the soft tissue thickness, measured 11 cm from midline on 12th rib of the carcass; ## Fat thickness measured with vernier caliper. aOn empty body weight basis; bincludes heart, liver kidney, pancreas, spleen and testes; CW carcass weight; SW slaughter weight. 111 384 SINGH ET AL. only in GR measurement (T1, 2.16 mm and T2, 2.08 mm). Kids from the pellet fed group had better type chevon carcass with larger loins and enhanced back and breast fat thickness. Hot carcass weight and dressing percentage on slaughter weight basis was also numerically higher in T1 than T2, however, difference in both the group for carcass and noncarcass traits was nonsignificant (Table 3). Kidney and cod fat depot were similar in both the groups, whereas, pelleted feed resulted higher (P<0.05) omental fat deposit in T1 than T2. Due to higher body weight in T1, the weight of different edible organs (testis, spleen, kidney, liver and heart) tended to be higher in T1 than T2. Different cut weights like leg cut, loin cut, rack cut, neck and shoulder cut and breast and shank cut were higher in T1 than T2. Sealable meat yield was 44.03 in T 1 and 40.76% in T 2 , however, differences were nonsignificant. The complete feeds in this experiment contained 50% concentrate mixture, therefore, contain higher protein (CP 14.52 and 14.38%) and energy (estimated TDN 60.65% in both treatments) contents. The intake of nutrients was increased by the kids under T1 due to reduced particle size of pelleted feed, which triggered into more fat deposit in the carcass as compared to TMR fed kids. Higher proportion of cod and kidney fat for intensively fed kids were probably brought about by higher intake of protein and energy (Shinde et al. 2000 and Abdullah and Musallan 2007). Additional DM intake of 229 g/day/kid in complete pellet fed group resulted into 3.02 kg higher meat yield in the same feeding period than TMR fed kids of Jamunapari breed. The growth rate (90.23 g) of Jamunapari kids under T1 was better than the reported weight gain/day (50.33 g) in the same breed reared under intensive feeding. Therefore, complete pelleted feed prepared with 50% concentrate mixture and 50% roughage (C. cajan) can be used for higher growth and chevon production in Jamunapari kids under commercial goat production as well as conservation of this endangered breed of goat. [Indian Journal of Animal Sciences 80 (4) back and breast fat thickness. Hot carcass weight and dressing percentage on slaughter weight basis was numerically higher in T1 than T2. Additional DM intake of 229 g/day/kid in complete pellet fed group on account of better palatability resulted into higher body weight by 4.87 kg, weight gain by 25.34 g/day and 3.02 kg higher meat yield over total mixed ration in the same period. Results indicate that Jamunapari goat farmers could utilize complete pelletd feed for higher growth and sustainable conservation of the unique Jamunapari goat breed. REFERENCES Abdullah A Y and Musallam H S. 2007. Effect of different levels of energy on carcass composition and meat quality of lame black goat kids. Livestock Science 107: 70–80. AOAC. 1984. Official Methods of Analysis, 14th edn. Association of Official Analytical Chemists, Washington DC, USA. Chowdhury S A and Orskov E R. 1997. Protein energy relationship with particular references to energy under nutrition: A review. Small Ruminant Research 26: 1–7. Dhanda J S, Taylor D G, Murray P J and McCosker J E. 1999. The influence of goat genotype on capretto and chevon cacasses. Chemical composition of muscle and fatty acid profile of adipose tissue. Meat Science 52: 375–79. Dutta T K, Singh Nawab, Rekhate D H, Reddy T J and Settar V B. 2003. Utilization of by-products of pulses, oilseeds along with coarse cereals grain for intensive goat production. Final Report of NATP-RPPS–12. Central Institute for Research on Goats, Makhdoom, Farah, Mathura, Uttar Pradesh 281 122 (India), pp 61–93. Goering H K and Van Soest P J. 1970. Forage fiber analyses. Apparatus, reagents, procedures and some application. U.S.D.A, A.R.S. Agriculture Handbook, No. 379, Washington, D.C. Prakash B, Dutta T K and Siddiqui I A. 2006. Effect of plane of nutrition on nutrient utilization and performance of Barbari kids. Indian Journal of Animal Nutrition 23: 29–33. Prasad V S S and Agnihotri M K. 1992. Relevance of frame size and fatness indices to liv animal and carcass classification in goats. Indian Journal Animal Sciences 62: 461–66. Rekhate D H, Madavi V B, Dhok A P and Patil. 2004. Evaluation of arhar and gram straw based pelleted complete feed in goat. Indian Journal of Animal Nutrition 21: 257–60. Saini A L, Pakash B and Khan B U. 1986. System of management in relation to growth performance in Jamunapari kids. Indian Journal of Animal Production and Management 2: 26–29. Shinde A K, Singh N P, Sen A and Verma D L. 2000. Evaluation of kids rearing system for meat production. Indian Journal of Animal Sciences 70: 200–02. Singh M K, Rai B, Singh Pallavi, Singh P K and Singh N P. 2008. Status of goat production in different agro-climatic regions of India: An Overview. Indian Journal of Small Ruminants 14: 48–70 Snedecor G W and Cochran W G. 1980. Statistical Methods. 6th edn. Oxford and IBH Publishing Co. Pvt. Ltd., New Delhi. SUMMARY Post-weaned Jamunapari males at 3 months of age were fed either with ad lib. complete pelleted feed (CPF) (T1) or with total mixed ration (TMR) (T 2 ). The rations were prepared with 50% concentrate mixture and 50% Cajanus cajan straw. Kids consumed numerically higher DM in T1 than T2, however, the difference was nonsignificant. Whereas, CP and NDF intakes/kg W0.75 were significantly (P<0.05) higher in T1 as compared to T2. Average daily gain (ADG) was significantly higher (P<0.05) in T 1 than in T 2 . Biometrical and carcass measurements were statistically similar in both the groups. Kids from the pellet fed group had better type chevon carcass with larger loins and enhanced 112 Indian Journal of Animal Sciences 80 (4): 385–387, April 2010 Housing and feeding managemental practices for goats followed in South Gujarat G P SABAPARA 1, S B DESHPANDE 2, V B KHARADI 3 and P K MALIK4 Navsari Agricultural University, Navsari 396 450 India Received: 3 July 2009; Accepted: 20 November 2009 Key words: Feeding management, Goats, Housing management Goat husbandry is a popular enterprise among the artisan people and back word communities in South Gujarat region comprising seven districts, viz. Bharuch, Surat, Narmada, Navsari, Valsad, Tapi and Dangs, where goats mainly thrive well on zero or minimum inputs system with high prolificacy. The managemental practices for goat followed by their keepers are entirely different in various geographical regions and even it is somewhat different in the South Gujarat than the followed in other region of Gujarat. Therefore, an attempt was made to study the housing and feeding pattern of goat followed by the owners in South Gujarat region. For the purpose, a field survey was conducted on 1243 goat keepers of 45 villages in three districts, viz. Navsari, Surat and Bharuch of South Gujarat region to study the prevailing housing and feeding practices followed by them. The data were analyzed and tabulated to draw the meaningful Table 1. Housing management practices on caste basis Particulars Type of housing Open Close Kachcha Pacca Roof Kachcha Pacca Flooring Kachcha Pacca Manger Yes No Watering Well Pond Others Place of housing Near Residence Inside Residence Others Goats Keepers SC (n =124) ST (n =565) OBC (n =164) Other (n =390) Overall (n =1243) 121(97.58) 3 (2.42) 3 (100) 0 (0) 129 (22.83) 436 (77.17) 434 (99.54) 2 (0.36) 91 (55.49) 73 (44.51) 68 (93.15) 5 (6.89) 226 (57.95) 164 (42.05) 141 (85.98) 23 (14.02) 567 (45.62) 676 (54.38) 646 (95.56) 30 (4.44) 3 (100) 0 (0) 433 (99.31) 3 (0.69) 68 (93.15) 5 (6.85) 138 (84.15) 26 (15.85) 642 (94.97) 34 (5.03) 3 (100) 0 (0) 433 (99.31) 3 (0.69) 68 (93.15) 5 (6.85) 138 (84.15) 26 (15.85) 642 (94.97) 34 (5.03) 0 (0) 3 (100) 0 (0) 436 (100) 0 (0) 73 (100) 0 (0) 164 (100) 0 (0) 676 (100) 100 (80.65) 16 (12.90) 8 (6.45) 75 (13.28) 139 (24.60) 351 (62.12) 26 (15.85) 30 (18.29) 108 (65.86) 19 (4.87) 193 (49.49) 178 (45.64) 220 (17.70) 378 (30.41) 645 (51.89) 118 (95.16) 5 (4.03) 1 (0.81) 143 (25.31) 350 (68.95) 72 (12.74) 81 (49.39) 56 (34.15) 27 (16.46) 337 (86.41) 19 (4.87) 34 (8.72) 679 (54.63) 430 (34.59) 134 (10.78) Figures in parenthesis indicate per cent. inferences. Among the 1243 goat keepers surveyed, the castewise distribution indicated that maximum goats keeping was practised by scheduled tribes (45.45%) in the region followed by general category (31.39%) and OBC (13.19% and results Present address: 1Assistant Professor, 2 Associate Professor and Head, 3Professor and Head, Animal Genetics and Breeding, Veterinary College, 4Assistant Professor, Department of Animal Sciences (4email: [email protected]). 113 386 SABAPARA ET AL. [Indian Journal of Animal Sciences 80 (4) Table 2. Feeding practices followed by goat keepers. Particulars Grazing pattern Semi-intensive Extensive Grazing duration 0–4 hrs 4–8 hrs Green fodder feeding Yes No Dry fodder feeding Yes No Concentrate feeding (A) Lactating does Yes No (B)During pregnancy Yes No (C) Growing kids (I) male Yes No (II) Female Yes No Type of concentrate Home made Purchased Mineral mixture feeding Yes No Goats Keepers SC (n =124) ST (n =565) OBC (n =164) Other (n =390) Overall (n =1243) 00 (00) 124 (100) 00 (00) 565 (100) 98 (59.76) 66 (40.24) 248 (63.59) 142 (36.41) 346 (27.84) 897 (72.16) 00 (00) 124 (100) 00 (00) 565 (100) 98 (59.76) 66 (40.24) 248 (63.59) 142 (36.41) 346 (27.84) 897 (72.16) 124 (100) 00 (00) 560 (99.12) 05 (0.88) 152 (92.68) 12 (7.32) 376 (94.41) 14 (3.59) 1212 (97.51) 31 (2.49) 00 (00) 124 (100) 00 (00) 565 (100) 32 (19.51) 132 (80.49) 92 (23.59) 298 (76.41) 124 (9.98) 1119 (90.02) 32 (25.81) 92 (74.19) 262 (46.37) 303 (53.63) 84 (51.22) 80 (48.78) 266 (68.21) 124 (31.79) 644 (51.81) 599 (48.19) 04 (3.23) 120 (96.77) 32 (5.66) 533 (94.34) 12 (7.32) 152 (92.68) 28 (7.18) 362 (92.82) 76 (6.11) 1167 (93.89) 124 (100) 00 (00) 532 (94.16) 33 (5.84) 152 (92.68) 12 (7.32) 358 (91.79) 32 (8.21) 1166 (93.81) 77 (6.19) 22 (17.74) 102 (82.26) 84 (14.87) 481 (85.13) 16 (9.76) 148 (90.24) 48 (12.31) 342 (87.69) 170 (13.68) 1073 (86.32) 124 (100) 00 (00) 565 (100) 00 (00) 164 (100) 00 (00) 390 (100) 00 (00) 1243 (100) 00 (00) 00 (00) 124 (100) 00 (00) 565 (100) 00 (00) 164 (100) 00 (00) 390 (100) 00 (00) 1243 (100) Figures in parentheses indicate per cen.t of the study and least preferred by SC. The results of the study are well supported by the findings of Tanwar et al. (2008), reported that the goat keeping is most popular (90.83%) among tribes of Mavli and Jhadol talukas of Udaipur district. Housing management: The animals 45.62% surveyed were kept under open housing system and rest of the animals were maintained under close housing system in the region and among this scheduled tribes were stood on the top (77.17%) for giving the shelter to their goats however, least shelter (2.42%) was provided to the goats by SC (Table 1), which indicated that the goats are the major source of livelihood for scheduled tribes in South. Gujarat region: Similar to the findings, Tanwar et al. (2008) also reported same pattern tribes in the region. 95.56% of goat keepers built kachcha type house under close housing system and among the pacca housing system, 94.97% goat keepers had kachcha type roof and floor. Only 5.03% of goat keepers provided pacca floor and roof to their goats in the category. Manger was not built in shed by any of the respondent in south Gujarat. The common feeling is to provide much natural comfort and conditions to the animals by providing kachcha flooring among the goat keepers of South Gujarat also strengthen the observation reported by Tanwar et al. (2008) in Rajasthan. Majority of goat keepers use bore well/hand pump or tap water (51.89%) followed by Pond (30.41%) and well (17.70%) as source of drinking water however, Tanwar et al. (2008) reported that goat keepers provide pond water to the goats for drinking purpose in Rajasthan. This difference in the pattern of type of drinking water may be attributed to the availability of water, as plenty of fresh water is available in South Gujarat region. During the heavy rainfall, goat keepers kept their goats in a Machan type houses made up 114 April 2010] HOUSING AND FEEDING MANAGEMENTAL PRACTICES FOR GOATS IN SOUTH GUJARAT 387 Figs 1–2. Housing (Machan Type) of goats in Heavy Rainfall Zone of South Gujarat of wooden logs and bamboo sticks to protect them from adversity of rain (Figs 1–2). The males and females housed separately in machan type system prevail in the south Gujarat region however, kids are kept along with mothers. Results of the study are corroborated with the findings of Jayashree (2009) in heavy rainfall zone (Malnad area) of Karnataka. Feeding management: Majority of the goat keepers follow extensive grazing (72.16%) for 4 to 8 hrs in a day (Table 2). Dhuppe et al. (2008) reported similar pattern of the grazing in Sangamner Tahsil of Maharashtra. Singh and Rai (2006) also reported same kind of the grazing pattern in the home tract of Barbari goat. The feeding of green fodder as tree lopping from Neem (Azadirachta indica), Tamarind (Tamarindus indica), Goras Amli (Pithecellobium dulce), Babul (Acasia niloticaica), Subabul (Leucaena leucocephala), Samadi (Prosopis cineraria), Banyan tree leaves (Ficus benghalensis L), Peepal (Ficus religigosa L), Ber (Zizyphus rotundifolia), Kavathi (Limonoa acidissima L.) etc. is very common and practised by 97.51% goat keepers. Similar observations have been reported by Dixit et al. (2007) in the breeding tract of Marwari goat of Rajasthan and Singh et al. (2009) in the breeding tract of Zalawadi goats in Surendranagar district of Gujarat. The feeding of dry fodder to the goats is uncommon in the region and practised only by 9.98% goat keepers. The concentrate mixture comprising of bajra, jowar, wheat, tuer chuni, gram chuni and babul pod etc. Feeding of home made concentrate to the pregnant does practised in the region by 6.11% owners, only however, 51.81% goat keepers fed concentrate to lactating does for better nourishment of kids. The feeding of concentrate is very common to the male kids (93.81%) rather than the female kid (13.68%) for achieving the better growth rate resulting good return. The feeding of common salt was general practice in the area but animal holders did not provide the mineral mixture to the goats, irrespective of the category. Concentrate feeding was not practice by goat keepers in the breeding tract of Zalawadi goats of Gujarat as reported by Singh et al. (2009), which showed the variation within the state for feeding pattern. SUMMARY Peculiar characteristic of goats to thrive well on zero or minimum input system makes the goat enterprise very popular among the artisan people and back word communities of the South Gujarat. The managemental practice in the region is somewhat better than the other parts of the Gujarat but not up to the mark. Results of the study indicated that there is an urgent need to make aware the goat keepers for the scientific feeding as well as the housing so that the owners can achieve maximum return from the enterprise to improve their socioeconomic status. REFERENCES Dhuppe S U, Shinde S B, Mote M G and Chavan K D. 2008. Study of Management Practices of Goats in Sangamner Tahsil in Maharashtra. Journal of Maharashtra Agricultural Universities, 33 (3): 377–80. Dixit S P, Verma N K, Aggarwal R A K, Patel A K, Kumar D, Sharma R and Ahlawat S P S. 2007. Phenotypic and Genetic Characterization of Marwari Breed of Goat in Hot-Arid Region of India. Indian Journal of Animal Sciences 77 (5): 395–99. Jayashree R. 2009. Housing Practices Adopted for Local Goats in Malnad Area of Karnataka. National Seminar on Goat Biodiversity Conservation: Challenges and Opportunity held at M.P.K.V. Rahuri (Maharashtra) during March, 20–21, 2009 pp.194. Singh M K and Rai B. 2006. Barbari Bred of Goat: Reasons of Dilution in its Home Tract. Indian Journal of Animal Sciences 76 (9): 716–19. Singh M K, Rai B, Kumar A, Simaria M B and Singh N P. 2009. Performance of Zalawadi Goats under Range Conditions. Indian Journal of Animal Sciences 79 (1): 68–72. Tanwar P S, Vaishnava C S and Sharma V. 2008. A Study on Socio-economic Aspects of Goat keepers and Management Practices Prevailed in the Tribal Area of Udaipur District of Rajasthan. Indian Journal of Animal Research 42 (1): 17–74 . 115 388 SABAPARA ET AL. [Indian Journal of Animal Sciences 80 (4) The Indian Journal of Animal Sciences ARTICLE CERTIFICATE F.No. Article Entitled : ............................................................................................................................................................. ............................................................................................................................................................. Title changed to : ............................................................................................................................................................. ............................................................................................................................................................. Authors (names) : ............................................................................................................................................................. ............................................................................................................................................................. It is certified that: 1. The article has been seen by all the authors (signatures given below), who are satisfied with its form and content. 2. The sequence of names of authors in the by-line is as per their relative contribution to this experiment, giving due credit to all scientists who made notable contribution to it. 3. The address of the organization where the research was conducted is given in the by-line (changes of author’s address is given the footnote). 4. The experiment was carried out during 200.............. and the article is submitted soon after completion of the experiment. 5. The article is exclusive for this journal, and the results reported here have not been sent (and not be sent during its consideration by this journal) for publication in any other journal. 6. The article has not been rejected for publication in any other journal/rejected in ................................................................. whose comments are attached (such a rejection does not disqualify the article for consideration in this journal). 7. It is based/not based on a part/complete M.Sc/Ph.D thesis of the first author, who submitted the thesis in 200......... to the ........................................................................ university, place). 8. Correct data and facts are presented in the article. 9. I/we agree to abide by the objective comments of referees and do agree to modify the article into a shortnote as per the recommendation, for publication in The Indian Journal of Animal Sciences. 10. If published in The Indian Journal of Animal Sciences, the copyright of this article would vest with the Indian Council of Agricultural Research who will have the right to enter into any agreement with any organization in India or abroad engaged in reprography, photocopying, storage and dissemination of information contained in it, and neither we nor our legal heirs will have any claims on royalty. Signature Name in full and designation Present official address Subscription with date number 1. ....................................................................................................................................................................................... ....................................................................................................................................................................................... 2. ....................................................................................................................................................................................... ....................................................................................................................................................................................... 3. ....................................................................................................................................................................................... ....................................................................................................................................................................................... 4. ....................................................................................................................................................................................... ....................................................................................................................................................................................... 5. ....................................................................................................................................................................................... ....................................................................................................................................................................................... OPTIONAL The above certificate is correct to the best of my knowledge and I have no objection to the publication of the article cited above in The Indian Journal of Animal Sciences Signature and address of Head of the department /Head of the Institute 116