Mar - International Buffalo Information Center

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International Buffalo Information Center (IBIC)
BUFFALO BULLETIN
ISSN : 0125-6726
Aims
IBIC is a specialized information center on water buffalo. Established in 1981 by Kasetsart University
(Thailand) with an initial financial support from the International Development Research Center (IDRC) of
Canada. IBIC aims at being the buffalo information center of buffalo research community through out the
world.
Main Objectives
1. To be world source on buffalo information
2. To provide literature search and photocopy services
3. To disseminate information in newsletter
4. To publish occasional publications such as an inventory of ongoing research projects
Buffalo Bulletin is published quarterly in March, June, September and December. Contributions on
any aspect of research or development, progress reports of projects and news on buffalo will be considered
for publication in the bulletin. Manuscripts must be written in English and follow the instruction for authors
which describe at inside of the back cover.
Publisher
International Buffalo Information Center, Office of the University Library, Kasetsart University
Online availible
http://ibic.lib.ku.ac.th/e-Bulletin
Advisory Board
Prof. Dr. Charan Chantalakhana
Prof. Dr. John Lindsay Falvey
Prof. Dr. Metha Wanapat
Mr. Antonio Borghese
Dr. Aree Thunkijjanukij
Miss Wanphen Srijankul
Editorial Member
Dr. Pakapan Skunmun
Dr. Kalaya Bunyanuwat
Prof. Dr. Federico Infascelli
Thailand
Faculty of Veterinary and Agricultural Science, University
of Melbourne, Australia
Department of Animal Science, Faculty of Agriculture,
Khon Kaen University, Thailand
International Buffalo Federation, Italy
International Buffalo Information Center, Office of the
University Library, Kasetsart University, Thailand
Thailand
Department of Livestock Development, Thailand
Department of Veterinary Medicine and Animal Science,
University of Naples Federico II, Italy
Dr. Rafat Al Jassim
Prof. Dr. Nguyen Van Thu
Prof. K. Sarjan Rao
Prof. Dr. Masroor Ellahi Babar
Asst. Prof. Dr. Asif Nadeem
Prof. Dr. Raul Franzolin
School of Agriculture and Food Sciences, Faculty of
Science, The University of Queensland, Australia
Department of Animal Sciences, Faculty of Agriculture
and Applied Biology, Can Tho University, Vietnam
Department of Livestock Production and Management,
College of Veterinary Science, India
Virtual University of Pakistan, Pakistan
Institute of Biochemistry and Biotechnology, University of
Veterinary and Animal Sciences, Pakistan
Departamento de Zootecnia, Universidade de São Paulo,
Brazil
Editor
Dr. Sunpetch Sophon
Journal Manager
Mr. Chalermdej Taterian
Assistant Journal Manager
Miss Jirawadee Wiratto
Faculty of Veterinary
Technology, Thailand
Medicine,
Mahanakorn
of
BUFFALO BULLEITN
IBIC, KASETSART UNIVERSITY,
P.O. BOX 1084, BANGKOK 10903, THAILAND
E-mail : [email protected]
Tel
: 66-2-9428616 ext. 344
Fax
: 66-2-9406688
Buffalo Bulletin (March 2015) Vol.34 No.1
CONTENTS
Page
Case Report
An outbreak of trypanosomosis in buffaloes caused by diminazene
resistant Trypanosoma evansi
G. Ponnudurai, S. Sivaraman, N. Rani and C. Veerapandian.........................................................1
Original Article
Evaluation of urea molasses multi-nutrient blocks containing
alternate feed resources in buffaloes
M. Choubey, M. Wadhwa and M.P.S. Bakshi...................................................................................5
Effect of supplementation of Tinospora cordifolia on lactation
parameters in early lactating Murrah buffaloes
N.A. Mir, P. Kumar, S.A. Rather, F.A. Sheikh and S.A. Wani............................................................17
Prevalence and seasonal variation in ixodid ticks on buffaloes of
Mathura district, Uttar Pradesh, India
Geeta Patel, Daya Shanker, Amit Kumar Jaiswal, Vikrant Sudan
and Santosh Kumar Verma...............................................................................................................21
Sedative, analgesic and cardiopulmonary effects of midazolam-butorphanol premedication
in water buffaloes (Bubalus bubalis)
Deepti Bodh, Kiranjeet Singh, Jitender Mohindroo, Sashi Kant Mahajan
and Narinder Singh Saini.................................................................................................................29
Prevalence and antibiogram of bacterial pathogens from subclinical mastitis in buffaloes
Z. Ali, U. Dimri and R. Jhambh........................................................................................................41
Macro and micro mineral profile in forage and blood plasma of water buffaloes
with respect to seasonal variation
Sushma Chhabra, S.N.S. Randhawa and S.D. Bhardwaj.................................................................45
CONTENTS
Page
Original Article
A study on the prevalence of pathological abnormalities of the ovaries
and oviducts diagnosed at post mortem of buffaloes in Mosul
O.I. Azawi and A.J. Ali...................................................................................................................51
Effect of vitamin E and mineral supplementation on biochemical profile
and reproductive performance of buffaloes
H.M. Khan, T.K. Mohanty, M. Bhakat, A.K. Gupta, A.K. Tyagi and G. Mondal...........................63
Effect of vitamin E and mineral supplementation during peri-partum period
on BCS, body weight and calf performance in Murrah buffaloes
H.M. Khan, T.K. Mohanty, M. Bhakat, A.K. Gupta and G. Mondal..............................................79
Study on micro-mineral status of buffaloes during peripartum period in different season
H.M. Khan, T.K. Mohanty, M. Bhakat, A.K. Gupta, A.K. Tyagi and G. Mondal...........................86
Lifetime performance of Murrah buffaloes hot and humid climate of Tamil Nadu, India
A.K. Thiruvenkadan, S. Panneerselvam and R. Rajendran...........................................................92
Effect of season on semen quality parameters in Murrah buffalo bulls
M. Bhakat, T.K. Mohanty, A.K. Gupta, S. Prasad, A.K. Chakravarty and H.M. Khan.................100
Milk yield and composition and efficiency of nutrients for milk production in
Jaffrabadi buffaloes on rations supplemented with varying levels of bypass fat
H.H. Savsani, K.S. Murthy, P.U. Gajbhiye, P.H. Vataliya, A.R. Bhadaniya,
V.A. Kalaria, S.N. Ghodasara and S.S. Patil.................................................................................113
Real time PCR- an approach to detect meat adulteration
Rajni Kumari, D.N. Rank, Sanjay Kumar, C.G. Joshi and S.V. Lal...............................................124
The use of tropical of multiproposes trees as a feed supplement to
Thai swamp buffaloes (Bubalus bubalis) reciving a basal diet of pangola hay
Thongsuk Jetana, Sunworn Usawang and Sunpetch Sophon........................................................130
Case Report
AN OUTBREAK OF TRYPANOSOMOSIS IN BUFFALOES CAUSED BY DIMINAZENE
RESISTANT TRYPANOSOMA EVANSI
G. Ponnudurai*, S. Sivaraman, N. Rani and C. Veerapandian
ABSTRACT
trypanosomosis. Consequent to this all the animals
were treated with Antrycide Pro-salt as prophylactic
measures.
An outbreak of trypanosomosis caused
by diminazine resistant Trypanosoma evansi was
recorded in 6.9 percent of buffaloes in an organised
government farm during the month of August’2012.
A total of 144 buffaloes are being maintained at
the district livestock farm, Orathanadu, Thanjavur
district of Tamil Nadu. Initially, 2 animals
had developed the clinical symptoms of fever
(104oC), oedema of the legs, pale visible mucous
Keywords: trypanosomosis, buffaloes, diminazene
resistant strain, India
INTRODUCTION
Trypanosomosis is one of the important
haemoprotozoan diseases affecting wide range of
domestic and wild animals in India. Horse has been
incriminated as natural host for this haemoflagellate,
while cattle, buffalo and camel act as reservoir
hosts and they usually exhibit subclinical form
of disease. However, the reservoir hosts may also
suffer with clinical trypanosomosis, if they are
subject to stress. Since the disease is endemic
throughout India, it causes heavy economic losses
to the farmers in terms of morbidity, mortality,
abortion, infertility, reduced milk yield and various
neurological disorders resulting into death of the
affected animals. In India, diminazene aceturate,
Quinapyramine sulphate and chloride (Antrycide
Prosalt) and Quinapyramine sulphate (Antrycide)
are currently available drugs for treatment and
prophylactic use against trypanosomosis in domestic
animals. But drug resistance is now a severe and
increasing problem in trypanosome (Witola et al.,
2005 and Shaba et al., 2006). The present paper
membrane, frequent micturition and anorexia.
The examination of thin blood smear showed the
presence of Trypanosoma evansi with parasitaemia
level of +++. Subsequently the affected animals
were first treated with diminazene aceturate at the
rate of 3.5 mg /Kg body wt i/m. The examination
of blood smear on the next day of diminazene
aceturate treatment showed the presence of
Trypanosoma evansi without any reduction in the
parasitaemia level. But, blood smear obtained after
Antrycide Prosalt, at the dose rate of 7.4 mg/ Kg
b.wt- s/c , treatment free of T.evansi and hence it
was presumed that the buffaloes might have been
infected with diminazene aceturate resistant strain
of Trypanosoma evansi. In addition, examination of
blood smears collected from the remaining animals
revealed that eight animals were found to carry
Trypanosoma evansi with a moderate parasitaemia
level of ++, without showing any clinical signs of
Department of Veterinary Parasitology, Veterinary College and Research Institute, Orathanadu, Tamil Nadu,
India, *E-mail: [email protected]
1
reports an outbreak of trypanosomosis in buffaloes
caused by Diminazine aceturate resistant strain of
T. evansi.
showed typical clinical symptoms of clinical
trypanosomosis with parasitaemia level of +++,
while the remaining 8 animals though they
harboured moderate parasitaemia level of ++, did
not exhibit any symptoms (Figure 1). The findings
of the present investigation are in consonance
with Lang (2001) who recorded trypanosomosis
in an average of 7.97 percent buffaloes in delta
areas in Vietnam by blood smear examination
and immunodiagnostic method. Lang (1984) also
reported that buffaloes suffered with surra had
heavy clinical signs and died more when they meet
a lot of environmental stress and the light infection
rates in buffaloes could be associated with the
environmental factors rather than host factors,
but this observations do not corroborate with the
findings of the present study. Because, the hot and
humid climatic conditions prevailed here during
month of August might definitely have caused
much stress to animals, despite of this barring two
animals others did not exhibit any clinical signs.
This observation is in agreement with Aulakh
(2003) who reported that buffaloes exhibited latent
infection and more than 50-80 percent of infections
are cryptic and undetectable by direct microscopy.
In this case, intriguingly Trypanosoma
evansi with parasitaemia level of +++ was observed
in the blood smear obtained after diminazene
treatment (Figure 2). But the drug Antrycide
Prosalt, given on subsequent day, was able to
clear the parasites clearly. These observations
have prompted to suspect that animals might have
been infected with diminazene resistant strain of
Trypanosoma evansi. The observations recorded in
the present case are akin to the findings of Elamin
et al. (1982) who stated that single doses of 3.5 mg/
HISYORY AND OBSERVATIONS
The buffalo unit of District Livestock
Farm (DLF), Orathanadu, Thanjavur district,
is located inside the newly started Orathanadu
Veterinary College campus in Tamil Nadu. A total
of 144 buffaloes are being maintained there. The
department of Parasitology received the blood
smears obtained from buffalo with a history of fever
(104°C), oedema of the legs, frequent micturition
and pale visible mucus membrane. The blood smears
were stained with Giemsa stain and examined under
oil immersion. The affected animals were initially
treated with Diminazine aceturate 3.5 mg / kg b.wt
– i.m and then with Antrycide Prosalt 7.4 mg/kg
b.wt s/c. After each treatment blood smears were
collected and examined to ascertain that whether
parasites are eliminated or not. The blood smears
were also collected from remaining animals and
screened for Trypanosoma evansi. Animals those
found to be harboured T.evansi without clinical
signs and animals which diagnosed negative for
T.evansi as well were treated with Antrycide Prosalt
7.4 mg/kg b.wt s/c. A day after the treatment, the
blood smears were collected from T.evansi infected
animals and examined to monitor post treatment
parasitaemia level.
RESULTS AND DISCUSSIONS
In the present investigation, of the 144
buffaloes 10 animals (6.9 %) were found positive
for trypanosomosis. But, only two animals
kg of berenil were less effective against T.evansi in
mice. Gill (1991) also stated that there are variable
reports on the therapeutic efficacy of diminazene
2
Figure 1. Parasitaemia before Diminazene aceturate treatment.
Figure 2. Parasitaemia after Diminazene aceturate treatment.
3
aceturate in buffaloes. In a similar vein Singh
and Joshi (1991) observed that prophylactically
single dose of diminazene (10 mg/kg) was not
effective as there was persistence of T. evansi in
buffaloes 48 and 30 days after treatment. They also
reported that Quinapyramine and isometamedium
were good therapeutic agents but prophylactically
Quinapyramine proved better than isometamedium.
In contrast, Aulakh (2003) reported that there was
progressive decrease in number of trypanosomes
immediately after treatment and blood smear was
cleared of trypanosomes within eight hours of
treatment with berenil (Diminazene aceturate) 5
mg/kg body weight.
(caused by T. evansi) in the northern provinces
of Vietnam, p. 165-172. In Results of study
on Veterinary Science and Technique from
1979-1985 of NIVR. Agriculture Publishing
House, Hanoi, Vietnam.
Lang, P.S. 2001. Studies on incidence and control
of Trypanosomiasis in buffaloes caused by
Trypanosoma evansi steel 1885 in North
Vietnam, p. 1-8. In Proceedings of Buffalo
Workshop, Vol. 1. Hanoi, Vietnam.
Shaba, P., O.P. Sharma, N.P. Kurade, J.R. Rao, R.K.
Singh, N.N. Pandey and Bhanu Prakash.
2006. In vitro antitrypanosomal activity
and cytotoxicity of methanolic extract of
Plumbago zeylanica against Trypanosoma
evansi. J. Vet. Pub. Health, 4: 31-36.
Singh, B. and S.J. Joshi. 1991. Epidemiology,
clinicopathology and treatment of clinical
Trypanosoma evansi infection in buffalo
(Bubalus bubalis). Indian Vet. J., 68: 975979.
Witola, W.H., A. Tsuda, N. Inoue, K. Ohashi and
M. Onuma. 2005. Acquired resistance to
berenil in a cloned isolate of Trypanosoma
evansi is associated with upregulation of
a novel gene, TeDR40. Parasitology, 131:
635-646.
ACKNOWLEDGEMENT
Authors thank Deputy Director and
Veterinary Assistant Surgeons working in the DLF,
Orathanadu for the support extended to carry out
the study.
REFERENCES
Aulakh, G.S. 2003. Haemato-biochemical and
therapeutic studies on haemoprotists
in bovines. M.V. Sc., Thesis, Punjab
Agricultural University, Ludhiana, India.
Elamin, E.A., A.M. Homeida and S.E. Adam.
1982. The efficacy of berenil (diminazene
aceturate) against Trypanosoma evansi
infection in mice. J. Vet. Pharmacol. Ther.,
5: 259-265.
Gill, B.S. 1991. Trypanosomes and Trypanosomiosis
in Indian Livestock. ICAR Publication, New
Delhi, India. p. 2-12.
Lang, P.S. 1984. Epidemiology of Trypanosomiasis
4
Original Article
EVALUATION OF UREA MOLASSES MULTI-NUTRIENT BLOCKS CONTAINING
ALTERNATE FEED RESOURCES IN BUFFALOES
M. Choubey, M. Wadhwa and M.P.S. Bakshi*
ABSTRACT
compared to control group. The blood urea nitrogen
(BUN) was higher (P<0.05) in animals offered
UMMBs as compared to animals in control group.
The purine derivatives (PDs) excreted in the urine
were comparable in all the groups. All the animals
gained weight, but the differences were statistically
non significant. It was concluded that WB and TP
could be incorporated into UMMBs without any
adverse effect on palatability, nutrient utilization,
rumen metabolites or health of buffaloes.
The present study was undertaken to
formulate and compare the nutritional worth of
conventional urea molasses multinutrient block
(UMMB) with UMMB containing waste bread
(WB) and/or tomato pomace (TP) in buffaloes.
Wheat flour in the UMMB was replaced with
WB and oiled mustard cake with TP. The in vitro
digestibility of nutrients, release of ammonia and
partitioning factor were statistically comparable.
UMMB containing WBTP resulted in higher
total volatile fatty acids (VFAs) production and
availability of metabolizable energy (ME). 20
male Murrah buffaloes (442.1±6.3 kg BW) were
randomly distributed into five equal groups.
The animals in control group were offered 2 kg
conventional concentrate mixture supplemented
with 5 kg green fodder and 9 kg wheat straw.
Same feeding schedule was followed for animals
in the experimental groups, except that in place
of 2 kg conventional concentrate mixture, only 1
kg conventional concentrate mixture was offered
with ad lib conventional UMMB or the one
containing WB, TP or WBTP. The daily intake of
block varied from 1.08 kg (conventional) to 1.84
kg (TP). The DM intake was comparable in all the
groups. Supplementation of UMMBs in the diet
of experimental animals improved (P<0.05) the
digestibility of CP and TCA-N concentration in the
rumen resulted in higher (P<0.05) N-retention as
Keywords: in-vitro/in-vivo, nutrient availability,
rumen metabolites, tomato pomace, urea molasses
multinutrient block, waste bread
INTRODUCTION
The poor quality crop residues constitute
the bulk of dry matter consumed by the ruminants
under field conditions (Bakshi and Wadhwa, 2011).
The enrichment of such poor quality roughages
with urea and other NPN resources like UMMB
improved the nutrient utilization (Bakshi et al.,
1986; Wadhwa and Bakshi, 2011a,b) and improved
milk production (Lamba et al., 2002), thus helped
to save oil seed cake/concentrate for vulnerable
species. The demand and cost of conventional
energy (starch/wheat flour) and protein (groundnut/
mustard cake) supplements have escalated due
Department of Animal Nutrition, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana,
5
to dynamic explosion of human population and
urbanization. There is a dire need to exploit alternate
energy and protein supplements for ruminants.
Waste bread (left over, unsold, fungal infested etc.)
available in abundance, is an excellent source of
cooked bypass starch and protein. Tomato pomace
is another potential feed resource (consisting of
tomato peels, seeds and damaged tomatoes) a good
source of lycopene, a pigment that gives colour
WB, while mustard cake was replaced with TP on
nitrogen basis. The required quantity of molasses
and urea were weighed and mixed in a 25 kg
capacity iron pan. The guar gum was added to the
urea-molasses mixture as a binder in UMMBs. A
premix of other ingredients was prepared (CaO was
the last ingredient added to this premix) and added
to iron pan with rapid stirring. Heat generated at
this stage, converted the contents into a semi-solid
mass, which was put into rectangular die of block
making machine. The solidified UMMBs were
packed into polythene bag.
to meat and is a known antioxidant. However,
till date neither WB nor TP has been used in the
formulations of UMMB. This study was therefore,
planned to formulate UMMBs containing alternate
energy and protein supplements like WB and/or TP
and compare these with conventional UMMB and
to assess the effect of such UMMBs on nutrient
utilization in buffaloes.
In-vitro and in-vivo evaluation of different
UMMBs
The net gas production, digestibility of
nutrients and availability of ME from different
UMMBs was assessed by in vitro gas production
technique (Menke et al., 1979).
For in vivo evaluation 20 male Murrah
buffaloes (5-6 yr old of 442.1±6.3 kg body
weight) were randomly distributed into five
equal groups. The animals in the control group
were fed 2 kg conventional concentrate mixture
(maize 30, mustard cake 10, solvent extracted
mustard cake 20, rice bran 15, solvent extracted
rice bran 22, mineral mixture 2 and common salt
1 percent each) supplemented with 5 kg green
fodder and 9 kg wheat straw (NRC, 2001). Same
feeding schedule was followed for animals in the
experimental groups, except that in place of 2
kg conventional concentrate mixture, only 1 kg
conventional concentrate mixture was offered with
ad lib conventional UMMB or the one containing
WB, TP or WBTP. The animals were weighed for 3
consecutive days at 15 days interval before feeding
and the feeding schedule was adjusted accordingly.
At the termination of experimental period, a 7-day
metabolism trial was conducted. The samples of
MATERIALS AND METHODS
Procurement of alternate energy and energy
cum protein supplements
Waste bread procured from Cremica
Industries, Phillaur, was sun dried for 14 h in order to
eliminate the aflatoxin, if any (Gowda et al., 2005).
The sundried, ground waste bread was got tested
for the level of mycotoxins, from the Department
of Veterinary Microbiology, GADVASU. The
tomato pomace (containing tomato peels, seeds
and damaged tomatoes) was procured free of cost
from Nijjar Agro Industries, Amritsar. The tomato
pomace was sun dried and finely ground.
Preparations of UMMBs
Iso-nitrogenous and iso-caloric UMMBs
were prepared by manipulation of feed ingredients
(Table 1) in a block-making machine. In
experimental blocks wheat flour was replaced with
6
from each animal were collected for 3 consecutive
days at 2 hourly intervals, starting from zero and
continuing up to 12 h post-feeding. The rumen
liquor samples were strained through four layered
muslin cloth and few drops of saturated mercuric
chloride solution were added to arrest the microbial
activity. The samples of strained rumen liquor
(SRL) were pooled for the respective animal and
the pH was measured immediately and the samples
were stored in a refrigerator till analyzed. The
SRL samples were analyzed for TCA- precipitable
nitrogen, non-protein nitrogen (NPN), ammonical
nitrogen (AOAC, 2005) and total VFAs (Barnett
and Reid, 1957).
WB, TP, different UMMBs, concentrate mixture,
wheat straw, green fodder, feed residue and faeces
were analyzed for their proximate constituents
(AOAC, 2005), cellulose (Crampton and Maynard,
1938) and other cell wall constituents (Robertson
and Van Soest, 1981). The urine sample (10 ml)
was kept in a vial containing 0.5 ml of 20% H2SO4
to keep the pH below 3 and analyzed for allantoin
(Young and Conway, 1942), uric acid (Trivedi et
a1., 1978) and creatinine (Folin and Wu, 1919).
Purines absorbed were calculated from the daily
urinary PD excreted (IAEA, 1997).
Collection and analysis of rumen liquor
samples
The rumen studies were conducted on
three rumen fistulated male buffaloes for assessing
the effect of supplementing different blocks on
the rumen metabolites. After 30 days adaptation
on a particular ration, the rumen liquor samples
Collection and analysis of blood samples
At the termination of metabolism trial
blood samples were collected (in heparin and
sodium flouride + oxalate vials) from the juglar
vein of animals at 4 h post parandial. The serum
Table 1. Ingredient composition of different urea molasses multinutrient blocks (UMMBs), g/3 kg lick.
Ingredients
Conventional
WB
TP
WBTP
Molasses
900
900
900
900
Urea
Mustard cake
Deoiled rice
bran
Wheat flour
Waste bread
Tomato pomace
Mineral
mixture
Calcium oxide
Salt
Guar gum
300
300
300
304
300
300
315
300
319
300
450
450
450
450
450
300
450
450
300
450
120
120
60
120
115
60
120
105
60
120
95
60
WB- Waste bread-UMMB; TP- Tomato pomace-UMMB; WBTP-Waste bread tomato pomace-UMMB.
7
was separated and stored at 0oC till analyzed. The
analysis was conducted on Erba (Mannheim) Chem
5X (Transasia). The serum collected with sodium
fluoride and oxalate was used for assay of blood
glucose (Trinder, 1969), total protein (Henry et al.,
1974), albumin (Doumas et al., 1971), globulin
was calculated by difference between total protein
and albumin, urea (Evans, 1968), calcium (Henry
and Dryer, 1963) and phosphorus (Amador and
Urban, 1972).
The data were analyzed by simple ANOVA
(Snedecor and Cochran, 2004) by using SPSS
(2007) version 16 and means were compared by
using Tukey’s b test.
feedstuffs.
In-vitro evaluation of different UMMBs
The net gas production and digestibility of
OM and NDF; release of ammonia and partitioning
factor were similar in all the blocks (Table 3).
The TVFAs production varied from 8.95 (TPUMMB) to 9.75 meq/dl (Conventional-UMMB).
Replacement of cereal grains with WB (both in WB
and WBTP blocks) showed no significant effect on
the production of TVFA, and level was statistically
comparable (P>0.05) to that of ConventionalUMMB. However, replacement of mustard cake
with TP alone (TP-UMMB) resulted in depression
(P<0.05) in TVFAs as compared to that produced
from the Conventional-UMMB. The availability of
ME from different blocks varied from 5.71 (WBUMMB) to 6.03 MJ/kg DM (WBTP-UMMB).
A combination of WB and TP (WBTP-UMMB)
proved to be a better option as far as production
of VFAs and availability of ME was concerned.
These results showed that the incorporation of WB
and/or TP in the blocks would not affect nutrient
utilization.
RESULTS AND DISCUSSION
Chemical composition of the feedstuffs
The WB used in this study had negligible
level of mycotoxins. The WB and TP contained 12.5
and 20.9% CP, 1.3 and 11.0% EE respectively on
dry matter basis (Table 2). WB is an excellent source
of bypass starch (Bhargava, 2008). Besides energy
and protein, TP is a good source of phosphorus,
essential fatty acids (linoleic acid), lysine, vitamin
E and lycopene, a pigment which gives a typical
colour to meat and acts as an antioxidant (Wenli
et al., 2001; Kravchenko et al., 2003). The cell
wall constituent i.e. NDF, ADF, cellulose and hemi
cellulose were much higher in TP as compared to
that in WB. The total ash content was comparable
in all UMMBs. The high ash content in the blocks
could be due to high level of mineral mixture used
(150 g vs. 10 g/kg). The comparable CP (41.2 to
41.7%) and EE (1.2 to 1.83%) in different UMMBs
revealed that the blocks were iso nitrogenous
and iso caloric. The WB had the lowest cell wall
constituents followed by different blocks and other
Impact on feed consumption and digestibility of
nutrients
The daily intake of concentrate mixture
was higher (P<0.05) in the control group as
compared to those offered UMMBs (Table 4). But
within the UMMB groups, it was comparable.
The daily intake of UMMBs varied from 1.08 kg
(Conventional-UMMB) to 1.84 kg (TP-UMMB).
The comparable DM intake in all the experimental
groups suggested that incorporation of WB and/or
TP did not have any negative effect on palatability of
blocks, rather improved the consumption of wheat
straw. Tiwari et al. (1990) and Toppo et al. (1997)
also observed increased consumption of DM in the
8
9
2.8
97.2
12.5
1.3
10.0
2.0
1.5
8.0
Total ash
OM
CP
EE
NDF
ADF
Cellulose
Hemicellulose
93.1
20.9
11.0
68.0
53.0
38.0
15.0
6.9
TP
90.5
21.4
4.1
30.0
14.0
8.0
15.5
9.5
Concentrate
mixture
WB- Waste bread; TP- Tomato pomace.
WB
Constituents
72.4
41.2
1.4
11.0
6.3
2.0
4.8
27.6
Conventional
Table 2. Chemical composition of waste bread and tomato pomace, % DM.
72.8
41.4
1.2
10.0
6.0
2.0
4.0
27.2
WB
UMMBs
72.9
41.6
1.83
13.5
7.5
3.0
6.0
27.1
TP
73.5
41.7
1.83
12.0
7.3
2.5
4.8
26.5
WBTP
86.8
20.8
2.0
46.0
35.0
18.0
11.0
13.2
Green
92.5
3.4
1.0
78.0
50.5
41.0
27.5
7.5
Wheat
straw
Table 3. In vitro utilization of nutrients from different UMMBs.
Parameter
Conventional
WB
TP
WBTP
PSE
NGP, ml/g
DM/24h
OMD, %
NDFD, %
PF
95.70
94.49
93.64
93.44
0.51
69.87
10.39
3.84
69.57
10.27
3.89
69.26
10.15
3.83
69.45
10.39
3.85
0.10
0.11
0.03
NH3-N, %
0.058
0.057
0.057
0.056
0.00
TVFA, meq/dl
ME, MJ/kg DM
9.75
5.78ab
9.50
5.71a
8.95
5.89b
9.50
6.03c
0.12
0.04
b
ab
a
ab
NGP- Net gas production; D-Digestibility; PF- Partitioning factor; Figures with different
superscripts in a row differ significantly (P<0.05).
Table 4. Consumption of different feedstuffs, kg/d.
Feedstuffs
Conc.
Mixture
UMMB
Wheat straw
Green fodder
Total DM
Control
UMMBs
PSE
Conventional
WB
TP
WBTP
1.84b
0.92a
0.92a
0.92a
0.92a
0.09
-7.34
1.08
7.78
1.30
8.15
1.84
8.10
1.22
7.88
0.06
0.16
1.01
0.80
0.80
0.80
0.80
0.04
10.98a
12.06b
12.01b
12.12b
11.96b
0.12
62.10
b
62.31
58.40
59.58
2.11
18.88
81.12
9.33
90.67
19.82
80.18
8.89
91.11
18.52
81.48
8.94
91.06
19.70
80.30
9.17
90.83
0.40
0.40
0.65
0.64
52.62
55.81
66.83b
47.75
41.44
51.64
55.89
65.79b
48.25
40.56
53.64
56.52
64.06b
48.25
41.43
51.70
55.74
62.77b
47.94
41.08
1.43
1.25
2.53
1.44
1.64
Water, l/d
46.58
Roughage to concentrate ratio
Concentrate
18.25
Roughage
81.75
Green
12.44
Straw
87.56
Digestibility of nutrients, %
DM
48.10
OM
51.18
CP
41.30a
NDF
46.00
ADF
38.08
a
b
ab
Figures with different superscripts in a row differ significantly (P<0.05).
10
ab
UMMB supplemented groups. The higher water
intake (P<0.05) in animals offered UMMBs as
compared to animals in the control group could be
due to higher intake of urea and minerals through
blocks. The water consumption by animals offered
blocks was statistically comparable.
The supplementation of UMMBs improved
(P<0.05) the digestibility of crude protein in
comparison to un-supplemented control group.
Tiwari et al. (1990) and Toppo et al. (1997) also
observed similar results for CP digestibility in
UMMB supplemented groups. The supplementation
of UMMBs improved (P>0.05) the digestibility of
DM, OM and cell wall constituents in comparison to
that of control, but the differences were statistically
non significant.
was higher (P<0.05) in animals offered UMMB
supplemented diets as compared to conventional
control group. But within the UMMB supplemented
groups the differences were statistically non
significant. Although, BUN was higher in treatment
groups, but no symptoms of urea toxicity was
observed during the study period and all animals
were found to be active and in good health. The
possibility of less heat generation (by CaO during
mixing) required to produce maillard product,
could not be ruled out. The plasma concentration of
different parameters was within the range of values
(Jain, 1996; Kaneko, 1997).
Impact on urinary excretion of purine
derivatives
Allantoin, uric acid and the total purine
derivatives excreted in urine of animals were
comparable in all the groups (Table 6). Allantoin
constituted the major (83-91%) proportion of total
PD excreted in urine. The purine derivates absorbed
and microbial protein synthesized in the rumen and
in turn utilized in the lower gastro-intestinal tract
were also comparable in all the groups, indicating
that nutrients from different UMMBs were utilized
effectively.
Impact on rumen metabolites and blood profile
The TVFA concentration in the rumen was
comparable in all the groups while pH remained
almost constant throughout the study (Table 5).
It indicated that higher consumption of different
blocks did not have any adverse effect on rumen
environment. The NPN concentration was highest
(P<0.05) in the rumen liquor of animals offered
control diet, while it was lowest in the animals
offered diet supplemented with UMMB containing
WB and TP. Ammonia-N as expected was higher
(P<0.05) in rumen liquor of animals offered
diet supplemented with UMMBs (except that in
WBTP-UMMB), confirming the earlier reports
(Toppo et al., 2000; Jain et al., 2005). The efficient
utilization of NPN resulted in higher concentration
of TCA-N in WBTPL group as compared to other
groups, confirming that WBTPL provided nutrients
synchronized in energy and protein.
Supplementing the control diet with different
UMMBs did not have any significant impact on the
blood profile of animals. However, the BUN level
Nitrogen utilization and body weight changes
The N-intake was higher (P<0.05) in the
animals offered different iso-nitrogenous blocks
as compared to control group (Table 7). It could
be due to higher N content in blocks than that of
conventional control concentrate mixture and
higher licking of block (1.08 to 1.84 kg/animal/
day) as compared to expected intake (500 g/
animal/d). The urinary-N excretion was higher
(P<0.05) in animals offered UMMBs as compared
to those in control group. The urinary-N excretion
was statistically comparable in animals offered
11
Table 5. Supplementation of UMMBs and rumen metabolites.
Parameter
Control
Rumen metabolites
pH
6.83
TVFA, meq/dl
9.10
TCA-N, mg/dl
48.60
NPN, mg/dl
35.91b
NH3-N, mg/dl
11.14
Blood profile, mg/dl
Glucose
48.14
BUN
21.32a
Total protein,
6.64
g/dl
Albumin (A),
1.90
g/dl
Globulin (G),
4.75
g/dl
A:G
0.40
Calcium
10.45
Phosphorus
8.18
UMMBs
WB
Conventional
TP
WBTP
PSE
6.85
9.47
41.83
33.13ab
15.46
6.90
9.00
38.29
23.88a
12.97
6.88
9.00
42.16
24.40a
12.10
6.80
9.13
48.32
26.11ab
10.39
0.01
0.28
1.59
1.60
0.72
49.75
47.48b
47.52
44.28b
58.52
38.25b
50.55
44.20b
1.74
2.54
7.30
7.66
7.54
6.76
0.25
2.08
2.30
2.16
2.12
0.07
5.22
5.36
5.38
4.56
0.21
0.40
10.82
9.31
0.45
11.62
10.22
0.41
11.59
11.36
0.51
10.96
11.06
0.02
0.32
0.66
12
Table 6. Supplementation of blocks and urinary purine derivatives in adult buffaloes.
Parameter
Allantoin (A),
mM/d
Uric acid (UA),
mM/d
Purine
derivatives
(PD), mM/d
Creatitine,
mM/d
A as % of PD
UA as % of PD
Purines
absorbed,
mM/d
MNS, g/d
Control
UMMBs
WB
Conventional
TP
WBTP
PSE
35.0
31.86
27.43
28.24
31.92
1.76
7.50
7.74
2.59
4.42
3.12
1.04
42.50
39.60
30.02
32.66
35.04
2.70
37.26
44.19
37.43
48.29
36.72
1.98
84.32
15.68
83.26
16.74
91.12
8.88
86.00
14.00
90.97
9.03
1.41
1.41
186.65
164.70
83.58
103.16
119.02
23.49
135.70
119.75
60.76
75.01
86.53
17.07
Table 7. Supplementation of blocks and nitrogen retention in adult buffaloes, g/d.
Parameter
Control
Nitrogen balance, g/day
N-Intake
131.57a
Faecal-N
77.00
Urinary-N
33.41a
N-outgo
110.41
N-Retained
21.16a
BV
15.68
Body weight changes
Initial BW, kg
437.98
Final BW, kg
450.64
Gain in BW
350.18
g/d
Conventional
UMMBs
WB
TP
WBTP
PSE
201.69b
66.93
59.71b
126.64
75.04b
36.88
220.31b
74.47
53.33ab
127.8
92.51b
41.52
207.14b
74.77
56.71ab
131.48
75.57b
36.62
213.10b
79.32
51.94ab
131.26
81.83b
38.44
8.03
2.45
3.10
5.55
7.24
3.32
441.05
451.65
445.58
457.50
438.28
454.25
447.85
466.52
6.39
4.64
386.74
409.72
603.85
359.55
107.58
13
UMMBs. The N retention was higher (P<0.05) in
animals offered UMMBs as compared to control
group. The apparent biological value was also
higher (P>0.05) in the animals supplemented with
blocks as compared to those in control group.
Bakshi, M.P.S. and M. Wadhwa. 2011. Nutritional
status of dairy animals in different regions of
Punjab State in India, Indian J. Anim. Sci.,
81: 52-58.
Bakshi, M.P.S., V.K. Gupta and P.N. Langar. 1986.
Fermented straw as a complete basal ration
for ruminants, Agr. Wastes, 16: 37-46.
Barnett, A.J.G. and R.L. Reid. 1957. Studies on the
production of volatile fatty acids from the
grass in artificial rumen.1. Volatile fatty acid
production from fresh grass, J. Agr. Sci., 13:
315-321.
Bhargava, A. 2008. Study on waste bread as non
conventional energy supplement for buffalo
calves. M.V. Sc. Thesis, Guru Angad Dev
Veterinary and Animal Sciencies University,
Ludhiana, India.
Crampton, E.W. and L.A. Maynard. 1938. The
relation of cellulose and lignin content to
the nutritive value of animal feeds. J. Nutr.,
15: 383-395.
Doumas, B. T., W. A. Watson and H. G. Briggs. 1971.
Albumin standards and the measurements
of serum albumin with bromocresol green.
Clin. Chim. Acta, 31: 87-96.
Evans, R.T. 1968. Manual and automated methods
for measuring urea based on a modification
of its reaction with diacetyl monoxime and
thiosemicarbazide. J. Clin. Pathol., 21: 527529.
Folin, D. and H. Wu. 1919. A system of blood
analysis. J. Biol. Chem., 38: 81-110.
Gowda, N.K.S., V. Malathi, R.U. Suganthi and
A. Raghvendra. 2005. Effect of dry heat
and sunlight on the aflatoxin content in
compounded feed. Indian J. Anim. Nutr.,
22: 132-134.
Henry, R. J. and R. L. Dryer. 1963. Standard Method
of Clinical Chemistry, Vol. 4, Academic
Changes in live weight
The average daily gain in weight of all the
animals offered diet supplemented with UMMB
was higher than that of control group, but the
differences were statistically non significant. The
animals offered diet supplemented with UMMB
containing TP gave the highest gain/d (603.85
g/d) while gain was lowest (350.18 g/d) for unsupplemented control group.
CONCLUSION
It was concluded that non-conventional
feed resources like waste bread and tomato pomace
could be incorporated into UMMBs without any
adverse effect on palatability, nutrient utilization,
rumen metabolites or health of animals. Above all
the preparation of UMMB could be economized
and conventional ingredients could be spared for
more vulnerable species.
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16
Original Article
EFFECT OF SUPPLEMENTATION OF TINOSPORA CORDIFOLIA ON LACTATION
PARAMETERS IN EARLY LACTATING MURRAH BUFFALOES
N.A. Mir1,*, P. Kumar1, S.A. Rather2, F.A. Sheikh3 and S.A. Wani4
ABSTRACT
SNF % however significant change was observed
in milk protein % of treatment group compared
to control group. No significant difference in total
milk Ig was observed between control and treatment
group. The DMI showed an increasing trend with
significant difference from day 11 up to day 75 of
lactation between control and treatment.
The present study was conducted to study
production parameters of lactating Murrah buffaloes
supplemented with Tinospora cordifolia. Twelve
lactating Murrah buffaloes in early stage of lactation
were selected from the herd of National Dairy
Research Institute Karnal, Haryana. The buffaloes
were divided into two groups of six animals each.
One group was taken as control and the other group
supplemented with Tinospora cardifolia 120 g/
animal/day from day 3 to day 75 of lactation was
taken as treatment group. All the buffaloes were
hand milked throughout the experimental period.
Keywords: Murrah buffaloes, Bubalus bubalis,
Tinospora cordifolia, lactating
INTRODUCTION
Buffalo is the major source of milk
production and contributes more than 54% of
annual milk production in India. The buffalo has
evoked worldwide interest as an animal with
potential for meeting the emerging demand for
meat, milk and work in developing countries.
Further, in countries like India, the buffalo is the
major milch animal, accounting for more than 50%
of the milk produced. However it is well known fact
that large amount of milk produced is not because
of higher productivity but because of the higher
population of animals. The low productivity of
buffaloes is primarily due to poor genetic potential,
inadequate supply of nutrients and unscientific
Milk samples from mixed whole milking were
collected early in the morning in sterilized milk
sampling bottles from all the animals upto 75th
day of lactation. The milk samples were analyzed
for somatic cell count, composition and milk total
immunoglobulin’s. The milk yield was recorded
daily. Significant increase (P<0.05) in milk yield
of treatment group as compared to control group
was obtained. The milk somatic cell count was
significantly lower in treatment group as compared
to control group. The milk composition (fat %,
protein %, lactose % and SNF %) was estimated
using LactoScan milk analyzer. No significant
change was observed in milk fat %, lactose % and
DCP Division, 2ABC Division, 3DCN Division, 4 DX Division, National Dairy Research Institute,
Karnal, Haryana, India, *E-mail: [email protected]
1
17
approach in feeding. Hence in order to improve
the productivity of buffaloes, there is need to adopt
scientific feeding strategies.
Guduchi (Tinospora cordifolia) is a large
glabrous deciduous climbing shrub belonging to
family Menispermaceae. It is one of the most versatile
rejuvenating herbs found throughout tropical Indian
subcontinent. Commonly known as a rasayan
plant, it is widely used in veterinary folk, ayurveda
and other systems of medicine for its general tonic,
immunomodulatory, antioxidant, antibacterial,
hepatoprotective and anti-inflammatory properties
(Krishna et al., 2009). Guduchi itself means the
“one which protects our body” and Amrita means
“the nectar that confers immortality”. In Hindi
the plant is commonly known as ‘giloya’, which
is a Hindu mythological term that refers to the
heavenly elixir that has saved celestial beings from
old age and keeps them eternally young. Though
every part of plant has therapeutic value the stem
is used in most of the medicinal preparations. It is
claimed that the plant climbing up the Neem tree
is said to be the best as synergy between these two
bitter plants enhances guduchi’s efficacy. A variety
The experiment was conducted in cattle
yard of National Dairy Research Institute, Karnal,
Haryana, India. Twelve early lactating murrah
buffaloes were selected from institute herd. The
animals were in 2nd lactation number with the
mean body weight of 480 kg. The experiment
was conducted during the months of april to
june. Animal experimentation was performed in
compliance with regulations set by the cattle yard,
NDRI and approved by the Institutional Animal
Ethics committee.
The nutrient requirements of animals
were met by feeding concentrate mixture and
green fodders as per NRC 1989 .Animals had
round the clock access to ad libitum fresh water.
The dried cylindrical stem pieces of Tinospora
cordifolia were collected from the local ayurvedic
shop. Authentication of the stem was performed
by the ayurvedic doctor in the institute health
complex. The stems were ground to powder form
in a medicinal herb grinding machine, weighed and
packed in polythene. The animals under treatment
group were fed dried guduchi stem powder by
mixing it in small amount of concentrate 120g/day
for a period of 72 days after calving(from day 3rd
postpartum upto 75th day postpartum). The control
animals were fed equal amount of concentrate
without guduchi powder for similar period. Both
control and treatment buffaloes were hand milked
throughout experimental period. Milk samples
from mixed whole milking were collected early
in the morning in sterilized milk sampling bottles
from all the animals upto 75th day of lactation on
days 3, 11,19, 27, 35, 43, 51, 59, 67 and 75 of
lactation. Milk samples were analyzed for somatic
cell count by the method of (Singh and Ludri
2001). Milk composition (Fat %, SNF %, Lactose
of constituents belonging to different classes such
as alkaloids, diterpenoid lactones, glycosides,
ecdysteroids, sesquiterpenoids, phenolics, aliphatic
compounds and polysaccharides have been isolated
from Tinospora cordifolia ( Singh et al., 2003).
In present times, Tinospora cordifolia has been
subjected for numerous chemical, Pharmacological,
Pre-clinical and clinical investigations and many
new therapeutic applications have been indicated,
however in buffaloes no study has been conducted
regarding supplementation of Tinospora cordifolia.
Thus present study was undertaken to study the
effect of supplementation of Tinospora cordifolia
on lactation parameters of murrah buffaloes during
early lactation.
18
infusion of hydromethanolic extract of Tinospora
cordifolia in bovine subclinical mastitis initially
enhanced somatic cell count but a significant
reduction in somatic cell count was observed on
day 15 of the treatment period.
% and protein %) was estimated using Lacto Scan
milk analyzer (Netco Company), The milk samples
were maintained at 28-32oC at the time of analysis
,which is the calibration temperature of analyzer.
Milk total immunoglobulin’s were estimated by
zinc sulphate turbidity method of( McEvan and
Fisher, 1970). Dry matter intake was estimated at
weekly intervals.
Milk composition
There was no significant difference (P>0.05)
in the milk fat%, SNF% and lactose% of control
and treatment groups of lactating Murrah buffaloes,
however there was significant (P.<0.05) difference
in milk protein % of control and treatment groups,
being higher in treatment group as compared to
control group. However no literature is available in
large animals for comparison of our study.
Milk yield
The overall average milk yield of control
and treatment group of lactating murrah buffaloes
was 7.19±0.10 and 8.00±0.12 kg/day. The
milk yield of treatment group of buffaloes was
significantly (P<0.05) higher from 12-19th day
Dry matter intake
The overall average dry matter intake
in control and treatment groups was 11.27 and
11.88 (kg/day). The dry matter intake showed an
increasing trend with significant difference between
control and treatment group from day 15th up to
75th day postpartum. The percent increase in dry
matter intake of treatment group as compared to
control group was 5.13%. The increasing trend of
dry matter intake during the period of our study is
supported by Ingvartsen and Anderson, 2000; they
reported that increase in dry matter intake during
the lactation is the result of greater sensation of
hunger caused by the rapid increase in nutrient
demand. However no literature is available in large
animals for comparison of our study.
of lactation. There was 10.10% increase in milk
yield of treatment group as compared to control
group of buffaloes. (Mallick and Prakesh, 2011
a)also reported significant increase in milk yield
of guduchi supplemented cows as compared to
untreated control group.
Somatic cell count
Milk somatic cell count was higher on day
3rd of lactation in both control and treatment group
and decreased thereafter, but the reduction was
more in treatment group as compared to control
group. The overall average of somatic cell count
was significantly lower (P<0.05) in treatment group
as compared to control group of buffaloes. Similar
results were reported by (Mallick and Prakash,
2011b), they reported that somatic cell count was
significantly higher in untreated control cows
through the period of experiment as compared to
guduchi supplemented group of cows. (Mukherjee
et al, 2006) also reported that intramammary
Milk total immunoglobulin’s
The overall average of milk total
immunoglobulin level of control and treatment
group of lactating murrah buffaloes was 2.00±1.55
and 2.01±1.54 mg/ml. No significant difference
(P>0.05) was observed in milk total immunoglobulin
19
levels in neonatal calf serum. Clin. Chim.
Acta., 17: 155.
Mukherjee, P.K., K. Maiti, K. Mukherjee and
P.J. Houghton. 2006. Leads from Indian
medicinal plants with hypoglycemic
potentials. J. Ethnopharmacol., 106: 1-28.
National Research Council. 1989. Nutrient
Requirements of Dairy Cattle, 6th revised ed.
National Academy of Science, Washington,
DC.
Singh, M. and R.S. Ludri. 2001. Influence of stages
of lactation, parity and season on somatic
cell counts in cows. Asian Austral. J. Anim.,
14(12): 1775-1780.
Singh, S.S., S.C. Pandey, S. Srivastava, V.S. Gupta,
B. Patro and A.C. Ghosh. 2003. Chemistry
and medicinal properrties of Tinospora
cordifolia (Guduchi). Indian J. Pharmacol.,
35: 83-91.
level of control and treatment group of lactating
murrah buffaloes during the experimental period.
CONCLUSION
Supplementation of Tinospora cordifolia
increased milk production (10%) and milk quality
in terms of reduction in somatic cell count.
Supplementation of Tinospora cordifolia also
enhanced dry matter intake (5%) of lactating
murrah buffaloes, however there is need to carry
out study in large group of animals and isolation
of various constituents of Tinospora cordifolia for
studying their pharmacological actions in bovines
at cellular level.
REFERENCES
Ingvartsen, K.L. and J.B.Andersen. 2000. Integration
of metabolism and intake regulation: a
review focusing on periparturient animals.
J. Dairy Sci., 83: 1573-1597.
Krishna, K., B. Jigar and P. Jagruti. 2009. Guduchi
(Tinospora cordifolia): Biological and
Medicinal properties, a review. Int. J. Alt.
Med., 6(2): 1-12.
Mallick, S. and B.S. Prakash. 2011a. Effects of
supplementation of Tinospora cordifolia to
crossbred cows peripartum. Anim. Reprod.
Sci., 123(1-2): 5-13.
Mallick, S. and B.S. Prakash. 2011b. Influence of
feeding Tinospora cordifolia on lactation
parameters in crossbred cows. J. Anim.
Physiol. An. N. doi:10.1111/j.14390396.2011.01228.x.
McEvan, A.D. and E.W. Fisher. 1970. A turbidity
test for estimation of immunoglobulin
20
Original Article
PREVALENCE AND SEASONAL VARIATION IN IXODID TICKS ON BUFFALOES OF
MATHURA DISTRICT, UTTAR PRADESH, INDIA
Geeta Patel1, Daya Shanker1, Amit Kumar Jaiswal1, Vikrant Sudan1,* and Santosh Kumar Verma2
ABSTRACT
INTRODUCTION
Considering the economic impact of
various ticks species on livestock, the present study
was projected for epidemiological characterize
of common ticks infesting water buffaloes. The
present study was conducted between July 2010
and June 2011 period at various locations of
Mathura region. A total of 635 water buffaloes were
examined randomly. The overall prevalence of ticks
infestation among buffaloes alone was found out
to be 51.81%. The highest and lowest prevalence
was reported in month of September (69.09%) and
January (37.74%), respectively. Based on seasonal
prevalence, highest tick infestation was found
in rainy season (61.14%), followed by summer
(50.95%) while lowest in the winter (43.46%).
Overall highest age wise prevalence was noticed
in the young ones (74.17%) followed by grownups
(60.93%) and lowest in adults (36.33%).
Buffalo-the incredible Asian dairy animal,
is commonly known as ‘Black Diamond’, for its
versatile role in socioeconomic upliftment of its
owners from the rural agricultural communities.
The major constraints in achieving maximum
financial gain from these animals are the diverse
disease conditions caused by ecto and endo
parasites (Bianchin et al., 2007). A single female
engorged tick is responsible for daily loss of 0.5 to
2 ml of blood, 8.9 ml of milk and 1 gram of body
weight. Losses attributable to ticks are caused either
directly, through tick worry, blood loss, damage to
hides and udders, injection of toxins (and loss of
body weight gain or indirectly through transmission
of disease pathogens, milk yield reduction, stunted
growth (FAO, 2004). The global economic losses
due to tick infestation has been estimated as 14000
to 18000 million US $ annually in which India has
a share of 498.7 million US $ (Minjauw and Mc.
Lead, 2003). A large amount of data is available
for the ecto and endo parasites of cattle, but when
it comes to buffaloes and that too ectoparasites, the
Keywords: buffaloes, Bubalus bubalis, prevalence,
ticks
Department of Parasitology, College of Veterinary Sciences and Animal Husbandry, U. P. Pandit Deen Dayal
Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan (DUVASU), Mathura,
2
Department of Pathology, College of Veterinary Sciences and Animal Husbandry, U. P. Pandit Deen Dayal
Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan (DUVASU), Mathura,
India
1
21
the season wise prevalence are given in Graphs 1
and 2, respectively. During the study of age-wise
tick infestation, overall maximum percentages of
positive cases (74.17%) were noticed in the group
I (up to 1 year) followed by 60.93 % in group II
(1–3 years) and minimum tick infestation (36.33%)
was observed in group III (> 3 years) (Graph 3).
During the study period, ixodid ticks belonging —
Hyalomma anatolicum anatolicum and Boophilus
microplus were recorded both in pure and mixed
infestation in different seasons (Figure 1).
Hyalomma spp. infestation was observed in 294
buffaloes (46.29%) examined for tick infestation.
Pure infestation of Hyalomma spp. was seen in 245
buffaloes (38.58%) and mixed with Boophilus spp.
in 49 (7.71%) cases. Pure Boophilus spp. infestation
was seen in 84 buffaloes (13.23%). Besides these,
H. marginatum issaci and H. dromedarii were also
collected from some of the buffaloes. The most
common feeding sites for adult ticks were neck,
axilla, belly, groin, udder, perineal regions and tail
(Figure 2, 3).
During study period, a total of 635 buffaloes
were examined from different localities of Mathura
district for the presence of ixodid ticks and their
prevalence was found out to be 51.81%. Contrary to
this, Mishra (1984); Sharma (1984); Kumar (1996)
and Vatsya et al. (2007) had earlier reported that
prevalence of ixodid ticks in buffaloes to be 61.0%,
33.50% and 38.06% respectively, from various
agro climatic regimes across India. Difference
among the results might be due to variation in
geographical locations, climatic conditions of the
experimental area, region and method of study and
selection of samples (Patel et al., 2012).
Month wise prevalence of ticks in buffaloes
was found maximum in September (69.09%) and
minimum in the month of January (37.74%). The
difference in tick infestation in different month was
literature seems restricted to finger tips. Therefore,
the present study was undertaken to know the
prevalence of ticks in relation to the different month
of the year, different seasons of the year, age of the
animals, sites of their attachment and identification
of ticks up to species level.
Area of study
Systematic survey on ixodid ticks of
buffaloes was undertaken at various locations of
Mathura district (Uttar Pradesh, India) during the
period from July 2010 to June 2011. The selected
areas were visited once a week to determine the
seasonal pattern of tick infestation and to observe
variation in prevalence of tick infestation with
respect of host (age, species) and environmental
determinants.
Collection and identification of ixodid ticks
The adult ticks were gently plucked up
from the body of the host by hand manipulation
or with the aid of blunt pointed forceps without
damaging their mouth parts. The specimens were
kept in separate plastic containers and the date, host,
age, locality and site of collection were entered on
the label of each container. These samples were
transported to the laboratory for further studies
and identification using standard keys (Sen and
Fletcher, 1962; Walker et al., 2003).
The overall prevalence of ticks during the
study period was found to be 51.81%. The month
wise prevalence of ticks throughout the year and
22
Graph 1. Month wise variation in the prevalence of ticks.
Graph 2. Season wise variation in prevalence of ticks.
23
Graph 3. Age wise variation in prevalence of ticks.
may be due to the change in the climatic condition.
The present study revealed that the prevalence rate
of ticks is highest in rainy season (61.14%) followed
by summer (50.95%) and least in winter season
(43.46%). Although the animals were infested with
ticks throughout the year but their number increased
following rains. Thus, rainfall (humidity) seemed
to be an important macroclimatic factor influencing
seasonal variation in tick infestation (Vatsya et
al., 2007). The decrease infestation rates during
extreme winters in the month of December, January
and February was sup-positively due to the drop in
the temperature (13.02oC). At low temperature ticks
(36.33%). Lower rate of tick infestations in adults
could be attributed to acquired resistance incidental
to repeatedly exposed of host to low grade field
infestations during the prolonged growth and
development period (Mishra,1984; Das, 1994).
It is important to note that the cattle
are mostly infested with Boophilus spp., while
buffaloes are mostly infested with Hyalomma spp.
(Papadopoulos et al., 1996; Patel et al., 2012).
Buffaloes have less dense hair coat and have access
to mud for wallowing which might cause dropping
of ticks and hence less infested with Boophilus spp.
(Khan, 1986). In present study, four species of ticks
were identified as B. microplus, H. a. anatolicum,
H. dromedari and H. marginatus issaci. Pure
Hyalomma spp. infestation was found to be 38.58%
and pure Boophilus species infestation was 5.51%.
Aberrant infestation with H. dromedarii (a camel
tick) and. H. marginatus issaci (a small ruminant
tick) might be attributed to frequent contact of
buffaloes and grazing on forest land having free
access of camels and small ruminants (Chhabra et
al., 1983).
try to protect themselves by entering in diapauses
leading to delayed morphogenesis and reduced
behavioural activities (Gray, 1991; Denlinger,
1985).
The infestation rate of ticks was found
maximum in group I animals consisting of young
ones below 1 year of age (74.17%) followed by
group II animals consisting of between 1-3 years
of age (60.93%) and minimum in group III animals
consisting of animals of more than 3 years of age
24
a) B. microplus (anterior end)
b) B. microplus (posterior end)
d) H. anatolicum anatolicum (posterior end)
c) H. anatolicum anatolicum (anterior end)
e) H. marginatum issaci (anterior end)
f) H. marginatum issaci (posterior end)
g) H. dromedarii (anterior end)
h) H. dromedarii (posterior end)
Figure 1. Various species of ticks on buffaloes identified in the present study.
25
Figure 2. Buffalo calf infested with ticks.
Figure 3. Tail of buffalo infested with ticks.
26
Denlinger, D. 1985. Hormonal control of diapause,
p. 353-412. In Kerkutt, G.A. and L.I. Gilbert
(eds.) Comprehensive Insect Physiol.,
Biochemistry and Pharmacol. Vol. 8. New
York Pergamon Press.
F.A.O. 2004. Resistance Management and Integrated
Parasite control in Ruminants- Guidelines,
Module I- Ticks: Acaricide Resistance:
Diagnosis, Management and Prevention.
Food and Agriculture Organization, Animal
Production and Health Division, Rome: 2577.
Gray, J.S. 1991. The development and seasonal
activity of the tick Ixodes ricinus: A vector
of Lyme borreliosis. Med. Vet. Entomol., 79:
323-333.
Kumar, R. 1996. Studies on tick infestations in
cattle and buffaloes. M.V.Sc. Thesis, C.S.A.
University of Agriculture and Technology
Kanpur, (U.P.). p. 1-178.
Minjauw, B. and A. McLeod. 2003. Tick-borne
diseases and poverty. The impact of ticks
and tick born diseases on the livelihood of
small scale and marginal livestock owners
in India and eastern and southern Africa.
Research Report, DFID Animal Health
Programme, Centre for Tropical Veterinary
Medicine, University of Edinburg, UK.
Mishra, S.C. 1984. A note on the incidence and
control of ixodid ticks at Bhubaneswar.
Cheiron, 13(1): 5-8.
Papadopoulos, B., P.C. Morel and A. Aeschlimann
1996. Ticks of domestic animals in the
Macedonia region of Greece. Vet. Parasitol.,
63(1/2): 25-40.
Patel, G., D. Shanker, A.K. Jaiswal, V. Sudan and
S.K. Verma. 2012. Prevalence and seasonal
variation in ixodid ticks on cattle of Mathura
district, Uttar Pradesh. J. Parasit. Dis., DOI
In conclusion, management practices and
animal holdings influence the tick infestations
on the body of the host. Evidently, in tropics
and sub tropics, distribution of ixodid ticks is
mainly governed by the rainfall and precipitation.
Effective ixodid tick control strategies ought to be
mainly focused upon the seasonal periodicity of
the dominant tick species and their susceptibility
to the acaricide, based on in vitro testing, to
minimize production losses incidental to ixodid
tick infestations, besides scientific management
of grazing lands and other strategies most suited
in the endemic areas of ambient temperature and
rainfall request to be evolved.
ACKNOWLEDGMENT
The authors are very grateful to Hon’ble
Vice Chancellor DUVASU for making the facilities
available.
REFERENCES
Bianchin, I., J.B. Catto, A.N. Kichel Torres and
M.R. Honer 2007. Effect of the control of
endo and ectoparasites on weight gains in
crossbred cattle (Bos Taurus taurus× Bos
Taurus indicus) in the central region of
brazil. Trop. Anim. Health Pro., 39(4): 287296.
Chhabra, M.B., N.S. Ruprah and S.K. Gupta.
1983. Ixodid ticks on bovines in Haryana- A
preliminary report. Cherion, 12: 298-303.
Das, S.S. 1994. Prevalence of ixodid ticks
infestation on farm animals in Pantnagar,
tarai of Uttar Pradesh. J. Parasit. Appl.
Anim. Biol., 3: 71- 73.
27
10.1007/s12639-012-0154-8.
Sen, S.K. and T.B. Fletcher 1962. Veterinary
Entomology and Acarology for India.
Indian council of Agricultural Research,
New Delhi.
Soulsby, E.J.L. 2006. Helminths, Arthropods and
th
Protozoa of Domesticated Animals, 7 ed.
Bailliere Tindall and Cassel Ltd., London.
p. 444-475.
Vatsya, S., C.L. Yadav, R.R. Kumar and R. Garg.
2007. Seasonal activity of Boophilus
microplus on large ruminants at an organised
livestock farm. J. Vet. Parasitol., 21(2):
125-128.
Walker, A.R., A. Bouattour, J.L. Camicas, A. Estrada
Pena, I.G. Horak, A.A. Latif, R.G. Pegram
and P.M. Preston. 2003. Ticks of Domestic
Animals in Africa: A Guide to Identification
of Species. Bioscience Reports. 221p.
28
Original Article
SEDATIVE, ANALGESIC AND CARDIOPULMONARY EFFECTS OF MIDAZOLAMBUTORPHANOL PREMEDICATION IN WATER BUFFALOES (BUBALUS BUBALIS)
Deepti Bodh1,*, Kiranjeet Singh1, Jitender Mohindroo2, Sashi Kant Mahajan2 and
Narinder Singh Saini2
ABSTRACT
in water buffaloes as this combination provided
adequate sedation, analgesia and muscle relaxation
with only transient changes in cardiopulmonary
parameters.
The study was conducted in 12 water
buffaloes of either sex, aged 3 to 8 years and
weighing 400-500 kg, to evaluate and compare the
sedative, analgesic and cardiopulmonary effects of
intravenous midazolam-butorphanol combination
with midazolam. Animals were randomly divided
into two groups: Group I (midazolam) and group
II (midazolam-butorphanol) having six animals in
each group. Midazolam (0.2 mg/kg, i.v.) in group
I and midazolam-butorphanol (0.2 mg/kg and 0.02
mg/kg, i.v.) combination in group II was used for
premedication. Thiopentone sodium (5%) (10 mg/
kg, i.v) was used as induction agent in both the
groups. Better degree of sedation, analgesia and
muscle relaxation was observed in midazolambutorphanol group. Heart rate decreased
significantly after premedication in midazolambutorphanol group. Respiratory rate decreased nonsignificantly while rectal temperature decreased
significantly (p<0.01) after premedication in both
the groups. Halothane concentration required to
maintain adequate depth of anaesthesia was lower
in midazolam-butorphanol group. The results
showed that midazolam-butorphanol combination
(0.2 mg/kg and 0.02 mg/kg) can be used safely
for premedication during halothane anaesthesia
Keywords: midazolam, butorphanol, halothane,
thiopentone sodium, water buffaloes
INTRODUCTION
Use of sedatives before induction of
anaesthesia is well established in veterinary practice.
Preoperative use of sedatives improve the quality
of induction and decrease drug related adverse
effects by reducing the amount of injectable and
inhalant anaesthetics (Kojima et al., 2002; Sano et
al., 2003). Midazolam has mild cardiovascular and
respiratory effects and is commonly used as a mild
tranquillizer, muscle relaxant and anticonvulsant
(Lemke, 2007). It has supra-additive effect with
opioids and barbiturates. The combined effect of
barbiturate and benzodiazepine is mediated through
benzobarbiturate-GABA receptor supramolecular
complex in which each site when occupied
modulates the other (Tverskoy et al., 1988; Vinik
et al., 1994). Butorphanol, an opioid agonistantagonist has good analgesic, antitussive and
1
Division of Surgery, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India,
*E-mail: [email protected]; [email protected]
2
Department of Veterinary Surgery and Radiology, Guru Angad Dev Veterinary and Animal Sciences
University, Ludhiana, Punjab, India
29
with the help of pulse oxymeter. Systolic, diastolic
and mean arterial pressure was measured with
the help of non-invasive blood pressure (NIBP)
monitor whose cuff was applied around the base
of tail.
The degree of sedation, analgesia and
muscle relaxation was graded on 1 to 4 scoring
scale. Onset of sedation was recorded by observing
behavioural changes after premedication and was
graded as: 1 (no sedation) = animal standing alert
with its head high and all reflexes present, 2 (mild
sedation) = decreased alertness with no reduction
in palpebral and pin prick reflexes; 3 (moderate
sedation) = animal calm, minimal restraint needed,
eyelids partially closed, sluggish palpebral reflex
and partial ventromedial rotation of eye; 4 (deep
sedation) = animal completely calm, no restraint
needed, eyelids closed, very weak palpebral reflex,
complete ventromedial rotation of eye.
The quality of induction was evaluated five
min after administration of thiopentone sodium and
was graded as 1 (poor) = animal excited, frequent
attempts to stand after recumbency, massive
regurgitation and inability to intubate trachea; 2
(moderate) = mild excitement, mild regurgitation,
slightly longer tracheal intubation time and slightly
prolonged induction; 3 (good) = no excitement, no
regurgitation, no gag reflex, 4 (excellent) = smooth
and rapid induction, easy and quick tracheal
intubation, no regurgitation. Quality of analgesia
was recorded by observing animal’s response to
deep pin prick with a 22 G hypodermic needle at
every 15 minutes interval. Analgesia was graded as
1 (no analgesia) = strong response to pin prick; 2
(mild analgesia) = weak response to pin prick; 3
(Moderate analgesia) = occasional response to pin
prick; 4 (Excellent analgesia) = no response to pin
prick.
Extent of muscle relaxation was recorded
sedative effect (Pfeffer et al., 1980). It induces only
mild sedation and has minimum adverse effects to
the cardiovascular system (Greene et al., 1990;
Trim, 1983). There are only few reports available
on the use of midazolam-butorphanol combination
as preanaesthetic in water buffaloes. The present
study was therefore, designed to evaluate the
sedative, analgesic and cardiopulmonary effects
of midazolam-butorphanol combination in water
buffaloes.
The study was conducted in 12 water
buffaloes of either sex, aged 3-8 years and weighing
400-500kg. Buffaloes were randomly divided
into two groups: group I (M) and group II (MB)
having 6 animals in each group. All buffaloes were
kept off feed for 24 h and water was withheld for
12 h prior to onset of anaesthesia. Different surgical
procedures like ventral hernia (n=3), plating (n=3),
diaphragmatic hernia (n=3), prepubic tendon rupture
repair (n=2), excision of large tumorous mass on
neck (n=1) were performed with buffaloes restrained
either in lateral or dorsal recumbency. Midazolam
(0.2 mg/kg i.v.) in group I (M) and midazolam
(0.2 mg/kg, i.v.) + butorphanol (0.02mg/kg, i.v.)
combination mixed in a single syringe in group II
(MB) was used for premedication. Thiopentone
sodium (5%) was used as induction agent in both
the groups. Following induction, jaw was opened
using a mouth gag and endotracheal intubation
was performed. Anaesthesia was maintained with
halothane and oxygen mixture via a semi-closed
rebreathing system. The vaporizer setting was
adjusted according to depth of anaesthesia after
monitoring animal’s response to various reflexes.
Haemoglobin oxygen saturation value was obtained
30
between the two groups. Paired‘t’ test was used
to compare the means at different intervals with
their respective base values in each group. For non
parametric observations the Kruskal-Wallis one
way test (Stiegel and Castellan, 1988) was used to
compare the means between the two groups.
by observing relaxation of muscles of limb, jaw and
tail and was graded as: 1 (no relaxation) = tightly
closed jaws and stiff limbs; 2 (mild relaxation) =
moderate resistance to opening of jaw and bending
of limbs; 3: moderate relaxation (mild resistance
to opening of jaw and bending of limbs; 4 (good
relaxation) = no resistance to opening of the jaw
and bending of the limbs.
The degree of abolition of palpebral,
corneal, pin prick and rectal pinch reflex was
graded as: 1 (intact and strong reflex); 2 (mildly
depressed reflex); 3 (sluggish reflex); 4 (complete
loss of reflex). The extent of salivation was graded
as 1 (no salivation); 2 (mild salivation); 3 (moderate
salivation); 4 (profuse salivation).
Quality of recovery was graded as 1
(poor) = prolonged struggling, premature attempts
to stand; 2 (moderate) = transient excitement
along with some struggling; 3 (good) = smooth,
easy transition to alertness, resumption of sternal
position, 4 (excellent) = smooth, excitement free,
animal standing of its own.
Physiological parameters like heart rate
(HR) (beats/min), respiratory rate (RR) (breaths/
minute) and rectal temperature (RT) (ºC) was
recorded at base i.e. 0 minute, 5 minutes after
premedication and at 5, 15, 30, 45, 60, 90 and
120 minutes after induction of anaesthesia.
Haemodynamic parameters like systolic blood
pressure (SBP) (mm of Hg), diastolic blood
pressure (DBP) (mm of Hg), mean arterial pressure
(MAP) (mm of Hg) and haemoglobin oxygen
saturation (SpO2) (%) were recorded at the above
time interval in both the groups.
The study was conducted to evaluate and
compare the sedative, analgesic and cardiopulmonary
effects of midazolam-butorphanol combination
with midazolam in water buffaloes. Addition of
butorphanol to midazolam brought about clinically
appreciable changes in sedation, analgesia and
muscle relaxation without any adverse effects
on cardiopulmonary parameters. The quality of
sedation was better in group II (MB) as compared
to group I (M) and the difference between two
groups was statistically significant (p<0.05). Mean
sedation scores of 2.75±0.40 and 3.75±0.25 were
reported in group I (M) and II (MB), respectively.
In group I, out of six buffaloes, two gained score
4, three scored 3 and remaining one scored 2 while
in group II(MB), out of six buffaloes, four gained
score of 4 and remaining two scored 3 (Figure 1).
Signs of sedation appeared early in buffaloes of
group II (MB) (30 sec to 1 minute) as compared
to group I (M) (1 to 2 minutes). Excitement
immediately after premedication was observed in
2 buffaloes belonging to group I (M) while none
of the buffaloes in group II (MB) showed any
signs of excitement. Better sedation produced
by midazolam (0.2 mg/kg b.wt.) + butorphanol
(0.02 mg/kg b.wt.) combination in group II (MB)
might be due to combined sedative effect of both
the drugs. Midazolam as a single agent has a mild
sedative effect but it shows additive or synergistic
Statistical Analysis
Analysis of variance (ANOVA) and
Duncan’s multiple range test (DMRT) was used
to compare the means at different time intervals
31
activity when administered with other sedatives
(Cwiek et al., 2009).The analgesic and sedative
effect of butorphanol is due to two subtypes of μ
receptors: μ1-receptors that act above the level of
the spinal cord, and μ2-receptors that act within the
spinal cord (Boothe, 2001).
Moderate quality of induction was
observed in buffaloes of group I (M), with a
mean induction score of 3.25 ± 0.25. In group II
(MB), the quality of induction was good, with
mean induction score of 3.75 ± 0.25 (Figure 2).
Laryngeal reflex diminished early in group II (MB)
and the time taken for endotracheal intubation was
(MB) as compared to group I (M). Analgesia with
complete abolition rectal pinch reflex for longer
duration was observed in buffaloes of group II (MB)
as compared to group I (M) (Figure 5). Midazolam
does not have analgesic property, however addition
of μ opioid agonist butorphanol in group II (MB)
might have resulted in deeper and adequate level of
analgesia (Pfeffer et al., 1980). Midazolam is found
to have a considerable effect on the nociceptive
transmission in superficial dorsal horn (Kohno
et al., 2006) and cause pain relief (Akhlaghi and
Rajaei, 2008). Similar findings were reported by
Itamoto et al. (2000) in dogs, where addition of 0.1
significantly (p<0.05) less in group II (MB) (26.25
± 1.75) as compared to group I (M) (30.00 ± 3.39).
A synergistic interaction between midazolam and
butorphanol could be responsible for the abolition
of laryngeal reflex and better conditions for
endotracheal intubation in group II (MB). Similar
synergism between opioid and midazolam was
observed in humans (Ben Shlomo et al., 1990) and
animals (Kissin et al., 1986).
Significant (p<0.05) increase in the
score for palpebral reflex was observed after
premedication in group II (MB) while in group I
(M), the increase was non-significant (Figure 4).
A non-significant increase in the score for corneal
reflex followed by a significant (p<0.05) increase
in the score for rotation of eyeball was observed
within 5 minutes of preanaesthetic administration
in both the groups. Moderate abolition of palpebral
and corneal reflex observed after premedication in
both groups was found similar to the observations
of Kaur and Singh (2004) who reported loss of
eyelash reflex, mild to moderate palpebral reflex and
full corneal reflex after midazolam administration
(0.2 mg/kg i.v.) in bovines. Completely ventral
and ventromedial rotation of eyeball for a longer
duration was observed in buffaloes of group II
mg/kg butorphanol to medetomidine or midazolam
produced adequate analgesic effect.
The extent of muscle relaxation was
assessed by relaxation of jaw tone and limbs,
respectively. The score for limb relaxation
increased significantly (p<0.05) in group II(MB)
compared to a non-significant (p>0.05) increase
in group I(M) (Figure 6). Better degree of muscle
relaxation observed in buffaloes of group II (MB)
might be attributed to the synergistic interaction
between midazolam and butorphanol. Midazolam
is a benzodiazepine derivative known to have good
muscle relaxant action (Hellyer et al., 1991; Ilkew et
al., 1998). Although, opioids by themselves do not
induce muscle relaxation, however, their additive
or synergistic interaction with benzodiazepine
might have caused enhanced muscle relaxation in
buffaloes of group II (MB).
No statistically significant (p>0.05)
difference between the two groups regarding degree
of salivation was reported. However, salivation
was moderate following midazolam administration
in group I (M) while it was mild after midazolambutorphanolpremedication in group II (MB). Court
and Greenbalt (1992) and Butola and Singh (2007)
reported similar findings in dogs where drooling
32
during maintainence period in both groups may be
attributed to the hypotensive effect of halothane.
Similar observation was reported in cattle (Short
et al., 1968) and buffaloes (Gahlawat et al., 1986).
No significant difference in respiratory rate was
observed after premedication in both the groups.
Significant (p<0.01) hypoxemia observed after
induction in both the groups might be due to
the respiratory depressant effect of thiopentone
sodium, as the barbiturates can cause significant
cardiovascular and respiratory depression
(Carpenter et al., 2005).
Respiratory
rate
decreased
nonsignificantly after premedication followed by a
significant (p<0.01) decrease after induction in
buffaloes of both the groups (Figure 9).
Rectal temperature decreased significantly
(p<0.05)
after
midazolam
butorphanol
premedication in group II (MB) while in group I
(M), the decrease was non-significant (Figure 10).
Reduced muscle activity along with deep sedation
induced by midazolam-butorphanol combination
in group II (MB) might have led to the decrease
in rectal temperature in this group. Significant
(p<0.05) hypothermia observed after induction and
throughout maintainence period in both the groups
could be due to reduced basal metabolic rate and
muscle activity on one hand and depression of
thermoregulation on the other which might have
resulted in hypothermia (Ponder and Clarke,
1980).
A non significant decrease in blood
pressure was recorded after premedication in
both the groups while after induction and during
maintenance, blood pressure was found to decrease
significantly (p<0.05) at few intervals in both the
groups (Figure 11). However, in both the groups
hypotension was a consistent finding during
maintainence with halothane and the severity
of saliva was seen after midazolam administration.
Opioids, on the other hand decrease the production
of saliva in mouth and this may explain for mild
salivation reported after midazolam butorphanol
premedication in group II (MB).
Significant (p<0.01) difference in the
amount of halothane used was observed between
the two groups. Halothane concentration required
to maintain adequate depth of anaesthesia was
3.25 ±0.50 % and 2.75 ±0.25 % in group I(M)
and group II (MB), respectively (Figure 7). More
halothane sparing effect of midazolam butorphanol
combination in group II (MB) might be due
combined analgesic and/or sedative effects of
both the drugs. Midazolam decreases the MAC of
potent inhaled anesthetics in humans (Inagaki et
al.,1993) and animals (Hall et al., 1988). Studies
in humans suggested that midazolam produced
marked reduction of halothane MAC at serum
concentration lower than that required to cause
sleep (Inagaki et al., 1993). A plasma midazolam
concentration of 539 ng mL-1 reduced halothane
MAC by up to 70% in the same study.
Heart rate decreased significantly (p<0.05)
after midazolam-butorphanol administration in
group II (MB) while in group I (M), the decrease
in heart rate was non-significant (Figure 8).
Midazolam has minimal cardiovascular depressant
effects (Gross et al., 1990; Tranquilli et al., 1991)
and although butorphanol posses less cardiovascular
effect than classical opiate agonists, it can cause
a decrease in cardiac rate secondary to increased
parasympathetic tone and mild decrease in arterial
blood pressure (Taylor et al., 1988). Decrease in
heart rate after thiopentone administration in both
groups of the present study supported the findings
in buffaloes administered with thiopentone sodium
and glyceryl guiacolate (Agrawal et al., 1983).
Significant (p<0.05) decrease in heart rate observed
33
of Teaching Veterinary Clinical Complex, Guru
Angad Dev Veterinary and Animal Sciences
University, Ludhiana (Punjab), India for providing
the facilities of Diagonostic Clinical Complex. We
would also like to thank Dr. Ravi, Senior Scientist,
Department of Biotechnology, Indian Veterinary
Research Institute, Izatnagar, Uttar Pradesh, India
and Dr. Samita, Associate Professor, Department
of Biostatistics, Guru Angad Dev Veterinary and
Animal Sciences University, Ludhiana (Punjab),
India for their great help in statistical analysis.
of hypotension was closely related to the depth
of anaesthesia. Similar hypotensive effect of
halothane has also been demonstrated in cattle
(Short et al., 1968), dogs (Steffey et al., 1975) and
horses (Smith, 1969).Significant (p<0.05) decrease
in SpO2 after premedication in group II (MB) might
be due to the respiratory depression caused by the
combined effect of sedatives used. A decrease in
SpO2 value after medetomidine and butorphanol
anaesthesia was noticed in buffalo calves (Malik,
2008; Ahmed, 2009). Decrease in SpO2 at few
intervals was observed after induction in both the
groups (Figure 12). However, at other intervals,
decrease in SpO2 was only transient and was fairly
Declaration
A prospective, randomized, blinded study
protocol was used and was approved by the State
Veterinary Authorities and informed consent
was obtained from the owners. The experiments
performed comply with the current laws of the
country.
maintained throughout most of the observation
period.
The scores for recovery quality in buffaloes
of group I (M) and group (MB) were 3.75±0.25 and
3.25±0.25, respectively. No statistically significant
difference in the time of recovery was observed
between the two groups.
The results showed that the combination
of midazolam with butorphanol (0.2 mg/kg and
0.02 mg/kg) for the purpose of premedication in
water buffaloes induces high-quality sedation,
analgesia and muscle relaxation.The combination
considerably reduces the amount of inhalant
anaesthetics used and produces transient changes
in cardiopulmonary parameters. Midazolambutorphanol combination can be used safely for
premedication during halothane anaesthesia in
water buffaloes.
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Ben-Shlomo, I., H. abd-el-Khalim, J. Ezry, S. Zohar
AKNOWLEDGEMENTS
The authors are grateful to Dr. S. K. Uppal,
Professor, Department of Veterinary Medicene and
Dr. S. Prabhakar, Professor-cum-head, Department
34
Figure 1. Mean ± SE of score for sedation quality in group I (M) and group II (MB).
Figure 2. Mean ± SE of score for induction quality in group I (M) and group II (MB).
Figure 3. Mean ± SE of score for Intubation time in group I (M) and group II (MB).
35
Figure 4. Mean ± SE of score for palpebral reflex in group I (M) and group II (MB).
Figure 5. Mean ± SE of score for rectal pinch reflex in group I (M) and group II (MB).
Figure 6. Mean ± SE of score for limb relaxation in group I (M) and group II (MB).
36
Figure 7. Mean ± S.E. of halothane (%) used at various intervals group I (M) and group II (MB).
Figure 8. Mean ± SE of heart rate (beats/min) at different time intervals in group I (M) and group II (MB).
Figure 9. Mean ± SE of respiratory rate (breaths/min) in group I (M) and group II (MB).
37
Figure 10. Mean ± SE of rectal temperature (°C) in group I (M) and group II (MB).
Figure 11. Mean ± SE of mean arterial pressure (mm Hg) in group I (M) and group II (MB).
Figure 12. Mean ± SE of hemoglobin oxygen saturation (mm Hg) in group I (M) and group II (MB).
38
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40
Original Article
PREVALENCE AND ANTIBIOGRAM OF BACTERIAL PATHOGENS FROM
SUBCLINICAL MASTITIS IN BUFFALOES
Z. Ali, U. Dimri and R. Jhambh*
may reduce the chances of treatment failure and the
economic losses.
ABSTRACT
The present investigation was carried out
to study the prevalence of bacterial pathogens
responsible for subclinical mastitis in buffaloes and
their antibiogram pattern to selected antibiotics.
The study was carried out on lactating buffaloes
maintained at the Cattle and Buffalo farm, Indian
Veterinary Research Institute, Izatnagar, India.
Screening for subclinical mastitis was done by
California Mastitis Test (CMT) and Somatic Cell
Count (SCC) in milk. The buffaloes showing
CMT score ≥ 2 and SCC ≥ 0.5 million/ml of milk
were considered for isolation and identification
of bacterial pathogens by cultural examination
and requisite biochemical tests. Fifteen out of
48 lactating buffaloes were found positive for
subclinical mastitis, affecting one or two quarters,
giving a prevalence of 31.25%. Out of them, 9 (60%)
were found positive for Staphylococcus aureus,
3 (20%) positive for Streptococcus agalactiae, 1
(7.69%) for other streptococci and 2 (13.33%) for
E. coli. The antibiogram of the bacterial isolates
to standard antibiotic discs determined by disc
diffusion method revealed the highest sensitivity
to ciprofloxacin and enrofloxacin followed by
cefotaxime, cloxacillin, erythromycin, amoxycillin
in decreasing order and least sensitivity to penicillin
G. Judicious use of antibiotics based on antibiotic
sensitivity and pharmacokinetics in mastitis control
Keywords: antibiogram, bacterial pathogens,
buffaloes, prevalence, subclinical mastitis
INTRODUCTION
Mastitis, defined as inflammation of the
mammary gland, is a major disease affecting dairy
animals worldwide. Based upon severity of the
inflammation, it can be classified into sub-clinical,
clinical and chronic forms. Out of which subclinical
form as difficult to detect due to the absence of
any visible indications has major cost implications
associated with decreased milk production (Viguier
et al., 2009). In India, annual economic loss to dairy
industry due to subclinical mastitis is estimated
to be Rs. 43653 million (Dua, 2001). It primarily
occurs in response to intramammary bacterial
infection (Zhao and Lacasse, 2008). Therefore, a
bacteriological diagnosis, prevalence study in the
herd and proper selection of antibiotic based on
antibiotic sensitivity are critical for rational and
effective control of mastitis. Based upon these
facts, the present investigation was carried out
to study the prevalence of bacterial pathogens
responsible for subclinical mastitis in buffaloes and
the antibiogram pattern of the isolates to selected
Division of Medicine, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India,
E-mail: [email protected]
41
factors such as herd size, milking parlor hygiene,
variation in systems of feeding and management
etc. Out of total 15 cases of subclinical mastitis,
9 (60%) were found positive for Staphylococcus
aureus based on growth characteristics on Mannitol
salt agar, 3 (20%) positive for Streptococcus
agalactiae based on growth characteristics on
Edward’s media and Hotis test, 1 (7.69%) for other
streptococci and 2 (13.33%) for E. coli based on
growth characteristics on MacConkey’s agar and
EMB agar plates. The present study revealed a
predominance of Staphylococcus aureus followed
by Streptococcus agalactiae in subclinical mastitis
in buffaloes, which is similar to the finding by
Khan and Muhammad (2005). Staphylococcus
aureus and Streptococcus agalactiae are the
most common contagious pathogens of bovine
mammary gland. S. aureus is a major pathogen
responsible for subclinical mastitis (Radostits et
al., 2007) while S. agalactiae is still a significant
cause of chronic mastitis where control measures
for contagious mastitis are not used (Keefe, 1997).
Thus, the present study reveals the predominance
of contagious form of subclinical mastitis at the
farm that needs to be controlled with appropriate
measures to prevent further spread. On the other
hand, a low prevalence of subclinical mastitis due
to E. coli and other streptococci infection which
are considered environmental pathogens (Radostits
et al., 2007) suggests the improved sanitation and
hygienic practices at the farm.
The antibiogram of the bacterial isolates
revealed highest sensitivity to ciprofloxacin and
enrofloxacin followed by cefotaxime, cloxacillin,
erythromycin, amoxycillin in decreasing order and
least sensitivity to penicillin G. Similar antibiogram
pattern of bacterial isolates has been recorded by
Awandkar et al. (2009) and Harini and Sumathi
(2010) from bovine clinical and subclinical
antibiotics.
The present study was carried out on
lactating buffaloes under different phases of
lactation maintained at the Cattle and Buffalo farm,
Indian Veterinary Research Institute, Izatnagar,
India. Screening of the buffaloes for subclinical
mastitis was done by estimation of Somatic Cell
Count (SCC) in milk from individual quarters
indirectly by California Mastitis Test (CMT) as
per Schlam and Noorlander (1957) and directly as
per Schlam et al. (1971). The buffaloes showing
CMT score ≥ 2 and SCC ≥ 0.5 million/ml of milk
were considered for isolation and identification
of bacterial pathogens. Milk samples collected in
sterile vials from affected quarters was subjected
to bacterial isolation and identification by cultural
examination and requisite biochemical tests by
the method of Quinn et al. (2004). Further, the
sensitivity of each bacterial isolate to standard
antibiotic discs viz. penicillin (10 units/disc),
amoxycillin (10 μg/disc), cloxacillin (30 μg/disc),
erythromycin (15 μg/disc), ciprofloxacin (5 μg/
disc), enrofloxacin (10 μg/disc) and cefotaxime (10
μg/disc) was determined by disc diffusion method
(Bauer et al., 1966).
Out of 48 lactating buffaloes, 15 were found
positive for subclinical mastitis affecting one or
two quarters giving a prevalence of 31.25%. Joshi
& Gokhle (2006) documented a lower (5 to 20%)
prevalence of subclinical mastitis in buffaloes.
Higher prevalence may be dependent on many
42
REFERENCES
mastitis respectively. Poor sensitivity to penicillin
G and amoxycillin may be due to production of
β-lactamase enzyme by resistant strains of isolates
owing to their frequent use at the farm for mastitis
control. On the other hand, higher sensitivity to
ciprofloxacin, enrofloxacin, cefotaxime might be
explained on the basis of their less frequent use at
the farm. Antimicrobial susceptibility determined
in vitro has been considered as a prerequisite for
treatment. However, activity in vitro does not
guarantee efficacy in vivo as pharmacokinetics
of the antimicrobial substance greatly affects its
suitability for mastitis treatment (Pyöräla, 2009).
Enrofloxacin is an antibacterial under class
fluoroquinolones, exclusively developed for use in
animals. Ciprofloxacin is a major, active metabolite
of enrofloxacin formed by the de-ethylation of
enrofloxacin. Both of them tend to be concentrated
in milk of lactating animals (Idowu et al., 2010),
so presents the potent options for management of
subclinical mastitis in buffaloes.
Awandkar, S.P., N.V. Khode, V.M. Sardar and
M.S. Mendhe. 2009. Prevalence and current
antibiogram trend of mastitic agents in
Udgir and its Vicinity, Maharashtra State,
India. Int. J. Dairy Sci., 4(3): 117-122.
Bauer, A.W., W.M.M. Kieby, J.C. Shrenis and M.
Turck. 1966. Antibiotic susceptibity testing
by a standardized single disc diffusion
method. Am. J. Clin. Pathol., 45: 453-496.
Dua, K. 2001. Incidence, etiology and estimated
economic losses due to mastitis in Punjab
and in India- An update. Indian Dairyman,
53(10): 41-48.
Harini, H. and B.R. Sumathi. 2010. Screening of
bovine milk samples for sub-clinical mastitis
and antibiogram of bacterial isolates. Vet.
World, 4(8): 358-359.
Idowu, O.R., J.O. Peggins, R. Cullison and
J.V.
Bredow.
2010.
Comparative
pharmacokinetics of enrofloxacin and
ciprofloxacin in lactating dairy cows and beef
steers following intravenous administration
of enrofloxacin. Res. Vet. Sci., 89: 230-235.
Joshi, S. and S. Gokhale. 2006. Status of mastitis
as an emerging disease in improved and
periurban dairy farms in India, Ann. N. Y.
Acad. Sci., 1081: 74-83.
Keefe, G.P. 1997. Streptococcus agalactiae mastitis:
A review. Can. Vet. J., 38: 429-437.
Khan, A.Z. and G. Muhammad. 2005. Quarterwise comparative prevalence of mastitis in
buffaloes and crossbred cows. Pak. Vet. J.,
25(1): 9-12.
Pyöräla, S. 2009. Treatment of mastitis during
lactation. Irish Vet. J., 62: 40-44.
Quinn, P.J., M.E. Carter, B. Markey and G.R. Carter.
2004. Clinical Veterinary Microbiology,
CONCLUSION
The present study revealed a high prevalence
of subclinical mastitis in lactating buffaloes with
the predominance of Staphylococcus aureus
followed by Streptococcus agalactiae which are
the most common contagious pathogens of bovine
mammary gland. The antibiogram revealed the
highest sensitivity to ciprofloxacin and enrofloxacin
followed by cefotaxime, cloxacillin, erythromycin,
amoxycillin, penicillin G in decreasing order
of sensitivity. Judicious use of antibiotics based
on antibiotic sensitivity and pharmacokinetics
in mastitis control may reduce the chances of
treatment failure and the economic losses.
43
Mosby. Elsevier Limited, Philadelphia,
USA.
Radostits, O.M., D.C. Blood, C.C. Gay and P.D.
Constable. 2007. Veterinary Medicine:
Diseases of Cow, Buffalo, Horse, Sheep,
Goat and Pig, 10th ed. Saunders Elsevier
Limited, Philadelphia, USA.
Schlam, O.W. and D.O. Noorlander. 1957.
Experiments and observations leading to
development of the California mastitis test.
J. Am. Vet. Med. Assoc., 130: 199-204.
Schlam, O.W., E.J. Carrol and N.C. Jain.
1971. Bovine Mastitis. Lea and Febiger,
Philladelphia. p. 128-129.
Viguier, C., S. Arora, N. Gilmartin, K. Welbeck and
R. O’Kennedy. 2009. Mastitis detection:
current trends and future perspectives.
Trends Biotechnol., 27(8): 486-493.
Zhao, X. and P. Lacasse. 2008. Mammary tissue
damage during bovine mastitis: Causes and
control. J. Anim. Sci., 86(1): 57-65.
44
Original Article
MACRO AND MICRO MINERAL PROFILE IN FORAGE AND BLOOD PLASMA OF WATER
BUFFALOES WITH RESPECT TO SEASONAL VARIATION
Sushma Chhabra, S.N.S. Randhawa and S.D. Bhardwaj
ABSTRACT
INTRODUCTION
The present study was carried out to assess
the levels of some macro and micro minerals
in blood plasma of water buffaloes and forage
consumed by these ruminants in the central region
of Punjab, India using apparently health animals
during two consecutive seasons of the year. Blood
plasma samples were obtained from the animals
twice during each season, and analyzed for
calcium, inorganic phosphorous, copper, inorganic
iodine, zinc, manganese and iron levels. The results
showed that concentrations of all the minerals
studied in plasma were comparable in both the
seasons with the exception of zinc and phosphorus
which were significantly higher in winter. Analysis
of forages collected showed that the variations in
the fodder mineral concentrations corresponded to
the plasma mineral variations, indicating a direct
plant-animal relationship and showing the need of
supplementation of the deficient minerals during
summer season for these animals at the place where
the livestock were being reared.
Farm animals derive most of their mineral
requirements from their feed and fodder. Therefore,
all the factors that influence mineral content of the
fodder determine the mineral intake of animals,
especially the agro-climatic and environmental
factors like climate, soil type, species and stage
of maturity (Suttle, 2010) and the adequacy of the
diet in essential minerals can be determined by
analysis of animals’ plasma mineral status and of
forages which are the sole sources of minerals for
the requirements of the animals.
Mineral imbalances have been established
in many parts of Punjab, India (Singh, 2002),
where intensive agricultural practices having been
practiced for over decades but only fragmentary
data is available concerning the mineral status
of livestock and forages. There is need for
information on this aspect, in which problems of
mineral nutrition exist. The main objectives of this
investigation were, therefore, to determine mineral
imbalances and particularly to find out the effect of
season on the levels of some essential minerals in
forages and plasma of animals
Keywords: buffaloes, minerals, profile, plasma,
forage
Department of Veterinary Medicine, College of Veterinary Science, Guru Angad Dev Veterinary and Animal
Sciences University (GADVASU) , Ludhiana, Punjab, India
45
animals was collected, oven dried (overnight at
65ºC) and ground fodder samples were digested
on hot plate with sulphuric acid and hydrogen
peroxide and their mineral contents (Cu, Mn, Zn,
Fe, Ca) were estimated by Atomic Absorption
Spectrophotometer. Phosphorus content of fodder
was estimated by Vanado molybdate phosphoric
yellow colour method in nitric acid system using
Spectronic-20.
A base line survey on mineral (Ca, Pi, Zn,
Cu, Fe, Mn and PII) status of dairy animals and
fodder was conducted in a total of 67 dairy units
of 29 villages of central Punjab during months of
June in summer and January in winter. Average
temperature during the experimental year was 38
± 5ºC during summer and 14 ± 5ºC during winter.
A total of 188 buffaloes were selected randomly
without considering any health problem or mineral
deficiency symptoms.
Statistical analysis
Statistical analysis of the data was done by
method described by Singh et al. (1998).
Chemical Analyses
Blood
Blood samples from 188 buffaloes were
collected in sterile test tubes containing anticoagulant
(heparin). The samples were centrifuged at 3000
rpm fro 30 minutes at room temperature to separate
plasma. The plasma samples were stored in small
aliquots in mineral free glass vials at -10º C until
analysis. Two milliliters of plasma was digested
with nitric acid and perchloric acid and after
digestion the volume was made upto 10 ml with
double distilled water for micro-mineral analysis,
whereas plasma was used as such for Ca and Pi
of estimations. Concentrations of various plasma
minerals viz. Zn, Cu, Fe and Mn were measured by
Atomic Absorption Spectrophotometer (SpectraAA
20 plus, Varian, Melbourne, Australia) and plasma
Pi was estimated by method of Taussky and Shorr
(1953). Plasma Ca was estimated by Autoanalyser
using diagnostic reagent kits (Bayer Diagnostic
India Ltd., Baroda) by cresolphthalein complexone
method whereas plasma inorganic iodine (PII) was
determined by the method of Aumont and Tressol
(1987).
Fodder
Fodder which was being fed to these
RESULTS
The mineral contents of blood plasma and
forage samples are summarized in Table 1 and
2, respectively. The plasma samples contained
higher levels (P<0.05) of Zn and Pi during winter
as compared to summer. The variations in Zn and
P in the forage were also found to be significant
(P<0.05), with higher levels of these elements
during winter compared to during summer. Forage
had statistically non-significant (P>0.05) levels of
Ca, Fe, Cu and Mn during both seasons; contents
of the Ca, Fe, Cu were higher during winter than
those during summer though the difference was
not statistically significant. The concentrations of
other minerals in the plasma between the seasons
was not statistically significant (P>0.05), with nonstatistically significant higher levels of Ca, Fe and
PII minerals during winter than during summer.
DISCUSSION
The functions of the minerals in animal
46
Table 1. Mean plasma concentrations (Mean ± SE) and deficiency rate of different minerals during summer
and winter seasons in buffaloes.
Element
Zn (μmol/l)
Fe (μmol/l)
Cu (μmol/l)
Mn (μmol/l)
PII (ng/ml)
Ca (mg/dl)
Pi (mg/dl)
Summer
10.90 ± 0.45
(76.19)
47.53 ± 2.07
(4.17)
12.68 ± 0.35
(23.81)
0.82 ± 0.04
(20.83)
43.44 ± 3.15
(88.69)
8.92 ± 0.26
(41.07)
4.27 ± 0.20
(56.55)
Winter
22.20*± 2.53
(21.81)
51.93 ± 5.92
(9.57)
12.68 ± 1.45
(23.40)
0.80 ± 0.09
(13.30)
49.09 ± 4.05
(84.57)
9.74 ± 0.30
(26.06)
4.99*± 0.20
(37.77)
Figures in parenthesis show prevalence rate of deficiency .
* Winter v/s summer difference (P<0.05)
Table 2. Mean concentrations (Mean ± SE) of different minerals in summer and winter fodders.
Element
CLa
Cu(ppm)
Mn (ppm)
Zn(ppm)
Fe(ppm)
Ca(%)
P(%)
<10.0
<30.0
<30.0
<30.0
0.30
0.25
Summer
Meanb ±SEc
3.03 ± 0.17
19.25 ± 1.19
7.93 ± 0.61
239.18 ± 12.88
0.46 ± 0.05
0.24 ± 0.01
Winter
Meanb ±SEc
3.83 ± 0.29
15.60 ±1.03
17.39*±1.77
294.81 ± 40.48
0.77 ± 0.11
0.31*± 0.01
a = Critical level (McDowell 2003)
b = Least square mean from samples from all the districts in both the seasons
c = SE of least square means
* = Winter v/s summer difference (P<0.05)
47
both seasons were within the normal physiological
range as per McDowell (1992). Baruah and Baruah
(1997) also recorded no seasonal variation in mean
plasma Cu levels of Jersey heifers. On the other
hand, Goswami et al (1993) observed significant
seasonal changes in plasma Cu levels. The overall
prevalence of hypocuprosis among buffaloes was
23.40 and 23.81percent in winter and summer,
respectively (Table 1).
Similar overall mean plasma Mn levels in
buffaloes during both the seasons (Table 1) were
above the critical level of 0.37 μmol/l (Hidiroglou
1979). There was a wide variation in the plasma Mn
levels (0 – 2.48 μmol/l) among buffaloes whereby
many of the buffaloes were found to have nondetectable plasma Mn values in both the season.
The overall deficiency percentage of Mn during
winter was lower (13.30%) than that in summer
(20.8%). These results corroborated with findings
of Sawhney and Kehar (1961) who reported that
Mn stores in liver and kidneys got depleted in hot
and dry season and got repleted in winter.
Mean plasma inorganic iodine (PII) level
was higher during winter. The values during both
the seasons were well below the critical level of
104.9 ng/ml (Rogers,1992). Lundgren and Johnson
(1964) had reported that the low levels of PII
could be due to higher environmental temperature.
Jain (1990) found low iodine content of soil in
Punjab which supports the present findings. High
percentage of sub-clinical iodine deficiency was
observed during summer (88.7) as well winter
(84.6) seasons.
Overall mean plasma Ca level in winter
was higher. Higher plasma Ca levels in winter
could be attributed to the significantly (P<0.01)
higher levels of Ca found in winter fodder (Table
2). Earlier, Behera et al (2005) had also reported
higher plasma Ca during winter season in sheep.
physiology are interrelated: seldom can they be
considered as single minerals with independent
and self-sufficient roles (Ozdemir et al., 2006). The
mineral elements are not synthesized in the body
but are supplied by the feed. Their concentrations
in the body fluids will therefore depend on the
mineral contents of feed and forage, the level of
dietary sources intake, and the availability of
minerals (Suttle, 2010). Plant forages make up the
bulk of the diet consumed by the livestock. Many
environmental and plant factors affect the mineral
concentrations of forage plants; these include
,species or strain/ variety, soil type, the climatic of
seasonal conditions during plant growth, stage of
maturity of forage plants and other management
practices.
The data reported here indicate that most
of the macro and micro minerals studied are higher
in the winter season both in the animal blood and
forages compared to those during summer season
indicating a direct plant-animal relationship. Mean
plasma Zinc (Zn) level was significantly higher in
winter (Table 1). Considering the critical limit of
12.2 μmol/l, the prevalence rate of Zn deficiency
among buffaloes was 21.81 and 76.19 percent in
winter and summer, respectively (Table 1). These
findings could be correlated to significantly higher
plasma Zn levels in winter season. Yadav et al
(2002) had also reported lower incidence of Zn
deficiency among buffaloes during winter.
Plasma iron levels varied non-significantly
among both the seasons but the mean plasma Fe
values in the present study were much higher than
the normal range of 17.9 - 35.8 μmol/l (Radostits
et al., 2000). High Fe contents of fodder (Table 2)
against the dietary requirement of 50 ppm (NRC
1989) appeared as the main factor behind elevated
plasma Fe concentrations.
Overall mean plasma Cu levels during
48
Tuli, V.P. Dixit and S.L. Bhela. 1993.
Blood and seminal plasma trace mineral
concentrations during different seasons in
crossbred bulls Indian J. Anim. Sci., 63:
430-433.
Hidiroglou, M. 1979. Manganese in ruminant
nutrition. Can. J. Anim. Sci., 59: 217.
Jain, R. 1990. Dietary intakes of iodine in selected
goitre endemic and non-endemic areas
of Punjab. Ph.D. Dissertation, Punjab
Agricultural University, Ludhiana, India.
Lundgren, R.G. and H.D. Johnson. 1964. Effects
of temperature and feed intake on thyroxine
Overall mean plasma inorganic phosphorus
(Pi) level in winter was significantly (P<0.05)
higher than the summer season (Table1). Higher
winter levels could be attributed to the significantly
(P<0.05) higher fodder levels (Table2) The overall
prevalence of hypophosphataemia among buffaloes
was 37.77 and 56.55 percent in winter and summer,
respectively (Table 2). These findings of low
prevalence in winter could be due to significantly
higher Pi levels in plasma and fodder in the
winter season. Similar prevalence rate (38.0%) of
deficiency had been reported by Yadav et al (2002)
among buffaloes of Haryana during winter.
Thus, it can be concluded that alteration
in the levels of different micro and macro
minerals in the plasma corresponded to the fodder
mineral variations, indicating a direct plantanimal relationship and showing the need of
supplementation of the deficient minerals during
summer season for these animals at the place where
the livestock were being reared.
I131 disappearance rates of cattle. J. Anim.
Sci., 23: 28-31.
McDowell, L.R. 1992. Minerals in Animal and
Human Nutrition. Academic Press Inc, New
York.
NRC, 1996. National Academy of Sciences-National
Research Council, 7th ed. Washington DC.
Ozdemir, M., M. Cinar, S. Haliloglu and A.
Eryavuz. 2006. Effects of defaunation and
dietary nitrogen source on plasma and wool
of lambs. Turk. J. Vet. Anim. Sci., 30: 367373.
Radostits, O.M., C.C. Gay, D.C. Blood and K.W.
Hinchcliff. 2000. Veterinary Medicine: A
Textbook of Diseases of Cattle, Sheep, Pig,
Goat and Horses. W.B. Saunders Harcourt
Publishers Ltd., London.
Rogers, P.A.M. 1992. Iodine deficiency in cattle.
Irish Vet. News, September issue, p. 14-17.
Sawhney, P.C. and N.D. Kehar. 1961. Investigation
of trace element manganese. Part III.
Variations in the manganese content of
blood and tissues of cattle. Ann. Biochem.
Exptl. Med., 21: 125-128.
Singh, R. 2002. Epidemiological, clinicobiochemical and therapeutic studies on
REFERENCES
Aumont, G. and J.C. Tressol. 1987. Rapid method
for the direct determination of inorganic
iodine in plasma using ion-exchange
chromatography and the Sandell and
Kolthoff reaction. Analyst, 112: 875-877.
Baruah, A. and K.K. Baruah. 1997. Studies on
serum micro-minerals in Jersey heifers
during different seasons. Indian Journal of
Animal Health, 236: 115-116.
Behera, P.C., N. Sharma and P.C. Bisoi. 2005.
Seasonal variation of clinically important
blood biochemical constituents of lambs.
Indian Vet. J., 82: 26-28.
Goswami, S.C., S.N. Mehta, G.C. Georgie, R.K.
49
trace elements deficiency in dairy animals
and sheep of sub-mountainous region.
Ph. D. Dissertation, Punjab Agricultural
University, Ludhiana, India.
Singh, S., M.L Bansal, T.P. Singh and R. Kumar.
1998. Statistical Methods for Research
Workers. Kalyani Publishers, New Delhi. p.
287-301.
Suttle, N.F. 2010. Mineral Nutrition of Livestock,
4th ed, Midlothian, UK.
Taussky, H.H. and E. Shorr. 1953. A micro
colorimetric method for the determination
of inorganic phosphorus. J. Biol. Chem.
202: 675-685.
Yadav, P.S., A.B. Mandal and D.V. Dahiya, 2002.
Feeding pattern and mineral status of
buffaloes in Panipat district of Haryana
state. Anim. Nutr. Feed Techn., 2: 127-138.
50
Original Article
A STUDY ON THE PREVALENCE OF PATHOLOGICAL ABNORMALITIES OF THE
OVARIES AND OVIDUCTS DIAGNOSED AT POST MORTEM OF BUFFALOES IN MOSUL
O.I. Azawi* and A.J. Ali
major problems in buffalo herds in Mosul leading
to slaughtered and economic losses.
ABSTRACT
The objective of the present study was to
determine the prevalence of ovarian abnormalities
and oviduct abnormalities of Iraqi buffaloes.
Buffalo cow reproductive tracts were collected at
random intervals slaughtered at Mosul abattoir,
from January 2006 to August 2010. A total of 405
of mature primiparous and pluriparous genital
tracts were examined. Ovaries were inspected for
cross lesions and oviductal lesions were included
in this study. Hydrosalpinx and pyosalpinx were
diagnosed and evaluated by measurement using
ruler and caliper. Salpingitis was classified into
chronic, subacute and acute according to histological
examination. Out of the 405 buffalo genital tracts
examined, various abnormalities with different
degrees of severity were observed in 216 (53.3%)
of cases. Twenty two (5.4%) were pregnant and the
remaining 41.2% (167/405) were macroscopically
normal. Follicular cyst, luteal cyst, cystic corpus
luteum, paraovarian cyst, ovarian sarcoma,
inactive ovaries, senility anestrous, pyosalpinx,
hemosalpinx, obstruction of oviduct, salpingitis,
double oviduct were recorded. In a conclusion, the
current study disclosed that, ovarian and oviductal
abnormalities seem to be an important problem
with possible subsequent infertility and sterility
in buffalo cows in Mosul. The high proportions
of hydrosalpinx and ovarobursal adhesions are the
Keywords: ovarian abnormalities, follicular
cyst, luteal cyst, ovarobursal adhesions, oviductal
abnormalities, hydrosalpinx, pyosalpinx, buffalo
cow
INTRODUCTION
Genital organ disorders are important cause
of infertility and sterility in buffalo cows causing
high economic losses (Azawi 2006, 2008; Azawi et
al. 2008a). The ovaries and oviducts are important
for controlling estrous cycle, hormonal production,
fertilization and the maintenance of the embryo
until its arrival in the uterus. Ovarian pathology and
oviductal abnormalities are common diseases in
domestic mammals, especially cattle and buffaloes
(McEntee 1990; Azawi et al. 2008b). Abnormalities
of the buffaloes reproductive organs had been
reported in surveys in Iraq (Alwan et al. 2001; AlFahad et al. 2004; Azawi et al., 2008c), India (Rao
and Sreemannarayana 1983; Sar et al. 1996), Egypt
(Ghaneem et al. 2002) and Iran (Moghaddam
and Mamoei 2004). The animals presented in
all the above surveys did not included detailed
pathological causes of certain abnormalities of the
ovaries and oviducts. The aim of this study was to
Department of Surgery and Theriogenology, College of Veterinary Medicine, University of Mosul, Mosul,
Iraq, *E-mail: [email protected]
51
was no CL or CH and without > 5 mm diameter
follicle (s) was regarded as being in anestrous due
to old age or senility. Specimens with oviductal
lesions were included in this study. Hydrosalpinx
and pyosalpinx were diagnosed and evaluated by
measurement using ruler and caliper. Salpingitis
was classified into chronic, subacute and acute
according to histological examination. The patience
of each uterine tube was checked by injecting 5 ml
of colored fluid (Indian ink) near the junction of the
uterine tube with the corresponding uterine horn.
Biopsies (approximately 1 cm3) were obtained
from each oviducts affected with hydrosalpinx,
pyosalpinx, salpingitis and oviductal obstruction
of samples included in this study. The biopsy
was immediately placed into bottle containing
10% formal saline solution and stored at 4°C till
preparation for sectioning, which was included
dehydration, clearing, embedding, sectioning and
staining were performed as the methods described
study the prevalence of ovarian abnormalities and
oviduct abnormalities of buffaloes.
Buffalo cow reproductive tracts were
collected at random intervals slaughtered at Mosul
abattoir, from January 2006 to August 2010. A
total of 405 of mature primiparous and pluriparous
genital tracts were examined. The specimens were
transported to the college of veterinary medicine,
university of Mosul. Each specimen was examined
grossly in the laboratory in order to exclude any
specimen containing reproductive abnormality.
Pregnant specimens were discarded. All cases
were examined for presence of fetuses. Then the
vagina, uterus, uterine tubes and ovaries were
visually inspected for cross lesions. The vagina
and uterus were opened up to utero-tubal junction
and examined. Ovaries were inspected for cross
lesions and the number of corpora albicantia (CA)
and side of the ovary with corpus luteum (CL)
recorded. A pair of ovaries with either a corpus
hemorrhagicum (CH), a large CL and > 5 mm
follicle (s) in diameter or a regressing CL with
follicle (s) > 6 mm in diameter were classified
as active and the animals as cycling. When there
was no CL or CH or the presence of a regressed
CL without > 5 mm in diameter follicle (s), such
ovaries were classified as inactive and the animals
as noncycling. A regressing CL coupled with an
incomplete involuted uterus was classified as postparturient anestrous. Corpora albicantia replacing
the corpora lutea of pregnancy are large and tend to
persist indefinitely (Roberts, 1986). They are more
prominent in buffaloes, and can therefore be used
to estimate the parity of an animal (Jainudeen et al.,
1983). An animal with more than 7-10 CA and there
by Luna (1968).
Reproductive organs from 405 animals
were examined; 5.4% (22/405) of the animals were
pregnant, 41.2% (167/405) were cycling. Various
abnormalities with different degrees of severity
were observed in 216 (53.3%) of cases. The
prevalence of the various ovarian and oviductal
abnormalities of buffaloes is presented in Table 1.
Ovarian abnormalities
Follicular cysts (Figure 1) are recorded in
six (1.5%) cases. The average diameter was 32.8 ±
1.3 mm. Luteal cysts (Figure 2) was diagnosed in
one (0.2%) buffalo cow. Cystic corpora lutea were
encountered unilaterally in five (1.2%) cases. The
52
corpora lutea had an average diameter of the cystic
cavity in the center of the corpora lutea varied
considerably from 6 to 18 mm. Twenty six (6.4%)
cases had ovarobursal adhesions (Figure 3 and 4).
The severity of ovarobursal adhesions ranged from
mild strands of connective tissue between the ovary
and the bursa (34.6%) to severe adhesions (65.4%),
when the ovary was completely encapsulated
in fibrous tissue. Paraovarian cysts were found
in 18 (4.4%) of the cases, they were generally
single (Figure 5), but double and triplet were also
recorded. These cysts were filled with thick mucoid
fluid. One ovarian tumor (Figure 6) was examined
histologically and was confirmed to be ovarian
sarcoma. Out of 405 examined genital tracts, 6
(1.5%) were found to be inactive ovaries. While
five (1.2%) were found as senility anestrous.
Oviductal abnormalities
Hydrosalpinx (Figure 7 and 8) was found
in 20 (4.9%) cases. In these cases dilatation of
oviduct due to clear amber fluid accumulation
were detected. In eight cases extreme dilatation
were observed with the oviduct having maximum
diameter of 30 mm. Pyosalpinx (Figure 9) was
recorded in nine (2.2%) characterized by dilatation
of the oviduct due to thick whitish-yellowish
pyogenic fluid. Three cases (0.7%) of oviducts
Table 1. Prevalence of various kinds of abnormalities in ovaries and oviducts in buffaloes.
Abnormalities
Follicular cyst
Luteal cyst
Cystic corpus luteum
Paraovarian cyst
Ovarobursal adhesions
Ovarian sarcoma
Inactive ovary
Senility anestrous
Total
Abnormalities
Hydrosalpinx
Pyosalpinx
Hemosalpinx
Obstruction
Salpingitis
Adhesions-salpingitis
Double oviducts
Total
No.
6
1
5
18
26
1
6
5
Ovary
%
1.5
0.2
1.2
4.4
6.4
0.2
1.5
1.2
Right
Left
4 (0.9)
1 (0.2)
1 (0.2)
14 (3.5)
15 (3.7)
2 (0.5)
0
4 (0.9)
4 (0.9)
11 (2.7)
68
Oviducts
No.
%
20
4.9
9
2.2
3
0.7
6
1.5
5
1.2
7
1.7
1
0.2
Right
Left
15 (3.7)
6 (1.5)
5 (1.7)
3 (0.7)
51
53
filled with bloody discharge were recorded.
Obstruction of oviduct was observed in six (1.5%)
cases and salpingitis was found in five (1.2%) cases.
Adhesions between mesosalpinx and perisalpingeal
tissues were observed seven (1.7%). One case
(0.2%) of double oviduct (Figure 10) was found
in the left side of the tracts examined. Histological
examination confirmed the diagnosis of double
oviduct. Microscopic examination of the oviducts
with hydrosalpinx showed mucosal atrophy and
dilatation of oviduct lumen without any signs of
inflammation characterized by no infiltration of
inflammatory cell (Figure 11). While pyosalpinx
showed mucosal atrophy and dilatation of uterine
tube lumen with signs of severe inflammation
including higher infiltration of lymphocytes and
sloughing of the mucosa epithelial layer lining
uterine tubes.
The data obtained from this study reflect the
high incidence of gross lesions in buffalo ovaries
and oviducts. Most of these lesions were acquired as
manifested by the high incidence of follicular cyst,
luteal cyst, cystic corpus luteum, paraovarian cyst,
ovarobursal adhesions, ovarian sarcoma, inactive
ovary, senility anestrous hydrosalpinx, pyosalpinx,
adhesions and obstruction of the oviduct. The
prevalence of ovarian cyst recorded in this study is
comparable with those of (Al-Dahash and David,
cows (Kesler and Garverick, 1982). The frequency
of paraovarian cyst in the present study is similar
to these (Fathalla et al., 2000), who reported 4%
in Jordan cattle. In some cases of paraovarian
cyst were observed on the surface of the oviduct
and extended pressure on it. This is in line with
the reports of Roine (1998), who checked for the
blockage of the lumen by flushing. The prevalence
of ovarobursal adhesions obtained in this study is
in agreement with those of (Alwan et al., 2001; AlFahad et al., 2004), who reported 5% and 10.4%,
respectively. However, it is lower than those of
(Assey et al., 1998; Feyissa, 2004), who reported
11% and 11.6%, respectively. Although the exact
mechanism by which adhesions develop is unclear
(Roberts, 1986), extreme adhesions have probably
resulted from pregnancy complications that
include retained fetal membranes and endometritis
(Hatipoglu et al., 2000). Mild adhesions could result
from non-infectious conditions such as physical
trauma as a result of rough manipulation (Abalti et
al., 2006). Localized abdominal infections such as
omphalophlebitis and peritonitis are also suggested
cause this condition (Noakes et al., 2002). The
adhesions involved the right ovary more than the
left but bilateral cases were also observed this
study. This is in agreement with the findings of
several workers (Herenda, 1987; Fathalla et al.,
1977; Hatipoglu et al., 2000; Azawi, 2009) who
reported 5.4% and 3.8%, respectively. However,
the prevalence in the present study is lower than
that of other previous reports, which varied from
6% to 30% (Herenda, 1987; Roine, 1998; Feyissa,
2004). Breed, age, level of milk production, feeding,
management and exercise are factors, suggested
as influencing the prevalence of cystic ovaries in
cattle (Noakes et al., 2002). In dairy cattle cystic
ovaries prolongs the postpartum interval to first
estrous and conception in about 10-30% of dairy
2000; Hatipoglu et al., 2002; Abalti et al., 2006).
This difference may be attributed to the more
active of right ovary (Roberts, 1986). Extensive
adhesions leading to the obliteration of the ovarian
bursa, blockage of the abdominal opening of the
infundibulum or extensive coverage of the ovarian
surface with fibrous tissue will certainly interfere
with ovulation. This in turn may lead to infertility or
even sterility depending on extent and on whether
the adhesions are unilateral or bilateral.
In the present study, hydrosalpinx and
54
Figure 1. Follicular cyst in the right ovary.
Figure 2. Luteal cyst in the left ovary.
55
Figure 3. Complete ovarobursal adhesions in the right ovary.
Figure 4 Bilateral ovarobursal adhesions with a follicular cyst in the left ovary.
56
Figure 5. Paraovarian cyst near the right oviduct.
Figure 6. Ovarian sarcoma.
57
Figure 7. Hydrosalpinx in the left oviduct highly enlarged than right oviduct that was also affected with
hydrosalpinx with lower enlargement.
Figure 8. Hydrosalpinx in both sides highly enlarged.
58
Figure 9. Pyosalpinx in both oviducts.
Figure 10. Double oviducts.
59
pyosalpinx were accompanied with ovarobursal
adhesions and chronic endometritis. Results of
the present study indicated a high prevalence of
hydrosalpinx when compared to the Iraqi southern
breeds (Alwan et al., 2001; Al-Fahad et al., 2004).
However, this disagreement can be accounted
for largely, due to the high prevalence of toxic
puerperal metritis and chronic metritis as founded
by previous studies (Azawi et al., 2007; Azawi
et al., 2008). These two reasons may explain the
high prevalence of hydrosalpinx in Iraqi northern
buffaloes. The obstruction in the lumen of the
oviducts resulted in accumulation of fluid. It is
tempting to attach some special significance to the
association of endometritis with the occurrence of
hydrosalpinx, and to suggest some contributing
role in the production of severe inflammation
in the endometrium extended to the utero-tubal
junction. This theory could be confirmed by the
results of the present study as all obstructions of
the uterine tubes examined were near the uterotubal junction or in the end part of isthmus. These
observations are in agreement with Miller and
Campbell (1978) who claimed that hydrosalpinx
is a sequel to salpingitis. In addition, Mastroianni
(1999) reported hydrosalpinx as a result of some
inflammatory process in or around the uterine tubes.
While, Ellington and Schlafer (1993) opinion that
is hydrosalpinx may be congenital disease.
In a conclusion, the current study disclosed
that, ovarian and oviductal abnormalities seem to
be an important problem with possible subsequent
infertility and sterility in buffalo cows in Mosul. The
high proportions of hydrosalpinx and ovarobursal
adhesions are the major problems in buffalo herds
in Mosul leading to slaughtered and economic
losses.
REFERENCES
Abalti, A., M. Bekana, M. Woldemeskel and
F. Lobago. 2006. Female genital tract
abnormalities of Zebu cattle slaughtered at
Figure 11. Hydrosalpinx, mucosal atrophy and dilatation of oviduct lumen with low infiltration of leukocytes
(H&E X 100).
60
Bahir-Dar Town, north-west Ethiopia. Trop.
Anim. Health Pro., 38: 505-510.
Al-Dahash, S.Y.A. and J.S.E. David. 1977. The
incidence of ovarian activity, pregnancy
and bovine genital abnormalities shown by
abattoir survey. Vet. Rec., 101: 296-299.
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Original Article
EFFECT OF VITAMIN E AND MINERAL SUPPLEMENTATION ON BIOCHEMICAL
PROFILE AND REPRODUCTIVE PERFORMANCE OF BUFFALOES
H.M. Khan1, T.K. Mohanty2, M. Bhakat2, A.K. Gupta2, A.K. Tyagi3 and G. Mondal3
ABSTRACT
vitamin E values obtained were statistically not
significant. However, the supplemented group had
lower levels than the control group at all the stages
but vitamin E values had higher levels than the
control group at all the stages. Cervical and uterine
involution was completed in lesser days, involutory
The experiment was designed to
provide higher plane of nutrition, vitamin E and
mineral supplementation for augmenting the
improvement in reproductive performance. In
the present investigation, 10 Murrah buffaloes
each in two groups, expected to calve in winter
season were selected during prepartum period.
None of the buffaloes during periparturient period
suffered from any clinical metabolic disease or
reproductive disorders. Plasma Ca and Plasma
inorganic P concentration showed significant
difference on day 15 prepartum (P<0.01); Zn on
day 15 prepartum (P<0.05) and day 15 postpartum
(P<0.01); Cu on day 30 prepartum (P<0.05) and
Mn on day 30 prepartum (P<0.05), however, the
differences in concentrations at all other stages
were non-significant but the supplemented group
had higher levels than the control group at all the
stages. The concentration of the plasma glucose
exhibited significant difference (P<0.01) at 45 days
postpartum but the supplemented group had higher
levels than the control group at all the stages. The
plasma BUN showed significant difference (P<0.05)
at days 30 and 15 prepartum, calving day and on
day 30 postpartum (P<0.01) and Plasma NEFA and
changes took place at a faster pace and there were
lesser percent of cows suffering from abnormal
uterine changes in supplemented compared to
control group. Supplemented group showed better
reproductive performance considered in the study
than control group. In total, around 12 days could
be saved in days to first service if vitamin E and
minerals were supplemented. Supplemented group
showed early initiation of cyclicity (32 days
postpartum) compared to control group (35 days
postpartum). Cyclicity in most of the animals
might have been initiated earlier than 30days as
was evident from progesterone concentration (>1
ng/ml). Short and long luteal phases were observed
on appraisal of progesterone concentration in
both the groups which delayed the days to first
service in these animals. It can be concluded that
mineral and vitamin E supplementation improved
the reproductive performance of buffalo during
periparturient period.
Sheep Research Station, Faculty of Veterinary Science and Animal Husbandry, Sher-e-Kashmir University
of Agricultural Sciences and Technology of Kashmir (SKUAST-K), E-mail: [email protected], bhakat.
[email protected]
2
Artificial Breeding Research Centre, National Dairy Research Institute, Karnal, Haryana, India
3
Dairy Cattle Nutrition Division, National Dairy Research Institute, Karnal, Haryana, India
1
63
Keywords: Murrah buffalo, mineral, vitamin E,
reproductive performance, periparturient period
Balagopal, 1994; Prajapati et al., 2005), metabolic
disorders, retention of foetal membranes (Gupta
et al., 2005), dystocia, abortion (McDowell 1992;
Dutta et al., 2001; Sharma et al., 2005), weak calf
syndrome (Logan et al., 1990), milk fever, vulval
discharge (Husband, 2006) and poor conception
rate (Khasatiya et al., 2005). Thus have negative
impact on the subsequent fertility of the cow. Such
disorders could probably be prevented by addressing
to the basic etiology through balanced feeding
and mineral supplementation during advanced
pregnancy and early post-partum period, when the
animals are highly prone to stress of heavy nutrient
demand and drain (Mandali et al., 2002). Thus
nutritional supplementations play important role
to improve general, productive and reproductive
health of animals (Kleczkowski et al., 2003). Further
mineral (Sharma et al., 2003; Hussain et al., 2004;
Borghese, 2005; Yildiz et al., 2006) and vitamin
E supplementation (LeBlanc et al., 2004; Panda
et al., 2006) improves reproductive performance
because of their positive effect on steroid synthesis,
release, follicular growth and symptoms of
ovulatory oestrus (Srivastava, 2008). The impact
of minerals and vitamins supplemented in the peripartum period in buffaloes on subsequent fertility
is lacking and needs due importance for prevention
of periparturient problems and improvement of
fertility. In order to deal with above problems
and to improve overall reproductive efficiency in
buffaloes, the present investigation was undertaken
to fill up the gaps in knowledge in Murrah buffaloes
with the objective to investigate the role of
prepartum mineral and vitamin E supplementation
on postpartum reproductive performance.
INTRODUCTION
Nutritional management during the
dry period is the main factor which may affect
susceptibility of cows to metabolic and infectious
diseases during the periparturient period (Dann et
al., 2005; Campanile et al., 1997). Besides general
nutritional status, deficiencies or imbalance with
respect to specific nutrients (minerals and vitamins)
have been found to have drastic effects on various
determinants of reproductive performance leading
to infertility. Vitamins and minerals (macro
and microelements) in minute quantity play
a decisive role in overall metabolism, normal
growth, production and reproduction. Excess or
even imbalance of some minerals and vitamins
may have deleterious effect on health (Borghese,
2005). Further the impacts of such disturbances
on general health including insidious sub-clinical
diseases/ disorders have been recognized as the
most important but covert factors with deleterious
consequences for reproductive performance.
Minerals and vitamins have direct or indirect
relationship with productive and reproductive health
of animals. Vitamin E is important for maintaining
optimal immune function (Sikka et al., 2002; Sikka
and Lal, 2006) and as anti-stress factors (Kahlon et
al., 2006) and requirements are higher compared
to production or reproduction requirements
(Xin et al., 1991; Weiss, 1998). Deficiencies and
imbalance of minerals during peri-parturient period
are either solely incriminated for or associated
with anestrous (Patil and Deshpande, 1979; Naidu
and Rao, 1982; Agarwal et al., 1985; Singh and
Vadnere, 1987), repeat breeding (Balakrishnan and
64
Scoring of uterine discharges and uterine
involution
The present study was conducted on 20
pregnant dry Murrah buffaloes maintained at Cattle
Yard of National Dairy Research Institute (NDRI),
Karnal, India. Twenty Murrah buffaloes 60 days
prepartum were selected and randomly assigned
to two experimental groups with 10 animals in
each group; group 1 (C) was provided 20% higher
nutrients than Kearl’s Feeding Standard (Chauhan et
al., 2000) and group 2 (T) was provided 20% higher
nutrients than Kearl’s Feeding Standard (Chauhan
et al., 2000) along with vitamin E { (2000 IU from
60 days prepartum to 30 days postpartum and 1500
IU from 30 to 60 days postpartum) vitamin E 50%
powder, Vet Chem } supplementation (Panda et al.,
2006) and 50 gm of commercial mineral mixture
(Agrimin, Agrivet Farm Care Division) to meet
the expected requirements of the minerals. The
buffaloes used for the investigation were kept in
conventional barns throughout the prepartum
period and were shifted to calving pens 2 weeks
prior to expected date of parturition for extra care
and attention upto 5 days after parturition. After
that they were shifted to loose housing and group
management system where other lactating buffaloes
were kept. The parameters (time of parturition,
days required for cessation of lochia, days required
for complete uterine involution, days at first heat,
days at first service) were recorded.
All the experimental buffaloes were
monitored regularly for estrus by visual observation
and parading of vasectomised bull in the morning
and evening hours. Animal were rectally confirmed
for heat and inseminated with frozen semen by
two inseminations at 12 h intervals. Buffaloes
not returning to estrus after inseminations were
examined per rectum on 45 for pregnancy
confirmation.
The
experimental
animals
under
investigation were scored for uterine discharges
and Uterine Involution on 7, 14, 21, 28 and 35 days
postpartum as per Sheldon and Noakes (1998) for
early diagnosis and treatment of uterine infections.
Plasma Biochemical Assay
The blood samples were collected from
jugular vein into heparinized (20 IU heparin/ ml
blood) tubes from all experimental animals at
fortnightly interval from 60 days prepartum to
60 days postpartum. Immediately after sampling
the blood was centrifuged at 3000 rpm for 15 to
20 minutes and the plasma was separated and
stored frozen (-20°C) until analyzed. Following
micronutrients and metabolites in control as well
as in experimental groups were estimated:
Plasma Mineral and Metabolite Estimation
Plasma Ca, inorganic P, Zn, Cu and Mn
were estimated at fortnightly intervals in both the
groups. Plasma minerals (Ca, Zn, Mn and Cu)
except phosphorous were estimated with the help
of Atomic absorption Spectrophotometer (Model
PU9100X Atomic absorption Spectrophotometer,
Philips). The procedure described in AAS (1988)
manual for preparation of stock and standard
solutions and choice of instrumental conditions
were followed. Plasma inorganic phosphorus
was estimated following the method of Fiske
and Subbarow (1925). An HPLC method for
simultaneous estimation of α-tocopherol in plasma
was adopted (Chawla and Kaur, 2001). The plasma
urea was estimated according to Rahmatulla and
Boyde in 1980. The copper soap extraction method
modified by Shipe et al. (1980) was adopted for the
determination of plasma NEFA and the standard
65
Table 1 for interpretation from 45 days prepartum
to 45 days postpartum taking 0 day as the day of
calving. The supplemented group had higher levels
of plasma mineral (Ca, inorganic P, Zn, Cu and Mn),
Plasma glucose, Plasma Vitamin E and lower level
Plasma BUN and Plasma NEFA than the control
group at all the stages reflecting improvement
and beneficial effect due to vitamin E and mineral
supplementation.
curve was prepared with palmitic acid as specified
by Koops and Klomp (1977). Glucose in blood
plasma was estimated by end-point o-Toluidine
method.
Blood plasma analysis for progesterone
quantification
The method of Kamboj and Prakash
(1993) was followed through RIA for blood plasma
progesterone estimation.
Plasma Ca
Plasma Ca concentration did not follow
a specific pattern in both the groups. Plasma
Ca decreased on days 30 and 15 prepartum in
control and day 30 only in supplemented group
during the prepartum period. These changes
were attributed to growing needs of the fetus and
changes in the available fodder. At parturition and
15 day postpartum there was a substantial increase,
whereas days 30 and 45 showed a decrease in Ca
level in both the groups. The changes reflected in
both the groups were not significant except day 15
prepartum (P<0.01).
The increasing and decreasing trend of
plasma Ca not following a specific pattern may
be attributed to changes in available fodder. The
drop in Ca concentration on day 30 and 45 might
be due to its increased diversion for foetal growth
and more secretion of Ca through colostrums and
milk; however the concentrations were within the
normal range. The normal values in cows vary
between 8-12 mg/dl and hypocalcaemia occurs
when value decreases to 3-7 mg/dl (Hidiroglou,
1979; McDowell, 1992; Shah et al., 2003). Buffalo
calcium blood levels have been reported to show
limited variability during lactation and dry milk
period. Campanile et al. (1997) found constant
values of about 10 mg/dl. Higher levels have been
found in the last month of pregnancy and lower ones
Statistical analysis
Effect of vitamin E and mineral
supplementation on mineral and metabolic status
and reproductive performance was calculated by t
test using Systat 6 software package.
Feeding plays an important role in the
performance of the animals. The experiment was
designed to provide higher plane of nutrition
(Chauhan et al., 2000) and vitamin E and mineral
supplementation for augmenting the improvement
in reproductive performance. In the present
investigation, 10 buffaloes each expected to calve
in winter season were selected during prepartum
period for investigating the role of vitamin E and
mineral supplementation supplemented through
prepartum to postpartum period. None of the
buffaloes during periparturient period suffered
from any clinical metabolic disease or reproductive
disorders. Two of the buffaloes, one each from both
the groups due to chronic problem were removed
from the experimental study.
Mineral profile of buffaloes
The results obtained are presented in the
66
A
67
Supplemented
Control
Supplemented
Control
Supplemented
Control
Supplemented
Control
Supplemented
Control
-45d
10.06±0.21
9.97±0.26
5.59±0.22
5.51±0.15
2.46±0.27
2.05±0.16
0.96±0.08
0.93±0.11
1.40±0.10
1.44±0.13
- mg%; B- ppm; * - Significant (P<0.05); ** - Significant (P<0.01)
CuB
MnB
ZnB
PA
Ca
A
Prepartum
-30d
-15d
9.6±0.23
10.19±0.29**
9.15±0.26
9.06±0.22**
5.65±0.18
5.77±0.10**
5.42±0.22
5.13±0.14**
2.34±0.34
2.74±0.20*
2.08±0.20
2.20±0.14*
1.02±0.03*
0.80±0.08
0.87±0.05*
0.71±0.04
1.55±0.07*
1.29±0.15
1.24±0.13*
1.21±0.09
0d
10.22±0.33
9.47±0.32
5.65±0.15
5.27±0.18
1.89±0.23
1.72±0.09
0.65±0.07
0.54±0.05
0.70±0.09
0.63±0.08
15
9.96±0.14
9.46±0.23
5.68±0.15
5.22±0.20
1.92±0.12**
1.46±0.08**
0.91±0.10
0.72±0.08
1.17±0.19
0.80±0.17
Table 1. Mineral status of vitamin E and mineral supplemented and control buffaloes (Mean ± S.E.).
Postpartum
30
10.09±0.19
9.62±0.24
5.72±0.14
5.44±0.12
1.98±0.08
1.81±0.08
0.73±0.07
0.64±0.08
1.22±0.09
0.92±0.18
45
9.26±0.14
8.95±0.16
5.99±0.23
5.55±0.24
2.62±0.40
2.10±0.09
0.76±0.07
0.71±0.03
1.23±0.09
1.12±0.15
at the end of the lactation period (Montemurro et
al., 1997). A seasonal variation has been evidenced
with higher values during the winter as leguminous
fodder contains more calcium. Ca deficiency in
cow causes reproductive disorders viz. prolapse
of uterus, retained placenta, difficult delivery and
delay in uterine involution. In buffalo, calcium
excesses could alter the Ca/P ratio during the dry
period, inducing parathyroid hypoactivity which
would cause magnesium to increase and calcium to
decrease at the beginning of the lactation due to a
non immediate calcium mobilization by the bones.
The altered Ca/Mg ratio has been incriminated for
atony of uterus and eventually uterine prolapse
(Campanile et al., 1997).
P i.e. maintaining normal level (Campanile et al.,
1997).
Plasma Zn
Plasma Zn concentration was lowest on
the day of calving in both the groups. The Zn
concentration dropped on day 30 prepartum and
day of calving and showed an increasing trend
thereafter in supplemented buffaloes, whereas it
showed an increasing trend at all stages except
day of calving and day 15 postpartum in control
group. There was significant difference in plasma
concentration of the Zn on day 15 prepartum
(P<0.05) and day 15 postpartum (P<0.01). The
differences in concentration at all other stages
was non significant. The results regarding plasma
Zn concentration in consonance with Panda
(2003), who also reported decrease in plasma Zn
concentration in buffaloes during late gestation and
parturition.
Plasma inorganic P
Plasma inorganic P showed a specific
trend. It increased in the supplemented group upto
days 15 prepartum and declined on calving day and
showed an increasing trend thereafter, whereas in
control group it declined upto day15 prepartum
and thereafter showed an increasing trend. The
differences in concentration at all stages was non
significant except day 15 prepartum (P<0.01).
The drop in concentration of plasma
P during prepartum may be due to increasing
demands of growing foetus and also due to changes
in concentration of available fodder. Phosphorus
levels in buffaloes have been found to be quite
stable at 6 mg/dl (Campanile et al., 1997). An
increasing trend has been evidenced starting from
the pre-partum period (6.3 mg/dl) to 160 days of
lactation (7.9 mg/dl) (Montemurro et al., 1997).
Phosphorus deficiency during dry period has been
recognized as the most frequent causes of vaginal
and/or uterine prolapse. Dietary deficiency of P
before calving has been found to cause decreased
calcium levels at calving without altering serum
Plasma Cu and Mn
Plasma Cu levels followed a particular
trend in both the groups. It started declining upto
calving and thereafter there was an increase in its
concentration, except at day 30 in the supplemented
group wherein it showed an increasing level. The
extent of decrease in Cu concentration at parturition
was more in comparison to other minerals. There
was significant difference in plasma concentration
of the Cu on day 30 prepartum (P<0.05), however,
the differences in concentration at all other stages
were non significant.
Plasma Mn levels followed a decreasing
trend upto parturition and followed increasing trend
following parturition except on day 30 in both the
groups. The drop in the Mn concentration during
prepartum might be due to the increasing demands
of growing foetus and utilization for improving
68
antioxidant status. The drop following parturition
on day 30 might be due to variation in available
fodders. There was significant difference in plasma
concentration of the Mn on day 30 prepartum
(P<0.05), however, the differences in concentration
at all other stages were non significant. Panda
(2003) also found the similar trend in Cu and Mn
concentration but reported higher levels than the
present findings.
and highly significant difference (P<0.01) on day
30 postpartum. The differences in concentration
at other periods was non significant. Plasma blood
urea concentrations were close to the normal ranges
in buffaloes (Borghese, 2005). Urea concentration
is an indicator of energy protein balance (Dhali,
2001; Campanile et al., 1998; Dhali et al., 2006)
and is typically increased in cows deficient in
energy.
Metabolic profile of buffaloes
Metabolic profile reflects the nutritional
and physiological status of the animal. In the present
study, the metabolic profile of the animals in terms
of blood glucose, BUN and NEFA were evaluated to
delineate their effects on reproduction performance
in control and supplemented groups. The metabolic
profile in control and supplemented buffaloes is
presented (Table 2) for ease of interpretation.
Plasma NEFA
Plasma NEFA showed decreasing and
increasing trend in both the groups and the values
obtained were statistically non significant. During
postpartum increasing trend was followed during
30 days signifying slight negative energy balance
but the values are much lower than in cattle.
Buffaloes have higher protein and energy utilizing
efficiencies as compared to cattle at similar fat
corrected milk production level, plane of energy
and protein nutrition, body size and weight change
(Paul et al., 2003) which could be the reason for
less negative energy balance reflected in buffaloes
during postpartum period.
Plasma glucose
The concentration of the plasma glucose
exhibited a highly significant difference (P<0.01)
among the treatment groups at 45 days postpartum
(Table 2). Plasma glucose followed an increasing
trend throughout the experiment period except on
day 30 when it showed slight decrease in both the
groups which might be due to change or variation
in the fodder supplied or other managemental and
environmental effects. However, the differences in
concentration at other periods was non significant.
Plasma vitamin E
The plasma vitamin E values obtained
showed a decreasing trend in the control group
during prepartum period and thereafter increased
upto day 45 postpartum. In Supplemented group,
there was slight decrease in the concentration on days
15 prepartum, calving day and 15 day postpartum.
The difference in the concentration of two groups
was statistically non significant. Campanile et al.
(1997) reported that in buffaloes average value
of vitamin E is 175 g/l and it increased with the
distance from calving and reduced after 120 days
of lactation. An increase in serum α-tocopherol
to 1 μg/ml in the last week prepartum has been
Plasma BUN
The plasma BUN values obtained were
lower in the prepartum than postpartum period.
Plasma BUN decreased during prepartum period
and increased during postpartum period. Plasma
blood urea nitrogen showed significant difference
(P<0.05) at days 30 and 15 prepartum, calving day
69
Supplemented
Control
Supplemented
Control
Supplemented
Control
Supplemented
Control
-45d
57.53±2.73
63.89±5.63
15.03±1.25
24.95±5.64
305.89±10.98
305.41±11.35
1.42±0.15
1.40±0.19
– mg%; B - μmol/l; C -μg/ml; * - Significant (P<0.05); ** - Significant (P<0.01)
BUN – Blood urea nitrogen; NEFA – Non-esterified fatty acids
A
Vitamin E C
NEFA B
BUN A
Glucose
A
Prepartum
-30d
-15d
62.06±1.72
65.46±3.44
67.18±2.20
69.80±2.44
15.13±1.28*
12.19±1.38*
24.71±4.11*
21.26±2.96*
275.64±19.31 298.81±10.76
273.81±10.07 295.22±7.17
1.44±0.21
1.24±0.16
1.25±0.27
1.23±0.17
0d
71.67±3.67
72.19±3.66
16.70±2.81*
22.81±2.27*
327.73±16.93
338.10±14.82
1.30±0.22
0.93±0.11
15
76.26±2.27
73.41±2.68
16.63±2.81
25.65±4.13
337.04±13.39
351.31±11.35
1.23±0.14
1.04±0.17
Table 2. Metabolic status of vitamin E and mineral supplemented and control buffaloes (Mean ± S.E.).
Postpartum
30
75.53±3.01
72.21±2.70
20.16±1.10**
28.55±2.57**
331.04±13.52
365.64±10.31
1.53±0.14
1.28±0.12
45
79.13±2.06**
71.03±1.40**
21.18±1.96
25.42±2.16
325.17±6.99
347.66±15.05
1.68±0.17
1.42±0.07
70
found to reduce the risk of retained placenta by
20% (LeBlanc et al., 2004). Peripartum decreases
in serum concentrations of vitamins A and E
have been incriminated for impaired immune
function in dairy cows (LeBlanc et al., 2004). Oral
administration of selenium along with vitamin E to
anoestrus buffaloes is more beneficial in increasing
the antioxidant status as revealed by the increase
in the level of vitamin E, β-carotene and decrease
in lipid per oxidation (Nayyar et al., 2002); higher
levels glucose, cholesterol, triiodothyronine and
thyroxine (Nayyar et al., 2003) and improvement
of blood biochemical composition (Anita et al.,
2004).
in both the groups which may be attributed to
better nutrition available to both groups. This is in
agreement with findings of Chauhan et al. (2000).
They also reported no case of RFM if buffaloes
are fed at a higher rate than recommended by
Kearl (1982). The buffaloes with abnormal
uterine involutory changes particularly having
foul smelling discharges or purulent discharge
(puerperal metritis) and RFM cases were treated as
soon as detected and were declared free of disease
as per the score card after completion of uterine
involution. The presence of mildly purulent uterine
discharge in the first month postpartum likely
reflects a successful immune response by the cow
to a bacterial challenge. Uterine involution largely
depends on the intrauterine contamination with
pathogenic bacteria. However, presence of bacteria
in the uterus of postpartum cows does not always
indicate a disease condition. Bacterial presence in
the uterus for the first 10-14th day postpartum has
been considered usual and could be detected in
more than 90% of the cows, regardless of disease
signs (Sheldon and Dobson, 2004). The presence
of bacteria has been found sporadic on 28-35 days
after calving, and in normal healthy conditions the
uterine cavity has to be sterile thereafter (Paisley
et al., 1986; Hussain, 1989; Hussain and Daniel,
1991a,b). However the condition may be either
clinical or sub-clinical as well as the overall effects
vary depending upon the immune status of the
host (Gilbert et al., 1998; LeBlanc et al., 2002;
Kasimanickam et al., 2004). Although the role
of host’s humoral immune response in disease
remains poorly defined, in a physiological situation
the self-defence mechanisms of the uterus are able
to counteract the bacterial infection (Foldi et al.,
2006). Sheldon (2004) propounded that 90% cows
postpartum develop a mild, nonpathological form
of endometritis. In majority of cases the local
Uterine and cervical involution
During postpartum period, the reproductive
organs were palpated transrectal on days
7,14,21,28,35 and 42 for observing cervical and
uterine involutory changes, abnormal discharges
if any as per the score card (Sheldon and Noakes,
1998) with slight modifications. The general goal
for postpartum reproductive health is for the uterus
to be completely involuted and free of infection,
and for cows to be cyclic by the time they enter
the breeding period (after 50 to 60 DIM) (LeBlanc
et al., 2002). There was no significant difference
in days to completion of cervical and uterine
involution. In supplemented group, involuntary
changes took place at a faster pace than the control
group (Table 3). However, cervical and uterine
involution was completed in shorter days (Table 3)
and lesser percent of cows suffering from abnormal
uterine changes in supplemented group than control
group (Table 4) signifies the role of vitamin E and
mineral supplementation which can further be
substantiated by the mineral and metabolic profile
of the supplemented group.
There were no cases of RFM and metritis
71
Table 3. Effect of vitamin E and mineral supplementation on involutory changes.
Days
7
14
21
28
35
42
Control
4.89
3.56
2.33
1.33
0.33
0
Supplemented
5.55
3.22
1.33
0.22
0
0
Table 4. Effect of vitamin E and mineral supplementation on abnormal uterine involutory changes as per
score card.
Days
7
14
21
28
35
42
Control (%)
22.22
11.11
33.33
55.56
11.11
0
Supplemented (%)
33.33
11.11
0
0
0
0
Table 5. Effect of vitamin E and mineral supplementation on reproductive parameters.
Parameter
Control
6.37±1.11
34.22±2.17
73.25±10.82
32
44.44
66.67
33.33
PDD (h)
Uterine involution (Days)
DFS
Initiation of cyclicity P4 > 1 ng/ml (Days)
Risk to First service <60 days (%)
Risk to First service <90 days (%)
Risk to First service >90 days (%)
* - Significant (P<0.05); ** - Significant (P<0.01)
PDD- Placental Delivery Duration; DFS- Days to First Service
72
Supplemented
4.53±0.72
28.78±1.40
61.88±6.92
35
44.44
87.5
12.5
antimicrobial defence mechanisms eliminates the
pathogens and this mild non-pathological form of
endometritis resolves within some days.
Supplementation of Vitamin E and selenium
to Murrah buffaloes during prepartum period has
been shown to shorten expulsion time of foetal
membranes and early uterine involution (Qureshi
et al., 1997; Mavi et al., 2006; Panda et al., 2006)
and decrease metritis cases. Similar observations
have been reported for Dairy cows supplemented
with Vitamin E and Zinc (Campbell and Miller,
1998). However, LeBlanc et al. (2002) reported
that supplementation caused only conditional
benefit of treatment for reduction of the incidence
of retained placenta and no significant effects could
be observed in the incidence of retained placenta,
clinical mastitis, metritis, endometritis, ketosis,
displaced abomasum, or lameness. Metabolic
disorders like hyperketonaemia and deficiency
conditions such as selenium, vitamin E and vitamin
A deficiency have been incriminated for altered
competence of cellular self-defence mechanisms,
which in turn increases the risk for developing
metritis (Lewis, 1997; Reist et al., 2002; Sheldon
and Dobson, 2004). Oral administration of selenium
along with vitamin E in buffaloes is more beneficial
as it increases the antioxidant status as revealed by
the increase in the level of vitamin E (Nayyar et
al., 2002); higher levels glucose (Nayyar et al.,
2003) and improvement of blood biochemical
composition (Anita et al., 2004). It has been opined
that the effects of vitamin E and Se on neutrophils
promote uterine modeling and involution.
5). Risk to first service on days 60, 90 and >90 was
calculated by the number of animals receiving first
service by days 60, 90 and >90 divided by the total
number of experimental animals.
Vitamin E and mineral supplemented
group showed better reproductive performance in
all the reproductive parameters considered in the
study than control group. Mineral and metabolic
status as already discussed substantiates the better
performance of the supplemented group in the
study regarding reproductive performance. In total
around 12 days could be saved in days to first
service if vitamin E and minerals are supplemented.
The better performance in winter season of this
experiment could be attributed mainly to high
plane of nutrition provided in the prepartum
period which is substantiated by the mineral and
metabolic profile which was optimum required for
better performance.
Progesterone estimation was done from 30
days postpartum to evaluate the effect of vitamin
E and mineral supplementation on the initiation
of cyclicity in the experimental buffaloes. Plasma
progesterone concentration more than 1 ng/ml was
used as criteria for assessing initiation of cyclicity.
Supplemented group showed early initiation of
cyclicity (32 days postpartum) compared to control
group (35 days postpartum). Cyclicity in most
of the animals might have been initiated earlier
than 30 days as was evident from progesterone
concentration. Short and long luteal phases were
observed on appraisal of progesterone concentration
in both the groups which delayed the days to first
service in these animals.
Campbell and Miller (1998) also reported
improvement of reproductive performance for dairy
cows supplemented with vitamin E and Zn. Changes
in immune function can contribute to improved
reproductive efficiency. Immunopotentiation in
Reproductive performance
Reproductive performance of the buffaloes
in two different groups was evaluated on the basis
of PDD, uterine involution, days to first service,
risk to first service on days 60, 90 and >90 (Table
73
late gestation with vitamin E and Selenium has
been shown to reduce the calving to first oestrus
interval and the length of the postpartum service
period (Qureshi et al., 1997; Panda et al., 2006).
Supplementation of vitamin E and selenium to
Murrah buffaloes during prepartum period has
been shown to cause a significant increase in
conception rate, decrease in service per conception,
early initiation of postpartum ovarian activity and
early exhibition of first postpartum heat (Mavi
et al., 2006). The progesterone levels remained
at basal levels from day 5 to day 30 postpartum
and started rising thereafter (Bahga and Ganwar,
1988). Progesterone levels of 0.30, 1.43, 3.29
and 0.88 were reported by Qureshi et al. (2000)
at estrus, developing, developed and regressing
corpus luteum respectively. Progesterone analysis
revealed that 23-70% of the postpartum buffaloes
had one or more covert estruses before the first
overt estrus (Batra and Pandey, 1983; Usmani et
al., 1984; Sharma and Kaker, 1990). Also, duration
of first progesterone rise of over I ng/ ml of plasma
was found to be significantly longer (Mavi et al.,
2006) in supplemented buffaloes. Progesterone
monitoring of postpartum buffaloes for detection
of first ovulation offers an objective and accurate
method for assessment of the reproductive
potential of buffaloes (El-Wishy, 2007). Similar
observations have been reported for Dairy cows
supplemented with vitamin E and Zinc (Campbell
and Miller, 1998). Shorter days to first service have
been attributed to combined effect of mineral and
vitamin supplementation because of their positive
effect on steroid synthesis, release, follicular growth
and symptoms of ovulatory oestrus (Srivastava,
2008).
Immunocompetence has been suggested
as a useful tool for determining the requirements
of some vitamins. Requirements that are based on
measures of immune function have been reported to
be higher than those that are based on production or
reproduction (Weiss, 1998). It has been emphasised
that the amount of micronutrients needed for
optimal immune function may exceed that amount
which will prevent more classical deficiency
signs. In general, mineral deficiencies have been
associated with altered metabolic profile leading
to most periparturient disorders in buffaloes. Thus,
such disorders could probably be prevented by
addressing to the basic etiology through balanced
feeding and mineral supplementation during
advanced pregnancy and early post-partum period,
when the animals are highly prone to stress of
heavy nutrient demand and drain (Mandali et al.,
2002).
CONCLUSION
Higher plane of nutrition needs to be
followed during prepartum for meeting the
requirements of buffaloes. Additionally vitamin
E and mineral supplementation should be
provided during the prepartum period to meet
the requirements of critical elements for better
reproductive performance.
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78
Original Article
EFFECT OF VITAMIN E AND MINERAL SUPPLEMENTATION DURING PERI-PARTUM
PERIOD ON BCS, BODY WEIGHT AND CALF PERFORMANCE IN MURRAH BUFFALOES
H.M. Khan1, T.K. Mohanty2, M. Bhakat2, A.K. Gupta2 and G. Mondal3
ABSTRACT
that vitamin E and mineral supplementation during
peripartum period improves the performance of
Murrah buffalo and their calves.
The present study was conducted on
twenty Murrah buffaloes 60 days prepartum and
randomly assigned to two experimental groups
with 10 animals in each group; Control group was
provided 20% higher nutrients than Kearl’s Feeding
Standard and group 2 was provided 20% higher
nutrients than Kearl’s Feeding Standard along with
vitamin E { (2000IU from 60 days prepartum to
30 days postpartum and 1500IU from 30 to 60
days postpartum) vitamin E 50 % powder, Vet
Chem } supplementation and 50 gm of commercial
mineral mixture (Agrimin, Agrivet Farm Care
Division) to meet the expected requirements of the
minerals. Body condition score (BCS) increased
upto parturition and thereafter decreased in both
the groups. The prepartum and postpartum changes
in body weights (BW) were not apparently marked
to be reflected in BCS changes which were almost
similar (0.12 vs 0.16 prepartum and 0.39 vs 0.35
postpartum). Calves born to mineral and vitamin E
supplemented buffaloes performed well in terms of
their birth weight, body weight gain upto 90 days
and calf weight to dam weight ratio. However,
the differences between the two groups were
statistically not significant. It can be concluded
Keywords: BCS, BW, mineral, Murrah buffalo,
vitamin E
INTRODUCTION
Vitamins and minerals (macro and
microelements) play a vital role in metabolism,
normal growth, production and reproduction.
Requirement of these elements are very less and
depends on the system of rearing, agronomic status
and physiological status of the animal. Under
tropical climatic conditions, mineral and vitamin
deficiency problems have been recognized to be
very common causing production and reproduction
problems unless proper dietary supplementations
are provided to save huge economic losses (Sharma
et al., 2003; Yildiz et al., 2006). Prepartum cows
undergo a number of changes from the end of
lactation until subsequent parturition. Lactation
ceases, and cows experience changes in type of
diet, amount of dry matter intake, body condition,
body weight, and fetal development. Kertz et al.
[email protected]
2
3
1
79
to two experimental groups with 10 animals in
each group; group 1 (C) was provided 20% higher
nutrients than Kearl’s Feeding Standard (Chauhan et
al., 2000) and group 2 (T) was provided 20% higher
nutrients than Kearl’s Feeding Standard (Chauhan
et al., 2000) along with vitamin E { (2000 IU from
60 days prepartum to 30 days postpartum and 1500
IU from 30 to 60 days postpartum) vitamin E 50%
powder, Vet Chem } supplementation (Panda et al.,
2006) and 50 gm of commercial mineral mixture
(Agrimin, Agrivet Farm Care Division) to meet
the expected requirements of the minerals. The
buffaloes used for the investigation were kept in
conventional barns throughout the prepartum
period and were shifted to calving pens 2 weeks
prior to expected date of parturition for extra care
and attention upto 5 days after parturition. After
that they were shifted to loose housing and group
management system where other lactating buffaloes
were kept. The animals under investigation were
body condition scored on entry in the experimental
groups, on the day of parturition and at the end of the
experiment. The condition-scoring chart formulated
by Prasad (1994) was adopted in the present study.
The fortnightly body weight of each animal was
recorded early in the morning between 7.30 a.m.
to 8.30 a.m. before providing the animals with any
feeding stuff or water, using electronic weighing
machine during the experimental period. Weight of
calves at birth and 90 days was recorded.
Effect of vitamin E and mineral
supplementation BCS, body weight and
performance of calves was calculated by t test
using Systat 6 software package.
(1997) reported loss of BW at parturition. Previous
studies have showed that body condition scores
(BCS) at calving and body condition loss in early
lactation were related to health (Dann et al., 2005),
reproductive performance (Baruselli et al., 2001;
Pryce et al., 2001; Buckley et al., 2003; Shrestha
et al., 2005), fertility (Balakrishnan et al., 1997;
Contreras et al., 2004; Roche, 2006) and milk yield
(Ramasamy and Singh, 2004; Holter et al., 1990).
The maintenance of an optimal body condition
score relative to lactation stage, milk yield,
nutrition and health status is perhaps the most
important aspect of dairy buffalo management
that facilitates a healthy transition from pregnancy
to lactation. Supplementation of dams has been
observed to enhance secretion of immune proteins,
immunoglobulin (Ig) in colostrum by 80%, and
improve growth and immune status and growth
performance of the calves (Sikka et al., 2002; Sikka
and Lal, 2006). In general, mineral deficiencies
have been associated with altered metabolic profile
leading to most periparturient disorders in buffaloes.
Thus such disorders could probably be prevented
by addressing to the basic etiology through
balanced feeding and mineral supplementation
during advanced pregnancy and early post-partum
period, when the animals are highly prone to stress
of heavy nutrient demand and drain (Mandali et
al., 2002). There is lack of information regarding
vitamin E and mineral supplementation on BCS,
body weight and calf performance, therefore, the
present study was conceived to fulfill the gap.
Twenty Murrah buffaloes 60 days
prepartum were selected and randomly assigned
80
adnexia (membranes and foetal fluids). It seemed
that vitamin E and mineral supplementation tended
to improve body reserves which resulted in lesser
body weight loss postpartum than the control
animals.
Similar trends in body weight changes
following UMMB supplementation have been
reported in buffaloes (Brar and Nanda, 2007) and
feeding cationic or anionic diets in cattle (Gulay
et al., 2008). Body weight loss at parturition is
physiological owing to expulsion of foetus, foetal
fluids and placenta (Brar and Nanda, 2007) and
stress of parturition. The weight loss thereafter
is primarily due to mobilization of body reserves
for fulfilling the demands for maintenance and
production of milk (Grummer, 2006). Body weight
loss could be curtailed and an early body weight
rise could be commenced through supplementary
feeding in both pre and postpartum period (Sharma
et al., 1993). Adequate nutrition and management
are recommended during the last trimester of
pregnancy to minimize body weight loss or enhance
body weight recovery after calving (Prakash et al.,
1990; Chauhan et al., 2000).
Body weight changes from pre to postpartum
period
The supplemented buffaloes had higher
body weight gains during 60 days prepartum period
than the control group (Table 1 and 2). Thereby it
fell sharply at parturition and continued to decline
over the next 2 months. Compared to controls, the
cumulative body weight loss in the supplemented
buffaloes was less during the postpartum period.
However, the differences in body weight at all
stages were non significant but the supplemented
group had higher body weight gains and per day
body weight gain in the prepartum period and
lower body weight losses and per day body weight
loss postpartum than the control group at all the
stages, reflecting improvement and beneficial effect
due to Vitamin E and mineral supplementation.
Supplementing prepartum Vitamin E and minerals
appears to have helped in modulating pre and
postpartum body weight changes. Also, it was
observed that out of the total body weight loss
(60.11 ± 3.71 vs. 62.67 ± 2.80) at parturition, calf
birth weight was only 62.35 ± 2.33% and 57.19
± 2.29%, respectively in the supplemented group
and control group, reflecting rest loss to foetal
Body condition score
Body condition score reflected changes in
Table 1. Performance of vitamin E and mineral supplemented buffaloes.
Parameters
Bwt gain prepartum (Kg)
Per day gain prepartum (kg)
Wt loss parturition (Kg)
Calf wt % parturition loss
Calf wt % buffalo wt
Bwt loss postpartum (Kg)
Per day loss postpartum (Kg)
Supplemented
45.44 ± 4.94
0.76 ± 0.08
60.11 ± 3.71
62.35 ± 2.33
6.06 ± 0.25
33.22 ± 11.04
0.67 ± 0.19
81
Control
39.44 ± 2.99
0.66 ± 0.07
62.67 ± 2.80
57.19 ± 2.29
5.70 ± 0.23
41.11 ± 8.56
1.04 ± 0.05
Initial
625.22±9.93
650.67±21.39
3.89±0.14
3.88±0.18
36.89±1.20
35.56±1.33
Treatment
Supplemented
Control
Supplemented
Control
Supplemented
Control
BCS – Body Condition Score
Calf weight (kg)
BCS
Body weight (Kg)
Parameters
Just before
parturition
670.67±7.14
690.11±21.95
4.01±0.11
4.04±0.17
-
Just after
parturition
610.56±8.14
627.11±21.38
576.00±9.36
593.11±22.99
-
30 days
577.33±10.32
583.56±21.90
3.62±0.15
3.69±0.17
-
60 days
Table 2. Effect of vitamin E and mineral supplementation on body weight, BCS, calf weight and milk yield in buffaloes.
71.83±4.13
65.57±4.43
90 days
82
CONCLUSION
the body weight in both supplemented and control
buffaloes (Table 2). BCS increased upto parturition
and thereafter decreased in both the groups. The
prepartum and postpartum changes in body weights
were not apparently marked to be reflected in BCS
changes which were almost similar (0.12 vs. 0.16
prepartum and 0.39 vs. 0.35 postpartum) in both
supplemented and control groups, respectively.
Lack of any gross apparent difference of changes
could be due to better and high plane of nutrition
available to both groups. Lower milk production
compared to cattle may be a reason for not being
affected by negative energy balance to be reflected
in BCS. Also it may be attributed to higher protein
and energy utilizing efficiencies in buffaloes as
compared to cattle at similar fat corrected milk
production level, plane of energy and protein
nutrition, body size and weight change (Paul et al.,
2003) which could be the reason for less negative
energy balance reflected in buffaloes during
postpartum period.
During peripartum period vitamin E
and mineral supplementation seems to improve
the performance of buffaloes and their calves.
Body condition score (BCS) is a logistic tool
for assessment of nutritional status of animal
and management for optimal performance. The
maintenance of an optimal body condition score
relative to lactation stage, milk yield, nutrition and
health status is perhaps the most important aspect
of dairy cow management that facilitates a healthy
transition from pregnancy to lactation.
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Indian J. Dairy Sci., 50(5): 393-397.
Baruselli, P.S., V.H. Barnabe, R.C. Barnabe, J.A.
Visintin, J.R. Molero-Filho and R. Porto.
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Brar, P.S. and A.S. Nanda. 2007. Effect of
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Buckley, F., K. O’Sullivan, J.F. Mee, R.D. Evans
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Chauhan, T.R., N.D. Sharma, S.S. Dahiya, B.S.
Performance of the calves
Calves born to mineral and vitamin E
supplemented buffaloes performed well in terms
of their birth weight and body weight gain upto
90 days and calf weight to dam weight ratio.
However the difference between the two groups
was non-significant but the supplemented group
had higher values than the control group which
could be attributed to vitamin E and mineral
supplementation. Supplementation of dams has
been observed to enhance secretion of immune
proteins, immunoglobulin (Ig) in colostrum by
80%, and improve growth and immune status of the
calves (Sikka et al., 2002; Sikka and Lal, 2006).
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Panda, N., H. Kaur and T.K. Mohanty. 2006.
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supplemented with varying levels of vitamin
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Paul, S.S., A.B. Mandal, A. Kannan, G.P. Mandal
and N.N. Pathak. 2003. Comparative
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Prasad, S. 1994. Body condition scoring and feeding
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relationship between body condition score
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Ramasamy, M. and M. Singh. 2004. Effect of
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Murphy and J.K. Drackley. 2005. Prepartum
intake, postpartum induction of ketosis, and
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period energy status for improved health
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World Buiatrics Congress, Nice, France.
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Head. 2008. Prepartum feeding of cationic
and anionic diets to Holstein cows given 30
to 60 day dry periods: comparison of dry
matter intake, physiological measures and
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21(1): 83-89.
Holter, J.B., M.J. Slotnick, H.H. Hayes and C.K.
Bozak. 1990. Effect of prepartum dietary
energy on condition score, postpartum
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3502-3511.
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Vet. J., 70: 1165.
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T. Higaki. 2005. Relationships between
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dairy cows in a subtropical region in Japan.
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Sikka, P., D. Lall, U. Arora and R.K. Sethi. 2002.
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to micronutrient supplementation in newborn calves of Murrah buffaloes given fat
soluble vitamins during late pregnancy.
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Yildiz, H., E. Kaygusuzoglu and M. Aydin. 2006.
Serum mineral status during pregnancy in
85
Original Article
STUDY ON MICRO-MINERAL STATUS OF BUFFALOES DURING PERIPARTUM PERIOD
IN DIFFERENT SEASON
H.M. Khan1, T.K. Mohanty2, M. Bhakat2, A.K. Gupta2, A.K. Tyagi3 and G. Mondal3
components of various enzymatic systems and
important for good health and requirements vary with
the physiological status of the animal (Borghese,
2005). In buffaloes minimum but long-term
deficiencies during dry period have been known to
cause impaired health during lactation (Campanile
et al., 1997). Mineral status of animals is a direct
reflection of their presence, absence, deficiency or
excess in soil and fodder. Under tropical Indian
conditions, mineral deficiency problems have been
recognized to be very common causing production
and reproduction problems unless proper dietary
supplementations are provided to save economic
losses (Sharma et al., 2003). The Cu and Zn
deficiencies have been incriminated for loss of
production and reproduction, irregular cycles, cycle
extension, subestrus, difficult delivery, retained
placenta, abortions, estrus prevention, congenital
abnormalities/disorders, repeat breeding and early
embryonic deaths (McDowell, 1992; Balakrishnan
and Balagopal, 1994; Dutta et al., 2001; Sharma et
al., 2005; Prajapati et al., 2005). The normal blood
values of Mn in cattle have been established to be
18-19 μg/dl (Sharma et al., 2005). Mn deficiencies
have been found to suppress conception rates,
delay estrus, cause abortions, deformed calves at
birth and increase occurrence of cystic ovaries
ABSTRACT
Two groups of 15 Murrah buffaloes each,
expected to calve in winter and summer season were
selected for monitoring during peripartum period
to have an overview of the herd micro-mineral
status. There was significant difference in plasma
concentration of the Zn on day 30 prepartum,
calving day and day 60 postpartum (P<0.05); Cu
on day 60 prepartum (P<0.05) and Mn at all the
stages (P<0.01).The summer season calvers had
higher levels at all the stages. After evaluating the
herd status, it was clear that buffaloes were either
deficient or had imbalance in nutrients in winter
season calvers resulting into wide variation in
reproductive performance. Herd status regarding
mineral status need to be evaluated from time to
time in different seasons to achieve set targets in
terms of reproduction and production performance
by adjusting feeding schedule.
Keywords: Murrah buffalo, Zn, Cu, Mn, season
INTRODUCTION
Micro minerals are critical functional
[email protected]
2
3
1
86
per day for body maintenance) and ad libitum
green fodder (berseem, oat, mustard and maize).
All the experimental buffaloes were monitored
regularly for estrus by visual observation and by
parading of vasectomized bull in morning and
evening hours. Animal were confirmed for heat
by rectal palpation and inseminated with frozen
semen by two inseminations at 12 h intervals.
Buffaloes not returning to estrus after 21 days of
insemination were examined per rectum on 45th
(Sharma et al., 2005). A lower level of manganese
and copper has been reported in anestrus buffaloes
when compared with those exhibiting estrus (Patil
and Deshpande, 1979; Naidu and Rao, 1982;
Agarwal et al., 1985; Singh and Vadnere, 1987).
Thus such disorders could probably be prevented
by addressing to the basic etiology through
balanced feeding and mineral supplementation
during advanced pregnancy and early post-partum
period, when the animals are highly prone to stress
of heavy nutrient demand and drain (Mandali et
al., 2002). Satisfactory conception rates have been
attributed to combined effect of mineral and vitamin
supplementation because of their positive effect on
steroid synthesis, release, follicular growth and
symptoms of ovulatory oestrus (Srivastava, 2008).
During different seasons availability of feed and
fodder changes thereby changing the availability
of certain nutrients which may have direct or
indirect effect on productive and reproductive
performance of the buffaloes. Therefore envisages
evaluation of herd status in terms of micro-mineral
profiling of buffaloes during peripartum from time
to time for further decisions for improving the herd
performance.
day for pregnancy confirmation.
Plasma Biochemical Assay
The blood samples were collected from
jugular vein into heparinized (20 IU heparin/ ml
blood) tubes from all experimental animals at
fortnightly interval from 60 days prepartum to
60 days postpartum. Immediately after sampling
the blood was centrifuged at 3000 rpm for 15 to
20 minutes and the plasma was separated and
stored frozen (-20°C) until analyzed. Following
micronutrients in control as well as in experimental
groups were estimated with the help of Atomic
absorption Spectrophotometer (Model PU9100X
Atomic absorption Spectrophotometer, Philips).
The procedure described in AAS (1988) manual
for preparation of stock and standard solutions and
choice of instrumental conditions were followed.
Effect of season of calving on micro-mineral status
was calculated by t test, using Systat 6 software
package.
The present study was conducted on 30
pregnant dry Murrah buffaloes maintained at Cattle
Yard of National Dairy Research Institute (NDRI),
Karnal and divided into 15 animals each as per
expected day of calving in winter(January to March)
and summer (April to July) season. The herd was
kept under loose housing and group management
system following standard managemental practices.
The nutrient requirements of all the animals were
mostly met through limited concentrates (1.5 kg
In the present investigation 15 buffaloes
each expected to calve in winter and summer season
were randomly selected during prepartum period
to have an overview of micro-mineral status of the
87
herd. Mineral status of plasma is a direct reflection
of their presence, absence, deficiency or excess in
soil and fodder. Under tropical Indian conditions
mineral deficiency and imbalance problems have
been recognized to be very common causing
production and reproduction problems unless proper
dietary supplementations are provided. Correction
of deficiencies and imbalance by balanced mineral
supplementation has been shown to produce a
marked response saving huge economic losses due
to production and reproduction losses (Sharma et
al., 2003).
Plasma Zn, Cu and Mn were estimated
at monthly interval in both the groups. The
results obtained are presented in the Table 1 for
interpretation from 60 days prepartum to 60 days
postpartum taking 0 day as the day of calving.
day 30 postpartum in summer season. There was
significant difference in plasma concentration
of the Zn on day 30 prepartum, calving day and
day 60 postpartum (P<0.05).The differences in
concentration at all other stages was non significant
but summer season had higher levels than the winter
season at all the stages reflecting better availability
of fodders having sufficient nutrients required for
optimum performance.
Decrease in plasma Zn concentration in
buffaloes during late gestation and parturition has
also been reported by House and Bell (1993) and
Panda (2003). Zn accretion rate in the conceptus
in late pregnancy is 11.7 mg/day which may be
reason for lower level of Zn before parturition.
Zn deficiency has been incriminated for impaired
reproductive performance, decreased fertility
and abnormal estrus in cows and decreases cell
mediated immunity (Sharma et al., 2005). Zinc is
a critical nutrient of immunity, being involved in so
many immune mechanisms including cell-mediated
and antibody-mediated immunity, thymus gland
function and thymus hormone action. When zinc
levels are low, the number of T cells is reduced and
many white blood functions critical to the immune
Plasma Zn
Plasma Zn concentration was lowest
on the day of calving in winter season. The Zn
concentration dropped on day 30 prepartum and day
of calving and showed an increasing trend thereafter
in winter season, whereas it showed an increasing
trend at day 30 prepartum and then decreased upto
Table 1. Seasonal effect on mineral status in Murrah buffaloes.
Micro
Mineral
Zn†
Mn†
Cu†
Season
Winter
Summer
Winter
Summer
Winter
Summer
Prepartum
-60d
-30d
1.46±0.27
1.13±0.17*
1.88±0.29
2.01±0.36*
0.26±0.04** 0.25±0.03**
0.60±0.05** 0.45±0.05**
0.90±0.10*
0.809±0.072
1.45±0.18*
1.006±0.224
0d
0.90±0.21*
1.73±0.32*
0.17±0.03**
0.43±0.04**
0.749±0.061
0.833±0.094
† – ppm; * - Significant (P<0.05); ** - Significant (P<0.01)
88
Postpartum
30d
60d
0.91±0.10
1.00±0.09*
1.05±0.21
1.40±0.15*
0.24±0.02** 0.29±0.02**
0.49±0.06** 0.54±0.05**
0.984±0.119 1.047±0.086
1.002±0.172 1.076±0.119
Plasma Mn
Plasma Mn levels followed a decreasing
trend upto parturition and followed increasing
trend following parturition in both seasons. The
drop in the Mn concentration during prepartum
due to increasing demands of growing fetus (House
and Bell, 1993) and utilization for improving
antioxidant status. There was highly significant
difference (P<0.01) in plasma concentration of
the Mn at all the stages and summer season had
higher levels than the winter season at all the stages
reflecting better availability of feeds and fodders
having sufficient nutrients required for optimum
performance. This is in agreement with the findings
of Panda (2003), who has reported similar trend in
Mn concentration but reported higher levels than
the present findings. Mn deficiency has been found
to suppress conception rates, delay estrus, cause
abortions, deformed calves at birth and increase
occurrence of cystic ovaries (Sharma et al., 2005).
The deficiency or imbalance of critical
factors has either immediate effects on health,
productive and reproductive processes or the effects
may be covert and recognized after a prolonged
period. The effects depend on the nature of the
factor, extent of deficiency or imbalance, duration
and physiological status of the animal. In buffaloes
minimum but long-term deficiencies during dry
period have been known to cause impaired health
during following lactation (Campanile et al.,
1997). Various workers have revealed a direct
correlation of mineral status of the animals with
their physiological status and observed disorders
and incriminated altered levels of minerals and
enzymes for loss of production and reproduction
(Patil and Deshpande, 1979; Naidu and Rao, 1982;
Agarwal et al., 1985; Singh and Vadnere, 1987).
Inactive ovaries, anestrus and poor conception
rates have been recognized as the most common
response are severely lacking. Like vitamin
C, zinc also possesses direct antiviral activity,
including activity against several viruses. It is also
present in members of a class of proteins called
the metallothioneins that are believed to provide
antioxidant protection by scavenging free radicals
(Borghese, 2005).
Plasma Cu
Plasma Cu levels followed a particular
trend in both the groups. It started declining upto
calving and thereafter there was an increase in
its concentration. The extent of decrease in Cu
concentration at parturition was more in comparison
to other minerals due to more accretion rate of Cu
in the conceptus (House and Bell, 1993). There
was significant statistical difference in plasma
concentration of the Cu on day 60 prepartum
(P<0.05), however, the differences in concentration
at all other stages was non significant but summer
season had higher levels than the winter season at
all the stages reflecting better availability of fodders
having sufficient nutrients required for optimum
performance.
Panda (2003) also found the similar trend
in Cu concentration but reported higher levels
than the present findings. Cu deficiency has been
incriminated for poor performance, reduced fertility
in animals attributed to poor conception rates,
anoestrus and foetal resorption. It has also been
associated with impaired immune response and
failure to respond to treatment (Sharma et al., 2005).
The Cu and Zn deficiencies have been incriminated
for loss of production and reproduction, irregular
cycles, cycle extension, subestrus, difficult delivery,
retained placenta, abortions, estrus prevention,
congenital abnormalities/disorders and early
embryonic deaths (McDowell, 1992; Dutta et al.,
2001) and repeat breeding (Dhami et al., 2003).
89
expressions consequent upon the deficiency of Cu,
Zn and Mn (Khasatiya et al., 2005). Also, imbalance
between minerals has been incriminated as a
possible cause for repeat breeding (Balakrishnan
and Balagopal, 1994; Prajapati et al., 2005; Kalita
and Sarmah, 2006). Requirements that are based on
measures of immune function have been reported
to be higher than those that are based on production
or reproduction (Weiss, 1998). The amount of
minerals required for optimal immune function
may exceed that amount which will prevent more
classical deficiency signs.
Profilo metabolico nel bufalo. Bubalus
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profile of blood plasma in relation to age
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Dutta, A., B. Baruah, B.C. Sharma, K.K. Baruah and
R.N. Goswami. 2001. Serum macromineral
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profile of postpartum fertile and infertile
Surti buffaloes. Indian J. Anim. Reprod.,
26(2): 145-148.
Reprod., 23(2): 130-134.
McDowell, L.R. 1992. Minerals in Animal and
Human Nutrition. Academic Press London.
Naidu, K.V. and A.R. Rao. 1982. A study on the
etiology of anestrus in crossbred cows.
Indian Vet. J., 59: 781.
Panda, N. 2003. Optimisation of vitamin E doses
for improved immunity and udder health in
Murrah buffaloes. Ph. D. Thesis, National
CONCLUSION
There was significant difference in plasma
concentration of the Zn on day 30 prepartum,
calving day and day 60 postpartum (P<0.05); Cu
on day 60 prepartum (P<0.05) and Mn at all the
stages (P<0.01).The summer season calvers had
higher levels at all the stages.
REFERENCES
Agarwal, S.K., N.N. Pandey and U. Shanker. 1985.
Serum protein, inorganic phosphorous and
blood glucose in relation to different phases
of reproduction in crossbred cattle. Indian J.
Anim. Reprod., 6: 23-25.
Balakrishnan, V. and R. Balagopal. 1994. Serum
calcium, phosphorous, magnesium, copper
and zinc level in regular breeding buffaloes.
Indian Vet. J., 71: 23-25.
Borghese, A. 2005. Buffalo Production and
Research. REU technical series 67. FAO,
United Nations, Rome.
Campanile, G., R. Di Palo and A. D’Angelo. 1997.
90
Dairy Research Institute, Karnal, India.
Patil, R.V. and B.R. Deshpande. 1979. Changes
in body weight, blood glucose and serum
proteins in relation to the appearance of
postpartum oestrus in Gir cows. J. Reprod.
Fertil., 57: 25-27.
Prajapati, S.B., D.J. Ghodasara, B.P. Joshi,
K.S. Prajapati and V.R. Jani. 2005. Etiopathological study of endometritis in repeat
breeder buffaloes. Buffalo J., 2: 145-165.
Sharma, M.C., C. Joshi and M. Kumar. 2005. Micro
mineral deficiency disorders and treatment:
A review. Indian J. Anim. Sci., 75(2): 246257.
519-528.
Singh, S. and S.V. Vadnere. 1987. Induction of
oestrus by supplementation of different
minerals in postpartum anestrus crossbred
cows. Indian J. Anim. Reprod., 8: 46.
Srivastava, S.K. 2008. Effect of mineral supplement
on oestrus induction and conception in
anoestrus crossbred heifers. Indian J. Anim.
Sci., 78(3): 275-276.
Weiss, W.P. 1998. Requirements of fat-soluble
vitamins for dairy cows: a review. J. Dairy
Sci., 81: 2493-2501.
91
Original Article
LIFETIME PERFORMANCE OF MURRAH BUFFALOES HOT AND HUMID CLIMATE
OF TAMIL NADU, INDIA
A.K. Thiruvenkadan*, S. Panneerselvam and R. Rajendran
Keywords: herd life, Murrah, lifetime performance,
tropical climate
ABSTRACT
Study on lifetime production traits Murrah
buffaloes was carried out at Central Cattle Breeding
Farm, Alamadhi, Tamil Nadu, India by collecting
milk production and reproduction records of
Murrah buffaloes over a period of 28 years (i.e.,
from 1976 to 2003). The overall means (± SE)
for longevity, productive herd life, lifetime milk
production, milk yield per day of longevity, milk
yield per day of productive herd life and lifetime
calf crop of Murrah buffalo cows were 3078.4 ±
46.3 kg, 1520.7 ± 46.2 kg, 5441.6 ± 206.0 kg, 1.41
± 0.04 kg, 2.89 ± 0.05 and 3.00 ± 0.08 respectively.
The study revealed that the age at first calving had
significant (P<0.05) effect on longevity and highly
significant (P<0.01) effect on productive herd life,
lifetime milk production, milk yield per day of
longevity and lifetime calf crop. The coefficients
of variation of most of the lifetime production traits
were very high and they generally ranged between
34.0 and 78.2 percent. Therefore, selection for
reducing the age at first calving through better
feeding and breeding management practices
would result in improvement in different lifetime
production traits in Murrah buffaloes. Based on the
lifetime performance study, it may be concluded
that Murrah buffaloes performed satisfactorily at
this hot and humid climatic conditions of Tamil
Nadu.
INTRODUCTION
Dairy animal production viz. buffalo and
cattle rearing are now considered as one of the
important income generating activities in rural
India. As a policy the system of grading up of
non-descript buffaloes and cross breeding of cattle
have been adopted to alter the genetic makeup of
the native stock in India. This tool has had India,
transverse a long way in the global scenario of
milk production. Murrah breed is the finest genetic
material of milk producing buffalo not only in
India but also probably in the world. In India, the
buffalo is the principal dairy animal. Although, the
breedable buffaloes are almost one-third in number
as compared to cattle, buffaloes contribute in
excess of 50 percent (i.e. 54.47 percent) of the total
milk produced in the country. Their contribution in
terms of meat is also significant (Report, 2006). The
national buffalo breeding policy envisages selective
breeding for conservation and improvement of
buffalo breeds in their home tract and grading up
of non-descript buffaloes with recognised buffalo
breeds viz. Murrah, Nili-Ravi and Surti. Murrah
buffaloes are originally from Haryana and Punjab
and they have been used extensively throughout
Department of Animal Genetics and Breeding, Veterinary College and Research Institute, Orathanadu, Tamil
Nadu, India, *E-mail: [email protected]
92
approximately 20 km from the farm. The mean
annual maximum and minimum temperatures were
33.0o and 24.7o C respectively. The mean relative
the country to upgrade the non-descript buffalo
stock to improve the milk production. Although,
no scientific evaluation of the grading up scheme
has been made, large increase in share of buffalo
milk to total milk production over time (from 40.03
percent in 1995-96 to 54.47 percent in 2003-2004)
suggest the effectiveness of grading up scheme for
the genetic improvement of non-descript buffaloes
(Taneja, 1998; Report, 2006).
The age at first calving had appreciable
effect on the lifetime production traits in Dairy
cattle and buffaloes (Lin et al., 1988). Thus
dairyman would have a considerable interest in
the relationship of these traits in lifetime milk
production and other lifetime production traits. In
addition, the information on lifetime production
parameters of Murrah buffaloes at variable climatic
conditions is vital for overall assessment of breed
performance. Hence, this study has been made at
Central Cattle Breeding Farm, Alamadhi, Tamil
Nadu, India to assess the lifetime performance
of Murrah buffaloes at hot and humid climatic
conditions of Tamil Nadu.
humidity ranged from 69.2 to 76.2 percent. This
region recorded an average annual rainfall of
1446.6 mm received in 58.6 rainy days. The data
for the estimation of lifetime production traits were
available for 664 Murrah heifers and cows born
and bred in this farm. The different categories of
Murrah buffaloes were maintained under intensive
system of management and roughage in the form
of green fodder and paddy straw was provided. In
addition, concentrate mixture was provided as per
the standard requirements. Calves were weaned at
birth and pail feeding was practised. The lifetime
production traits considered in the study were
longevity (number of days from birth to disposal
of the heifer/ cow either due to culling or death),
productive herd life (number of days from first
calving to till death or disposal of the cow from
the herd), lifetime milk production (total milk
produced by the cow during its productive herd
life), milk yield per day of productive herd life,
milk yield per day of longevity and lifetime number
of calf crop. The different lifetime production traits
were analysed by including age at first calving as
a fixed effect. Few workers (El-Arian and Tripathi,
1988; Kuralkar and Raheja, 2000) considered the
influence of period and season of first or last calving
as a fixed non-genetic factors in addition to age at
first calving. Since, lifetime traits pass through
different periods and seasons and classification
based on either first or last period and season of
calving had no appreciable effect, therefore, the
same was not considered for the present analysis.
The classification was made as ≤1250, >1250 to
1400, >1400 to 1550, >1550 to 1700, >1700 to 1850
and >1850 days. The following fixed effect model
was used for the analysis of lifetime production
Milk production and reproduction records
of Murrah buffaloes were collected over a period
of 28 years (1976 to 2003) from Central Cattle
Breeding Farm, Alamadhi, Tamil Nadu, India. This
farm is located approximately at 13o N latitude and
80o E longitude at Alamadhi village about 30 km
from Chennai city on Red hills to Tiruvallur road.
It is situated at an altitude of about 20 metres above
mean sea level. The farm covers an area of 397.73
hectares with a total cultivated area of about 84
hectares. The climate is generally hot, humid and
tropical in nature. The coast of Bay of Bengal is
93
The longevity observed in ≤1250 days age group
was significantly different from >1400 to 1550
and >1700 and 1850 days groups. The significant
influence of age at first calving on the variation
of longevity is in agreement with the findings of
Kuralkar and Raheja (2000). However, Gowane
and Tomar (2007) reported non-significant effect
of age at first calving on this trait.
traits: Yij = μ + pi + eij . Where, Yij=lifetime lactation
character of the jth buffalo cow that calved in the ith
age at first calving group, μ= overall mean when
equal subclass frequencies exist, pi=effect of ith age
at first calving group (i =1 to 6) and eijk=random
errors NID (0, σ2e). LSMLMW and MIXMDL
PC-2 VERSION computer programme of Harvey
(1990) was used to study the effect of age at first
calving on different lifetime production traits and
the means were compared using Duncan’s multiple
range test. The estimation of heritability and genetic
correlation between different lifetime production
traits were also obtained by REML method using
Derivative Free Restricted Maximum Likelihood
(DFREML) software package of Meyer (1997).
Productive herd life
The overall mean productive herd life of
1520.7 ± 46.2 days observed in Murrah buffaloes
in the present herd was within the range of values
reported for other Murrah buffalo herds located at
different places in India (Kalsi and Dhillon, 1982;
El-Arian and Tripathi, 1988). However, higher than
the present estimates were also observed in some
studies (El-Arian and Tripathi, 1988; Rao and Rao,
1996; Sasidhar et al., 2000). Raheja (1998) and
Gowane and Tomar (2007) reported lower values
than the present estimates. The age at first calving
had highly significant (P<0.01) effect on this trait.
The productive herd life generally reduced as the
age at first calving increases. The productive herd
life decreased sharply between >1400 to 1550 and
>1550 and 1700 days and again between >1700 to
1850 and >1850 days groups. It was the lowest in
>1850 days group and they differed significantly
(P<0.05) with others.
Heifers
with
lower
age at first calving had an advantage over those
freshening at an older age throughout all six
opportunities groups. Lin et al. (1988) also found
similar finding in dairy cattle and concluded that by
reducing the age at first calving the profitability of
the farm could be improved by increasing lifetime
milk production and milk yield per day of herd life.
Hence, every effort should be made to reduce the
age at first freshening either by reducing the age at
breeding alone or in combination with increasing
The means (±SE) for different lifetime
production traits of Murrah buffaloes are presented
in Table 1.
Longevity
Longevity is one among the lifetime
production traits and higher longevity is one of
the indications of fit and healthy herd. A longer
mean longevity increases profitability, because
it decreases replacement costs and increases the
proportion of the most productive mature age
groups in the herd. The average longevity (3078.4
± 46.3 days) observed in Murrah buffaloes in
the current study was comparable to the values
reported earlier for Murrah buffaloes (Kuralkar and
Raheja, 2000; Sasidhar et al., 2000). However, ElArian and Tripathi (1988) observed higher value
of 3531.29 ± 109.68 days for Murrah buffaloes
maintained at MDF, Ambala. The age at first
calving had significant (P<0.05) effect on this trait.
94
95
Productive herd
life (days)
1520.7 ± 46.2
(664)
**
1721.8 ± 105.5b
(113)
1625.4 ± 90.3b
(154)
1726.7 ± 86.2b
(169)
1550.8 ± 125.3b
(80)
1526.7 ± 124.6b
(81)
972.6 ± 137.0a
(67)
Longevity (days)
3078.4 ± 46.3
(664)
*
2873.1 ± 105.7a
(113)
2954.2 ± 90.5ac
(154)
3198.2 ± 86.4bc
(169)
3175.8 ± 125.6ab
(80)
3294.7 ± 124.8b
(81)
2974.6 ± 137.3ab
(67)
5441.6 ± 206.0
(553)
**
6130.3 ± 454.0d
(95)
5734.8 ± 383.7c
(133)
6158.9 ± 366.2d
(146)
5293.6 ± 553.1b
(64)
6223.0 ± 517.9d
(73)
3109.1 ± 682.8a
(42)
Lifetime milk
production (kg)
1.41 ± 0.04
(509)
**
1.66 ± 0.08b
(95)
1.56 ± 0.07b
(123)
1.61 ± 0.07b
(132)
1.39 ± 0.10b
(61)
1.40 ± 0.10b
(59)
0.85 ± 0.13a
(39)
2.89 ± 0.10
(95)
2.95 ± 0.09
(123)
2.96 ± 0.09
(132)
2.85 ± 0.13
(61)
2.98 ± 0.13
(59)
2.69 ± 0.16
(39)
2.89 ± 0.05
(509)
Milk yield
per day of
productive herd
life (kg)
Figures in parentheses are the number of observations.
Means with at least one common superscript within classes do not differ significantly (P≥0.05). ** P<0.05, ** P<0.01.
>1850 days
>1700 to 1850 days
>1550 to 1700 days
>1400 to 1550 days
>1250 to 1400 days
≤ 1250 days
Age at first calving
Overall mean
Effect
Milk yield
per day of
longevity
(kg)
Table 1. Least-squares means (±SE) for different lifetime production traits of farmbred Murrah buffaloes.
3.00 ± 0.08
(755)
**
3.56 ± 0.19b
(119)
3.27 ± 0.16b
(172)
3.31 ± 0.15b
(193)
2.93 ± 0.22b
(91)
2.96 ± 0.21b
(102)
1.95 ± 0.24a
(78)
Lifetime calf
crop
Table 2. Estimates of genetic (rG), phenotypic (rP) and environmental (rE) correlations among different traits.
Character
rG ± SE
Age at first calving with
Longevity
Lifetime milk production
Milk yield per day of longevity
Milk yield per day of productive herd life
Number of calf produced
Longevity with
Number of calf crop
Productive herd life with
Number of calf crop
Lifetime milk production with
Number of calf crop
Milk yield per day of longevity with
Number of calf crop
Milk yield per day of productive herd life with
Number of calf crop
96
Correlation
rP ± SE
rE
0.253 ± 0.648
-0.503 ± 0.705
0.181 ± 0.937
-0.706 ± 0.766
-0.454 ± 0.363
-0.456 ± 0.286
0.099 ± 0.043
-0.119 ± 0.043
-0.070 ± 0.049
-0.215 ± 0.049
-0.087 ± 0.049
-0.164 ± 0.040
0.094
-0.069
-0.140
-0.138
0.074
-0.017
1.024 ± 0.225
0.675 ± 1.426
0.999 ± 0.002
0.930 ± 0.019
0.849 ± 0.027
0.434 ± 0.046
0.918 ± 0.017
0.998
0.922
0.692 ± 1.651
0.930 ± 0.019
0.851 ± 0.027
0.432 ± 0.046
0.920 ± 0.017
0.923
-
0.935 ± 0.018
0.630 ± 0.039
0.926 ± 0.019
-
1.439 ± 2.502
-
0.781 ± 0.031
0.881 ± 0.024
0.776
-
-
0.539 ± 0.043
-
prepubertal average daily weight gain.
(Tomar and Ram, 1992; Gowane and Tomar, 2007).
The age at first calving group had highly significant
(P<0.01) effect on this trait. The Murrah buffaloes
calving at younger ages produced more number
of calves than those calved later. The highest and
lowest lifetime calf crops were produced in ≤1250
days and >1850 days age at first calving groups
and they differed significantly (P<0.05) with each
other.
The average lifetime milk production of
the Murrah buffaloes estimated (5441.6 ± 206
kg) in the present herd was lower than the values
reported by earlier researchers (Rao and Rao, 1996;
Kuralkar and Raheja, 2000; Sasidhar et al., 2000).
However, Kumar et al. (2006) reported comparable
values of 5381.07 ± 66.63 kg for Murrah buffaloes
maintained at different MDFs. The age at first
calving had highly significant (P<0.01) effect on the
trait, however, no conclusive pattern was observed
between different ages at first calving groups. The
lowest lifetime milk production was observed in
heifers that calved after 1850 days of age and they
differed significantly (P<0.05) with rest.
Estimation of genetic parameters
The heritability estimates for longevity,
productive herd life, lifetime milk production
and milk yield per day of longevity were either
zero or nearly zero. Among the different lifetime
production traits, milk yield per day of productive
herd life had higher heritability value but with high
standard error. The heritability estimates observed
for the different traits were lower than the reports
of Narula et al. (1994), Raheja (1998), Dutt and
Taneja (2000) and Galeazzi et al., (2010). The
lower estimates of heritability for different lifetime
milk production traits in the present study suggested
that direct selection for the traits would not bring
much genetic improvement. Moreover selection
on lifetime performance traits is not practical
because of long generation interval and high cost
of maintaining potential replacement stock.
Age at first calving had a medium to
high negative genetic correlation with lifetime
production traits except with longevity and lifetime
milk yield, where the associations were positive and
low which is not desirable. However, the negative
association with milk yield per day of longevity
and productive herd life was in the expected and
desired manner but with high standard error. This
pointed out that by reducing age at first calving,
the productive herd life of the herd would be
improved and also improvement in milk yield per
Milk yield per day of longevity and Milk yield
per day of productive herd life
The overall least-squares means for milk
yield per day of longevity and milk yield per day
of productive herd life in the present study in
Murrah buffaloes at Central Cattle Breeding Farm,
Alamadhi was found to be on the lower side of the
range of values reported in most of the studies (Rao
and Rao, 1996; Raheja, 1998; Kumar et al., 2006).
The age at first calving had highly significant
(P<0.01) effect on milk yield per day of longevity
and gradual decline in yield was observed with the
increase of age at first calving up to 1850 days.
Whereas, it had no effect on milk yield per day of
productive herd life and the values generally ranged
between 2.69 ± 0.16 and 2.96 ± 0.09 kg.
Lifetime calf crop
The mean lifetime calf production (3.00 ±
0.08) of Murrah buffaloes in the herd studied was
comparable to those reported by earlier workers
97
of lactation length as selection criteria for
maximizing lifetime milk yield. Indian J.
Anim. Sci., 70: 87-88.
El-Arian, M.N. and V.N. Tripathi. 1988. Studies on
the herd life and productive life of Murrah
buffaloes. Buffalo J., 2: 225-229.
Galeazzi, P.M., M.E.Z. Mercadante, J.A.I.I.V.
Silva, R.R. Aspilcueta-Borquis, G.M.F.
de Camargo and H. Tonhati. 2010. Genetic
parameters for stayability in Murrah
buffaloes. J. Dairy Res., 77: 252-256.
Gowane, G.R. and S.S. Tomar. 2007. Genetic and
non-genetic factors affecting selective value
in a herd of Murrah Buffaloes. Indian J.
Dairy Sci., 60: 25-29.
Harvey, W.R. 1990. Least-Squares Analysis of Data
with Unequal Subclass Numbers. USDA,
Science and Education AdministrationAgricultural Research, Beltsville, USA.
Kalsi, J.S. and J.S. Dhillon. 1982. Performance of
buffaloes in first three lactations. Indian J.
Dairy Sci., 35: 218-219.
Kumar, S., M.C. Yadav, B.P. Singh and R.B. Prasad.
2006. Relative importance of reproductive
traits on herd life milk production and profit
in buffaloes. Buffalo Bull., 25: 90-94.
Kuralkar, S.V. and K.L. Raheja. 2000. Factors
affecting first lactation and lifetime traits in
Murrah buffaloes. Indian J. Dairy Sci., 53:
273-277.
Lin, C.Y., A.J. McAllister, T.R. Batra, A.J. Lee,
G.L. Roy, J.A. Vesely, J.M. Wauthy and K.A.
Winter. 1988. Effect of early and late breeding
heifers on multiple lactation performance of
dairy cows. J. Dairy Sci., 71: 2735-2743.
Meyer, K. 1997. Derivative Free Restricted
Maximum Likelihood Programme-Version
3.0 α. User Notes. University of New
England, Armidale, Australia.
day of longevity and milk yield per day productive
herd life. The genetic and phenotypic correlation
estimates between different lifetime production
traits indicated that all the traits were controlled by
similar sets of genes and an increase or decrease in
any one of them will have similar effect on other
traits because of positive association.
Proper nutritional management, protection
against inclement weather through provision
of comfortable housing and special concentrate
mixture supplements/balanced ration just before
and at the time of milking and feeding roughage,
especially straw, at night will certainly provide
remedial measures and reduce age at first calving
and increase lifetime milk production (Singh and
Dangi, 2011).
CONCLUSION
The analysis of effect of age at first calving
on different lifetime production traits revealed
that the optimum age at first calving group in hot
and humid climatic conditions of Tamil Nadu for
higher longevity, productive herd life and lifetime
milk production was >1550 days. On the basis of
present finding it may be concluded that the age
at first calving should be given due weightage
for improving the lifetime production traits of
Murrah buffaloes. Proper nutritional management
and protection against inclement weather through
provision of comfortable housing will reduce age at
first calving to the desired level for better lifetime
performance.
REFERENCES
Dutt, T. and V.K. Taneja. 2000. Milk yield per day
98
Narula, H.K., B.S. Chhikara, A.S. Kanaujia and
A. Saini. 1994. Factors affecting some
economic traits of production in Murrah
buffaloes. Journal of Dairying, Foods and
Home Sciences, 13: 98-102.
Raheja, K.L. 1998. Multivariate restricted
maximum likelihood estimates of genetic
and phenotypic parameters of lifetime
performance traits for Murrah buffalo. In
Proceedings of the 6th World Congress on
Genetics Applied to Livestock Production,
Armidale, Australia, 24: 463-466.
Rao, A.V.N. and H.R.M. Rao. 1996. Longevity,
lactation efficiency and culling pattern of
Murrah buffaloes in Andhra Pradesh. Indian
Vet. J., 73: 1196-1197.
Report. 2006. Basic Animal Husbandry Statistics.
AHS Series-10. Government of India.
Department of Animal Husbandry, Dairying
and Fisheries, New Delhi. pp. ix+162+14.
Sasidhar, P.V.K., B.S. Rao and R.V.S. Kumar.
2000. Calving pattern and some lifetime
performance attributes of buffaloes. Indian
J. Dairy Sci., 53: 239-241.
Singh, R. and K.S. Dangi. 2011. Higher age at first
calving and long calving interval: limitations
and remedies. Indian Dairyman, 63: 44-48.
Taneja, V.K. 1998. Buffalo breeding research in
India. Indian J. Anim. Sci., 67: 713-719.
Tomar, S.S. and R.C. Ram. 1992. Inheritance
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e:919443565565.
99
Original Article
EFFECT OF SEASON ON SEMEN QUALITY PARAMETERS IN
MURRAH BUFFALO BULLS
M. Bhakat, T.K. Mohanty1, A.K. Gupta1, S. Prasad2, A.K. Chakravarty1 and H.M. Khan3
volume (2.69, 2.71 and 2.56), mass activity (2.56,
2.40 and 2.67) and total sperm output (2510.08,
2812.44 and 2923.49) in Murrah buffalo bulls,
but it had a significant (P<0.05) effect on pH
(6.85, 6.77 and 6.71) of semen. The present data
clearly indicated that there was highly significant
(P<0.01) effect of season on seminal attributes
such as initial motility (56.18, 59.64 and 65.95),
sperm concentration (870.62, 1028.20 and
1151.20), non-eosinophilic count (61.91, 65.65
and 73.74), HOST reacted sperm percent (47.05,
48.32 and 62.67), acrosome integrity (65.04, 68.64
and 76.28), sperm abnormalities (HEAD: 2.79,
ABSTRACT
The present study was undertaken to know
the seasonal influence on various seminal attributes
in Murrah buffalo bulls. Data on 156 ejaculates
of eight Murrah buffalo bulls (nearly 30 to 58
months) maintained under identical nutrition and
management conditions were selected randomly
from Artificial Breeding Complex, NDRI, Karnal,
India, from May, 2006 to April, 2007. The
information on 156 ejaculates was subjected to least
square analysis to quantify the effect of non genetic
factor (summer, rainy and winter) on various semen
quality parameters. The overall least square mean
values of the 8 Murrah buffalo bulls for ejaculate
volume (ml), mass activity, Initial motility (%),
Sperm concentration (106/ml), Total sperm output
2.68 and 1.53; MP: 2.15, 1.58 and 1.19; TAIL:
7.11, 5.61 and 4.01 and TOTAL: 12.11, 9.90 and
6.76) and osmolality (288.05, 279.81 and 265.48)
of semen. During hot dry (summer) season, the
highest values of VOL, sperm abnormalities, pH
and OSMOL and lowest MA, IM, SPC, SPCE,
LIVE, HOST and AI were observed. During hothumid (rainy) season, intermediate values of all
the seminal attributes were observed. During cold
(winter) season highest magnitude of MA, IM,
SPC, SPCE, LIVE, HOST and AI and lowest value
of VOL, sperm abnormalities, pH and OSMOL
were observed. Thus, it may be concluded that the
hot-dry season adversely affect the various bio-
(106), Non-eosinophilic count (%), HOST (%),
Acrosome integrity (%), Head abnormality (%),
Mid-piece abnormality (%), Tail abnormality
(%), Total abnormality (%), pH and Osmolality
(mOsmol/Kg) were 2.66±0.10; 2.54±0.70;
60.64±0.02; 1016.68±21.25; 2748.67±122.86;
67.20±0.03; 40.88±0.03; 52.72±0.01; 70.10±0.02;
2.30±0.001; 1.62±0.01; 5.50±0.002; 9.47±0.002;
6.78±0.20 and 277.78±2.40, respectively. Seasonal
variations had no significant effect on ejaculate
2
Livestock Research Centre, National Dairy Research Institute, Karnal, Haryana, India
3
of Agricultural Sciences and Technology of Kashmir (SKUAST-K), Shuhama, Alusteng, Srinagar, Kashmir
(J&K), India
1
100
animal productivity. Investigations carried out
under carefully controlled conditions have shown
that both the magnitude and duration of stress
induced by the adverse environmental condition
influences the animal’s physiological reactions
and production, in the experimental location. The
maximum and minimum temperature during the
summer months varies from 30 to 46oC and the
annual rainfall is about 760 to 960 mm which
is mostly received during the month of July and
August and the relative humidity ranges from 45 to
99 percent. The stressful effect of such a severity
of weather condition was further aggravated by
the stress imposed by direct and reflected solar
radiations. Many places in northern India such
an environment characteristics of the summer is
encountered for a period of 50 to 100 days and
animal in the field, during summer, is not only
burdened with the above heat load but also the
stress is further aggravated by exposure to the sun
for over 12 h a day foraging in terrains which are
sandy and devoid of sufficient vegetation cover. It is
under such environment that the farm animal exists
in the semi-arid zones of northern India (Sengupta
et al., 1963).
An extraordinary long calving interval in
buffalo is a result of many factors of which the
seasonality in the breeding of females and its effect
on the libido and semen production of the bulls is
the most distinct one (Zafar et al., 1988). In farm
animals though the spermatogenesis activity is a
continuous process with the attainment of puberty,
many investigation have shown that the quality and
quantity of semen may vary during different season
of the year. In buffalo bulls it is not conclusive due
to the lack of sufficient reports and the variable
results may be particularly due to different agro
climatic conditions under which the experiments
were carried out (Gupta et al., 1978; Zafar et al.,
physical characteristics of semen in Murrah buffalo
bulls. Winter was the most favourable season for
good quality semen production and the rainy season
might be considered as the intermediate between
the two extremes.
Keywords: season, semen quality parameters,
Murrah buffalo bull
INTRODUCTION
In recent years animal climatology as a
subject of systemic study has gained recognition
and the results achieved so far emphasize the
importance of further concerned and intensive study
of the subject in relation to animal productivity. In
addition to the fundamental aspect of temperature
adaptation by farm animals the subject has been
approached from its applied aspect as well namely
the economy of production, e.g., milk production,
rate of survival, growth and fertility. As an offshoot of these studies has grown newer concepts
of management- feeding and handling of animals
and designs for housing or shelter for the livestock
as well as the lay-out of the immediate surrounding
to the animal houses. In the tropical climate heatstress is largely responsible for the low animal
productivity. Tropical countries like India where
ambient temperature remains above the thermoneutral zone of the farm animals for a large part
of the year. Nearly 90% of the rainfall takes
place in northern regions during three successive
months of the year. Since productivity is primarily
determined by the extent of the utilization of the
available natural resources as well as the direct
effects of climatic components on the physiology
of the animals, it is necessary to ascertain the
detrimental influences of climatic condition on
101
8 Murrah bulls (30 to 58 months of age and 518.58
to 782.50 kg body weight) maintained at Artificial
Breeding Complex, NDRI, Karnal, India. Data
were collected from 156 ejaculates over a period
of one year (May, 2006 to April, 2007). The farm is
situated at an altitude of 250 meters above the mean
sea level on 29.43°N latitude and 72.2°E longitude.
The bulls were maintained identical and optimal
conditions of feeding and management during the
entire course of the experiment. The bulls were
healthy, free from diseases, sexually mature, good
libido and clinically normal, randomly selected
from the herd. The year was subdivided into three
seasons: Hot Dry or summer (April to June); Hot
Humid or Rainy (July to October) and Cold or
winter (November to March). Semen was collected
in the morning once a week from the bulls using
sterilized bovine artificial vagina (IMV model005417) (temp 42-45°C), using dummy bull. Soon
after collection volume was measured and each
ejaculate was placed in a water bath at 30°C and
various standard laboratory tests for semen were
recorded. Semen was assessed for mass activity
and individual motility using DIC phase contrast
microscope (Nikon Eclipse E600, Tokyo, Japan)
with Tokoiheat thermal stage as per standard
method. Mass motility was expressed qualitatively
in (0-5) scale as per the description given by Tomar
et al. (1966). Sperm concentration was estimated
by Haemocytometer (Improved Neubauer’s
chamber) method. pH of the fresh semen was
determined within 15 minutes of collection with
Cyberscan 510 pH meter (Eutech Instrument,
Singapore) and osmolality by WESCOR vapour
pressure Osmometer (WESCOR model 5500, INC,
USA). The live and dead spermatozoa count was
determined as per the method of Bloom (1950)
and Hancock (1951) and the same slide was used
to determine the sperm abnormalities. The hypo-
1988; Bhosrekar et al., 1992b; Prajapati, 1995;
Bhat et al., 2004; Koonjaenak et al., 2007). Among
different seasons hot-dry and hot-humid season
reported to be unfavourable for production as well
as reproduction. The effect of season is both direct
and indirect. It affects the animal directly through
macro and micro climatic factors, like temperature,
humidity, rainfall and photoperiod. Indirectly it
acts by affecting the vegetation, forage quality
and soil-plant-animal interaction. The magnitude
of variations differs from breeds, location,
prevailing climatic conditions, feeding and general
management (Mandal et al., 2000).
Information regarding the effect of seasons
on semen characteristics in Murrah buffalo bulls had
been of conflicting nature. Some research workers
had reported ill effects of heatstress (Gupta et al.,
1978; Bhosrekar et al., 1992b; Prajapati, 1995),
while others observed (Bhat et al., 2004) similar
findings during the winter season; whereas such
effects had been reported in the spring season by
Sengupta et al. (1963). Ravimurugan et al. (2003)
and Bhat et al. (2004) reported monsoon proved
to be best season for production of quality semen
in Murrah buffalo bulls. The knowledge of trend
of seasonal influence on semen characteristics
would help to know the requirement of bulls to
meet the demand of frozen semen and to provide
any suitable additional managerial requirements
time to time. Hence, the present study was
undertaken to investigate the effect of seasons
on various characteristics of semen production in
Murrah buffalo bulls keeping in view the specific
consideration of climatic components.
The present experiment was conducted on
102
various seminal attributes of Murrah buffalo bulls
during different season are presented in Table 1.
osmotic swelling test was performed according to
the methods described by Correa and Zavos (1994).
Staining was carried out as described by Hancock
(1952) for acrosome integrity.
To study the effect of season on the semen
quality parameters (volume, mass activity, sperm
concentration, total sperm production, motility,
non-eosinophilic count, HOST reacted sperm count,
intact acrosome, sperm abnormality studies) the
following least square model has been used. Prior to
the analysis proportionality data (motility, percent
non-eosinophilic count, HOST, acrosome integrity
and abnormality data) were transformed using the
arcsine transformation [asin (sqrt (percent/100))]
(Snedecor and Cochran, 1994) with adjustment to
allow for zero values.
Yik = μ + Si + eik
Where,
Yik = kth record of seminal parameter collected
on a bull in ith season
Si =
Effect of ith season of collection
[i=1 (Hot Dry (Summer): April to June),
2 (Hot Humid (Rainy): July to October)
& 3 (Cold (Winter): November to March)]
eik =
Random error associated with Yik which
is assumed to be normally and independently
distributed with mean zero and constant variance.
The recorded data were subjected to
statistical analysis using LSML-91 software
package, Walter Harvey.
Seminal attributes
Seasonal variations had no significant
effect on ejaculate volume (VOL), mass activity
(MA) and total sperm output (SPCE) in Murrah
buffalo bulls, but it had a significant (P<0.05)
effect on pH of semen. The present data clearly
indicated that there was highly significant (P<0.01)
effect of season on seminal attributes such as initial
motility (IM), sperm concentration (SPC), noneosinophilic count (LIVE), HOST reacted sperm
percent (HOST), acrosome integrity (AI), sperm
abnormalities (head, mid-piece, tail and total) and
osmolality (OSMOL) of semen.
In the present study the overall least squares
mean of ejaculate volume of Murrah buffalo bulls
was found to be 2.66 ± 0.10. The ejaculate volume
was highest during rainy and lowest during winter
season (2.71 vs. 2.56). Bhattacharya et al. (1978)
and Mandal et al. (2000) reported highest semen
volume in Murrah bulls during summer season and
lowest during winter season. However, Rao et al.
(1991) and Ravimurugan et al. (2003) obtained
highest ejaculate volume during rainy season;
Sengupta et al. (1963) and Singh and Singh
(1993) reported highest semen volume during
spring season. No significant seasonal difference
was observed in ejaculate volume which is in
agreement with the findings of Oloufa et al. (1959)
in Egyptian buffalo bulls; Tomar et al. (1966)
in Murrah bulls; Manik and Mudgal (1984) in
Murrah buffalo bulls and Koonjaenak et al. (2007)
in Swamp buffalo. Several factors such as, age of
the animal, differences between species, number of
specimens, level of nutrition, management practice
and environment conditions etc. may be responsible
for the differences in results.
RESULTS AND DISCUSSIONS
Although spermatogenesis is a continuous
process in male once it has reached reproductive
maturity, the semen of farm animals exhibits a
distinct climatic pattern with respect to its quality
and fertilizing efficiency. Least squares means of
103
104
Hot Humid (Rainy)
(N=53)
Mean
S.E.
2.71
0.17
2.40
0.11
a
59.64
0.05
B
1028.20
34.08
2812.44
197.03
a
65.65
0.07
A
48.32
0.04
a
68.64
0.06
B
2.68
0.001
B
1.58
0.001
B
5.61
0.004
B
9.90
0.005
AC
6.77
0.03
A
279.81
3.84
Cold Humid (Winter)
(N=69)
Mean
S.E.
2.56
0.15
2.67
0.10
b
65.95
0.04
C
1151.20
31.34
2923.49
181.20
b
73.74
0.06
B
62.67
0.03
b
76.28
0.05
C
1.53
0.001
C
1.19
0.001
C
4.01
0.004
C
6.76
0.004
BC
6.71
0.03
B
265.48
3.53
Least squares means bearing different alphabets as superscripts differ significantly row-wise (abP<0.05, ABP<0.01).
Ejaculate volume (ml)
Mass activity (0-5 Scale)
Initial motility (%)
Sperm concentration (106/ml)
Total sperm output (106)
Non-eosinophilic count (%)
HOST (%)
Acrosome integrity (%)
Head abnormality (%)
Mid-piece abnormality (%)
Tail abnormality (%)
Total abnormality (%)
pH
Osmolality (mOsmol/Kg)
Parameters
Hot Dry (Summer)
(N=34)
Mean
S.E.
2.69
0.21
2.56
0.14
a
56.18
0.08
A
870.62
42.23
2510.08
244.19
a
61.91
0.11
A
47.05
0.06
a
65.04
0.10
A
2.79
0.001
A
2.15
0.001
A
7.11
0.006
A
12.11
0.008
A
6.85
0.41
A
288.05
4.76
Table 1. Least squares means ± S.E. for effect of season on semen quality parameters of Murrah buffalo bulls.
Mean
2.66
2.54
60.64
1016.68
2748.67
67.20
52.72
70.10
2.30
1.62
5.50
9.47
6.78
277.78
S.E.
0.10
0.70
0.02
21.25
122.86
0.03
0.01
0.02
0.001
0.01
0.002
0.002
0.20
2.40
Overall (N=156)
The overall least squares mean of mass
activity (MA) was found to be 2.54 ± 0.70. Mass
activity was the maximum during winter, followed
by summer and rainy season (2.67, 2.56 and 2.40).
The highest mass activity during winter season
had been reported in Murrah buffalo bulls (Manik
and Mudgal, 1984; Mandal et al., 2000); Mehsana
(Prajapati, 1995) and Surti buffalo bulls (Bhosrekar
et al.,1992b), however, in Murrah bulls Bhosrekar
(1980) and Dhami et al. (1998) observed its
highest value during rainy season, whereas Zafar et
al.(1988) obtained no significant seasonal variation
in mass activity in Nili-ravi bulls as it was found in
the present study. Results are conflicting because
mass motility was subjectively determined by
microscopic examination of a drop of fresh semen;
these data should be considered with caution.
Significant (P<0.01) seasonal difference
was observed in percent initial motility (IM) which
is in agreement with the findings of Tuli and Singh
(1983) in Murrah buffalo bulls and Bhosrekar et al.
(1992b) in Surti buffalo bulls and Ravimurugan et
al. (2003) in Murrah buffalo bulls. On the contrary,
Oloufa et al. (1959) in Egyptian buffalo bulls; Gupta
et al. (1978) in Surti buffalo; Zafar et al. (1988) in
Nili- ravi buffalo bulls; Bhosrekar et al. (1991) in
Murrah buffalo bulls; Prajapati (1995) in Mehsana;
Mandal et al. (2000) in Murrah buffalo bulls and
Koonjaenak et al. (2007) in Swamp buffalo did
not obtain any significant seasonal variation in
IM. The initial motility was found to be maximum
during winter (65.95 vs. 56.18 and 59.64 %) which
varied significantly (P<0.05) in summer and rainy
season.
Sperm concentration per unit volume of the
semen is perhaps one of the most studied seminal
attributes in relation to seasonal variation of
semen quality. The results revealed that the sperm
concentration per ml (SPC) varied significantly
(P<0.01) among seasons being maximum during
winter followed by rainy and summer season
(1151.20, 1028.20 and 870.62 × 106/ml) which is
corroborate with the report of Mandal et al. (2000)
and Ravimurugan et al. (2003) in Murrah bulls. The
difference in SPC between seasons was significant
(P<0.01). Bhosrekar (1980) and Manik & Mudgal
(1984) reported highest SPC during summer season
in Murrah, where as Prajapati (1995) and Dhami
et al. (1998) obtained highest value during rainy
season; however, in Egyptian buffalo bulls Oloufa et
al. (1959) observed its highest value during spring.
On the contrary, Zafar et al. (1988) in Nili- Ravi
buffalo bulls; Bhosrekar et al. (1992a) in Murrah;
Bhosrekar et al. (1992b) in Surti and Koonjaenak et
al. (2007) in Swamp buffalo obtained no significant
seasonal variation in sperm concentration. The
differences between this and other studies might be
the result of length of the study period, as well as
differences in the age and breed of the bulls. In the
present finding lower concentration of spermatozoa
during the summer may be due to significant
reduction in the feed intake and increase in dead
and abnormal spermatozoa. Dead and abnormal
spermatozoa, which are absorbed by leucocytes
through phagocytosis (Mann and Mann, 1981).
The increased resorption of abnormal spermatozoa
leads to reduction in epididymal sperm reserves
(Rao et al., 1980), thus decreasing concentration.
This finding support to the conclusion that the
spermatozoa produced during summer were either
intrinsically less active and vigorous at the time
of production or that they, though normal at the
time of genesis, suffered deterioration at some
stage of their passage down the male reproductive
tract prior to their release in the ejaculate under
sustained impact of a climatically stressful summer
environment.
Total sperm output (SPCE) was maximum
105
have an opportunity to come out in the ejaculate.
Significant (P<0.01) seasonal variation
in hypo-osmotic swelling test (HOST) reacted
spermatozoa was observed in the present
investigation being maximum during winter season
followed by rainy and summer season (62.67,
48.32 and 47.05%). However the variation between
summer and rainy season was non-significant. The
results obtained here are quite similar to those
reported by Mandal et al. (2000) in Murrah buffalo
bulls. Whereas, Koonjaenak et al. (2007) reported
PMI was highest in summer and lowest in winter
(P< 0.05).
Percentage of spermatozoa with intact
acrosome were found to be significantly (P<0.01)
affected by seasons. The lowest acrosome integrity
percent was observed during summer and highest
during winter season (65.04 vs. 76.28%). However
the variation between summer and rainy season
was non-significant (65.04 and 68.64 %). However,
Manik and Mudgal (1984) and Mandal et al. (2000)
reported lowest value during summer, whereas,
Singh and Singh (1993) reported lowest value
during winter season.
The variation in head, mid-piece, tail and
total abnormality percent were highly significant
(P<0.01) among the seasons. All the abnormalities
were found to be higher during summer followed
by rainy and winter seasons (HEAD- 2.79, 2.68
and 1.53; Mid-piece- 2.15, 1.58 and 1.19; TAIL7.11, 5.61 and 4.01; TOTAL- 12.11, 9.90 and 6.76
%). Significant (P<0.01) variation between season
were also observed in case of all the abnormalities.
Bhavsar et al. (1990) in Mehsana buffalo bulls and
Mandal et al. (2000) in Murrah buffalo obtained
almost similar types of results, but Manik and
Mudgal (1984) and Bhosrekar et al. (1991) reported
higher abnormality during winter season. Whereas,
Koonjaenak et al. (2007) reported significant
during winter followed by rainy and summer
season (2923.49, 2812.44 and 2510.08×106).
As the sperm concentration per ml was highest
during winter season resulted in maximum sperm
output per ejaculate. However, Prajapati (1995) in
Mehsana; Singh et al. (1992) in Mehsana; Gupta
et al. (1978) in Surti; Rao et al. (1991) in Murrah
and Mandal et al. (2000) in Murrah buffalo bulls
obtained maximum output during rainy season,
whereas Bhavsar et al. (1986) obtained highest
sperm per ejaculate in autumn season in Mehsana
bulls. However, Koonjaenak et al. (2007) in Swamp
buffalo observed no significant seasonal variation
in total sperm output. These variations may be due
to managemental conditions, laboratory method of
estimating sperm concentration.
Significant (P<0.01) seasonal variation
in non-eosinophilic sperm percent was observed.
Highest live sperm percent was observed during
winter, followed by rainy and summer season
(73.74, 65.65 and 61.91%). The live sperm percent
was found to be maximum during winter which
varied significantly (P<0.05) with summer and
rainy season. Our results are in agreement with the
findings of Gupta et al. (1978) in Surti and Mandal
et al. (2000) in Murrah buffalo bulls. Sengupta et
al. (1963); Manik and Mudgal (1984) and Dixit et
al. (1984) also reported lowest live sperm count
during summer season. However Bhosrekar (1981)
and Singh and Singh (1993) observed lowest
value of live sperm percent in Murrah bulls during
winter season and Prajapati (1995) reported lowest
value during rainy season in Mehsana bulls. The
occurrence of lowest percentage of live spermatozoa
synchronizing with the part of year characterized
by the highest mean ambient temperature
suggests that the summer environment becomes
instrumental in causing death and abnormality to
a high percentage of spermatozoa even before they
106
seasonal influence on sperm morphology. He also
found that among morphological abnormalities,
only proportions of tail defects were affected by
season, being highest in the rainy season and lowest
in summer (P < 0.001).
Significant (P<0.05) seasonal difference in
pH was observed in the present study. This may
be due to seasonal fodder changes. During summer
pH was high may be due to silage feeding. On
the contrary, Koonjaenak et al. (2007) in swamp
buffalo reported no significant seasonal variation
in pH. Mandal et al. (2000) also observed similar
results in Murrah buffalo bulls. Highly significant
(P<0.01) variations due to season were observed in
case of osmolality, being maximum during summer,
followed by rainy and winter seasons (288.05,
279.81 and 265.48 %). Heat stress increases loss of
body fluid due to sweating and panting, if the stress
continues for a longer period the fluid loss can
reach critical level (Kadzere et al., 2002) and may
be reflected in the seminal plasma which is evident
from the above finding that osmolality was highest
during the hot dry season. The reason for variation
in osmolality of seminal plasma is not clearly
known, however, it may be due to fluctuation in
core body temperature, seasonal fodder changes
and changes in thermodynamics of body. On the
other side may be variation of environmental
temperature has effect on the osmolality reading
by the vapor pressure machine to the extent of 5
to 7%. Individual variation might be attributed
to the genetic makeup, age, nutrition and the
influence of the climatic components, which might
have transduced variably into endocrine messages
controlling
hypophyseal-hypothalamo-gonadal
axis.
Haryana is the home track of the Murrah
buffalo and they are very much adaptable to the
environment. Normal management practices
in the field condition to ameliorate heat stress is
by wallowing in the village pond or in irrigation
canal for longer part of the day, when the solar
radiation and heat is maximum, but in case of our
farm condition we are not able to provide such
type of facilities for breeding bulls. Sprinkling
facility is available during this period. In case of
female buffaloes, providing mist, forced cooling
and wallowing improves productivity. In general
summer may be regarded as the season exerting
relatively more adverse effect on the overall semen
quality than the other seasons. This appears quite
likely in view of the high ambient temperature,
a relatively long spell of that high temperature,
hot blast of wind (Loo) and a continuous stream
of radiation impinging directly and indirectly
through reflection from terrains or shed on the
animal’s body and thereby precipitating a really
distressing challenge to the animal’s thermoregulatory mechanism. The buffalo bulls seem to
be susceptible to either extremes of heat and cold.
But under the experimental conditions it appeared
that they were more tolerant towards the colder
months as compared to the hotter months. It will be
apparent from the climatic table that winter in this
part can be considered neither extreme nor severe
to the extent to being definitely detrimental. The
semen picture during winter was better to that of
rainy and summer season.
Semen quality in bull reflects the degree
of normality of the function of their testes, ducti
epididymides and genital tract (including the
accessory sex glands). The normality of the genital
system also depends on the hormonal balance of
the bull, which is sensitive to changes in health
status, nutrition and management. Changes in these
conditions influence sperm output, accessory sex
gland secretion and epididymal function, all of
which are reflected in the in the semen quantitative
107
(ejaculate volume, sperm concentration and
total sperm output per ejaculate) and qualitative
characteristics (mass motility, individual motility,
non-eosinophilic, abnormal sperm percent, intact
acrosome and spermatozoa with intact plasma
membrane percent). The sperm quality in the
ejaculate, regarded as the sum of these variables.
Furthermore, external cues such as seasonality also
appear to influence sexual function, either through
photoperiod (Barth and Waldner, 2002) or through
changes in ambient temperature (Fayemi and
Adegbite, 1982; Sekoni and Gustafsson, 1987).
The time course of spermatogenesis in the bull
is as follows: the transformation of committed A
spermatogonia to B2 spermatogonia takes 20 days,
of B2 spermatogonia to panchytene spermatocyte
takes 10 days, of panchytene spermatocyte to early
round spermatid takes 13.5 days, of early round
spermatid until release into the lumen takes 17.5
days, epididymal transit time is six to eight days
(Amann and Schanbacher, 1983). The numbers
of sperm and morphology of the semen finally
ejaculated are determined by the numbers of stem
cells recruited to undergo spermatogenesis and by the
mortality throughout spermatogenesis, particularly
at the pachchytene spermatocyte and early round
spermatid stage. Hence semen quality at any time is
likely to reflect the environmental influences upon
the sensitive stages of spermatogenesis, which is
highly sensitive to even short increases in scrotal
temperature, as has been recorded in Bos taurus
AI sires kept in temperate regions (Januskauskas
et al., 1995). From the findings of the present
investigation, it could be inferred that the hot
seasons was the worst for semen production in
Murrah buffalo bulls. It might be attributed to the
fact that heat stress reduces the release of GnRH,
which in turn affected the release of hormones
responsible for spermatogenesis. Heat stress might
have also increased the release of ACTH, which
inhibits the effect of LH, an important hormone
responsible for spermatogenesis. Clarke and
Tilbrooke (1992) reviewed the effects of heat and
various other environmental stresses in different
species of livestock. It indicated that the stressors in
general affect the normal process of reproduction in
a multi-dimensional way by reducing feed intake,
impairing either the release or response to the
important hormones of reproduction, like GnRH,
LH and increased levels of plasma corticosteroids,
have inhibitory effect on LH. However, the exact
effects on gonadal functions on dairy bulls need
to be investigated. Besides, decline in thyroxin
level during hot-dry and hot-humid seasons as
compared to winter (Madan, 1985) impaired
the general metabolism and feed intake and
could be instrumental in causing reproductive
dysfunctions (Zafar et al., 1988). The temperature
sensitive muscles of the testis- tunica dartos and
external cremester muscles get relaxed to its
maximum limit to keep testicles cool, after that
core temperature goes on increasing. Nonetheless,
increased core temperature of testes might have
reduced the activity of enzymes responsible for
spermatogenesis and impaired the normal process
of reproduction. A few investigators (Kushwaha
et al., 1955; Gopalakrishna and Rao, 1978) have
attributed the low breeding efficiency of buffaloes
during summer due to deterioration of semen
quality, but this has been disputed by others (Tomar
et al., 1964; Chaudhary and Gangwar, 1977).
As the sexual activity of each species
including human beings, is highly influenced by the
environment of the surroundings, the variation in
these parameters are quite obvious with reference
to time, place and subject concerned. In summer,
extreme heat stress causes physical exhaustion,
which might reduce the eagerness of the bulls and
108
functions of the breeding bulls. In general it is
suggested that during summer, breeding bulls
should be kept cool and comfortable by splashing
water at least 3-4 times a day, protected from direct
wind blasts, housed in a place with comfortable
micro-environment with least humidity, fed during
cool hours and have a free access to cool drinking
water.
thus, result in higher reaction time and total time
for successful ejaculation, thus having an ultimate
effect on production of sperms Mandal et al. (2000).
Reasons for good quality seminal ejaculates during
winter might be attributed to the congenial weather
condition which affects the activity and secretions
of accessory reproductive glands, since the
secretions are dependent on testosterone liberated
by interstitial cells during this season which might
have favoured the process of spermatogenesis
Mandal et al. (2005). Madan (1985) found that
thyroxin was higher during cold as compared to
hot-dry and hot-humid seasons. Thyroxin is one
of the primary metabolic hormones which bear the
significance in this regard. Application of Vaseline
during winter season prevented the cracking of the
skin as result damage to testicular tissue may be
very minute.
ACKNOWLEDGEMENT
We are thankful to Director, NDRI for
providing the necessary funding to carry out the
research work.
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112
Original Article
MILK YIELD AND COMPOSITION AND EFFICIENCY OF NUTRIENTS FOR MILK
PRODUCTION IN JAFFRABADI BUFFALOES ON RATIONS SUPPLEMENTED WITH
VARYING LEVELS OF BYPASS FAT
H.H. Savsani1,*, K.S. Murthy2, P.U. Gajbhiye2, P.H. Vataliya1, A.R. Bhadaniya1, V.A. Kalaria1,
S.N. Ghodasara2 and S.S. Patil1
10 g/kg milk supplemented group of buffaloes
exhibited positive effect in influencing lower DMI,
CPI, DCPI and TDNI per kg milk production.
Overall results indicated higher level of bypass
supplementation has no beneficial effect and prepartum supplementation of bypass fat tends to
counter balance negative energy in early lactation.
ABSTRACT
Negative energy balance in early lactation
can be counterbalanced by supplementing bypass
fat in lactating animals. An experiment was
conducted to evaluate the effects of supplementing
daily rations with 0, 10, 20 and 30 g bypass fat/
kg milk production in 24 lactating Jaffrabadi (1-4
lactations 425-727 kg B.Wt.) buffaloes in four
groups of six each in CRD. All the experimental
buffaloes were offered bypass fat 150 g/day/animal
15 days prior to prepartum to nullify negative
energy problem in early lactation. Milk and FCM
production was not affected significantly(p>0.05)
due to bypass fat supplementation, though 10 g/
kg milk supplementation resulted in 22.70% and
18.93% more milk and 28.21% and 20.51% more
FCM, respectively, than control group. There
was significant (p<0.05) and linear increase in
milk fat percent and total solids percent in bypass
supplemented group of buffaloes. Milk SNF,
Protein and Ash percent were not influenced by
the level of supplementation significantly. Total
solids percent increased by 2.26, 5.73 and 4.93%,
respectively in supplemented groups compared to
control. This assumes significance due to the fact
that buffalo milk is used for Khoa making. Though
the nutrients efficiency for milk production was
non significant among experimental groups,
Keywords: bypass fat, Jaffrabadi buffaloes,
nutrients efficiency, milk composition, milk yield
INTRODUCTION
Increasing energy density of the ration in
early lactation to counterbalance negative energy is
critical in high yielding buffaloes to optimize milk
production. Unprotected fat in the total diet should
not exceed 4% (Palmquist, 1988) as its affects
Cellulose digestibility. Bypass fat availability in
the lower gut not only enhances energy density
of the ration but also supplies additional source
of calcium (Anonymous, 2002). Different
technologies like prilled fats, calcium salts of fatty
acids, Formaldehyde treated fats to circumvent
rumen degradation to enhance availability in the
lower gut are available. Beneficial effects of bypass
feeding as a supplement in early lactation are also
established.. However, practical feeding of bypass
College of Veterinary Science and Animal husbandry, Junagadh Agricultural University (JAU), Junagadh,
Gujarat, India, *E-mail: [email protected]
2
Cattle Breeding Farm, Junagadh Agricultural University (JAU), Junagadh, Gujarat, India
1
113
of negative energy balance in early lactation. In
the second week after calving, rations of buffaloes
according to the treatment were enriched with
bypass fat for twenty six weeks of lactation. Ration
schedules were adjusted every fort night according
to changes in milk yield and fat percent and body
weight. Standard managerial conditions were
provided during the experimental study .
Animals were hand milked twice daily
(5:00 h and 17:00 h) and the yields were recorded.
Milk samples were drawn at fortnight intervals
from individual animals during both the times of
milking. Milk fat (Gerber’s method (ISI 1961)),
Total solids (gravimetric or evaporation method),
SNF (difference method) and Milk protein
(AOAC,1995) were estimated. For FCM conversion
formula of Rice et al., (1970) was adopted (0.4x
Milk yield in kg + 15 x weight of fat content/1.3).
Data were analyzed according to Snedecor and
Cochran, (1994).
fat to ruminants by dairy farmers in India is not
common. Jaffrabadi buffaloes are heavy (600-800
kg body weight ) animals producing 2500 litres of
milk per lactation with high far content (8.0%) . Fat
globules are larger in size and the milk is highly
preferred for ghee and khoa making. Feeding
requirements of these buffaloes seem to be on higher
side considering their body size. Present experiment
was undertaken to compensate the negative energy
balance of early lactation by 15 day pre-partum
feeding of bypass fat and to evaluate the effect of
varying levels of bypass fat supplementation postpartum on milk yield, fat content and composition
of milk in Jaffrabadi buffaloes.
An experiment was conducted in
Completely Randomized Design for twenty six
weeks on twenty four lactating Jaffrabadi buffaloes
(1-4 lactation, 6 to 8 kg average milk production of
previous lactation, BW 451-727 kg) in four groups
of six each to evaluate the effect of feeding varying
levels of bypass fat supplementation to the ration
(T1) 0, (T2) 10 g, (T3) 20 g and (T4) 30 g/kg of
milk) following ICAR(1998) feeding standards.
All the experimental buffaloes were individually
offered a basal diet of 10 Kg. seasonal green and
mature pasture grass hay ad lib. DCP requirement
was met 50% from concentrate mixture and 50%
from cotton seed cake. Commercially available
bypass preparation containing calcium soap of
Palm fatty acids (Myristic acid -1.5%, Palmitidc
acid -44%, Stearic acid-5%, Oleic acid-40%,
Lineoleic acid-9.5%, with NEl 5.75 Mcal/kg
and ED 7.95 Mcal/kg) was offered fifteen days
prepartum daily 150 g/buffalo, to nullify the effect
RESULT AND DISCUSSION
Milk and FCM production
Milk and FCM production during
different phases of the experiment did not differ
significantly except for periodic differences during
P11 and P12 (Table 1 and 2 and Figure 1) Overall
milk and FCM (6% corrected) production (kg/
day) was 6.43±0.44, 7.96±0.67, 6.15±0.56, and
7.05±0.36 and 6.63±0.49, 8.50±0.72, 6.86±0.64
and 7.99±0.48, respectively for T1, T2, T3 and
T4. Whole milk and FCM production during
the period of 182 days was 1170.10±80.6,
1448.80±122.7, 1118.77±102.5 and 1282.79±66.4
kg and 1238.40±89.3, 1519±132, 1217.53±114.7
and 1472.77±87.2 kg respectively under T1, T2, T3
and T4. Milk and FCM production increased from
114
P1 to P7 and more or less maintained throughout
the experimental period. The differences in daily
and overall milk and FCM production were nonsignificant. However, bypass fat offered buffaloes
under T2 and T4 produced 22.70 % and 18.93 %
more daily milk and 23.79 and 9.64% more total
than control group. There was an increase of
28.21% and 20.51% in daily FCM production in
T2 and T4 over control group of animals. Grummer
(1988), Kent and Arambel (1988) , Schneider et al.
(1988) , Jenkins and Jenny (1989), Schauff and
Clark (1989) , Klusmeyer et al. (1991), Kim et
al. (1993), reported no beneficial effect of feeding
of bypass fat in in increasing the milk and FCM
production. Supplementation of 10 g bypass fat per
kg milk production produced beneficial results.
period wise and increase in fat percent was also
linear from T1 to T4 indicating positive response
of milk fat percent to graded levels of bypass fat
supplementation i.e. 10 g, 20 g and 30 g per kg of
liter milk production. Under Indian conditions milk
prices are solely determined on fat percent and
hence, any increase in milk fat percent will fetch
higher prices for milk producer.
Mean SNF percent during different periods
from P1 to P13 and overall SNF is presented in
Table 3 and Figure 2. Enriching the ration with
bypass fat had no significant effect on SNF during
different periods of milk production and also on
overall SNF percent during entire study. It might
be seen that SNF percent gradually declined from
P1 to P13 and the decline was linear, opposite to
that of milk fat percent. Feeding of protected tallow
(Sharma et al., 1978), CSFA (Grumer, 1988, Kent
and Arambel, 1988, Schauff and Clark, 1989,
Klusmeyer et al.,1991, Schauff and Clark, 1992,
Kim et al.,1993, Wu et al.,1994 and Sirohi et al.,
2007) and bypass fat (Shankhpal et al., 2009) did
not have any significant effect on SNF percent in
milk. Andrew et al. (1991) reported that feeding of
calcium salts of fatty acid reduced SNF percent in
milk.
Total solids content in milk was at par
under four treatment groups during different
periods, except P8, P9 and P10. Effect of bypass fat
on total solid percent in milk was non-significant.
However during a period 8, 9 and 10, differences
among treatments (P<0.05) were significant.
Overall total solid percent during entire experiment
period was different significantly (P<0.05) among
the treatments. During P8, P9 and P10 and overall
total solid percent in milk was at par in T1 and
T2, which, differed significantly from T3 and T4
which are again at par. Supplemental by pass fat
feeding increased the percent total solids in T2, T3
Milk Composition
Overall milk fat percent was significantly
(P<0.05) higher in T4 followed by T3, T2 and T1
and they were 6.26±0.15, 6.58±0.08, 7.04±0.20 and
7.12±0.60, respectively for T1, T2, T3 and T4(Table
3 and Figure 2). T1 was at par with T2 while T2 was
at par with T3 but lower than T4. Period effect on
milk fat was linear though non-significant, except,
during P6, P7, P8, P9 and P10. Total milk fat yield
during the experimental period was 73.42±5.07,
95.42±8.18, 78.31±7.29 and 91.84±8.85 kg. in T1,
T2, T3 and T4 respectively with non-significant
differences. Sharma et al. (1978) and Schneider et
al. (1988) reported significant (P<0.05) increase in
milk fat when protected tallow and calcium salts
of fatty acids were fed to Holstein cows. Grummer
(1988), Kent and Arambel (1988), Schauff and
Clark (1989), Canale et al. (1990), Klusmeyer et
al. (1991), Andrew et al. (1991) did not find any
significant effect of bypass fat feeding on milk fat
percent. Response of lactating buffaloes to bypass
fat feeding in the present experiment was linear
115
and T4 by 2.26%, 5.73% and 4.93%, respectively
over control groups of animals. Kim et al. (1993)
did not find any significant difference in percent
total solid in milk in lactating dairy cows that were
offered calcium salts of fatty acid.
Protein percent in milk was also nonsignificant during 13 fortnights(Table 3 Figure 2).
Overall protein percent during the experimental
period was non-significant and is in consonance
with the findings of Sirohi et al., (2007). Fat, SNF
and total solids are highly variable constituents in
milk, while protein, ash and lactose remain more
or less same and remain unaffected by feeding
different regimens. The present study indicated
non-significant effect of bypass fat feedings at 10,
20 and 30 g per liter of milk and no significant
effect on percent total protein and ash content.
and clark (1989) observed that feeding of Ca salts
or prilled fat did not affect DM required for kg
milk production. However, Moallem et al. (2000)
observed significantly higher DMI per kg FCM
production in CSFA fed cows than in control group
and still higher DMI in BST treatment offered
cows compared to other cows. DMI per kg milk
production recorded in present experiment appears
to be on the higher side than the findings of above
research workers. Reason could be higher body
weight of Jaffrabadi buffaloes requiring higher
maintenance requirement and high fat percent
in milk again requiring higher level of nutrients
intake. However CP intake, DCP intake and TDN
intake required per kg milk production appeared
to be in agreement with values reported by Tyagi
and Thakur (2007) and Shankhpal et al. (2009).
Supplementation of bypass fat 10 g per liter milk
production appeared to be bearing positive effect
in influencing lower DMI, CPI, DCPI and TDNI
per kg of milk production in lactating Jaffrabadi
buffaloes.
Present experimental results indicate that
supplementation of bypass fat10 g/kg milk resulted
in higher milk yield, milk fat % . Feeding of
supplementary fat 10, 20 and 30 g increased total
solids content and yield in Jaffrabadi buffaloes. Prepartum feeding of bypass fat seems to be beneficial
in eliciting positive response in lactating buffaloes
throughout the experimental period of interest to
this study is the fact that buffalo milk in India is
mainly used for Ghee and Khoa making and hence
highly total solids yield is economically beneficial
to dairy farmers.
Efficiency of nutrients for milk production
DMI, CP, DCP and TDN required for
kg milk production is given in Table 4 and
Figure 3a and 3b for T1, T2, T3 and T4 groups.
Varying levels of bypass fat supplement did not
influence significantly DMI required for 1 kg milk
production during different periods and during the
entire experiment. However CP and DCP intakes
required for 1 kg milk production significantly
(P<0.05) differed during P3, while, remaining at par
during other periods. During P3, T1 and T2 group
of buffaloes required significantly (P<0.05) lower
CP and DCP per kg milk production in comparison
to T3 and T4 groups. TDN intake per kg milk
production was not influenced by supplemental by
pass fat feeding during different periods as well as
during the entire experimental period. Kent and
Arambel (1988) recorded that supplementation of
223 g of Ca salt of long chain fatty acid offered
daily as a top dress did not influence DMI in
lactating dairy cows compared to control. Schauff
REFERENCES
Andrew, S.M., H.E. Tyrrell, C.K. Reynolds and
116
117
P2
6.04
±
0.43
7.27
±
0.77
6.85
±
0.79
6.81
±
0.52
0.64
NS
23.52
P1
4.58
±
0.39
5.42
±
0.51
4.73
±
0.37
5.12
±
0.52
0.45
NS
22.34
6.59
±
0.37
8.48
±
0.74
6.82
±
0.85
5.69
±
0.95
0.76
NS
27
P3
6.97
±
0.36
8.57
±
0.90
6.85
±
0.96
7.19
±
0.46
0.72
NS
23.86
P4
6.71
±
0.53
8.45
±
0.79
6.58
±
0.76
7.68
±
0.35
0.63
NS
21.07
P5
6.76
±
0.50
8.60
±
0.68
6.56
±
0.78
7.63
±
0.44
0.61
NS
20.38
P6
P7
6.75
±
0.61
8.39
±
0.82
6.69
±
0.86
7.70
±
0.39
0.69
NS
23.01
Means in a column with different superscripts differ significantly (p<0.05).
S.Em.±
C.D. at 5 %
C.V. %
T4
T3
T2
T1
Treatment
6.79
±
0.57
8.42
±
0.72
6.80
±
0.92
7.65
±
0.49
0.69
NS
22.92
P8
6.75
±
0.46
7.74
±
0.71
6.92
±
0.95
7.56
±
0.46
0.67
NS
22.83
P9
6.50
±
0.58
7.86
±
0.80
6.19
±
0.74
7.51
±
0.37
0.64
NS
22.49
P10
Table 1. Average daily milk yield (kg) of lactating Jaffrabadi buffaloes during different phase of experiment.
6.41bc
±
0.49
8.39a
±
0.80
4.68c
±
0.87
7.47ab
±
0.25
0.65
1.92
23.71
P11
P13
6.22
±
0.51
7.79
±
0.71
5.14
±
0.88
6.55
±
0.26
0.63
NS
24.15
P12
6.52abc
±
0.51
8.10a
±
0.68
5.09c
±
0.93
7.08ab
±
0.36
0.65
1.92
23.9
6.43
±
0.44
7.96
±
0.67
6.15
±
0.56
7.05
±
0.36
0.52
NS
18.64
Overall
118
P1
4.35
±
0.32
5.58
±
0.52
5.04
±
0.44
5.42
±
0.75
0.52
NS
25.44
P2
5.74
±
0.37
7.07
±
0.75
6.87
±
0.86
6.82
±
0.59
0.66
NS
24.75
P3
6.48
±
0.42
8.65
±
0.74
7.18
±
1.18
5.92
±
1.03
0.89
NS
31.1
P4
7.05
±
0.39
8.87
±
0.96
7.24
±
1.18
7.54
±
0.54
0.82
NS
26.29
P5
6.76
±
0.57
8.70
±
0.88
7.36
±
1.12
8.03
±
0.35
0.78
NS
24.99
P6
6.69
±
0.46
8.64
±
0.70
7.13
±
0.88
8.20
±
0.55
0.66
NS
21.24
P7
6.79
±
0.70
8.61
±
0.88
7.29
±
0.91
8.59
±
0.57
0.77
NS
24.35
P8
6.67
±
0.53
8.92
±
0.80
7.59
±
0.97
9.00
±
0.65
0.75
NS
23.07
Means in a column with different superscripts differ significantly (p<0.05)
S.Em.±
C.D. at 5 %
C.V. %
T4
T3
T2
T1
Treatment
P9
6.90
±
0.52
8.14
±
0.78
7.87
±
1.06
8.44
±
0.65
0.77
NS
24.25
P10
6.66
±
0.61
8.37
±
0.93
7.04
±
0.76
8.99
±
0.49
0.71
NS
22.58
Table 2. Average daily FCM yield (kg) of lactating Jaffrabadi buffaloes during different phase of experiment.
P11
6.96abc
±
0.75
9.34a
±
0.97
5.57c
±
1.02
9.02ab
±
0.59
0.85
2.50
26.98
P12
7.37abc
±
0.90
9.44a
±
0.83
6.15c
±
1.11
8.90ab
±
0.37
0.84
2.49
26
P13
7.79
±
0.61
10.17
±
0.96
6.81
±
1.18
9.02
±
0.47
0.84
NS
24.63
Overall
6.63
±
0.49
8.50
±
0.72
6.86
±
0.64
7.99
±
0.48
0.59
NS
19.3
Fat %
6.26c ± 0.15
6.58bc ± 0.08
7.04ab ± 0.20
7.12a ± 0.16
0.15
0.45
5.63
SNF %
8.76 ± 0.07
8.77 ± 0.12
8.83 ± 0.09
8.64 ± 0.09
0.09
NS
2.7
Total solid %
15.02c ± 0.16
15.36bc ± 0.16
15.88a ± 0.14
15.76ab ± 0.12
0.14
0.42
2.28
Protein %
4.13 ± 0.08
4.23 ± 0.05
4.18 ± 0.05
4.26 ± 0.04
0.05
NS
3.34
Ash %
0.94 ± 0.00
0.94 ± 0.01
0.91 ± 0.01
0.93 ± 0.00
0.01
NS
2.93
119
DMI (kg) / kg milk
production
2.80 ± 0.20
2.38 ± 0.19
3.19 ± 0.33
2.46 ± 0.08
0.21
NS
19.86
TREATMENT
T1
T2
T3
T4
S.Em.±
C.D. at 5 %
C.V. %
261.69 ± 12..28
234.97 ± 13.78
308.78 ± 32.60
252.22 ± 5.83
18.96
NS
17.57
CPI (g/day) / kg milk
production
132.11 ± 4.58
122.24 ± 5.91
158.61 ± 16..96
133.46 ± 2.67
9.4
NS
16.79
DCPI (g/day) / kg milk
production
1.18 ± 0.09
1.02 ± 0.07
1.35 ± 0.14
1.05 ± 0.03
0.09
NS
19.36
TDNI (kg/day) / kg milk
production
Table 4. Overall DMI (kg), CPI (g/day), DCPI (g/day) and TDNI (kg/day) per kg milk production of lactating Jaffrabadi buffaloes.
Means in a column with different superscripts differ significantly (p<0.05).
Treatment
T1
T2
T3
T4
S.Em.±
C.D. at 5 %
C.V. %
Table 3. Overall Fat, SNF, total solid, protein and ash percent of lactating Jaffrabadi buffaloes.
Figure 1. Average dairy milk yield (kg) of lactating Jafrabadi buffaloes during different phase of
experiment.
Figure 2. Overall fat, SNF, total solid, protein and ash percent of lactating Jafrabadi buffaloes.
120
Figure 3a. Overall DMI (kg) and TDNI (kg) per kg milk production of lactating Jafrabadi buffaloes.
Figure 3b. Overall CPI (g) and DCPI (g) per kg milk production of lactating Jafrabadi buffaloes.
121
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123
Original Article
REAL TIME PCR- AN APPROACH TO DETECT MEAT ADULTERATION
Rajni Kumari1, D.N. Rank2, Sanjay Kumar2, C.G. Joshi2 and S.V. Lal2
ABSTRACT
melting peaks on DNA templates from sheep, goat
and chicken. Thus, Real Time PCR was found to
be successful in differentiating cattle and buffalo
mixed meat samples.
The present study was carried out for the
identification of cattle and buffalo meat from a
mixed meat sample using cyt b gene variability by
Real Time PCR. In Real Time PCR, the common
forward primer with cattle specific reverse primer
showed melting peak 76.2oC on cattle DNA while
Keywords: cattle, buffalo, meat speciation,
cytochrome b gene, duplex Real Time PCR
the common forward primer with buffalo specific
reverse primers showed melting peak at 78.2oC on
buffalo DNA. Even in duplex PCR it showed only
species specific melting peaks in respective species
DNA. But when duplex PCR was evaluated on
cattle- buffalo mixed DNA template in equal
proportion it exhibited two peaks, a major buffalo
specific and a minor cattle specific, merging into one
broader peak at 78.2oC. However it was possible to
know presence of mixed DNA by Real Time PCR
duplex primers. The duplex Real Time PCR showed
only a single broader peak at 78.2oC at 1: 10 and all
further ratios. Hence all independent cattle specific
Real Time PCR was run on mixed DNA which
produced cattle specific melting peak at 76.2oC upto
1:1000 dilutions. Thus, it was possible to detect and
differentiate cattle meat mixed in buffalo meat upto
1:1000 fraction i.e. 9 pg of cattle DNA adulterated
in buffalo DNA by running a duplex PCR followed
by cattle specific Real Time PCR. Duplex Real
Time PCR did not produce any amplification and
INTRODUCTION
The determination of food authenticity
and the detection of adulteration are major issues
in the food industry which attract immense
attention. Species identification of animal products
especially meat (Ahmed et al., 2007) has always
been in demand. The prime concern remains with
adulteration of cattle meat with the buffalo meat
in countries like Australia, America, and India
because of health and religious reasons. The buffalo
meat is very similar to beef but it is considered a
healthier alternative because buffalo meat contains
less fat 1.8%, lowest cholesterol level of all
domestic meats -46 mg per 100 grams and more
protein than beef. It contains significant amounts of
omega-3 polyunsaturated fats, which are believed
to be protective against heart disease and other
inflammatory disorders.all this makes buffalo
meat consumers choice mainly because of health
1
Animal Biotechnology, ICAR Research Complex for Eastern Region (ICAR- RCER), Patna, P.O.- BVCC.,
Patna, India, *E-mail: [email protected]
2
Animal Biotechnology, Anand Veterinary College, Anand, Gujarat, India
124
reasons. Buffalo meat fat is white and buffalo meat
is always darker in color than beef because of more
pigmentation or less intramuscular fat. So, both
meats can be differentiated easily based upon gross
appearance. But, differentiation of cooked meat is
a challenge.
Hence, proper meat identification methods
are required especially for cattle and buffalo for
preventing the illegal practice.
Until now, a vast array of techniques has
been developed for this purpose, each beset with
its own limitations. These can be broadly divided
into protein-based methods and nucleic acid based
techniques.
Nucleic acid based techniques; popularly
known as molecular techniques involve the DNA
analysis. Analysis of DNA, rather than protein has
been exploited for species identification due to its
stability at high temperatures and its conserved
structure within all tissues of an individual. DNA
based techniques have been further simplified and
benefited from introduction of PCR.
Number of strategies has been employed in
PCR including use of repitive sequences, multigene
family and use of cytochrome b gene (Fairbrother
et al., 1998; Matsunaga et al., 1999; Girish et al.,
2005; Haunshi et al., 2009). However, the method
was not able to differentiate cattle and buffalo meats.
Only a few studies have addressed differentiation
of cattle meat and buffalo meat (Rajapaksha
et al., 2003; Rastogi et al., 2007; Gupta et al.,
2011). But the detection and assessment of cattle
meat adulteration in buffalo meat still remains a
challenge. All these studies based on traditional
PCR based methods are very sensitive but they
suffer from some limitations. The methods require
Time PCR, the technique is simplified. Real-Time
PCR does not only depict amplification in real time
(on line or live), but is also a quantitative one. Real
Time PCR detects PCR products using fluorescent
probes or a DNA binding dye, such as SYBR
Green. Real-time PCR assays can be automated
and are sensitive and rapid. They can quantify
PCR products with greater reproducibility while
eliminating the need for post-PCR processing, thus
preventing carryover contamination (Jothikumard
et al., 2002).
The present study focused on identification
of cattle and buffalo meat from a mixed meat
sample using cyt b gene variability by Real Time
PCR. Further the study also targeted the assessment
of adulteration of cattle meat in buffalo meat.
DNA extraction from muscle samples
Meat samples (twenty each) from cattle,
buffalo, sheep, goat and chicken were procured
from slaughter house /market or obtained through
biopsy and were processed immediately or stored
frozen at -40oC.
DNA from meat samples was extracted as
per the standard protocol described by Ausubel et
al., (1987) with some modifications.
Muscle tissue (0.25 g) was taken and ground
thoroughly with the frequent additions of liquid
nitrogen. The tissue homogenate was transferred
into a 15 ml sterile tube and mixed with 0.5 ml Lysis
buffer - ST (50 mM Tris-HCl, 10 MmM EDTA,
100 mM NaCl) along with 20 mg /ml proteinase
K and SDS (10%) to make final concentration
to 2%. The homogenate was incubated for
12-16 h or overnight at 55oC. The incubated lysate
post-PCR product separation by gel electrophoresis
which is time-consuming and increases the chances
of carryover contamination. With the advent of Real
was transferred to an autoclaved 15 ml tube and
125
duplex Real Time PCR.
equal volume 0.5 ml of Tris saturated phenol
(pH-8.0) was added and mixed gently for 10
minutes. The lysate was then centrifuged for 10
minutes at 10,000 rpm at 15oC. The supernatant
was collected into the 2 ml tube and added half
the volume of Tris saturated phenol : chloroform:
isoamyl alcohol (25:24:1) and mixed gently for 10
minutes and centrifuged for 10 minutes at 10.000
rpm at 15oC. Again, the supernatant was collected
into 2 ml centrifuge tube and equal volume of
chloroform: isoamylalcohol (24:1) was added and
mixed gently for 10 minutes and centrifuged for
10 minutes at 10,000 rpm at 15oC. The supernatant
was collected into a fresh 2 ml centrifuge tube and
1/10th volume of 3 M sodium acetate (pH 5.5) and
DNA amplification was detected by use
of the Real Time sequence detection system 7500
from Applied Biosystems.
On the completion of duplex Real Time
PCR, characteristic peaks were observed for cattle
and buffalo species. Peak was observed at the
melting temperatures of 76.2oC and 78.2oC in case
of cattle and buffalo DNA respectively (Graph 1).
Targeted amplification on Real Time PCR
was confirmed by agar gel electrophoresis of PCR
products. PCR products 5 μl were mixed with 1 μl
gel loading dye solution and loaded in a 2% agarose
gel containing 1% ethidium bromide 5 μl/ 100 ml
in tris- borate EDTA (TBE) buffer. Electrophoretic
separation of DNA fragments was done at 100 V
for 60 minutes.
A characteristic band pattern was obtained
for cattle and buffalo species in duplex PCR.
The PCR products showed species specific
DNA fragments of 113 and 152 bps from cattle and
buffalo respectively.
By using the same set of primers detection
of other species viz., sheep, goat and poultry
meat DNA was also attempted. No amplification
occurred in other species, and so no band pattern
was obtained for goat, sheep and chicken species
(Graph 2 and 3) eliminating the chances of cross
amplification.
Further, Real Time PCR was used to assess
the level of adulteration of cattle meat in buffalo
meat. DNA samples of cattle and buffalo meat
were mixed in ratios of 1:1, 1:10, 1:100, 1:1000,
and 1:10000 respectively. On melt curve analysis
of meat sample containing cattle and buffalo meats
equal volume of isopropyl alcohol was added. The
DNA was precipitated by slowly swirling the tube.
Precipitated DNA was washed thrice with 70%
ethanol to remove excess salt and air dried and
dissolved in 200 μl volume of 0.3 x TE. The quality
and quantity of extracted DNA was checked by
nanodrop and by running on 0.8% agarose gel.
Gene amplification by Real Time PCR
Amplification was performed in 25 μl
reaction volume containing 12.5 μl QuantiTect TM
SYBR Green PCR master mix (2X), 2 μl primer
mix (0.4- 10 pm each), 3 μl (90 ng template DNA)
and 7.5μl QuantiTect DNAse Free water. Speciesspecific oligonucleotide primers reported by (Rea
et al., 2001 ) consisting of common forward primer
CONP- F-2 (5’- CTT CTT ATT CGC ATA CGC
AAT CTT ACG ATC- 3’) and reverse primers, cattle
primer BOVP- R- 2 (5’- TGG AGG TGT GTA GTA
GGG GGA TTA GAG CA- 3’) and buffalo primerBUFP- R-2 (5’- GGC ATT GGC TGA ATG GCC
GGA ACA TCA TA-3’) were used. These primers
were mixed in ratio of 1: 1: 0.4 for CONP- F-2:
BOVP- R-2: BUFP- R-2 and used together for the
126
developed to quantify bovine contamination in
buffalo products.
A quantification procedure is proposed and
involves amplifying a sample with both primer
sets and then normalizing the total DNA using the
total non-normalized bovine and buffalo DNA. To
correct for the potential deviations between the real
and measured DNA quantity caused by biological
differences between species, the use of calibration
curves generated from each analyzed matrix is
proposed. This method is yet to be checked on
meat derived products.
The present study based on Real-Time
chemistries allow for the detection of PCR
amplification during the early phases of the
reaction. Real Time PCR overcomes the limitations
of end point PCR. Many times meat samples
brought to the laboratory are cooked, putrified or
semiputrified in nature. So, Real Time PCR was
used for these samples and was found very sensitive
and successfully amplified small fragment of
cytochrome b gene from these cooked and putrified
meat samples. The use of mt DNA in this study
further increases the chances of achieving a positive
result even in the case of samples suffering severe
DNA fragmentation due to intense processing
conditions (Bellagamba et al., 2001).
Detection
of
meat
adulteration
simultaneously creates the need of the assessment
of adulteration level. Andreo et al., (2006) assessed
the level of adulteration in a series of DNA
mixtures, containing (in percentage) 1/99, 5/95,
10/ 90, 40/60, 50/50, 60/40, 90/10, 95/5, and 99/1
ratios of cattle/ horse, cattle/wallaroo, pork/horse,
and pork/wallaroo by using duplex Real Time PCR.
The smaller percentage allowing identification of
the peaks in double-species duplex reactions were
established as follows: 5% (cattle or wallaroo) in
cattle/ wallaroo mixtures, 5% pork and 1% horse in
mixed in the ratio of 1:1, cattle (76.2oC) and buffalo
(78.2oC) specific peaks were found merged to give
a broader peak (Graph 4). But on all further ratios
(i.e. 1:10 to 1:1000) cattle specific amplicon peaked
much lower and hence shadowed under major
buffalo specific peak at 78.2oC (Graph 5). This posed
a limit in detecting mixed meats beyond 1:10 ratios.
To overcome this problem, another Real-Time PCR
using only cattle specific reverse was required to
amplify cattle genome in these ratios. The cattle
specific Real-Time PCR successfully amplified
cattle sequences at all the ratios except 1:10,000
in quantitative manner (Graph 6). At 1:10,000
ratio only buffalo specific peak was obtained when
duplex Real-Time PCR was attempted (Graph 7).
To verify presence of cattle specific template, the
mixed template was amplified with cattle specific
primers. However, this yielded no amplification.
This confirmed that both of the species continued to
give amplification up to 1:1000 ratios of admixture.
However, at 1:10000 ratios, only buffalo species
gave amplification. Thus, duplex Real Time PCR
was found successful in detecting upto 1:1000 ratio
of cattle into buffalo DNA admixture. In absolute
quantity this comes to detection of 9 pg of cattle
DNA adulterated in buffalo DNA. The duplex Real
Time PCR could detect upto 1:10000 ratio of DNA
admixture i.e. up to 9 pg of cattle DNA adulterated
in buffalo DNA. Thus, it was possible to detect and
differentiate cattle meat mixed in buffalo meat upto
1: 1000 fraction i.e. 9 pg of cattle DNA adulterated
in buffalo DNA by running a duplex PCR followed
by cattle specific Real Time PCR. Similar results
were obtained by (Rea et al., 2001 ; Gupta et al.,
2011) but the study was carried out on milk samples
and meat samples respectively which was based
on duplex PCR and required post PCR processing
and was not quantitative. Recently, (Drummond et
al., 2013) Real Time PCR based method has been
127
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CONCLUSION
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This research was supported by the
Department of Animal Biotechnology, Department
of Animal Genetics and Breeding and Department
of Surgery, College of Veterinary Science and
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Anand, Gujarat.
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129
Original Article
THE USE OF TROPICAL OF MULTIPROPOSES TREES AS A FEED SUPPLEMENT
TO THAI SWAMP BUFFALOES (BUBALUS BUBALIS)
RECIVING A BASAL DIET OF PANGOLA HAY
Thongsuk Jetana1,*, Sunworn Usawang1 and Sunpetch Sophon2
supplementary diet was more worthy of improving
feeding systems than the use single high proportions
of TMPTs containing readily soluble carbohydrates
for buffaloes due to each tropical multipurpose
trees have its own limitation for using as a feed in
particularly when gives it to animals.
ABSTRACT
The effects of tropical multipurpose trees
(TMPTs) on digestibility of nutrients, nitrogen
(N) balance, and ruminal microbial production,
rates of passage and blood metabolites in four
swamp buffaloes were studied. Animals were fed
with pangola hay as a basal diet and one of the
four TMPTs supplements: i) urea+cassava meal
(UCSM), ii) sun-dried leucaena leaves (SDLL), iii)
SDLL+Sun-dried pod of rain tree (SRTP) [LLRT]
and iv) SRTP. Dry matter (DM) and organic matter
(OM) digestibility in buffaloes supplemented with
UCSM and SRTP respectively, increased higher
(P<0.05) than in buffaloes fed other supplements,
but NDF digestion decreased (P<0.05). N
digestibility in animals improved (P<0.05) when
supplemented UCSM. There was no difference PD
excretion in the urine and affects the passage rate
parameters of buffaloes fed different supplements.
Plasma urea-N and glucose were higher (P<0.05) in
animals fed SRTP supplements than in animals fed
other supplements. None of supplement affected
plasma none-esterified fatty acids (NEFA) and
beta- hydroxy butyrate (β-HBA).
The study demonstrated the use low
proportions of TMPTs containing readily soluble
carbohydrates (starch or sugar) in combination of
Keywords: cassava meal, leucaena, pangola hay,
rain tree pod, swamp buffalo
INTRODUCTION
The use of tropical multiple purpose
trees as supplements (TMPTs) is a suitable and
worthwhile method to improve the quality of
livestock feeding systems (Jetana et al., 2010;
Jetana et al., 2011), in addition it saves costs of
other expensive ingredients (e.g., maize, soybean,
and molasses and fish meal). Cassava (Manihot
esculenta, Crantz) is vastly planted in tropical
countries, particularly in Thailand. Cassava meal
contains high level of readily soluble carbohydrates,
but low N content and is highly degradable in the
rumen comparing with other energy sources. Urea
therefore is a highly rumen degradable non proteinnitrogen, always used as an N source when cassava
meal is added in ruminant feed. At the present,
Faculty of Veterinary Science, Chulalongkorn University, Henri Dunant street, Phathumwan, Bangkok,
Thailand, *E-mail: [email protected]
2
Faculty of Veterinary Medicine, Mahanakorn University, Chueamsamphan Road, Khrtumrai, Khet Nong
Chok, Bangkok, Thailand
1
130
the concentrates are expensive, therefore the use
of local protein-rich legume or available multiple
purpose trees are possible methods, not only saving
cost of feeds, in particularly concentrates, but also
being suitable to smallholder farmers and the large
scale farms. One of the most widely used legumes is
leucaena (Leucaena leucocephala). The appropriate
proportions of leucaena can be used only 25%
as in feed for buffaloes (Jetana et al., 2012a),
however leucaena leaves might be fully used as a
protein supplement in ruminants when animals are
inoculating with Synergistes Jonesii (Palmer et al.,
2010). The rain tree (Samanea Saman), is a tropical
legume, with pods of rain tree easily to be found in
dry season, it is shown that these have been used
as an animal feed (Staples and Elevitch, 2006 and
Jetana et al., 2008). Studies demonstrated that the
high sugar and protein content in the rain tree pod
has the advantage of increasing the efficiency of
microbial growth in the rumen of buffaloes (Jetana
et al., 2011), cattle (Jetana et al., 2010) and goats
(Jetana et al., 2012b).
The present study therefore, was
undertaken to determine which of these tropical
multiples proposes trees (TMPTs) supplements
containing similar metabolizable energy (ME) are
suitable to be fed as a protein supplement to Thai
swamp buffalo fed a basal diet of pangola hay. The
objectives of the experiments were to determine
and compare the effects of supplementation with
either three types of TMPTs; leucaena leaves,
leucaena leaves plus RTP and RTP or urea plus
cassava meal on whole tract apparent digestibility
of DM, OM and fibre, rates of passage, N balance,
ruminal microbial production and blood metabolite
values.
The experiment was conducted using four
male swamp buffaloes (mean initial 274±1.24 kg,
18-24 month). The animals were daily fed pangola
hay as a based diet on 1.0% of body weight and
supplemented 1.0% of body weight with one
of the four combination dietary supplements:
(i). urea+cassava meal (UCSM); (ii). Sun-dried
leucaena leaves (SDLL); (iii). Sun-dried leucaena
leaves rain tree pods (LLRT) and (iv). Sun-dried
rain tree pod (SRTP). Each supplement was
formulated to provide similar the proportions of
N and metabolizable energy (ME). Sun-dried
leucaena and pods of rain tree were purchased from
the farmer surrounding the Kasatsert University,
Kampengseang campus, Narkorn Pathom and the
cassava meal and other ingredients were purchased
from the HungHong Company, Narkorn pathom.
After purchasing the pods of rain tree were firstly
ground through a 14-mm grinding plate prior to
grinding twice passing through an 8-mm grinding
plate using ≥5-Horsepower electrical meat grinder.
The ground rain tree pods were drying by the Sun
for 12-18 h and keeping in air-tight storages before
using them. Whilst pangola hay was purchased from
Animal Nutrition Cooperation, Chainat Province.
The animals were fed equally amounts
twice daily, 07.00 and 17.00 h. with four dietary
supplements and pangola hay. Each buffalo was
daily fed with 3.0 kg supplements; one of four dietary
supplements and 3.0 kg of pangola hay. Total feed
allocations were 6 kg fed basis (3 kg pangola hay
+ 3 kg supplement)/animal (ani)/d. Four animals
were used over four cycles in a double 4×4 Latin
square with four dietary supplements (Table 1). In
each period, the animals were kept in individual
pens and fed with the experimental diets. On the
first day and the last day (d 20) of each experiment
131
Cr analysis.
Blood samples from the jugular vein were
collected at 0 h and at 3, 6 and 9 h after morning
feed (d 15, 06:00h). Blood samples were drawn
using a 2-way blood collection needle (Vacuette®
AUSTRIA, Model 18 G × 1½) and transferred into
two heparinised vacutainers (9 ml/tube) and one
tube containing sodium fluoride and potassium
oxalate (for non-esterified fatty acids analyses).
The tubes were gently inverted a couple of times
and immediately centrifuged at 3500 × g for 25
min. Individual plasma was stored in tubes (3-3.5
ml/tube) at -20oC for further analysis.
The DM content of the feed and faecal
samples was determined by drying to a constant
weight in an oven at 105oC for 48 h. The ash in
period, the animals were weighed before the
morning feed (06.00 h). The study was conducted
in 4 periods; each period lasted for 20 days, where
10 days were for dietary adaptation and 10 days
for sample collection. The animals were housed in
individual pens during the adaptation period, but
were transferred to individual metabolic cages 2
days prior to urine and faeces collection.
Whole tract in vivo digestibility was
determined by collecting all faeces from day 1115 of the sample collection period. Sub-samples
of the daily offered feed and faecal samples were
collected and stored at -20ºC. Total daily feces were
weighed and samples (10%) were then stored at 4
ºC for further chemical analysis. There was no feed
refusal for each animal and fresh drinking water
was supplied throughout the experiments. At the
end of each sampling period, samples from each
animal was bulked, and then dried in a hot air oven
at 65 ºC for 48 h., prior to analysis for dry matter
(DM), ash, nitrogen (N) and neutral detergent
fibre (NDF). Urine was collected in plastic bag
containing 200 ml of 20% H2SO4 to maintain a pH
below 3. Total daily urine was weighed and subsamples were taken, diluted 5times with distilled
water, and stored at 4ºC for purine derivatives (PD)
analysis.
Chromium (Cr) mordanted fiber was
prepared from pangola hay. The pangola hay
was ground through a 2-mm sieve following the
method that was described by Uden et al. (1980).
On day 16 each animal was dosed with 40 g of Cr
III-mordanted (Cr = 560 mg) fiber by mixing it
with 1.5 kg supplements fed in the morning, before
the pangola hay were offered. Grabs samples of
faeces (200-300 g) were collected at 0, 3, 6, 9, 16,
24, 32, 40, 48, 56, 63, 72, 79, 87, 96, 104, 112 and
120 h., after the marker administration. Grab faecal
samples were kept at –20oC prior to ash DM and
the feed and the faecal samples was determined by
combustion in a muffle furnace at 550oC for 8 h.
The N content in the feed, the urine and the faecal
samples was determined by the micro Kjeldahl
method (AOAC, 2000 method no. 995.04).
NDF was analysed according to Van Soest et al.
(1991; structure A). The content of total phenolic
compounds and condensed tannins in samples
were assayed by the procedures of Makkar (2000).
Total non-structural carbohydrates in the feed
were determined according to Nelson’s reducing
sugar procedure (Hodge and Hofreiter, 1962).
Matabolisable energy for ruminants (ME) were
calculated according to Menke et al. (1979).
Allantoin and uric acid were determined
by the method described by IAEA-TECDOC-945
(1997). Plasma urea nitrogen (PUN) was measured
by using a commercial kit (Urea liquiUV, Human
GMbH-D 65205 Wiesbaden, Germany). Plasma
glucose concentrations were measured by using
a commercial kit (Glucose liquicolor, Human
GMbH-D 65205 Wiesbaden, Germany). Plasma
non-esterified fatty acid (NEFA) concentration
132
was analyzed using a commercial diagnostic kit
(No. 279-75401, Wako Pure Chemical Ind. Osaka,
Japan). Plasma β-HBA was analyzed using a
commercial diagnostic kit (Randox Laboratories
Ltd. Ardmore, Diamond Road, Crumlin, Co. Antrim,
UK, BT29 4QY). Plasma insulin concentrations
were determined by the kit manufacturer (Coat-ACount®-Insulin, Diagnostic Products Corporation,
Los Angeles, CA, USA).
Chromium (Cr) concentrations in the grab
faecal samples were determined by the method
described by Le Du and Penning (1982). Grab faecal
samples were dried in an oven at 65ºC for 72 h and
105ºC for 48 h, then ashed in a muffle furnace at
450ºC, for 8 h. The ashes were then, digested with
acid mixture and diluted before being analyzing Cr
in an Atomic Absorption Spectrophotometer.
The means of each parameter measured in
this study were analysed by Analysis of Variance
(ANOVA) using the procedures of the Statistical
Analysis System Institute (SAS, 1998). The
differences between means were compared by a
least significant difference method (LSD).
et al. (1979).
The DM content of CSM, SDL and RTP
were similar (890-913 g/kg as fed-basis) and
the N contents of SDL and RTP diets were quite
similar (39.4-39.6 g/kg DM), but they were higher
than CSM (5.06 g/kg DM). Including, the content
of condensed tannins varied widely among the
different TMPTs, ranging from 4.49, 18.4, 13.5 and
4.45 g/kg DM in CSM, SDL, RTP and pangola hay,
respectively. Similarly, the total sugar content also
varied between SDL and RTP, being from 66.0 and
200 g/kg DM, respectively. However, the ME of
CSM, SDL and RTP were the same value 10.0 MJ/
kg DM in among main materials.
The ingredients (g/kg DM) and chemical
composition of the supplements are presented in
Table 3. Three different TMPTs mixed with other
ingredients were used as protein supplements. The
DM content of all supplements was similar (917920 g/kg as fed-basis) and the N contents of UCSM,
SDLL, LLRT and SRTP supplemental diets were
quite similar (19.1-19.8 g/kg DM). However, the
content of condensed tannins varied widely among
the different TMPTs, ranging from 0, 9.15, 7.92 and
6.68 g/kg DM in UCSM, SDLL, LLRT and SRTP,
respectively. Similarly, total sugar content also
varied among the different types of LPT, ranging
from 0, 32.9, 65.4 and 98.9 g/kg DM in UCSM,
SDLL, LLRT and SRTP, respectively. However,
the ME of UCSM, SDLL, LLRT and SRTP were
quite similar, within 9.16-9.20 MJ/kg DM.
Chemical composition in ingredients of diets
and supplements
The ingredients (g/kg DM) of the feeds
are presented in Table 2. The pangola hay was
used as the basal diet and three different TMPTs;
cassava meal, Sun-dried leaves, and Sun-dried rain
tree pod were used as a main ingredient of protein
supplements. The pangola hay contained 922 g DM/
kg as fed-basis, it contained (g/kg DM) 14.0 g N, 706
g NDF and 7.92 mega joules (MJ) of matabolisable
energy for ruminants ME. Matabolizable energy
for ruminants were calculated according to Menke
Nutrients digestibility
The DM and OM digestions increased
(P<0.05) in swamp buffaloes fed UCSM and SRTP
supplemental diets, but the NDF digestion decreased
(P<0.05) when compared with buffaloes fed SDLL
and LLRT supplemental diets. This was according to
the supply of readily soluble carbohydrates, starch
133
from UCSM and sugar from SRTP in the rumen.
However, OM digestion was higher in animals
fed UCSM than in animals fed STRP. It may be
the proportions of starch were higher (P<0.05)
in UCSM than the proportions of sugar in SRTP;
therefore OM digestion was greater (P<0.05) in
buffaloes fed UCSM than in buffaloes fed SRTP.
As a result of rapid fermentation of readily soluble
carbohydrates was the pH reduction in the rumen,
the decreased pH in the rumen has sequent a main
impact on fiber digestion. In agreement with Jetana
et al. (1998), who reported the depression in fiber
digestion due to readily soluble carbohydrates have
always been associated with rapid fermentation of
readily soluble carbohydrates and the subsequent
depression of ruminal pH when sheep fed guinea
grass and supplemented with corn flour. While NDF
digestion decreased (P<0.05) in buffaloes fed SRTP
supplement, in similarly to the earlier reported that
fibre digestion depressed in buffaloes when fed
rice straw and supplemented with oven-dried rain
tree pods (Jetana et al., 2010). The low pH in the
rumen affected to ruminal microbial activities, in
particularly cellulolytic bacteria (Stewart, 1977),
therefore depressed fibre digestion reported by
Cheng et al. (1984), who indicated low pH in the
rumen prevented strong attachment of bacteria to
plant cell wall. Besides of the low pH in the rumen,
the longer lag time may be explained that the
utilization of readily soluble carbohydrates before
the degradation of fibre by rumen micro-organisms
(Mertens, 1977).
This was in agreement with Jetana et al. (2009b),
they indicated there were no differences for k1, k2
and TMRT in swamp buffaloes fed diets containing
different the proportions between pineapple waste
silage and concentrates.
In the present study, there was no
significance different in k1 of the animals fed the
different sources of TMTPs. This could be due to
the particle size of TMTPs and rate of degradation
in the rumen, which may be similar to all of the
supplemental diets. However, the estimated for k1
in the present study 3.23-3.58 % h-1 were lower
than the ranges of 7.70-11.3 % h-1 and 6.05-7.96 %
h-1 in swamp buffaloes fed rice straw (Abdullah et
al., 1990) and pineapple waste silage (Jetana et al.,
2009b) as the basal diets, respectively.
The passage rate (k2) of the marker solid
particles in caecum-proximal colon (second
compartment), which can be considered the
digestive tract, the abomasum, in which the blend
of digested feed take place showed that values
were similar (6.55-7.72 % h-1) to all buffaloes fed
different supplemental diets. These values were
faster than in the range k2 of 2.90-4.10 % h-1
(Abdullah et al., 1990) and 4.04-4.21 % h-1 (Jetana
et al., 2009).
The time between the administration of the
marker of solids and its first appearance in the faces
(transit time, TT) did not result to be significantly
difference in swamp buffaloes when fed different
TMTPs in supplemental diets. Therefore, these
implied that the different sources of TMTPs in
supplemental diets in swamp buffaloes did not
affect to the values of TT (1.29-1.57 h) and TMRT
(44.1-50.1h).
Rates of passages
In present study, the reticulo-rumen passage
rate in the first compartment (k1) of the Cr marker
of the solid particles in the whole gastrointestinal
tract evaluated by the multi-compartment model
was not significantly different by treatment diets.
N balance and N digestiblity
The urinary-N excretion was higher
134
given a diet containing a high proportions of readily
soluble carbohydrate (sucrose).
(P<0.05) in animals supplemented with UCSM
and SDLL than in animals supplemented with
LLRT. This demonstrated that the over amount of
ammonia-N in the rumen was absorbed passing
rumen wall to blood circulation, but due to ammonia
toxicity, thus ammonia would be converting to urea
at the liver, then was readily cleared from renal,
indicating that excess fermentable N sources always
loss through the urine (Nolan, 1993). Whislt the
urinary-N excretion decreased (P<0.05), but the
faecal-N excretion increased (P<0.05), indicating
the proteins may be attached with tannins before it
appeared in the fecaes when animals supplemented
with LLRT. This reflected that those N sources
in the diets were excreted more via the faeces or
hind gut fermentation. In the present study, none of
supplements affected to N balance in buffaloes, this
may imply that animals fed all supplemental diets
showed similar synchrony between carbohydrate
and N compounds in the rumen, due to the fact
that the amount of CP (578-583 g/ani d-1) and ME
intakes (47.1-47.3 MJ/ani d-1) were not different
among diets.
The N digestibilty improved (P<0.05)
in swamp buffaloes supplemented with UCSM,
as a result of urea is an effective degradation
to ammonia-N in the rumen, and some rapidly
degraded, were absorbed passing through the rumen
wall and easily were loss by excreting into the urine.
The effect of SRTP also improved N digestibilty
in swamp buffaloes as a result of rain tree pod
is a readily soluble carbohydrate, the increase in
apparent ruminal N digestibility due to sugar is
effective for capturing ammonia-N and N in the
rumen, therefore, increased hind gut fermentation
(high faecal-N output). This observation was in
agreement with Howard et al. (2007) and Owens
et al. (2008), who reported that generally the
digestibility of N increased when the animal was
Urinary purine derivatives excretion
Excretion of PDs in the urine were no
differences in swamp buffaloes fed the different
supplemental diets. This was in contrast with Obara
et al. (1994), Chamberlain et al. (1993) and Khalili
and Huhtanen (1991), who found PDs excretion
and microbial protein synthesis increased in sheep
and cattle supplementation with sugar (sucrose).
On the other hand, this was also contrast with the
observations of Hall and Herejk (2001) and Hoover
et al. (2006), they showed that the starch resulted in
more microbial growth and microbial N production
than sugar did. In present study, daily PDs excretion
in the urine ranged from 0.64-0.80 μmol d-1/
BW0.75 in swamp buffaloes. This value was in the
range of 0.54-1.76 mmold-1/BW0.75 that reported
by Liang et al. (1999) but higher than the range
of 0.19-0.24 mmol d-1/BW0.75 reported by Jetana
et al. (2009b, 2011). The PDs excretion rates in
swamp buffaloes (13.8-15.6 mmol PD/kg DOMI)
were not different. The values for swamp buffaloes
in the present study were about 2.5 times higher
than those (4.6-6.34 mmol PD/kg DOMI) reported
by Jetana et al. (2009b) and Chen et al. (1996). The
lower excretion rate of PD per DOMI in swamp
buffaloes as compared to other studies, which was
recorded in the present study was in agreement
with Vercoe (1976), Liang et al. (1994) and Jetana
et al. (2009b) who earlier reported the lower in the
rate of PD excretion in swamp buffaloes.
The measurement of PDs excretion in
the urine is a simple method which always uses
for estimating microbial protein production from
the rumen. Therefore, the results of calculated
microbial supply can be expressed as a g microbial
N per kg digestible organic matter in the rumen
135
(DOMR) (Chen and Gomez, 1995). However, this
technique was not suitable to use for estimating
ruminal microbial production in swamp buffaloes,
surprisingly, it is different from others species such
as sheep, goats and cattle. Due to rate excretion
of PDs in the urine was always low in buffaloes
differing from others animal species. It was
possible that its directly related to the lower of renal
clearance rate of plasma PD into the urine (Jetana et
al., 2006) and the lower glomerular filtraation rates
(GFR) in buffaloes than the other animal species
(Norton et al., 1979). In addition to the differences
in enzyme activities involved in the degradation and
utilisation of purine in swamp buffalo, therefore
xanthine and hypoxanthine in the urine were not
determined in swamp buffaloes (Chen and Ørskov,
2003). On the other hand, swamp buffaloes
might have a greater ability to recycle PDs in the
blood and other tissues that degrade uric acid and
allantoin into other metabolites prior to 1) excreting
their metabolites in the urine, or 2) recycling as N
sources for supplying in the rumen, therefore this
is possible that level of NH3 in the rumen is always
high, even buffoles were fed with low quality diets,
they can be survine when compared with other
animal species (Kennedy, 1990).
blood systems. However, none of the supplements
affected to plasma NEFA, indicating there was no
any difference from the utilization of energy and
the mobilization of reserve energy in all animals
fed different supplements. It is probably due to
i) the total ME intakes equaled among diets as
mentioned above and ii) the total ME intakes did
not only meet ME requirements of buffaloes in
developing countries, but also higher than buffaloes
requirement (Kearl, 1982). The concentrations of
β-HBA in plasma showed the same level in swamp
buffaloes when the animals were fed with different
supplemental diets, this may be explained the
proportions of butyrate in the rumen did not differ.
Subsequently, butyrate in the rumen from rumen
fermentation was similar converted to β-HBA
in rumen epithelium, before passing into blood
systems. Plasma insulin concentration tended to
be lower (P<0.1) in animals fed supplemented
diet containing UCSM. This may also explain the
better capacity for protein/fat reserve mobilization
for meat synthesis in animals supplemented with
UCSM.
The high starch but low N containing
in cassava meal are the main factors affecting
digestibility when this material is used as a
feedstuff in ruminants, urea thus is required to
fulfill N sources. At the present, both cassava meal
and urea were rather expensive, as a consequence
of cassava meal being not only use as a main raw
material in starch industry, but also being use as a
raw material in producing bio-fuels and urea being
use as a fertilizer. Therefore, enhancing the cost
of feed as well, it might be substitution of chicken
dung or urine for urea so that saving cost, but N
contents in chicken dung and urine are variable
and also required time of process, improvement
of techniques and cost of chicken dung. Thus,
the use of cassava meal and urea as a feed for
Blood metabolites
Plasma urea-N was higher (P<0.05) in
buffaloes fed SRTP supplements than in buffaloes
fed other supplements. It may be that supply of
sugar from RTP in the rumen resulted in increased
proteolysis in the rumen; therefore some urea-N
loss was higher by absorption through the rumen
wall to the blood system. Plasma glucose was
higher (P<0.05) in buffaloes fed SRTP supplements
than in buffaloes fed other supplements. Due to the
supply of sugar containing SRTP in the resulting
in higher absorption through rumen wall into the
136
Table 1. The experimental design with four different tropical multipurpose trees (TMTPs) supplements and
four animals of each species and body weight of each animal and period.
Periods
1
2
3
4
no. 1
UCSM
SRTP
LLRT
SDLL
Swamp buffaloes
no. 2
no. 3
SDLL
LLRT
UCSM
SDLL
SRTP
UCSM
LLRT
SRTP
no. 4
SRTP
LLRT
SDLL
UCSM
UCSM = urea plus cassava meal; SDLL = Sun-dried leucaena; LLRT= Sun-dried leucaena plus rain tree pod
and SRTP = Sun-dried rain tree pod
Table 2. Chemical composition in ingredients of diets.
Dry matter (DM)
Ash
Nitrogen (N)
Crude protein (N × 6.25)
Neutral Detergent fibre (NDF)
Phenolic compounds
Condensed tannins (CT)
Total starch1
Total sugar
Reducing sugar
Sucrose
Metabolizable energy
(MJ/kg DM)
CSM
890
47.8
5.06
31.6
165
0.0
4.49
696
0.0
0.0
0.0
Ingredients of diets (g/kg DM basis)
SDL
RTP
Pangola Hay
913
910
922
60.4
85.1
84.3
39.4
39.6
14.0
246
247
87.4
394
357
706
0.0
0.0
0.0
18.4
13.5
4.45
66.0
200
0.0
33.3
108
0.0
32.7
92.4
0.0
9.99
10.0
10.0
CSM = cassava meal; SDL = Sun-dried leucaena; RTP = Sun-dried rain tree pod
Determination of starch as the procedures described by Southgate (1976)
1
137
7.92
Table 3. Ingredients of dietary supplements (kg on as fed basis) and chemical composition (g/kg DM basis).
Supplements (kg as-fed basis)
UCSM
SDLL
LLRT
SRTP
Urea
30.0
Sun-dried leucaena
500.0
250.0
Sun-dried rain tree pod
250.0
500.0
Cassava meal
797.5
327.5
327.5
327.5
Corn meal
50.0
50.0
50.0
50.0
Soybean meal
50.0
50.0
50.0
50.0
Di calcium
30.0
30.0
30.0
30.0
Lime
20.0
20.0
20.0
20.0
Sulphur
2.5
2.5
2.5
2.5
Sea salt
15.0
15.0
15.0
15.0
Premixes
5.0
5.0
5.0
5.0
Chemical composition (g/kg DM basis)
Dry matter (DM)
917
918
919
920
Nitrogen (N)
19.1
19.5
19.7
19.8
Neutral Detergent fibre (NDF)
467
600
589
576
Phenolic compounds
11.3
117
97.4
77.7
Condensed tannins (CT)
0.0
9.15
7.92
6.68
Total starch
494
203
203
203
Total sugar
32.9
65.4
98.9
Reducing sugar
16.6
35.1
53.7
Sucrose
16.3
31.0
45.8
Metabolizable energy (MJ/kg DM)
9.16
9.18
9.19
9.20
Ingredients
Premixes contained (g/kg DM basis): vitamin A 40,000,000 units, vitamin D3 4,000,000 units, vitamin E
40,000 Unit, vitamin B12 0.02 g, Mn 160 g, Fe 240 g, Zn 100 g, Cu 20 g, Se 0.5 g, Co 2 g and I 5
g.
138
Table 4. Intakes and the coefficients of digestion, nitrogen balance, nitrogen utilization and daily urinary purine
derivative excretion, the ratios of purine derivatives to digestible nutrient intakes in Thai swamp
buffaloes fed pangola hay as a basal diet and supplemented with different tropical multipurpose
trees (TMPTs).
Diet supplementation
SEM1
UCSM
SDLL
LLRT
SRTP
Body weight2
291
290
289
289
7.91
Metabolic body weight (kg)
70.4
70.2
70.1
70.1
0.85
Dry matter (DM)
78.4
78.5
78.8
78.9
1.60
Organic matter (OM)
73.3
72.8
72.7
72.2
1.48
Neutral detergent fibre (NDF)
Intakes(g /BW 0.75d-1)
33.6
43.3
42.6
41.8
0.81
ME intakes3 (MJ/ani d-1)
47.1
47.1
47.3
47.3
0.96
CP intakes (g/ani d )
578
578
581
583
11.8
DM
0.71a4
0.61b
0.62b
0.67a
0.03
OM
0.74
a
0.65
0.66
c
0.70b
0.03
NDF
0.73
b
0.79
a
0.78
a
0.75
b
0.02
Passage rate from rumen (k1% h-1)
3.23
3.58
3.40
3.38
0.04
Passage rate through caecum and colon (k2% h-1)
6.58
7.43
6.55
7.72
0.04
Transit time (TT, h)
1.46
1.47
1.29
1.57
0.17
Total mean retention time (TMRT, h)
50.1
44.1
49.5
45.3
4.70
N intake
1.41
1.41
1.42
1.43
0.03
N in urine
0.22
0.19
0.17
0.16
ab
0.03
N in faeces
0.52b
0.64a
0.65a
0.60ab
0.08
3
-1
The coefficient of digestion
c
Passage rates
Nitrogen balance (g/BW
0.75
d )
-1
N-balance
Digestibility of N (g/kg)
a
a
b
0.68
0.58
0.62
0.67
0.08
632a
541b
541b
578a
6.51
594
551
598
488
87.1
PD in urine (μmol/BW0.75 d-1)
Allantoin
Uric acid
214
165
138
160
113
PD
808
716
737
647
123
Total PD/kg DDMI
14.1
13.8
15.3
13.9
3.30
Total PD/kg DOMI
14.5
13.9
15.6
14.4
3.34
Total PD (mol)/digestible nutrients
Standard error of mean 2Averaged throughout experiments, Nutrient requirements for domestic buffalo in
developing countries, body weight 300 kg maintenance required ME = 37.7 (MJ/d), CP = 377 (g/d) and gain
0.25 kg/d required ME = 49.2 (MJ/d), CP = 579 (g/d) [Kearl, 1982] 4 abcValues within the same column with
different superscripts are significantly (P<0.05) different. Values within the same column without different
superscripts are not significantly (P<0.05) different.
1
139
Table 5. Solid digesta flow kinetics in the gastrointestinal tract and plasma metabolite concentration of Thai
swamp buffaloes fed pangola hay as a basal diet and supplemented with different different tropical
multipurpose trees (TMPTs).
Body weight2
Metabolic body weight (kg)
In plasma
Urea-N (mg/dl)
Glucose (μmol/ml)
Non-esterified fatty acid (μmol/ml)
β-hydroxybutyrate (μmol/ml)
Insulin (μ IU/ml)
Diet supplementation
UCSM
SDLL
LLRT
SRTP
291
290
289
289
70.4
70.2
70.1
70.1
23.2b2
50.7b
81.5
306
6.33
23.6b
57.0b
71.9
263
7.38
31.7b
50.0b
65.9
332
7.70
50.2a
60.7a
78.0
334
7.09
SEM1
7.91
0.85
10.8
5.58
19.7
81.0
1.02
Standard error of mean 2Averaged throughout experiments, 3 abcValues within the same column with different
superscripts are significantly (P<0.05) different. Values within the same column without different superscripts
are not significantly (P<0.05) different.
1
animals must carefully consider when smallholder
farmers required to use them. Leucaena, providing
a good protein source is possibly more suitable
to farmers, in addition to the fact that this plant
grows easily and it can be used more than 25%
in diet when inoculating bacteria (Synergist
joneses). Whilst, the rain tree pod is another good
choice, it is only required to be ground by using
an electrical grinder, then drying by the Sun, prior
to keep for them long times. It is considered to
be a superiority product, providing both readily
soluble carbohydrate and protein sources but high
readily soluble carbohydrate content, it always
depresses fibre digestion. Thus, the further study
must investigate to find out appropriate proportions
of rain tree pods, anti-nutritional factors and toxic
substance compounds in rain tree pods, so that they
can be used as a feed in buffaloes for enhancing
fibre digestion and microbial yields in the rumen.
CONCLUSIONS
The DM and OM digestions improved in
swamp buffaloes when supplemented with cassava
meal or rain tree pods, but NDF digestion reduced
in animals supplemented with high proportions of
cassava meal or high proportions of rain tree pods
in diet, even the amount of sugar in SRTP was less
than that of starch in UCSM. The NDF digestion
increased in animals fed leucaena mixed with rain
tree pods. Plasma urea-N and glucose in animals
supplemented with SRTP was higher than in animals
supplemented with the other supplements. The
present study indicated feeding different TMPTs as
supplemental diets with high proportions of readily
140
soluble carbohydrates, in particularly, cassava meal
or rain tree pods for swamp buffaloes depressed
fibre digestions, but did not affect to, the rate of
passage, N-balance, the rate of PD excretion in
the urine and the mobilization of energy reserves.
The study demonstrated the use low proportions of
TMPTs containing readily soluble carbohydrates
(starch or sugar) in combination of supplementary
diet was more worthy of improving feeding
systems than the use single high proportions of
TMPTs containing readily soluble carbohydrates
for buffaloes due to each tropical multipurpose
trees have its own limitation for using as a feed in
particularly when gives it to animals.
17th
ed. AOAC, Washington, D.C.,
Association Official Agriculture Chemists.
Chamberlain, D.G., S. Robertson and J. Choung.
1993. Sugars versus starch as supplements to
grass silage: effects on ruminal fermentation
and the Supply of Microbial Protein to
Small Intestine, Estimated from the Urinary
Excretion of Purine Derivatives, in Sheep.
J. Sci. Food Agr., 63: 189-194.
Chen, X.B. and M.J. Gomez. 1995. Estimation
of Microbial Protein Supply to Sheep and
Cattle Based on Urinary Excretion of
Purine Derivatives- An Overview of The
Technical Details. Occasional Publication
1992, International Feed Resources Unit,
Rowette Research Institute, Aberdeen, UK.
Chen, X.B. and E.R. Ørskov. 2003. Research on
urinary excretion of purine derivatives
in ruminants: past, present and future, p.
180-210. In Makkar, H.P.S. and X.B. Chen
(eds.) Estimation of Microbial Protein
Supply in Ruminants Using Urinary Purine
Derivatives. Kluwer Academic Publishers.
Chen, X.B., L. Samaraweera, D.J. Kyle, E.R.
Ørskov and H. Abeygunawardene. 1996.
Urinary excretion of purine derivatives and
tissue xanthine oxidase (EC 1.2.3.2) activity
in buffaloes (Bubalis bubalis) with special
reference to differences between buffaloes
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Cheng, K.J., C.S. Stewart, D. Dinsdale and J.W.
Costerton. 1984. Eletron microscopy of
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ACKNOWLEDGEMENTS
The authors wish to thank to Professor Dr. S.
Chanpongsang, Department of Animal Husbandry,
Faculty of Veterinary Science, for providing
metabolic cages and facilities for the present
study. The funds provided by a Thai government
budget under the Project of increasing Efficiency
of Food and Agricultural Productivity by Nuclear
Technology (EFF01/49) are acknowledged.
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