Posters Posteri - Università di Bologna

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Posters
Posteri
XVI Congress of the Mediterranean Federation for Health and Production of Ruminants (FeMeSPrum), Zadar, Croatia, 2008
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XVI. kongres Mediteranske federacije za zdravlje i produktivnost preživača (FeMeSPrum), Zadar, Croatia, 2008
SPINAL CORD TISSUE DETECTION IN COMMINUTED BEEF IN A
PRIVATE ABATTOIR IN SOUTHERN TURKEY
DETEKCIJA TKIVA LEĐNE MOŽDINE U USITNJENOJ GOVEDINI IZ
PRIVATNE MESNICE U JUŽNOJ TURSKOJ
M. Kale a*, Ö. Kurşunb, A. S. Akcan Kalec, F. Pehlivanoğlud, S. Hasırcıoğlua, S. Yavrue
Department of Virology a, Food Hygiene and Technology b, Microbiology d, Faculty of Veterinary Medecine,
University of Mehmet Akif Ersoy, 15100, Burdur, TURKEY. c Ministry of Agriculture, State Control Laboratory, 15100, Burdur, TURKEY. Department of Virology e, Faculty of Veterinary Medecine, University of Selcuk,
42075, Konya, TURKEY. *Corresponding author. [email protected]
Abstract
In this study, the distribution of Central Nervous System (CNS) tissue contamination on the joints of beef
during cutting of 88 carcass halves into edible meat parts in a private slaughterhouse was investigated. The high
level contamination (>0.4) was detected at the highest ratio on the neck (75.0%), antre cote steak (62.5%) and
thick flank (50.0%) areas. The moderate level contamination (< 0.4) was detected at the highest ratio on the
contre-filet (87.5%), sirloin steak (50.0%) and silver side (50.0%) areas. The low level contamination (< 0.3)
was detected at the highest ratio on the rump (62.5%) and contre-nuar (75.0%) areas. Both the moderate and
the low level contamination were found at the similar and highest ratio on the topside (37.5%) areas. The high
level contamination (>0.4) was found at the highest ratio on the knives (62.5%), cutting tables (62.5%), moving
cutting tables (100.0%) and aprons (100.0%). In conclusion, by this study the first time in Turkey, the level
of contamination of spinal cord tissue as CNS tissue on the joints of beef during cutting the carcasses into the
parts was shown. Additionally, the high level contamination risk was found on the knives used for cutting the
carcasses, cutting tables, moving cutting tables and aprons.
Keywords: SRM; spinal cord; joints of beef; edible meat; ELISA
Sažetak:
U ovom istraživanju ispitana je rasprostranjenost tkiva središnjeg živčanog sustava kroz zagađenost
zglobova toplih polutki 88 goveda, koja su rasječena u konzumno meso u privatnoj klaonici. Visok stupanj
prisutnosti živčanog tkiva (>0.4 uočen je na vratu (75,0% antre cote odresku ( 62,5% i području flama ( 50,0%.
Umjereno prisustvo tkiva SŽS-a (<0.4 je uočeno kod contre-fileta ( 87.5%, i mjestu rasjeka ( 50,0% . nizak
stupanj prisutnosti SŽS tkivom uočen je na sapima ( 62,5% i kontranuar području (75,0% . I umjerena i niska
kontaminacija utvrđena su u najvećem omjeru na površinskim područjima ( 37,5% toplih polutki. Visok stupanj
kontaminacije ( >0,4 je uočen najviše na noževima (62.5% , stolovima za rasijecanje ( 62,5%, pokretnim
radnim površinama (100%. Ovim istraživanjem smo po prvi put u Turskoj pokazali kontaminaciju tkivom
leđne moždine (SŽS) u zglobovima prilikom rasijecanja goveda u konzumnu govedinu. Dodatno je uočen visok
stupanj rizičnosti kontaminacije na noževima, radnim plohama, pokretnim radbim plohama i pregačama.
Ključne riječi:Leđna moždina, goveđe meso, ELISA
INTRODUCTION
Contamination of beef with CNS tissue including spinal cord has been a concern from the food
safety standpoint because of potential human health implications in relation to bovine spongiform
encephalopathy (BSE). Introducing CNS tissue (i.e., brain and spinal cord materials) to the edible
portion of the carcass is suspected of increasing the risk of human infection with new variant
Creutzfeldt-Jakob disease (vCJD). In an effort to prevent CNS tissue from being introduced into the
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human food chain, the European Commission (EC) in 1997 endorsed a proposal to regulate the use
of specified risk materials (SRMs) such as brain and spinal cord presenting a BSE risk, and decided
that specified risk material (SRM) must not to be used in food effective October 1, 2000 (Hajmeer
et al., 2006).
The conventional slaughter practices at present primarily include the following critical processes:
captive bolt stunning, removal of head from the carcass and carcass splitting (Troeger, 2004). Current
slaughter methods may allow brain or spinal cord tissue to contaminate the carcass and enter the
food chain (Helps et al., 2002). Studies on the spreading of CNS tissue have been conducted by
many researchers (Helps et al., 2002; Hajmeer et al., 2003). These previous studies focused on that
the slaughter practice could disseminate spinal cord material over the carcass, the operator, and the
environment during the splitting process. But, current practices in the butchery of cattle have led to
concerns regarding the possible dispersion of CNS material to edible meat parts. Therefore, in the
present study, distribution of contamination of spinal cord tissue as CNS tissue on the joints of beef
during cutting the carcasses into the parts, the knives used for cutting the carcasses, cutting tables,
moving cutting tables and aprons were shown.
MATERIALS AND METHODS
Sample collection and preparation for analysis The joints of beef during cutting of eighty
eight carcass halves into edible meat parts were used from a commercial beef abattoir in Burdur,
Southern Turkey. Samples were collected from edible meat parts of cattle carcass halves slaughtered
older than 30 months during twelve visits between August 2007 and January 2008. The samples were
collected from nine edible meat parts (neck, antre cote steak, contre-filet, sirloin steak, rump, silver
side, topside, contre-nuar and thick flank) on the carcass halves. The sample collection was performed
from knives, cutting tables, moving cutting tables and aprons after each cutting the carcasses into
the edible parts. Edible meat samples were prepared for this analysis by dipping a Dacron® fibertipped sterile swab (Fisher Scientific, Houston, TX, USA) five times into the sample while rotating
the swab. The swab, containing approximately 50 mg of meat, was removed, and squeezed in a test
tube containing 1 ml sample dilution buffer containing sodium dodecyl sulfate (SDS). Also, the swap
samples were collected from knives, cutting tables, moving cutting tables and aprons, with the premoisten the swabs (Fisher Scientific, Houston, TX, USA) into diluted 1 ml sample dilution buffer
containing SDS from 10 x 10 cm a representative areas, and before ELISA, the swabs were squeezed
thoroughly in test tubes.
Enzyme immunoassay for the quantitative analysis of risk material (CNS) in raw meat and
on contaminated surfaces A 100 μl sample was transferred from each test tube to an assay well. A
sufficient number of antibody-coated wells was inserted into the micro-well holder to accommodate
the number of samples and all standards tested. Enzyme conjugate (50 μl) was added to each well
containing sample, and the plate was incubated for 10 min. at room temperature. After incubation, the
liquid was poured out to empty the wells, and the micro-well holder tapped upside down thoroughly
against absorbent paper, so that the liquid added earlier was completely removed from the wells. The
wells were washed with 250 μl washing buffer and emptied as described earlier. A 100 μl volume
of substrate/chromogen was added to each well and mixed thoroughly; the plate was incubated for
5 min. at room temperature in the dark. The reaction was stopped by adding 100 μl stop solution to
each well. Optical density was determined by ELISA reader (MR-96A, Hamburg, Germany) with
a filter at 450 nm. Results were interpreted as indicated in the Ridascreen®risk material 10/5 kit
information booklet.
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RESULTS AND DISCUSSION In this study, for the Ridascreen risk material 10/5, the samples
were accepted positive if the absorbance was equal to or more than the absorbance of standard 0.1%,
and we determined the cutoff value as 0.100. The low level contamination (< 0.3) and the high level
contamination (>0.4) were detected at the ratio on the neck as 25.0% and 75.0%, on the antre cote
steak as 12.5% and 62.5%, on the sirloin steak as 25.0% and 25.0%, on the rump as 62.5% and
25.0%, on the silver side as 12.5% and 37.5%, on the topside as 37.5% and 25.0%, on the thick
flank as 25.0% and 50.0%, respectively. Even though the low level contamination was not detected
in contre-filet, the high level contamination was found at the ratio as 12.5%. Although the high level
contamination was not detected in contre-nuar, the low level contamination was found at the ratio as
75.0%. The moderate level contamination (< 0.4) were detected at the ratio on the antre cote steak as
25.0%, on the contre-filet as 87.5%, on the sirloin steak as 50.0%, on the rump as 12.5%, on the silver
side as 50.0%, on the topside as 37.5%, on the contre-nuar as 25.0%, on the thick flank as 25.0%. But,
the moderate level contamination was not detected on the neck.
Troeger (2004) stated that further measures for minimizing the risk during head boning; meat
should not be cut from the parts with potentially high contamination risk (neck meat). In a research
project, ten different ground beef patties were obtained per plant, which were then sampled raw
and analyzed for Glial Fibrillary Acidic Protein (GFAP). As the initial data for this project was
being collected, it was clear that the major problem with contamination with CNS tissue occured
during the Advanced Meat Recovery (AMR) processing of beef neck bones. Well over 50% of the
samples of AMR tissue from beef neck bones had detectable to high levels (>5 ng/mg) of CNS
tissue as determined by the GFAP/ELISA method. In the study also, low level contamination in antre
cote steak (mean GFAP 0.140 ng/mg tissue), high level contamination in contre-filet (mean GFAP
0.495 ng/mg tissue), low level contamination in sirloin steak (mean GFAP 0.227 ng/mg tissue), low,
moderate and high level contaminations in rump, silver side, topside and contre-nuar parts (mean
GFAP 0.264, 0.306 and 0.478 ng/mg tissue as respectively), and high level contamination in thick
flank parts (mean GFAP 1.464 ng/mg tissue) were detected (National Cattlemen’s Beef Association,
2002). The results of our study in neck, rump, silver side, topside, contre-nuar and thick flank parts
are similar to the results of the study mentioned above.
Steaks produced from bone-in loin subprimals where the vertebral column bone is first
cut out may inherently leave residual SRMs on steak surfaces when undercut. Lopes et al. (2005)
evaluated current and alternative cutting methods that could be used to minimize the transfer of
SRM tissue during preparation of steaks from bone-in short loins. They indicated that surface areas
along the vertebral column cut line had detectable (0.100 and 0.215% / 100 cm2) and, thus, higher
SRM contamination compared to resulting steak surfaces or the cutting blade. These results imply
that steaks may be cut from bone-in subprimals prior to removal of vertebral column bone without
affecting transfer of SRM tissue at concentrations <0.100% / 100 cm2 to resulting steaks. In our
study, CNS contamination in contre-filet part of short loin was moderate (<0.4; 87.5%) level and high
level (>0.4; 12.5%). Also, in sirloin steak part of short loin, CNS distribution was low, moderate and
high level, and the highest ratio was detected for moderate level (<0.4; 50.0%). But, another study
had assessed contamination in the boning hall on the carcass. This indicated a significant reduction
in contamination in the boning hall and a much lower level (personal communication H. Anil). 1%
of the contamination on the carcass will be transferred to meat during boning and packing operations
(Comer and Huntly, 2003). Similary, Daly et al. (2002) reported that the primal cuts, forerib and
striploin receive the highest level of cord material during splitting. The level of contamination of
these primals is greatly reduced after de-boning.
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Dorsal Root Ganglia (DRG) can be removed with meat during boning of beef carcasses. Reduced
risk equates to lower yield and more SRM. CNS tissue adheres to the split vertebral face after splitting
carcasses and contaminates boneless meat. To avoid carcass self contamination by CNS tissue,
alternatives to medial plane splitting must be implemented. Cross contamination of beef sides by
CNS tissue may occur if contact is made with other sides (e.g. during transfer to the chiller) (personal
communication H. Anil). Coore et al. (2004) have attempted to more accurately define the position of
the DRG by dissection of fresh carcasses. The dissection work has revealed a level of variation between
specimens with respect to the location of the DRG. Following sheet boning, all DRG were found to
remain with the vertebral column in contrast to that of traditional butchery in which several DRG
were found to have been removed with the meat. Pikse (2005) estimated the risk from these ganglia,
their way during slaughter and cutting procedures were observed. For this aim, thirty seven cutting
plants in Northern Germany were visited. For reasons of reproducibility, the trunk was separated into
five parts during observation (T1, T2-T6, T7-T13, L1-L6, S1-S5). Whereas the rear thoracic ganglia
(T7- T13) and sacral ganglia (S1-S5) mostly remained in natural connection with the column bones
and were littered as SRM, the ganglion stellatum (T1), the front thoracic ganglia (T2-T6) and also
the lumbal ganglia (L1-L6) went into the food chain in different percentages. For the other thoracic
ganglia (T1-T13) and the sacral ganglia (S1-S5) standard operation prescriptions for the removal of
the musculature that is associated with the spine and the removal of the transverse processes of the
vertebraes are needed. The lumbal ganglia (L1-L6) are hidden between the connective and fat tissues
of the filet chain. They could not be taken away in practise. In the current study, SRM ratios were
similar to SRM ratios detected in antre cote steak (moderate level) and rump (low level) parts.
Limited trials suggest that the DRG would not be removed from the bone during normal deboning
(Comer, 2003). In Turkish abattoirs the spinal cord is removed manually and the cavity is cleaned
using a steam spray device. The half carcasses are then cut into pieces for transport to incineration
plant. The carcasses may be cut into primals and sent to work stations where operators cut and trim
for specific cuts (“boneless” or “bone-in” meat). Removal of the spinal cord is performed manually
with a knife in Turkish abattoirs. When the spinal cord is removed, there is a risk that the remains of
the cord may be left on the vertebral column and the muscle of the back. In addittion, the vertebral
column is removed and cut through each joint into individual vertebrae with the aim of improving the
yield. This procedure creates a greater risk of contamination of SRM from the exposed DRG.
The low level contaminations (< 0.3) were detected at the ratio on the knives as 37.5% and on
the cutting tables as 12.5%. But, it was not determined on the moving cutting tables and aprons. In
addition, the moderate level contamination was detected only on the cutting tables as 25.0%. The high
level contamination (>0.4) were detected at same the ratio on the knives and cutting tables as 62.5%.
However, it was detected at same the ratio on the moving cutting tables and aprons as 100.0%.
If the spinal cord is not removed, it remains in certain cuts of beef and is hence available for
potential human consumption (e.g., T-bone steak). In addition, spinal cord left in the carcass can
contaminate the boning table. For older animals, such as bulls or cows, all vertebrae are likely
to be processed using AMR. If the facility does not process the spinal column using AMR, other
technology, such as vibration or hand held knives (e.g., Whizzard knives), are used to recover the
remaining meat attached to the bones (Cohen et al., 2001). During carcass processing and boning, the
amount of nervous tissue dropped onto the abattoir and boning room floors is estimated to be around
10% or 20 g from each animal (The Bureau of Rural Sciences, 2001).
The danger therefore exists of both cross-contamination due to spinal cord tissue that may adhere
to the knife, and direct contamination of the exposed areas of meat at the neck due to release of
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cerebrospinal fluid and spinal cord tissue particles (Ramantanis, 2006). The best practice for control
of cross contamination during the slaughter process is a “two-knife” technique. For all knife cuts
with SRM contact, separate knives have to be used, with the best practice being to use a knife of a
different colour (e.g. red for SRM contact) (FAO, 2007). Prendergast et al. (2003) investigated the
concenrations of GFAP on operative’s aprons/knives, and meat being processed in the boning hall,
boning hall equipment and surfaces before, during, and at the end of boning operations. Significantly
higher concentrations of GFAP were recovered from the aprons than were recovered on knives. In the
current study similar results were found. In this study, although different knife is used in each halves
of carcasses, low and high level CNS contaminations were detected. If the same knife is used for
cutting other parts of the carcass there is a risk of cross contamination (Troeger, 2004). If we consider
the use of single knife for each carcass cutting and deboning in Turkey abattoirs, there is stil a risk for
contamination of edible meats for human consumption.
Troeger et al. (2002) investigated both 60 split carcasses which cut after removal of the whole
vertebral column and 60 split carcasses which cut without carcass splitting. In this study, they found no
CNS protein (GFAP) on all of the carcasses, protecting gloves and cutting tables in both applications.
But, in the abattoirs where we conducted this study in, since this kind of application is not performed,
high level SRM contamination was present in cutting tables, moving cutting tables and aprons. For
this reason, the regular presence of CNS tissue needs to be considered in relation to hazard analysis
and critical control point plans in Turkish abattoirs (e.g. we observed that the workers transport to
carcasses to cutting/deboning hall by embracing carcasses. Thus, we think that contamination of
aprons with SRM can occurs at this point).
We think that the potential contamination risk determined in this study may be caused by
dispersal of spinal cord material during the splitting of beef carcasses, during separation of spinal
cord and DRG from vertebral column by a knife, during transfer of carcasses to chilling rooms
after washing or carcass halves to deboning/cutting halls by embracing carcasses. Also, another
way of contamination with very low probability is dissemination of brain tissue during the stunning
procedure.
REFERENCES
1. ANIL, M.H., LOVE, S., HELPS, C.R., HARBOUR, D.A. (2002). Potential for carcass contamination with
brain tissue following stunning and slaughter in cattle and sheep. Food Control, 13, 431-436.
2. COHEN, J.T., DUGGAR, K., GRAY, G.M., KREINDEL, S., ABDELRAHMAN, H., HABTEMARIAM, T.,
ORYANG, D., TAMERU, B. (2001). Evaluation of the potential for bovine spongiform encephalopathy
in the United States. Research Project, Harvard Center for Risk Analysis Harvard School of Public Health
and Center for Computational Epidemiology College of Veterinary Medicine Tuskegee University. USA,
pp. 1-101.
3. COMER, P. (2003). Assessing exposure to BSE infectivity: The impact of the over thirty month rule in the
UK. CSL/JIFSAN Joint Symposium Food Safety and Nutrition: Risk Analysis. June 11-13, University of
Maryland, Maryland.
4. COMER, P.J., HUNTLY, P.J. (2003). TSE Risk Assessments-A decision support tool. Statistical Methods in
Medical Research, 12, 279-291.
5. COORE, R.R., ANIL, M.H., FISHER, A.V. (2004). Carcass contamination, dorsal root ganglia and common
butchery practice. Conference on Farm to Fork Food Safety: A Call for Common Sense Abstract Book. May
12th- 14th. Athens, Greece, pp.12.
6. DALY, D.J., PRENDERGAST, D.M., SHERIDAN, J.J., BLAIR, I.S., MCDOWELL, D.A. (2002). Use of a
marker organism to model the spread of central nervous system tissue in cattle and the abattoir environment
387
during commercial stunning and carcasses dressing. Applied and Environment Microbiology, 68, 791798.
7. FAO (2007). Management of transmissible spongiform encephalopathies in meat production. Course Manual
Project Capacity Building for Surveillance and Prevention of BSE and Other Zoonotic Diseases. Rome,
pp. 39-68.
8. HAJMEER, M., CLIVER, D.O., PROVOST, R. (2003). Spinal cord tissue detection in comminuted beef:
comparison of two immunologicalmethods. Meat Science, 65, 757–763.
9. HAJMEER, M., CLIVER, D.O., MARSDEN, J.L. (2006). Central nervous system tissue detection in meat
from advanced meat recovery systems. Meat Science, 72, 656-659.
10. HELPS, C.R., HINDELL, P., HILLMAN, T.J., FISHER, A.V., ANIL, M.H., KNIGHT, A.C., WHYTE, R.T.,
O’NIELL, D.H., KNOWLES, T.G., HARBOUR, D.A. (2002). Contamination of beef carcasses by spinal
cord tissue during splitting. Food Control, 13, 417-423.
11. LOPES, M., STOPFORTH, J., SUCRE, K., MIKSCH, R., GIDDENS, E., SAMADPOUR, M. (2005).
Alternative cutting methods to minimize transfer of specified risk materials during steak preparation from
bone-in short loins. IAFP Congress Abstract Book, August 15, Baltimore, Maryland, pp. 161.
12. NATIONAL CATTLEMEN’S BEEF ASSOCIATION (2002). Presence of central nervous system (CNS)
tissue in advanced meat recovery (AMR) products. http://www.beefusa.org/uDocs/amrreport.pdf (accessed
on August 15, 2005).
13. PISKE, K. (2005). BSE and cutting: The sympathetic trunk ganglia and their fate in northern German cutting
plants. PhD Thesis. Fachbereich Veterinärmedizin, Freie Universität Berlin, pp. 1-164.
14. PRENDERGAST, D.M., SHERIDAN, J.J., DALY, D.J., MCDOWELL, D.A., BLAIR, I.S. (2003).
Dissemination of central nervous system tissue from the brain and spinal cord of cattle after captive bolt
stunning and carcass splitting. Meat Science, 65, 1202-1209.
15. RAMANTANIS, S.B. (2006). Alternative cattle slaughtering technologies and/or measures reducing
the dissemination of central nervous system tissue during head handling, harvesting of cheek meat and
tongue and carcass splitting - A review. Veterinarski Arhiv, 76, 19-36.
16. THE BUREAU OF RURAL SCIENCES (2001). Risk assessment of abattoir effluent should BSE be found
in cattle in Australia. The NHMRC Special Expert Committee on TSEs, Commonwealth Department of
Agriculture, Fisheries and Forestry-Australia. June, Kingston, pp. 1-24.
17. TROEGER, K., SCHURR, B., WACHMANN, G., KOLB, R., BEHRSCHMIDT, M. (2002). Vorbeugende
maßnahmen gegen eine mögliche BSE-Gefährdung. Fleischwirtsch, 82, 129-135.
18. TROEGER, K. (2004). Overview of current and alternative slaughter practices. Biotechnology, Agronomy,
Society and Environment, 8, 275-281.
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INVESTIGATION OF BOVINE VIRAL DIARRHEA VIRUS (BVDV)
INFECTION IN RELATION TO FERTILITY IN CATTLE
ISTRAŽIVANJE UTJECAJA BVD ( BOVINE VIRAL DIARRHEA) NA
PLODNOST U GOVEDA
Mehmet KALE1,†, Ayhan ATA2, Sibel YAVRU3, Orhan YAPKIÇ3, Oya BULUT3, Uğur
BÜYÜKYÖRÜK4, Mehmet Şükrü GÜLAY5, Oğuzhan AVCI3
Department of Virology 1,†, Theriogenology and Artificial Insemination2, Physiology5, Faculty of Veterinary
Medecine, Mehmet Akif Ersoy University, 15100, Burdur, TURKEY. 3 Department of Virology, Faculty of
Veterinary Medecine, Selçuk University, 42075, Konya, TURKEY.4 Holstein Breeding Associations, 15100,
Burdur, TURKEY. † Corresponding author: [email protected] This Project was supported by TUBITAK
foundation (TOVAG-106O366)
Abstract
In this study, the blood serum and leukocyte samples were collected from 150 Holstein cows and 150
Holstein heifers with clinically healthy appearance. Antibodies (Ab) against BVDV were detected in 126 cow
serum samples and 102 heifer serum samples. BVD viral antigens (Ag) were detected in 32 cow leukocyte
samples and 11 heifer leukocyte samples. Viral genome was detected in 42 of the cow leukocyte samples and
24 of heifer leukocyte samples which were passaged on cell cultures.The differences of average conception
rates (CR) between BVDV (Ag-/Ab+ = seropositive) seropositive heifers and BVDV (Ag-/Ab- = seronegative)
seronegative heifers, and average age (day) between BVDV seropositive pregnant cows and BVDV seronegative
cows were found statistically important (P<0.05).No significant differences were detected in terms of average
service period (SP), age (day), CR and first insemination time (FSA) between the animals (both cows and
heifers) with persistently infected (PI) and non infected (P>0.05). In this group no pregnant cows and heifers
were present. Also, no significant differences were detected between non pregnant PI BVDV (Ag+/Ab-) cows
and heifers, and BVDV negative cows and heifers in terms of SP, age and FSA. CR of Ag+/Ab+ heifers for
BVDV was significantly different from the noninfected heifers (P<0.01). Average SP of Ag+/Ab+ pregnant
cows for BVDV was significantly different from the noninfected cows (P<0.05). PCR (+) cows and heifers for
BVDV (both pregnant and nonpregnant animals) were not significantly different from the noninfected animal
groups in terms of SP, age, CR and FSA (P>0.05). In conclusion, only fertility of BVDV (Ag-/Ab+) and BVDV
(Ag+/Ab+) heifers were affected from the infection. None of the cows and heifers with PI BVDV (Ag+/Ab-)
and BVDV PCR (+) test results had any effect on fertility parameters.
Keywords: Dairy cattle, bovine viral diarrhea virus, fertility, ELISA, PCR
Sažetak
U ovom radu analizirani su uzorci seruma i leukocita koji su uzeti od 150 krava i 150 zdravih bikova
pasmine Holstein.Protutijela protiv BVDV nađena u 126 uzoraka seruma krava i 102 uzorka seruma junica.
Virusni antigen nađen je u 32 uzorka leukocita krava i 11 uzoraka leukocita kod junica. Genom virusa nađen je
u 42 uzorka leukocita krava i u 24 uzorka kod bikova. Razlike od prosječnog stupnja koncepcije između BVDV
( Ag_/Ab+=seropozitivni) seropozitivnih junica i BVDV (Ag-/Ab-=seronegativni) seronegativnih bikova, te
između seropozitivnih bređih krava i BVDV seronegativnih krava statistički su značajne (p<0.05).Nije utvrđena
statistički značajna razlika u trajanju prosječnog servis perioda, stupnja koncepcije i vremena prve inseminacije
između životinja ( krava i junica) sa prisutnom infekcijom te neinficiranih životinja ( P>0.05). Stupanj
koncepcije Ag+/Ab+ junica za BVDV bio je statistički značajan u odnosu na neinficirane junice (P<0.01).
Prosječni servis period kod Ag+/Ab+ bređih krava bio je statistički značajan u odnosu na neinficirane krave
(P<0.05). PCR metodom za BVDV nisu uočene značajne razlike između inficiranih i neinficiranih životinja.
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U ovoj skupini nisu bile ispitivane gravidne krave niti junice. Također nisu uočene značajne razlike između
negravidnih PI BVDV (Ag+/Ab-) krava i junica, te BVDV negativnih krava i junica u odnosu na SP, životnu do
bi FIT. Stupanj gravidnosti Ag+/Ab+ junica u odnosu na BVDV su se signifikantno razlikovale od neinficiranih
junica. Prosječni servis period Ag+/Ab+ gravidnih krava za BVDV se značajno razlikovao od neinficiranih
krava (p>0.05). PCR(+) krave i junice se po BVDV ( gravidne i negravidne životinje) se nisu signifikantno
razlikovale od neinficiranih skupina što se tiče SP. Zaključujemo da je jedino plodnost BVDV (Ag-/Ab+) I
BVDV (Ag+/Ab+) junica bila ugrožena infekcijom. Niti jedna od krava i junica s PI BVDV (Ag+/Ab-) i BVDV
PCR(+) rezultatima pretzrage nije pokazala promjene u bilo kojim pokazateljima plodnosti.
Ključne riječi:Mliječna goveda, virus bovine virusne diareje, plodnost, ELISA, PCR
INTRODUCTION
In general, cows that have recovered from an acute BVD infection develop a strong and usually
long-lasting immunity (MOERMAN et al., 1993). Infection with BVDV yields different outcomes
depending on the stage of pregnancy (ROEDER et al., 1986) such as fertilization problems, immunetolerant or PI calves, different degrees of malformations, fetal deaths, aborts or mummifications and
stillbirths (FRAY et al., 2000). BVDV infections negatively affect the calf health and reproductive
performance of the herd (MOERMAN et al., 1994). There are number of reports about effect of
BVDV infection on reproductive parameters such as pregnancy rates and calving (MCGOWAN
et al. 1993, RUFENACHT et al. 2001, VALLE et al. 2001). The economic significance of BVD
infections is enormous due to culling infected or PI cows (TAYLOR and Rodwell, 2001). It has been
concluded that BVDV has negative affect on fertility of cows (FRAY et al., 2000). Previous field
studies reported upto 44% decline in pregnancy rates (VIRAKUL et al., 1988, LARSSON et al.,
1994). However, there are also opposing studies (MEYLING and Mikel-Jensen, 1988).
In the current study 300 blood and leukocyte samples from multiparous cows (n=150) and heifers
(n=150) were collected in Burdur region (Turkey). BVDV-ELISA (Ab) test was used to detect BVDV
antibody in blood samples, whereas BVDV-ELISA (Ag) test was used for BVDV antigen detection.
BVDV RT-PCR test was also used for viral genome detection. Afterwards, fertility parameters
were evaluated between seropositive and seronegative, viral antigen (+) and viral antigen (-), and
viral genome (+) and viral genome (-) groups. Moreover, BVDV prevalence in the area and relation
among the tests were studied.
MATERIALS and METHODS
Animals: Three hundred Holstein-Freisian cows (n=150; 2 to 10 year old) and heifers (n=150; 13 to 24 months
old) kept at 120 different dairy farms in the province of Burdur, Turkey, and brought to The Holstein Breeding
Association of Burdur for AI was assigned to the trial. Body condition scores (BCS) were taken prior to artificial
insemination (1 to 5; FERGUSON et al., 1994) and animals with BCS lower than 2.5 were not included in
the study. Blood and leukocyte samples were collected from all animals. All dairy cows and heifers were examined
by experienced veterinarian vaginally per rectum, and were healthy and free of anatomical abnormalities of the
reproductive tract. No animals in the trial were vaccinated against BVDV prior to sampling period.
Estrus detection, artificial insemination (AI), and pregnancy checks: AI dates were recorded
by the inseminator and pregnancy diagnosis was performed by rectal palpation 6-8 weeks after AI. The records of
AI, pregnancy diagnosis and calving dates were obtained from the Holstein Breeding Associations data base. The
AI coincided with middle of estrus, as evidenced by CMD, high myometrial tone and contractility. In addition to
secondary signs of estrus, the stage of estrus cycle was confirmed by the presence of fluctuant Graafian Follicles and
the absence of corpus luteum by rectal palpation for all animals prior to AI. The AI was performed for the first time on
all unmated heifers and was the first time after postpartum in all cows. AI was performed using frozen-thawed semen
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from a single bull containing at least ten million of motile spermatozoa (Alsole Benchmark Birbo, Consorzio
Semenzo-Italy via Masaccio, 11- 42010 Mancasale/Italy) with proven fertility. Semen was placed at the
corpus uteri for all cows and heifers. Breeding day (day 0) was accepted as equal to the day of onset of strong estrus
signs. Pregnancy was diagnosed by rectal palpation of heifers and cows at 8 weeks after AI. When AI
led to positive pregnancy check, it was defined as successful. If an animal declared non-pregnant by
rectal palpation or returned to heat, the AI was coded as unsuccessful.
Antibody detections in serum samples: Presence of antibodies BVDV in serum samples were
detected by BVD/MD/BD P80-ELISA kits (Institut Pourquier, France).
Antigen detection in leukocyte samples: ELISA kit (Bio-X Diagnostics, Belgium) was used to
detect Antigens against BVDV in leukocyte.
Preparation of cell culture: Madine Darby Bovine Kidney (MDBK) cell cultures were prepared
in Konya Selcuk University, Department of Virology. BVDV negative fetal calf serum (FCS; 10%;
Gibco, Paisley-Scotland, UK) was used for cell production. Prior to use, MDBK cell cultures were
checked for presence of BVDV antigens by BVD/BD Antigen Capture ELISA kits (Laboratory
Service International, France).
Pre par ati on of l eu koc yte sam p le s in P olym era se C h ai n R eact ion ( P C R ) :
Leukocytes were passaged one times in MDBK cell cultures prior to use in Reverse TranscriptasePolimerase Chain Reaction (RT-PCR). NucleoSpin® RNA Virus Kits (Macherey-Nagel, Germany)
were used for Viral RNA isolation from leuk ocy te samples. F or d etection of B V D V, an
ampl if ica ti on ki t (Genekam Biotechnology AG, Germany) was used; cDNA and One-Step
PCR steps were carried by this kit. The end products from PCR were evaluated by electrophoresis
according to the procedures of Genekam Biotechnology AG®. Presence of bands after electrophoresis
was examined by white ultraviolet Tranilluminator (UVP Inc., Upland CA, USA).
Statistical Analysis of Data: Sensitivity, specificity and correlations among the tests used in this trial were
calculated with MINITAB by Pearson correlation. The differences between BVDV (Ag- /Ab+) / BVDV (Ag/Ab-), BVDV (Ag+ /Ab-) / BVDV (Ag- /Ab-), BVDV (Ag+ /Ab+) / BVDV (Ag- /Ab-) and BVDV (PCR+)
/ BVDV (PCR -) in average days open (OD), and age for cows and FSA for heifers were compared by Proc Mixed
procedure of SAS . CR for cows and heifers were compared by FREQ and LOGISTIC procedure of SAS.
RESULTS
Determination of Pregnancy rates and test results in cows and heifers: In pregnant cows
and heifers the highest seropositivity and lowest seropositivity-antigen rates were in ELISA (Ag/Ab+) and ELISA (Ag+/Ab+), respectively. On the other hand, the highest seropositivity and lowest
seropositivity-antigen rates for open cows and heifers were determined by ELISA (Ag-/Ab+) and
ELISA (Ag+/Ab-), respectively. No correlation was found between ELISA (Ab) and ELISA (Ag) or
ELISA (Ab) and PCR results. However, there was a positive correlation between ELISA (Ag) and
PCR (Correlation coefficient: + 0.770).
Reproductive parameters in BVDV (Ag- /Ab+) / BVDV (Ag- /Ab-) cows and heifers: In the
current study, average ages at the time of AI between BVDV (Ag- /Ab+) and BVDV (Ag- /Ab-) cows
differed significantly (P<0.01). Morover, pregnant BVDV (Ag- /Ab+) cows were older than pregnant
BVDV (Ag- /Ab-) cows (P<0.05). However, no differences for OD or CR were detected between
BVDV (Ag- /Ab+) and BVDV (Ag- /Ab-) cows. No difference for OD detected between pregnant or
open BVDV (Ag- /Ab+) and pregnant or open BVDV (Ag- /Ab-) cows. Pregnancy rates of BVDV
(Ag- /Ab+) heifers were higher than BVDV (Ag- /Ab-) heifers (P<0.05). However, FSA was similar
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in two groups. In addition, FSA of pregnant or open BVDV (Ag- /Ab+) heifers was similar to FSA
of pregnant or open BVDV (Ag- /Ab-) heifers.
No difference in any of the reproductive parameters evaluated was different between BVDV
(Ag+ /Ab-) and BVDV (Ag- /Ab-) cows and heifers. In the current study, all BVDV (Ag+ /Ab-)
cows and heifers were open. In open BVDV (Ag+ /Ab-) cows and heifers, reproductive parameters
studied did not differ from BVDV (Ag- /Ab) cows and heifers.
Reproductive parameters for BVDV (Ag+ /Ab+) / BVDV (Ag- /Ab-) cows and heifers: No
difference in average OD, age (days) and CR of BVDV (Ag+ /Ab+) and BVDV (Ag- /Ab-) cows
were detected. In addition, difference in FSA for BVDV (Ag+ /Ab+) and BVDV (Ag- /Ab-) heifers
were not significant. However, CR of BVDV (Ag+ /Ab+) and BVDV (Ag- /Ab-) heifers were found
statistically important (P<0.01). There was a significant difference in OD of pregnant BVDV (Ag+ /Ab+)
and BVDV (Ag- /Ab-) cows (P<0.05). However, the difference in age was not significant between the
pregnant groups. In addition, no difference was detected between nonpregnant (Ag+ /Ab+) and BVDV
(Ag- /Ab-) cows in any of the parameters studied. No statistical difference was seen any of the reproductive
parameters between neither pregnant nor nonpregnant BVDV (Ag+ /Ab+) and BVDV (Ag- /Ab-) heifers.
Reproductive parameters for BVDV (PCR+) / BVDV (PCR-) cows and heifers: Average OD, age
(day) and CR between BVDV (PCR+) and BVDV (PCR-) cows were not significant. In addition,
no differences for FSA and CR in heifers were detected between BVDV (PCR+) and BVDV (PCR-)
groups. None of the reproductive parameters for pregnant and nonpregnant cows and heifers studied differed
significantly.
DISCUSSION
The prevalence of BVDV infected herds in individual countries most often ranges from 50%
to 90% in the world (HOUE, 1995). The prevalence of herds in Turkey has ranged from 14.3% to
100% (ÇABALAR and Karaoğlu, 1999). In the current study, prevalence of BVDV infection was
76% (228/300), and the prevalence was higher among the multiparous cows (84%). The infection
appears highly prevalent (60-85%) in mature dairy cows (HOUE, 1999). NIZA-RIBEIRO et al.
(2005) reported that non-vaccinated herds have prevalence of 23%. The prevalence was higher in
multiparous cows (42%) than heifers (14%). Another study in Sweden (n=711) the prevalence of
BVDV infection was 41% (BJÖRKMAN et al., 2000), and 35.3% of pregnant heifers had BVDV
(Ag-/Ab+).
There are numbers of studies reporting different prevalence rates for BVDV. Different rates
in different studies could be due to diverse management practices in different farms and countries
(VEGA et al., 1997, HOUE, 1999). In the current study, the prevalence of BVDV [ELISA (Ab), ELISA
(Ag) and PCR] was high. This could be due to different factors: 1) the number of animals housed
per square meter is high in Burdur, 2) cows, heifers and calves are housed in the same area without
separation, 3) new animals are introduced to the herds without screening for BVDV infection, 4)
herds do not monitored for BVDV infection regularly, 5) there are no control over animal movement
inside and/or outside the herds, and 6) no eradication program is present for PI animals.
Similar to the previous studies, prevalence of BVDV was higher in multiparous cows in the
current study. MOCKELIUNIENE et al. (2004) reported that rate of BVDV infection increases with
age because older cows have more chance to face with the infection. Similarly, MOCKELI et al.
(2003) compared the age of the animals and Open days (day), BVDV infection rates. In their study
439 cows divided to 8 different groups by age. The least amount of seropositive animals were in age
1. The cows that were 3 and 4 years old had higher prevalence rates. The highest groups were age
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5 and older. They concluded that there was a positive correlation with age and prevalence rates for
BVDV.
It has been reported that BVDV infection affects fertility and therefore pregnancy rates (GRAHN
et al., 1984). BVDV infection also causes early aborts (LARSSON et al., 1994). In preplanned
experiments, animals infected with BVDV 9 days prior to their estrus had 50% lower CR than
controls. In addition, embrio quality and quantity was decreased significantly (KAFI et al., 1997).
MCGOWAN et al. (1993) infected Bos indicus cattle during the artificial insemination by using
BVDV contaminated sperms, and the CR of infected animals were lower than immune positive
ones. This could be due to the embryo-toxic effect of BVDV or direct effect on follicular function
and/or ovaries (ovaritis) (GROOMS et al., 1998). On the other hand, RUFENACHT et al. (2001)
showed that CR in BVDV seropositive and seronegative dairy cows did not differ significantly.
They also concluded that BVDV infection did not cause early embryonic deaths in first 45 days of
pregnancy. However, aborts were apparent from 46 to 120 days of pregnancy. In the current study CR
of seropositive and seronegative heifers were higher that that of seropositive and seronegative cows.
In addition CR were different in of seropositive and seronegative heifers (p<0.05). MCGOWAN et
al. (1993) also reported that BVDV seropositive and seronegative heifers had higher CR compared to
multiparous cows in the same herd. This is not surprising since there is an inverse relation between
age and CR (OLA REFSDAL, 2007).
In the current study CR between BVDV (Ag+/Ab+) and infection free cows were different
(p<0.05). In dairy cows, OD increases due to lower CR (MCDOUGALL, 2006). SWASDIPAN et al.
(2004) reported that OD of BVDV infected cows was 60 day longer than healthy cows. Moreover,
treatment methods used against fertility could also have negative effect on OD length (ROCHE et al.,
1998). The current study, on the other hand, did not detect any difference in FSA between infected
and healthy heifers. Similarly, VALLE et al. (2001) showed that BVDV infection had no effect on
OD in cows or FSA in heifers. In addition, BVDV had no affect on calving intervals. No difference
in FSA, service intervals, and average service for conception was detected between healthy and
BVDV seropositive heifers.
The current study concluded that presence of BVDV (Ag-/Ab+) and BVDV (Ag+/Ab+)
affected fertility only in heifers. Moreover no effect on fertility was detected for PI BVDV (Ag+/
Ab-) and PCR BVDV (+) cows and heifers. Previously, preplanned controlled studies have been
conducted to determine the effects of BVDV infection on fertility. Moreover, limited knowledge is
also available on naturally occurring BVDV infection in herds and its effect on fertility. Thus, it is
hard to give any conclusion about BVDV and fertility. Our current study suggests further studies
should be performed on specially BVDV seropositive and BVDV (Ag+/Ab+) cows and heifers in
different herd management practices. These studies should also include the animals showing clinical
symptoms of BVDV. We also think that studies should also distinguish the difference in different
BVDV serotypes.
REFERENCES
1. BJÖRKMAN, C., Alenius, S., Emanuelsson, U., Uggla, A., Neospora caninum and Bovine Virus Diarrhoea
Virus Infections in Swedish Dairy Cows in Relation to Abortion, The Veterinary Journal, 159, 201-6,
(2000).
2. ÇABALAR, M., Karaoğlu, T., Sığırlarda Bovine Viral Diarrhea (BVD) Virus Enfeksiyonuna Karşı Antikor
Varlığının Araştırılmasında Nötralizasyon Immunoperoksidaz (NPLA) ve Serum Nötralizasyon (SN)
Testlerinin Karşılaştırılması, Ankara Üniversitesi Veteriner Fakültesi Dergisi, 46, 249-55, (1999).
393
3. FERGUSON, J.D., Galligan, D.T., Thomsen, N., Principal Descriptors of Body Condition Score in Holstein
Cows, Journal of Dairy Science, 77, 2695-703, (1994).
4. FRAY MD, Paton DJ, Alenius S, The Effects of Bovine Viral Diarrhoea Virus on Cattle Reproduction in
Relation to Disease Control, Animal Reproduction Science, 60, 615-27, (2000).
5. GRAHN, T.C., Fahning, M.L., Zemjanis, R., Nature of Early Reproductive Failure Caused by Bovine Viral
Diarrhea Virus, Journal of the American Veterinary Medical Association, 185, 429-32, (1984).
6. GROOMS, D., Brock, K., Pate, J., Day, M., Changes in Ovarian Follicles Following Acute Infection with
Bovine Viral Diarrhea Virus, Theriogenology, 49, 595-605, (1998).
7. HOUE, H., Epidemiology of Bovine Viral Diarrhea Virus, Veterinary Clinics of North America: Food Animal
Practice, 11, 521-47, (1995).
8. HOUE, H., Epidemiological Features and Economical Importance of Bovine Viral Diarrhoea Virus (BVDV)
Infections, Veterinary Microbiology, 64, 89-107, (1999).
9. KAFI, M., McGowan, M., Kirkland, P., Jillella, D., The Effect of Bovine Pestivirus Infection on the
Superovulatory Response of Friesian Heifers, Theriogenology, 48, 985-96, (1997).
10. LARSSON, B., Niskanen, R., Alenius, S., Natural Infection with Bovine Viral Diarrhea Virus in a Dairy
Herd: A Spectrum of Symptoms Including Early Reproductive Failure and Retained Placenta, Animal
Reproduction Science, 36, 37-48, (1994).
11. MCDOUGALL, S., Reproduction Performance and Management of Dairy Cattle, The Journal of Reproduction
and Development, 52, 185-94, (2006).
12. MCGOWAN, M.R., Kirkland, P.D., Rodwell, B.J., Kerr, D.R., Carroll, C.L., A Field Investigations of the
Effects of Bovine Viral Diarrhoea Virus-Infection Around the Time of Insemination on the Reproductive
Performance of Cattle, Theriogenology, 39, 443-9, (1993).
13. MEYLING, A., Mikel-Jensen, A., Transmission of Bovine Viral Diarrhoea Virus (BVDV) by Artifical
İnsemination with Semen from a Persistently-Infected Bull. Veterinary Microbiology, 17, 97-105, (1988).
14. MOCKELI, R., Salomskas, A., Mockeli, V., 2003, Epidemiological Studies of BVDV and Herpes
Virus Infections in AI Centers in Lithuania, Farm Animal Reproduction: Reducing Infectious Diseases
Symposium, Jelgava-Latvia, (2003) pp: 32-4.
15. MOCKELIUNIENE, V., Salomskas, A., Mockeliunas, R., Petkevicius, S., Prevalence and Epidemiological
Features Of Bovine Viral Diarrhoea Virus Infection in Lithuania, Veterinary Microbiology, 99, 51–7,
(2004).
16. MOERMAN, A., Straver, P.J., De Jong, M.C., Quak, J., Baanvinger, T., Van Oirschot, J.T. A Long Term
Epidemiological Study of Bovine Viral Diarrhoea Infections in a Large Herd of Dairy Cattle, Veterinary
Record, 132, 622-6, (1993).
17. MOERMAN, A., Straver, P.J., De Jong, M.C., Quak, J., Baanvinger, T., Van Oirschot, J.T., Clinical
Consequences of a Bovine Virus Diarrhoea Virus Infection in a Dairy Herd: A Longitudinal Study,
Veterinary Quarterly, 16, 115-9, (1994).
18. NIZA-RIBEIRO, J., Pereira, A., Souza, J., Madeira, H., Barbosa, A., Afonso, C., Estimated BVDVPrevalence, Contact and Vaccine Use in Dairy Herds in Northern Portugal, Preventive Veterinary Medicine,
72, 81-5, (2005).
19. OLA REFSDAL, A., Reproductive Performance of Norwegian Cattle from 1985 to 2005: Trends and
Seasonality, Acta Veterinaria Scandinavica, 49, 1-7, (2007).
20. ROCHE, J.F., Austin, E., Ryan, M., O’Rourke, M., Mihm, M., Diskin, M., Hormonal Regulation of the
Oestrous Cycle of Cattle, Reproduction in Domestic Animals, 33, 227-31, (1998).
21. ROEDER, P.L., Jeffrey, M., Cranwell, M.P., Pestivirus Fetopathogenicity in Cattle: Changing Sequelae with
Fetal Maturation, Veterinary Record, 118, 44-8, (1986).
394
22. RUFENACHT, J., Schaller, P., Audige, L., Knutti, B., Küpfer, U., The Effect of Infection with Bovine Viral
Diarrhea Virus on the Fertility of Swiss Dairy Cattle, Theriogenology, 56, 199-210, (2001).
23. SWASDIPAN, S., McGowan, M.R., Bock, R., Estimating the Economic Impact of Bovine Viral Diarrhoea
Virus (BVDV) Infection During Pregnancy in Commercial Dairy Cattle, Revista Portuguesa de Ciéncias
Veterinarias, 127, 41, (2004).
24. TAYLOR, L., Rodwell, B., Outbreak of Foetal Infection with Bovine Pestivirus in a Central Queensland
Beef Herd, Australian Veterinary Journal, 79, 682-5, (2001).
25. VALLE, P.S., Martin, S.W., Skjerve, E., Larssen, R.B., Age at First Calving and Calving Interval in Bovine
Viral Diarrhoea Virus (BVDV) Sero-Converted Dairy Herds-A Longitudinal Study, Preventive Veterinary
Medicine, 51, 17-36, (2001).
26. VEGA, S., Bayon, M.C., Jiminez, T., Asensio, A., Mirat, F., Cid, D., Fuente, R., Prevalence of Bovine Viral
Diarrhoea Virus in the Cattle Population of Communidad de Madrid (Spain), The Third ESVV Symposium
on Pestivirus Infections, Lelystad-Netherlands, (1997) pp: 116-9.
27. VIRAKUL, P., Fahning, M.L., Joo, H.S., Zemjanis, R., Fertility of Cows Challenged with a Cytopathic
Bovine Viral Diarrhea Virus during an Outbreak of Spontaneous Infection with a Non-Cytopathic Strain,
Theriogenology, 29, 441-9, (1988).
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DETECTION OF SPINAL CORD TISSUES AS BOVINE SPONGIFORM
ENCEPHALOPATHY SPECIFIED RISK MATERIAL (BSE-SRM) IN BEEF
CARCASSES DURING SPLITTING IN TURKEY
OTKRIVANJE TKIVA RIZIČNOG ZA PRIJENOS BSE PRILIKOM
ODVAJANJA GOVEĐIH POLOVICA U TURSKOJ
M. KALE1,*, O. KURSUN2, A. S. AKCAN KALE3, F. PEHLIVANOGLU4, A. GUNER5,
R. BASKAYA6, Y. DOGRUER5, C. OZTURK6
Department of Virology 1, †, Food Hygiene and Technology 2, Microbiology 4, Faculty of Veterinary Medecine,
Mehmet Akif Ersoy University, 15100, Burdur, TURKEY. 3 Ministry of Agriculture, State Control Laboratory,
15100, Burdur, TURKEY. 5Department of Food Hygiene and Technology, Faculty of Veterinary Medecine,
Selçuk University, 42075, Konya, TURKEY. 6School of Military, Food Control Laboratory, Selimiye, 34668,
Istanbul, TURKEY. †Corresponding author: [email protected]
ABSTRACT
We have used an enzyme-linked immunosorbant assay (ELISA) to detect central nervous system (CNS)specific glial fibrillary acidic protein (GFAP) to show the presence of CNS material on the medial surface of each
half (left and right) of washed carcasses after splitting with a reciprocating saw. The high level contamination (≥
0.4) was detected at the highest ratio on the brisket + plate area surface of carcasses (33.3%). The moderate level
contamination (> 0.2) was detected at the highest ratio on the brisket + plate and loin areas surface of carcasses
(11.1%). The low level contamination (≥ 0.1) was detected at the highest ratio on the flank and hip areas surface
of carcasses (100.0%). The similar level contamination risk was found on both the right and left half of 72 beef
carcasses. Although no contamination was detected on the reciprocating saw, the low level contamination (≥
0.1) was detected on the floor where the splitting of the carcasses was performed. Even though the low level
contamination (≥ 0.1) was detected on all areas of the carcasses, even after washing, the moderate and high level
contamination were detected only on the chuck + rib, brisket + plate and loin areas.
SAŽETAK
U radu je primjenjena ELISA metoda u pretraživanju GFAP (glialni fibrilarni acidni proteini) u središnjem
živčanom sustavu kako bi se dokazalo da nakon odvajanja polovica goveda, još uvijek ima rezidua živčanog tkiva
na obje isprane tehnološki odvojene polovice. najveća kontaminacija bila je na plečkama, nešto manja na trupu
a najmanja na butinama polovica (33,3%, prema 11,1% i ispod 0,1%). u radu je pretraženo 72 goveđe polovice
pri čemu je ustanovljeno da su jednako kontaminirane i lijeve i desne. na strojevima za odvajanje polovica
nakon pranja nije bilo kontaminacije, dok je na podu prostorije u kojima se odvajaju polovice kontaminacija
bila neznatna.
INTRODUCTION
Bovine spongiform encephalopathy has been diagnosed first in Great Britain in 1986 (DEFRA
2004). The BSE agent has been shown to cause variant Creutzfeldt–Jakob Disease (vCJD) in humans
(Bruce et al. 1997). The infectious agent, most likely the pathological isoform of the prion protein
(PrPsc) (Collins et al. 2004) is mainly located in the CNS of infected animals (Dormont 2002). As a
consequence, the presence of CNS tissue in beef and beef products is banned in many countries, such
as USA, Canada, the countries of the European Commission, and Switzerland (Dormont 2002).
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The most critical process stage, in terms of edible meat contamination with SRM, is the current
common practice of the longitudinal splitting of the carcass (Ramantanis 2006). Many researchers
(Anil et al. 2002; Helps et al. 2002; Prendergast et al. 2003; Lim et al. 2007) studied the contamination
of beef carcasses by spinal cord tissue during splitting. They demonstrated that the slaughter practice
could disseminate spinal cord material over the carcass, the operator, and the environment during
the splitting process. The conventional slaughter practices at present in Turkey primarily include the
following processes: removal of head from the carcass and carcass splitting. Studies on the spreading
of CNS tissue have been conducted by only two researchers on meat and meat products in Turkey
(Yesilbag and Kalkan 2005; Kale et al. 2007). But there has been no study conducted in Turkey on
the contamination of beef carcasses by spinal cord tissue during splitting in the abattoirs. This study,
therefore, was conducted to determine the level of contamination of beef carcass surfaces with CNS
tissue slaughtered with conventional method in the abattoirs in Turkey.
Sample Collection and Preparation A total of seventy-two beef carcasses were used from
two commercial beef abattoirs in Burdur, Southern Turkey. Samples were collected from cattle
carcasses slaughtered older than 30 months during six visits between March 2007 and August 2007.
All carcasses were splitted in approximately 45 min after slaughter with a reciprocating saw (built-in
washing function) [EFA 44 splitting saw, Maulbronn, Germany], with two sides of each carcass being
spray washed with water. Afterwards spray washing, samples were collected using a Dacron® fibertipped sterile swab (Fisher Scientific, Houston, TX, USA), placed into a 2 ml test tube containing 1
ml of 0.5% SDS sample dilution buffer (Ridascreen, R-Biopharm, Darmstadt, Germany) and mixed.
The aliquoted samples were stored at 4 °C for 2 days. 48 h later the samples were brought to room
temperature and mixed shortly before the ELISA. Samples were collected from five defined areas
(chuck + rib was sagittal section of dorsal spines and centra of thoracic vertebrae, brisket + plate
was ventral section of costae and internal of thoracic cavity, loin was centra of lumbar vertebrae,
flank was ventral section of internal abdominal cavity, hip) on the medial surface of each half of the
split carcass, yielding 5 samples per carcass side. After each splitting, the reciprocating saw and floor
were washed and then 72 swab samples from the reciprocating saw and 72 floor swab samples (17
m2) were taken. All workroom floors were constructed of waterproof concrete.
ELISA for Raw Meat Samples A commercially available ELISA kit (Ridascreen risk material
10/5, R-biofarm GmbH), which detects GFAP as marker, was used. At the first step, 50 μl sample
extract or standard was left to separate wells. Four standards (0, 0.1, 0.2 and 0.4%) were used as
controls. After adding 50 μl of conjugate to the each well, the test plates were incubated at room
temperature for 10 min. Three-times washing and tapping were followed by the addition of 100 μl
of substrate/ chromogen solution. After second incubation at room temperature for 5 min in the dark,
100 μl of stop solution was added to each well and the ODs were measured at 450 nm.
RESULTS AND DISCUSSION
For the Ridascreen risk material 10/5, the samples were accepted positive if the absorbance was
equal to or more than the absorbance of standard 0.1%, and we determined the cutoff value as 0.100.
The similar level contamination risk was found on both the right and left half of 72 beef carcasses.
The low level contamination (≥ 0.1) was detected at the highest ratio on the flank and hip areas
surface of carcasses (100%). The low level contamination (≥ 0.1) was detected at the lowest ratio on
the brisket + plate area surface of carcasses (55.5%). The moderate level contamination (> 0.2) was
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detected at the highest ratio on the brisket + plate and loin areas surface of carcasses (11.1%). The
moderate level contamination (> 0.2) was not detected on the flank and hip areas surface of carcasses
(11.1%). The high level contamination (≥ 0.4) was detected at the highest ratio on the brisket + plate
area surface of carcasses (33.3%). The high level contamination (≥ 0.4) was not detected on the flank
and hip areas surface of carcasses.
Two proteins which are specific to the CNS have been identified. One of them is GFAP, a major
constituent of nonneural cells, and this makes up 10% of total spinal cord protein (Eng 1985). Daly et
al. (2002) investigated spread of spinal cord material during beef slaughter in three Irish commercial
beef abattoirs. In the study, 4 areas were sampled after splitting and washing of carcasses. The results
indicated that there was dispersal of GFAP onto meat as a result of splitting of the spinal column
during beef carcass dressing. In general, the highest concentrations of GFAP on carcasses were found
at sites brisket + plate, chuck + rib and loin along the vertebrae, after washing. Similarly in other
study, Lim et al. (2007) determined the higher level of contamination occured on the interior surface
of a total of sixty beef carcasses along the vertebral area. In their study, it was indicated that parts
chuck + rib, brisket + plate and loin of carcass were the most highly contaminated parts with specific
risk material. Also in our study, the high level contamination (≥ 0.4) was detected at highest ratio on
the brisket + plate area surface of carcasses (33.3%) and the moderate level contamination (> 0.2) was
detected at the highest ratio on the brisket + plate and loin areas surface of carcasses (11.1%). Some
researchers (Prendergast et al. 2003; Helps et al. 2002), also observed greatest CNS contamination
on areas chuck + rib and loin. But they determined less contamination on brisket + plate, flank and
hip areas. Wenther (2004) recommended that bones and SRM must be removed from beef cuts prior
to fabrication into steaks and roasts, from cattle 30 months of age and older. The most critical point
in terms of contamination of the meat surface with SRM is the currently common practise of sawing
the spine vertically in the middle with hand-guided belt-type saws. During carcass splitting, areas
closest to the vertebrae had the greatest risk of contamination by spinal cord material from the action
of the saw (Troeger 2004). The major source of CNS contamination on the carcass is from splitting
the spinal column. Most of the CNS material is spread on the medial cut surface of the carcass in the
vicinity of the vertebral column (Daly et al. 2002).
Baeuerle (2005) in his study for the evaluation of CNS contamination collected 1500 swab
samples from the inner left side of 375 carcasses. His results showed that of the four carcass regions
sampled, the sacral area represented the region with the lowest degree of contamination. Also, in
our study, both moderate and high level contamination was not detected on the hip area surface of
carcasses. The spinal cord is usually cut, on occasions along its length, spreading cord tissue along
the whole cut surface of the split carcass. This particularly affects the internal neck musculature
(Schwaegele et al. 2002). Schwaegele et al. (2002) found CNS protein (GFAP) positive in 82% of
internal surface of neck muscules of 107 half carcasses, and 70% of them had a high contamination
ratio. Low level contamination risk in sacral region may be due to the splitting of the carcasses start
from this region.
The percentage of carcasses with the low level contamination (≥ 0.1) was higher than the
percentage of the moderate and high level contaminated carcasses and this result proved that the
carcasses in the abattoirs are contaminated with CNS. This reveals that washing carcasses after
splitting by saw can not prevent the contamination with CNS. Studies conducted by Helps et al. (2002)
have shown the presence of CNS material on carcasses after splitting with a conventional band saw.
This contamination was still present after the carcass had been washed or steam-vacuum cleaned.
They noticed that in some cases washing served only to spread the material on the carcass, thereby
399
contaminating previously uncontaminated areas. On the other hand, Daly et al. (2002) reported that
the highest level of spinal cord protein was found along the spinal cord tract and again washing
was not an efficient cleaning method. The average number of cases of spinal cord tissue fragments
adhering to the carcass was assumed five cattle or 20%. There is a 20% chance that spinal cord tissue
fragments have not been completely removed, although washing the carcass is believed to reduce
the possibility of any fragments remaining to 2% or less (The Food Safety Commission/The Prion
Expert Committee 2005). Knight (2002) reported that typically 25-50 mg of CNS material was found
on each of the medial carcass surfaces, particularly along the cut vertebral surface with less in the
body cavity. A total of 100mg of CNS tissue was present on the cut surfaces of each carcass following
splitting and washing (Comer and Huntley 2003). Based on data from experiments that measured the
amount of spinal cord associated protein deposited on the carcass during splitting (Harbour 2001), the
base case assumes that approximately 0.001% (2.5 mg) of the spinal cord contaminates edible meat.
It was also reported that considerable variation was found in carcass contamination levels between
abattoirs, both in the same country and between countries (Anil et al. 1999; Anil et al. 2002).
Cross contamination risk exists mostly through utensils, knives, saws and sucking devices at
spinal cord removal. In most abattoirs a band saw is used to divide carcases into sides. Band saws
have a fine blade and teeth and produce very little sawdust. The saws are generally fitted with water
sprays that wash dust down the carcass. The alternative to the band saw is the reciprocating saw,
used in smaller abattoirs. This has a wider blade and larger teeth than the band saw. It cuts a wider
path through the backbone and is more likely to damage the spinal cord because of the wider cut
(Comer and Huntley 2003). The blade is sprayed with water which will run down the carcass into the
abdominal and thoracic cavity, and onto the neck and floor, with the risk of spreading SRM (sawdust
from the vertebral column and spinal cord). There is also a risk of the spinal cord remaining in on
half of the carcass carcase due to uneven splitting of the vertebral column (Food Standards Australia
New Zealand 2002). Daly et al. (2002) collected the swab samples from the saw used at carcass
splitting. The results indicated that there was dispersal of GFAP onto carcass splitting saw as a result
of splitting of the spinal column during beef carcass dressing. In their study were determined GFAP
onto carcass splitting saw at mean value 28 ng/mg. Helps et al. (2004) reported that under controlled
conditions in an experimental abattoir, between 23 and 135 g of tissue accumulated in the saw after
splitting five to eight carcasses. Many researchers also confirmed that the carcass-splitting saw poses
a particular risk in terms of cross-contamination of carcasses, equipment, surfaces and operatives
(Helps et al. 2002; Prendergast et al. 2003). But in our study, no contamination was detected on the
reciprocating saw which was washed after each splitting of 72 carcasses. This may be due to the
fact that reciprocating saw was fitted with water spray (to decrease the splashing worked minimal
level) and after splitting the spinal cord was removed from reciprocating saw manually by scratching
out with a thumb knife and rewashed with hot water (85°C). But it is not ignored that, the BSE
agent is resistant to routine methods of disinfection. As a result, chemical disinfection of containers,
vehicles, work surfaces, floors etc. exposed to contamination by SRM using standard disinfectants
is not practicable. Although caustic agents are partially effective and used in certain situations (such
as the decontamination of rendering plants), thorough cleaning by dilution with large volumes of hot
water and detergent is recommended for most circumstances (Schuett-Abraham 2002). Additionally,
to prevent the cross contaminations between carcasses during splitting or washing the carcasses were
kept 10 meter apart from each other.
Specific Risk Material, along with brain and spinal cord tissue being released during the handling
and boning of head and carcass splitting, will be dispersed in the blood during exsanguination, onto
the floors and walls, equipment, work surfaces and meat surfaces and onto the personnel in the
400
abattoir. Over the course of the day the contamination will accumulate, especially on personnel, with
some having higher levels of exposure than others. If the operatives in abattoirs are to be safeguarded,
the regular presence of CNS tissue needs to be considered in relation to the abattoir’s hazard analysis
and critical control point plans (Prendergast et al. 2003; ABAS 2001). During carcass processing
and deboning, the amount of nervous tissue dropped onto the abattoir and deboning room floors is
estimated to be around 10% or 20 g from each animal. It is also estimated that 30% of Australian
abattoirs sweep the floor before the washdown, and that in these abattoirs only 10% of the dropped
material remains on the floor or 2 g of infective tissue. In the worst case scenario 20 g of infectious
tissue will end up in the effluent with an average of 14.6 g and a minimum of 2 g (BRS 2001). In
total 72 floor swab samples which were collected after each splitting of 72 carcasses, the low level
contamination (≥ 0.1) was detected (100%). In the abattoirs the floor was being cleaned with hot
water (approximately at 60°C). The swab samples from the floor were collected after running hot
water was cut.
In this study, the low level contamination with spinal cord tissues as BSE-SRM was found in
most of 72 carcasses. However, the low level contamination was found on the floor where carcass
splitting was performed on. But, no contamination was detected on reciprocating saw. Thus, the
use of new techniques, removal of spinal cord, extraction off the vertebral column without splitting
and boning out the unsplit (unquartered carcase), were advised to prevent the mechanical damage
of spinal cord during splitting of the vertebral column and the contamination of meat products and
environments with spinal cord tissues as BSE-SRM. Likewise, with the recent North American cases
of BSE contaminated cattle, the demand for control technologies that efficiently remove SRMs from
bovine carcasses is on the rise. Research engineers at Food Science Australia (FSA), the nonprofit
joint venture of Australian Commonwealth Scientific and Industrial Research Organisation (CSIRO)
and the Australian Food Industry Science Centre (AFISC) have developed a control system using
ultrasonic feedback that automatically separates a carcass into beef sides using a standard meat
industry split saw and a robot. Following the completion of preliminary testing, the system is now
undergoing production trials at an Australian processing facility (LaBudde 2004).
In conclusion, the carcasses in the abattoirs in Turkey are contaminated at low level with BSESRM and this may constitute a potential threat to public health.
REFERENCES
ABAS 2001. Spezielle maßnahmen zum schutz der beschäftigen vor infectionen durch BSE-Erreger.
Bundesgesundheitsbl-Gesundheitsforsch-Gesundheitsschutz. 44, 410-412.
ANIL, M.H., LOVE, S., WILLIAMS, S., SHAND, A., MCKINSTRY, J.L., HELPS, C.R., WATERMANPEARSON, A., SEGHATCHIAN, J. and HARBOUR, D.A. 1999. Potential contamination of beef carcasses
with brain tissue at slaughter. Veterinary Record, 145, 460-462.
ANIL, M.H., LOVE, S., HELPS, C.R. and HARBOUR, D.A. 2002. Potential for carcass contamination with
brain tissue following stunning and slaughter in cattle and sheep. Food Control, 13, 431-436.
BAEUERLE, B. 2005. Contamination of beef carcasses with central nervous tissue during splitting. PhD.
Thesis Dissertation, pp. 1-114, München University, Germany.
BRS 2001. Risk assessment of abattoir effluent should BSE be found in cattle in Australia. http://www.affa.gov.
au/output/ruralscience.html (accessed January 21, 2008).
BRUCE, M.E., WILL, R.G., IRONSIDE, J.W., MCCONNELL, I., DRUMMOND, D., SUTTIE, A.,
MCCARDLE, L., CHREE, A., HOPE, J., BIRKETT, C., COUSENS, S., FRASER, H. and BOSTOCK,
C.J. 1997. Transmissions to mice indicate that ‘new variant’ CJD is caused by the BSE agent. Nature, 389,
498-501.
COLLINS, S.J., LAWSON, V.A. and MASTERS, C.L. 2004. Transmissible spongiform encephalopathies. The
Lancet, 363, 51-61.
401
COMER, P.J. and HUNTLY, P.J. 2003. TSE Risk Assessments–a decision support tool. Statistical Methods in
Medical Research, 12, 279-291.
DALY, D.J., PRENDERGAST, D.M., SHERIDAN, J.J., BLAIR, I.S. and MCDOWELL, D.A. 2002. Use of a
marker organism to model the spread of central nervous system tissue in cattle and the abattoir environment
during commercial stunning and carcasses dressing. Applied and Environment Microbiology, 68, 791798.
DEFRA 2004. BSE Statistics. http:// www.defra.gov.uk/animalh/bse/index.html (accessed January 21, 2008).
DORMONT, D. 2002. Prions, BSE and food. International Journal of Food Microbiology, 78, 181-189.
ENG, L.F. 1985. Glial fibrillary acidic protein (GFAP): The major protein of glial intermediate filaments in
differentiated astrocytes. Journal of Neuroimmunology, 8, 203-214.
FOOD STANDARDS AUSTRALIA NEW ZEALAND 2002. BSE risk assessment and risk management
strategy. Final Assessment Report, 06/03, pp. 1-142, Canberra, Wellington, Australia, New Zealand.
HARBOUR, D. 2001. Measures to reduce contamination of meat and environment with CNS tissue during
slaughter and processing of cattle and sheep. http://europa.eu.int/comm/research/press/1998/pr2710en.html
(accessed January 21, 2008).
HELPS, C.R., HINDELL, P., HILLMAN, T.J., FISHER, A.V., ANIL, M.H., KNIGHT, A.C., WHYTE, R.T.,
O’NIELL, D.H., KNOWLES, T.G. and HARBOUR, D.A. 2002. Contamination of beef carcasses by spinal
cord tissue during splitting. Food Control, 13, 417-423.
HELPS, C.R., FISHER, A.V., HARBOUR, D.A., O’NEILL, D.H. and KNIGHT, A.C. 2004. Transfer of spinal
cord material to subsequent bovine carcasses at splitting. Journal of Food Protection, 67, 1921-1926.
KALE, M., KURŞUN, Ö. and PEHLIVANOĞLU, F. 2007. Detection of central nervous system tissues as
Bovine Spongiform Encephalopathy specified risk material in processed and raw meat products in Turkey.
Journal of Food Safety, 27, 56-65.
KNIGHT, A.C. 2002. Dispersion of CNS material during splitting of cattle carcasses. In The OTM risk assessment
group on the 20th, September (O-RR/RAG3/20).
LABUDDE, R.A. 2004. BSE in the USA. Redux: How made are we getting? http://www.foodscience.afisc.
csiro.au (accessed January 21, 2008).
LIM, D.G., ERWANTO, Y. and LEE, M. 2007. Comparison of stunning methods in the dissemination of central
nervous system tissue on the beef carcass surface. Meat Science, 75, 622-627.
PRENDERGAST, D.M., SHERIDAN, J.J., DALY, D.J., MCDOWELL, D.A. and BLAIR, I.S. 2003.
Dissemination of central nervous system tissue from the brain and spinal cord of cattle after captive bolt
stunning and carcass splitting. Meat Science, 65, 1202-1209.
RAMANTANIS, S.B. 2006. Alternative cattle slaughtering technologies and/or measures reducing the
dissemination of central nervous system tissue during head handling, harvesting of cheek meat and tongue
and carcass splitting - a review. Veterinarski Arhiv, 76, 19-36.
SCHUETT-ABRAHAM, I. 2002. BSE-Präventivmaßnahmen bei der schlachtung von rindern. Berliner und
Münchener Tierarztliche Wochenschrift, 115, 125-130.
SCHWAGELE, F., MUELLER, E., FISCHER, K., KOLB, R., MOJE, M. and TROEGER, K. 2002. Nachweis
von gewebe des ZNS auf rinderschlachttierkörpern nach absaugen des rückenmarks. Fleischwirtschaft, 6,
1118-1120.
THE FOOD SAFETY COMMISSION/THE PRION EXPERT COMMITTEE 2005. Food safety risk assessment
related to measures against Bovine Spongiform Encephalopathy (BSE) in Japan. Prion Expert Committee
Report, 6, 1-54.
TROEGER, K. 2004. Overview of current and alternative slaughter practices. Biotechnology, Agronomy,
Society and Environment, 8, 275-281.
WENTHER, J. 2004. Bones that are designated as specified risk material (SRM) and must be removed from
cuts originating from animals of 30 months of age or older. http://www.aamp.com (accessed January 21,
2008).
YESILBAG, K. and KALKAN, A. 2005. Detection of central nervous system tissues as BSE specified risk
material in meat products in Turkey. Food Control, 16, 11-13.
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THE METABOLIC PROFILE OF BOER GOATS DURING PUERPERIUM
THE METABOLIC PROFILE OF BOER GOATS DURING PUERPERIUM
Dobranić1, T., M. Samardžija1, D. Đuričić2, I. Harapin1, S. Vince1, D. Gračner1, N.
Prvanović1, J. Grizelj1, M. Karadjole1, Lj. Bedrica1, D. Cvitković1
Faculty of Veterinary medicine, Zagreb, Croatia1
Veterinary practice Đurđevac2Croatia1
Summary
The aim of this research was to establish metabolic profile of Boer goats during puerperium. For this
reason 14 Boer goats and 8 cross breed dairy goats in a German improved fawn type (control group) 2 to 4 years
old were used. Blood samples were taken every 72 hours from the 3rd untill 40th day after delivery. Following
parameters were determined: aspartate amminotransferase, gamma glutamiltransferase, alkaline phosphatase,
creatine kinase, glucose, total proteins, albumins, triglycerides, cholesterole, urea, creatinine, bilirubin i ßhidroxybutirate, Ca, P, Na, K, Cl and Mg. In the Boer goats significantly higher (P<0,05) levels of creatine kinaze,
GGT, alkaline phosphatase, glucose were noticed, whereas in the dairy goats significantly higher level (P<0,05)
of ß-hidroxybutirate, cholesterole, urea, phosphorus and AST were found. However, values of mentioned
parameters were within physiological levels in both groups. Statisticaly significant difference (P>0,05) between
the groups for any other biochemical parameter was not established. The conducted tests have shown that
group of Boer goats had no metabolic disorders although levels of some liver enzymes were increased. Since
Boer goats are poliestrus breed of goats, regarding obtained results we can assume that hypothalamus-pituitaryovarian axis would be reestablished in Boer goats during physiological time postpartum.
Sažetak
Istraživanjem smo željeli odrediti metabolički profil tijekom puerperija burskih koza. U tu svrhu
smo koristili 14 koza burske pasmine i 8 koza križanih u tipu njemačke srnaste koze (kontrolna skupina) u
dobi 2-4 godine. Uzorci krvi su uzimani svaka tri dana počevši od trećeg pa sve do 40. dana nakon poroda.
Određivali smo sljedeće pokazatelje: aspartat aminotransferazu, gama-glutamiltransferazu, alkalnu fosfatazu,
kreatin kinazu, glukozu, ukupne bjelančevine, albumine, trigliceride, kolesterol, ureju, kreatinin, bilirubin
i ß-hidroksibutirat, te Ca, P, Na, K, Cl i Mg. Ustanovili smo značajno višu (P<0,05) razinu kreatin kinaze,
GGT, alkalne fosfataze, glukoze i kalcija kod burskih koza, dok smo kod muznih koza ustanovili značajno
višu razinu (P<0,05) ß-hidroksibutirata, kolesterola, ureje, fosfora i AST-a. Za ostale biokemijske pokazatelje
nismo ustanovili značajnu razliku između skupina (P>0,05). Međutim, vrijednosti svih pokazatelja bile su u
normalnim fiziološkim granicama za obje skupine koza. Provedenim istraživanjem smo ustanovili da skupina
burskih koza nije imala metaboličke poremećaje neovisno o povišenoj razini nekih jetrenih enzima. Budući
je burska koza poliestrična pasmina koza, glede dobivenih rezultata možemo zaključiti da će u njih osovina
hipotalamus- hipofiza-jajnici biti uspostavljena tijekom fiziološkog razdoblja puerperija.
Uvod
Koza je sezonski poliestrična životinja u krajevima umjereno kontinentske klime, što znači da se
tjera samo u određeno vrijeme godine i to više ciklusa zaredom (Zarrouk i sur., 2001.). U postojbini
burske koze na južnoj polutki nije zabilježeno vrijeme potpune spolne neaktivnosti, a vrhunac
spolne aktivnosti je u jesen, dok je kasno proljeće i sredina ljeta vrijeme smanjene spolne aktivnosti.
(Greyling i Van Der Nest, 1990.). Smatra se da razina specifičnih metabolita u plazmi ne pokazuje
samo poremećaj, nego i daje signal osovini hipotalamus-hipofiza-jajnici o metaboličkom statusu
403
(Opsomer et al., 1999). Djelovanje enzima aspartat aminotransferaze (AST) je pokazatelj oštećenja
jetre (Reid i Roberts, 1982.), ali se javlja i kod bolesti kosturnog mišićja pa je potrebno kod životinje
odrediti i djelovanje kreatin kinaze (Forenbacher, 1993.). Kreatin kinaza ima tri izoenzima MM (u
mišićju), MB (miokard) i BB (mozak), pa je i pokazatelj oštećenja tih tkiva. Gama-glutamil transferaza
(GGT) je enzim vezan uz membranu stanice većine parenhimatoznih organa (jetra, slezena, bubrezi,
gušterača i tanka crijeva), a klinički se koristi isključivo za dokazivanje hepatobilijarnih bolesti.
Alkalna fosfataza je enzim prisutan u skoro svim tkivima (kosti, jetra, bubrezi, prostata, slezena,
mukoza duodenuma i neka krvna tjelešca), a važan je pokazatelj bolesti hepatobilijarnog sustava,
ostitisa, osteomalacije. (Ramadan i Harapin, 1998.). Razina glukoze u plazmi je niska tijekom ranog
puerperija u preživača (i do 30-tog dana), da bi se kasnije povećala, pa se smatra da razina glukoze u
krvi nije povezana s energetskim stanjem (Lucy i sur., 1991.). Određivanje razine ß-hidroksibutirata
(BHB) u serumu nam služi kao najpouzdaniji test za otkrivanje ketoze (Geishauser i sur., 2000.).
Početak ciklične aktivnosti jajnika u preživača u suodnosu je s povećanjem razine nezasićenih
masnih kiselina, BHB-a i ureje u plazmi (Grummer, 1993.). Cijelo vrijeme puerperija kod koza,
ukupni kolesterol je imao srednje vrijednosti 2,06-2,75 mmol/L, dok su vrijednosti ukupnih lipida
bile najniže do 7. dan post partum, a zatim su do 40. dana puerperija vrijednosti rasle (Krajničakova i
sur., 1999.). Poremećaji mijene tvari imaju najvažniju ulogu u patologiji puerperalnog razdoblja kod
preživača. Manjak energije često uzrokuje atoniju i hipotoniju maternice, a posljedica je usporena
involucija maternice. Razina glukoze je u fiziološkim granicama zbog visoke razine kortizola
(Subandrio i Noakes, 1997.). Atonija maternice i imunosupresija mogu biti posljedica fiziološkog i
metaboličkog stresa u preživača (Dobson i Smith, 2000.). Razina ketonskih tijela i slobodnih masnih
kiselina je na gornjoj fiziološkoj granici u puerperiju preživača (Kaczmarowski i sur., 2006.) što
ukazuje na manjak energije, lipolizu, supkliničku ketozu i masnu degeneraciju jetre (Grummer,
1993.; Bobe i sur., 2004.). Cilj našeg istraživanja bio je ustanoviti pouzdanost metaboličkog profila u
svrhu praćenja ciklične aktivnosti jajnika tijekom puerperija u burskih koza.
Materijal i metode
Životinje. Ovim su istraživanjem obuhvaćene 22 koze, 14 burskih i 8 križanih u tipu njemačke
srnaste koze u dobi od dvije do četiri godine. Koze su držane ekstenzivno na sjevernim obroncima
Bilogore: Čepelovečki breg, općina Đurđevac. Životinje su smještene u skupne nastambe i hranjene
jednako: livadno sijeno po volji, te oko 0,7 kg koncentrata po kozi dnevno. Obje su skupine koza
držane zajedno s jaradi, od jarenja do završetka istraživanja. Jarčevi su izdvojeni na nekoliko
kilometara udaljenosti kako bi se izbjegao učinak nazočnosti mužjaka («male effect»).
Skupljanje uzoraka krvi. Uzorke krvi smo uzimali s pomoću vacutainer pribora iz vene jugularis
svaka tri dana počevši od trećeg završno do četrdesetog dana puerperija. Krv je ostavljena najmanje
15 minuta na sobnoj temperaturi te smo je, prema Bage i sur., (2002), centrifugirali 15 minuta na
3.000 okretaja u minuti. Izdvojeni smo serum vrlo brzo (15 minuta) pohranili na temperaturu od -20
°C, sve do pretraga.
Određivanje razine biokemijskih parametara u krvi. Biokemijskim su analizama iz seruma
određivani: aspartat aminotransferaza, kreatin kinaza, gama-glutamiltransferaza, alkalna fosfataza,
glukoza, ukupne bjelančevine, ß-hidroksibutirat, albumini, trigliceridi, kolesterol, ureja, kreatinin,
bilirubin, kalcij, fosfor, natrij, kalij, klor i magnezij, a određivali smo ih standardno spektrofotometrijski
u laboratoriju Klinike za unutarnje bolesti na Veterinarskom Fakultetu, Sveučilišta u Zagrebu na
Olympus AU 600 analyser (Olympus Diagnostica GMBH, Hamburg, Germany), koristeći Olympus
reagense i standardne metode spektrofotometrije.
404
Statistička obrada podataka. Svi rezultati istraživanja obrađeni su statističkom metodom ANOVA
i Tukeyevim testovima post-hoc analize. Rezultati s P<0,05 smatrali su se statistički značajnima.
Rezultati
Četrdeset dana puerperija srednja vrijednost AST-a je iznosila 102,8 ± 2,32 U/L u burske, a
118,4 ± 3,10 U/L u koza križanih u tipu njemačke srnaste koze. Kod burskih koza srednje vrijednosti
Slika 1a. Usporedba srednjih vrijednosti biokemijskih pokazatelja u obje skupine koza (burske n=14; križane
n=8). Unutar istog stupca se značajno razlikuju vrijednosti različitog eksponenta
Slika 1b. Usporedba srednjih vrijednosti biokemijskih pokazatelja u obje skupine koza. (burske n=14; križane
n=8)Unutar istog stupca se značajno razlikuju vrijednosti različitog eksponenta
405
alkalne fosfataze (167,7 ± 18,13 U/L), kreatin kinaze (194,1 ± 13,09 U/L), GGT-a (53,25 ± 1,01 U/L)
bile su više od srednjih vrijednosti koza križanih u tipu njemačke srnaste koze (AP 52,9 ± 2,3 U/L,
CK 178,0 ± 8,2 U/L i GGT 41,9 ± 1,2 U/L) Vrijednosti glukoze kod burskih koza su iznosile od 3,18,3 mmol/L (4,21 ± 0,07 mmol/L), a koza križanih u tipu njemačke srnaste koze od 2,3-4,6 mmol/L
(3,47 ± 0,06 mmol/L). Ukupne bjelančevine (70,8 ± 0,38 g/L kod burskih, a 70,6 ± 0,60 g/L kod
križanih) i albumini (33,33 kod burskih, te 35,4 ± 0,37 g/L kod križanih) su gotovo istih vrijednosti u
obje skupine koza. Srednja vrijednost triglicerida kod burskih koza je iznosila 0,28 ± 0,02 mmol/L, a
kod križanih 0,17 ± 0,01 mmol/L, te srednje vrijednosti kolesterola 2,31 ± 0,06 mmol/L kod burskih
i 3,31 ± 0,09 mmol/L križanih. Vrijednosti ß-hidroksibutirata su iznosile od 0,1 do 0,8 mmol/L (s
prosjekom od 0,28 ± 0,01 mmol/L) kod burskih koza i 0,1-2,9 mmol/L (0,62 ± 0,04 mmol/L) kod
koza križanih u tipu njemačke srnaste koza. Srednja vrijednost bilirubina je iznosila 4,04 ± 0,06
μmol/L kod burskih koza, a 4,16 ± 0,07 μmol/L kod koza križanih u tipu njemačke srnaste koze.
Vrijednost ureje se kretala od 0,83 do 11,85 mmol/L (4,39 mmol/L ± 0,18 mmol/L) u burskih koza,
a u kontrolne skupine od 2,41 do 16,36 mmol/L (6,71 ± 0,31 mmol/L). Srednja vrijednost kreatinina
je iznosila 72,35 ± 1,12 μmol/L u burskih koza, a 67,84 ± 1,30 μmol/L u kontrolne skupine koza.
Vrijednosti kalcija (2,39 ± 0,02 mmol/L) i fosfora (2,02 ± 0,06 mmol/L) su više kod burskih koza
nego kod koza križanih u tipu njemačke srnaste koze (Ca 2,17 ± 0,04 mmol/L i P 2,59 ± 0,10 mmol/
L). Ustanovili smo da su razine kalija (4,76 ± 0,08 mmol/L kod križanki, a 4,69 ± 0,07 mmol/L kod
burskih), magnezija (kod križanki 1,13 ± 0,02 mmol/L, a kod burskih koza 1,12 ± 0,02 mmol/L),
natrija (kod križanki 147,45 ± 1,02 mmol/L, a kod burskih koza 147,61 ± 0,65 mmol/L) i klora (kod
križanki 111,75 mmol/L, a kod burskih koza 113,19 ± 0,36 mmol/L) podjednake bez značajnih
razlika. (Grafikoni . i 2.).
Rasprava
Razina glukoze u krvi je važan pokazatelj funkcioniranja mijene tvari organizma (Ramadan i
Harapin, 1998.). Neki autori navode nižu razinu glukoze u postpartalnom periodu preživača kod
kojih je kasnio povratak ciklične aktivnosti jajnika (Huszenica i sur., 1988.), dok neki navode da je
razina glukoze u granicama normale (Samardžija i sur., 2006.). Mi smo ustanovili da postoji značajna
razlika između razine glukoze u serumima mliječnih koza kojima se nije vratila ciklična aktivnost u
puerperiju (ojarene u veljači i ožujku) prosječne razine 3,47 mmol/L, i burskih koza, koza s nižom
produkcijom mlijeka (mesna pasmina) kod kojih je razina glukoze značajno (P<0,05) viša 4,21
mmol/L. Djelovanje enzima AST-a je pokazatelj oštećenja jetre (Reid i Roberts, 1982.), ali se javlja
i kod bolesti kosturnog mišićja pa je potrebno odrediti i djelovanje kreatin kinaze (Forenbacher,
1993.). Herak i sur., (2000.) su pratili koncentraciju AST-a u krvi preživača tijekom puerperija, a
kako je aktivnost AST-a bila u fiziološkim granicama ustanovili su da nije bilo većih oštećenja jetre
kod preživača korištenih u istraživanju. Ustanovili su da preživači s cistama na jajnicima kod kojih
je kasnio povratak ciklične aktivnosti imaju značajno višu (P<0,05) razinu AST-a od preživača s
uspostavljenom cikličnom aktivnošću. Sukladne rezultate u preživača je dobio i Huszenicza i sur.
(1988.). U našem istraživanju smo dobili slične rezultate jer smo kod križanih koza dobili višu
(P<0,05) razinu AST-a od burskih koza, kod kojih se trebala uspostaviti ciklična aktivnost. Djelovanje
enzima alkalne fosfataze (AP), kreatin kinaze (CK) i gama-glutamil transferaze (GGT) bilo je više
u burskih koza nego u križanki kod kojih nije došlo do uspostave ciklične aktivnosti jajnika. Razina
ovih enzima je varirala od jedinke do jedinke. Sve vrijednosti su u fiziološkim granicama, osim GGTa, čije su granice od 20 U/L do 50 U/L. Prosječna vrijednost kod križanih koza iznosi 41,85 U/L, a
prosječna vrijednost kod burskih koza 53,26 U/L što možemo pripisati, najvjerojatnije, netoksičnom
406
zamašćenju jetre (Forenbacher, 1993.), jer iako su koze obiju skupina hranjene jednako, smatramo
da su burske koze ušle u istraživanje u boljoj tjelesnoj kondiciji, a moramo uzeti u obzir i pasminska
obilježja, iako u literaturi sličnih podataka nema. Krajničakova i sur., (2003.) i Vrzgula i sur., (1985.)
su ustanovili da je razina mikroelemnata Mg, Na, K i Cl u puerperiju koza u fiziološkim granicama. U
našim istraživanjima smo ustanovili da su razine K, Mg, Na i Cl podjednake kako kod burskih, tako i
kod križanih koza, bez značajnih razlika. Prosječna vrijednost Ca u serumu preživača u puerperalnom
razdoblju pada, a razina kalcija je obrnuto proporcionalna količini mlijeka (Ivanov i sur., 1990.).
Ustanovili smo, i u našem istraživanju, da koze križane u tipu njemačke srnaste koze, koje
imaju veću proizvodnje mlijeka, imaju 2,17 mmol/L prosječnu razinu kalcija u serumu, što je ispod
donje fiziološke granice dok burske koze (mesna pasmina) imaju značajno višu (P<0,05) prosječnu
razinu kalcija u serumu od 2,4 mmol/L. Prosječna razina fosfora kod križanki u tipu njemačke
srnaste koze je u našem istraživanju iznosila 2,58 mmol/L, a kod burskih znatno niža 2,02 mmol/
L. Krajničakova i sur., (2003.) ustanovili su da su ukupne bjelančevine u puerperiju koza iznosile
65,00 do 71,79 g/L, kolesterol 2,06 do 2,75 mmol/L, a ukupni lipidi od 1,76 do 2,27 g/L. Iako je
kolesterol osnova za sintezu steroida jajnika, ne smijemo zaboraviti njegovu ulogu nosača masnih
kiselina kod sinteze mlijeka, što smo i mi potvrdili u našem istraživanju, jer je razina u serumu
kod križanih koza značajno viša (P<0,01) nego kod burskih koza. Statistički se značajno (P<0,01)
snižavala razina triglicerida u puerperiju koza do 7. dana puerperija (Krajničakova i sur., 2003.), a
smatra se da je to povezano s kočenjem sinteze apoproteina i brojem njegovih receptora, koji su važni
za formiranje VLD-lipoproteina (very low density lipoproteins) kod sinteze progesterona (Grummer
i Caroll, 1988.). Nazifi i sur., (2002.) su odredili kod klinički zdravih koza koncentraciju triglicerida u
serumu od 0,11 do 0,22 mmol/L. Ustanovili smo da burske koze imaju značajno višu (P<0,05) razinu
triglicerida u serumu tijekom puerperija, nego križane koze u tipu njemačke srnaste koze. Nadalje,
ustanovili smo da je prosječna razina ukupnih bjelančevina u puerperiju koza gotovo jednaka kako
kod burskih koza, tako i kod križanki u tipu njemačke srnaste koze, a prosječna razina albumina
iznosila 33,33 g/L kod burskih, a kod križanih 35,40 g/L. Svako ispitivanje funkcije jetre započinje
određivanjem razine bilirubina u krvnom serumu, bez obzira postoji li žutica ili ne. Kod nekrobioze i
teške masne degeneracije jetre razina ukupnog bilirubina je 61,56 μmol/L, a spregnutog 44,46 μmol/
L, a fiziološke vrijednosti za kozu iznose 1,7-3,4 μmol/L (Forenbacher, 1993.). Mi smo u našem
istraživanju dobili prosječnu vrijednost bilirubina 4,16 μmol/L za križanke u tipu njemačke srnaste
koze, a u burskih koza je ta vrijednost iznosila 4,04 μmol/L, što bi potvrdilo druge autore (Kaneko i
sur., 1997.) koji navode znatno višu gornju fiziološku granicu (4,3 μmol/L, čak 6,5 μmol/L) za ukupni
bilirubin u serumu koza, jer uspoređujući jetrene enzime možemo reći da nema oštećenja jetre, a
vrijednosti bilirubina u našem istraživanju prelazile bi gornju fiziološku granicu. The conducted
tests have shown that group of Boer goats had no metabolic disorders although levels of some liver
enzymes were increased. Since Boer goats are poliestrus breed of goats, regarding obtained results
we can assume that hypothalamus-pituitary-ovarian axis would be reestablished in Boer goats during
physiological time postpartum.
Literatura
Bage, R., H. Gustafsson, B. Larsson, M. Forsberg, H. Rodriguez-Martinez (2002): Repeat breeding in dairy
heifers: follicular dynamics and oestrous cycle characteristics in relation to sexual hormone patterns.
Theriogenology, 57, 2257-2269.
Bobe, G., J. W. Young, D. C. Beitz (2004.): Invited review: Pathology, etiology, prevention, and treatement of
fatty liver in dairy cows. J. Dairy Sci. 87, 3105-3124.
407
Dobson, H., R. F. Smith (2000.): What is stress, and how does it affect reproduction? Anim. Reprod. Sci. 60,
743-752.
Forenbacher, S. (1993.): Klinička patologija probave i mijene tvari domaćih životinja, Svezak II, Jetra, HAZU,
Školska knjiga.
Geishauser, T., K. Leslie, J. Tenhag, A. Bashiri (2000.): Evaluation of eight cow-Side ketone tests in milk for
detection of subclinical ketosis in dairy cows. J. Dairy Sci. 83, 296-299.
Greyling, J. P. , M. Van der Nest (1990.): Ovulation in the Boer goat doe. Small Rumin. Res. 3, 457-464.
Grummer, R. R. (1993.): Etiology of lipid-related metabolic disorders in periparturient dairy cows. J. Dairy Sci.
76, 388-394.
Grummer, R. B., D. J. Caroll (1988.): A review of lipoprotein cholesterol metabolism importance to ovarian
function. J. Anim. Sci. 66, 3160-3173.
Herak, M., T. Dobranić, Melita herak, S. Milinković-Tur, V. Dolar (2000.): Krvna koncentracija glukoze,
bjelančevina, bilirubina i ureje te aktivnost aspartat transaminaze u krvi krava tijekom puerperija. Zbornik,
Drugi hrvatski veterinarski kongres, Cavtat, 267-272..
Huszenicza, G., J. Haraszti, L. Molnar, L. Solti, S. Fekete, K. Ekes, A. C. Yaro (1988.): Some metabolic
characteristics of dairy cows with different postpartum ovarian function. J. Vet. Med. A. 35, 506-515.
Ivanov, I., I. Rajić, M. J. Jovanović, M. Lalić (1990.): Concentration of calcium in the blood serum in highpregnant and lactating cows in intensive breeding. Vet. Glasnik 44, 359-364.
Kaczmarowski, M., E. Malinowski, H. Markiewicz (2006.): Some hormonal and biochemical blood indices in
cows with retained placenta and puerperal metritis. Bull. Vet. Inst. Pulawy 50, 89-92.
Kaneko, J. J., W. Harvey, M. L. Bruss (1997.): Clinical Biochemistry of Domestic Animals, 5 th edition Academic
Press, San Diego London Boston New York Sydney Tokyo Toronto.
Krajničakova, M., G. Kovač, M. Kostecky, I. Valocky, I. Maraček, I. Šutiakova, L. Lenhardt (2003.): Selected
clinico-biochemical parameters in puerperal period of goats. Bull. vet. Inst. Pulawy 47, 177-182.
Krajničakova, M., E. Bekeova, L. Lenhardt, V. Cigankova, I. Valocky, I. Maraček (1999.): Microscopic analysis
of the uterine endometrium in post parturient ewes. Acta Vet. Brno 68, 9-12.
Lewis, G. S. (1997.): Uterine health and disorders. J. Dairy Sci. 80, 984-994.
Lucy, M. C., C. R. Staples, F. M. Michel, W. W. Thatcher (1991.): Energy balance and size and number of
ovarian follicles detected by ultrasonography in early postpartum dairy cows. J. Dairy Sci. 74, 473-482.
Nazifi, S., H. R. Gheisari, F. Shaker (2002.): Serum lipids and lipoproteins and their correlations with thyroid
hormones in clinically healthy goats. Vet. Arhiv 75, 249-257.
Opsomer, G., Y. T. Grohn, J. Hertl, H. Laevens, M. Coryn, A. de Kruif (1999): Protein metabolism and the
resumption of ovarian cyclicity potpartum in high yielding dairy cows. Reprod. Dom. Anim., (Suppl. 6),
Proceedings of the 3rd Conference of the European Society for Domestic Animal Reproduction 54-57.
Ramadan, P., I. Harapin (1998.): Interna klinička propedeutika domaćih životinja.
Veterinarski fakultet
Sveučilišta u Zagrebu.
Reid, I. M., J. Roberts (1982.): Fatty liver in dairy cows. In Practice 4, 164-169.
Samardžija, M., T. Dobranić, S. Vince, M. Cergolj, A. Tomašković, K. Đurić, J. Grizelj, M. Karadjole, D.
Gračner, Ž. Pavičić (2006): Beziehung zwischen Progesteron P4, IGF-I, Blutparameter und zyklischer
Ovarienaktivität der Kühe im Puerperium. Tierärztliche Umschau 61, 8; 421-427.
Subandrio, A. J., D. E. Noakes (1997.): Neutrophil migration into the uterine lumen of the cow: the influence
of endogenous and exogenous seks steroid hormones using two intrauterine chemoatractants. Theriogen.
47, 825-835.
Vrzgula, L., H. Seidel, J. Gardaš (1985.): Yearly dynamycs of haematological and biochemical indices in blood
serum of goats. Folia Vet. 29, 53-69.
Zarrouk, A., O. Souilem, P. V. Drion, J. F. Beckers (2001.): Caracteristiques de la reproduction de lèspece
caprine. Ann. Med. Vet. 145, 98-105.
408
NIVELES DE METALES TÓXICOS (AS, CD, HG Y PB) EN HÍGADOS
DE BOVINOS CRIADOS EN INTENSIVO EN LA PROVINCIA DE LEÓN
(N.O. ESPAÑA).
TOXIC METAL CONCENTRATIONS (AS, CD, HG Y PB) IN LIVER OF
BEEF CATTLE FROM PROVINCE OF LEON (NW SPAIN).
KONCENTRACIJE TOKSIČNIH METALA (AS, CD, HG I PB) U
JETRI TOVNE JUNADI S PODRUČJA LEONA (SJEVEROZAPADNA
ŠPANJOLSKA)
Escudero A1, González JR1, Miranda M2, Hernández J3, Lechuga A1, Castillo C3, Benedito JL.3
Department of Medicine, Surgery and Veterinary Anatomy, University of Leon, 24071,
Spain.
2
Department of Clinical Veterinary Sciences, University of Santiago de Compostela, Lugo
27002, Spain.
3
Department of Animal Pathology, University of Santiago de Compostela, Lugo 27002,
Spain.
1
Abstract
The objective of this study was to determine the concentrations of four toxic elements (arsenic,
cadmium, mercury and lead) in the liver of beef cattle between 6 months and 2 years old from feedlots
localized in Leon, NW Spain. Sixty-two liver samples of caudal lobule (200 g approximately) from
all animals were analyzed by ICP-MS. The arithmetic mean concentrations in all animals (age and
sex combined) were 6.86 µg/kg/w.wt.; 19.9 µg/kg/w.wt.; 1.08 µg/kg/w.wt. and 9.53 µg/kg/w.wt. for
arsenic, cadmium, mercury and lead, respectively. Geometric mean concentrations were 5.10 µg/kg/
w.wt for As), 17.9 µg/kg/w.wt for Cd, 0.68 µg/kg/w.wt for Hg and 8.59 µg/kg/w.wt for Pb. Knowledge
of toxic metal concentrations in livestock is important for assessing the effects of pollutants on domestic animals
and protection of consumers.
Abstract
Svrha rada je odrediti koncentracije 4 toksična elementa (arsena, kadmija, žive i olova) u jetri tovne junadi
starosti 6 mjeseci do dvije godine iz tovilišta u Leonu, Španjolska. Analizirana su 62 uzorka kaudalnog lobula
jetre (200 g) od svake životinje primjenom ICP-MS tehnike. Srednja aritmetička koncentracija (s obzirom na
starost i spol) iznosila je 6.86 µg/kg/w.wt.; 19.9 µg/kg/w.wt.; 1.08 µg/kg/w.wt. i 9.53 µg/kg/w.wt. za arsen,
kadmij, živu i olovo.Geometrijska srednjakoncentracija je iznosila 5.10 µg/kg/w.wt za As), 17.9 µg/kg/w.wt za
Cd, 0.68 µg/kg/w.wt za Hg i 8.59 µg/kg/w.wt za Pb. Spoznaje o prisutnosti toksičnih metala u mesu vrlo su
važne zbog sigurnosti potrošaća.
409
Introducción
Está perfectamente demostrado que algunos metales como el arsénico (As), cadmio (Cd),
mercurio (Hg) o el plomo (Pb) pueden ejercer un efecto tóxico, disminuyen la productividad y los
índices reproductivos de los animales de abasto y afectan a la salud pública (Jensen, 1991; WHO,
1995; WHO, 2001).
Los metales se pueden incorporar al organismo humano y al de los animales a través de su
inhalación y por la ingestión de agua y alimentos, dependiendo en gran medida del grado de
contaminación del sitio de localización, así como los hábitos y recursos alimenticios de los individuos
(Alberti-Fidanza et al, 2003; Chen et al, 2003; Prankel et al, 2004). Estos elementos son tóxicos para
el ser humano y los animales, debido a su elevada concentración en el ambiente, pudiendo aumentar
sus efectos nocivos como consecuencia de su bioacumulación (Santos et al, 2004).
Material y Métodos
Muestras.La recogida de las muestras se llevó a cabo en el matadero municipal de León entre
Septiembre 2006 y Marzo 2007. Se recogieron muestras de un total de 62 animales, con edades
comprendidas entre los 6 meses y 2 años, descartando aquellos animales que por cuestiones sanitarias
fueran objeto de decomiso. Se registró la procedencia exacta de todos los animales mediante la
utilización del Documento de Identificación Bovina (DIB), garantizando la identificación de la
muestra (hígado) con el animal al que correspondía.
Las muestras de hígado se tomaron del lóbulo caudado (200 g aproximadamente), y conservaron
en bolsas de polipropileno debidamente identificadas, manteniéndolas refrigeradas hasta su transporte
en el mismo día al laboratorio.
Una vez en el laboratorio, las muestras se limpiaron de grasa, tejido conectivo y principales
vasos sanguíneos; de cada muestra de tejido se tomaron 3 submuestras de 10 g aproximadamente que
se colocaron en bolsas de polipropileno debidamente identificadas. Las muestras de cada animal se
introdujeron en una única bolsa identificada con el código del animal, congelándolas a -18ºC hasta
su posterior análisis laboratorial.
Digestiones.Todas las muestras de hígado (2 g aproximadamente) se pesaron de forma precisa en
los vasos de digestión utilizando una balanza electrónica SALTER-AND, modelo ER-60 A. A cada
muestra se le añadieron 5 ml de ácido nítrico concentrado, (Suprapur Grade, Merck 69%), 2 ml de
peróxido de hidrógeno 30% p/v y 1 ml de agua ultrapura. A continuación se cerraron los vasos y se
sometieron a un proceso de digestión en un sistema microondas (marca Milestone Ethos Plus) La
solución resultante se diluyó con agua ultrapura hasta un volumen final de 25 ml y se almacenó en
tubos de polipropileno hasta su posterior análisis químico.
Determinación laboratorial.La determinación de los niveles de los metales (As, Cd, Hg y
Pb), se llevó a cabo por Espectroscopia de Masa con Fuente de Plasma Acoplado (ICP-MS). Los
análisis se realizaron en el Laboratorio FISQUITECNAL perteneciente a los servicios centrales de la
Universidad de Santiago de Compostela.
Durante todo el estudio se llevó a cabo un estricto programa de control de calidad analítica. En
cada lote de 10 muestras se incluía un blanco y una muestra de material de referencia certificado.
El límite de detección de la digestión ácida se calculó como tres veces la desviación estándar de
los blancos. Los límites de cuantificación, expresados como la concentración de cada analito en el
tejido, se calcularon teniendo en cuenta el peso de la muestra y la disolución empleada. Los estudios
de recuperación analítica se llevaron a cabo empleando un Material de Referencia Certificado (Pig
410
Kidney CRM 186, BCR Reference Materials) con una concentración certificada de As (7,98 µg/l);
Cd (2572 µg/kg); Hg (1,97 µg/lg) y de Pb (306 µg/kg), obteniendo un porcentaje de recuperación de
98,7% para todos los metales, a excepción del mercurio en las muestras de uno de los lotes (29%).
Resultados y discusión
En la tabla 1 se encuentran representados los niveles de As, Cd, Hg y Pb en hígados de ganado
vacuno procedentes de la provincia de León. En nuestro estudio encontramos las siguientes niveles
medios: para arsénico (6,86 µg/kg); cadmio (19,9 µg/kg); mercurio (1,08 µg/kg) y plomo (9,53
µg/kg) en peso fresco. La concentración hepática de los diferentes metales tóxicos tienden a estar
sesgadas a la derecha, tal y como se demuestra por la presencia de una mediana y media geométrica
inferior a la media aritmética. Se determinaron unos valores de media geométrica de 5,10 µg/kg (As);
17,98 µg/kg (Cd); 0,68 µg/kg (Hg) y 8,59 µg/kg (Pb) de peso fresco en hígado. El comportamiento
de los datos en el caso de los niveles de metales en los tejidos, por ser tan variables los niveles de
concentración, resulta más adecuado el uso del valor de la mediana o de media geométrica para este
tipo de información (Langlands y col., 1987).
En la tabla 2 se presentan las concentraciones de metales obtenidas en otros estudios. Como se
puede observar, es amplio el rango de concentración de los diferentes metales en los diversos estudios
consultados. Son múltiples los factores que están relacionados con la variación de los niveles de
metales en el organismo como: la edad, raza, especie, sexo, estado reproductivo y nutricional, área
geográfica, nivel local de contaminación ambiental, grado de exposición al contaminante, sistema de
producción, entre otros; que justifican su concentración en los tejidos, como es el caso del hígado
(Al-Awadi et al, 2000; Reilly, 2002; Vahter et al 2006).
Los niveles de metales observados en nuestro estudio en general son más bajos que los observados
en estudios previos llevados a cabo en otros países y en España (ver tabla 2). Si lo comparamos con
un trabajo previo llevado a cabo en ganado vacuno en extensivo en la provincia de León (LópezAlonso et al., 2004) observamos que el nivel de contaminación por arsénico y mercurio sigue siendo
muy bajo, y que la contaminación por cadmio y plomo es menor, concretamente un 66 y un 69%
respectivamente.
La importancia de la valoración de los niveles metales en vísceras y tejidos de origen animal,
radica en la existencia de un riesgo potencial de que los diversos productos de origen animal y
fórmulas lácteas usados por el hombre, provengan de ganado alimentado y localizado en regiones
contaminadas. La determinación de la concentración residual de metales en los diferentes tejidos de
origen animal, es un importante indicador directo del grado de contaminación de estos productos; así
como un indicador indirecto del grado de contaminación ambiental local o periférica, principalmente
el suelo, agua, aire y flora de la zona en donde se localice el ganado (Prankel et al, 2004; Santos et
al, 2004; Calafat et al, 2006). Por lo anterior, se considera necesario el monitoreo periódico de las
concentraciones de los diferentes metales tóxicos en los alimentos y el ambiente para asegurar una
calidad y protección alimentaria.
411
Tabla 1. Niveles de As, Cd, Hg y Pb (µg/kg, peso fresco) en hígado de ganado vacuno en la provincia de León,
NO España.
As
62
Cd
62
Hg
18
Pb
62
6,86
19,9
1,08
9,53
Muestra
N
Media
D.E.
7,87
9,92
1,19
5,42
Media Geométrica
5,10
17,9
0,679
8,59
Mediana
4,09
16,8
0,656
7,94
Valor mínimo
2,06
7,14
0,081
4,29
Valor máximo
47,7
56,7
5,02
35,9
Tabla 2. Niveles de As, Cd, Pb y Hg en hígado de ganado vacuno en diferentes países. Representado como
medias aritméticas (µg/kg, peso fresco) y rango de valores entre paréntesis.
Autor
As
Amodio-Cocchieri et al, 1987 (Italia)
Kramer et al, 1983 (Australia)
Vos et al, 1987 (Holanda)
Hg
<20,0
13
Kottferova y Koréneková, 1995 (República Eslovaca)
Doganoc, 1996 (Eslovenia)
(100-1600)
60,0
50,0 (<20-530)
(5-570)
105,0 (7-360)
(3,0-6,0)
170,0 (10-640)
120,0 (5-850)
4,2
160,0 (10-910)
316,0
465,0
90,0 (<3-620)
100,0 (<50-660)
(50,0-790,0)
(750-1020)
30,0
Farmer y Farmer, 2000 (Kazajstán)
Abou-Arab, 2001 (Egipto)
López-Alonso et al, 2004 (León, España)
López-Alonso et al, 2000 (Galicia,
España)
Miranda et al, 2005 (Asturias, España)
Pb
405
119,0 (38-320)
Falandysz, 1993 (Polonia)
Salisbury et al, 1991 (Canadá)
Cd
(121,0-556,0)
ND
59,6* (13-564)
9,58-10.2*
7,56-83,3*
(ND-7990)
(ND-401)
11,7-12,4*
(ND-363)
ND
28,0* (ND-320)
31,1-47,5*
22,9-29,6*
(ND-446,0)
20,7-38,1*
(3.39-221)
(ND-411)
ND: < límite detección; *: media geométrica
Bibliografía
Abou-Arab AAK. Heavy metal contents in Egyptian meat and the role of detergent washing on their levels. Food
Chem Toxicol 2001;39:593-599.
Al-Awadi FM, Srikumar TS. Trace-element status in milk and plasma of Kuwaiti and non-Kuwaiti lactating
mothers. Nutrition 2000;16:1069-1073.
412
Alberti-Fidanza A, Burini G, Perriello G, Fidanza F. Trace elements intake and status of Italian subjects living
in the Gubbio area. Environ Res 2003;91:71-77.
Amodio-Cocchieri A, Fiore P. 1987. Lead and cadmium concentrations in livestock breed in Campania, Italy.
Bull Environ Contam Toxicol 39:40-464.
Calafat AM, Ye X, Silva MJ, Kuklenyik Z, Needham LL. Human exposure assessment to environmental
chemicals using biomonitoring. Int J Andrology 2006;29:166-171.
Chen H, Lee C, Liao P, Guo Y, Chen C, Su H. Associations between dietary intake and serum polychlorinated
dibenzo-p-dioxin and dibenzofuran (PCDD/F) levels in Taiwanese. Environ Res 2003;91:172-178.
Doganoc D. 1996. Lead and cadmium concentrations in meat, liver and kidney of Slovenian cattle and pigs from
1989 to 1993. Food Add Contam 13(2):237-241.
Falandysz, J. 1991. Manganese, copper, zinc, iron, cadmium, mercury and lead in muscle meat, liver and
kidneys of poultry, rabbit and sheep slaughtered in the northern part of Poland, 1987. Food Additives and
Contaminants. 8 (1): 71-83.
Kottferova J, Korenekova B. The effect of emissions on heavy metals concentrations in cattle from the area of
an industrial plant in Slovakis. Arch Environ Contam Toxicol 1995;29:400-405.
Kramer HL, Steiner JW, Vallely PJ. 1983. Trace element concentrations in the liver, kidney and muscle of
Queensland cattle. Bull Environ Contam Toxicol 30:588-594.
Langlands, J.P.; Donald, G.E.; Smith, A.J. 1987. Analysis of data collected in a residue survey: copper and zin
concentrations in liver, kidney and muscle in Australian sheep and cattle. Aust. J. Exp. Agric. 1987. 27:
485-491.
López Alonso, M.; Benedito, J.L.; Miranda, M.; Castillo, C.; Hernández, J.; Shore, R.F. 2000. Toxic and trace
elements in liver, kidney and meat from cattle slaughtered in Galicia (NW Spain). Food Additives and
Contaminants. 17(6): 447-457.
López Alonso, M.; Prieto Montaña, F.; Miranda, M.; Castillo, C.; Hernández, J.; Benedito, J.L. 2004. Interactions
between toxic (As, Cd, Hg, and Pb) and nutritional essential (Ca, Co, Cr, Cu, Fe, Mn, Mo, Ni, Se, Zn)
elements in the tissues of cattle fron NW Spain. Biometals. 17: 389-397.
Miranda M, López-Alonso M, Castillo C, Hernández J, Benedito JL. 2005. Effects of moderate pollution on
toxic and trace metal levels in calves from a polluted area of Northern Spain. Environ Int 31:543-548.
Prankel SH, Nixon RM, Phillips CJC. Meta-analysis of feeding trials investigating cadmium accumulation in
the livers and kidneys od sheep. Environ Res 2004;94:171-183.
Reilly C. Metal contamination of food, 2nd Ed. Elsevier Applied Science. New York, 2002.
Salisbury, C.D.C.; Chan, W.; Saschenbrecker, P. 1991. Multielement concentrations in liver and kidney tissues
from five species of Canadian slaughter animals. J. Assoc. Off. Anal. Chem. 74 (4): 587-591.
Santos EE, Lauria DC, Porto da Silveira CL. Assesment of daily intake of trace elements due to consumption of
foodstuffs by adult inhabitants of Rio de Janeiro city. Sci Tot Environ 2004;327:69-79.
Vahter M, Akesson A, Lidén C, Ceccatelli S, Berglund M. Gender differences in the disposition and toxicity of
metals. Environ Res 2006.
Vos G, Hovens JPC, Delft WV. 1987. Arsenic, cadmium, lead and mercury in meat, livers and kidneys of cattle
slaughtered in The Netherlands during 1980-1985. Food Add Contam 4(1):73-88.
World Health Organization (WHO). International Program for Chemical Safety Inorganic Lead. Environmental
health criteria 165. Inorganic lead. Geneva, 1995.
Este trabajo ha sido subvencionado por la Junta de Castilla y León (España) Referencia: LE035A06.
Agradecemos su colaboración al Departamento de Bioquímica de la Universidad de Santiago de Compostela y
a la Empresa Ganadera de D. Laurentino Marcos.
413
414
STARVED PERIOD IN LATE GESTATION: EFFECTS ON EWES AND
LAMBS
RESTRICCIÓN DEL ALIMENTO EN LA GESTACIÓN AVANZADA:
EFECTOS SOBRE LA OVEJA Y SOBRE EL CORDERO
UTJECAJ GLADOVANJA TIJEKOM KASNE GRAVIDNOSTI NA OVCU I
JANJAD
Benech A1, Cal L2, Martín A2, Da Silva S1, Lataste V1, Torío R2, Fernández Rodríguez
F3, González Montaña JR2, Prieto Montaña F2 y Rodas E1.
1.
Faculty of Veterinary Science. University de la República. Montevideo, Uruguay. E-mail: alebenech@
hotmail.com
2.
Department of Veterinary Medicine, Surgery and Anatomy, University of León, 24071, Spain.
3. Regional laboratory of Animal Health. Junta de Castilla y León. León, Spain.
SUMMARY
The extensive ovine farming depends too much on the weather conditions and especially on the availability
of food. It was evaluated the effect of food restriction in the final pregnancy stage. It was used double purpose
ewes (meat-wool) maintained in extensive conditions, which it was known breed date. At the 142-d of pregnancy,
animals were divided in three groups. The first group animals had a starved period for 24 h; second one for 48
h and the other group was considered as testing group. It was determined the glycemia, seric cortisol, ketonuria
and urinary pH twice at day in all ewes, as well as glycemia and seric cortisol in lambs at the moment of birth.
Whereas, was determined the vitality and evolution in each lamb during two weeks. All starving ewes showed
significant decrease of glycemia and urinary pH, and increased of ketonuria. It was observed an increase of
blood cortisol level due to stress period for food restriction. All the lambs from starving ewes showed lower
weight than the control group, but those differences disappeared at 15 days-old. There were no differences in
glycemia, blood cortisol, neither in the open field test parameters. There is not difference for mortality rate
among the groups. We can conclude that 24 or 48 hours of starvation from 142nd day of pregnancy caused low
weight in stillbirth, but this condition they could recovered into the two first weeks of life. Mortality rate and
open field test parameters were not affected in the first week.
RESUMEN
Las explotaciones extensivas ovinas son muy dependientes de las condiciones climáticas y sobre todo de
la disponibilidad de alimento. Hemos evaluado la repercusión, tanto sobre las ovejas como sobre las crías, de
la restricción de alimento realizada en la fase final de gestación. Utilizamos ovejas de doble aptitud carne-lana
explotadas en régimen extensivo con fecha de cubrición conocida. En el día 142 de la gestación las ovejas
se dividieron en tres grupos. Un grupo se sometió a un ayuno de 24 horas, otro grupo se mantuvo en ayuno
durante 48 horas y el resto de las ovejas permanecieron en el pasto como grupo control. En las ovejas se midió
la glucemia, el cortisol sérico, la cetonuria y el pH urinario cada 12 horas, así como la glucemia y el cortisol
sérico de los corderos en el momento del nacimiento. Asimismo se valoró la vitalidad y la evolución de los
corderos durante las dos primeras semanas de vida. En los grupos sometidos a ayuno las ovejas presentaron
una disminución significativa de la glucemia y del pH urinario, así como un incremento de la cetonuria. El
incremento del cortisol sanguíneo parece tener su origen en la respuesta de adaptación de la oveja a la restricción
alimentaria. Los corderos de las ovejas sometidas a ayuno presentaron menor peso que los del grupo control,
esas diferencias desaparecieron a los 15 días del parto. No se encontraron diferencias en la glucemia ni el cortisol
415
sanguíneo en el momento del parto, ni en los períodos parto-estación, ni parto-succión; así como tampoco en
la prueba de actividad de los corderos. No se observaron diferencias entre los grupo en la mortalidad. Podemos
concluir que en las ovejas un ayuno de 24 ó 48 h a partir del día 142 de gestación conduce al nacimiento de
corderos más livianos, si bien estos logran una recuperación del peso dentro de los primeros 15 días de vida.
Además no se vio aumentada la mortalidad, ni disminuyó la actividad de los corderos a la semana de vida.
SAŽETAK
Ekstenzivno ovčarenje umnogome zavisi o klimatskim uvjetima te posebice o raspoloživosti krmiva.
Proučili smo utjecaj restriktivne prehrane tijekom završne faze gravidnosti, kako na ovce tako i na janjad.
Korištene su ekstenzivno držane ovce, kombiniranih proizvodnih svojstava (meso-vuna) sa poznatim datumom
parenja. Točno 142. dana gravidnosti životinje su podijeljene u 3 skupine. Jedna je grupa podvrgnuta 24, druga
48 satnom postu, dok su preostale ovce nastavile pasti kao kontrolna skupina. U ovaca je svako 12 sati mjerena
glikemija, serumski kortizol, ketonurija i ph mokraće, kao i glikemija te serumski kortizol u janjadi neposredno
nakon poroda. Jednako tako procjenjivala se vitalnost i razvoj janjadi tijekom prva dva tjedna života. U grupama
ovaca podvrgnutih gladovanju utvrđeno je znakovito smanjenje glikemije i ph urina, kao i porast ketonurije.
S druge pak strane, čini se da je porast krvnog kortizola nastao kao adaptacijski odgovor ovce na restrikciju
prehrane. Janjad ovaca podvrgnutih postu je imala manju težinu, nego janjad kontrolne skupine. Razlike su
nestale 15 dana nakon poroda. Nisu utvrđene razlike u glikemiji i krvnom kortizolu u vrijeme poroda, kao
ni u trajanju razudoblja porod-ustajanje, porod-sisanje te u probama aktivnosti janjadi. Nisu utvrđene razlike
između grupa u mortalitetu. Možemo zaključiti kako u ovaca post u trajanju od 24 ili 48 sati počevši 142.
dana gravidnosti dovodi do rađanja janjadi manje tjelesne mase, koja nadoknadi isti manjak unutar prvih 15
dana života. Nadalje, nije utvrđen povećani mortalitet kao ni smanjena aktivnost janjadi tijekom prvog tjedna
života.
INTRODUCTION
Las condiciones de cría extensiva en que se producen los ovinos en Uruguay hacen que la
alimentación de la oveja gestante sea muy dependiente de las condiciones climáticas. Por ello es
posible que al final de la gestación el aporte de las praderas naturales no sea suficiente para las
demandas energéticas que requiere el feto en crecimiento.
La tasa de crecimiento fetal está influenciada significativamente por el ambiente uterino, quien
a su vez es modulado por hormonas, factores de crecimiento y nutrientes. Por tal motivo, el estado
nutricional de la madre puede afectar el desarrollo y crecimiento de la placenta y del feto (Robinson,
1983; Robinson, 1996) ya que una mala nutrición de la madre durante 7 a 21 días entre los días 124 y
138 lleva a una reducción del 50 % en la captación neta de glucosa a nivel umbilical y de la glucemia
fetal (Leury et al, 1990).
Aunque muchos autores coinciden en que las necesidades nutricionales del feto son bajas al
comienzo y en la mitad de la gestación (Wallace, 1948; Geenty, 1997; Père, 2003; Rhind, 2004;
Montossi, 2005), se ha encontrado que una baja nutrición de la madre en todos los estadios de la
preñez pueden afectar al crecimiento fetal (Rhind et al, 1989; Abecia et al, 1997; Bloomfield et al,
2003; Fahey et al, 2005).
Los efectos de una baja nutrición han sido largamente estudiados en los animales de producción,
particularmente en los rumiantes ya que muchas veces son explotados en zonas marginales en las
cuales el aporte de nutrientes es bajo (Rhind, 2004). Sin embargo existe escasa bibliografía sobre la
evaluación del efecto del ayuno agudo sobre la viabilidad de los corderos.
El objetivo del presente trabajo es evaluar el efecto de la restricción alimenticia, al final de la
gestación, tanto sobre la oveja como sobre el cordero.
416
El protocolo experimental del presente trabajo fue aprobado por la Comisión Honoraria de
Experimentación Animal (CHEA) de la Universidad de la República, Uruguay y se realizó en el
Campo Experimental Nº 2 de laFacultad de Veterinaria (Libertad, Km 42,5 ruta Nº1, San José,
Uruguay, 34º, 38´ S y 56º,39´º W).
Se utilizaron 30 ovejas preñadas de raza Corriedale obtenidas de una rebaño de 50 animales
cuyos celos fueron sincronizados mediante el uso de esponjas intravaginales conteniendo 60 mg/
esponja de medroxiprogesterona (Sincrocel®, Santa Elena) durante 12 días (Romano et al, 1993).
La cubrición se realizó con dos carneros de raza Corriedale a los que se les preparó con arneses
marcadores. Los carneros se introdujeron en el corral donde se encontraban las ovejas el mismo día
en que se retiraron las esponjas intravaginales y dos veces por días se controló la monta. Las ovejas
marcadas en la grupa fueron separadas, registrándose el día en que fue cubierta cada oveja como día
cero de la gestación, la que se confirmó mediante ecografía transabdominal entre los días 50 y 60 de
la monta (Buckrell, 1988). En el día 142 de gestación los animales se dividieron en tres grupos al
azar: Grupo A) 10 ovejas se sometieron a un ayuno total para sólidos de 24 h, Grupo B) 10 ovejas se
sometieron a un ayuno total para sólidos de 48 h y Grupo C) 10 ovejas que permanecieron a campo
como grupo control.
En las ovejas se registró el peso corporal y se tomaron muestras de sangre y orina cada 12 h para
determinar glicemia y cortisol en sangre y cuerpos cetónicos (CC) y pH en orina. En los corderos se
registró el peso al nacimiento, a los 15 días y al momento del destete (2 meses de edad), la mortalidad
dentro de los 3 primeros días de vida, el tiempo que tardaron en erguirse sobre las 4 patas y que
mamaron calostro por primera vez. También se obtuvo una muestra de sangre de cada cordero al
momento del nacimiento para determinar glucosa y cortisol y se realizó una prueba de actividad de
“campo abierto” (open field) a la semana de vida.
Para el estudio estadístico se utilizó el programa informático SPSS 13.0.
RESULTS AND DISCUSSION
Al final del protocolo el grupo control registró un aumento de peso del 1,2% (p<0,001) con respecto
a su peso inicial. Las ovejas del grupo sometido a ayuno durante 24 h presentaron una pérdida de peso
de un 5% de su peso inicial, mientras que las ovejas del grupo encerrado durante 48 h perdieron en
promedio un 11% de su peso inicial.
A las 12 horas de ayuno los grupos A y B mostraron un descenso significativo de la glucemia (p
< 0,001) que se vio reflejado en la presencia de altos niveles de CC y descenso significativo del pH
en orina. Este hecho fue descrito por Dufield (2000) quien afirma que la presencia de CC en la orina
se corresponde con cetosis subclínica. Una oveja del grupo B debió ser retirada por presentar signos
de Toxemia de la Gestación, lo que demuestra el alto riesgo de producción de esta enfermedad por
la restricción de comida al final de la gestación. También se observó aumento del cortisol en suero,
posiblemente como respuesta a la hipoglucemia causada por el ayuno (Cal y col, 2006).
Los corderos nacidos de los grupos A y B fueron significativamente más livianos que los corderos
nacidos del grupo control (p < 0,05). Posiblemente la hipoglucemia provocada por restricción de
comida repercutió en la glucemia fetal, llevando al feto a utilizar menos glucosa con el consecuente
retraso en el crecimiento (Liggins y Thorburn, 1994). Sin embargo el peso a los 15 días y al destete
no mostró diferencias entre los grupos, mostrando la capacidad de crecimiento compensatorio ya
descrito por Banchero y col (2005) en los corderos mellizos con respecto a los partos simples.
417
La restricción de comida al final de la gestación no afectó los períodos parto-estación ni partosucción, al contrario de lo encontrado por Dwyer y col (2003) de que los corderos más pesados tienen
un mayor porcentaje de éxito en encontrar la teta, así como tampoco se registraron diferencias en la
prueba de actividad de “campo abierto” realizada a la semana de vida. La glucemia y el cortisol en
suero al nacimiento no mostraron diferencias entre los grupos. En los grupos tratados no aumentó
la mortalidad de los corderos, que se encontró dentro de los valores registrados históricamente en el
país (del 10 al 35 %) (Mari, 1987).
REFERENCES
Abecia, J.A; Lozano, J.M; Forcada, F; Zarazaga, L (1997). Effect of level of dietary energy and
protein on embrio survival and progesterona production on day 8 of pregnancy in Rasa Aragonesa
ewes. Anim. Reprod. Sci. (48): 209-218.
Banchero, G; Quintans, G; Milton, J; Lindsay, D (2005). Comportamiento maternal y vigor de los
corderos al parto: Efecto de la carga fetal y la condición corporal. Reproducción ovina: recientes
avances realizados por el INIA, Treinta y Tres, Uruguay,pp: 85-102.
Bloomfield, F.H; Oliver, M.H; Hawkins, P; Campbell; M; Phillips, D.J; Gluckman, P.D; Challis,
J.R; Harding, J.E (2003). A periconceptional nutritional origin for noninfectious preterm birth.
Science (300): 561–562.
Buckrell, B.C (1988). Application of ultrasonogrphy in reproduction in sheep and goats.
Theriogenology (29): 11-20.
Cal, L; Benech, A; Abreu, M.N; Borteiro, C; Cruz, J.C; Ricciardi, L; Godiño, L; Nievas, C; Rodas,
E; González Montaña, J.R (2006). Evaluaciónpreliminar del riesgo de aparición de toxemia de
la gestación en ovejas bajo diferentes manejos nutricionales y sometidas a ayuno de 48 horas.
VETERINARIA (Montevideo), vol. 41 (161-162): 39-44.
Duffield, T (2000). Subclinical ketosis in lactating dairy cattele. Vet. Clin. North Am.:Food An.
Pract., vol. 16 (2): 231-251.
Dwyer, C.M; Lawrence, A.B; Bishop, S.C; Lewis, M (2003). Ewe-lamb behaviours at birth are
affected by maternal undernutrition in pregnancy. British J. Nutr. (89): 123-136.
Fahey, A.J; Brameld, J.M; Parr, T; Buttery, P.J (2005). The effect of maternal undernutrition before
muscle differentiation on the muscle fiber development of the newborn lamb. J Anim Sci. vol.83,
(11):2564-2571.
Geenty, K.G (1997). A guide to improve lambing percentage of farmers and advisors. Rev 200 by
2000.
Leury, B.J; Bird, A.R; Chandler, K.D; Bell, A.W (1990). Glucose partitioning in the pregnant ewe:
effects of undernutrition and exercise. British J. Nutrition (64): 449-462.
Liggins, G. C.y Thorburn, G. D (1994). Initiation of parturition. In: Marshall´s Physiology of
Reprodaction, Pregnancy and lactation, Part Two, Fetal Physiology, Parturition and Lactation G.
E. Lamming (Editor),4th edn., Vol. 3,. Chapman and Hall, London.
Mari, J (1987) Enfermedades que afectan la supervivencia del cordero. En: Enfermedades de los
lanares. Tomo III. J. Bonino (Ed). Hemisferio Sur, Montevideo – Uruguay.
Montossi, F; De Barbieri, I; Digiero, A; Martinez, H; Nolla, M; Luzardo, S; Mederos, A; San Julián,
R; Saint, W; Levratto, J; Furgoni, J; Lima, G; Costales, J (2005) La esquila preparto temprana:
Una nueva opción para la mejora reproductiva ovina.En: Seminario de actualización técnica.
418
Reproducción ovina: recientes avances realizados por el INIA, Treinta y Tres, Uruguay, pp: 85102
Père, M.C (2003). Materno-foetal exchanges and utilisation of nutrients by the foetus: comparision
between species. Reprod. Nutr. Dev. (43): 1-15.
Rhind, S.M (2004). Effect of maternal nutricio non fetal and neonatal in reproductive development
and function. Anim Reprod Sci 82-83: 169-181.
Rhind, S.M; McKelvey, W.A.C; McMillen, S.R; Gunn, R.G; Elston, D.A. (1989). Effect of restricted
food intake, before and/or after mating, on the reproductive performance on greyface ewes.
Anim. Prod. (48): 149-155.
Robinson J J. (1983) Nutrition of the pregnant ewe. En: Sheep Production. London, Butterworths.
Robinson, J.J. (1996). Nutrition and reproduction. Am. Reprod. Sci. (42): 25-34.
Romano, J.E y Benech, A (1996). Effects of service and vaginal-cervix anaesthesia on estrus duration
in dairy goats. Theriogenology (45): 691-696.
Wallace, L.R (1948). The growth of the fetal lamb before and after birth in relation to the level of
nutrition. J.Agric. Sci. (38): 17-302.
419
420
BONE METABOLISM IN DAIRY EWES DURING LACTATION
KOŠTANI METABOLIZAM U MLIJEČNIH OVACA TIJEKOM
LAKTACIJE
Filipović, Natalija1, Nikica Prvanović2, T. Mašek3, Ž. Mikulec3, Z. Stojević1
1
Department of Physiology and Radiobiology, Faculty of Veterinary Medicine University of Zagreb
2
Clinic for Obstetrics and Reproduction, Faculty of Veterinary Medicine University of Zagreb
3
Department of Animal Nutrition, Faculty of Veterinary Medicine University of Zagreb
Summary
Pregnancy and lactation induce changes in mineral metabolism and may compromise the skeletal integrity.
Due to a high milk production of dairy ewes lactation is a metabolically demanding process. A rapid increase
in milk production in the beginning of lactation causes increase the need for calcium and phosphorus, which
cannot be adequately satisfied by absorption from the digestive tract. The consequence is an elevated rate of
bone resorption. The aim of this study was to investigate the changes in concentration of bone remodelling
markers in relation to the hormonal changes and milk production during lactation in dairy ewes. The research
was conducted on ten crossbred Istrian X East Friesian dairy ewes. During the experimental period the ewes
received 1 kg/ewe/day of concentrate mixture, 0.3 kg/ewe/day Lucerne hay and were allowed to graze from 7:00
a.m. to 15:00 p.m. and from 18:00 p.m. to 21:00 p.m. on rotational mixed grass pasture. The concentrate mixture
was composed of corn (35%), barley (20%), sunflower meal (16%), wheat bran (16%), soybean meal (8%),
and vitamin-mineral mixture (5%). Samples for blood analyses were taken in five times during the experiment:
10 days before lambing (D -10), on day 10 (D +10), day 20 (D +20), day 90 (D +90) and on day 130 after
lambing (D +130). Concentrations of crosslinked carboxyterminal telopeptide of type I collagen, a marker
of bone resorption (CTx); parathyroid hormone (PTH), estradiol 17β (E17 β), and activity of bone specific
alkaline phosphatase, a marker of bone synthesis (BAP) were determined in blood serum by using commercial
ELISA kits. A statistically significant influence of experimental period was observed for CTx (p<0.0001), BAP
(p<0.0001), E17 β (p<0.001), milk quantity (p<0.001) and milk protein yield (p<0.0001). The concentration of
CTx was significantly lower on D +90 in comparison with D -10 and D +20 (p<0.05 and p<0.001) and on D
+130 in comparison with D -10, D +10 (p<0.05) and D +20 (p<0.001). The activity of BAP was significantly
higher on D +90 in comparison with D -10 (p<0.01), D +10, D +20 and D +130 (p<0.001). The concentration of
E17 β was significantly higher on D -10 in comparison with D +10 (p<0.05), D +20, D +90 and D +130 (p<0.01).
The milk protein yield was significantly higher on D +20 in comparison with D+10 and D +130 (p<0.01) and
on D +90 in comparison with D +10 (p<0.05). A statistically significant correlation between the milk protein
yield and concentration of CTX (r = 0.75; p<0.0001) and activity of BAP (r = - 0.50; p<0.01); between the
activity of BAP and concentration of CTx (r = - 0.46; p<0.01) and PTH (r = - 0.33; p<0.05); and between the
concentration of PTH and E17 β (r = 0.66; p<0.0001). The changes in the CTx concentration indicate to high
rate of bone resorption in the late pregnancy and early lactation that decreased in the late lactation and was
directly related to the milk production rate. The changes in the activity of the BAP prove decreased rate of bone
synthesis in the late pregnancy and early lactation that increased in the late lactation and was inversely related to
the milk production rate. The bone resorption and the bone synthesis were inversely related during lactation in
ewes. The concentration of the PTH was inversely related to the rate of bone synthesis that is consistent to the
role of PTH in osteoblasts activity. The relation of PTH and E17 β concentrations shows that the regulation of
bone metabolism in late pregnancy and early lactation is, similarly to other species, at least in part, influenced
by estradiol changes. The presented data confirm a high mobilisation of minerals from skeleton during late
pregnancy and early lactation in dairy ewes. The level of bone loss was directly related to the milk production
rate. In comparison with data from literature for dairy cows, the similar influence of lactation on bone resorption
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was observed for ewes, but in dry period the rate of bone resorption was higher in ewes indicating relatively
higher need for minerals in the late pregnancy in ewes. Observed changes in levels of bone markers are consistent
to data from literature for East Friesian ewes, but earlier decrease of bone resorption rate was observed in the
present investigation that was related to shorter lactation and earlier fall of milk production rate.
Key words: bone metabolism, ewes, dry period, lactation, milk production
Sažetak
Istražen je utjecaj kasne gravidnosti i laktacije na koncentraciju biljega koštane pregradnje u krvnom
serumu ovaca. Istraživanje je provedeno na deset križanih mliječnih ovaca (istarske X istočnofrizijska). Uzorci
krvi uzimani su 5 puta tijekom pokusa: 10 dana prije janjenja (D -10), 10. dan (D +10), 20. dan (D +20),
90. dan (D +90) i 130. dan poslije janjenja (D +130). U uzorcima krvnog seruma određene su koncentracije
paratireoidnog hormona (PTH), križno povezanog C-terminalnog telopeptida kolagena tipa I (CTx) – pokazatelja
koštane resorpcije; estradiola 17 β (E17 β), te aktivnosti koštane alkalne fosfataze (BAP) – pokazatelja koštane
sinteze. Statistički značajan utjecaj istraživanih razdoblja utvrđen je za CTx (p<0.0001), BAP (p<0.0001), E17 β
(p<0.001), količinu mlijeka (p<0.001) i ukupnu količinu bjelančevina u mlijeku (p<0.0001). Koncentracija CTx
bila je značajno niža u razdoblju D +90 u usporedbi s D -10 i D +20 (p<0.05 i p<0.001), te u razdoblju D +130
u usporedbi s D -10, D +10 (p<0.05) i D +20 (p<0.001). Aktivnost BAP bila je značajno viša u razdoblju D +90
u usporedbi s D -10 (p<0.01), D +10, D +20 i D +130 (p<0.001). Koncentracija E17 β bila je značajno viša u
razdoblju D -10 u usporedbi s D +10 (p<0.05), D +20, D +90 i D +130 (p<0.01). Ukupna količina bjelančevina
u mlijeku bila je značajno viša u razdoblju D +20 u usporedbi s D+10 i D +130 (p<0.01) i u razdoblju D +90 u
usporedbi s D +10 (p<0.05). Statistički značajna korelacija utvrđena je između ukupne količine bjelančevina
u mlijeku i koncentracije CTX (r = 0.75; p<0.0001) i aktivnosti BAP (r = - 0.50; p<0.01); između aktivnosti
BAP i koncentracije CTx (r = - 0.46; p<0.01) i PTH (r = - 0.33; p<0.05); kao i između koncentracije PTH i E17
β (r = 0.66; p<0.0001). Rezultati istraživanja ukazuju na povećani opseg koštane resorpcije i smanjeni opseg
koštane sinteze tijekom kasne gravidnosti i rane laktacije u ovaca, koji je izravno povezan s količinom ukupnih
bjelančevina dnevno izlučenih mlijekom, kao pokazateljem proizvodnje.
Ključne riječi: koštani metabolizam, ovce, suhostaj, laktacija, proizvodnja mlijeka
Uvod
Gravidnost i laktacija induciraju promjene u metabolizmu minerala koje mogu utjecati na
integritet kostura. Visoka proizvodnja mlijeka u mliječnih krava, ovaca i koza metabolički je vrlo
zahtjevan proces. U početku laktacije dolazi do naglog porasta proizvodnje mlijeka. Navedeno
uzrokuje porast potreba za kalcijem i fosforom, koje se ne mogu u potpunosti zadovoljiti resorpcijom
ovih minerala iz probavnog trakta, već se moraju nadoknaditi iz zaliha u kosturu (Liesegang i sur.,
2000). Tako je u mliječnih krava tijekom prvih 6 – 8 tjedana laktacije bilanca kalcija negativna
i kostur gubi oko 13% svojeg sadržaja kalcija (Horst i sur., 2005). U laktaciji i zadnjoj trećini
gravidnosti kod žena također je utvrđen povećani obim koštane pregradnje (Kovacs i Kronenberg,
1997). Metabolizam minerala reguliran je djelovanje brojnih hormona, od kojih se najvažnija uloga
pripisuje paratireoidnom hormonu (PTH). PTH potiče razgradnju koštane mase, poticanjem osteocitne
osteolize, kao i posrednim povećanjem broja i aktivnosti osteoklasta. Osim što potiče resorpciju
kosti, PTH istodobno ima i anaboličke učinke na koštano tkivo (Swarthout i sur., 2002; Jilka, 2007).
U bubrezima PTH povećava reapsorpciju kalcija u distalnim kanalićima bubrega i koči reapsorpciju
fosfata u proksimalnim kanalićima bubrega, kočeći izlučivanje kalcija i potičući izlučivanje fosfata
mokraćom, te potiče tvorbu 1,25 dihidroksi vitamina D3, koji utječe na povećanu razgradnju koštane
mase i povećava apsorpciju kalcija i fosfata iz crijeva (Genuth, 1988). Opseg koštanog prijetvora
može se pratiti posredno, mjerenjem biljega koštane pregradnje u krvi i mokraći, kao pokazatelja
aktivnosti koštanih stanica (Delmas, 1995). Križni povezanog C-terminalni telopeptid kolagena tipa
422
I (carboxyterminal crosslinked telopeptide of type I collagen, CTx), je jedan od produkata razgradnje
kolagena tipa I (koji je glavna komponenta organskog dijela koštanog matriksa) i jedan od najčešće
korištenih biljega koštane razgradnje u ljudi (Delmas, 1995). Koštanospecifična alkalna fosfataza
(bone specific alkaline phosphatase, BAP), je enzim osteoblasta mjerenje čije aktivnosti se učestalo
koristi kao biljeg opsega koštane sinteze (Delmas, 1995). Osim u humanoj medicini, primjena
navedenih biljega sve više se koristi i u istraživanjima koštanog metabolizma u domaćih životinja
(Liesegang i sur., 2000; 2006; 2007; Lepage i sur., 2001; Holtenius i Ekelund, 2005; Seebeck i sur.,
2005). U vezi s navedenim, istražen je utjecaj kasne gravidnosti i laktacije na koštani metabolizam
mliječnih ovaca, te njegova povezanost s kretanjem proizvodnje mlijeka, kao i hormonalnim
promjenama karakterističnim za ovo razdoblje.
Materijal i metode
Istraživanje je provedeno na deset križanih mliječnih ovaca (istarske X istočnofrizijska). Tijekom
pokusnog razdoblja obrok se sastojao od 1 kg smjese i 0.3 kg sijena lucerne po ovci dnevno uz
ispaša od 7:00 do 15:00 h i od 18:00 do 21:00 h na pregonskom pašnjaku zasijanom djetelinskotravnom smjesom. Smjesa se sastojala od kukuruza (35%), ječma (20%), suncokretove sačme (16%),
pšeničnog brašna (16%), sojine sačme (8%), i vitaminsko mineralnog premiksa (5%). Uzorci krvi
uzimani su 5 puta tijekom pokusa: 10 dana prije janjenja (D -10), 10. dan (D +10), 20. dan (D
+20), 90. dan (D +90) i 130. dan poslije janjenja (D +130). Nakon grušanja i centrifugiranja (2000
x g/20 minuta/4°C), iz uzoraka je izdvojen krvni serum. Uzorci za određivanje aktivnosti BAP i
koncentracije PTH su pohranjeni na -60°C, a ostali na -20°C. Koncentracija/aktivnost pojedinih
pokazatelja koštanog metabolizma određena je metodom ELISA, korištenjem gotovih kompleta
reagensa: Intact PTH (Parathyroid Hormone) ELISA (Biomerica, Newport Beach, SAD) za PTH;
Serum CrossLaps® ELISA (Nordic Bioscience Diagnostics, Denmark) za CTx; Metra BAP EIA Kit
(Quidel Corporation, San Diego, USA) za BAP; i VITROS Estradiol Reagent Pack (Ortho-Clinical
Diagnostics, Amersham, UK) za E17β. Tijekom analiza nije se odstupalo od uputstava proizvođača.
Uzorci mlijeka uzimani su usporedno s uzorcima krvi. Količine proizvedenog mlijeka do odbića
određene metodom po Benson-u i sur. (1999), a nakon odbića vaganjem količine mlijeka ispuštene
u menzuru iz uređaja za mužnju. Koncentracija bjelančevina u mlijeku određena je metodom
infracrvene spektrofotometrije (Milcoscan FT 120, Foss, Danska). Rezultati su statistički obrađeni
korištenjem računalnog paketa Statistica 7 (StatSoft, USA): analizom varijance (One way ANOVA),
post-hoc Tukey testom i određivanjem koeficijenta linearne korelacije. Razlike su smatrane statistički
značajnima ako je p<0.05.
Rezultati
Statistički značajan utjecaj istraživanih razdoblja utvrđen je za CTx (p<0.0001), BAP (p<0.0001),
E17 β (p<0.001), količinu mlijeka (p<0.001) i ukupnu količinu bjelančevina u mlijeku (p<0.0001).
Koncentracija CTx bila je značajno niža u razdoblju D +90 u usporedbi s D -10 i D +20 (p<0.05 i
p<0.001), te u razdoblju D +130 u usporedbi s D -10, D +10 (p<0.05) i D +20 (p<0.001). Aktivnost
BAP bila je značajno viša u razdoblju D +90 u usporedbi s D -10 (p<0.01), D +10, D +20 i D +130
(p<0.001). Koncentracija E17 β bila je značajno viša u razdoblju D -10 u usporedbi s D +10 (p<0.05),
D +20, D +90 i D +130 (p<0.01). Ukupna količina bjelančevina u mlijeku bila je značajno viša u
razdoblju D +20 u usporedbi s D+10 i D +130 (p<0.01) i u razdoblju D +90 u usporedbi s D +10
423
(p<0.05). Statistički značajna korelacija utvrđena je između ukupne količine bjelančevina u mlijeku
i koncentracije CTX (r = 0.75; p<0.0001) i aktivnosti BAP (r = - 0.50; p<0.01); između aktivnosti
BAP i koncentracije CTx (r = - 0.46; p<0.01) i PTH (r = - 0.33; p<0.05); kao i između koncentracije
PTH i E17 β (r = 0.66; p<0.0001).
Tablica 1. Deskriptivna statistika za istraživane pokazatelje u mliječnih ovaca tijekom kasne gravidnosti i laktacije
Table 1. Descriptive statistics for investigated parameters in dairy ewes during late pregnancy and lactation
CTx (ng/mL)
Days
before/
after
calving
-10
+10
+20
+90
+130
1,38
5
1,31
4,5
1,69
1,3
0,54
1,2,3
0,43
4,5
±0,57
±0,53
±0,60
±0,15
±0,19
BAP
PTH
E17 β (pmol/
L)
(U/L)
(pg/mL)
21,00 ±10,96 151,80 ±75,96 2,3,4,557,80
4
11,50 ±5,48 161,00 ±141,65 128,20
4
12,10 ±4,46 170,30 ±68,80 125,90
1,2,3,5
35,20 ±10,74 144,80 ±102,40 125,40
4
19,80 ±9,69 171,30 ±82,22 125,90
4
milk protein
(g/day)
±62,29
±12,90
±11,99
±10,66
±7,50
3,49
5,08
2,3
2,27
2,3
1,95
3,4,5
2,4,5
±0,95
±1,49
±0,52
±0,50
Superskripti označavaju prema kojoj skupini postoji statistički značajna razlika.; Superscriptes denotes
statistically significant differences between groups.
Rasprava
Istražen je utjecaj kasne gravidnosti i laktacije na koštani metabolizam ovaca, praćenjem
koncentracija biljega koštane pregradnje u krvnom serumu. Kretanje koncentracija CTx ukazuje
na visoki opseg koštane resorpcije u kasnoj gravidnosti i ranoj laktaciji, koji se smanjio u
kasnoj laktaciji i bio izravno povezan s proizvodnjom mlijeka, izraženom kroz količinu ukupnih
bjelančevina dnevno izlučenih mlijekom. Kretanje aktivnosti BAP ukazuje na smanjeni opseg
koštane sinteze tijekom kasne gravidnosti i rane laktacije, koji je porastao u kasnoj laktaciji i bio
obrnuto proporcionalan s količinom bjelančevina izlučenih mlijekom. Navedeno je u suglasju s
podacima iz literature dobivenim istraživanjima na mliječnim kravama, ovcama i kozama, u kojima
je u početku laktacije utvrđen je povećani opseg koštane resorpcije i smanjeni opseg koštane sinteze
(Liesegang i sur., 2000; 2006; 2007; Holtenius i Ekelund, 2005; Filipović i sur., 2008). Opseg
koštane resorpcije i sinteze bili su obrnuto proporcionalni tijekom laktacije u ovaca. Koncentracija
PTH negativno je korelirala s opsegom koštane sinteze, što je u suglasju s ulogom PTH u poticanju
aktivnosti osteoblasta (Swarthout i sur., 2002; Jilka, 2007). Povezanost koncentracija PTH i E17 β
pokazuje da je regulacija koštanog metabolizma u kasnoj gravidnosti i ranoj laktaciji kod ovaca,
slično ostalim vrstama (Kovacs i Kronenberg, 1997; Filipović i sur., 2008), barem djelomično pod
utjecajem promjena koncentracije estradiola u cirkulaciji. Prikazani rezultati potvrđuju visoki opseg
mobilizacije minerala iz kostura tijekom kasne gravidnosti i rane laktacije u mliječnih ovaca. Opseg
gubitka koštane mase bio je izravno povezan s proizvodnjom mlijeka. Kao pokazatelj proizvodnosti
korištena je ukupna količina bjelančevina izlučena dnevno mlijekom. U usporedbi s podacima iz
literature za mliječne krave (Liesegang i sur., 2000; Holtenius i Ekelund, 2005; Filipović i sur.,
2008), utvrđen je sličan utjecaj laktacije na koštanu resorpciju kod ovaca, ali u suhostaju je opseg
koštane resorpcije bio visok u ovaca, što ukazuje na relativno visoke potrebe za mineralima u kasnoj
gravidnosti kod ovaca. Uočene promjene razine koštanih biljega u suglasju su s podacima iz literature
za istočnofrizijske ovce (Liesegang i sur., 2006; 2007), ali je u našem istraživanju utvrđen raniji pad
opsega koštane resorpcije tijekom laktacije, što je najvjerojatnije bilo povezano s kraćom laktacijom
i ranijim padom produkcije mlijeka.
424
Literatura
Benson, M. E., M. J. Henry, R. A. Cardellino (1999): Comparison of weigh-suckle-weigh and machine milking
for measuring ewe milk production. J. Anim. Sci. 77, 2330-2335.
Delmas, P. D. (1995): Biochemical markers of bone turnover. Acta Orthop. Scand. 66 (Suppl 266), 176-182.
Filipović, N., Z. Stojević, M. Zdelar-Tuk, V. Kušec (2008): Plasma parathyroid hormone-related peptide and
bone metabolism in periparturient dairy cows. Acta Vet. Hung. 56, 235–244.
Genuth, S. M. (1988): Endocrine System. U: Berne, R. M., M. N. Levy, Physiology, 2nd edition, The C. V.
Mosby Company, St. Louis, Washington, D. C., Toronto, Hrvatsko izdanje, Medicinska naklada, Zagreb,
pp 819-1023.
Holtenius, K., A. Ekelund (2005): Biochemical markers of bone turnover in the dairy cow during lactation and
the dry period. Res. Vet. Sci. 78, 17-19.
Horst, R. L., J. P. Goff, T. A. Reinhardt (2005): Adapting to the Transition Between Gestation and Lactation:
Differences Between Rat, Human and Dairy Cow. J. Mammary Gland Biol. Neoplasia. 10, 141-156.
Jilka, R. L. (2007): Molecular and cellular mechanisms of the anabolic effect of intermittent PTH. Bone. 40,
1434-1446.
Kovacs, C. S., H. M. Kronenberg (1997): Maternal-Fetal Calcium and Bone Metabolism During Pregnancy,
Puerperium, and Lactation. Endocr. Rev.18, 832-872.
Lepage, O. M., B. Carstanjen, D. Uebelhart (2001): Non-invasive assessment of equine bone: an update. Vet.
J. 161, 10-22.
Liesegang A., R. Eicher, M. L. Sassi, J. Risteli, M. Kraenzlin, J. L. Riond, M. Wanner (2000): Biochemical
markers of bone formation and resorption around parturition and during lactation in dairy cows with high
and low standard milk yields. J. Dairy Sci. 83, 1773-1781.
Liesegang A., J. Risteli, M. Wanner (2006): The effects of first gestation and lactation on bone metabolism in
dairy goats and milk sheep. Bone. 38, 794-802.
Liesegang A., J. Risteli, M. Wanner (2007): Bone metabolism of milk goats and sheep during second pregnancy
and lactation in comparison to first lactation. J. Anim. Physiol. Anim. Nutr. (Berl) 91, 217-225.
Seebeck P, Bail HJ, Exner C, Schell H, Michel R, Amthauer H, Bragulla H, Duda GN. (2005): Do serological
tissue turnover markers represent callus formation during fracture healing? Bone. 37, 669-677.
Swarthout, J. T., R. C. D’Alonzo, N. Selvamurugan, N. C. Partridge (2002): Parathyroid hormone-dependent
signaling pathways regulating genes in bone cells. Gene. 282, 1-17.
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ACTIVITY OF GLUTATHIONE PEROXIDASE IN SPERM AND BLOOD
SERUM OF SIMMENTAL BREEDING BULLS
AKTIVNOST GLUTATION PEROKSIDAZE U SPERMI I KRVNOM
SERUMU BIKOVA SIMENTALSKE PASMINE
I. Majić-Balić1, S. Milinković-Tur2, J. Piršljin2, B. Beer Ljubić2, A. Orak1, P. Božić3
Centar for reproduction and animal breeding, Potočka 20, Križevci, Croatia
1
2
Department of physiology and radiobiology, University of Zagreb, Faculty of veterinary medicine, Heinzelova
55, Zagreb, Croatia
Centar for reproduction and animal breeding, Planinska 2b, Zagreb, Croatia
3
Sažetak
Glutation peroksidaza je enzim koji ima ključnu ulogu u zaštiti stanica od oksidativnih oštećenja
uklanjajući vodikov peroksid kao i druge hidroperokside. Porodicu glutation peroksidaza čine četiri izoenzima
koji u aktivnom centru sadrže element selen i jedan izoenzim koji ne sadrži selen. Membrana spermija bogata
je višestrukonezasićenim masnim kiselinama koje su vrlo osjetljive na oksidaciju pri čemu nastaju lipidski
hidroperoksidi. Aktivnost antioksidativnih enzima u citoplazmi spermija je ograničena, te je stoga sastav sjemene
plazme vrlo važan u očuvanju integriteta i oplodne sposobnosti spermija. Mnogobrojni okolišni, fiziološki i
genetski čimbenici djeluju na smanjenje kvalitete sperme i mogu dovesti do neplodnosti životinje te je stoga
cilj rada bio utvrditi promjene aktivnosti glutation peroksidaze u spermi i krvnom serumu rasplodnih bikova
simentalske pasmine različite dobi. Uzorci sperme uzimani su pomoću umjetne vagine, a uzorci krvi punkcijom
v. jugularis od dvije skupine bikova: devet mlađih bikova starih između dvije i četiri godine i osam bikova starih
između šest i deset godina. Volumen ejakulata, koncentracija spermija te progresivna pokretljivost određeni su
u ejakulatu bikova. U izoliranim spermijima i krvnom serumu bikova određena je aktivnost izoenzima glutation
peroksidaze ovisne o selenu (SeGSH-Px), dok je u sjemenoj plazmi određena aktivnost ukupne glutation
peroksidaze (ukupna GSH-Px), SeGSH-Px kao i aktivnost glutation peroksidaze neovisne o selenu (neSeGSHPx). Aktivnost SeGSH-Px u krvnom serumu starijih bikova bila je značajno viša u odnosu na aktivnost kod
mlađih životinja (144,63 ± 48,79 U/L odnosno 80,67 ± 31,14 U/L; P<0,01). Usporedbom aktivnosti ukupne
GSH-Px i njenih izoenzima u spermijima i sjemenoj plazmi bikova utvrđene su vrlo ujednačene vrijednosti u
promatranim skupinama. Ocjenom volumena i koncentracije ejakulata kao i progresivne pokretljivosti spermija
u navedene dvije dobne skupine bikova nisu utvrđene značajne razlike. Navedeni rezultati ukazuju da dob utječe
na aktivnost glutation peroksidaze u krvnom serumu, no ne i na aktivnost ovog enzima u spermi rasplodnih
bikova simentalske pasmina.
Abstract
Glutathione peroxidase is an enzyme which plays a key role in protection of cells from oxidative damages
by removing hydrogen peroxide and organic peroxides. Family of glutathione peroxidase consists of four
isoenzymes and et the active site they contain selenium and one isoenzyme which does not contain selenium.
Sperm membrane is rich in polyunsaturated fatty acids susceptible to oxidation, which leads to creation of
lipid hydroperoxides. Activity of antioxidant enzymes in sperm cytoplasm is limited, so the composition of
seminal plasma is of crucial importance in preserving integrity and fertilization capacity of the sperm. Several
environmental, physiological and genetic factors affect reduction of sperm quality and can lead to infertility
of an animal, so the objective of this study was to define variations in activity of glutathione peroxidase in
sperm and blood serum of breeding bulls of Simmental breed of different ages. Sperm samples were collected
with artificial vagina and blood samples through punction v. jugularis from two groups of bulls: nine younger
bulls aged between two and four years and eight bulls aged between six and ten years. Volume of ejaculate,
sperm concentration and progressive motility were analyzed in the samples. Activity of selenium dependent
glutathione peroxidase (SeGSH-Px) was determined in isolated sperms and blood serum of bulls, whereas in
427
seminal plasma the activity of total glutathione peroxidase (total GSH-Px), SeGSH-Px was measured, as well
as activity of non-selenium dependent glutathione peroxidase (nonSeGSH-Px). Activity of SeGSH-Px in blood
serum of older bulls was significantly higher when compared to the activity of younger animals (144,63 ± 48,79
U/L, vs. 80,67 ± 31,14 U/L; P<0,01). Comparison of activity of total GSH-Px and its isoenzymes in sperm and
seminal plasma of bulls indicated very homogenous values in observed groups. No significant differences were
observed in evaluation of volume and concentration of ejaculate, as well as progressive motility of sperms in the
two age groups of bulls. Obtained results indicate that age influences the activity of glutathione peroxidase in the
blood serum, but it does not affect the activity of this enzyme in the sperm of Simmental breeding bulls.
Uvod
Razmnožavanje je temeljni proces koji omogućuje životinjama i ljudima prijenos gena na
potomstvo. Postoji veliki broj radova koji upućuju na to da stvaranje reaktivnih spojeva kisika
(ROS, eng. reactive oxygen species) i njihovo uklanjanje pomoću antioksidansa igra veliku ulogu
u plodnosti. U etiologiji mnogobrojnih bolesti pa tako i kao uzrok neplodnosti u ljudi navodi se
oksidativni stres. Tijekom oksidativnog stresa ne dolazi samo do poremećaja fluidnosti membrane
spermija zbog oksidativnih oštećenja lipida nego i do oštećenja integriteta DNA u jezgri spermija
(Agarwal i sur., 2003). Ova oštećenja mogu ubrzati proces apoptoze zametnih stanica dovodeći do
smanjenja broja spermija, smanjenja kvalitete sjemena i neplodnosti.
Neka od opisanih oksidativnih oštećenja mogu biti uklonjena, no spermiji su stanice s
vrlo ograničenim antioksidativnim kapacitetom zbog svoje specifične građe. Naime, aktivnost
antioksidativnih citoplazmatskih enzima je vrlo niska (Aitken i sur., 1989) što spermije čini
jedinstvenim s obzirom na stupanj osjetljivosti na oksidativni stres. Nadalje, plazmatska membrana
spermija sisavaca bogata je višestrukonezasičenim masnim kiselinama te je osjetljiva na oštećenja
izazvana ROS pri čemu se razvija lipidska peroksidacija (Sikka, 1996).
U antioksidativnoj zaštiti spermija sudjeluje veliki broj antioksidativnih spojeva u samoj stanici
kao i u sjemenoj plazmi koji kompenziraju mali antioksidativni kapacitet samih spermatozoa (Agarwal
i sur., 2003). Naročitu važnost imaju enzimi glutation peroksidaza (Imai i sur., 2001; Vaisberg i sur.,
2005), superoksid dismutaza, katalaza kao i glutation (Imai i sur., 2001).
Glutation peroksidaza je enzim koji ima ključnu ulogu u zaštiti stanica od oksidativnih oštećenja
uklanjajući vodikov peroksid i organske hidroperokside (Michiels i sur., 1994). Porodicu glutation
peroksidaza čine četiri izoenzima koji u aktivnom centru sadrže element selen i jedan izoenzim koji
ne sadrži selen (Halliwell i Gutteridge, 1999).
Mnogobrojni okolišni, fiziološki i genetski čimbenici djeluju na smanjenje kvalitete sperme i
mogu dovesti do neplodnosti životinje te je stoga cilj rada bio utvrditi promjene aktivnosti glutation
peroksidaze u spermi i krvnom serumu rasplodnih bikova simentalske pasmine različite dobi.
Materijali i metode
Istraživanje je provedeno na 17 bikova simentalske pasmine. Životinje su grupirane u dvije
skupine: devet mlađih bikova starih između dvije i četiri godine i osam bikova starih između šest i
deset godina. Bikovi su držani u jednakim hranidbenim i smještajnim uvjetima tijekom istraživanja.
Hranjeni su dva puta dnevno sa 8 kg krmne smjese, oko 10 kg livadnog sijena, 6 kg sjenaže, slame po
volji, te im je na raspolaganju bila stočna sol (MISSOL KR) za lizanje. Bikovi su napajani pomoću
automatskih pojilica.
Uzorci sperme uzimani su pomoću umjetne vagine, a uzorci krvi punkcijom v. jugularis.
Serum je dobiven centrifugiranjem krvi na 1500 g/15 minuta. Sjemena plazma je odvojena nakon
428
centrifugiranja uzoraka sperme na 1000 g/15 minuta. Talog spermija je resuspendiran u destiliranoj
vodi kako bi se izazvala liza spermija te je uzorak centrifugiran 3000 g/15 minuta.
Volumen ejakulata, koncentracija spermija te progresivna pokretljivost određeni su u ejakulatu
bikova. Volumen polučenih ejakulata određivao se očitavanjem na graduiranom spermoprimaču.
Koncentracija spermija u ejakulatima određena je elektronskim brojačem «PHOTOMETAR SDM 5mini tub». Progresivna pokretljivost spermija ocjenjena je binokularnim mikroskopom «OLYMPUS»
model BX 50 F s ugrađenim spermotermom.
U izoliranim spermijima i krvnom serumu bikova određena je aktivnost izoenzima glutation
peroksidaze ovisne o selenu (SeGSH-Px) metodom po Paglia and Valentine (1967), dok je u sjemenoj
plazmi određena aktivnost ukupne glutation peroksidaze (ukupna GSH-Px) metodom po Lawrence
i Burku (1976), SeGSH-Px kao i aktivnost glutation peroksidaze neovisne o selenu (neSeGSH-Px).
Aktivnost neSeGSH-Px izračunata je kao razlika ukupne GSH-Px i SeGSH-Px.
Aktivnost glutation peroksidaze u izoliranim spermijima i sjemenoj plazmi izražena je po
gramu bjelančevina koje su određene metodom po Lowry i sur. (1951) s goveđim albuminom kao
standardom.
Rezultati su prikazani kao srednja vrijednost ± standardna devijacija. Značajnost razlika među
rezultatima provjerena je t-testom i Mann-Whitney U testom, korištenjem računalnog programa
Statistica 7.1. Razlike se smatraju statistički značajnim ako je p < 0,05.
Rezultati
Rezultati aktivnosti glutation peroksidaze u spermijima, sjemenoj plazmi i krvnom serumu
bikova su prikazani su tablici 1.
Aktivnost SeGSH-Px u krvnom serumu starijih bikova bila je značajno viša u odnosu na aktivnost
kod mlađih životinja (144,63 ± 48,79 U/L odnosno 80,67 ± 31,14 U/L; P<0,01). Usporedbom
aktivnosti ukupne GSH-Px i njenih izoenzima u spermijima i sjemenoj plazmi bikova utvrđene su
vrlo ujednačene vrijednosti u promatranim skupinama.
Tablica 1. Aktivnost glutation peroksidaze u spermijima, sjemenoj plazmi i krvnom serumu bikova
Spermiji
(U/g bjelanč.)
Sjemena plazma
(U/g bjelanč.)
Krvni serum (U/L)
Stariji bikovi (n=8)
Mlađi bikovi (n=9)
P<
SeGSH-Px
833,42 ± 495,42
602,33 ± 150,26
ns
Ukupna GSH-Px
1359,96 ± 337,97
1368,03 ± 179,89
ns
SeGSH-Px
418,72 ± 163,08
448,89 ± 93,47
ns
neSeGSH-Px
941,23 ± 239,82
919,14 ± 156,79
ns
SeGSH-Px
144,63 ± 48,79
80,67 ± 31,14
0,01
Rezultati volumena ejakulata, koncentracije i progresivne pokretljivosti spermija bikova
prikazani su tablici 2. Ocjenom volumena i koncentracije ejakulata kao i progresivne pokretljivosti
spermija u navedene dvije dobne skupine bikova nisu utvrđene značajne razlike (p>0,05).
429
Tablica 2. Volumen ejakulata, koncentracija i progresivna pokretljivost spermija bikova
Stariji bikovi (n=8)
Mlađi bikovi (n=9)
P
Volumen ejakulata (ml)
4,73 ± 2,72
2,84 ± 1,08
ns
Koncentracija spermija (*109/ml)
1,25 ± 0,28
1,18 ± 0,30
ns
Progresivna pokretljivost (%)
80,00 ± 9,01
79,50 ± 11,41
ns
Rasprava
Pod utjecajem brojnih okolišnih, genetskih čimbenici kao i dobi dolazi do smanjenje
kvalitete sperme i neplodnosti kod ljudi (Sikka, 1996). Rutinske analize sjemena podrazumijevaju
makroskopsku (volumen, boja, miris, konzinstencija) i mikroskopsku (pokretnjivost, morfološke
osobitosti, koncentracija spermija) ocjenu. Na osnovi rezultata ovih analiza uzorci sjemena bikova
se uzimaju za duboko smrzavanje ili se odbacuju kao nedovoljno kvalitetni. U ovom istraživanju
rezultati volumena ejakulata, koncentracije spermija i progresivne pokretljivosti u obje skupine
bikova su bile ujednačene.
Neka istraživanja pokazuju da je ocjena kvalitete sjemena samo na osnovi makroskopskog i
mikroskopskog pregleda nedostatna te se predlaže određivanje dodatnih biokemijskih pokazatelja
u spermijima i sjemenoj plazmi. Kako oksidativni stres igra veliku ulogu u plodnosti životinja za
procijenu kvalitete uzoraka sperme raspoldnih bikova u radu smo određivali aktivnost glutation
peroksidaze.
Za održavanje plodnosti naročito je važna fosfolipid hidroperoksidna glutation peroksidaza
(Vaisberg i sur., 2005), izoenzim ovisan o selenu koji ima ulogu u spermatogenezi i funkciji
spermija, a smanjena aktivnost dovodi do smanjene plodnosti (Imai i sur., 2001; Imai i Nakagawa,
2003.). Aktivnost glutation peroksidaze u spermi rasplodnih bikova u korelaciji je s progresivnom
pokretljivošću spermija (Vaiseberg i sur., 2005).
U ovom istraživanju u sjemenoj plazmi bikova utvrđena je dvostruko veća aktivnost izoenzima
glutation peroksidaze neovisne o selenu u usporedbi s izoenzimima ovisnim o selenu što je u skladu
s rezultatima Bilodeau i sur. (2000).
U radu je određena aktivnost glutation peroksidaze ovisne o selenu u krvnom serumu te je
utvrđeno da njena aktivnost sa dobi raste.
Navedeni rezultati ukazuju da dob utječe na aktivnost glutation peroksidaze u krvnom serumu,
no ne i na aktivnost ovog enzima u spermi rasplodnih bikova simentalske pasmina.
Literatura
Agarwal, A., A. Ramadan, M. D. Saleh, M. A. Bedaiwy (2003): Role of reactive oxygen species in the
pathophysiology of human reproduction. Fert. Steril. 4, 829-843.
Aitken, R. J., J. S. Clarkson, S. Fishel (1989): Generation of Reactive Oxygen Species, Lipid Peroxidation, and
Human Sperm Function. Biol. Reproduct. 40, 183-197.
Bilodeau, J. F., S. Chatterjee, M. A.´ Sirard, C. Gagnon (2000): Levels of antioxidant defenses are decreased in
bovine spermatozoa after a cycle of freezing and thawing. Mol. Reproduct evelopm 55, 282–288.
Halliwell, B., J. M. C. Gutteridge (1999): Free radicals in biology and medicine. 3rd edition. Oxford University
Press, 134-140.
430
Imai, H., K. Suzuki, K. Ishizaka, S. Ichinose, H. Oshima, I. Okayasu, K. Emoto, M. Umeda, Y. Nakagawa (2001):
Failure of the Expression of Phospholipid Hydroperoxide Glutathione Peroxidase in the Spermatozoa of
Human Infertile Males. Biol. Reproduct. 64, 674–683.
Imai, H., Y. Nakagawa (2003): Biological significance of phospholipid hydroperoxide glutathione peroxidase
(PHGPx, GPx4) in mammalian cells. Free Rad. Biol. Med. 2, 145-169.
Lawrence, R. A., R. F. Burk (1976): Glutathione peroxidase activity in selenium-deficient rat liver. Biochem.
Biophis. Res. Com. 4, 952-958.
Lowry, O. H., N. J. Rosebrough, A. L. Farr, R. J. Rendal (1951): Protein measurement with the Folin phenol
reagent. J. Biol. Chem. 193, 265-267.
Michiels, C. M., M. Raes, O. Toussaint, J. Remacle (1994): Importance of Se-lutathione peroxidase, catalase and
Cu/Zn-SOD for cell survival against oxidative stress. Free Rad. Biol. Med. 17, 235-248.
Paglia, D. E, W. N Valentine (1967): Studies on the quantitative and qualitative characterization of erythrocyte
peroxidase. J Lab Clin Med 1, 158-169.
Sikka, S. C. (1996): Oxidative stress and role of antioxidants in normal and abnormal sperm function. Frontiers
in Bioscience 1, 78-86.
Vaisberg, C. N., L. V. Jelezarsky, B. Dishlianova, T. A. Chaushev (2005): Activity, substrate detection and
immunolocalization of glutathione peroxidase (GPx) in bovine reproductive organs and semen.
Theriogenology 2, 416-428.
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INTERACTIVE FAT SUPPLEMENTATION CHANGED THE MILK
QUALITY AND PLASMA THYROID HORMONE CONCENTRATION IN
SIMMENTAL COWS
DODATAK NEŽAŠTIĆENE MASTI U OBROK MIJENJA KVALITETU
MLIJEKA I KONCENTRACIJU HORMONA ŠTITNJAČE U
SIMENTALSKIH KRAVA
Marenjak1*, T. M., Nina Poljičak-Milas1,Jasna Piršljin2, Blanka Beer Ljubić2, Ivančica
Delaš3
1
2
Faculty of Veterinary Medicine University of Zagreb, Department of Pathophysiology
Faculty of Veterinary Medicine University of Zagreb, Department of Physiology and Radiobiology
3
School of Medicine University of Zagreb, Department of Chemistry and Biochemistry
ABSTRACT
Ten healthy multiparous Simmental cows in mid-lactation, with average 4,34 % of milk fat and 3,61 % of
milk protein, were appointed for five weeks simple change-over feeding trial. The cows were randomly assigned
in two groups, one treatment per group. Treatment 1 (WSO-diet) consisted of 41% concentrate, 33% perennial
ryegrass haylage, 26% corn silage in DM, and Treatment 2, (SO-diet) comprised of WSO diet supplemented with
200 g of non-protected sunflower oil per cow and day. Milk production and the majority of milk components did
not change significantly regarding the treatment, apart the milk urea concentration that increased in Treatment
2. The milk fatty acid profile was modified with the significant decrease in lauric, miristic and palmitic acid
(4,23 vs. 3,64; 12,52 vs. 10,85; 31,21 vs. 24,50), and the increase in stearic, vaccenic, linoleic and conjugated
linoleic acid (CLA) (10,70 vs. 15,26; 0,61 vs. 0,96; 2.55 vs. 3,61; 0,71 vs.1,57) content. No remarkable changes
in eicosapentaenoic and docosahexaenoic acid milk content, as well as in the index of milk lipid peroxidation
were identified. The concentration of total tetraiodothyronine (T4) in plasma increased in Treatment 2 whereas
no changes in total triiodothyronine (T3) concentration were detected. The positive correlation between CLA
and T4 was determined (r=0,38; p<0,05). We may suspect that supplementation of low content of interactive
sunflower oil in the diet influenced the thyroid hormone secretion and partially modified the fatty acid profile
of milk fat, with lowering effect on unfavourable lauric and palmitic acid, and raising the dietary source of
bioactive CLA in raw milk.
SAŽETAK
U istraživanje je bilo uključeno deset zdravih višetelki simentalske pasmine s prosječnim udjelom
mliječne masti od 4,34 % i mliječnim bjelančevinama od 3,61 %. Životinje su nasumce odabrane za provođenje
jednostavnog “change-over” hranidbenog tretmana tijekom pet tjedana, te su bile podijeljene u dvije skupine
sa po jednim hranidbenim tretmanom po skupini. Tretman 1 (WSO-obrok) sadržavao je 41% koncentrata, 33%
sjenaže engleskog ljulja i 42% kukuruzne silaže u ST obroka, a Tretman 2 (SO-obrok) je sadržavao WSO obrok
kojemu je dodano 200 g nezaštićenog suncokretova ulja po kravi dnevno. Proizvodnja mlijeka i većina mliječnih
sastojaka nije se značajnije mijenjala, izuzev ureje koja je značajno porasla u Tretmanu 2. Sadržaj masnih
kiselina u mlijeku se značajno promijeno s obzirom na tretman s utvrđenim značajno smanjenim udjelom
laurinske, miristinske i palmitinske kiseline (4.23 vs. 3.64; 12.52 vs. 10.85; 31.21 vs. 24.50) te uvećanim udjelom
stearinske, vakcenske, linolne i konjugirane linolne kiseline (CLA) (10.70 vs. 15.26; 0,61 vs. 0.96; 2.55 vs. 3.61;
0.71 vs.1.57) u Tretmanu 2. Nisu utvrđene značajne promjene sadržaja eikozapentaenske i dokozaheksaenske
kiseline, kao ni indeksa lipidne peroksidacije u mlijeku krava u pokusu. Koncentracija ukupnog tiroksina (T4) u
plazmi se povećala, dok je koncentracija trijodtironina ostala nepromijenjena u Tretmanu 2. Utvrđena je značajna
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pozitivna korelacija između CLA i T4 (r=0,38; p<0,05). Na temelju ovog istraživanja možemo pretpostaviti da
je dodatak interaktivnog suncokretovoga ulja u obrok simetalskih krava utjecao na sekreciju hormona štitnjače
i djelomično promijenio masnokiselinski profil mlijeka, smanjivši udio nepovoljnih kiselina (palmitinske i
laurinske) te povećao udio bioaktivne konjugirane linolne kiseline u sirovom mlijeku.
*Corresponding author: Tel. +385 1 2390 183; fax; +385 1 2390 184; E- mail: [email protected]
Introduction
In order to change the milk fatty acid composition and increase the polyunsaturated fatty acid
content in milk fat, development of protected fats and fatty acids are intensively studied for the
application in the dairy cows’ diet. On the other hand, the dietary fat may influence the dairy cows’
health and production. The energy metabolism in animals and humans is regulated with both fatty
acids and thyroid hormones. Thyroid hormones play a key role in the up-regulation of a variety of
metabolically-related processes such as body temperature, heart rate, cellular repair, metabolism
of energy stores, and milk production (Lima et al., 2001). It should be emphasised that thyroid
hormones influence the fatty acid synthesis and metabolism (Blennemann et al., 1992), stimulate de
novo liver cholesterol synthesis (Müller and Seitz, 1984), and vice versa, fatty acids may affect the
thyroid hormones plasma level. There are some evidences, from the experiments with rodents and
humans, about the influence of the dietary fat on the thyroid hormone activity (Rosołowska-Huszcz
and Lachowicz, 2006). Rather less data are available from the experiments with ruminants. The
results of Marenjak et al. (2007) indicate that the supplementation of non-protected polyunsaturated
fatty acids (PUFA) in concentrate mixture changed the milk fatty acid composition and increased
the total blood cholesterol level in mid-lactating Simmental cows. The influence of the interactive
fats on the blood lipid concentration and milk fatty acid profile was detected but the level of action
was not clear. The objective of our research was to extend the knowledge in the area of dairy cows
energy metabolism and milk production, and to clarify possible model of action of interactive fat
supplementation and its effect on the milk fatty acid composition.
Material and methods
Ten multiparous mid-lactating and healthy Simmental cows, were appointed to simple change
over feeding trial in duration of five weeks with four weeks pre-trial period. The ingredients and the
chemical composition of the diet are presented in Table 1. The basal diet (WSO- treatment 1) and
the diet in pre-trial period comprised of 26% corn silage, 33% perennial ryegrass haylage, and 41%
concentrate in DM, and in the experimental diet (SO - treatment 2) where the concentrate mixture in
the basal diet was partly replaced with 2 % of non-protected sunflower oil. The concentrate mixture
was prepared on the daily bases on the farm. The cows were housed indoors with ad libitum water
supply in two separate feeding lots at the same stable. After four weeks of the same feeding procedure,
cows were randomly selected in two groups and fed one of the previously described diets. After
seven days wash out period the change over was applied. The milk and blood samples were collected,
once in pre-trial period, and four times during the trial period, 14th and 21st day after the introduction
of sunflower oil to the concentrate mixture of each animal group. The cows were milked and fed two
times daily each day, at 7 a.m. and 7 p.m.
The blood samples were collected from the v. jugularis in BD vacutainer tubes (LH 85 I.U.)
with lithium heparin as the anticoagulant agent, 3-4 hours after the feeding. The blood samples
were transported at +4 °C to the laboratory where plasma was separated after the centrifugation at
3000 rpm for 15 min. and stored at -20 ºC until analyses. The thyroid hormone concentration was
determined by time-resolved fluoroimmunoassay (Perkin Elmer Life and Analytical Sciences, Turku,
434
Finland) on a 1234 Delfia Research Fluorimeter, Wallac, Turku, Finland. Intra-assay precision within
run was CV= 6,8%, inter-assay precision between run was 5,9%, for both analyses.
Table 1. Ingredients and chemical composition of the basal (WSO) and experimental (SO) diet
Diet
Ingredients (% of DM)
Perennial ryegrass haylage
Corn silage
Concentrate mixa
Sunflower oil
Chemical composition
Dry matter (%)
Crude protein (CP)
RUP % DM
RUP % CP
Ether extract
Acid detergent fiber
Neutral deterget fiber
Ca
P
Mg
NEL Mcal/kgb
ME Mcal/kgb
WSO
SO
33
26
41
33
26
39
2
63,1
16,9
4,87
31,29
4,4
14,1
24,5
0,44
0,34
0,38
1,81
4,30
63
16,8
4,85
31,24
5,2
13,6
23,6
0,47
0,35
0,40
1,83
6,82
Concentrate mix. consisting of: 33,3 % barley, 44,5 % corn, 11,1% soybean meal (WSO) and 9,1 % of
soybean meal (SO), 5,6 % Protamilk, 5,5 % Profisan Sano, GmbH) bEstimated using NRC (2001) for dietary
ingredients DM-dry matter;: RUP-rumen undegradible protein;NEL-net energy of lactation; ME-metabolisable
energy
a
The milk samples for the standard milk components analyses, index of milk lipid peroxidation,
and somatic cell count (SCC) were taken from the composite of the morning milking for each
cow separately. The milk was placed in the 40 ml plastic container, and transported at 4 °C to the
laboratory where milk constituents (protein, fat, lactose, and solid-non fat; SNF) were regularly
controlled by the automatic analyser (MilcoScan 4000, Denmark), and somatic cell count (SCC) was
determined by Fossomatic 5000 (Denmark). The index of milk lipid peroxidation was estimated by
determination of thiobarbituric acid reactive substances (TBARS) concentration and milk superoxide
dismutase (SOD) activity in raw milk. The TBARS concentration was determined with the TROTTA
et al. method (1982) on the Helious Thermospectronic, Helios Delta spectrophotometer (Unicam,
Cambridge, UK) at 25 °C. The absorption peak was measured at 532 nm and the concentration was
calculated using molar extinction coefficient of 1.5 x 105. Total SOD activity (E.C. 1.15.1.1) was
measured using commercial kit (RANSOD, “Randox”, Ireland) on automatic analyser SABA 18
(AMS, Italy) at 37 °C.
The milk samples for the fatty acid analyses were collected as a 1:1 composite of the morning
and evening milk samples from each cow separately. The milk samples were stored at – 20 °C until
analysed for the fatty acid composition by GC procedure (GC chromatograph SRI 8619C, Qadrex
435
Corporation, New Haven). Lipid extraction and washing of the extract was done according to the
method of Folch et al. (1957). The preparation of fatty acid methyl esters (FAME) was carried out
according to the ISO 14156-IDF 172:2001 and ISO 15884-IDF 182:2002. Aliquots of FAME in
chloroform (1 µL) were fractionated with a fused silica capillary column (Carbowax 0212051; 30 m
x 0.25 mm i.d. x 0.25 µm filter thickness) using H2 as a carrier gas with a flow of 60 mL/min. The
injector and detector temperatures were 160 °C and 230 °C, respectively. The initial oven temperature
was held at 100 °C for 10 min, then increased to 160 °C at 10 °C per min and held for 7 min, and
finally increased to 220 °C at 5 °C per min and held for 15 min. All fatty acids determined in this trial
were identified by comparing the retention times with methylated standards (Sigma Aldrich Chemie,
GmbH and Supelco, USA). The CLA standards were purchased from Matreya INC., PA, USA. The
quantification of FAME was done using nonadecanoic acid as an internal standard. The milk urea
concentration was measured by colorimetric method using the commercial kit (Herbos Dijagnostika,
Sisak, Croatia) on the
The data were analysed as a change over using the ANOVA/MANOVA model of Statistica
(version 7; 2005, StatSoft, Inc. USA) with the treatment as a class variable. Correlation coefficients
were obtained using linear regression models.The significance of the difference between
means was evaluated by Tukey LSD test taking p < 0,05 as significant, unless indicated otherwise.
Results
The data of the milk yield, milk components and oxidative stability in milk of Simmental cows
included in the trial were presented in Table 2. Although, the milk production was not significantly
different between two treatments, slightly higher milk production was noted in the SO treatment.
The similar could be perceived for the milk protein, SNF, and lactose content, whereas the milk fat
content was slightly lower. The only significant shift was determined for the milk urea concentration,
that was higher in the SO treatment (Table 2.), but still in the normal range for respective group of
cows. The SOD activity was higher and TBARS lower in the SO treatment, but not at the significant
level.
Table 2. Production and oxidative stability of milk in Simmental cows according to the treatment (WSO-basal
diet; SO-experimental diet)
Treatment
Milk production L/d
Milk fat (%)
Milk protein (%)
Milk urea (mmol/L)
Lactose (%)
SNF1 (%)
SOD2 U/g prot
TBARS3 µmol/g prot
1- WSO (N=30)
2- SO (N=20)
18,2 ± 0,96
4,32 ± 0,09
3,61 ± 0,08
3,06 ± 0,20
4,66 ± 0,03
9,02 ± 0,06
254,44 ± 21,27
0,13 ± 0,01
20,4 ± 1,32
4,29 ± 0,15
3,72 ± 0,03
3,72 ± 0,14*
4,70 ± 0,04
9,13 ± 0,09
279,98 ± 19,59
0,12 ± 0,01
The results were expressed as mean ± SEM;*p<0,05; 1SNF-solid-non fat;2SOD-superoxide
dismutase;3TBARS- thiobarbituric acid reactive; substances
The milk fatty acid profile changed significantly between the treatments in the study. The major
changes were perceived in the group of fatty acids with 18 carbon atoms in the chain and medium
chain fatty acids (Table 3.). The content of lauric (C12:0), miristinic (C14:0) and particularly palmitic
acid (C16:0) decreased, whereas stearic (C18:0) linoleic (LA), trans- vaccenic (TVA) and conjugated
linoleic (cis-9, trans-11 C18:2; CLA) acid increased significantly in the SO treatment. Higher, but
436
not significant increase in alfa-linolenic (α-LNA), arachidonic (AA), eicosapentaenoic (EPA) and
docosahexaenoic (DHA) acids concentration was remarked.
The plasma level of total thyroxine (T4) and triiodothyronine (T3) had opposite trend in the two
treatments (Figure 1.). The significant increase in T4 was detected in SO treatment (Figure 1.). The
positive correlation between CLA and T4 was presented in Figure 2.
Table 3. Fatty acid composition in Simmental cows milk according to the treatment (WSO-basal diet; SOexperimental diet)
Treatment
Fatty acids
C8:0
C10:0
C12:0
C14:0
C16:0
C18:0
C18:1 n 9
C18:1 n 7 t11 (TVA)
C18:2 n 6 (LA)
C 18:2 n7 cis-9, trans-11 (CLA)
C18:3 n 3 (α-LNA)
C20:4 n6 (AA)
C20:5 n3 (EPA)
C22:6 n3 (DHA)
1- WSO (N=30)
2- SO (N=20)
(g/100g of fatty acids)
1,04 ± 0,29
0,67 ± 0,16
2,71 ± 0,14
2,43 ± 0,20
4,23 ± 0,11
3,64 ± 0,24*
12,52 ± 0,32
10,85 ± 0,81*
31,21 ± 1,03
24,50 ± 2,03**
10,70 ± 0,88
15,26 ± 1,66**
20,59 ± 0,79
19,7 ± 1,43
0,61 ± 0,08
0,96 ± 0,09*
2,55 ± 0,18
3,61 ± 0,25***
0,71± 0,08
1,57± 0,24***
0,48 ± 0,13
0,78 ± 0,33
0,15 ± 0,05
0,27 ± 0,11
0,35 ± 0,07
0,52 ± 0,19
0,16 ± 0,07
0,32 ± 0,13
The results were expressed as mean ± SEM;*p<0,05; **p<0,01; ***p<0,001
Discussion
Many attempts have been made in order to produce advantageous meat and milk fatty acid
composition for human consumption. The various feeding procedures were applied and different
sources of dietary fat where tested. Three major groups of fats are available for the dairy cows or
beef steers nutrition: rumen-inert, rumen-active and protected fats. In this study we have chosen the
rumen-active fats of plant origin with higher content of polyunsaturated fats (PUFA), mostly linoleic
acid, that theoretically provide the precursor for the CLA and trans-C18:1 fatty acid production. The
linoleic acid may have toxic effect on the rumen bacteria, but according to Koppova et al. (2006)
it may be a stimulus for the bacterial growth. Therefore, our scope was to test the influence of nonprotected sunflower oil on the milk production and fatty acid composition of milk fat, and also to
determine the oxidative stability of raw milk. On the other hand, our objective was to determine
the possible effect of sunflower oil treatment on the concentration of plasma thyroid hormones and
possible interrelationship with the milk fatty acid composition.
In our study, the supplementation of interactive fat did not affect the animal production and cows
remained healthy during and after the trial, with no remarkable changes detected in milk fat and other
milk components content, except for the milk urea concentration that was significantly higher in SO
treatment. It is known that the measuring of blood or milk urea is good supplementary tool to control
the adequacy of protein fed to dairy cows (Oltner and Wiktorsson, 1983; Marenjak et al. 2000).
We may suspect that the linoleic acid stimulated the growth of rumen bacteria which are important
source of dietary protein for the dairy cattle and the milk production. Therefore, the significant
437
tre a tm e n t; L S Me a n s
V e rti c a l b a rs d e n o te 0 ,9 5 c o n fi d e n c e in te rva l s
2 ,2
2 ,1
2 ,0
T3 nmol/L
1 ,9
1 ,8
1 ,7
1 -W S O
2- S O
tre a tm e n t
tr eatment; LS M eans
T ukey H SD test; var iable T 4 nmol/L; p= 0,023
Appr oximate Pr obabilities for Post H oc T ests
Er ror : Between M S = 136,66, df = 26,000
90
*
85
80
75
T4 nmol/L
70
65
60
1- WSO
2- SO
tr eatment
Figure 1. Concentration of total thyroxine (T4) and triiodothyronine (T3) in the blood plasma of Simmental
cows regarding the treatment
Scatter plot: T 4 nmol/L vs. C LA ,cis9 tr ans11 ( C asewise M D deletion)
C LA ,cis9 tr ans11 = - ,3973 + ,01897 * T 4 nmol/L
C or r elation: r = 0,370;p< 0,05
3,5
3,0
2,5
2,0
1,5
CLA ,cis9 trans11
1,0
0,5
0,0
30
40
50
60
70
80
90
100
T 4 nmol/L
Figure 2. Correlation matrix between the plasma total thyroxine (T4) and the conjugated linoleic acid (cis-9,
trans-11 C18:2; CLA) concentration in Simmental cows’ milk
438
increase of milk urea concentration and less remarkable increase of milk protein content (Table 2)
could be a consequence of the sunflower oil supplementation in the diet. Furthermore, the fatty acid
composition of milk fat was significantly changed regarding the treatment (Table 3.) with no influence
on the milk fat content, thus we may expect on the one hand, the improvement in healthfulness and
functional properties of milk, and on the other hand, the retaining of the economic value for the milk
producer. Several authors demonstrated the alterations in the duodenal flow and plasma fatty acids
concentration in the milk fat and the modification of milk fatty acid composition due to the various fat
source infusion or supplementation to the dairy cattle (Kalscheur et al., 1997; Petit, 2003; Bell et al.,
2006). In our study, the incorporation of the non-protected sunflower oil into the concentrate mixture
resulted in the lower milk content of miristic, lauric and palmitic acids, that are nutritionally derived,
LDL- cholesterol raising factors for humans. At the same time the significantly higher concentration
of stearic, linoleic and bioactive conjugated linoleic acid (cis-9, trans-11 C18:2; CLA) was observed
with slightly, but not significantly lower content of oleic acid (Table 3.). The five fold increase of
the stearic acid content was expected because of the biohydrogenation in the rumen. In contrast, the
significant increase in the vaccenic acid (trans -11 C18:1; TVA) content was determined due to the
incomplete biohydrogenation processes of linoleic acid by rumen bacteria of group A. It is partially
transferred to the mammary tissue and via activity of ∆9-desaturase converted to cis-9, trans-11
C18:2 (CLA). Therefore, the increase of the CLA content in milk resulted from two possible sources:
one from the rumen synthesis and another comes from the endogenous synthesis by the desaturation
of TVA in the mammary gland that accounts for 64 % of the overall milk CLA synthesis according
to Grinarii et al. (2000). Therefore, from our study we may conclude that the interactive sunflower
oil supplementation resulted in the incomplete biohydrogenation and consequently the increase in
CLA and TVA content was observed. The active pathway of the synthesis of CLA from TVA was
also documented in humans (Adolf et al., 2000) with several intestinal bacterial species recognised
for such ability (Alonso et al., 2003, Coakley et al., 2003). Among all milk fatty acids analysed in
our study, only CLA content was positively correlated with T4 (r = 0,370; p<0,05; Figure 2.) and
both values increased in the SO treatment (Table 3.; Figure 1.). Hence synergy in action on the lipid
metabolism of both CLA and T4 might be hypothesised. The influence of thyroid hormones on the
lipid metabolism is well documented and various thyroid states can modulate the lipid metabolism
by activating the lipogenic enzymes, such as lipoprotein lipase and fatty acid synthetase, leading
to the increase of fatty acids concentration in plasma (Müller and Seitz, 1984). In our study, the
concentration of thyroxin was increased during the SO treatment (Figure 1.) and the increase in
the total blood cholesterol level was also determined during the supplementation of non-protected
sunflower oil in the Simmental cows diet (Marenjak et al., 2007). The concentration of T4 in our
study was higher compared to the values reported by Kaneko (1997) for both treatments, whereas
the concentration of T3 ranged over the referent values (from 2,01 nmol/L in WSO to 1,99 nmol/L
in SO treatment). The increase in PUFA content provided in the free form from the sunflower oil,
may directly affect the liver, adipose tissue and mammary gland fatty acid metabolism through the
expression of the lipogenic genes as it was presented by Harvatine and Bauman (2006). The higher
concentration of T4 may enhance the mitochondrial oxidation of fatty acids in many tissues, but
according to the Müller and Seitz (1984), the increase of cytosolic de novo synthesis, desaturation
and elongation of fatty acids could also be expected. The positive correlation between the milk
CLA content and the plasma T4 concentration revealed the potentially same regulatory mechanism
involved in the particular fatty acid synthesis and thyroid hormone responsiveness to the dietary
PUFA in Simmental cows from our study. Regulatory mechanism of PUFA on the metabolically
related proceses in ruminants should be futher studied.
439
Acknowledgement
The research was supported by the grant of Ministry of Science, Education and Sports, Republic of Croatia.
References
Adolf, R. O., S. Duval, E. A. Emken (2000): Biosythesis of conjugated linoleic acid in humans. Lipids 35, 131135.
Alonso, L., E. P. Cuesta, S. E. Gilliland (2003): Production of Free Conjugated Linoleic Acid by Lactobacillus
acidophilus and Lactobacillus casei of Human Intestinal Origin. Journal of Dairy Science 86, 1941-1946.
Bell, J. A., J. M. Griinari, J.J. Kennelly (2006): Effect of safflower oil, flaxseed oil, monensin, and vitamin E on
concentration of conjugated linoleic acid in bovine milk fat. Journal of Dairy Science 89, 733-748.
Blennemann, B., Y. K. Monn, H. C. Freake (1992): Tissue-specific regulation of fatty acid synthesis by thyroid
hormone. Endocrinology 130, 637-643.
Coakley, M., Ross, R. P., Nordgren, M., Fitzgerald, G., Devery, R., Stanton, C. (2003): Conjugated linoleic acid
biosynthesis by humanderived Bifidobacterium species. Journal of Applied Microbiology 94,138–145.
Folch, J., M. Lees, G. H. Sloane-Stanley (1957): A simple method for the isolation and purification of total lipids
from animal tissues. Journal of Biological Chemistry 226, 497-509.
Griinari, J. M., B. A. Corl, S. H. Lacy, P. Y. Chouinard, K.V. Nurmela, D. E. Bauman (2000): Conjugated linoleic
acid is synthesized endogenously in lactating dairy cows by Delta (9)-desaturase. Journal of Nutrition
2285- 2291.
Harvatine, K. J., D. E. Bauman (2006): SREBP1 and thyroid hormone responsive spot 14 (S14) are involved in
the regulation of bovine mammary mipid synthesis during diet-dnduced milk fat depression and treatment
with CLA. The Journal of Nutrition 136, 2468-2474.
Kalscheur, K. F., B. B. Teter, L. S. Piperova, R. A. Erdman (1997): Effect of fat source on trans fatty acid flow
in lactating dairy cows. Journal of Dairy Sciece 80, 2115-2126.
Kaneko, J. J., J. W. Harvey, M. L. Bruss (1997): Clinical biochemistry of domestic animals. Fifth Edition.
Academic Press, Inc. San Diego. CA.
Koppova, I., F., Lukash, J. Kopečny (2006): Effect of fatty acids on growth of conjugated linoleic acid producing
bacteria in rumen. Folia microbiologica 51, 291-293.
Lima, F. R. S., A. Gervais, C. Colin (2001): Regulation of microglial development: A novel role for thyroid
hormone. Journal of Neuroscience 21, 20028-2038.
Marenjak, T. S., N. Poljičak-Milas, R. Turk, B. Kampl (2000): Possibility of metabolic control in cows by milk
protein and urea determination. Veterinarski arhiv 70 (Suppl.), 251-257.
Marenjak, T. S., N. Poljičak Milas, I. Delaš (2007): Blood lipids and proportion of fatty acids, especially CLA
in milk of Simmental cows fed with unprotected sunflower oil. 5th Euro Fed Lipid Congress, Gothenburg,
Sweden, 16. - 19. 09. 2007, p.215.
Müller, M. J., H. J. Seitz (1984): Thyroid hormone action on intermediary metabolism. Part II: Lipid metabolism
in hypo- and hyperthyroidism. Klinische Wochenschrift 62, 49-55.
Oltner, R., H. Wiktorsson (1983): Urea concentration in milk and blood as influenced by varying amounts of
protein and energy to dairy cows. Livestock Production Science 10, 457-467.
Petit, H.V. (2003): Digestion, milk production, milk composition, and blood composition of dairy cows fed
formaldehyde flaxseed or sunflower seed. Journal of Dairy Science 86, 2637-2646.
Rosołowska-Huszcz, D., K. Lachowicz (2006): Influence of dietary fat on thyroid hormone plasma concentrations.
New Medicine 1, 15-18.
Trotta, R. J., S. G. Sullivan, A. Stern (1982): Lipid peroxidation and hemoglobin degeneration in red blood cells
exposed to T-utyl hydroperoxide. Biochemical Journal, 204 (1982), 405-415.
440
SEASONAL CHANGES IN LIPID COMPOSITION OF BOVINE SERUM
AND SEMINAL PLASMA
UTJECAJ GODIŠNJIH DOBA NA SASTAV MASNIH TVARI U SERUMU I
SJEMENOJ PLAZMI BIKOVA
Blanka Beer Ljubić1, Renata Laškaj2, Jasna Piršljin1, Ivanka Majić-Balić3, Terezija
Silvija Marenjak4, Suzana Milinković-Tur1
1
Zavod za fiziologiju i radiobiologiju, Veterinarski fakultet, Zagreb, Hrvatska
2
3
Klinika za infektivne bolesti, Zagreb, Hrvatska
Centar za reprodukciju u stočarstvu, Križevci, Hrvatska
Zavod za patološku fiziologiju, Veterinarski fakultet, Zagreb, Hrvatska
4
Abstract
A key feature in the function of the spermatozoa is the lipid composition of seminal plasma and sperm
membrane. Lipid molecules are transferred from blood plasma to the accessory sex glands through bloodseminal plasma barrier. Cholesterol is the major free sterol in ejaculated bovine sperm. Importance of
cholesterol in process of spermatozoa capacitation is well known. The aim of this study was to investigate
seasonal variations of the bovine blood serum and seminal plasma lipid concentrations. The experiment was
carried out on 19 Simmental breeding bulls at the age of 2 -10 years. Blood samples were collected from the v.
jugularis and bovine semen with an artificial vagina once per season. Serum concentrations of total cholesterol,
high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), triacylglycerols,
free fatty acids (FFA) and lipoprotein electrophoretic patterns were determined. Seminal plasma concentrations
of total cholesterol, HDL-C and LDL-C were assayed. Serum concentrations of total cholesterol and LDLC were significantly higher in the winter (2,51±0,46 mmol/L;. 0,56±0,11 mmol/L; ( x ±SD) compared with
values determined in blood samples collected in the spring period (2,13±0,38 mmol/L; 0,42 ± 0,08 mmol/L).
Triacylglycerol concentrations in bovine serum were higher in the winter (0,23±0,06 mmol/L) than in the summer
(0,18±0,05 mmol/L) or in the autumn period (0,17±0,05 mmol/L). There were significant differences between
serum FFA concentrations in blood samples collected in the autumn (0,19±0,07 mmol/L) compared with those
collected in the summer (0,14±0,05 mmol/L) or in the winter (0,14±0,03 mmol/L). The absolute concentrations
of serum HDL and LDL lipoproteins in electrophoretic tracings in the winter (2,49±0,89 mmol/L; 0,36±0,21
mmol/L) were significantly higher than in the spring period (1,82±0,54 mmol/L; 0,17±0,09 mmol/L). Seasonal
variations were found in seminal plasma total cholesterol and HDL-C with higher concentrations in the winter
(1,02±0,47 mmol/L; 0,09±0,04 mmol/L) than in the summer (0,61±0,30 mmol/L; 0,06±0,01 mmol/L). The
results of this study demonstrate seasonal variation in bovine serum and seminal plasma lipid levels. Observed
changes of cholesterol concentrations in seminal plasma are associated with extracellular lipid utilisation and
appear to be useful biochemical marker of sperm quality.
Sažetak
Građa i uspješnost spermija u oplodnji jajne stanice ovisi o sastavu masnih tvari u staničnoj
membrani i sjemenoj plazmi. Molekule masnih tvari prenose se do akcesornih spolnih žlijezda.
Kolesterol je osnovni, slobodni sterol u spermi bikova i ima ključnu ulogu u procesu kapacitacije
spermija. Cilj ovog istraživanja je utvrditi utjecaj godišnjih doba na koncentracijue masnih tvari u
serumu i sjemenoj plazmi bikova. Istraživanje je provedeno na 19 bikova simentalske pasmine u dobi
od 2 do 10 godina. Uzorci krvi uzimani su punkcijom v. jugularis, dok su uzorci sperme uzimani
441
pomoću umjetne vagine jednokratno tijekom četiri godišnja doba. U serumu je određena koncentracija
ukupnog kolesterola, kolesterola vezanog na lipoproteine visoke (HDL-C) i niske gustoće (LDL-C),
triglicerida, slobodnih masnih kiselina (SMK) i elektroforeza serumskih lipoproteina. U sjemenoj
plazmi je određena koncentracija ukupnog, HDL-C i LDL-C kolesterola. Koncentracija ukupnog
kolesterola i LDL-C u serumu bikova bila je značajno veća u zimi (2,51±0,46 mmol/L;. 0,56±0,11
mmol/L; ( x ±SD) u odnosu na vrijednosti dobivene u uzorcima seruma prikupljenim u proljeće
(2,13±0,38 mmol/L; 0,42±0,08 mmol/L). Tijekom zime, koncentracija triglicerida u serumu bikova
bila je veća (0,23±0,06 mmol/L) od vrijednosti dobivenih u ljetnom (0,18±0,05 mmol/L) i jesenskom
(0,17±0,05 mmol/L) periodu. Koncentracija SMK u serumu bila je značajno veća u jesen (0,19±0,07
mmol/L) u odnosu na ljetne (0,14±0,05 mmol/L) i zimske (0,14±0,03 mmol/L) uzorke. Analizom
elektroforetograma utvrđeno je da su apsolutne koncentracije HDL i LDL frakcija bile značajno
veće u zimskim (2,49±0,89 mmol/L; 0,36±0,21 mmol/L) u odnosu na proljetne uzorke (1,82±0,54
mmol/L; 0,17±0,09 mmol/L). Koncentracija ukupnog kolesterola i HDL-C u sjemenoj plazmi bikova
bila je veća u zimi (1,02±0,47 mmol/L; 0,09±0,04 mmol/L) u odnosu na ljeto (0,61±0,30 mmol/
L; 0,06±0,01 mmol/L). U istraživanju su uočene promjene koncentracija masnih tvari u serumu i
sjemenoj plazmi bikova tijekom godišnjih doba. Koncentracija kolesterola u sjemenoj plazmi bikova
povezana je s iskorištavanjem masnih tvari u spermi te bi njeno određivanje moglo postati koristan
biokemijski pokazatelj u procjeni kvalitete sperme.
Uvod
Proizvodnja i reproduktivne kvalitete usko su povezane sa zdravljem životinja. Određivanje
masnih tvari u serumu životinja, pa tako i bikova, dio je pretraga u sklopu metaboličkog profil
testa kojim se prati zdravlje životinja (PAYNE i PAYNE, 1987). Masne tvari, naročito trigliceridi i
slobodne masne kiseline, važni su pokazatelji energetskog statusa monogastričnih životinja, dok u
preživača mjerenje koncentracije slobodnih masnih kiselina (SMK) te u stanovitoj mjeri određivanje
koncetracije slobodnog i estera kolesterola mogu ukazivati na opskrbu energetskim supstratom i
metabolički obrat masti (HERDT, 1991; KATOH, 2002; SEIFI i sur., 2007).
Umjetno osjemenjivanje u stočarskoj proizvodnji ima veliko zdravstveno i ekonomsko
značenje. U govedarskoj proizvodnji, važno je za selekciju i unapređivanje govedarstva jer se njime
mogu unaprijediti proizvodne sposobnosti goveda. Kvaliteta sjemena procjenjuje se na osnovi
makroskopskih (volumen, boja, miris, konzinstencija) i mikroskopskih (pokretljivost, morfološke
osobitosti, koncentracija) analiza. Uzorci sjemena visoke kvalitete čuvaju se duboko smrznuti.
Procesi zamrzavanja i odmrzavanja dovode do trajnih oštećenja spermija čime se smanjuje njihova
pokretljivost i sposobnost oplodnje (CEROLINI i sur., 2001; JANETT i sur., 2003) tako da se već
dugi niz godina nastoji poboljšati tehnika dubokog zamrzavanja spermija kao i sastav razrijeđivača
sjemena. Dosadašnja istraživanja su pokazala da na kvalitetu sjemena osim vrste, dobi i individualnih
razlika, utječe i promjena godišnjih doba. Utjecaj godišnjih doba na kvalitetu sjemena utvrđen je i
kod ljudi (CHEN i sur., 2003) i kod životinja (ARGOV i sur., 2007; JANETT i sur., 2003).
Građa i uspješnost spermija u oplodnji jajne stanice ovise o sastavu masnih tvari u staničnoj
membrani spermija i sjemenoj plazmi. Oplodnji jajne stanice prethodi proces kapacitacije praćen
promjenama u sastavu masnih tvari u membrani spermija. Promjene su vezane uz izlazak kolesterola
iz membrane spermija i vezanje na bjelančevine u sjemenoj plazmi i spolnom traktu ženskih životinja
(WITTE i SCHÄFER-SOMI, 2007). Osim kolesterola, u sjemenoj plazmi se nalaze i trigliceridi
čijom oksidacijom spermiji zadovoljavaju svoje energetske potrebe. Prisustvo VLDL receptora na
membrani omogućava spermijima unos i iskorištavanje triglicerida (ARGOV i sur., 2007). Intenzivan
442
metabolizam lipida nastavlja se i nakon ejakulacije, a dodatak razrijeđivača bogatog lipidima
osigurava bolju kavalitetu spermija nakon odmrzavanja (CEROLINI i sur., 2001).
Cilj ovog istraživanja bio je utvrditi promjene u sastavu masnih tvari u serumu i sjemenoj plazmi
bikova tijekom godišnjih doba, te utvrditi postoji li povezanost promjena masnih tvari u serumu s
promjenama u sjemenoj plazmi bikova.
Materijali i metode
Istraživanje je provedeno na 19 bikova simentalske pasmine u dobi od 2 do 10 godina
prosječne tjelesne težine 1177±100 kg. Tijekom istraživanja bikovi su držani u zasebnim ležištima s
pregradama od cjevastih metalnih prečki na vezu pomoću kožnog ovratnika. Prije svakog polučivanja
sjemena bikovi se izvode iz staje, privezuju za vrtuljak i kreću oko 30 minuta u svrhu pripreme za
polučivanje.
Bikovi su hranjeni dva puta dnevno. Količinu i kemijski sastav krmne smjese i krmiva prikazuje
tablica 1. Bikovi su napajani pomoću automatskih pojilica. zorci krvi i sperme uzimani su jednokratno
tijekom svakog godišnjeg doba. Uzorci krvi uzeti su iz v. jugularis, u BD Vacutainer SST. Serum
je dobiven centrifugiranjem krvi na 1500 g/15 minuta. Uzorci sperme dobiveni su pomoću umjetne
vagine. Sjemena plazma je odvojena nakon centrifugiranja uzoraka sperme na 1000 g/15 minuta.
Serum i sjemena plazma su do analize pohranjeni na -80 oC. Uzorci krvi i sperme uzimanu su od 8:00
do 10:00 sati.
U krvnom serumu određena je koncentracija ukupnih masnih tvari, triglicerida, slobodnih
masnih kiselina (SMK), kolesterola vezanog na lipoproteine visoke gustoće (HDL-C), kolesterola
vezanog na lipoproteine niske gustoće (LDL-C) i slobodnog kolesterola gotovim kompletima tvrtke
Randox (Irska), na biokemijskom analizatoru SABA 18 (AMS, Italija). Elektroforeza serumskih
lipoproteina je obavljena reagensima tvrtke MALTA Chemetron (Italija), a međusobni odnos i
apsolutna koncentracija lipoproteinskih frakcija određena je na Glob-Al Scan denzitometru (MALTA
Chemetron, Italija). U sjemenoj plazmi bikova određena je koncenracija HDL-C, LDL-C i slobodnog
kolesterola gotovim kompletima tvrtke Randox (Irska), na biokemijskom analizatoru SABA 18
(AMS, Italija).
Tablica 1. Količina i kemijski sastav obroka za bikove u pokusu
krmna
smjesa*
sjenaža
livadno sjeno
slama
misol**
voda
KEMIJSKI
sir.
bjelančevine
(%)
KOLIČINA
SASTAV
sir.
vlakna
(%)
sir. pepeo
(%)
sir. mast
(%)
vlaga
(%)
8 kg
18,0
15,0
10,0
-
13,5
6 kg
10 kg
po volji
po volji
po volji
10,2
5,8
2,3
32,4
28,5
39,8
6,15
6,02
5,3
1,8
2,1
1,1
31,5
14,4
15,6
* žitarice, sporedni proizvodi u proizvodnji ulja, sporedni proizvodi industrije škroba, alkohola i vrenja, mlinarstva,
sušeni biljni proizvodi, mineralna krmiva te mineralno-vitaminski dodaci (Ca 0,80%, P 0,35%, Na 0,20%, vitamin A 7500
U/kg, vitamin D3 1000 U/kg, vitamin E 10 mg/kg) * 1% Mg, 39% Na, 3000 mg/kg Zn, 1200 mg/kg Mn, 24 mg/kg Co, 20
mg/kg Se, 20-30 mg/kg J
443
Rezultati su obrađeni statističkim programom Statistika (data analysis software system) version
7.1 (Stat Soft, Inc. 2005) i prikazani kao srednja vrijednost ± standardna devijacija ( x ±SD).
Značajnost razlika među rezultatima tijekom godišnjih doba provjerena je analizom varijance i
Tukey testom ukoliko se radilo o normalnoj razdiobi, a Kruskall-Wallisovom analizom varijance i
testom sume rangova za sve grupe ukoliko je nulta hipoteza odbačena. Razlike se smatraju statistički
značajnim ako je p<0,05. Međusobna povezanost promatranih pokazatelja ispitana je linearnom
korelacijom uz korištenje navedenog statističkog programa.
Rezultati
Na slici 1. prikazane su koncentracije triglicerida i slobodnih masnih kiselina u serumu bikova
tijekom četiri godišnja doba. Koncentracija triglicerida u serumu bikova bila je značajno veća u
zimskom razdoblju (0,23±0,06 mmol/L) (p<0,05) u odnosu na vrijednosti dobivene u ljeto (0,18±0,05
mmol/L) i jesen (0,17±0,05 mmol/L). Najveća koncentracija slobodnih masnih kiselina određena je
u uzorcima prikupljenim u jesen (0,19±0,07 mmol/L), te je bila statistički značajno veća (p<0,05)
u odnosu na vrijednosti dobivene u ljetnom (0,14±0,05 mmol/L) i zimskom razdoblju (0,14±0,03
mmol/L).
0,25
a
abc
mmol/L
0,20
bc
a
b
abc
0,15
bc
c
0,10
0,05
0,00
SMK
trigliceridi
proljeće
ljeto
jesen
zima
Slika 1. Koncentracije triglicerida i slobodnih masnih kiselina u serumu bikova tijekom četiri godišnja doba.
Histogrami s različitim abcsuperskriptom statistički se značajno razlikuju (p<0,05).
Koncentracije ukupnog kolesterola, HDL-C i LDL-C u serumu bikova tijekom četiri godišnja
doba prikazane su na slici 2. Koncentracija ukupnog kolesterola i LDL-C u serumu bikova bila
je statistički značajno veća (p<0,05) u zimskom razdoblju (2,51±0,46 mmol/L; 0,56±0,11 mmol/
L) u usporedbi s vrijednostima dobivenim u proljeće (2,13±0,38 mmol/L; 0,42±0,08 mmol/L).
Koncentracija HDL-C u serumu bikova nije pokazala značajne promjene tijekom različitih godišnjih
doba (p>0,05).
444
3,00
2,50
mmol/L
a
ab ab
b
2,00
1,50
1,00
a
0,50
ab ab
b
0,00
HDL-C
kolesterol
proljeće
ljeto
LDL-C
zima
jesen
Slika 2. Koncentracije ukupnog kolesterola, HDL-C i LDL-C u serumu bikova tijekom četiri godišnja doba.
Histogrami s različitim abcsuperskriptom statistički se značajno razlikuju (p<0,05).
Koncentracije lipoproteinskih frakcija u serumu bikova tijekom četiri godišnja doba prikazane
su na slici 3. Usporedbom koncentracija HDL i LDL lipoproteina u serumu bikova, utvrđene su
značajno veće (p<0,05) vrijednosti u uzorcima dobivenim u zimskom (2,49±0,89 mmol/L; 0,36±0,21
mmol/L) u odnosu na proljetno razdoblje (1,82±0,54 mmol/L; 0,17±0,09 mmol/L).
3,00
2,50
g/L
2,00
ab ab
b
a
1,50
1,00
0,50
a
ab ab b
0,00
HDL
proljeć e
V LDL
ljeto
LDL
jes en
z im a
Slika 3. Koncentracije lipoproteinskih frakcija u serumu bikova tijekom četiri godišnja doba. Histogrami s
različitim abcsuperskriptom statistički se značajno razlikuju (p<0,05).
Na slici 4. prikazane su koncentracije ukupnog kolesterola, HDL-C i LDL-C u sjemenoj plazmi
bikova tijekom četiri godišnja doba. Koncentracija ukupnog kolesterola i HDL-C u sjemenoj plazmi
bikova bila je statistički značajno veća (p<0,05) u zimskom razdoblju (1,02±0,47 mmol/L; 0,09±0,04
mmol/L) u odnosu na vrijednosti dobivene u ljeto (0,61±0,30 mmol/L; 0,06±0,01 mmol/L).
445
1,20
mmol/L
1,00
a
ad
0,80
0,60
b
bc
0,40
0,20
abc a
b bc
0,00
HDL-C
kolesterol
proljeće
ljeto
LDL-C
jesen
zima
Slika 4. Koncentracije ukupnog kolesterola, HDL-C i LDL-C u sjemenoj plazmi bikova tijekom četiri godišnja
doba. Histogrami s različitim abcsuperskriptom statistički se značajno razlikuju (p<0,05).
Rasprava
Rezultati načinjenog istraživanja pokazali su da su u krvnom serumu rasplodnih bikova najveće
koncentracije triglicerida, ukupnog kolesterola, LDL-C kao i LDL i HDL lipoproteinske frakcije
izmjerene u zimskom razdoblju, a SMK u jesen. Također, u sjemenoj plazmi raspodnih bikova u zimi
su izmjerene najveće koncentracije ukupnog i LDL kolesterola.
Koncentracija masnih tvari u serumu važna je u procijeni kvalitete obroka, odnosno energetskog
statusa životinja. Oksidacijom triglicerida oslobađaju se masne kiseline koje se u stanici mogu
esterificirati i pohraniti ili oksidirati pri čemu povećana koncentracija SMK ukazuje na mobilizaciju
masnih rezervi uzrokovanu negativnom energetskom bilancom obroka. U odnosu na fiziološke
vrijednosti za SMK u mliječnih krava, koje navodi WHITAKER (2000) te u usporedbi sa vrijednostima
WENSVOORT i sur. (2004) u deva i ostalih preživača u uvjetima negativne energetske bilance, u
ovom istraživanju vrijednosti SMK u serumu bikova bile su u granicama fizioloških vrijednosti,
iako je utvrđeno da su u uzorcima dobivenim u jesen vrijednosti bile značajno veće nego u ostalim
godišnjim dobima. Navedeni nalaz bi mogao biti posljedica nešto veće mobilizacije tjelesnih rezervi
nakon ljetnog razdoblja, odnosno neiskorištavanja bikova u rasplodne svrhe.
S obzirom da je netopiv u vodi, kolesterol se u sjemenoj plazmi i u serumu nalazi vezan s
molekulama bjelančevina u komplekse lipoproteina no njihov udio u ova dva medija je različit
(OCHOA i MARCHELLO, 1991). Koncentracija HDL-a u serumu bikova u ovom je istraživanju
bila najveća, nešto manja bila je koncentracija VLDL-a, dok je najmanje bilo LDL-a. Ovi su rezultati
u skladu s istraživanjem CHAPMAN i FORGEZ (1985) i OCHOA i MARCHELLO (1991). Frakcije
HDL i LDL u serumu bikova mijenjale su se tijekom istraživanja te su tako utvrđene značajne razlike
između proljetnog i zimskog razdoblja (slika 3.).
Osnovna funkcija HDL i LDL u serumu je prijenos kolesterola. Frakcija HDL prenosi kolesterol
iz perifernih stanica u jetru, a LDL prenosi kolesterol do stanica perifernih tkiva. I dok se koncentracija
HDL-C u serumu bikova tijekom godine nije značajno mijenjala, koncentracija LDL-C bila je veća
u zimi u odnosu na sva ostala godišnja doba, a značajno veća nego u proljeće. Koncentracija LDLkolesterola u serumu pokazala je sezonske promjene i kod ljudi (OCKENE i sur., 2004; KELLY,
446
2005) najvjerojatnije zbog promjena načina prehrane, aktivnosti tijekom godina i temperature okoline
kao i kombinacije svih pobrojanih utjecaja.
Na osnovi volumena, koncentracije i pokretljivosti spermija odabiru se uzorci sjemena za duboko
smrzavanje. Dosadašnja istraživanja su pokazala da se kvaliteta sjemena mijenja tijekom godine i
kod ljudi (CHEN i sur., 2003) i kod životinja (FUERST-WALTL i sur., 2006), a javljaju se i značajne
individualne razlike (JANETT i sur., 2003). CHEN i sur. (2003) su utvrdili da je kod ljudi najveća
koncentracija spermija i najveći postotak morfološki normalnih u zimskom razdoblju. JANETT i sur.
(2003) su jesenske uzorke sjemena pastuha proglasili najkvalitetnijim jer su i nakon zamrzavanja
zadržali dobra morfološka svojstva dok su ARGOV i sur. (2007) sjeme najlošije kvalitete dobili u
ljetnom uzimanju uzoraka sperme bikovima. FUERST-WALTL i sur. (2006) su istraživanjem na
bikovima utvrdili različitu osjetljivost na temperaturu okoline, kako u vrijeme uzimanja uzoraka,
tako i u vrijeme sazrijevanja spermija.
Neka istraživanja pokazuju da je ocjena kvalitete sjemena samo na osnovi makroskopskog
i mikroskopskog pregleda nedostatna (AGROV i sur., 2007; TURBA i sur., 2007) te se predlaže
određivanje dodatnih biokemijskih pokazatelja u spermijima i sjemenoj plazmi (BRINSKO i sur.,
2007). Analizom uzorka sjemena pastuha sa smanjenom plodnošću nepoznatog uzroka utvrđeno je
da imaju smanjenu koncentraciju kolesterola u sjemenoj plazmi (BRINSKO i sur., 2007). Smanjena
koncentracija kolesterola potvrđena je i u poznato lošim ljetnim uzorcima bikova čak i onda kada
su ocijenjeni kao dobri na temelju broja i morfoloških osobitosti spermija (ARGHOV i sur., 2007).
Ovakove rezultate potvrđuje i ovo istraživaje. Koncentracija ukupnog kolesterola u sjemenoj plazmi
bikova bila je značajno veća u uzorcima uzetim u zimskom u odnosu na ljetno razdoblje. Kolesterol
zajedno s fosfolipidima čini osnovu fizikalnog integriteta stanica i daje fluidnost staničnoj membrani.
Specifičnu ulogu kolesterol ima u membrani spermija jer odvajanjem s membrane spermija započinje
ključni korak u procesu kapacitacije i akrosomske reakcije bez kojih nema oplodnje jajne stanice
(WITTE i SCHÄFER-SOMI, 2007). Osim toga, utvrđeno je da dodatak kolesterola u razrijeđivače
prije procesa smrzavanja povećava otpornost spermija na stres izazvan procesima zamrzavanja i
odmrzavanja čime spermij zadržava svoju pokretljivost i sposobnost oplodnje jajne stanice (MOORE
i sur., 2005; ZAHN i sur., 2002).
U ovom istraživanju je utvrđeno da se kolesterol u sjemenoj plazmi nalazi raspoređen u dvije
lipoproteinske frakcije, HDL i LDL, pri čemu je koncentracija LDL-C višestruko veća od HDL-C.
Veću koncentraciju LDL-C utvrdili su ARGOV i sur. (2007) kod bikova kao i YOUSEF i sur. (2006)
kod kunića. Koncentracija LDL-C se nije značajno mijenjala tijekom različitih godišnjih doba, no
bila je znatno veća u zimsko doba (slika 4). Ovi rezultati potvrđuju da kvaliteti sperme doprinosi
povećana koncetracija LDL-C jer su zimski uzorci poznato dobre kvalitete (CHEN i sur., 2003) dok
je najniža u ljeto kad je sperma i najlošija. Lipoproteini niske gustoće štite membrane spermija od
oštećenja nastalih tijekom odmrzavanja, no iako se točan mehanizam još uvijek istražuje, MOUSSA
i sur. (2002) su utvrdili da dodatak LDL iz žumanjka jajeta u razrijeđivač za spermu bikova značajno
doprinosi zadržavanju pokretljivosti spermija nakon odmrzavanja. Isto tako, poznato je da je
sperma nerasta najosjetljivija na stres izazvan zamrzavanjem-odmrzavanjem, a dodatkom LDL-a u
razrijeđivač postiže se značajano poboljšanje kvalitete sjemena nakon odmrzavanja (JIANG i sur.,
2007).
447
Slika 5. Linearna korelacija LDL-C u sjemenoj plazmi i SMK u serumu (lijevo) i HDL-C u sjemenoj plazmi i
VLDL u serumu (desno) bikova tijekom četiri godišnja doba.
U istraživanju je utvrđena povezanost koncentracija masnih tvari u serumu i u sjemenoj plazmi.
Koncentracija slobodnih masnih kiselina u serumu bikova bila je u negativnoj korelaciji s LDLC u sjemenoj plazmi bikova (r=-0,30, p<0,05; slika 5) tako da mobilizacija masnih rezervi može
nepovoljno djelovati na kvalitetu sjemena dovodeći do smanjenja koncentracije LDL-kolesterola u
sjemenoj plazmi koja je uočena u spermi smanjene kvalitete.
Uočena je korelacija VLDL frakcije lipoproteina u serumu i HDL-C u sjemenoj plazmi (r=0,25,
p<0,05; slika 5). VLDL donosi trigliceride u periferne stanice preko vlastitih receptora (ARGOVi
sur., 2007). BRITES i sur. (1998) su utvrdili da u serumu postoji povezanost VLDL-triglicerida s
HDL-kolesterolom jer metabolizam lipoproteina znači međusobnu izmjenu lipidnih i proteinskih
sastojaka. Pa tako, djelovanjem bjelančevine koja prenosi estere kolesterola (eng. cholesterol ester
transfer protein, CETP) dolazi do prijenosa kolesterol estera s HDL na VLDL pri čemu nastaju
subfrakcije VLDL u serumu vrlo bogate kolesterolom (BRITES i sur., 1998). S druge strane, u
sjemenoj je plazmi koncentracija HDL-C izrazito mala dok je za proteinski dio utvrđeno da sudjeluje
u početku procesa kapacitacije (THÉRIEN i sur., 1998). Daljnjim istraživanjima treba utvrditi na koji
način VLDL utječe na količinu HDL-C.
Rezultati ovog istraživanja pokazuju da se sastav masnih tvari u serumu i sjemenoj plazmi bikova
mijenja tijekom godišnjih doba. Promjene koncentracije kolesterola u sjemenoj plazmi povezane su s
iskorištavanjem izvanstaničnih lipida u smislu očuvanja integriteta membrane spermija. Određivanje
koncentracije kolesterola u sjemenoj plazmi bikova moglo bi postati koristan dodatak standardnoj
procjeni kvalitete sjemena.
Literatura
ARGOV, N., D. SKLAN, Y. ZERON, Z. ROTH (2007): Association between seasonal changes in fatty-acid
composition, expression of VLDL receptor and bovine sperm quality. Theriogenology. 67, 878-885.
BRINSKO, S. P., C. C. LOVE, J. E. BAUER, M. L. MACPHERSON, D. D. VARNER (2007): Cholesterol-tophospholipid ratio in whole sperm and seminal plasma from fertile stallions and stallions with unexplained
subfertility Anim. Reprod. Sci. 99, 65-71.
448
BRITES, F. D., C. D. BONAVITA, M. CLOËS, M. J. YAEL, J. C. FRUCHART, G. R. CASTRO, R. W.
WIKINSKI (1998): VLDL compositional changes and plasma levels of triglycerides and high density
lipoprotein Clin. Chim. Acta. 269,107-124.
CEROLINI S., A. MALDJIAN, F. PIZZI, T. M. GLIOZZI (2001): Changes in sperm quality and lipid composition
during cryopreservation of boar semen. Reproduction. 121, 395-401.
CHAPMAN, M. J., P. FORGEZ (1985): Lipid transport systems: some recent aspects in swine, cattle and trout
during development. Reprod. Nutr. Develop. 25, 217-226.
CHEN, Z., T. TOTH, L. GODFREY-BAILEY, N. MERCEDAT, I. SCHIFF, R. HAUSER (2003): Seasonal
variation and age-related changes in human semen parameters. J. Androl. 24, 226-231.
FUERST-WALTL, B., H. SCHWARZENBACHER, C. PERNER, J. SÖLKNER (2006): Effects of age and
environmental factors on semen production and semen quality of Austrian Simmental bulls. Anim. Reprod.
Sci. 95, 27-37.
HERDT, T. H. (1991): Relationship of fat metabolism to health and performance in dairy cattle. Bov. Pract. 26,
92–95.
JANETT, F., R. THUN, S. BETTSCHEN, D. BURGER, M. HASSIG (2003): Seasonal changes of semen quality
and freezability in Franches-Montagnes stallions Anim. Reprod. Sci. 77, 213-221.
JIANG, Z. L., Q. W. LI, J. H. HU, W. Y. LI, H. W. ZHAO, S. S. ZHANG (2007): Improvement of the quality
of boar cryopreservation semen by supplementing with low density lipoprotein in diluents. Cryobiol. 54,
301-304.
KATOH, N. (2002): Relevance of apolipoproteins in the development of fatty liver and fatty liver-related
peripartum diseases in dairy cows. J. Vet. Med. Sci. 64, 293–307.
KELLY, G. S. (2005): Seasonal variations of selected cardiovascular risk factors. Alter. Med. Rev. 10, 307320.
MOORE, A. I., E. L. SQUIRES, J. K. GRAHAM (2005): Adding cholesterol to the stallion sperm plasma
membrane improves cryosurvival. Cryobiol. 51, 241-249.
MOUSSA, M., V. MARTINET, A. TRIMECHE, D. TAINTURIER, M. ANTON (2002): Low density
lipoproteins extracted from hen egg yolk by an easy method: cryoprotective effect on frozen-thawed bull
semen. Theriogenology. 57, 1695-1706.
OCHOA, M. F., J. A. MARCHELLO (1991): Bovine lipoprotein and apolipoprotein profiles as influenced by
sex and growth. J. Anim. Sci. 69, 4030-4038.
OCKENE, I. S., D. E. CHIRIBOGA, E. J. STANEK, M. G. HARMATZ, R. NICOLOSI, G. SAPERIA, A. D.
WELL, P. FREEDSON, P. A. MERRIAM, G. REED, Y. MA, C. E. MATTHEWS, J. R. HEBERT (2004):
Seasonal variations in serum cholesterol. Arch. Intern. Med. 164, 863-870.
PAYNE, J. M., S. PAYNE (1987): Indicators of energy status. In: The metabolic profile test. Oxford university
press, , Oxford, New York, Tokio. Pp. 17-26.
SEIFI, H. A., M. GORJI-DOOZ, M. MOHRI B. DALIR-NAGHADEH, N. FARZANEH (2007): Variations
of energy-related biochemical metabolites during transition period in dairy cows. Comp. Clin. Pathol. 16,
253- 258.
THÉRIEN, I., R. MOREAU, P. MANJUNATH (1998): Major proteins of bovine seminal plasma and highdensity lipoprotein induce cholesterol efflux from epididymal sperm. Biol. Reprod. 59, 768-776.
TURBA, M. E., P. FANTINATI, C. BERNARDINI, F. GENTILINI, M. L. BACCI, M. FORNI (2007):
Relationships between innovative and traditional parameters to investigate semen quality in pigs. Anim.
Reprod. Sci. 99, 72-81.
WENSVOORT, J., D. J. KYLE, E. R. ORSKOV, D. A. BOURKE (2004): Biochemical adaptation of camelids
during fasting. J. Camel Science. 1, 71-75.
449
WHITAKER, D. A. (2000): Use and interpretation of metabolic profiles. In: The health of dairy cattle. (A. H.
Andrews ed.) Blackwell Science Ltd, Oxford, UK.
WITTE, T. S., S. SCHÄFER-SOMI (2007): Involvement of cholesterol, calcium and progesterone in the
induction of capacitacion and acrosome reaction of mammalian spermatozoa. Anim. Reprod. Sci. 102,
181-193.
YOUSEF, M. I., F. M. EL-DEMERDASH, K. I. KAMIL, F. A. M. ELASWAD (2006): Ameliorating effect of
folic acid on chromium(VI)-induced changes in reproductive performance and seminal plasma biochemistry
in male rabbits. Reprod. Toxicol. 21, 322-328.
ZAHN, F. S., F. O. PAPA, J. A. DELL’AQUA (2002): Cholesterol incorpotation on equine sperm membrane:
effects on post-thaw sperm parameters and fertility. Theriogenology. 58, 237-240.
450
EFFECT OF DONOR BREED ON BOVINE OOCYTE RECOVERY AND
IN VITRO EMBRYO DEVELOPMENT AFTER OVUM PICK UP
UTJECAJ PASMINE GOVEDA NA JAJNE STANICE DOBIVENE
POSTUPKOM TRANSVAGINALNE ULTRAZVUČNE PUNKCIJE I
EMBRIONALNI RAZVOJ IN VITRO
M. Karadjole1, I. Getz1, N. Macesic1, M. Samardzija1, T. Karadjole1, G. Bacic1, Z.
Makek1, T. Dobranic1, M. Knezevic2, G. Perculija2, D. Cvitkovic1
1
Faculty of Veterinary Medicine, University of Zagreb
Faculty of Agriculture, University of Zagreb
2
Abstract
Ultrasound guided transvaginal oocyte recovery, also known as Ovum Pick-Up (OPU), and in vitro embryo
production (IVP) provides an alternative mean to increase the number of offspring from genetically valuable
cows. The objective of this study was to investigate the effect of two different cattle breeds on oocyte recovery
rate, quality and subsequent embryo development after transvaginal ovum pick up. Transvaginal ultrasound
guided (Pie Medical, the Netherlands) oocyte collection was performed in 8 nonlactating donor cows (4
Holstein-Friesian and 4 Charolais). Cows were synchronized with PGF2α and stimulated with pFSH, twice a
day during two days (Folltropin®, Bioniche, a total dose: 200 mg NIH-FSH-P1). OPU was performed 24 hours
after the last FSH injection and procedure was repeated every second week for eight consecutive weeks. The
number of aspirated follicles, the number of retrieved oocytes and oocyte recovery rate were recorded. The
collected oocytes were morphologicaly evaluated and subsequently submitted to in vitro embryo production.
The cleavage rates on Day 2, the total number of morulas (M) and blastocysts (Bl) on Day 7 and the numbers
of hatched blastocysts (hBl) on Day 9 were recorded. Significantly more follicles were aspirated from Charolais
cows (p<0.05). The same group also yielded higher quality oocytes than the Holstein-Friesian cows (p<0.05).
No differences in recovery rates, cleavage rates, blastocyst rates and hatched blastocyst rates were observed
between two breeds. Although we observed some breed differences related to the number of follicles and
oocyte quality, further investigations are necessary to provide more data on breed influences on superovulatory
response, oocyte number, quality and developmental competence.
Key words: cow, breed, ovum pick up, oocyte recovery, in vitro embryo development.
Sažetak
Transvaginalna ultrazvučna punkcija jajnih stanica, kratko nazvana Ovum Pick-Up (OPU) i proizvodnja
zametaka in vitro (IVP) pruža mogućnost dobivanja većeg broja potomaka od genetski vrijednih krava. Cilj
ovog istraživanja bio je usporediti utjecaj dviju pasmina goveda na postotak aspiriranih jajnih stanica nakon
transvagnalne ultrazvučne punkcije, njihovu kvalitetu i embrionalni razvoj in vitro. Transvaginalna ultrazvučna
puncija (Pie Medical, Nizozemska) provedna je na 8 zasušenih krava davateljica (4 Holštajn-frizijske i 4
Charolais pasmine). Krave su sinkronizirane sa PGF2α i stimulirane sa pFSH, dva puta dnevno tijekom dva
dana (Folltropin®, Bioniche, a total dose: 200 mg NIH-FSH-P1). OPU je izeden 24 sata nakon zadnje FSH
injekcije a postupak je ponavljan svaka dva tjedna tijekom dvomjesečnog razdoblja. Bilježen je broj aspiriranih
folikula, broj aspiriranih jajnih stanica i uspjeh aspiracije. Aspirirane jajne stanice morfološki su ocjenjene
te je praćena njihova sposobnost dozrijevanja, oplodnje i uzgoja in vitro. Ocjena razvoja goveđih zametaka
provedena je po fazama: drugog dana uzgoja zabilježen je broj brazdanih zametaka; sedmog dana broj morula
i blastocista te devetog dana broj izvaljenih blastocista. U krava Charolais pasmine aspiriran je značajno veći
broj folikula (p<0.05) što je rezultiralo i većim brojem jajnih stanica dobre kvalitete (p<0.05). Unatoč tome, nisu
451
utvrđene razlike u uspjehu aspiracije, postotku brazdanja te broju formiranih blastocista. Iako su ustanovljene
razlike vezane uz broj folikula i kvalitetu aspiriranih jajnih stanica, potrebna su daljnja istraživanja kako bi
dobili više podataka o pasminskom utjecaju na superovulacijski odgovor, broj jajnih stanica, njihovu kvalitetu
te sposobnost razvoja in vitro.
Ključne riječi: krava, pasmina, aspiracija jajnih stanica, embrionalni razvoj in vitro
Introduction
Ultrasound guided transvaginal oocyte recovery, also known as Ovum Pick-Up (OPU) is a
reliable technique for harvesting immature bovine oocytes from live donors. In conjunction with in
vitro embryo production (IVP), this technique provides an alternative mean to increase the number
of offspring from genetically valuable cows (Galli et al., 2001). OPU could be applyed in animals
of various physiological state (cyclic, non cyclic, early pregnancy) and of all ages, including calves,
prepubertal heifers and older cows with reproductive disorders or those who fail to respond to
superovulation protocols (Hasler, 2003). The success of OPU considerably depends on the number
and the quality of retrieved oocytes per session. A key factor confounding the predictability and
reliability of in vitro embryo production is variability in the quality or competence of harvested
oocytes (Reis et al., 2002). The in vitro developmental competence of oocytes is related to follicle size,
estrous cycle stage and the level of atresia influenced by other follicles, mainly the dominant follicle
(Machatkova et al., 2004). Numerous factors can affect the number of oocytes recovered by Ovum
Pick-Up such as genetic background (Roschlau et al., 2003), age, nutrition, parity, lactational status
and milk yield (Snijders et al., 2000). The proportion of antral follicles yielding developmentally
competent oocytes can also be influenced by external factors such as gonadotropine administration
(Fry et al., 1994). Also, significant effect of breed on follicle sizes, oocyte number, morphological
quality of the oocytes retrieved by OPU and subsequent embryo development was reported (Lopes
et al, 2006).
The aim of the present study was to investigate the effect of two different cattle breeds on number
of follicles, oocyte recovery rate, quality and subsequent embryo development after transvaginal
Ovum Pick-Up.
The study was carried out using 8 healthy, normally cycling cows (mean age 4,6 years), divided
into two groups according to the breed. Group A was Holstein-Friesian (HF) and group B Charolais
(CH) breed. Cows were synchronized with two prostaglandin F2α treatments at 11-day interval.
The gonadotropine treatment was initiated between 9 and 10 days after induced oestrus. Treatment
consisted of pFSH (Folltropin®, Bioniche Animal Health, Ireland), twice a day, during two days. The
total amount of pFSH administered was 200 mg NIH-FSH-P1 in 4 equivalent doses. Ultrasoundguided Ovum Pick-Up was performed 24 hours after the last FSH injection. Superovulation and
OPU/IVF procedures were repeated every 14 days during 2 months (a total of 4 aspirations per cow).
All media and reagents, unless otherwise stated, were from Sigma, Germany.
The OPU procedure and oocyte recovery
Oocyte collection using OPU technology was performed according to standard procedure
described by Pieterse et al. (1988) and Getz (2004). Briefly, xylazine hydrocloride was administered
i.m. (Xyilapan, Chassot; 0,25 ml/100 kg) to all animals, following an epidural anaesthesia (4 ml of
2% lidocaine hydrocloride) prior to transvaginal aspiration of oocytes. A 7.5 MHz sector transvaginal
452
transducer of an ultrasound scanner (Pie Medical, the Netherlands) mounted on a holder with a grip
was used to guide the needle (18G disposable needle, attached to the stainless steel guidance) through
the vagina into the ovary. All visible follicles (≥2mm) were punctured and the contents collected in
a sterile 15 mL tube (Falcon) with aspiration medium (TCM hepes supplemented with antibiotics,
heparine and bovine serum albumine) attached to a suction pump to ensure constant aspiration
pressure. The follicular fluid obtained by aspiration were transfered into an embryo transfer filter
(Minitub™, Germany). The recovered oocytes were identified under a stereomicroscope, counted
and morfologically classified into four categories (Makek et al., 1998). The number of aspirated
follicles, the number of retrieved oocytes and oocyte recovery rate were recorded.
In vitro embryo production
Oocytes were recovered from the embryo filter and transferred into a culture dish with TCM
hepes supplemented with 10% of FCS. Oocytes of grade 1 and 2 with intact cumulus investment and
evenly granulated cytoplasm were matured in vitro in TCM 199 bicarbonate medium supplemented
with 10% FCS, FSH/LH (Pergonal, 75/75 IU, Serono), 1 µg/ml estradiol-17ß and 100 µM cysteamine.
Oocytes were incubated in groups of 10 in 50 µL droplets of maturation media under mineral oil for
24 h at 38.5°C with 5% CO2 in humidified air. The expanded COCs were washed in HEPES-TALP
medium supplemented with 6 mg/mL BSA-FAF and transferred into 40 µL droplets of IVF medium
under mineral oil. In vitro fertilization was performed in modified Tyrode’s bicarbonate buffered
solution supplemented with 10 µg/mL heparin, 5 µg/mL hypotaurine, 5 µg/mL epinephrine and 6
mg/ml BSA-FAF. In all experiments, frozen semen from the same bull was used. Sperm preparation
for IVF on BoviPure® gradient was accomplished according to Samardzija et al., (2006). The final
concentration was adjusted to 1×106 spz/mL. Incubations were carried out at 39°C in 5% CO2 in air
for 24 h. After the sperm-oocytes co-incubation, the presumptive zygotes were denuded from cumulus
cells and spermatozoa and washed in HEPES-TALP medium and in culture medium. Fertilized
oocytes were cultured in vitro in synthetic oviductal fluid (SOF) with amino acids and 8 mg/mL
BSA without glucose for 48 h and then transferred in SOF with 1.5 mM glucose. The embryos were
cultured in vitro until Day 9 at 39°C in 5% CO2, 5% O2 ,90% N2 atmosphere with maximum humidity.
Cleavage was assessed 48 hours post insemination (hpi) and blastocyst development was recorded on
Days 7 and 9 (Day 0= day of IVF). Evaluation of embryos was performed under a stereomicroscope
using standard morphological criteria.
All results were statistically analyzed with ANOVA (StatSoft, Statistic, 7.1.) and with Tukey’s
tests post-hoc analysis.
Results
For the four Holstein-Friesian cows, a total of 126 follicles were punctured during four OPU
sessions and 75 oocytes were collected. For the four Charolais cows, a total of 198 follicles were
punctured during four OPU sessions and 115 oocytes were collected.
There were significantly more follicles on the ovaries of Charolais cows (13.2 versus 8.4,
P<0.05). This was reflected in significantly higher number of grade 1 and 2 oocytes recovered from
Charolais cows (6.3 versus 3.7, P<0.05) (Table 1.).
453
Table 1. Number of follicles punctured and oocyte morphology in Holstein-Friesian and Charolais cows (mean
± S.E.M.)
Animal breed
HolsteinFriesian
Charolais
Number
of follicles
punctuired per
animal
Number of
recovered
oocytes per
animal
Oocyte
recovery rate
per animal
Number of
grade 1-2
oocytes per
animal
Number of
grade 3-4
oocytes per
animal
8.4 ± 0.55a
5.0 ± 0.63
59.6 ± 0.06
3.7 ± 0.51a
1.2 ± 0.27
13.2 ± 1.32b
7.6 ± 1.23
59.2 ± 0.05
6.3 ± 1.02b
1.0 ± 0.22
Values with different superscripts differ between columns
ab
A total of 58 oocytes from HF and 100 oocytes from CH cows were submitted to IVM/IVF/IVC.
There were no significant differences regarding to cleavage rate, blastocyst rate or hatched blastocyst
rate between two breeds (Table 2.).
Table 2. Developmental competence of OPU derived oocytes after in vitro maturation, fertilization and culture
in Holstein-Friesian and Charolais cows (mean ± S.E.M.)
Animal breed
Holstein-Friesian
Charolais
Number of oocytes
Cleavage rate (%)
Blastocyst yield
(%)
Day 7
Hatched blastocyst
yield (%) Day 9
58
88.7 ± 0.04
51.0 ± 0.14
24.3 ± 0.1
100
86.5 ± 0.01
40.6 ± 0.07
16.6 ± 0.05
Discussion
Our study about efficiency of Ovum Pick-Up in cows from two different breeds shows that
Charolais donor cows yielded higher mean number of follicles punctured and higher number of good
quality oocytes recovered by OPU than Holstein-Friesian donor cows. However, irrespective to the
breed, oocytes of similar quality (grade 1-2) had a similar developmental ability and no differences
were found related to the proportion of good quality oocytes undergoing in vitro fertilization and
subsequent development to the blastocyst stage.
Numerous factors can affect the number of oocytes recovered by Ovum Pick-Up. Merton et
al. (2003) demonstrated that the oocyte yield was dependent on the OPU team, in particular the
technitian manipulating the ovaries. The proportion of antral follicles yielding developmentally
competent oocytes can also be influenced by external factors such as gonadotropine administration.
Also, different biological aspects such as genetic background (Roschlau et al., 2003.), breed (Duc
et al., 2003; Lopes et al., 2006) or external factors such as nutrition, menagment practice, parity,
lactational status, milk yield (Mac-Millan et al., 1996; Snijders et al., 2000) can affect the oocyte
recovery rate after OPU.
454
Breed differences have previously been reported for slaughterhouse derived oocytes (Domingez,
1995) and for the oocytes collected by OPU (Schernthaner et al., 1999; Duc et al., 2003; Faber et
al., 2003). The study about efficiency of OPU in cows from five different breeds (Schernthaner et al.
,1999.) reported that Charolais breed showed the best results in average of all parameters investigated,
which were number of follicles, number of oocytes recovered, number of embryos produced,
traferred and the percentage of pregancies. On contrary, Holstein-Friesian donor cows showed poor
results concerning the number of follicles, oocytes and IVP-useful oocytes per puncture session. This
finding was in agreement with our results where Charolais donor cows showed significantly better
results in number of follicles punctured (13.2 versus 8.4 follicles in Charolais and Holstein-Friesian,
respectively). The number of embryos of grade 1 and 2 was also higher in Charolais donor cows
(6.3 versus 3.7) which is related to the higher number of follicles aspirated. Lopes et al. (2006.) also
reported significant effect of breed on the morphological quality of the oocytes recovered by OPU.
In the present study the developmental competence of good quality oocytes (grade 1-2) to reach
the morula/blastocyst stage at day 7 and hatched blastocyst stage at day 9 of in vitro culture, was
similar between Charolais and Holstein-Friesian breed. There was no differences in the proportion
of good quality (grade 1-2) oocytes undergoing in vitro fertilization and subsequent development to
the blastocyst stage.
In conclusion, we reported that Charolais donor cows showed significantly better results in
number of follicles punctured and number of grade 1 and 2 embryos recovered by OPU, but there
was no differences in developmental competence of retreived oocytes of similar quality between two
breeds. Although we observed some breed differences related to the number of follicles punctured
and quality of recoverd oocytes, further investigations are necessary to provide more data on breed
influences on superovulatory response, oocyte recovery, quality and developmental competence.
References
1.
Duc NH., Uoc NT., Ty LV., Hanh NV., Thanh NT., Bui LC., Anh NT., Huu QX., Nguyen BX.: Potential for
in vitro production of embryo from follicular oocyte from yellow and yellow-red sindhi crossbred cattle.
Theriogenology 2003, 59, 442.
2.
Fry RC., Simpson TL., Squires TJ., Parr RA., Damanik RM.: Factors affecting transvaginal oocyte pick up
in heifers: Theriogenology 1994, 41:197 (Abstract).
3.
Galli C., Crotti G., Notari C., Turini P., Duchi R., Lazzari G.: Embryo production by ovum pick up from
live donors. Theriogenology 2001, 55, 1341-1357.
4.
Getz I.: Uspješnost stimulacije jajnika krava u postupku transvaginalne ultrazvučne punkcije i uzgoja in
vitro goveđih zametaka. Doktorska disertacija, Zagreb, 2004.
5.
Hasler JF.:The current status and future of commercial embryo transfer in cattle. Anim Reprod Sci 2003,
79, 245-264.
6.
Lopes AS., Martinussen T., Greve T., Callesen H.: Effect of days post-partum, breed,and Ovum pick-up
scheme on bovine oocyte recovery and embryo development. Reprod Dom Anim 2006, 41, 196-203.
7.
Machatkova M., Krausova K., Jokesova E., Tomanek M.: Developmental competence of bivine oocytes:
effect of follicle size and the phase of follicular wave on in vitro embryo production. Theriogenology 2004,
61, 329-335.
8.
Makek Z., Getz I., Cergolj M., Herak M., Tomašković A., Dobranić T., Sušić V.: Selection of immature
bovine oocytes as the preliminary phase of in vitro fertilization. Vet. Arhiv 1998, 68, 109 -119.
9.
Merton JS., De Roos AP., Mullaart E., De Ruigh L., Kaal L., Vos PL.: Factors affecting oocyte quality and
quantity in commercial application of embryo technologies in the cattle breeding industry. Theriogenology,
2003, 59:651-674.
455
10. Pieterse MC., Kappen KA., Kruip ThAM., Taverne MAM.: Aspiration of bovine oocytes during transvaginal
ultrasound scanning of the ovaries. Theriogenology 1988, 30, 751-762.
11. Roschlau K., Kuwer A., Kuhnt C., Poppe P., Roschlau D., Johanning S., Michaelis U., Dexne U.: Individuelle
varianzen in der Ausbeute an Embryonen nach OPU/IVP. In: Proceedings of the 30th Ann.Meeting AET-D,
2003, Marieensee, Germany. German ET-Association (AET-d), Marieensee Neustadt, Germany, pp. 3-4.
12. Samardzija, M., Karadjole M., Getz I., Makek Z., Cergolj M., Dobranić T.: Effects of bovine spermatozoa
preparation on embryonic development in vitro. Reproductive Biology and Endocrinology 2006,4;58.
13. Snijders SEM., Dillon P., O’Callaghan D., Boland MP.: Effect of genetic merit, milk yielj, body condition
and lactation number on in vitro oocyte development in diary cows. Theriogenology 2000, 53, 981-989.
14. MacMillan KL., Lean IJ., westwood CT.: the effects of lactation on the fertility of diary cows. Aust Vet J
1996, 73, 141-147.
15. Domingez MM.: Effects of body condition, reproductive status and breed on follicular population and
oocyte quality in cows. Theriogenology 1995, 43, 1405-1418.
16. Faber DC., Molina JA., Ohlrichs CL., Vander Zwaag DF., Ferre LB.: Commercialization of animal
biotechnology. Theriogenology 2003, 59, 125-138.
17. Schernthaner W., Wenigerkind H., Stojkovic M., Palma GA., Mödl J., Wolf E., Brem G.: Pregnancy rate
after Ultrasoun guided Follicle aspiration in nonlactating Cows from different breeds. J. Vet. Med. A 1999,
46, 33-37.
18. Reis A., Staines ME., Watt RG., Dolman DF., McEvoy TG.: Embryo production using defined oocyte
maturation and zygote culture media following repeated ovum pick-up (OPU) from FSH stimulated
Simmental heifers. Anim Reprod Sci 2002, 72, 137-151.
456
THE ETIOLOGY AND PREVALENCE OF INTRAMAMMARY
INFECTION OF DAIRY GOATS IN REGIONS OF NORTH CROATIA
ETIOLOGIJA I PREVALENCIJA INTRAMAMARNIH INFEKCIJA KOZA
U SJEVERNOJ HRVATSKOJ
Nino Maćešić1, Marijan Cergolj1, Nikica Prvanović1, Marko Samardžija1, Tugomir
Karadjole1, Martina Karadjole1, Goran Bačić1, Tomislav Dobranić1, Antun Kostelić2,
Miroslav Benić3
1
Faculty of Veterinary Medicine, University of Zagreb
Faculty of Agriculture, University of Zagreb
2
3
Croatian veterinary institute
Abstract
The aim of this of study was to determine the etiology and prevalence of intramammary infections (IMI)
of dairy goats in north Croatia. Milk samples (n=606) were taken from 303 goats from randomly selected
sheep flocks (n=5). The herds ranged in size from 40 to 114 dairy goats in lactation. All goats were French
alpine breed. The goats were reared under intensive system, in stalls without dry lot. Milking was conducted
in parlor by milking machine in a bucket. Milk samples were tested with bacteriological analyses and 122
samples were positive. Prevalence of IMI was 20,1% (n=122) of all udder-half samples examined. The most
prevalent mastitis agent was Staphylococcus aureus 72,1% (n=88 udder-half samples). Other prevalence were
Bacillus spp. 13% (n=16 udder-half samples), coagulasa-negative Staphylococci (CNS) 9,8% (n=12 udder-half
samples), Streptococcus group D 4,1% (n=5). Salmonella spp. and Listeria monocytogenes were not detected.
The prevalence of IMI between left and right udder half were not significant.
Key words: goat, intramammary infection, bacteriological analysis, milk
Sažetak
Istraživanje je provedeno u svrhu određivanja etiologije i prevalencije intramamarnih infekcija mliječnih
koza u sjevernoj regiji Hrvatske. Uzeti su uzorci mlijeka (n=606) od 303 životinje iz nasumično odabranih stada
(n=5). Stada su se kretala od 40 do 114 mliječnih koza francuske alpske pasmine u laktaciji. Koze su držane
intenzivno, stajski, bez ispusta. Mužnja se obavlja u izmuzištu, strojno, u kantu. Bakteriološkom pretragom
600 uzoraka mlijeka dobili smo 122 bakteriološki pozitivnih uzoraka. Prevalencija intramamarnih infekcija
bila je 20,1% (n=122) od svi pregledanih uzoraka polovina vimena. Najučestaliji uzročnik mastitisa bio je
Stapylococcus aureus 72,1% (n=88 uzoraka polovina vimena). Bacillus spp. izoliran je iz 13% uzoraka (n=16),
koagulaza negativni Staphylococci (CNS) kod 9,8% (n=12), Streptococcus skupine D 4,1% (n=5). Salmonella
spp. I Listeria monocytogenes nisu izdvojene. Prevalencija intramamarnih infekcija između lijeve i desne
polovine vimena nije bila značajna.
Ključne riječi: koze, intramamarne infekcije, bakteriološka pretraga, mlijeko
Uvod
Europska unija (EU) ima 1,5% (11,730,164 grla u 2002) svjetske populacije koza. Četiri zemlje
(Italija, Grčka, Španjolska, i Francuska) u Mediteranskoj zoni imaju 91% cijelokupne europske
populacije koza i 95% proizvodnje kozijeg mlijeka (FAO, 2002).
457
U Republici Hrvatskoj uzgaja se oko 80.000 koza, a Hrvatski stočarski centar je temeljem
zahtjeva za ostvarivanjem državnih novčanih potpora za držanje rasplodnih koza tijekom 2006.
godine evidentirao ukupno 52.397 grla kod 1.072 uzgajivača, pri čemu su evidentirana samo stada
s najmanje 20 odraslih grla, što je u 2006. godini bio minimalno potreban broj grla da bi uzgajivač
ostvario novčanu potporu.
U tri priobalne županije (Zadarska, Šibensko-kninska i Splitsko dalmatinska županija)
evidentirano je ukupno 27.816 koza ili 53,09% od ukupnog broja evidentiranih koza. U pasminskom
sastavu na ovim prostorima dominira hrvatska šarena koza (HSC, 2006).
U kontinentalnom dijelu Republike Hrvatske dominiraju inozemne mliječne pasmine, a među
njima je najzastupljenija francuska alpina. Najveći broj koza u kontinentalnom dijelu Republike
Hrvatske evidentiran je u Varaždinskoj, Međimurskoj i Bjelovarsko-bilogorskoj županiji (9.675 grla
ili 18,46% od ukupnog broja registriranih koza).
U Hrvatskoj je higijenska ispravnost kozjeg mlijeka regulirana zakonom (Pravilnik o kakvoći
svježeg sirovog mlijeka, NN 102/00). Minimalni uvijeti kojima treba udovoljavati kozije mlijeko
određeni su prema ukupnom broju bakterija u mlijeku dok granična vrijednost za broj somatski
stanica nije definirana. Preduvijet za proizvodnju higijenski ispravnog mlijeka i sira je zdravo vime,
stoga intramamarne infekcije imaju veliku važnost u proizvodnji kozjeg mlijeka.
Materijali i metode
Uzorci mlijeka uzeti su od 303 mliječne koze sa pet različitih, nasumično odabranih farmi u
sjevernoj Hrvatskoj (Varaždinska županija, Međimurska županija ). Veličina stada kretala se od 40114 životinja. Sve koze bile su francuske alpske pasmine. Životinje su držane intezivno u stajama
bez ispusta. Hranjene su sijenom, koncentratom i vodom koji su bili dostupni ad libidum. Mužnja se
obavljala u izmuzištu pomoću stroja za mužnju. Koze su se muzle dva puta dnevno.
Uzorci mlijeka uzeti su od svake životinje jednokratno. Prije uzorkovanja vrhovi sisa su očišćeni
5% alkoholom. Prvi mlazovi mlijeka su izmuzeni, a slijedećih 10 ml mlijeka iz svake polovine
vimena je aseptički uzeto u odvojenu sterilnu epruvetu. Uzorci su se držali na +4 ºC do bakteriološke
pretrage.
Rezultati i rasprava
Ukupno je uzeto 606 uzoraka iz svih polovina vimena od 303 koze sa pet farmi. Bakteriološkom
pretragom uzoraka dobili smo 122 bakteriološki pozitivana uzorka, pa je prevalencija infekcije
kod svih pregledanih uzoraka iznosila 20,1%. Između koza koje su imale mastitis, 66,4 % je imalo
unilateralnu infekciju vimena, dok su ostale imale inficirane obje polovine vimena. Koze kod kojih
je zabilježena infekcija obje polovine vimena imale su 70,7% infekcija sa Staphylococcus aureusom
i 29,3% sa koagulaza negativnim satafilokokima.
Najučestaliji uzročnik mastitisa bio je Stapylococcus aureus 72,1% (n=88 uzoraka polovina
vimena). Bacillus spp. izoliran je iz 13% uzoraka (n=16), koagulaza negativni Staphylococci (CNS)
kod 9,8% (n=12), Streptococcus skupine D 4,1% (n=5). Salmonella spp. I Listeria monocytogenes
nisu izdvojene. Prevalencija intramamarnih infekcija između lijeve i desne polovine vimena bila je
potpuno jednaka 50% i 50% što je slično rezultatima drugih autora koji su istraživali mastitise koza
(Moroni i sur., 2005).
Prevalencija infekcije s obzirom na vrstu uzročnika i zahvaćenu četvrt prikazana je u tablici 1.
458
Tablica 1. Izolirani mikroorganizmi i prevalencija (% - odnos inficiranih polovica vimena) infekcije kod lijeve
i desne polovice vimena koza
Polovica vimena
Vrsta mikroorganizma
Lijeva polovica (n=61)
50%
75,4%
8,2%
16,4%
Svi uzročnici
Staphylococus aureus
CNS
Ostali
Desna polovica (n=61)
50%
68,9%
8,2%
22,9%
Tablica 2. Izolirani mikroorganizmi i prevalencija infekcije (%) od uzetih uzoraka mlijeka
Vrsta mikroorganizma
Ukupna prevalencija
Staphylococcus aureus
Bacillus spp.
CNS
Streptococcus tip D
Escherichia coli
A(n=106)
14,1
60
0
13,3
26,6
0
B(n=104)
31,7
100
0
0
0
0
Stado
C(n=80)
25
100
0
0
0
0
D(n=88)
26,1
100
0
0
0
0
E(n=228)
14
12,5
50
31,2
3,1
3,1
Najveća skupina patogena bila je Staphylococcus spp. koja je izolirana iz 100 bakteriološki
pozitivnih uzoraka mlijeka (82% pozitivnih uzoraka). Najzastupljeni iz te skupine bio je S.aureus
koji je izdvojen iz 88 uzoraka mlijeka a CNS su izdvojeni iz 12 uzoraka Na tri farme (B, C, D)
sa prevalencijom infekcije od 25% do 31,6% izoliran je S.aureus iz svih (100%) bakteriološki
pozitivnih uzoraka (Tablica 2.). Tako visoku prevalenciju infekcije sa S.aureusom možemo povezati
sa izostankom izlučivanja kronično oboljelih životinja te izostankom provođenja adekvatnih
higijenskih mjera tijekom mužnje. Zbog svoje patogenosti S. aureus je najznačajniji patogen
mliječne žlijezde koze. Infekcije vimena sa S. aureusom mogu biti subkliničke, kronične, akutne, a u
najtežem obliku i gangrenozne. Rezistentni su na antibiotsku terapiju zbog sposobnosti da obitavaju
intracelularno i u L-oblicima te zbog smještaja unutar mikroapscesa u vimenu (Sears i sur., 1987;
Smith i Sherman, 1994). Terapija životinja u suhostaju, izlučivanje kronično oboljelih životinja iz
stada te provedba higijenskih mjera tijekom mužnje osnovne su kontrolne mjere za smanjenjem
novih infekcija (Robertson, 1999). Drugi najzastupljeniji mikroorganizam u ovom istraživanju bio
je Bacillus spp. koji je izoliran iz 13% bakteriološki pozitivnih uzoraka a svi pozitivni uzorci kod
kojih je izoliran bili su sa farme E gdje su izolirani kod 50% uzoraka. Na farmi E prevalencija
infekcije sa S.aureusom je 12,5% što je u sukladno ranije provedenim istraživanjima (Manser, 1986;
East i sur, 1987; Hall i Rycroft, 2007). Pojavnost Bacillusa spp. u tolikoj mjeri može se povezati sa
lošim postupcima vođenja farme a slično je zabilježio i Kalogridou-Vassiliadou (1991). Koagulaza
negativni stafilokoki (CNS) izolirani su iz 9,8% pozitivnih uzoraka. Na farmi A izolirani su iz 13,3%,
a na farmi E 31,2% pozitivnih uzoraka dok nisu zabilježeni na farmama B, C, i D. Stafikokoki koji
su najčešće uzročnici supkliničkih mastitisa kod koza su uglavnom koagulaza negativni stafilokokiCNS. Prevalencija CNS-a raste kod nedostatka adekvatne higijene tijekom mužnje ili upotrebe
neispravnog stroja za mužnju. Na taj način dolazi do izražaja oportunistička priroda ove skupine
mikroorganizama. Iako su CNS manje patogeni od S.aureusa oni mogu dovesti do perzistentnog
supkliničkog mastitisa i čak do kliničkog mastitisa (Deinhofer i Pernthaner, 1995; Contreras i sur.,
1997.) kao i stvaranja termostabilnog enterotoksina (Meyrand i sur., 1999; Udo i sur, 1999). Glavni
459
CNS patogeni koji uzrokuju intramamarne infekcije nalaze se na koži vimena i sisa koza (Valle i
sur., 1991). Smatra se da su postupci kod mužnje pogodovni za njihov ulazak u vime u terenskim
uvijetima. Na isti način, različiti postupci sanitacije koji se koriste kod mužnje mogu objasniti razliku
u prevalenciji supkliničkih intramamarnih infekcija između stada mliječnih koza (East i sur, 1987).
Streptococcus skupine D izolirani su iz 4,1 % pozitivni uzoraka. Streptokoki kao uzročnici
mastitisa ne predstavljaju skupinu osobito učestalih patogena na farmama koza i obično se pojavljuju
u manje od 5-10% slučajeva mastitisa kod koza (Contreras i sur., 2003).
Zaključci
Stapylococcus spp. bila je najučestalija skupina bakterija koja je izolirana iz polovina vimena
mliječnih koza u sjevernoj Hrvatskoj. Unutar te skupine a i kao pojedinačni uzročnik najučestaliji je
bio S.aureus.
Literatura
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
460
CONTRERAS, A, CORRALES, J.C., SANCHEZ, A, SIERRA, D (1997): Persistence of subclinical
intramammary pathogens in goats throught lactation. J. Dairy Sci. 80, 2815-2819.
CONTRERAS, A., LUENGO, C., SANCHEZ, A., CORRALES, J.C (2003): The role of intramammary
pathogens in dairy goats. Livest. Prod. Sci. 79, 273–283.
DEINHOFER, M., PERNTHANER¸A. (1995): Staphylococcus spp.as a mastitis-related pathogens in goat
milk. Vet. Microbiol. 43, 161-166
EAST, N.E., BIRNIE, E.F., FARVER, T.B. (1987): Risk factors associated with mastitis in dairy goats. Am.
J. Vet. Res. 48 5 (1987), pp. 776–779.
HALL, S.M., RYCROFT, A.N. (2007): Causative organisms and somatic cell counts in subclinical
intramammary infections in milking goats in the UK, Veterinary Record 160 (1) (2007), pp. 19-22
KALOGRIDOU-VASSILIADOU (1991): Mastitis-related pathogens in goat milk. Small Rumin. Res. 4 2
(1991), pp. 203–212.
HRVATSKI STOČARSKI CENTAR (2006): Godišnje izvješće za 2006. godinu, Hrvatski stočarski centar,
str.127
FAOSTAT, 2002. Agriculture Statistic (online: http://www.fao.org.), FAO, Rome, Italy
MORONI,P., PISONI,G., RUFFO,G., BOETTCHER,PJ. (2005): Risk factors for intramammary infections
and relationship with somatic-cell counts in Italian dairy goats. Prev Vet Med 69(3-4): 163-73
MANSER, P.A. (1986): Prevalence, causes and laboratory diagnosis of subclinical mastitis in the goat. Vet.
Rec. 118 20 (1986), pp. 552–554.
MEYRAND, A., MONTET, M.P., BAVAI, C., RAY-GUENIOT, S., MAZUY, C., GASPARD, C.E.,
JAUBERT, G., PERRIN, G., VERNOZY-ROZAND, C. (1999): Risk linked to an enterotoxigenic strain of
Staphylococcus lentus during the manufacture and ripening of raw goats’milk Camembert-type cheeses.
Rev. Med. Vet. 150 (8–9), 703–708.
PRAVILNIK O KAKVOĆI SVJEŽEG SIROVOG MLIJEKA (2000), NN, 102/00
ROBERSON, J.R. (1999): Epidemiology of Staphylococcus aureus on dairy farms. In: Proceedings of the
38th Annual Meeting of the National Mastitis Council, 14–17 February, Arlington, VA, (1999), p. 38.
SEARS, P.M., FETTINGER, M., MARSH-SALIN, J. (1987): Isolation of Lform variants after antibiotic
treatment in Staphylococcus aureus bovine mastitis. J. Am. Vet. Med. Assoc. 191, 681±684
SMITH, M.C., SHERMAN, D.M. (1994): Mammary gland and milk production. In: Goat Medicine, Lea
and Febiger, Philadelphia, PA, USA, pp. 474±479.
UDO, E.E., AL-BUSTAN, M.A., JACOB, L.E., CHUGH, T.D. (1999): Enterotoxin production by
coagulase-negative staphylococci in restaurant workers from Kuwait City may be a potential cause of food
poisoning. J. Med. Microbiol. 48, 819–823.
Valle et al., J. Valle, S. Piriz, R. De la Fuente and S. Vadillo (1991): Staphylococci isolated from healthy
goats. J. Vet. Med. B. 38, pp. 81–89.
PHENOTYPE AND PRODUCTIVE CHARACTERISTICS OF DUBIAN
ZECKEL SHEEP
FENOTIPSKE I PROIZVODNE KARAKTERISTIKE OVCE DUBSKE
PRAMENKE
Šakić Vedad, Katica Velija, Crnkić Ćazim, Softić Almira, Brdarić Maja
Veterinary Faculty of Sarajevo University, Bosnia and Herzegovina
Summary
Sheep breeding is important part of animal breeding in Bosnia and Herzegovina. More than 1.000.000
hectars of pasturing area in highlands provide optimal conditions for production of food of high biological
value, including lamb and cheese. Sheep breeding, completely integrated in land and climatic resources, is
still under traditional way of production without significant changes in production technology. In post-war
period almost complete young female sheep population is reserved for breeding. According to the assessments
of breeding specialists there are over 1.000.000 sheep in Bosnia and Herzegovina today. Zeckel sheep is a
dominant local breed with a number of varieties with different phenotype and productive traits. According
to literature data, varieties of Zeckel sheep like Dubian, Kupreska, Privorian, Glasinacka, Kljucka-Vrcarska,
Podveleska, Stolacka-Humnjacka represents about 25% of total sheep population in pure breeding. Dubian
sheep is combined production sheep type milk-meat-wool, and represents a sheep with the highest milking
capacity among sheep species of Bosnia and Herzegovina. This research was preformed during the year 2006 at
Vlasic mountain in the central part of Bosnia and Herzegovina. Nine flocks divided in different age groups (2, 3
and over 3 years) were included in investigation of basic body measures and production performances. Results
were (mean±SD): body weight 75,85 ± 0,31 kg, height at withers 77,17 ± 0,11 cm, body length 85,07 ± 0,19 cm,
breast circumference 10,43 ± 0,04 cm. Average milk yield of all age groups were 47,05 l per head. With activities
on breeding selection and appropriate feeding it would be possible to improve production performances and
phenotype characteristics of sheep. Data on phenotype characteristics obtained in this study indicate a need for
changes of present standards of Dubian sheep.
Key words: dubian sheep, phenotype, production performances
Sažetak
Uzgoj ovaca zauzima značajno mjesto u stočarstvu Bosne i Hercegovine, budući da velika pašnjačka
prostranstva od preko 1.000.000 ha u brdsko-planinskom području pružaju optimalne uslove za proizvodnju
biološki vrijedne hrane, posebno jagnjećeg mesa i ovčijeg sira. Ovčarstvo je u potpunosti integrisano u zemljišne
i klimatske resurse, te je zadržalo tradicionalan način proizvodnje bez značajnijih tehnoloških promjena. U
postratnom periodu cjelokupan ženski podmladak ostavlja se za priplod, odnosno proširenu reprodukciju.
Danas je, na teritoriju Bosne i Hercegovine, prema procjenama u uzgoju preko 1.000.000 grla ovaca. Pramenka
je dominantna, jedina autohtona pasmina u uzgoju, i kao takva ima veći broj sojeva različitih fenotipskih i
proizvodnih osobina. Prema podacima iz literature, sojevi poput Dubske, Kupreške, Privorske, Glasinačke,
Ključke - Vrcarske, Podveleške, Stolačke - Humnjačke čine oko 25% ukupne populacije ovaca u čistom uzgoju.
Dubska pramenka proizvodno predstavlja kombinovani soj – mlijeko, meso, vuna i najmliječnija je od svih
bosanskohercegovačkih sojeva pramenke. Istraživanje po dobnim skupinama (2, 3 i preko 3 godine) osnovnih
tjelesnih parametara i proizvodnih osobina u 9 stada je provedeno tokom 2006. godine na planini Vlašić
– Srednjebosanski Kanton, Bosna i Hercegovina. Prosječni utvrđeni osnovni tjelesni parametri cjelokupnog
uzorka obje spolne skupine su: tjelesna masa 75,85 ± 0,31 kg, visina do grebena 77,17 ± 0,11 cm, dužina trupa
85,07 ± 0,19 cm, obim cjevanice 10,43 ± 0,04 cm. Prosječna proizvodnja mlijeka ovaca svih dobnih skupina
po grlu iznosila je 47,05 litara po laktaciji. Uzgojno selekcijskim radom i odgovarajućom ishranom, moguće je
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poboljšati prosječnu proizvodnju mlijeka ovaca i fenotipske osobine. Utvrđeni fenotipski parametri ukazuju na
potrebu promjene dosadašnjeg standarda dubske pramenke.
Ključne riječi: dubska pramenka, fenotip, proizvodne osobine
Uvod
Uzgoj ovaca zauzima značajno mjesto u stočarstvu Bosne i Hercegovine budući da velika
pašnjačka prostranstva od preko 1.000.000 ha u brdsko-planinskom području pružaju optimalne
uslove za proizvodnju biološki vrijedne hrane, posebno jagnjećeg mesa i ovčijeg sira namijenjenih
domaćem i inostranom tržištu. Ovčarstvo je u potpunosti integrisano u zemljišne i klimatske resurse,
te je zadržalo tradicionalan način proizvodnje bez značajnijih tehnoloških promjena.
Danas je, na teritoriji Bosne i Hercegovine, prema procjenama, u uzgoju preko 1.000.000 grla
ovaca. Pramenka je dominantna, jedina autohtona pasmina u uzgoju, i kao takva ima veći broj sojeva
različitih fenotipskih i proizvodnih osobina.
Najznačajniji soj je Dubska pramenka. Nastala je i danas najviše rasprostranjena u širem području
planine Vlašić (Srednja Bosna), na nadmorskoj visini od 800-1200 metara. Glavne odlike ove ovce,
kao i većine pramenki, su izražena otpornost i skromnost u zahtjevima, te prilagodljivost na različite
hranidbene i klimatske uvjete. Većina sojeva pramenke je kasnozrela, pa tako i dubska. Godine 1991.
brojno stanje ove ovce u Bosni i Hercegovini se procjenjivalo na 140.000 grla. Od ovog broja 54%
se odnosilo na priplodne ovce. Međutim, u toku agresije na Bosni i Hercegovinu (1992-95) taj broj se
smanjio usljed migracije u susjedne zemlje i prodaje koja izlazi van tržišnih kriterija. Danas je teško
utvrditi precizan broj dubske premenke jer grla nisu markirana niti umatičena, ali je procjena da je
broj sličan onome iz 1991. godine. Godine 1992/93 godine u Hrvatsku je preseljeno oko 10 000 grla
dubske ovce. Danas ih najviše ima u Zapadnoj Slavoniji i Lici (3).
Dubska pramenka spada među najkrupnije sojeve pramenke, duge i grube vune te, kao ostali
sojevi, pripada skupini ovaca kombiniranih proizvodnih svojstava meso-mlijeko-vuna. Prsa su joj
prilično duboka, ali relativno uska, što je opća odlika svih pramenki. Vrat je dug, slabije do osrednje
mišićav, a greben dobro izražen. Leđna linija je duga, ravna i pravilna, završava s dugim repom.
Trup se nalazi na čvrstim, jakim i pravilno postavljenim nogama. Papci su crni, pravilni, jaki i čvrsti.
Glava je srednje razvijena s polustršećim, najčešće crno pigmentiranim ušima. Ovce i ovnovi mogu
biti šuti i rogati. Glava je obično bijele boje s manje ili više crno pigmentiranim stranama lica od
usna do očiju. Duž čela i nosnog dijela do nozdrva i vrha gornje usne pruža se bijela traka u vidu lise.
Ovnovi imaju ispupčen profil glave, a ovce ravan. Tijelo je prekriveno otvorenim runom sastavljenim
od dugih (22.63 cm), šiljastih i bičastih pramenova koji nerjetko sežu sve do zemlje. Trbuh, donji
dijelovi nogu, a ponekad i vrat, nisu obrasli vunom, nego gustom dlakom. Runo je u najvećem broju
slučajeva bijele boje, iako ima pojava manjeg boja grla s crnim ili sivim runom.
Cilj ovog istraživanja je bio mjerenje nekih od osnovnih tjelesnih parametara kako bi se utvrdile
promjene tjelesnog okvira današnjeg tipa dubske pramenke koje bi poslužile kao osnov pri izradi
prijedloga Pravilnika o uzgojnim i morfološkim standardima dubske pramenke u cilju zaštite iste.
Materijal i metode
Istraživanje osnovnih tjelesnih parametara i proizvodnih osobina 20 ovnova i 284 ovce iz 9 stada
je provedeno na području planine Vlašić u naseljima Vitovlje, Mehurići, Han Bila, Ovnak, Maglići,
Rakite, Jankovići i okolini grada Jajce tokom 2006. godine. Broj grla u svih 9 stada je iznosio
1260 ovaca. Uzgajivači stada imaju dugu tradiciju uzgoja ovaca, ali niti jedno istraživano grlo nije
462
umatičeno i uključeno u uzgojno-selekcijski rad. Izvršena je procjena dobi po zubima svakog grla
pojedinačno. Nakon procjene dobi i utvrđivanja spola grla su podijeljena u sljedeće dobne grupe: do 2
godine, od 2-3 godine i preko 3 godine dobi. Uzimanje tjelesnih mjera (visine grebena, dužine trupa)
izvršeno je Lydtinovim štapom, a opseg cjevanice pomoću vrpce. Tjelesna masa ovaca utvrđena je
pojedinačnim vaganjem na vagi preciznosti ± 0,2 kg. Kontrolom mliječnosti standardnom metodom
(periodično mjerenje) utvrđena je dnevna produkcija mlijeka u intervalima od po 30 dana (raspon
26-33 dana ili svake 4 sedmice) tokom laktacije nakon dojnog perioda.
Dobiveni rezultati statistički su obrađeni primjenom analize varijanse uz testiranje razlika srednjih
vrijednosti pomoću Duncanovog testa višestrukih intervala. Odnosi između pojedinih parametara
ispitani su izračunavanjem koeficijenta korelacije, a cjelokupna statistička obrada urađena je u
programu MS Excel 2003.
Utvrđeni tjelesni parametri cjelokupnog uzorka obje spolne skupine bili su: tjelesna masa 75,85
± 0,31 kg, visina do grebena 77,17 ± 0,11 cm, dužina trupa 85,07 ± 0,19 cm i obim cjevanice 10,43
± 0,04 cm. Utvrđeni tjelesni parametri kod ovnova bili su: tjelesna masa 77,59 ± 3,73 kg, visina do
grebena 78,33 ± 0,74 cm, dužina trupa 87,33 ± 1,31 cm i obim cjevanice 11,59 ± 0,15 cm. Tjelesni
parametri kod ovaca bili su: tjelesna masa 74,89 ± 0,21 kg, visina do grebena 76,92 ± 0,11 cm, dužina
trupa 84,69 ± 0,16 cm i obim cjevanice 10,34 ± 0,03 cm. Statistički značajne razlike između ovnova
i ovaca utvrđene su za dužinu trupa na nivou p<0,05 i obim cjevanice na nivou p<0,01. Utvrđene
fenotipske karakteristike po dobnim skupinama prikazane su u tabeli 1.
Tabela 1. Utvrđene fenotipske karakteristike ovaca po dobnim skupinama
Table 1. Phenotype characteristics of sheep by age classes
Tjelesne mjere
Body measures
abc
Visina do grebena,
Dužina trupa, cm Obim cjevanice, cm
Tjelesna masa, kg
cm
Lenght of the trunk, Circumference of the
Body weight, kg Height of withers, cm
cm
leg, cm
Mean
SD
Min.
Max.
72,23a
8,37
50
90
Mean
SD
Min.
Max.
76,30b
6,53
65
120
Mean
SD
Min.
Max.
75,38b
3,37
65
84
< 2 godine < 2 years
76,55a
2,69
69
87
2-3 godine 2-3 years
77,11b
1,94
70
84
> 3 godine > 3 years
77,37ac
1,52
73
83
83,70a
3,43
75
92
10,45
0,67
10
13
85,25b
3,64
76
100
10,44
0,65
10
13
85,36b
2,37
78
89
10,45
0,50
10
11
srednje vrijednosti u istoj koloni sa različitom slovnom oznakom razlikuju se signifikantno na novou p<0,05
463
Jaki koeficijenti korelacije utvrdjeni su kod ovnova izmedju tjelesne mase i visine grebena
(r=0,836), tjelesne mase i dužine trupa (r=0,890), te tjelesne mase i obim cjevanice (r=0,709)
(Grafikoni 1, 2 i 3). Isti koeficijenti kod ženskih životinja nisu bili ovako visoki, a samo je u slučaju
korelacije između tjelesne mase i dužine rupa koeficijent bio statistički značajan (r=0,546) (Grafikon
1).
ženski
muški
110
dužina trupa, cm
y = 0,3118x + 63,143; r= 0,890
100
90
80
y = 0,4221x + 53,076; r= 0,546
70
40
60
80
100
120
tjelesna masa, kg
Grafikon 1. Odnos tjelesne mase i dužine trupa kod ovaca i ovnova
Graph. 1. Relationship between body weight and lenght of the trunk in sheep and ram
ženski
muški
visina grebena, cm
90
y = 0,1654x + 65,5; r=0,836
85
80
75
y = 0,0854x + 70,528; r=0,172
70
40
60
80
100
120
tjelesna masa, kg
Grafikon 2. Odnos tjelesne mase i visine grebena kod ovaca i ovnova
Graph. 2. Relationship between body weight and height of withers in sheep and ram
464
ženski
muški
Obim cjevanice, cm
14
y = 0,0292x + 9,3298; r= 0,708
13
12
11
y = -0,0193x + 11,783; r= -0,145
10
40
60
80
tjelesna masa, kg
100
120
Grafikon 3. Odnos tjelesne mase i obima cjevanice kod ovaca i ovnova
Graph. 3. Relationship between body weight and circumference of the leg in sheep and ram
Slična istraživanja vanjštine hrvatskih autohtonih pasmina rađena su od strane Pavić i sur. (7),
gdje su komparativno predstavljeni i dobiveni rezultati istraživanja tjelesnih mjera dubske pramenke.
Usporedbom rezultata Pavić i sur. (7) sa našim rezultatima istraživanja vanjštine dubske pramenke
dolazimo do zaključka da se naši rezultati bitno razlikuju, što potvrđuju vrijednosti tjelesne mase
odraslih ovaca od 70,74 kg, visine grebena 66,76 cm, dužine trupa 73,86 cm i opsega cjevanice 8,68
cm. Komparacijom naših rezultata sa rezultatima autora (2, 4, 5, 6, 8, 9, 11) koji su istraživali fenotip
drugih sojeva pramenke (Rabska, Lička, Paška i Istarska) dolazimo do već potvrđenih saznanja da
dubska pramenka predstavlja najkrupniji soj pramenke na ovim prostorima.
Prosječna utvrđena mliječnost po ovci u 9 istraživanih stada je iznosila 46,94 litara. Dobivena
vrijednost se odnosi na količinu pomuženog mlijeka od kraja dojnog perioda do kraja laktacije, dok
jedan dio nepomuženog mlijeka posišu jagnjad i ta količina je relativna i iznosi 50 – 60% ukupne
količine tokom laktacije. Preciznije rezultate (ukupnu mliječnost po grlu) moguće je utvrditi samo
u kontrolisanim uslovima uzgoja, a takve uslove nismo imali. Dobiveni rezultati se, obzirom na
vremensku distancu, teško mogu komparirati sa rezultatima Šmalcelja i sur. (12) koji su utvrdili
prosječnu mliječnosti stada od 87 kg mlijeka kod dubske pramenke. Antunec i sur. (1) pak navode da
tokom prosječne laktacije od 235 dana dubska pramenka proizvede čak 137 litara mlijeka.
Današnje stanje mlječnosti vlašićkih ovaca ukazuje da razlike među stadima dosta variraju, dok
su unutar istog stada prilično postojane iz godine u godinu. Prosječna dužina perioda muže je 130-140
dana, a veliki dio proizvedenog mlijeka posisaju jagnjad koja se mlijekom hrane po volji praktično
sve do mjeseca maja. Obzirom da najveći dio populacije ovaca na Vlašiću čini dubska pramenka čiji
proizvodni potencijal prelazi 240 litara mlijeka u toku laktacije, a period muže je moguće produžiti i
do 240 dana (10), navedeni podaci o današnjem stanju su poražavajući.
Zaključak
Utvrđeni fenotipski parametri (tjelesna masa, visina grebena, dužina trupa i obim cjevanice)
ukazuju na potrebu promjene dosadašnjeg standarda dubske pramenke jer su morfometrijske
parametri značajno povećani u odnosu na ranije literaturne podatke. Kvalitetnim uzgojno selekcijskim
radom i odgovarajućom ishranom, moguće je poboljšati prosječnu proizvodnju mlijeka i fenotipske
465
karakteristike dubske pramenke. Ovu ovcu potrebno je uzgajati u čistoj krvi i po mogućnosti njen
uzgoj prenijeti i na druga područja sa visokokvalitetnim pašnjačkim površinama.
Literatura
1.
Antunac N., i sur. (2002): The effect of stage of lactation on milk quality and number of somatic cells in
sheep milk. Milchwissenschaft 57 (6), 310-311.
2.
Mioč B., Pavić Vesna, Barać Z. (1998): Odlike eksterijera ličke pramenke. Stočarstvo 52 (1) 93-98.
3.
Mioč B., Pavić Vesna, Posavi M., Sinković Karmen (1999): Program uzgoja i selekcije ovaca u Republici
Hrvatskoj, Zagreb.
4.
Mioč B., Ivanković A., Pavić Vesna, Barać Z., Sinković Karmen, Marić I. (2003): Odlike eksterijera i
polimorfizmi proteina krvi dubrovačke ovce. Stočarstvo 57 (1) 3-11.
5.
Mioč B., Pavić Vesna, Ivanković A., Barać Z., Vnučec I., Čokljat Z. (2004): Odlike eksterijera i polimorfizmi
proteina krvi krčke ovce. Stočarstvo 58 (5) 331-341.
6.
Mioč B., Pavić Vesna, Sušić V.: Ovčarstvo. Hrvatska mljekarska udruga, Zagreb, S.423. Zagreb, 2007.
7.
Pavić Vesna, Mioč B., Barać Z. (1999): Odlike eksterijera travničke pramenke. Stočarstvo 53 (2) 83-89.
8.
Pavić Vesna, Mioč B. (1997): Creska sheep – sheep of the island Cres. Stočarstvo 51(1) 47-51.
9.
Pavić Vesna, Mioč B., Barać Z., Vnučec I., Sušić V., Antunac N., Samaržija Dubravka (2005): Vanjština
paške ovce. Stočarstvo 59 (2) 83-90.
10. Piplica Slavica: Hemijski sastav i energetska vrijednost sijena šire regije planine Vlašić. Specijalistički rad,
Veterinarski fakultet, Sarajevo 1999.
11. Posavi M., Ernoić M., Ozimec R., Poljak F. (2002): Hrvatske pasmine domaćih životinja. Ministarsvo
zaštite okoliša i prostornog uređenja Republike Hrvatske, Zagreb, 56-58.
12. Šmalcelj I. (1937): Vlašićka ovca. Poljoprivredni glasnik 2.
466
METASTATIC CHOLANGIOCARCINOMA IN A COW - CASE REPORT
METASTATSKI KOLANGIOCELULARNI KARCINOM KRAVE –
PRIKAZ SLUČAJA
Grabarević, Ž.1, Seiwerth, S.2, Čorić, M.2, Šoštarić-Zuckermann, I.C.1, Hohšteter, M.1,
Artuković, B.1, Beck, A1, Gudan-Kurilj, A.1, Džaja, P.3, Švić, L.4
1
Veterinary Faculty, University of Zagreb, Department of General Pathology and Pathological Morphology,
Heinzelova 55, Zagreb
2
3
Medical School, University of Zagreb, Institute of Pathology, Šalata 3b, Zagreb,
Veterinary Faculty, University of Zagreb, Department of Forensic Veterinary Medicine, Heinzelova 55,
Zagreb
4
Veterinary Ambulance Pazin, Pazin
Summary
The case of liver tumor with lung metastasis is presented. Based on the histopathological and
immunohistochemical (positive cytokeratine staining and negative anti-hepatocyte antigen) findings the tumor
was classified as anaplastic cholangiocellular carcinoma.
Key words: cholangiocellular carcinoma, histopathology, immunohistochemistry, cow.
Sažetak
U radu je prikazan tumor jetre s metastazama u jetri. Temeljem obavljenih histopatoloških i
imunohistokemijskih analiza (negativna reakcija na antihepatocitni antigen i pozitivna na citokeratine) tumor
je klasificiran kao anaplastični kolangiocelularni karcinom, što predstavlja vrlo rijedak nalaz u veterinarskoj
onkologiji.
Ključne riječi: Kolangiocelularni karcinom, histopatologija, imunohistokemija, krava.
Introduction
Bile duct carcinoma (cholangiocarcinoma) has been described in all domestic animals with the
exception of swine. From the literature it seems that this tumour although very uncommon, could
be the most frequent in cattle (Monlux et al., 1956.; Anderson and Sandison, 1967.). Because of
the scarcity of this tumor, estimates of its incidence are not accurate. This paper presents the gross,
histopathologic, and immunohistochemical findings of a bovine cholangiocarcinoma with metastasis
to the lungs, found in cowe during a routine postmortem inspection at an abattoir. The animal was of
normal appearance at the time of slaughter.
Material and Methods
In February 2006, two samples of the bovine tissue (liver and lungs) were submitted to
histopathological analysis. The samples were taken from abattoir during routine postmortem
inspection. During postmortem veterinary examination of the carcass, large numbers of small whitepink, firm, nonencapsulated nodules, several milimeters in diameter, were found multinodulary
distrubuted throughout all liver lobes and lung parenchyma.
467
Representative sections of the liver and lungs were fixed in 10% neutral-buffered formalin,
processed routinely, and embedded in paraffin. Tissue sections were cut to 4 µm thickness and stained
with hematoxylin and eosin, periodic acid–Schiff (PAS) and PAS diastasis.
A standard indirect immunoperoxidase protocol was used for immunohistochemistry (ABCElite; Vector Laboratories, Inc., Burlingame, CA). For immunohistochemical studies, the primary
antibodies (Dako) used were: monoclonal anti–hepatocyte (Hep Par 1) at a dilution of 1:25, CK 18
at a dilution 1:25, CK 19 at a dilution 1:50, carcinoembryonic antigen (CEA) at a dilution 1:25 and
alpha-feto protein at a dilution 1:300. A high-temperature (20 minutes in a pressure cooker) treatment
procedure with antigen unmasking solution (Vector Laboratories, Inc.) was used to enhance the
staining. The primary antibody was omitted for negative controls.
Results
Histopathologically, primary liver tumor and its metastases in the lungs were very similar. Tumor
was composed of predominately cords and islets of malignant cells which were heavily vacuolated
(Fig. 1). Some individual cells were PAS positive. In the liver, but not in the lungs, abundant
connective tissue was present with scirrhous response (Fig. 2) in some parts of tumor. In the lungs,
nests of neoplastic cells were surrounded with fine connective tissue stroma. Cells did not resemble
normal biliary epithelium, cytoplasm was vacuolated in most cells, and there were pleomorphism,
anisonucleosis and anisocytosis. Mitoses were rare. Nuclei were round with vesicular appearance
(Fig. 3). There was also a compression of the adjacent hepatic parenchyma at the tumor margins
(Fig. 1).
Immunohistochemical analysis showed that liver cells were positive for anti-hepatocyte antigen
while tumor cells had negative reaction (Fig. 4). Tumor cells were mildly to moderately positive (Fig.
5) on cytokeratine staining (CK 18 and CK 19). CEA and alpha-feto protein were negative.
Fig. 1. Cords and islets of predominately vacuolated tumor cells with abundant connective tissue stroma.
Liver. H&E.
468
Fig. 2. Scirrhous tumor response. Liver. H&E.
Fig. 3. Pulmonary metastasis with nests of tumor cells separated with delicate fibers. Visible cellular pleomorphism, anisonucleosis and anisocytosis. Lung. H&E.
469
Fig. 4. Anti-hepatocyte antigen. Hepatocytes are positive (brown reaction) and tumor cells are negative
(center). Liver. Immunohistochemistry, ABC method.
Fig. 5. CK 19. Mild to moderate positive cytokeratine reaction in tumor cells. Liver. Immunohistochemistry,
ABC method.
Discussion
Based on the gross findings, histopathological and immunohistochemical data it can be concluded
that this tumor was cholangiocellular metastatic anaplastic carcinoma. Histopathology of the tumor
was mostly consistent with the features of cholangiocellular carcinoma (Cullen and Popp, 2002.;
Cullen, 2007.) which was confirmed with immunostaining. Namely, tumor cells were negative and
hepatocytes were positive for anti-hepatocyte antigen, and in the same time there were positive
470
cytokeratine reaction which is normal finding in biliary epithelium cells. As Jeong et al. (2005)
already stated, the significance of the PAS positive cells needs further investigation.
Literature
Anderson LJ, Sandison AT. Tumors of the liver in cattle, sheep and goats. Cancer 21: 289-301; 1967.
Cullen JM, Popp JA. Tumors of the liver and gall bladder. In: Tumors in domestic animals, Fourt edition,
Meuten DJ, ed. Iowa State Press, Ames, 2002.
Cullen JM. Liver, Biliary System, and Exocrine Pancreas. In: Pathologica basis of veterinary disease, Fourth
edition, McGavin MD and Zachary JF, eds. Mosby Elsevier, St. Louis, 2007.
Jeong WI, Do SH, Sohn MH et al. Hepatocellular Carcinoma with Metastasis to the Spleen in a Holstein Cow.
Vet Pathol 42:230-232; 2005.
Monlux AW, Anderson WA, Davis CL. A survey of tumors occuring in cattle, sheep and swine. Amer J Vet Res
17: 646-677; 1956.
471
472
POLIMORFISMO DEL GENE CSN1S2 NELLA CAPRA SARDA
POLIMORFIZAM GENA CSN1S2 U SARDINIJSKE KOZE
Dettori1, M.L., G.M. Vacca1, V. Carcangiu1, M. Pazzola1, M. Sanna1, G. Maricosu2, P.P.
Bini1
Dipartimento di Biologia Animale, via Vienna 2, 07100 Sassari, Italy
1
2
Associazione Regionale Allevatori della Sardegna, via Cavalcanti 8, 09128 Cagliari, Italy
ABSTRACT
The gene codifying for αs2-casein, CSN1S2, is characterized for the presence of seven alleles associated
with three synthesis levels: the “strong” alleles A, B, C, E, F, associated with the production of about 2.5 g/l of
αs2-casein for allele; “intermediate” allele D, associated with 1,5 g/l of αs2-casein for allele; the “null” allele 0,
that in homozygosis determines the almost total absence of this protein in milk. The present research was carried
out in order to assess allele frequencies at the CSN1S2 locus in Sarda goat and to check possible effects of the
genotype on milk composition. Two hundred and twenty lactating goats belonging to 20 different farms located
in Central Sardinia, were utilized in this trial. In the middle of lactation, after having determined the daily milk
production of all the animals, a milk sample was collected in order to measure fat, protein and lactose percentage
(Milko-scan), freezing point (thermistore cryoscope), pH, SCC (Fossomatic) and CMT (Bacto-scan). At the
same time, a peripheral blood sample was collected. DNA was isolated from leucocytes using a commercial kit
(Puregene DNA isolation kit, Gentra, Qiagen) and then analysed with different methods based on PCR. Allele
Specific PCR was used to reveal the presence of the A, B, C alleles; the E, F, D and 0 alleles were investigated
by PCR-RFLP. The Genepop software was used to evaluate allele frequencies and the equilibrium according
to the Hardy Weinberg (HW) law. Milk production and milk composition data was submitted to GLM analysis
in order to highlight possible correlations with the genotype of this casein fraction. Genotype frequencies were
as follows: AA, 0.136; AB, 0.009; AC, 0.082, AE, 0.032; AF, 0.264; CC, 0.023; CE, 0.023; CF, 0.250; EF,
0.077; FF, 0.105. The F allele showed the highest frequency (0.400), followed by the A (0.330), C (0.200),
E (0.066) and in the end by the B allele (0.005). No subject was found carrying the D and 0 alleles for this
locus. The analysis of allele frequencies according to HW law showed the occurrence of disequilibrium in the
goat population; departure from HW equilibrium at the CSN1S2 locus was due to an excess of heterozygotes.
The goats we analysed had an average milk yield of 840 g/day, characterized by high fat (5.05%) and protein
content (4.19%). Lactose percentage showed the typical values of the species (4.89%) as well as the freezing
point (-0.568 °H) and the pH (6.72). The log TMC value (5.10), which is an indicator of the hygiene-sanitary
conditions of the farm, shows the existence of good breeding conditions, despite manual milking and farm
structural precariousness. The log SCC value (6.22) may appear high, when compared to the other domestic
species, but it is similar to that detected in other goat breeds, reared in different managerial realities. Statistical
analysis did not show any significant link between the detected genotypes and the mean values of milk chemical,
physical and cytological parameters and productive levels. It is likely that the αs2-casein fraction did not exert
any significant effect on the samples we analysed, because the intermediate and null alleles did not occur. This
confirms the Sarda goat aptitude to the production of milk suitable for cheese making and on the other hand, the
high variability of the analysed gene suggests further investigations aimed at finding the defective alleles, which
may allow the production of milk suitable for direct human consumption, in particular destined to those people
with cow milk intolerance.
Key words: goat, milk, casein, CSN1S2 locus.
473
Sažetak
Gen koji kodira αs2-kazein, CSN1S2, karakteriziran je prisutstvom 7 alela povezanih s 3 nivoa sinteze:
jaki aleli A, B, C, E i F povezani s produkcijom oko 2,5g/L αs2-kazeina po alelu; srednji alel D, povezan s 1,5
g/L αs2-kazeina po alelu; nulti alel 0 koji u homozigozi određuje gotovo potpuni nedostatak te bjelančevine u
mlijeku. Ovo istraživanje izvedeno je kako bi se odredilo frekvencije alela na CSN1S2 lokusu u Sardinijske koze
i provjerilo moguće učinke genotipa na sastav mlijeka. U ovom je istraživanju korišteno 220 koza u laktaciji
sa 20 različitih farmi iz središnje Sardinije. U sredini laktacije, nakon što je određena dnevna proizvodnja
mlijeka svih životinja, uzimani su uzorci mlijeka kako bi se odredio postotak masti, bjelančevina i laktoze
(Milko-scan), točka ledišta (thermistore cryoscope), pH, SCC (Fossomatic) i CMT (Bacto-scan). Istovremeno
su uzimani uzorci krvi. DNK je izolirana iz leukocita koristeći komercijalni kit (Puregene DNA isolation kit,
Gentra, Qiagen) i zatim analizirana različitim PCR metodama. Alelno specifičan PCR se koristio kako bi se
otkrilo prisutstvo A, B i C alela; E, F, D i 0 aleli su istraživani PCR-RFLP-om. Genepop software je korišten
u procjeni učestalosti alela i ravnoteže po Hardy Weinberg (HW) zakonu. Podatci o proizvodnji i sastavu
mlijeka podvrgnuti su GLM analizi kako bi se naglasilo moguće korelacije sa genotipom te kazeinske frakcije.
Učestalost genotipa bila je kako slijedi: AA 0,136; AB 0,009; AC 0,082; AE 0,032; AF 0,264; CC 0,023; CE
0,023; CF 0,250; EF 0,077 te FF 0,105. Najveću učestalost je imao F alel (0,400), a zatim A (0,330), C (0,200), E
(0,066) te na kraju B alel (0,005). Nisu nađeni uzorci koji bi nosili D i 0 alele na tom lokusu. Analiza frekvencija
alela po HW zakonu pokazala je pojavu disekvilibrija u populaciji koza; što odstupa od HW ravnotežja na
CSN1S2 lokusu zbog prekomjernih heterozigota. Proučavane koze imale su prosječnu produkciju mlijeka od
840 g/dan, karakteriziranu visokom masnoćom (5,05%) i sadržajem bjelančevina (4,19%). Postotak laktoze bio
je u tipičnim okvirima za koze (4,89%) jednako kao točka ledišta (-0,568°H) i pH (6,72). Log TMC vrijednost
(5,10) koja je pokazatelj higijensko sanitarnih uvjeta na farmi ukazuje na dobre uzgojne uvjete, unatoč ručnoj
mužnji i nesigurnoj strukturi farme. Log SCC vrijednost (6,22) se može činiti visokim, u usporedbi s drugim
domaćim životinjama, ali je sličan onome u drugih pasmina koza uzgajanih u različitim uvjetima. Statistička
analiza nije pokazala nikakve znakovite veze među utvrđenih genotipa i srednjih vrijednosti sastava mlijeka,
fizikalnih i citoloških parametara te nivoa proizvodnje. Čini se da αs2-kazein frakcija ne ispoljuje značajniji
učinak u analiziranim uzorcima jer se srednji i nulti aleli nisu pojavili. To potvrđuje sposobnost sardinijske
koze da proizvodi mlijeko prikladno za proizvodnju sira, a s druge strane, velika varijabilnost analiziranih gena
sugerira daljnja istraživanja s ciljem pronalaska defektivnih alela, koji bi mogli omogućiti proizvodnju mlijeka
prikladnog za izravnu ljudsku konzumaciju, posebice za ljude s netolerancijom kravljeg mlijeka.
Ključne riječi: koza, mlijeko, kazein, CSN1S2 loku
INTRODUZIONE
L’allevamento e la valorizzazione delle razze autoctone rappresenta un’efficace opportunità
sia per potenziare importanti settori zootecnici che per lo sviluppo delle aree di interesse. La
tutela di un biotipo animale e del luogo di allevamento, oltre alla conservazione della biodiversità
e dell’ambiente, è garanzia di tipicità e genuinità dei prodotti (Boyazoglu et al., 2005). La capra
Sarda, con circa 200.000 capi, rappresenta un enorme serbatoio di biodiversità dal quale è possibile
attingere per ottenere numerose tipologie di prodotto. Questo se utilizzato nel modo più idoneo, può
contribuire alla stessa salvaguardia della razza ed al mantenimento di un comparto che in Sardegna
ha notevole importanza economica e sociale (Carcangiu et al., 2006). Nei piani di selezione della
razza non vengono ancora presi in considerazione quegli aspetti qualitativi del latte che possono
far valorizzare meglio il prodotto. A differenza di quanto avviene in altri Paesi europei, infatti, non
esiste, se non come prospettiva futura, un programma di caratterizzazione genetica degli animali
in relazione, ad esempio, alle diverse caseine la cui variabilità genetica si associa ad importanti
variazioni delle caratteristiche chimiche e tecnologiche del latte (Barbieri et al., 1995). Le quattro
frazioni caseiniche, αs1, αs2, β e κ, sono codificate da altrettanti geni strettamente associati tra loro.
Nella capra, l’intero complesso genico è localizzato sul cromosoma 6, in un segmento di DNA di
474
circa 250 Kb, nel seguente ordine: CSN1S1 (codificante per l’; s1-caseina), CSN2 (codificante per la
; -caseina), CSN1S2 (codificante per l’; s2-caseina), CSN3 (codificante per la k-caseina) (Threadgill
e Womack 1990). I geni CSN1S1 e CSN2 sono separati da una distanza di 12 kb e sono trascritti
in maniera convergente (Leroux e Martin, 1996). L’organizzazione genomica del locus caseinico
è altamente conservata tra i mammiferi, sebbene da una specie all’altra sia stata osservata una
grande variabilità quantitativa e qualitativa (Rijnkels, 2002). Le indagini condotte finora su diverse
razze caprine hanno messo in evidenza che tutte e quattro le frazioni caseiniche sono polimorfiche.
L’esistenza di questa grande variabilità costituisce un elemento importante ai fini del miglioramento
genetico dei caprini, in quanto offre la prospettiva di diversificare la produzione in relazione alla
sua destinazione finale: trasformazione casearia, latte alimentare, latte ipoallergenico (Ramunno
et al., 2007). La comprensione dei meccanismi da cui dipende la produzione e la composizione
del latte possono contribuire a sviluppare strategie per migliorare o finalizzare la composizione
del latte. La caseina αs2 caprina, fosfoproteina costituita da 208 aminoacidi, si ritiene sia uno dei
maggiori allergeni presenti nel latte e che i minori rischi del manifestarsi di una reazione allergica,
nei soggetti alimentati con latte di capra, dipendano dalla elevata omologia con il latte umano, nel
quale alcune lattoproteine risultano assenti (El-Agamy, 2007). Nel latte umano, infatti, non è stata
rivelata la presenza di αs2-caseina (Martin et al., 1996), sebbene nel locus caseinico umano siano
stati identificati due geni αs2-simili, denominati CSN1S2A e CSN1S2B. Per la sequenza CSN1S2A
è stata rivelata la presenza di trascritti nella ghiandola mammaria in lattazione umana, ma non è noto
se questi siano tradotti in sequenze peptidiche stabili, mentre per CSN1S2B non è stato identificato
nessun trascritto specifico (Rijnkels, 2003). Il gene CSN1S2 è il più lungo tra i geni che codificano
per le caseine, si estende per un tratto di DNA di circa 18,5 Kb, ed è organizzato in 18 esoni di
dimensioni variabili tra 21 e 266 coppie di nucleotidi (Groenen et al., 1993). Nella capra, il locus
CSN1S2 si caratterizza per la presenza di 7 alleli associati a tre livelli di sintesi: alleli “forti” A,
B, C, E, F, associati ad una produzione di circa 2,5 g/l di αs2-caseina per allele; “intermedio” D,
associato ad una produzione inferiore (1,5 g/l) di αs2-caseina rispetto agli alleli forti; “nullo” 0 che
in omozigosi determina la quasi totale assenza di caseina αs2 nel latte (Ramunno et al., 2001b). Lo
scopo della presente ricerca è analizzare la struttura genetica della capra Sarda al locus CSN1S2 e di
valutare le eventuali correlazioni tra il genotipo e le caratteristiche produttive degli animali.
MATERIALI E METODI
La ricerca è stata condotta su 220 capre di razza Sarda, appartenenti a 20 allevamenti dislocati
in altrettanti comuni della Sardegna Centrale. In ciascun allevamento sono state scelte, con criterio
casuale, 11 capre in lattazione. Su tutti gli animali dopo aver misurato la produzione giornaliera
è stato raccolto un campione di latte per determinare la percentuale di grasso, proteine e lattosio
(Milko-scan), l’indice crioscopico (Crioscopio a termistori), il pH, il CCS (Fossomatic), l’urea e
il CMT (Bacto-scan). Contestualmente da ciascun animale è stato effettuato un prelievo di sangue
periferico. Il DNA, estratto dai leucociti con l’utilizzo del kit Puregene DNA isolation (GENTRA)
è stato analizzato con l’ausilio di diversi metodi basati sulla PCR. La variante CSN1S2 B è
caratterizzata, rispetto alla variante A, da una transizione G→A al nt 10 dell’esone 9, questo comporta,
nella sequenza proteica, la sostituzione dell’acido glutammico in posizione 64 con un residuo di
lisina, mentre l’evento mutazionale responsabile della comparsa della variante CSN1S2 C è una
transversione A→T, localizzata al quinto nucleotide dell’esone 16, che determina, a livello proteico,
la sostituzione Lys167→Ile (Bouniol et al., 1994). La ricerca di queste variazioni, nel DNA genomico,
è stata realizzata mediante AS-PCR (Ramunno et al., 2000). L’allele CSN1S2 F si caratterizza per
una transizione G→C al nucleotide 13 dell’esone 3, responsabile della sostituzione aminoacidica
475
Val7→Ile, la mutazione viene rivelata mediante PCR-RFLP (Ramunno et al., 2001b). La variazione
(C83G) che caratterizza l’allele CSN1S2 E, come avviene per la variante C, si verifica nell’esone 16,
e determina la sostituzione aminoacidica Pro193→Arg nella proteina matura, è rivelata mediante PCRRFLP (Veltri et al., 2000). Entrambi gli eventi molecolari che contraddistinguono gli alleli CSN1S2
D e 0 si trovano nell’esone 11. Il primo presenta una delezione di 106 nt, comprendente gli ultimi 11
nt dell’esone 11 e i primi 95 nt dell’introne successivo (Ramunno et al., 2001b), mentre l’allele nullo
presenta una transizione (G→A) al nucleotide 80 dell’esone 11, che determina la formazione di uno
stop-codon prematuro e di conseguenza l’apparente assenza di sintesi di αs2-caseina (Ramunno et
al., 2001a). La loro presenza è indagata mediante PCR-RFLP (Ramunno et al., 2001b). Le frequenze
alleliche e l’equilibrio secondo la legge di Hardy-Weinberg sono state valutate utilizzando il software
Genepop. I dati relativi alle produzioni ed alla composizione del latte sono stati sottoposti ad analisi
di varianza per evidenziare un eventuale effetto del genotipo relativo a questa frazione caseinica.
RISULTATI
Nella tabella 1 vengono illustrate le frequenze alleliche riscontrate al locus CSN1S2.
Tabella 1 – Frequenze genotipiche ed alleliche del gene CSN1S2 in capre di razza sarda
CSN1S2
AA
AB
AC
AE
AF
CC
CE
CF
EF
FF
n.
30
2
18
7
58
5
5
55
17
23
Frequenze Genotipiche
0,136
0,009
0,082
0,032
0,264
0,023
0,023
0,250
0,077
0,105
CSN1S2
A
B
C
E
F
Frequenze Alleliche
0,330
0,005
0,200
0,066
0,400
Complessivamente sono stati identificati 10 differenti genotipi; quelli più frequenti sono risultati
AF e CF che da soli rappresentano più del 50% del campione esaminato. L’allele B è risultato il
più raro essendo stato rivelato, allo stato eterozigote, solo in 2 soggetti, mentre gli alleli D e 0 sono
risultati assenti. Le frequenze alleliche mostrano decisa predominanza degli alleli F ed A. L’analisi
delle frequenze alleliche, secondo la legge di HW, ha dimostrato che la popolazione analizzata
presenta, per questo locus, una situazione di disequilibrio, dovuta ad un eccesso di eterozigoti. Nella
tabella 2 si riportano, per ciascun genotipo trovato i valori medi della produzione giornaliera e della
composizione chimica, fisica e citologica del latte.
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Tabella 2 - Valori medi (± d.s.) dei parametri lattei in funzione dei diversi genotipi al locus CSN1S2
Genotipo CSN1S2
n.
media
Peso
(g/d)
d.s.
AA
AB
30
2
826,4 1025,0
472,0 534,1
AC
18
860,4
347,4
AE
7
704,8
370,9
AF
CC
58
5
864,5 926,8
400,4 363,5
CE
CF
5
55
716,4 861,5
249,0 396,1
EF
17
800,0
339,9
FF
23
818,6
277,2
Grasso
(%)
media
d.s.
5,11
0,89
4,78
0,40
5,22
1,39
5,51
1,18
5,05
1,28
4,05
1,58
5,66
1,57
5,05
1,42
4,81
1,29
5,05
1,06
Proteine
(%)
media
d.s.
4,25
0,61
4,05
0,16
4,24
0,68
4,36
0,44
4,14
0,58
3,85
0,97
4,73
0,54
4,24
0,63
4,16
0,60
4,03
0,51
Lattosio
(%)
media
d.s.
4,89
0,32
4,62
0,12
4,90
0,23
4,87
0,27
4,91
0,26
5,01
0,10
4,88
0,22
4,93
0,29
4,73
0,28
4,84
0,26
Criosc.
(°H)
media
d.s.
pH
media
d.s.
6,69
0,07
6,81
0,13
6,70
0,08
6,71
0,08
6,73
0,07
6,70
0,06
6,80
0,04
6,71
0,09
6,70
0,09
6,74
0,07
CCS
(Log)
media
d.s.
6,31
5,56
6,62
5,75
6,13
5,62
5,92
5,18
6,20
5,64
6,05
5,54
6,21
5,59
6,23
5,95
6,24
5,99
6,22
5,92
CMT
(Log)
media
d.s.
4,36
4,08
4,83
4,32
4,26
4,04
4,74
4,23
5,51
5,17
4,00
3,48
4,56
4,18
4,95
4,58
4,74
4,32
4,60
4,34
-0,569 -0,551 -0,569 -0,567 -0,568 -0,569 -0,565 -0,568 -0,570 -0,567
0,007 0,022 0,008 0,003 0,007 0,006 0,004 0,006 0,014 0,008
L’analisi della varianza non ha evidenziato alcun legame significativo tra genotipo e parametri
chimici, fisici e citologici del latte, né con i livelli produttivi degli animali. Mediamente le capre da
noi esaminate producevano 840 g di latte al giorno, con un contenuto elevato in grasso e proteine pari
rispettivamente al 5,05% e al 4,19%. Il lattosio si attestava su valori tipici della specie (4,89%) così
come il punto crioscopico (-0,568 °H) ed il pH (6,72). I valori del CMT si mostravano mediamente
buoni (log 5,10), così come quelli del CCS (log 6,22).
DISCUSSIONE
Per i genotipi AF e CF, le frequenze ritrovate in questa popolazione di capre sarde sono più
elevate rispetto a quanto trovato da Ramunno et al., (2000) in una popolazione locale allevata nella
penisola Sorrentina, dove il genotipo più frequente risultava essere AA (22,5%). Nel presente lavoro
gli alleli F (0,40), A (0,33) e C (0,20) risultavano quelli con frequenza maggiore, rivelando valori
molto simili a quanto riscontrato da Sacchi et al., (2005) in diverse razze di origine mediterranea.
Al contrario, Bouniol et al., (1994) riportano che nelle razze Alpine e Saanen l’allele CSN1S2 A
è il più frequente (0,85) mentre per l’allele C è stata stimata una frequenza dello 0,11%. Gli alleli
B ed E presentavano frequenze particolarmente basse, inferiori a quanto trovato da Sacchi et al.
(2005) in diverse razze italiane. Nel campione esaminato non è emersa la presenza delle varianti
477
difettive D e 0, che finora si sono rivelate molto rare (frequenze inferiori al 5%: Sacchi et al., 2005;
Marletta et al., 2005), mentre Kusza et al., (2005) riportano, in capre da latte ungheresi, una frequenza
particolarmente elevata per l’allele 0 (15%). Affinché i polimorfismi relativi ad un singolo gene
del locus caseinico siano informativi, è necessario considerarli contestualmente ai geni a cui sono
associati. Infatti, la sintesi delle caseine risulta essere estesamente co-regolata, ovvero la regolazione
negativa dell’espressione proteica in seguito a mutazione in un gene caseinico può risultare nella
regolazione positiva delle altre caseine (Hayes et al., 2006). A causa di questo meccanismo di
compensazione, risulta difficile valutare gli effetti sui caratteri produttivi del latte di un unico SNP
(Single Nucleotide Polymorphism), per questa ragione sarebbe opportuno utilizzare le informazioni
derivate dall’intero aplotipo caseinico (Hayes et al., 2006). A questo proposito si ritiene opportuno
segnalare che nei soggetti del presente lavoro è stata rilevata anche la presenza degli alleli difettivi
delle frazioni αs1 (CSN1S1 F, E, 01 ed N) e della β-caseina (CSN2 01) (Vacca et al., dati non
pubblicati). La presenza di alleli difettivi della frazione β è stata riscontrata anche nei maschi della
stessa razza (Vacca et al., 2005). In relazione al test di HW la capra Sarda per il locus CSN1S2 ha
evidenziato una situazione opposta a quella descritta da Marletta et al., (2005) in capre appartenenti
alla razza Girgentana, dove il disequilibrio, nella distribuzione delle frequenze alleliche, era dovuto
ad un deficit, e non all’eccesso, di eterozigoti. Per quanto riguarda le misurazioni quali-quantitative,
le produzioni, in media risultano inferiori a quelle fornite da razze ad elevata attitudine lattifera, ma il
latte esaminato si caratterizza per gli elevati tenori in materia utile. Come già riscontrato in precedenti
indagini svolte su questa razza da Vacca et al. (2002), la capra Sarda si contraddistingue, quindi,
per gli elevati tenori in grasso e proteine. I dati relativi ai lipidi, infatti, dimostrano come questi
valori siano superiori a quelli riportati in letteratura per capre di razze maggiormente produttive
(Castagnetti et al., 1984; Pizzillo et al., 1994) e simile a quelli di altre capre rustiche (Casoli et al.,
1986). Anche per il tenore in proteine, il confronto con le altre razze depone a favore della capra
Sarda (Duranti et al., 1991; Freschi 1992). I valori del CMT, indicatore della situazione igienicosanitaria delle aziende, segnalano buone condizioni di allevamento, nonostante la mungitura venisse
effettuata manualmente ed in condizioni di precarietà strutturale. Sebbene il contenuto in cellule
somatiche possa sembrare elevato, se confrontato con altre specie domestiche, risulta simile a quello
che si rileva in diverse razze caprine allevate in differenti realtà manageriali (Fekadu et al., 2005).
Nel campione da noi analizzato, la frazione αs2-caseina non ha esercitato un’influenza significativa
sulla composizione del latte, presumibilmente per l’assenza degli alleli intermedio e nullo. Questo
dato se da un lato conferma l’attitudine delle capre di razza Sarda alla produzione di un latte da
destinare prevalentemente alla trasformazione casearia, dall’altro, vista l’elevata variabilità del gene
considerato, stimola ulteriori indagini volte ad individuare gli alleli deboli o nulli. Il ritrovamento
di questi permetterebbe la produzione di un latte da destinare al consumo diretto ed in particolare
rivolto agli individui con sensibilità allergica a questa proteina.
BIBLIOGRAFIA
Barbieri M.E., Manfredi E., Elsen J.M., Ricordeau G., Bouillon J. et al. (1995). Effects of the alpha s1-casein
locus on dairy performances and genetic-parameters of Alpine goats. Genet. Sel. Evol. 27: 263-265. Bouniol C.,
Brignon G., Mahé, M.F., Printz C. (1994). Biochemical and genetic analysis of variant C of caprine αs2-casein
(Capra hircus). Anim. Genet., 25: 173-177. Boyazoglu J, Hatziminaoglou I., Morand-Fehr P. (2005). The role
of the goat in society: Past, present and perspectives for the future. Small Rum. Res., 60: 13-23. Carcangiu V.,
Mura M.C., Vacca G.M., Bini P.P. (2006). Sardinian goat breeding and utilisation of environmental resources.
In Animal products from the Mediterranean area. EAAP Pubblication n. 119. Wageningen Academic Publishers,
249-254. Casoli C., Duranti E., Debenedetti A., Lucaroni A. (1986). Rilievi preliminari sulla produzione e sulle
caratteristiche chimiche del latte di capra di razza popolazione umbra. Il Latte, 11: 650-658. Castagnetti G.B.,
478
Chiavari C., Losi G. (1984). Caratteristiche chimico-fisiche ed attitudine tecnologica del latte di razze caprine ad
elevata potenzialità produttiva. Scienza e Tecnica Lattiero-Casearia, 35: 109-132. Duranti E., Bosi G., Casoli C.,
Costarelli S., Lucaroni A., Morgante M., Todini L. (1991). Valutazione di alcuni parametri chimici e tecnologici
del latte di capra di popolazione rustica umbra in relazione a diversi fattori di variabilità. IX Congr. ASPA, 10191027. El-Agamy E.I. (2007). The challenge of cow milk protein allergy. Small Rum. Res., 68: 64-72. Fekadu B.,
Soryal K., Zeng S., Van Hekken D., Bah B., Villaquiran M. (2005). Changes in goat milk composition during
lactation and their effect on yield and quality of hard and semi-hard cheeses. Small Rum. Res. 59: 55-63. Freschi
P., Sciaraffia F., Massari M. (1992). Caratteristiche chimiche di latti caprini della Basilicata. X Congr. SIPAOC,
256-258. Groenen M.A.M., Dijkhof R.J.M., Verstege A.J.M., van der Poel J.J. (1993). The complete sequence
of the gene encoding bovine αs2-casein. Gene, 123: 187-193. Hayes B., Hagesæther N., Ådnøy T., Pellerud G.,
Berg P.R., Lien S. (2006). Effects on production traits of haplotypes among casein genes in Norwegian goats
and evidence for a site of preferential recombination. Genetics, 174: 455-464. Kusza S., Veress G., Kukovics
S., Jávor A., Sanchez A., Angiolillo A., Bősze A. (2007). Genetic Polymorphism of αs1- and αs2-caseins in
Hungarian Milking Goats. Small Rum. Res., 68: 329-332. Leroux C., Martin P. (1996). The caprine alpha s1
and beta-casein genes are 12kb apart and convergently transcribed. Goat αs1 casein alleles. A potential tool
in selection of individuals carrying alleles associated with a high level protein synthesis. Anim. Genet., 27:
93. Marletta D., Bordonaro S., Guastella A.M., Criscione A., D’Urso G., (2005). Genetic polymorphism of
the calcium sensitive casein in sicilian Girgentana and Argentata dell’Etna goat breeds. Small Rum. Res., 57:
133-139. Martin P., Brignon G., Furet J.P., Leroux C. (1996). The gene encoding αs1-casein is expressed in
human mammary epithelial cells during lactation. Lait, 76: 526-37. Pizzillo M., Cogliandro E., Rubino R.,
Fedele V. (1994). Capacità produttiva e caratteristiche qualitative del latte delle principali razze caprine allevate
nel mezzogiorno. XI Congr. SIPAOC, 431-434. Ramunno L., Pauciullo A., Mancusi A., Cosenza G., Mariani
P., Malacarne M. (2007). Influenza del polimorfismo genetico delle caseine calcio-sensibili su caratteristiche
strutturali, nutrizionali, attitudine casearia e proprietà ipoallergeniche del latte di capra. Scienza e Tecnica
Lattiero-Casearia, 58: 257-271. Ramunno L., Longobardi E., Pappalardo M., Rando A., Di Gregorio P., Cosenza
G., Mariani P., Pastore N., Masina P. (2001a). An allele associated with a non-detectable amount of αs2-casein in
goat milk. Anim. Genet. 32: 19-26. Ramunno L., Cosenza G., Pappalardo M., Longobardi E., Gallo D., Pastore
N., Di Gregorio P., Rando A., (2001b). Characterization of two new alleles at the goat CSN1S2 locus. Anim.
Genet., 32: 264-268. Ramunno, L., Pappalardo M., Cosenza G., Pastore N., Gallo D., Grimaldi G., Rubino R.,
Calandrelli M., Rando A. (2000). Caratterizzazione genetica ai loci delle caseine αS1, β e αS2 di una popolazione
caprina allevata nella Penisola Sorrentina. XIV Congr. SIPAOC, Vietri sul mare (SA) Italy, 321-324. Rijnkels M.,
Elnitski L., Miller W., Rosen J.M. (2003). Multispecies comparative analysis of a mammalian-specific genomic
domain encoding secretory proteins. Genomics, 82: 417-432. Rijnkels M. (2002). Multispecies comparison
of the casein gene loci and evolution of casein gene family. J. Mammary Gland Biol. Neoplasia, 7: 327-345.
Sacchi P., Chessa S., Budelli E., Bolla P., Ceriotti G., Soglia D., Rasero R., Cauvin E., Caroli A. (2005). Casein
haplotype structure in five Italian goat breeds. J. Dairy Sci., 88: 1561-1568. Threadgill D.W., Womack J.E.
(1990). Genomic analysis of the major bovine milk protein genes. Nucleic Acid Res, 18: 6935-6942. Vacca
G.M., Carcangiu V., Ghibellini A., Galioto M., Cancedda M., Bini P.P. (2002). La qualità del latte della capra
Sarda. X Congr. FeMeSPRum., Tunis, Pubblicato su C.D. Vacca G.M., Dettori M.L., Sanna M., Porqueddu M.,
Carcangiu V. (2005). Polymorphism of the CSN1S1, CSN1S2 and CSN2 genes in Sarda bucks. It. J. Anim. Sci.
Proceedings of ASPA 16th Congress, Turin Italy, 116. Veltri C., Lagonigro R., Pietrolà E., D’Andrea M., Pilla F.,
Chianese L. (2000). Molecular characterisation of the goat αs2-casein E allele and its detection in goat breeds of
Italy. 7th International Conference on Goats, Tours France, 727.
Ringraziamenti - Ricerca eseguita con fondi CIPE: APQ ricerca Intervento P5a “Biodiversità animale”
479
480
ANALYSIS OF GROWTH CURVE OF CROSSES RUBIA GALLEGA X
NELLORE BY MEANS OF MICHAELIS-MENTEN EQUATION
ANALISIS DE LA CURVA DE CRECIMIENTO DE CRUCES DE RUBIA
GALLEGA X NELORE MEDIANTE LA ECUACION DE MICHAELISMENTEN
Sanchez, L1, Cantalapiedra, J.A. 1, Carril, J. A.2, Iglesias, A.1
1
Dpto. Anatomía y Producción Animal. Universidad de Santiago de Compostela. 27002. Lugo. lusaga@lugo.
usc.es
Asociación de Criadores de la Raza Rubia Gallega (ACRUGA) arrakis.es/~acruga
2
SUMMARY
The crosses of Galician Blond x Nellore are being systematically valued since, in the year 2000, the Breeders’
Association of Blonde Galician Breed (ACRUGA) designed the planning of the same ones to determine the
productive behavior and its capacity of adjustment to the systems of production of meat in Brazil. The aim of
this work consisted of the study of the growth of animals of Galician Blond x Nelore by using Michaelis-Menten
Equation. This study used data collected on 76 individuals of Rubia Gallega x Nellore born in the farm Mosquera
Grandal (Buri, Sao Paulo Brasil). The data considered were measurements of weight collected at birth, weaning
(7 months of age) and monthly from 8 to 11 months of age. The used function was Michaelis-Menten. Means of
the individual growth curve parameters for Body weight were calculated. By means of different technologies of
adjustment of this growth curve, we have verified that the not linear method presents a better quality represented
by minor values for the average square of the combined mistake and a greater facility of convergence, providing
estimations adjusted to the reality, which allow a suitable biological interpretation of the parameters of the
studied model.
Key Words. Rubia Gallega. Nellore.Growth curves. Michaelis-Menten equation.
RESUMEN
Las cruces de Rubio Gallego x Nelore se están valorando sistemáticamente puesto que desde el
año 2000, la Asociación de Criadores de la Raza Rubia gallega (ACRUGA) diseñó la planificación de
los mismos para determinar el comportamiento productivo y su capacidad del ajuste a los sistemas de
producción de carne en Brasil. El objetivo de este trabajo consistió en el estudio del crecimiento de
animales de Rubia Gallega x Nelore usando la ecuación de Michaelis-Menten. Este estudio utilizó los
datos recogidos de 76 animales de Rubia Gallega x Nelore en la granja Mosquera Grandal (Buri, Sao
Paulo Brasil). Los datos considerados fueron medidas del peso recogidas en el nacimiento, destete
(7 meses de la edad) y mensualmente a partir de los 8 a los 11 meses de la edad. La función usada
fue la de Michaelis-Menten. Se calcularon las medias de los parámetros individuales de la curva del
crecimiento para el peso corporal. Por medio de diversas tecnologías de ajuste hemos verificado que
el método no linear presenta una mejor calidad de ajuste, representada por menores valores para el
cuadrado medio del error combinado y una mayor facilidad para la convergencia, proporcionando
valoraciones ajustadas a la realidad, lo que permite una interpretación biológica conveniente de los
parámetros del modelo estudiado.
Palabras clave. Rubia Gallega. Nelore. Curvas de crecimiento. Ecuación Michaelis-Menten.
481
INTRODUCCION
Una vez introducida la raza bovina Rubia Gallega en Brasil, se están realizando cruzamientos
con la raza Nelore, a fin de combinar las cualidades de rusticidad y adaptación a medios hostiles de
esta última con las características de especialización para la producción de carne y precocidad de la
Rubia Gallega, a la vez que se mejora la precocidad sexual de la población cebuína.
En este sentido uno de los aspectos a abordar es el crecimiento, el cual puede ser expresado en
términos numéricos, ya que cuando se realizan sucesivas pesadas de un animal de forma sistemática
y regular, es posible construir una curva de peso en función de la edad. Conocer el crecimiento es
fundamental puesto que presenta una relación directa con la producción de carne en cantidad y
calidad, producto final de de la explotación del ganado vacuno de carne.
Diversas funciones de crecimiento no lineales relacionando peso-edad, han sido probadas tanto
en bovinos (Nobre et al., 1987; López et al., 2000; Machado et al., 2004; Mazzini et al., 2004);
asimismo se ha realizado la interpretación de algunos de sus parámetros biológicos contrastando
la dificultad de su ajuste, cuestión ésta referida también por diversos autores en diferentes especies
ganaderas, sobre todo cuando se utilizan pequeños conjuntos de datos lo que provoca ligeros desvíos
que influyen en la precisión de las curvas (Nobre et al., 1987; Machado et al., 2004 Mazzini et al.,
2004).
La ecuación de Michaelis-Menten (Michaelis & Menten,1913) es utilizada ampliamente en la
literatura para describir matemáticamente diversas reacciones químicas permitiendo determinar la
velocidad de reacción de las mismas. De acuerdo a lo descrito por López et al. (2000), si se adapta
convenientemente se puede utilizar para determinar el crecimiento de los animales ya que, dados
sus buenos resultados, resulta equiparable a funciones usuales como las de Gompertz y Richards.
Aunque los trabajos con esta ecuación son muy escasos, ha mostrado su eficacia para describir el
crecimiento del ganado vacuno (Manzini et al., 2004; Machado et al., 2004).
Este trabajo tiene como objetivo verificar la capacidad de ajuste de la ecuación de MichaelisMenten mediante metodologías lineales y no lineales, a observaciones de peso-edad de bovinos
cruzados de Rubia Gallega x Nelore
MATERIAL Y METODOS
Para el presente trabajo se utilizó un rebaño base de 76 animales de la raza Nelore. Se registraron
los pesos al nacimiento, 210, 240, 270, 300 y 390 días criados en la granja Mosquera & Grandal del
estado de Burí, Sao Paulo (Brasil) mantenidos en sistema extensivo sobre un pasto de Brizantia..
Las reproductoras fueron inseminadas con toros de la raza Rubia Gallega. Los partos se
sucedieron con normalidad en la primavera y otoño, destetándose todos los animales (machos y
hembras) a los 7 meses de edad. Estos animales, mantenidos con lactancia natural, recibieron la
siguiente alimentación complementaria:
Durante 209 días un pienso prestarter a razón de 700 gr / animal y día, con una composición
de 18% de PB, 2,5% de Extracto etéreo, 7% de fibra, 13% de humedad, 10% materia mineral, 1,2%
de calcio y 0,5% de fósforo. Después de 150 días de iniciado el anterior y durante 145 días se
complementó con 2.600 gr / animal y día de un pienso con el 40% de PB, 2% de EE, 15% de fibra y
18% de minerales. Los últimos 80 días de confinamiento recibieron además , por animal y día, 18 kg
de silo de maíz y 3.700 gr de harina de maíz. El total de alimento, sin contabilizar la leche materna y
el consumo de pasto, fue de 2.400 gr de concentrado y 18 kg de silo de maíz, por animal y día.
La ecuación de Michaelis-Menten (Michaelis & Menten, 1913), es ampliamente utilizada para
evaluar la cinética de procesos enzimáticos y se expresa como sigue:
482
Y = ρx/x+δ: siendo Y, la velocidad de reacción; x, la concentración del sustrato; ρ la velocidad
máxima de reacción y δ la concentración del sustrato una vez conseguida la velocidad máxima. Los
parámetros ρ y δ pueden ser estimados a partir del ajuste. Según López et al., (2000) y Machado et
al., (2005), en la utilización de esta ecuación para estudios de crecimiento es posible asumir que:
Y es el peso de los animales en la edad x, ρ sería el peso adulto del animal y δ sería una medida de
precocidad, que representaría el intervalo de tiempo transcurrido para conseguir la mitad del peso
adulto, es decir, cuanto menor sea el valor de éste parámetro mas precoz resultaría el animal.
El trabajo de crecimiento con esta ecuación realizado por Machado et al.,(2004), utiliza, entre
otras, las siguientes expresiones de ajuste que pueden ser representadas como sigue:
lineal I
1
(δ/ρ) 1
___
_____
___
1/Y =
+
+
ρ
x
e
donde el término e representa el error aleatorio, e ~N(0,σ2).
no lineal
Y = ρx / x+δ
lineal II
δ
___
x /Y =
+
x
___
ρ
+
xe
ρ
La comparación entre las expresiones fue probaba por Machado et al., (2005) en lo referente
a la calidad de ajuste, representada por el valor del cuadrado medio del error combinado y por la
interpretación biológica de los parámetros evaluados, con resultados satisfactorios en ganado Nelore,
por lo que decidimos incorporar esta metodología. Para el desarrollo de la misma se emplearon los
programas Simfit v. 5.5 y el software estatístico R (R Development Core Team, 2004), concretamente
las funciones nlme.nlsList (No lineal) y lm (Lineal).
RESULTADOS Y DISCUSION
La curva ajustada de la ecuación de Michaelis-Menten se muestra en la gráfica 1.
Grafica 1. Curva ajustada para la ecuación de Michaelis-Menten
Las estimaciones medias de los parámetros y la evaluación de la calidad de los ajustes se muestran
en la tabla 1.
483
Tabla 1. Estimaciones medias de los parámetros, intervalos de confianza (95%) y Cuadrado Medio del error
combinado para el ajuste de las distintas formas de la ecuación de Michaelis-Menten a datos de peso-edad de
animales del cruce RG X Nellore.
Ecuación
No lineal
Lineal I
Lineal II
Parámetro
ρ
δ
ρ
δ
ρ
δ
Media
132,12
111,91
99,36
90,63
136,66
110,00
CME
835,47
1326,98
1100,66
De acuerdo a la tabla 1, la forma de ajuste No lineal mostró valores menores para el CME
combinado, y en consecuencia una calidad de ajuste mayor. El modelo Lineal I es la de peor
comportamiento, lo que coincide con el trabajo de Machado et al., (2005) en Nelore.
Respecto a la evaluación de la calidad de los ajustes del modelo lineal de acuerdo con el test
de Durbin-Watson, el modelo no presentó autocorrelación residual pues los valores medios de este
estadístico (1,424) no fueron significativos.
CONCLUSIONES
Con el uso del modelo de Michaelis-Menten fue posible calcular los pesos de los animales con
una aceptable precisión. Este modelo parece deseable para explicar el crecimiento del cruzamiento
de Rubia Gallega x Nelore.
BIBLIOGRAFIA
López, S.; France, J.; Gerrits, W.J.J.; Dhanoa, M.S.; Hunphries, D.J.; Dijkstra, J. 2000. A generalized
Michaelis-Menten equation for the analysis of growth. Journal of Animal Science, v.78, p. 1816-1828.
Machado, E. J., Muniz, J. A., Fonseca, F. 2004. Análise da curva de crescimento por meio da equação deMichaelisMenten. Estatística e Experimentação Agropecuária.
Mazzini, A. R., Muniz, J. A., Fonseca, F., Henrique de Aquino, L. 2004. Equação de Michaelis-Menten
generalizada na análise de crescimento de bovinos. 49ª Reunião da Região Brasileira da Sociedade
Internacional de Biometría. Brasil.
Michaelis, L.; Menten, M.L.1913. Die kinetik der invertinwirkurg. Biochemische Zeitschrift,v. 49, 334-336.
R Development Core Team. R: A language and environment for statistical computing. R Foundation for Statistical
Computing, Vienna, Austria. ISBN 3-900051-00-3, 2004. URL http://www.R-project.org.
Santoro, K. R. 2005. Adequação de modelos não lineares a observações peso-idade de crescimento de bovinos
com ganho compensatório. 49ª Reunião da Região Brasileira da Sociedade Internacional de Biometría.
Brasil.
Santos, S. A., Souza, G.S., Oliveira, M.R., Sereno, J.R.1999.Using nonlinaer models to describe heigth growth
curves is Pantanero horses. Pesq. Agropec. Bras., v.34, n.7, p.1133- 1138.
Schinckel, A. P., Ferrell, J., Einstein, M. E., Pearce, S. A., Boyd, R.D. 2003. Analysis of Pig Growth From Birth
to Sixty Days of Age. Swine Research Report. Purdue University p. 57-67.
484
COMPARISON OF MINERAL LEVELS IN BONE AND BLOOD SERUM
OF CATTLE IN NORTHWESTERN TURKEY*
USPOREDBA RAZINE MINERALA U KOŠTANOM SUSTAVU I KRVI
KRAVA U SJEVEROZAPADNOJ TURSKOJ
Remzi GÖNÜL1, Abdullah KAYAR1, Tanay BİLAL2, M. Erman OR1, Çağla PARKAN1,
H. Tamer DODURKA1, Tevfik GÜLYAŞAR3, Bora BARUTÇU4
Department of Internal Medicine, Veterinary Faculty, Istanbul University,
1
34320, Avcilar, Istanbul/Turkey
2
Animal Nutrition and Nutritional Diseases Department, Veterinary Faculty, Istanbul University, 34320,
Avcilar, Istanbul, Turkey
3
Department of Biophysics, Trakya University Medicine Faculty, Edirne/Turkey
4
Department of Biophysics, Cerrahpasa Medicine Faculty, Istanbul University,
Cerrahpasa-Istanbul/Turkey
*
“The present work was supported by the Research Fund of Istanbul University. Project Number:
73/15052003.”
ABSTRACT
In order for the mineral levels in the body to be accurately determined, it is essential to determine the mineral
content of bones together with that of blood serum. In this study, blood serum and bone samples were collected
from cattle in and around the provinces of Istanbul, Tekirdağ, Kırklareli, and Edirne and it was attempted to
determine reference values. In each region, blood and bone samples were collected from 100 heads of cattle
and phosphorus (P), Calcium (Ca), and Magnesium (Mg) proportions were calculated. According to the results,
it was found that the levels of P, Ca, and Mg in bone samples of the cattle in Northwest of Turkey were 132.76
± 17.84, 333.23 ± 163.02 and 2.38 ± 0.32 mg/g, respectively, while mineral levels in blood serum were 7.88 ±
1.05, 10.80 ± 0.71 and 2.37 ± 0.68 mg/dL, in the same order. It was thought that these values might constitute
a reference point for serial measurements that would be made for keeping the cattle in our region under control
against metabolic diseases.
Key Words: Cattle, Blood Serum, Bone, Phosphorus, Calcium, Magnesium.
SAŽETAK
Kako bismo mogli objektivno procijeniti razine minerala u organizmu važno je odrediti razine minerala u
kostima i krvnom serumu. u ovom je radu analiziran serum i kosti goveda sa područja istanbul, tekirdağ, kirklareli
i edirne sa svrhom određivanja referentnih vrijednosti. na svakom području uzet je uzorak od 100 goveda na
kojima je određena razina fosfora, kalcija,i magnezija te su izračunati njihovi međusobni omjeri. pokazalo se da
razine fosfora, magnezija i kalcija u goveda u navedenim područjima iznose 132.76±17.84, 333.23±163.02 i
2.38±0.32 mg/g, dok su vrijednosti istih tih minerala u serumu iznosile 7.88±1.05, 10.80±0.71 i 2.37±0.68 mg/
dl. smatramo da bi navedene referentne vrijednosti mogle poslužiti kao polazišna točka za ozbiljnija mjerenja
koja bin mogla poslužiti kontroli metaboličkih poremećaja goveda u pretraženim područjima.
Introduction
In domestic animals, trace elements play an important role in growth, reproduction and their
livestock productivity. Despite this fact, mineral deficiency or imbalances are still frequently
encountered phenomena almost anywhere in the world that cause serious economic loss (4, 6, 13). In
485
cattle, diseases such as parturient paresis (hypocalcemia), grass tetany (hypomagnesemia), and leftside and right-side displacement of the abomasum (alkalemia, hypochloremia and hypocalcemia) are
characterised by electrolyte deficiencies and generally become obvious in the first phase of lactation.
Their negative economic impact is considerable (2, 4, 5, 8).
For this reason, elements such as calcium and phosphorus-containing minerals are of importance
for cattle both during their growth and after they have reached maturity (12, 13). Their exists also a
well-known relationship between the level of phosphorus deposits in the body and diseases such as
botulism. This connection has prompted scientists to investigate the mineral levels in body tissue and
body fluids (1). However, in quite a few cases, electrolyte imbalances are difficult to detect because
the animals affected show no clinical symptoms (4). Consulting organisations recommended by the
Working Groups of the Agricultural Research Commission (ARC) have also stated the necessity to
determine the major minerals in cattle breeding as a matter of priority (12). In case the increased
demand for electrolytes during pregnancy, birth and other physiologically demanding events cannot
be met by the animals’ ration, the skeletal system serves as an important source for the plasma calcium
demand in order to preserve its concentration (8, 9, 10, 12, 14). Because of this protective mechanism,
the mineral concentration in blood is no good indicator for the level of mineral concentrations in
animals (1). For a number of reasons, the blood samples taken for the determination of mineral
concentrations do not provide reliable results. A variety of factors such as exercise, excitation during
blood sampling, the hour of the day, the feeding hours, the season, the animal’s age and the point the
blood is drawn from have an influence on the results (1, 13). Mineral deficiency observed in the blood
serum is of doubtful value as an indicator of correct calcium and phosphorus levels in an animal due
to the fact that they are replenished from mineral depots in the bones (1). For this reason, researchers
emphasize the importance of measuring both the mineral levels in blood serum and in the bones in
order to obtain a correct picture of the mineral concentrations of the entire body. This guideline to
mineral content measurements of the body has led to a demand for new researches on this subject (1,
8, 10, 12). The ensuing investigations have yielded significant results for the clinical evaluation of
metabolic diseases (1).
Only a limited number of studies have so far been carried out in Turkey on the effects of trace
elements in animals (11). The objective of this investigation is the determination of the level of
minerals in both blood serum and bones of cattle under different physiological conditions in
Northwestern Turkey. With this research, reference values for cattle in the region would be obtained
which are necessary for the use of bone tissue mineral concentrations in routine clinical diagnostics.
The lack of such reference values currently poses the biggest obstacle to the application of such a
method. Evaluation of bone biopsy samples has become an important method for the detection of
a number of metabolic diseases in animals before they become acute, and offers the opportunity
of continuous monitoring of pregnant, lactating or growing animals (1). The establishment of this
method as a diagnostic tool in veterinary medicine would help in obtaining additional diseaserelated information on the levels of Ca, P and Mg in bones which would support the development of
successful treatments in cases of parturient paresis, grass tetany, phosphorus deficiency and a number
of other diseases where classical methods prove inconclusive.
Cattle in and around the provinces of Istanbul, Tekirdağ, Kırklareli and Edirne served as material
of this study. From each region older than 24 months female animals of holstein breed were analysed.
486
They were from different herds with different feeding regimes. From each region clinically healthy
100 heads of cattle delivered to the abattoir were examined. Blood samples were taken from the
life animals via juguler vena puncture and bone samples from the same animals in accordance with
approved techniques, after slaughtering (1, 6, 15, 16). In total 400 samples each of blood serum
and bone were collected. The Ca and Mg levels in dry bone samples were determined with the
aid of a Shimadzu atomic absorption spectrometer, model AA-680, while the concentration of dry
bone P was measured spectrophotometrically with the molybdenum blue method (1, 8, 10, 12, 15,
16). The minerals in the blood serum samples were determined with a Ciba Corning autoanalyser,
model Express Plus. When statistically evaluating the results obtained, the mean values and standard
deviations were calculated for every province with t test, the various provinces were compared, and
the statistically significant differences with respect to mineral concentrations in bones and blood
serum examined with one-way Anova using KyPlot package program.
Results
The concentrations of minerals in blood serum and bone samples taken from cattle in Northwest
of Turkey are given in Tables 1 and 2.
Table 1. Mean value, standard deviation and statistical significance of mineral concentrations in dry bone
samples of cattle in Northwest Turkey.
Province
N=100
P (mg/g)
Ca (mg/g)
Mg (mg/g)
Istanbul
X ± Sx
130.26 ± 20.17
310.8 ± 140.7c
2.6 ± 0.15c
bc
Tekirdağ
X ± Sx
140.21 ± 20.12
330.64 ± 170.4
2.27 ± 0.52c
Kırklareli
X ± Sx
130.28 ± 10.92
420.86 ± 220.46b
2.28 ± 0.29b
Edirne
X ± Sx
130.3 ± 20.18
270.63 ± 120.52a
2.38 ± 0.32a
Mean
X ± Sx
132.76 ± 17.84
333.23 ± 163.02
2.38 ± 0.32
In each group significant statistical differences were established between provinces designated with different
letters (for Ca and Mg: p<0,001).
Table 2: Mean value, standard deviation and statistical significance of mineral concentrations in blood serum
samples of cattle in Northwest Turkey.
Province
N=100
P (mg/dL)
Ca (mg/dL)
Mg (mg/dL)
Istanbul
X±S
7.95 ± 1.16
11.75 ± 0.5
2.54 ± 0.71d
b
Tekirdağ
X ± Sx
7.9 ± 1
9.17 ± 0.5
1.97 ± 0.8c
Kırklareli
X ± Sx
7.7 ± 1.05
10.25 ± 1.32ab
2.2 ± 0.4b
Edirne
X ± Sx
8±1
12.05 ± 0.52a
2.79 ± 0.83a
Mean
X ± Sx
7.88 ± 1.05
10.80 ± 0.71
2.37 ± 0.68
In each group significant statistical differences were established between provinces designated with different
letters (for Ca and Mg: p<0,001).
a
Mineral concentrations in the examined bone samples were found to be higher for calcium
and phosphorus (333.23 ± 163.02 and 132.76 ± 17.84 mg/g respectively), while the values for
magnesium, 2.38 ± 0.32 mg/g, were lower. Comparison between provinces showed statistically
significant differences in the levels for Ca and Mg in particular in the regions of Kırklareli and Edirne
(p<0.001). For phosphorus between provinces, however, no statistically significant difference could
be established.
487
Examination of mineral concentrations in blood revealed calcium concentrations in all regions
were in the normal reference value. Istanbul and Edirne provinces were statistically significantly
higher (p<0.001) than Tekirdağ province. The Mg concentrations were established to be higher in
İstanbul, Kırklareli and Edirne provinces than in Tekirdağ province with the levels in İstanbul and
Edirne higher than the reference values, and those in Tekirdağ and Kırklareli provinces within the
reference range. Differences between all regions were found to be statistically significant (p<0.001).
P levels, though higher than the reference value range showed no statistically significant difference.
Discussion
The bones impart mechanical strength to the muscle-skeleton system and provide functional
capabilities, they also serve as mineral storage and are of vital importance for a range of metabolic
activities such as growth, reproduction and livestock productivity (6, 12, 13, 14). The blood serum
data are insufficient for the determination of the mineral concentrations in animal bodies. It has
been reported that for a correct determination of the mineral concentrations in the body, bone
samples from the ribs, the hip or the feet could be used (1, 7, 10, 12; 16). In this study, the minerals
concentrations in cattle were investigated by measuring both blood serum and bone samples with the
objective of determining reference values in Northwest of Turkey. The study thus served the purpose
of establishing the criteria necessary to identify metabolic diseases at an early stage and to permit
continuous control of the level of livestock productivity of the animals.
The application of bone biopsy techniques has become very simple and samples can be taken
swiftly without causing any discomfort to the animals (1, 6, 7, 10, 12, 16). If this method would be
used routinely in veterinary medicine, a number of metabolic diseases seen frequently in animals
during pregnancy, lactation and growth could be detected early and the significant economic losses
they cause could be prevented while their productivity could be increased at the same time (1).
Researchers in this field also stress the necessity of a more extensive publication of reference values,
in order to permit a routine usage of the method, which has not yet been achieved despite the fact
that reference values for mineral concentrations in bones do exist in literature (1, 6). In this study, the
level of minerals in the blood and bones of animals have been determined and compared, and at the
same time reference values for mineral concentrations in bones established. This is the first time this
has been done in Turkey for cattle. A correlation of those levels with livestock productivity will then
permit the calculation of the economic loss suffered from mineral deficiency.
It is known that the phosphorus content in the animal ration effects bone development to a
large extent (15). It is also known, however, that besides chemical and physical properties, bone
development is influenced by range of other factors such as age, ration, hormones and diseases
(12, 15). Beighle et al. (1) reported important differences in the values of P, Ca and Mg of ashed
bone samples taken from bones of animals of different age, sex and breed. In the dry bone samples
analysed in this study an important influence of those factors was found in particular for Mg and P.
Gerloff and Swenson. (6) had stressed in their study that for the determination of body phosphorus
depots from bone phosphorus measurements, dry or defatted dry samples should be used; and they
had advised against the use of ashed samples. For this reason, in this study dry bone samples taken
from over 2-year-old female animals of culture breed were analysed. With this approach it was
attempted to reduce the above-mentioned variations and to obtain more reliable information about
metabolic diseases observed in Turkey. This restriction in terms of age, sex and breed was motivated
by the fact that economically important metabolic diseases usually appear in over 2-year-old animals
of high-yielding culture breeds (1, 14). Comparison of the results of this study with those reported
by Beighle et al.(1), who worked on animals of similar characteristics. Mineral concentrations in the
488
examined bone samples were found to be higher for calcium and phosphorus than those reported in
literature (203.68 ± 1.0 and 108.25 ± 0.41 mg/g respectively), while the values for magnesium were
lower than previously reported values (5.51 ± 0.05 mg/g) (2).
Examination of mineral concentrations in blood revealed calcium concentrations in all regions
were in the normal reference value of 9.7-12.4 mg/dL (3). The Mg concentrations in İstanbul and
Edirne were established higher than the reference values, and those in Tekirdağ and Kırklareli
provinces within the reference range of 1.8-2.3 mg/dL (3). P levels, though higher than the reference
value range (5.5-6.5 mg/dL) (3) showed no statistically significant differences.
With respect to the mineral concentrations in blood serum samples, it was found that the P
level was rather high while the Ca and Mg concentration were within the normal limits in cattle in
Northwestern Turkey. This was interpreted as a sign for ossification related to high P level in the
blood serum resulting in high Ca and P levels in the bones. This in turn points to a mineral rich
region or to high-dose mineral additives in the animal feed and a lower probability of the emergence
of metabolic diseases.
As has been pointed out by Beighle et al (1), bone mineral concentrations may serve as a powerful
tool for prognosis of the development of metabolic diseases. The ease with which bone samples can
be taken and the availability of serial sample collection techniques make it possible to keep animals
under observation during certain phases of their development (pregnancy, lactation, growth), and
help to avoid unnecessary culling.
Conclusions
As a result of this study on the level of minerals in bones of healthy cattle in Northwestern
Turkey, reference values were determined which may, however, exhibit certain regional differences.
On the basis of those reference values, the animals may be monitored for metabolic diseases in
various phases of their lives with serial measurements of bone samples. An approach that will help to
reduce economic losses to a minimum.
The difference between the results found in this investigation and those reported by other
researchers as well as the dearth of publications on this subject require more extensive research
including other regions in order to be able to establish reliable reference parameters for mineral
concentrations in bone samples.
References
Beighle, D.E., Boyazoğlu, P.A., Hemken, R.W., Serumaga-Zake, P.A.: Determination of calcium, phosphorus,
and magnesium values in rib bones from clinically normal cattle. Am. J. Vet. Res., 1994; 55: 85-89.
Bjorkman, C.: Concentrations of sodium, potassium, calcium, magnesium and chlorine in the muscle cells of
downer cows and cows with parturient paresis. Res. Vet. Sci., 1994; 57: 53-57.
Smith, B.P.: Large animal internal medicine. Third ed., Mosby, St. Louis, Missouri, USA, 2002.
Dakka, A.A., Abdel-All, T.H.S.: Studies on minerals picture in the blood sera of egyptien Sheep. Assiut. Vet.
Med. J, 1992;. 28: 242-249.
Fleming, S.A., Hunt, E.L., Brownie, C., Rakes, A., Mcdaniel, B.: Fractional excretion of electrolytes in lactating
dairy cows. Am. J. Vet. Res., 1992; 53: 222-224.
Gerloff, B.J., Swenson, E.P.: Acute recumbency nd marginal phosphorus deficiency in dairy cattle. J. Am. Vet.
Med. Assoc., 1996; 208:716-719.
Jones, H.C., Fontenot, J.P., Veit, H.P.: Physiological and pathological effects of feeding high levels of magnesium
to steers. J. Anim. Sci., 1990; 68: 4400-4413.
489
Mosel, M.V., Klooster, A.T.H., Malestein, A.: Effects of an inadequate dietary intake of magnesium on
osteogenesis in dairy cows during the dry period. Res. Vet. Sci., 1990; 48: 280-287.
Mosel, M.V., Klooster, A.T.H., Mosel, F.V., Kuilen, J.V.: Effects of reducing diaetary ((Na+K)-(Cl+SO4)) on the
rate of calcium mobilisation by dairy cows at parturition. Res. Vet. Sci., 1993; 54: 1-9.
Mosel, M.V, Wouterse, H.S., Vant Klooster, A.T.H.: Effects of reducing dietary ((Na + K)-(Cl+SO4)) on bone ‘n
da’ry cows at parturition. Res. Vet. Sci., 1994; 56: 270-276.
Sezer, K., Ozdemir, H.: Elazığ ve çevresindeki ineklerde osteomalasinin epizootiylojisinin araştırılması. F.Ü.
Sağlık Bil. Dergisi., 2002: 16: 95-105.
Shupe, J.L., Butcher, J.E., Call, J.W., Olson, A.E., Blake, J.T.: Clinical signs and bone changes associated with
phosphorus deficiency in beef cattle. Am.J.Vet.Res., 1988; 49: 1629-1636.
Uysal, A.:. Metabolizma ve Karans Hastalıkları, Veteriner İç Hastalıklar Ders Notu, 1998; No:79.
Van De Braak, A.E:, Vant Klooster, A. T.H., Goedegebuure, S.A., Faber, J.A.J.: Effects of calcium and magnesium
intakes and feeding level during the dry period on bone resorption in dairy cows at parturition. Res. Vet.
Sci., 1987; 43: 7-12.
Williams, S.N., Lawrence, L.A., Mcdowell, L.R., Wilkinson, N.S., Ferguson, P.W., Warnick, A.C.: Criteria to
evaluate bone minerilization in cattle: I. Effect of dietery phosphorus on chemical, physical, and mechanical
properties. J. Anim. Sci., 1991; 69: 1232-1242.
Williams, S.N., Mcdowell, L.R., Lawrence, L.A., Wilkinson, N.S., Ferguson, P.W., Warnick, A.C.: Criteria to
evaluate bone minerilization in cattle: II. noninvasive techniques. J. Anim. Sci., 1991; 69: 1243-1254.
490
FORENSIC OBSERVATION ON THE USE OF AURICULAR MARKS ON
THE SARDINIAN PUREBRED SHEEP
OSSERVAZIONI CLINICHE, MEDICO-LEGALI, LEGISLATIVE E DI
BENESSERE ANIMALE SULL’APPLICAZIONE DELLE MARCHE
AURICOLARI IN OVINI DI RAZZA SARDA
MEDICINSKO-PRAVNI OSVRT NA PRIMJENU UŠNIH MARKICA KOD
SARDINIJSKIH PASMINA OVACA
Cubeddu G.M., Coluccio P.*, Lai M.G.**, Pintori G.*,Vacca E.***
Istituto di Clinica Medica Veterinaria-Università di Sassari
*Dottorato di ricerca in “Normative dei paesi UE relative al benessere e protezione animale-Università di
Messina
** Veterinario libero professionista
***Direttore Servizio Sanità Animale ASL 6 Sanluri
Summary
The numberless complaints of the flock owners, the deforming lesions caused by the application of the
auricular marks, the loss of them, as well as the death of the animals in consequence of the possible clinic
complications in the various Sardinian provinces, have lead the authors to:
1) to weigh up the difficulties met owing to the application of the auricular marks with regard a new
group of lambs in Sardinia, in compliance with the EC Regulation 21/2004;
2) to make a set of forensic observations with regard to both the rising of pathologies resulting from the
application of the auricular marks, through a constant clinic monitoring of the concerned animals, and to the
request of compensation for damages, proposing some suggestions. It is also emphasized, in the light of the
increasing of the rules in the subject of animal wellbeing and protection, that the animal identification systems
of sheep and goats could be considerably improved employing electronic identifiers, on account that some
conditions pertinent to the measures of accompanying are satisfied.
In Italy it has been confirmed the feasibility of cattle, sheep and goats electronic identification, and
therefore, it is hoped for that the EU can issue a course of action to oblige the member States to adopt the
electronic identification system.
Sažetak
Mnogobrojna negodovanja vlasnika stada ovaca u Sardiniji, zbog povreda prouzrokovanih primjenom
ušnih markica, zbog njihovog gubljenjea te zbog mogućih kliničkih komplikacija koje mogu rezultirati i
smrću životinja, navela su autore na: 1) procijenjivanje nastalih komplikacija prilikom primjene ušnih markica
proporcionalno remontu stada ovaca na Sardiniji, prema Pravilniku CE n. 21/2004; 2) izvršiti promatranje niza
medicinsko –pravnih segmenata s obzirom na pojave patologija izazvane primjenom ušnih markica, putem
konstantnog kliničkog monitoringa istih životinja, kao i s obzirom na zatražene odštete, s predlaganjem mjera.
U vrijeme donašenja mnogih normativnih akata vezanih uz dobrobit i zaštitu životinja, naglašava se, da bi
za identifikaciju ovaca i koza bilo bolje korištenje elektronskih čipova. U Italiji je već potvrđena izvedivost
elektronske identifikacije goveda, ovaca i koza, te je moguće da će EU obvezati zemlje članice na primjenu
istoga.
491
Riassunto
Le numerose lamentele da parte dei proprietari delle greggi, le lesioni deformanti provocate
dall’apposizione delle marche auricolari, la perdita delle stesse, nonché la morte degli animali a
seguito di possibili complicanze cliniche nelle diverse province della Sardegna, hanno indotto gli
Autori a: 1) valutare le difficoltà incontrate dopo l’applicazione delle marche auricolari relativamente
alla quota di rimonta negli ovini in Sardegna, in ottemperanza al Regolamento CE n. 21/2004; 2)
effettuare una serie di osservazioni medico-legali relativamente sia all’insorgenza di patologie
conseguenti all’applicazione delle marche auricolari, attraverso un costante monitoraggio clinico
degli animali interessati, sia alla richiesta di risarcimento per danni, proponendo dei suggerimenti.
Viene sottolineato, altresì, alla luce del proliferare delle normative in materia di benessere e
protezione animale, che i sistemi di identificazione degli animali delle specie ovina e caprina in
Sardegna potrebbero essere sensibilmente migliorati con l’impiego di identificatori elettronici, a
condizione che siano soddisfatte talune condizioni relative alle misure di accompagnamento. In Italia,
è stata confermata la fattibilità dell’identificazione elettronica di bovini, ovini e caprini e, pertanto,
è auspicabile che l’U.E possa emanare una direttiva per obbligare gli stati membri ad adottare il
sistema elettronico di identificazione.
Introduzione: l’allevamento ovino rappresenta un cardine per l’economia della Sardegna, con
circa tre milioni e mezzo di capi, ubicati in oltre 14.000 aziende; il benessere degli animali ha assunto
al giorno d’oggi connotazioni fino a poco tempo fa impensabili. Le difficoltà incontrate con l’avvento
delle nuove normative, vengono oggi superate anche da quelle categorie, tra le quali gli allevatori
sardi, i quali si dimostrano sempre più propensi a tutelare la protezione ed il benessere dei loro animali,
sia in un’ottica di maggiori economie di scala, sia soprattutto per una mutata coscienza zoofila, oltre
all’incentivo dei premi comunitari. Nell’allevamento attuale si pongono numerosi problemi, non
solo di ordine sanitario e mesologico, ma anche in riferimento al benessere animale ( sofferenza ).
Inizialmente il tema del benessere e della protezione animale è stato affrontato principalmente sotto
le spinte di sensibilità nei confronti degli animali. Successivamente tale problematica è stata anche
valutata come fattore di produzione in grado di incidere sul prodotto zootecnico. Il legislatore ha
emanato una vasta ed articolata normativa sulla questione “benessere” per gli stretti rapporti esistenti
fra questa condizione degli animali e produttività quanti-qualitativa dell’allevamento. L’aspetto
giuridico del benessere animale, inteso semplicemente come assenza di maltrattamenti, è stato per
molti anni legato alle disposizioni dettate dall’ex Art. 727 del Codice Penale, che sanciva le pene per
atti di crudeltà o maltrattamenti verso animali. Nel 1985 è stata emanata la Legge n. 623, a ratifica
della Convenzione di Strasburgo del 10.3.1976 riguardo la protezione degli animali negli allevamenti
e nei macelli. Sono state, inoltre, promulgate numerose disposizioni normative, a recepimento anche
di Regolamenti o Direttive Comunitarie, concernenti il benessere e la protezione animale. Tra i fattori
di interesse sanitario relativi al Benessere Animale si annovera anche la prevenzione delle patologie
d’allevamento. Inoltre c’è grande attenzione dell’UE al raggiungimento e mantenimento del benessere
animale nelle produzioni zootecniche. Il benessere animale passa attraverso tre buone ragioni: al’articolo 9 della Costituzione italiana è in fase di revisione con l’inserimento della “tutela delle
esigenze in materia di benessere degli animali in quanto esseri senzienti” da parte della Repubblica;
b-la nuova PAC ( Politica Agraria Comunitaria ) pone “il benessere animale” fra le 4 condizioni per
l’accesso al regime di aiuti da parte dell’UE; c-il Consiglio dell’UE ha stabilito che “i proprietari o
i custodi adottino le misure adeguate per garantire il benessere dei propri animali e per far sì che a
detti animali non venga provocato dolore, sofferenze o lesioni inutili” (dir. 98/58, art. 3); ancora la
Legislazione italiana, all’art. 9 della Costituzione recita: “La Repubblica riconosce l’ambiente, i suoi
492
ecosistemi, le sue biodiversità, omissis…..; tutela le esigenze in materia di benessere degli animali
in quanto esseri senzienti.
Materiale e metodi
A partire dal 9 luglio 2005, come è noto, in forza del regolamento (CE) N. 21/2004, tutti gli ovini
e i caprini nati dopo tale data devono essere identificati, entro sei mesi a decorrere dalla nascita e
in ogni caso prima che l’animale lasci l’azienda, attraverso due distinti mezzi di identificazione. Il
primo di questi è rappresentato da un marchio auricolare in materiale plastico applicato all’orecchio
sinistro. In aggiunta alla marca auricolare di cui sopra, l’Autorità competente dovrà approvare un
secondo mezzo di identificazione tra i seguenti: un altro marchio auricolare ( orecchio destro ) con
le medesime caratteristiche del marchio costituente il primo mezzo di identificazione; un tatuaggio
(tranne per gli animali destinati agli scambi intracomunitari e all’esportazione verso paesi terzi); un
transponder elettronico (obbligatorio a partire dal 1° gennaio 2008) o anche un marchio al pastorale
unicamente per la specie caprina. Le marche auricolari, in plastica rigida con un’anima in metallo,
vengono pinzate a pressione sulle orecchie delle nuove leve ( quota di rimonta ). Nel corso degli
ultimi due anni sono stati effettuati numerosi sopralluoghi nelle aziende ovine di tutte le province
della Sardegna, con la stretta collaborazione dei colleghi delle A.S.L., oltre che dei veterinari
dell’Associazione regionale allevatori. Nel corso dell’indagine abbiamo sottoposto gli animali
identificati a visita clinica, nell’ottica di valutare gli effetti negativi provocati dall’applicazione delle
targhette, oltre a verificare se, in un contesto legislativo moderno sulla tutela del benessere animale,
le condizioni di benessere fossero realmente rispettate.
Risultati: in un’alta percentuale di ovini ( 30-40% ) sottoposti a visita clinica, erano presenti
dei sintomi come ipertermia, ottundimento del sensorio, linfoadenomegalia a carico dei linfonodi
regionali, facile affaticabilità e, in alcuni casi difficoltà alla masticazione; inoltre, già ad una prima
osservazione a distanza, erano facilmente evidenziabili a carico delle orecchie lesioni deformanti.
L’esame obiettivo particolare dell’orecchio confermava i sospetti a distanza e in particolare,
soprattutto nella parte interna del padiglione auricolare si evidenziavano: tumefazione, soluzioni
di continuo, presenza di essudato purulento-emorragico; nel periodo primavera-estate, le lesioni
erano complicate dalla presenza di parassiti esterni , larve di mosca e necrosi dei tessuti. In tutti
i casi veniva intrapresa la terapia del caso, a base di antibiotici, antisettici, oltre alla toelettatura
delle parti compromesse. Alcuni animali, nonostante la terapia, venivano a morte; nei restanti si
evidenziavano ritardi nello sviluppo morfologico e funzionale. In questo periodo numerose nel
frattempo erano state le sollecitazioni agli assessorati competenti delle anomalie, nel tentativo di
richiesta di risarcimento danni, a tutt’oggi non ancora presa in considerazione. Stante tale situazione,
i costi per l’allevatore subivano un incremento che andava a gravare sul bilancio aziendale già
compromesso dalla sfavorevole congiuntura del mercato delle carni, del latte e dei derivati; inoltre
la patologia suddetta, compromettendo sensibilmente le condizioni di salute e di benessere degli
animali, interferiva col prosieguo della catena alimentare con possibili conseguenze. Un esempio per
tutti è che nel corso degli anni 2006 e 2007 il Servizio di Sanità Animale della sola A.S.L di Sanluri
ha rilevato ufficialmente lesioni da marche auricolari in forma grave in 57 allevamenti ovini.
Osservazioni personali
Non è difficile immaginare le lamentele degli allevatori sardi allorché si trattava di applicare detto
regolamento, dal momento che la marca auricolare poteva comportare, oltre che aggravio dei costi,
un’importante causa di lesioni al padiglione auricolare; si ha la sensazione, a quest’ultimo riguardo,
493
che si sia sottovalutata la sensibilità degli allevatori per il benessere dei propri animali. La resistenza
degli allevatori si faceva sentire, inevitabilmente, anche sul grado di efficacia dell’identificazione
basata sulla “marca” la cui applicazione in passato aveva dimostrato risultati non soddisfacenti e
comunque inferiori rispetto agli interventi di tatuaggio. Si riteneva oramai, vista la non poca esperienza
accumulata, che il marchio auricolare facesse parte del passato; invece ci si ritrova nuovamente
alle prese con la ben nota “targhetta” con l’aggravante, stavolta, dell’obbligatorietà (per il DPR
317/1996, per lo meno, il marchio auricolare era un’opzione, ovvero un’alternativa al tatuaggio per
contrassegnare il numero progressivo individuale). Il paradosso di tutta la vicenda è che le norme
sul benessere animale impongono, tra le altre cose, anche l’utilizzo delle marche, pena la perdita dei
premi erogati dalla Comunità europea, oltre a quelli della misura F sul benessere. In virtù di questo
ci si attendeva dalla Regione Sardegna, forte della propria esperienza su questo specifico argomento,
una partecipazione più attiva e determinata alle decisioni dirette alla formazione di atti normativi
comunitari così importanti per il comparto ovi-caprino; in questo senso, non sarebbe stato velleitario
proporre a suo tempo un coinvolgimento della sanità pubblica veterinaria e del settore zootecnico nel
definire orientamenti tecnici e procedure specifiche in materia di identificazione e registrazione dei
piccoli ruminanti da sottoporre all’attenzione in sede Statale e Comunitaria. La suddetta esperienza
isolana e le eventuali proposte di nuovi orientamenti e procedure in tema di anagrafe degli animali,
sarebbero state probabilmente sufficienti a dissuadere il Legislatore europeo dal rendere obbligatorio
il marchio auricolare. Ma nel frattempo l’ansia da marche auricolari dilagava negli allevamenti,
sino al punto che molti allevatori pretendevano che le targhette fossero messe solo prima della
macellazione. E’ agevole ipotizzare il pericolo anche per le carni, ove un’infezione latente potesse
determinare alterazioni a carico dello stato di salute degli animali. Ma inoltre la problematica in
oggetto suscita notevole perplessità; infatti, proprio da chi è preposto a ricercare le migliori soluzioni
per la tutela del benessere animale, si aspettavano metodi rispettosi delle norme in vigore, anziché
applicazioni perentorie di tecniche che si erano già in precedenza dimostrate non del tutto consone
all’identificazione degli ovini.
Conclusioni
Le argomentazioni sin qui addotte impongono alcuni suggerimenti utili alla soluzione del
problema. In primis un’immediata azione politica deve essere attivata al fine di far valere le ragioni
di un doppio sistema di identificazione più accettabile per la nostra importante realtà zootecnica.
Già nell’anno 2006 la Commissione europea ha presentato al Consiglio della UE una relazione con
proposte appropriate sul sistema di identificazione elettronica e sull’eventuale aggiornamento degli
aspetti tecnici relativi alla sua attuazione. Gli allevatori svizzeri stanno portando avanti la richiesta di
modifica del succitato regolamento proponendo come primo mezzo di identificazione il transponder
elettronico e come secondo mezzo il marchio-collare. Considerando il sistema di identificazione
fin qui adottato in Sardegna, basato quasi esclusivamente sul tatuaggio auricolare, dimostratosi più
rigoroso e meno traumatico delle “marche”, si ritiene opportuno sollecitare le Autorità competenti
affinché sia riconosciuto il transponder quale primo mezzo di identificazione e il tatuaggio come
secondo sistema identificativo. Infine sembra improcrastinabile garantire un giusto ristoro economico
per i danni subiti dagli allevatori, sulla base dei dettami dell’art.1223 del Codice Civile, anche e
soprattutto per continuare con l’opera di sensibilizzazione nei riguardi del benessere animale in
queste categorie sotto certi aspetti “difficili”. Solo così potremo porre in essere le normative sul
moderno benessere animale. Se così non fosse, potremo tranquillamente considerare gli organismi
competenti responsabili del reato di maltrattamento di animali.
494
Bibliografia:
1) Benazzi P.: Il regolamento di Polizia Veterinaria-Ed. Esculapio-31/08/2002; 2) Cinotti S., Peccolo.G.:
Protezione animale-Utet-1997; 3) Codice Civile – Libro IV delle obbligazioni; 4) Codice Penale: articoli
638 e 727, legge 189/2004; 5) Cubeddu G.M., Pintori G., Bassu G., Maninchedda G.-Differenti valutazioni
sul danno e risarcimento in ovini di razza sarda. Atti del VIII Congresso SIPAOC., Sezione Patologia, pag.
397, 1988; 6) Gasparini U.: Appunti di Medicina Legale Veterinaria, Legislazione Veterinaria e DeontologiaEd. Esculapio 1983; 7) Ghisleni P., Ghisleni G.: Trattato di Medicina Veterinaria Legale. UTET, Torino
1925; Mainardi, D., Papalia, S., Etologia e protezione animale, Editoriale grasso (1991); 8) Nisini: Comp.
Infort. La tribuna 1978 tratato di diritto privato; 9) Pezza F.: Diritto e Legislazione Veterinaria-Utet-1997;
10) Pilla: Ricerca e miglioramento dell’allevamento ovino; 11) Ruffo G.: Legislazione Veterinaria-Poletto
Editore-luglio 2003; 12) Torrente A.: Manuale di Diritto Privato. Giuffrè, Milano 1981.
495
496
BIOMETRY AND HISTOMORPHOMETRY OF OVARY IN LORI LOCAL
SHEEP
BIOMETRIJA I HISTOMORFOMETRIJA OVARIJA OVCE PASMINE
LORI
GHARZI , A 1and ABBASI , M 2
1
Department of Biology , Faculty of Basic Sciences, University of Lorestan, Khorram-Abad, Iran. 2 School of
Veterinary Medicine, University of Lorestan,Khorram-Abad, Iran.
The purpose of this study was to investigate morphometry and histological structure of the ovary in lori
sheep. The lori sheep ovaries were collected from apparently healthy cyclic sheep in khorramabad abattoir. After
removing the non-ovarian tissues the specimens were underwent for biometrical measurements of parameters
and then processed for histological studies. The result of the biometrical studies has shown that the mean
length,width and diameter of ovaries were 1.71± 0.31,1.26 ± 0.24, 0.98 ± 0.23 cm respectively. The ovarian
mean weight was 1.23 ± 0.85 g. The histological study revealed that the majority of primordial follicles was
distributed in close contact to the tunica albuginea, but with the initiation of follicular growth at first, the distance
between follicles and surface epithelium was increased whereas with continuation of the growth this distance
were reduced. Our study revealed that zona pellucida formation in more pronounced in in secondary follicle.
Key words; Sheep, Ovary, Histology, Biometry
Sažetak
Namjera ovog istraživanja je bilo utvrditi histomorfometrijsku i histološku građu jajnika ovce pasmine
Lori. Jajnici su prikupljeni od klinički zdravih ovaca obrađeni u Khorramabadskoj klaonici. Nakon odvajanja od
ne-jajničkog tkiva uzorci su podvrgnuti biometričkim mjerenjima i proslijeđeni na histološku obradu. Rezultati
biometričkog istraživanja pokazuju da su prosječna duljina, širina i promjer jajnika bili1.71+/-0.31 cm, 1.26+/0.24 i 0.98+/-0.23 cm. Prosječna težina jajnika bila je 1.23+/-0.85 grama.Histološkim istraživanjem ustanovili
smo da je većina primordijalnih folikula bila raspoređena u neposrednom dodiru s tunikom albugineom, ali
s početkom folikularnog rasta isprva razlika između folikula i površine epitela raste dok se nastavak rasta
smanjivao. Naše istraživanje je pokazalo da je zona pellucida više izražena u sekundarnom folikulu.
Ključne riječi: ovca, jajnik, histologija
497
BIOCHEMICAL AND HEMATOLOGICAL PARAMETERS IN COWS
WITH TRP
BIOKEMIJSKI I HEMATOLOŠKI POKAZATELJI U GOVEDA SA TRP
Hassanpour, A.1, Amougli Tabrizi, B. 1
1- clinical science department- faculty of veterinary scince- Islamic azad university of Tabriz -Iran
TRP is one the of pervalent diseases in cows that most common in dairy cows feed by prepared foods but
cases occur infrequently in yearlings , beef cattle, dairy bulls , sheep and goats. In this disease cardiac arrythmias
are probably due to electrolyte imbalances .In this research 20 cows with TRP were studied. Firstly affected
cows were diagnosed based on clinical signs. The two blood samples were obtained from the related cows.
The first sample with anticoagulant for haematological tests and determining PCV, RBC, WBC and differential
count ofwhite blood cells, and the second sample was whole blood, in which after separation blood serum by
centrifuging, the biochemical tests, such as sodium , potassium , Glucose , phosphorus, Albumin and calcium
levels in serum were measured. The mean level PCV in these animals was 37.4±4.6 percent. The mean of RBC
and WBC numbers were 7.8×103 and 13.2×106 respectively. The mean concentrations of sodium, potassium,
glucose, phosphorus, albumin and calcium in serum were 117.7± 13.3 meq/l, 3.11±0.96 meq/l, 42.29±5.63 mg/
dl, 3.84±1.02 mg/dl, 544.60± 87.64 mg/dl and 8.60±2.06 mg/dl respectively.According to this study electrolyte
imbalance there was in the cows with TRP and balancing of electrolytes will need.
Sažetak:
TRP je učestala bolest u goveda ,a najčešće se javlja u mliječnih krava koje su hranjene gotovom hranom
ali su zapaženi rijetki slučajevi te bolesti u jednogodišnjih tovljenika, ovaca i koza. Kod ove bolesti srčane
aritmije su vjerojatno povezane pomanjkanjem elektrolita. U ovom istraživanju koristili smo 20 krava. Prva
zapažanja kod oboljelih krava temeljili smo na kliničkim znacima. Od svake oboljele životinje uzeli smo 2
uzorka krvi. Prvi uzorak sa antikoagulansom uzeli smo za hematološku pretragu i određivanje PCV, RBC,
WBC i diferencijalnu krvnu sliku. Drugi uzorak krvi koristili smo za biokemijsku pretragu te odredili količinu
natrija, kalija, glukoze, fosfora, albumina i kalcija u serumu. Srednja vrijednost PCV-a u testiranih životinja bila
je 37.4+/-4.6%. Srednje vrijednosti RBC-a i WBC-a bile su 7.8*10 i 13.2 *10 6. Prosječna koncentracija natrija,
kalija, glukoze, fosfora, albumina i kalcija u serumu bila je 117.7+/-13.3 meq/l, 3.11+/-0.96 meq/l, 42.29+/-5.63
mg/dl, 3.84+/-1.02 mg/dl, 544.60+/-87.64 mg/dl i 8.60+/-2.06 mg/dl. Naše istraživanje je pokazalo da kod
goveda sa TRP-om postoji pomanjkanje elektrolita.
498
THE SURVEY ON PREVALENCE OF E.COLI O157:H7 USING VT1, 2
AND EAE GENES FROM SHEEP IN SHIRAZ-IRAN
PREGLED POJAVNOSTI E.COLI O157:H7 UPOTREBOM VT 1,2 I EAE
GENA IZ POKRAJINE SHIRAZ U IRANU
Yahya Tahamtan*a, Seyyed Shahram Shekarforooshb, Seyyed Ali Pourbakhshc
a
Department of Microbiology, Razi Vaccine and Serum Research Institute, Shiraz, 71955-367, Iran.
b
Department of Food Hygiene, School of Veterinary Medicine, Shiraz University, 71345-1731 Shiraz, Iran.
c
Department of Avian Disease, Research & Diagnosis, Razi Vaccine and Serum Research Institute, Tehran,
31975/148, Iran. •Corresponding author. Tel.: +98 711 6240 331; fax: +98 711 6240 201. E-mail address
[email protected] (Yahya Tahamtan). [email protected]
Abstract
From September 2004 to August 2005, swab surfaces samples from sheep were collected at the major
slaughterhouses of Shiraz-Iran. The samples were revealed the occurrence and characterization of E. coli O157
strains in Iranian domestic sheep. Escherichia coli O157:H7 is a highly virulent food-borne pathogen and threat
to public health. Sheep faces may be an important source of Verotoxin-producing Escherichia coli. Verotoxins
(VT) 1, 2 and eae genes were tested for this propose. The monthly prevalence of E. coli O157 in sheep was
obtained 3.92-0.2% and was at its highest in spring and late summer. Six (3.92%) of sheep carcasses were
contaminated by E. coli O157:H7. Five (3.34%) of 115 and 1(2.63%) of 38 samples were from ewes and
lambs carcasses respectively. Only six samples were positive to PCR specific for the VT2 gene and produced
verocytotoxin VT2, whereas all isolates were negative for the presence of VT1 and eae virulence genes
considered. The detection limit of the PCR protocol for VT2 in E. coli O157:H7 without prior enrichment was
103 CFU ml-1, and with over night prior enrichment in mTSB was 102 CFUml-1. Geographical variations and
season may be influenced in prevalence. The distribution of VTEC in sheep and lamb in subject area is widely.
The composition of the gastrointestinal flora may be changed by different diet and, therefore O157 VTEC rate
in sheep and lamb was different. Iranian sheep indicated as a natural host of E. coli O157 strains therefore, may
be potentially pathogenic for humans. This is the first report of E. coli O157 detection from sheep in Iran.
Keywords: Escherichia coli O157:H7; Sheep; PCR ; Iran
Sažetak:
U ovom radu prikupljani su uzorci briseva ovaca u većim klaonicama u jednoj iranskoj pokrajni. Uzorci su
ukazali na učestalost i karakteristike sojeva E. coli O157 u iranskih domaćih ovaca. Escherichia coli O157:H7
je vrlo virulentan pathogen koji se prenosi hranom te kao takav ima značajan utjecaj na javno zdravstvo.Ovčji
izmet može biti važan izvor verotoksina kojeg stvara Escherichia coli.U ovom radu istraživali smo verotoksine
1 i 2. Mjesečno pojavljivanje E.coli O157 u ovaca je uočeno u 3.92+/-0.2 % i bilo je najviše u proljeće i kasno
ljeto.Šest (3.92 %) ovčjih lešina je bilo zaraženo E. coli O157:H7. Pet (3.34%) od ukupnog broja od 115 i jedna
( 2.63%) od 38 uzoraka uzeto je od ovaca i janjadi. Samo 6 uzoraka je bilo PCR specifično i pozitivno za VT2
gen, te su proizvodili VT2 verotoksin dok su svi ostali uzorci bili negativni na VT1. Zemljopisne raznolikosti
i doba godine mogli su utjecati na pojavnost stanja. Očigledna je široka rasprostranjenost VTEC u ovaca i
janjadi promatranog područja. Sastav gastrointestinalne flore se mijenjao ovisno o režimu prehrane, pa je stoga
i različita rtazina O157 VTEC u probavilu ovaca i janjadi. Iranske ovce su uočene kao prirodni domaćini E.coli
O157 koja je potencijalno patogena i za ljude. Ovo je prvi izolat E.coli O157 u iranskih ovaca.
Ključne riječi: Escherichia coli O157:H7; Ovca; PCR ; Iran
499
FIRST IDENTIFICATION OF ESCHERICHIA COLI O157:H7 BY
IMPROVED TRYPTICASE SOY BROTH AND SORBITOL MACCONKEY
AGAR CONTAINING CEFIXIME AND TELLURITE AND COMPARISON
WITH PCRS FROM CATTLE CARCASSES IN SHIRAZ-IRAN
PRVA IDENTIFIKACIJA E.COLI O157:H7 NA POBOLJŠANOJ PODLOZI
BUJONA SOJINE TRIPTIKAZE I SORBITOL MCCONKEY AGARU KOJI
SADRŽE CEFEXIM I TELURIT U USPOREDBI S PCR OD GOVEĐIH
LEŠINA U IRANSKOJ POKRAJINI SHIRAZ
Yahya Tahamtana1•, Ali Pourbakhsh c, Shahram Shekarforoosh b
a
b
c
Razi Vaccine and Serum Research Institute,Shiraz-Iran
Razi Vaccine and Serum Research Institute, Tehran-Iran
School of Veterinary Medicine, Shiraz University, Shiraz, Iran
Background: E. coli O157:H7 as food-borne pathogens and implicated in HC, HUS and may be lead
to death. Cattle are the major reservoir. Sorbitol-MacConkey (SMAC) agar is developed as the first selective
medium for isolation of E. coli O157:H7 through form colorless colonies. We investigated the efficiency of
modified CT-SMAC medium and PCRs to detection of E. coli O157:H7 in Shiraz-Iran abattoirs. Material
and methods: The Trypticase Soy Broth (TSB) and SMAC agar was supplemented with cefixime (0.25 mg
L-1) and potassium tellurite (12.5 mg L-1) (5 times more than general use) to create improved CT-SMAC. 153
slaughtered cattle were tested from December 2005 to August 2006. Results, Discussion: E. coli O157:H7 were
isolated from 24 (15.58%) of 154 cattle carcasses (by CT-SMAC), while 17(11.03%) of this was positive by
O157 antiserum and finally 14(9.09%) of them were significantly positive by PCR and then Nested PCR. The
sequencing result was shown 80% homology with some O157 isolated. The bacteria were isolated 12(10.52%)
of 114 cattle two or below two and 3(7.5%) of 40 cattle over two years old (P<0.05). We have improved TSB
and SMAC agar, which largely inhibits the physiological intestinal flora. This method has allowed the detection
of bacteria among coliform grown by using specific primers to detect a fragment of 779 bp and combined with
a nested PCR which have 372 bp stx2 fragments. Composition of the gastrointestinal flora may be result in high
frequency for younger animals.This is the first report of E.coli O157:H7 isolated from cattle in Iran.
Keyword: Escherichia coli O157:H7; Cattle; PCR
Sažetak: E. coli O157:H/ je patogena bakterija koja se prenosi hranom a posljedice trovanja tim patogenom
mogu dovesti do smrti.Glavni rezervoar te bakterije su goveda. Sorbitol-MacConkey (SMAC) agar je prvi
selektiva hranjiva podloga razvijena za uzgoj E. Coli O157:H7 pri čemu ona na njemu raste u vidu obojenih
kolonija.U ovom radu istraživana je učinkovitost modificirane CT-SMAC hranjive podloge i otkrivanja E.coli
O157:H/ pomoću PCR metode.Materijal i metode: U triptikaza bujon (TSB) I SMAC agar dodan je cefixim
(0.25mg L-1) I natrijev telurit (12.5 mg L-1) u pet puta većoj koncentraciji od uobičajene . Tako smo dobili
poboljšani CT-SMAC . Od listopada 2005. godine pa do kolovoza 2006 godine obrađeno je 153 uzorka od
zaklanih goveda.Rezultati i rasprava:E.coli O157:H7 je izolirana iz 24 uzorka ( 15.58%) od ukupno 154 uzorka (
pomoću CT-SMAC), dočim je 17 uzoraka ( 11.03%) bilo pozitivno na antiserum od O157 i konačno 14 uzoraka
(9.09%) od njih bilo je pozitivno detektirano uz pomoć PCR-a.??Bakterija je bila izolirana u 10.52% u krava
ispod dvije godine starosti i u 7.5% krava iznad dvije godine starosti (P<0.05). Koristili smo poboljšani TSB i
SMAC agar koji značajno usporava rasžmnožavanje fiziološke crijevne flore.Ovo je prvi slučaj izolacije E.coli
O157:H7 iz goveda u Iranu.
Ključne riječi: Escherichia coli O157:H7; krava; PCR
500
INDUCTION OF PARTURITION IN EWES (LOCAL BREEDS) AND
SUBSEQUENT SURVIVAL OF NEONATES
Saif A.Balios*, Muaffq S.Kassim* *and Rabea A.S.Al-Oramary**.
* Dept. of Surgery and Medicine. College of Veterinary Medicine. University of Dohuk.Kurdistan Region. Iraq
**Dept.of Animal Production .College of Agriculture. University of Dohuk.Kurdistan Region. Iraq.
Abstract
Twenty –four pregnant ewes of local breeds were used in two equal groups ina study of induction of
parturition and subsequent survival of neonates. Ewes were treated with either 15mg dexamethasone or saline
(CONTROL) intramuscularly on day 145 of gestation .There were significant differences between the 2
treatments in the mean of the interval from treatment to lambing ( p < 0.05 ) (162 and 59.7h for control and
dexamethasone respectively) and in the proportion of that lambed by 48 hr after treatment ( p < 0.01) .Injection
of 15mg dexamethasone resulted in the short and less variable interval from treatment to lambing. There were
no significant differencesbetween the two treatment in groups in birth weight (3.8 and 4.2 kg for control and
dexamethasone, respectively) andin the mean of the live weightat one month of age (9.4 and 10.2 kg for control
and dexamethasone, respectively).There were no significant differences between the two treatment groups in the
survival rate of lambs at birth(92.8 and 93.3% for controland dexamethasone, respectively )and at one month
of age (71.4 and 66.6% for control and dexamethasone, respectively ). This study confirms the effectiveness
of the dexamethasone to induce the parturition in ewes oflocal breeds with no adverse effect on the survival
ofsubsequent neonates.
Key Words: Ewe, Parturition, Dexamethasone
501
EFFECTS OF UNTREATED GASTROINTESTINAL NEMATODE
INFECTIONS ON PERFORMANCES AND DEVELOPMENT OF
EFFECTIVE IMMUNE RESPONSE IN LAMBS OF VALLE DEL BELICE
BREED
Brianti E., Abbene S., Gaglio G., Risitano A.L., Ferlazzo M., Chiofalo B*., Cavallaro
M*., Giannetto S.
Dipartimento di Sanità Pubblica Veterinaria, *Dipartimento MOBIFIPA – Università di Messina, Italy
Gastrointestinal nematode infections in sheep are typified by suboptimal animal performance caused by
reductions in both feed intake and nutrient utilization. On the other hand the maintenance of host-parasite
relationship is believed to be of importance for the development of an effective immune response. This immune
response is crucial for the control of pathogenic consequences of parasites infections (resilience) and for the
control of new parasite infections (resistance). However, the importance of these topics little information
is available for sheep in this field and most of the studies are carried out under controlled conditions with
experimental infections in wool breeds. Therefore, aims of this study were to evaluate the effects of untreated
gastrointestinal nematode infections, naturally acquired from pastures in dairy lambs of Valle del Belice breed,
on: i) animal health and performances and ii) capability to develop effective immune response such as resistance
and resilience. To achieve these goals 26 female lambs of Valle del Belice breed, 2-month-old, were enrolled
in the study and randomly dived into treated (13) and untreated (13) groups. Lambs were from the same flock
and were kept from the start to the end of the study in the same management and alimentary regimes. For one
year lambs weights, faeces for Faecal Egg Count (FEC) and blood for Cell Blood Count (CBC) were collected
monthly. Lambs from treated group received monthly anthelmintic treatment (Moxidectin®, Fort Dodge) for
four months from the beginning of the study to the next spring (march) when lambs started a new season of
grazing. During the middle of the study, only one animal from the treated group was excluded for a suspected
cerebral coenurosis; however no symptom of gastrointestinal helminthiasis was observed in all the study
animals. FEC in untreated lambs showed increasing trend the first four months of the study reaching the higher
mean values in April (325 e.p.g.) to after gradually decrease till the end of the study. In the treated group FEC
rapidly peaked when anthelmintic treatment was suspended reaching similar values to those of untreated group
from May to the end of the study. Study results did not show any significant difference in lambs growth rates and
in CBC values between treated and untreated groups from the beginning to the end of the study. Nevertheless,
significant difference were observed in the count of eosinophilic cells in the first four months of the study
been these higher in untreated than in treated group. This study provide the evidence that a moderate burden
of gastrointestinal nematodes is well tolerated by lambs of Valle del Belice breed and anthelmintic treatment
have not evident benefits on both animal health and performance when parasitic burden is in “equilibrium”
with the host. However, in our study we were not able to demonstrate that the maintenance of host-parasite
relationship especially during the first months is of crucial importance for the development of efficient resilience
and resistance to gastrointestinal nematode infections. This failure could be the result of numerous events such
as the low parasite burden of infected animals and the consequently low pastures contamination, the parasites
genera and the well-balanced alimentary regime. Nonetheless, this study represents a first experience and we
hope to achieve more useful information in future studies.
502
SEROLOGICAL SURVEY ON BOVINE VIRAL DIARRHEA VIRUS
INFECTION OF CATTLE IN HAMADAN AREA-IRAN
Aliasghar Bahari1, Mohammadreza Sadeghi1, Shamsedin Ghaemmaghami2, Ali
Sadeghi-nasab1
Junior School of Veterinary Medicine, Bu-Ali Sina University, Hamadan-Iran
1
2
Razi Vaccine & Serum Research Institute- Arak branch, Arak- Iran
Abstract
Infection with bovine viral diarrhea virus (BVDV) is endemic in cattle populations in most part of the
world. The high prevalence in combination with the negative effects on reproduction and the general health
condition in affected herds result in significant economic losses to the cattle industry globally. In this study,
serological investigation was performed to determine the prevalence of bovine viral diarrhea virus infection of
cattle in Hamadan area. A total of 399 blood samples were collected by jugular puncture. The sera were tested
by enzyme linked immunoabsorbent assay (ELISA) for BVDV antibodies. The results were analysed by chisquare statistical test. The prevalence rate was 77.6%. A total of 182 cases (76.8%) and 127 cases (78.4%) of
industrial and rural non-industrial farms were positive respectively. Statistical analysis of data were not shown
any significant differences between age groups and methods of husbandry (P<0.05).
Key words: Bovine viral diarrhea virus, Seroprevalence, ELISA.
503
SEROEPIDEMIOLOGY OF BOVINE LEUKEMIA VIRUS (BLV)
INFECTION IN THE NORTH EASTERN PROVINCES OF IRAN
Alireza Haghparast1, Gholamreza Mohammadi2, Shalaleh Mousavi1
1
Immunology Section, Department of Pathobiology and 2Department of Clinical Siences, Faculty of Veterinary
Medicine, Ferdowsi University of Mashhad, Iran.
Bovine leukemia virus (BLV) is an exogenous c-type oncovirus in the retroviridae family that causes a
chronic infection in cattle, which develops in three possible pathological forms: asymptomatic course, persistent
lymphocytosis and lymphosarcoma. Once infected, cattle remain virus carriers for life and start to show a
serological reaction within a few weeks after infection. This disease causes significant economic losses associated
with the costs of control and eradication, loss in milk production and difficulties in exports.
The Agar gel immuno diffusion (AGID) and ELISA tests have been the serological choice assays for routine
diagnosis of serum samples. However, due to the higher sensitivity of ELISA, in recent years this method has
replaced the AGID in seroepidemiological studies for control and eradication programe.
Prevalence studies on BLV infection has not been conducted in the eastern part of Iran. This study was
designed to determine the prevalence of BLV in Khorasan Razavi and Khorasan Shomali provinces which are
the main provinces located in the north east of Iran.
During summer 2007, 429 blood samples were randomly taken from industrial dairy herds of these two
provinces and subjected to ELISA test (indirect sandwich ELISA).25.41% of these samples showed positive
reaction. The positive results in khorasane razavi and khorasan shomali provinces were 29.83% and 1.5% ,
respectively. Moreover, 64.7% of the herds tested, had at least one positive cow. The overall true prevalence
was 24.63%.
Statistical analysis revealed a direct correlation between age and occurence of BLV. Significant correlation
was also found between herd size and BLV infection. In another words, the large herds had the most BLV
positive cases. Moreover, number of parity was also shown to have a direct correlation with BLV seropositivity.
Results of Odd ratios analysis which is an indication of possible risk factors, confirmed the above findings.
Taken together, the results presented in this study along with simillar studies in recent years in Iran confirm
an alarming increase in prevalence of BLV infection among dairy herds. Therefore, control strategies based
on test and implementation of corrective management should be taken in order to downgrade and possibly
eliminate this economically hardship disease.
504
GENETIC CARACTERIZATION OF SARDINIAN MOUFLON
POPULATION USING MICROSATELLITES AND MTDNA MARKERS.
Leoni G.G, °Bebbere D., Satta V., °Berlinguer F., Mereu P., Pirastru M., Manca L.,
°Naitana S.
Department ofPhysiological, Biochemical and Cellular Sciences and °Department of Animal Biology, University of Sassari, Italy.
A recent molecular genetic assessment of the genus Ovis based on mitochondrial genotypes has affirmed
the distinctiveness among ovine species and has suggested that European Mouflon (Ovis gmelini musimon) is an
ancestral line of modern sheep breeds (Hiendleder et al., 2002). Actually, European Mouflon is found in large
number and interbred with domestic sheep in several European countries (Cugnasse, 1994), but lives in natural
state only in Sardinia and Corsica, were is fragmented in a few distinct isolated areas.
This study aimed to investigate the genetic structure of mouflons living in three different areas of Sardiniausing
both microsatellites and mitochondrial DNA as markers.Thirty two mouflons living in Montelerno (n=15),
Orgosolo (n=8) and Lanusei (n=9) areashave been trapped and blood sampled. We analysed 16 microsatellite
loci polymorphism length and sequence polymorphisms of the mitochondrial D-Loop region to characterize the
structure of each group and to clarify the genetic relationshipsamong them.
To genotype microsatellites, Polymerase chain reaction (PCR) of the DNA fragments containing the
polymorphisms was carried out according to standard protocols using primers . Microsatellite products were
loaded on ABI PRISM 3100 DNA Analyser (Applied Biosystems) and data were processed by GENESCAN v3.1
and GENOTYPER v2.5 softwares. Standard intra-population indices, such as allele frequency, gene diversity
and deviation from Hardy-Weinberg Equilibrium (HWE), were tested with the software package Arlequin 1.1
(http://lgb.unige.ch/arlequin/). Genetic differentiation (Fst) and the test of genotype assignment have been
chosen as inter-population indices to assess the relationships among the three samples.
The total length of D-loop region of the mouflon mitochondrial D samples was amplified using described
primers according to Wood & Phua, (1996). The amplified products were sequenced using the dideoxi-terminator
method and loaded on ABI PRISM 3100 DNA Analyser (Applied Biosystems). Clustal X software was used for
the sequence multi-alignment. Phylogenetic molecular analyses were conducted using MEGA software.
All the primers pairs successfully amplified the microsatellites sequences in the mouflon genome. Of the
sixteen genotyped loci, fourteen are polymorphic in at least one of the three analysed groups, with a number of
alleles ranging from two to seven, with a mean of four alleles per locus. Eight microsatellites are polymorphic in
all the three populations, and twelve have private alleles.The observed heterozigosity shows, for every marker,
high variability among the three populations.Five microsatellite loci deviated from HWE in at least one group of
mouflons due to heterozygote deficit. The exact test for population differentiation (Fst) revealed strong genetic
differentiation among the three populations, since almost all the relative P values are significant (P<0.05). The
high Fst values are largely due to private alleles. The test of Genotype Assignment determines the effective
belonging of every individual to the population to which it was initially assigned. It does so by taking the three
sampled population as potential source.
We were able to sequence the mitochondrialD-Loop regions of all the 32sampled mouflons. After sequence
alignment 3 aplotipes were evidenced showing variations in 11 different nucleotides. These data allowed us to
make a neighbour joining-tree of individuals where it was possible to separate three distinct clades each contain
the samples deriving from only one populations.
All these results indicate strong genetic differentiation among the three studied groups, probably due to long
term isolation.Despite the low number of individuals sampled, each population showed considerable genetic
variation. This could be due to the fact that originally the founder of each group carried high genetic variability
and the populations have not been isolated for a period of time long enough to show the disadvantages of
inbreeding. (Supported by Fondazione Banco di Sardegna)
505
INVESTIGATION OF BOVINE VIRAL DIARRHEA VIRUS (BVDV)
INFECTION IN RELATION TO FERTILITY IN DAIRY COWS WITH
REPEAT BREEDING
Mesih KOCAMÜFTÜOĞLU 1,Ayhan ATA2, Mehmet KALE3, Sibel HASIRCIOĞLU 3, *
3
1
Dept. of Gynecology and Reproduction, Faculty of Veterinary Medecine, Mehmet Akif Ersoy University,
15100, Burdur, TURKEY.
2
Dept. of Artificial Inseminatiom, Faculty of Veterinary Medecine, Mehmet Akif Ersoy University, 15100,
Burdur, TURKEY.
Dept. of Virology, Faculty of Veterinary Medecine, Mehmet Akif Ersoy University, 15100, Burdur, TURKEY.
*
Corresponding author. Fax: +90 248 234 45 00, E-mail address:[email protected] (S.
HASIRCIOĞLU)
Abstract
In this study, the blood serum and leucocyte samples were collected from 250 cows with repeat
breeding and tested by ELISA. 250 cows were screened for the presence of BVDV (Ab). Out of 250
cows, 106 cows were found BVDV (Ab-). Out of 106 BVDV (Ab-) animals, 48 were BVDV (Ag+)
and 58 were BVDV (Ag-).
The differences for calving to service interval (CSI) (days) and age between BVDV (Ag+/Ab-)
and BVDV (Ag-/Ab-) cows found statistically important (p<0.01). Also, the differences between
pregnant BVDV (Ag+/Ab-) and BVDV (Ag-/Ab-) cows for CSI (days) (p<0.05) and age (p<0.001)
was found statistically important. Conception rate (CR) between these groups was statistically found
insignificant (P=0.37).
No significant difference for age between animals in this group were detected whereas the
difference for CSI (days) between non-pregnant BVDV (Ag+/Ab-) and BVDV (Ag-/Ab-) cows
found statistically important (p<0.05).
In conclusion, the results of this study showed that there was a close relation between BVDV
infections and fertility in dairy cows being repeat breeding problems and BVDV in these animals
affected the fertility of herds negatively.
Keywords: Dairy cattle, BVDV, repeat breeding, fertility, ELISA
506
INFLUENCE OF IMPROPRIATE MILKING ON UDDER HEALTH OF
DAIRY COWS
N. Prvanović, M. Cergolj, A. Tomašković, N. Maćešić, J. Grizelj, M. Samardžija, T.
Dobranić, N. Filipović, G. Bačić, M. Lipar
Clinic for obstetric and reproduction, Faculty of veterinary medicine, University Zagreb, Croatia
Department for physiology and radiobiology,Faculty of veterinary medicine, University Zagreb, Croatia
The aim of the study was to compare and analyse influence of different milking failures on udder health of
dairy cows. Two dairy farms in southwestern Croatia were observed and followed during five years. At the first
farm there were 430 cows and at the second farm there were 320 monitored cows. Cows of both breeds were
uniform according to breed, age and milk production; they all were holstein-frisian cows, approximatly 8 years
old, used in milk production for 5-6 years with everage milk production of 7 000 l per year. Cows from the first
farm were milked on the milking parlour 3 times daily using „BOU-MATIC“ system. Unfortunately, due to
some technical problems, part of the system didn’t work properly and it was necessary to replace it with manual
milking during the last two years of research. Due to high hygienic standards of milking preparation, they didn’t
notice any change of milk hygiene and quality. On the second farm cows were milked 2-3 times daily according
to lactation status using milking machines on the lair but presanitation and hygienic standards were very poor
and absolutely unsufficient. On both farms mastitis test was performed regularly and milk samples from all
positive cows were collected for bacteriological tests. During five years we analysed 1292 samples from the
first and 2504 samples from the second farm. After bacteriological test of 1292 samples from the first farm only
18,60% samples were positive versus 16,6% positive results from 2504 samples from the second farm.
Monitoring and analysis of results from the first farm has shown statistically significant influence of
mentioned technical problem on positive results of bacteriological tests. From the 9,10% positive results at the
first year it decreased on 25,26% at the last year of study. The most common isolate was Staphylococcus aureus
(66,99%), Streptococcus agalactiae (12,95%), Streptococcus dysgalactiae (4,88%), Streptoccocus faecalis
(8,06%) and Lactobacillus spp. (7,09%). Furthermore, number of positive Staphylococcus aureus isolates
increased predominatly year after year and caused already mentioned dramatic increase of positive samples.
Analysis of results from the second farm has shown that number of positive results decreased slowly from
18,74% to 16,66% during five years. There was no predominance of particular bacteria species. The most
common isolates were: Streptococcus faecalis (20,41%), Micoplasma spp. (19,16%), Streptococcus agalactiae
(17,92%), Staphylococcus aureus (17,92%), Lactobacillus spp. (9,58%) and Coliform spp. (8,75%). Comparation
of all positive samples from the front quarters versus rear quarters has shown predominance of the front quarters
(55,41% versus 44,59%). It could be explained with high incidence of S. aureus who predominantly attacks
front quarters. According to our results we concluded that poor sanitation and milking hygiene enables different
bacteria and also Micoplasma species to survive at the farm and cause mastitis and dairy losses but in the mean
time cows develop sort of resistence wich causes decrease of subclinical infections after a few years. In the mean
time, milking of cows using high hygienic standards but changing routine and type of milking ( machine milking
replaced with manual milking) causses decrease of immunity and slowly increases subclinical infections. It also
enables significant increase of very resistent bacteria like S. aureus to intrude and stay in the farm causing more
and more problems and dairy losses.
Key words: cow, mastitis, impropriate milking, sanitation, bacteriological test, S. aureus
507
ANTIMICROBIAL EFFECT OF EDTA_TRIS AND ANTIBIOTICS ON
SALMONELLA TYPHIMURIUM INVITRO AND ON BROILER CARCASE
Mohagheghzadeh Ali
Faculity member of islamic azad university, kahnooj branch_kerman_iran.
The effect of six antibacterial agents (ampicilin, chloramphenicol, oxytetracyclin, streptomycine, nalidixic
asid and sulfacetamid) on salmonella typhimurium phage type 49, invitro and on broiler carcase and combined
with edta_tris were investigated.
In vitro solutions of edta_tris markly potentiated the effect of oxytetracycline and ampicillin and lesser
potentiated the effect of chloramphenicol, nalidixic acid and sulfanamid. But no potential effect was abserved
with streptomycine.
Enumeration of salmonella typhimurium from the broiler carcase of short term (2 min) and long term (60
min), were investigated.
The mixture of edta-tris with nalidixic acid, chloramphenicol, sulfanamid and oxytetracycline at long term
gave the most significant reduction in the number of organism.
Ampicillin and streptomycine without edta-tris at long term also gave reduction in the number of
organism.
508
EFFECTS OF BLUETONGUE LIVE MODIFIED VACCINE ON EARLY
AND MID PREGNANCY IN SHEEP
Nicolussi P, Bonelli P, Pau S, Canalis M, Soru A, Sarria A, Patta C, Marongiu E, Di
Gennaro A*, Bonfini B*, Savini G*.
Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Italy
*Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise, Teramo, Italy
Fifty six pregnant Sarda ewes were arranged into 4 groups according to their different stages of gestation.
The first group included 27 ewes from 15 to 30 days of gestation, the second 13 ewes from 31 and 45 days, the
third 9 ewes from 45 to 55 days and the fourth included 7 ewes from 56 and 75 days of gestation. Twenty seven
ewes, 13 from the first group, 6 from the second, 4 from the third and 4 from the fourth were vaccinated with
the combination of the monovalent bluetongue virus (BTV) serotypes 2, 4, 16 modified live vaccines.Following
vaccination, BT related symptoms were recorded in 6 (18.7%) vaccinated animals. The percentage of vaccinated
animals (66.7%) showing lambing failures was higher (P<0.05) than that ofthe unvaccinated controls (17.2).
Abortion, stillbirth and agalactia were observed in10 ewes(76.9%) of the first group, 5 (83.3%) of the second, 2
(50%) of the third and 1 of the fourth (25%). To assess the presence of bluetongue viraemia and determine the
BTV serotype implicated, blood samples were also collected the day of vaccination and three times a week for
the following 30 days. BTV-16 was the only serotype isolated in the viraemic animals. It is concluded that BTV2, BTV-4 and BTV-16 monovalent vaccine combination caused reproductive failures in ewes vaccinated in the
first period of pregnancy and that the BTV-16 wasprobably the serotype responsible for them.
509
EPIDEMIOLOGICAL SURVEY OF INFECTIOUS CAUSES OF
DIARRHEA IN YOUNG SMALL RUMINANTS IN SICILY.
Vicari D., Mancuso R., Cicero A., Randazzo V., Martorana C., Chetta M., Ferrantelli V.,
Castiglione F.
Istituto Zooprofilattico Sperimentale della Sicilia “A. Mirri”, via G. Marinuzzi 3, Palermo, Italy,
[email protected]
Diarrhea is the most common and costly disease affecting neonatal small ruminants and is the major
cause of lamb mortality. It is a complex, multi-factorial disease involving the animal, the environment,
nutrition, and infectious agents. The four major causes of diarrhea in lambs and kids during the first
month of life are E. coli, rotavirus, Cryptosporidum sp. and Salmonella spp.E. coli scours are most
common. The aim of this study is the description of causes of diarrhea in young small ruminants in
120 flocks in occidental lands of Sicily. Data reported here were collected from rectal swabs and
from animals died for diarrhea submitted at our laboratory facilities during a two-years time extent.
The most common microorganism isolated are E. coli, Clostridium spp. and Campylobacter spp.
confirming their rule in this disease.
510
DEVELOPMENT OF “POTENCY TEST” FOR EVALUATION OF FARM
VACCINES AGAINST CONTAGIOUS AGALACTIA IN A NATURAL
OUTBREAK
RAZVOJ “TESTA UČINKOVITOSTI” ZA OCJENU FARMSKE VAKCINE
PROTIV KONTAGIOZNE AGALAKCIJE
Castiglione F., Tamburello A., Macrì D., Emanuele M.C., Alaimo C., Monteverde V.,
Puleio R., Manno C., Ferrantelli V., Nicholas R.A.J. *, Loria G.R.
Istituto Zooprofilattico Sperimentale of Sicily, Italy; *) VLA (Weybridge), UK
Introduction:
Contagious agalactia is the cause of more than 30% of sheep and goats mastitis in Sicily (data
from yearly reports of Istituto Zooprofilattico Sperimentale della Sicilia-IZSS ). Because of the
loss of farm production, severe sanitary restrictions and difficulties in treatment and control of the
infection in the farm, this infection represents a veterinary priority among problems of sheep farming
in Southern Italy. The aim of this long-term on-going study is to add a practical contribution to the
knowledge and evaluation of an inactivated (autogenous) vaccine as control tool against contagious
agalactia. The authors report clinical and laboratory data one year after experimental vaccination of
sheep severely affected by contagious agalactia in the Trapani district of Sicily.
Sažetak:
Kontagiozna agalakcije uzrok je više od 30% mastitisa u ovaca i koza na Siciliji (podatci iz godišnjeg
izviješća sicilijanskog eksperimentalno - zooprofilaktičnog instituta IZSS). Zbog gubitka proizvodnje na
farmana, ozbiljnih sanitarnih ograničenja i poteškoća u liječenju i kontroli infekcije na farmi, ova infekcija
predstavlja veterinarski prioritet među problemima u farmskom ovčarstvu južne Italije. Cilj ove dugotrajne
studije je praktični doprinos dosadašnjim saznanjima i ocjena inaktivirane (autogenske) vakcine kao kontrolne
mjere protiv kontagiozne agalakcije. Autori iznose kliničke i laboratorijske podatke godinu dana nakon što je
provedeno eksperimentalno vakciniranje stada ovaca ozbiljno napadnutog kontagioznom agalakcijom u regiji
Trapani na Siciliji.
Materials and methods:
The flock was selected in November 2006 following clinical and laboratory investigations of a
farm in the Trapani district where the disease is endemic. The flock showed clinical, microbiological
and serological positivity for contagious agalactia with more than 50% of excretion in monitored
milks and even higher antibody levels in sera. The veterinarian of the farm vaccinated all the sheep,
inoculating 2 ml/head with autogenous vaccine produced by the IZS of Sicily, using the local strain
of Mycoplasma agalactiae previously isolated from the farm during aetiological investigations. The
vaccine was obtained from a broth culture, harvested at the peak of growth and the whole cells
inactivated with 0.2% of formalin at 37° C for 15 hours, and adjuvanted with 300 mg/l commercial
saponin (Quil A®) supplied by Brentag Biosector, Denmark (Estrada et al. 2000). The vaccine
preparation contained a median value of 108 CFU/ml. In February, a group of eighteen 3-5 year old
healthy lactating ewes, belonging to the Comisana dairy breed originating from a flock with no history
511
of contagious agalactia, were introduced into the infected flock. This introduced group comprised
vaccinated animals and some unvaccinated control ewes. The unvaccinated ewes were included to
act as sentinels to enable us to measure the level of infection on the farm. The introduced sheep
were divided into three subgroups of 6 animals each: group 1 vaccinated once; group 2 vaccinated
twice, with the booster dose given after an interval of one month; and group 3 as negative controls
treated only with saponin solution without antigen and same adjuvant concentration. This group were
previously screened by microbiological and serological tests and shown to be free of the causative
agents of contagious agalactia. Over the year since vaccination, the sheep have been monitored daily
by the owner and fortnightly by IZSS vets with, in addition to clinical assessment, samples taken for
laboratory monitoring of M.agalactiae. Each animal of the introduced group showing severe signs or
other pathologies were properly treated with antibiotics and vaccines when necessary or eliminated
from the experiment on welfare grounds. The rest of the flock were under the responsibility of the
owner and he was requested to manage as usual. Periodically every 2 weeks, clinical investigations
were performed on the whole flock and on all 18 sheep of introduced group. Biological samples were
collected from all “introduced” sheep and from a representative sample of the home herd, generally
the youngest ewes. Biological samples taken were blood samples, milk, joint fluid (if there was
detectable arthritis) ocular and nasal swabs. For isolation of the pathogen we utilised selective media
and procedures as described by Nicholas and Baker (1998): milk, ocular and nasal swabs, drops of
joint fluids were cultured in broth and semi-solid media (Mycoplasma Experience, UK; Mycoplasma
broth and agar, Oxoid, USA). After 2-3 days incubation at 37°C 10% CO2, broths were plated onto
agar for colony observation. Sera were tested by ELISA method utilising commercial kit (Check-kit
Agalactiae (Pourquier) to monitor for specific antibodies against M. agalactiae.
The PCR technique (Bashiruddin, 1998; McAuliffe et al., 2003) was also used either to confirm
microbiological results or wherever possible to detect the presence of dead mycoplasmas in field
samples. The home sheep were re-vaccinated with the same dose every 4 months; the introduced
ELISA antibodies
180
160
140
120
2 times v ac c inated
100
1 time v ac c inated
Control
80
Random
60
40
20
Au
gu
st
Se
pt
em
be
r
O
ct
ob
er
N
ov
em
be
r
D
ec
em
be
r
Ja
nu
ar
y
Fe
br
20
08
Ju
ly
Ju
ne
M
ay
Ap
ril
07
M
ar
ch
0
Fe
br
ua
ry
group was maintained/ kept with a single or double vaccination in order to monitor the length of
immunity and protection.
512
Results:
Clinical signs: after the severe clinical syndrome detected in winter 2006, no more clear cases
have been observed in the flock. Only some occasional mild conjunctivitis not always correlating to
isolation of the pathogen.
Antibody level (Elisa Kit Pourquier®- France) during the trial (median values):
Antigen excretion during the trial:
December
2006
Group
1
2
March-May
2007
June-August
2007
2 times vaccinated
1 time vaccinated
Controls
Random
■■■■■■■■■■
■■■■■■
●●●●
Quarterly reports
2007/2008
3
■■■■■
4
SeptemberNovember
2007
December
2007February 2008
●
●
●▲
■
●▲
( *Legenda: ■ milk ● nasal swab ▲ ocular swab)
The vaccinated introduced sheep have not shown clinical signs of contagious agalactia with
the exception of occasional mild conjunctivitis. ELISA testing showed high serological antibody
presence in both vaccinated groups (1 dose and 2 doses), but with prolonged levels of reactivity
detected only in the booster vaccinated group which has lasted over 12 months (see tables below
reported). An increase in antibody levels has been observed in the last sampling in the introduced
unvaccinated controls suggesting that the pathogen is starting to infect again exposed ewes.
No mycoplasma pathogens have been isolated from two times vaccinated sheep whereas one
time vaccinated were found positive on one occasion (nasal swab) and controls for milk, nasal and
ocular swabs in the last six months of the trial. Random sampling from the home herd have shown
lower antibody levels and occasional positive microbiological and/ or PCR results in the last three
months of the experiment.
Discussion:
Even after one year study it is difficult to draw conclusions: at the beginning of the experiment
the disease seemed under control with no clinical cases or excretion of the pathogen. Nevertheless
513
non-vaccinated animals have not so far shown any sign of disease, which can be attributed, in the
outbreak, to a initial very “low risk” of infection.
Only in the fourth quarterly period, the presence of the pathogen started to be identified in the
random and control groups with some isolation of Mycoplasma agalactiae from nasal, ocular swabs
and from milk samples. This data could theoretically confirm (until now) the efficacy of the inactivated
vaccine in prevention of clinical disease and limiting the excretion of the pathogen but it is possible
that after the acute syndrome the infection survives at more chronic, subclinical levels with milder
clinical behaviour and rare bacterial excretion. Even if it is too early for final answer, this field trial
gave an interesting and practical data on the efficacy of the autogenous saponin vaccine. It is our aim
to continue to monitor the outbreak for more complete data. The proven safety and low costs of these
products make them the “chosen tool” of prophylactic measures against contagious agalactia.
References:
Bashiruddin, J.B. (1998) Extraction of DNA from mycoplasmas. In: Miles R.J. and Nicholas R.A.J. (Editors)
Mycoplasma protocols, Methods in molecular biology, vol. 104, Humana Books Press, Totowa, New Jersey,
USA, pp. 141-144.
Estrada, A., Katselis, G.S., Laarveld, B., Barl, B. (2000) Isolation and evaluation of immunological adjuvant
activities of saponins from Polygala senega L. Comparative Immunology, Microbiology & Infectious
Diseases 23, 27-43.
McAuliffe, L., Ellis, R., Ayling, R.D., Nicholas, R.A.J. (2003) Differentiation of Mycoplasma species by
16S ribosomal DNA PCR and denaturing gradient gel electrophoresis fingerprint. Journal of Clinical
Microbiology. 41, 4844-4847.
Nicholas, R.A.J., Baker, S. (1998) Recovery of Mycoplasma from animals. In: Miles R.J. and Nicholas R.A.J.
(Editors) Mycoplasma protocols, Methods in molecular biology, vol. 104, Humana Books Press, Totowa,
New Jersey, USA, pp. 37-43.
514
CLINICAL AND HISTOPATHOLOGICAL FINDINGS IN A CASE OF
OVINE OCULAR SQUAMOUS CELL CARCINOMA (OSCC)
Galia S., Pugliese M., De Majo M., Mazzullo G.
Dipartimento di Sanità Pubblica Veterinaria – Facoltà di Medicina Veterinaria – Università di Messina
– Polo Universitario SS. Annunziata, 98168, Messina
Abstract – Ocular squamous cell carcinoma (OSCC) is one of the most important primary neoplasms of the
eye. [1] It occurs more frequently in cattle and horses than in cats and dogs [2-4] and has been reported, although
uncommonly, in buffaloes, sheep, pigs and humans beings [1,5,6]. The tumour arises and develops at the level
of the epithelial surfaces of the conjunctiva (more frequently in the nictitans) and/or cornea (more frequently at
the level of the limbus). Invasive and highly malignant tumours can progressively involve the entire ocular globe
and metastases in regional lymph nodes and in lungs can occur. [7] Aim of this paper is to report the clinical
and histopathological examinations of a case of OSCC in a sheep, observed in a Sicilian farm. Clinically, in the
right eye a voluminous mass involving the entire ocular globe was detected. Lesion showed ulcerations on the
periorbital region, eyelids’ involvement, and mucopurulent discharge. The subject underwent to necropsy and
histological examination of tissue samples showed neoplastic transformation whose appearance were consistent
with well-differentiated squamous cell carcinoma, characterized by “horn-pearls” and cords/islands of squamous
epithelial cells. The observed findings underlines the possible relationships between the poorly pigmented ocular
skin, the extensive farming system employed and the exposure of the animals to ultraviolet radiations.
Introduzione – Il carcinoma oculare a cellule squamose (OSCC), meglio conosciuto con il
termine corrente di “cancro dell’occhio”, rappresenta una delle neoplasie più comuni e più importanti
degli animali da reddito, in quanto oltremodo incide sotto il profilo economico. [8] E’ il tumore
oculare di più frequente riscontro nella specie bovina [9] e, anche se con incidenza inferiore rispetto
al bovino, nella specie ovina [10]. Recente è la segnalazione e la descrizione di due casi di OSCC in
due capre [11].
Negli ovi-caprini l’incidenza della neoplasia, i fattori predisponenti che ne influenzano
l’insorgenza e l’evoluzione eziopatogenetica del processo carcinomatoso sono identici a quelli
descritti per il bovino. [10,11]
Dal punto di vista eziologico, le cause di OSCC restano ancora da definire; tuttavia è indubbia,
almeno nella specie bovina, l’influenza svolta da alcuni fattori predisponenti (razza, pigmentazione
cutanea, predisposizione genetica, età, raggi UVA, virus, stato di nutrizione, ecc.) nell’insorgenza di
tale neoplasia. [12,13]
L’incidenza dell’OSCC è significativamente più elevata nelle razze che presentano aree di
depigmentazione intorno agli occhi, ai margini palpebrali o sulla mucosa congiuntivale si presentano
depigmentate. [14] E’ opportuno ricordare che la pigmentazione delle palpebre e del bulbo oculare
(processo che normalmente comincia subito dopo la nascita e che si completa entro il quinto anno
d’età) costituisce un fattore altamente ereditabile e strettamente correlato a particolari meccanismi
genetici. [12,13]
Nella comparsa della lesione neoplastica l’età gioca un ruolo predisponente non trascurabile; la
neoplasia, infatti, si osserva con una certa frequenza nei soggetti anziani, raramente in soggetti d’età
inferiore ai tre anni. [15]
Altri fattori predisponenti spesso chiamati in causa sono riferibili soprattutto a particolari
condizioni ambientali e d’allevamento, quali prolungata esposizione ai raggi ultravioletti solari,
aumento dell’altitudine e diminuzione della latitudine. [16,17]
515
Per quanto riguarda la sede d’insorgenza, circa il 75% delle forme neoplastiche origina a livello
della congiuntiva bulbare e/o della cornea, e di queste il 90% interessa il limbo e il 10% la cornea.
Nel rimanente 25% dei casi la proliferazione neoplastica coinvolge la congiuntiva palpebrale, la
membrana nittitante e la cute delle palpebre. Le lesioni limbari si localizzano elettivamente nella
porzione mediale e/o laterale, probabilmente perché queste aree risultano essere le più esposte
all’azione dei raggi ultravioletti e/o all’azione irritante svolta da eventuali corpi estranei. [2]
Per quanto concerne l’aspetto clinico, l’OSCC evolve generalmente attraverso quattro stadi:
placca, papilloma, carcinoma non invasivo e carcinoma invasivo. [7,18,19]
Dal punto di vista microscopico, utilizzando i criteri istopatologici riportati nella classificazione
dei tumori del World Health Organization (WHO), il carcinoma invasivo può essere distinto in:
- OSCC ben differenziato: il tumore assume spesso un aspetto vorticoso, con intensa eosinofilia,
presenza di perle cornee e numerosi ponti intercellulari;
- OSCC moderatamente differenziato: le cellule sono spesso raccolte in cordoni o nidi, l’aspetto
vorticoso è poco rappresentato e solo in alcuni casi è possibile riscontrare piccole quantità di materiale
eosinofilico corneificato, raro è il rilevamento di ponti intercellulari;
- OSCC scarsamente differenziato: solo poche cellule mostrano segni di cheratinizzazione e le
formazioni cordoniformi sono frequenti; le cellule sono più piccole rispetto a quelle presenti nelle
altre forme di carcinoma squamoso e il tumore tende ad invadere i tessuti connettivi dell’occhio.
[20]
La diagnosi di OSCC si basa sulla valutazione dei riferimenti anamnestici e degli aspetti clinici
e istologici sopra menzionati. [14]
Materiali e Metodi – Il nostro studio è stato condotto su una pecora di razza Valle del Belice,
di sette anni, femmina. Nella primavera del 2006 l’animale presentava, a livello della commessura
mediale dell’occhio destro, una lesione delle dimensioni di un cece, umida ed iperemica.
Fino alla successiva stagione primaverile, e nonostante l’effettuazione di tre cicli di antibioticoterapia con tilosina ad alto dosaggio, la lesione aveva mantenuto dimensioni ed aspetto costante, ed
il soggetto si manteneva in buone condizioni di salute.
All’inizio dell’estate 2007 la lesione cominciava a presentarsi ulcerata, infestata da larve di ditteri
e aumentava rapidamente di volume fino a raggiungere, nell’arco di quaranta giorni, un volume pari
a dieci volte quello iniziale.
A questo punto, eseguita un’accurata visita clinico-oftalmologica del soggetto ed accertata,
mediante esame citologico del materiale prelevato, la natura neoplastica della lesione, si decideva
di abbattere l’animale, ormai, tra l’altro, in avanzato stato cachettico. In sede autoptica si procedeva
all’asportazione delle strutture oculari orbitali e periorbitali interessate dal processo neoplastico e al
prelievo dei relativi linfonodi.
I campioni tissutali prelevati venivano fissati in formalina tamponata al 10%, inclusi in paraffina,
sezionati in modo da ottenere preparati di 4 µm di spessore e infine colorati con Ematossilina-Eosina
(EE).
Risultati – Alla visita clinica si evidenziava un’estesa tumefazione a livello della commessura
mediale dell’occhio destro, costituita da un’ampia area centrale crateriforme d’aspetto sarcomatoso,
di consistenza fibrosa e ricoperta da lesioni di tipo ulcerativo-crostoso, talora sanguinolente. La
516
proliferazione neoplastica interessava gran parte della superficie cutanea della regione periorbitale
destra, le palpebre, gli strati cutanei più profondi e le strutture muscolo-scheletriche sottostanti.
(Figura 1,2).
Per quanto concerne i risultati dell’esame istologico, l’osservazione microscopica a piccolo
ingrandimento mostrava la presenza di una proliferazione neoplastica più o meno uniforme sia
nell’aspetto che nella distribuzione. A più forte ingrandimento si apprezzava la presenza di piccole
cellule non cheratinizzate, ipercromatiche e con scarsi ponti intercellulari, tra le quali s’interponevano
rare cellule fusiformi. Le figure mitotiche erano numerose, ben evidenti e uniformemente distribuite
in tutti i campi osservati. (Figura 3).
Per l’aspetto spiccatamente anaplastico che la neoplasia mostrava all’esame istologico si emetteva
la diagnosi istologica di carcinoma squamocellulare invasivo scarsamente differenziato.
Figura 1
Figura2
Figura 3
Discussione – Il caso da noi descritto rientrerebbe, sia dal punto di vista clinico (localizzazione
e morfologia della lesione) che patologico (aspetti anatomo-istopatologici della neoformazione) tra
quelli recentemente riportati in letteratura. [10]
Il reperto neoplastico descritto risulta essere particolarmente interessante soprattutto se si tiene
conto della razza ovina coinvolta, la Valle del Belice, mai segnalata nei dati bibliografici consultati.
Tale razza rappresenta un biotipo di pecora allevato in Sicilia e riprodotto per meticciamento
selettivo in consanguineità, impiegata per l’elevata attitudine alla produzione di latte e lana, per la
conformazione dell’apparato mammario, per l’elevata prolificità, per la resistenza alla siccità estiva e
ad altri fattori climatici avversi tipicamente presenti del territorio siciliano. [21] E’ proprio la notevole
resistenza mostrata dagli esemplari appartenenti a questa razza che ne ha incoraggiato l’allevamento
allo stato brado.
Pertanto, è lecito ipotizzare che il particolare tipo di allevamento, che permetterebbe agli animali
di rimanere esposti all’azione dei raggi ultravioletti solari per tempi prolungati, unitamente alle
particolari condizioni climatiche e ambientali presenti nel territorio siciliano, conforterebbero l’ipotesi
secondo cui la prolungata esposizione ai raggi UVA rappresenta uno dei principali fattori predisponenti
chiamati in causa nell’insorgenza e nello sviluppo dell’OSCC. [16,17] Inoltre, la colorazione sbiadita
517
del mantello e l’ipotricosi congenita che affligge tale razza, tra l’altro recentemente descritta [22],
potrebbero rappresentare fattori patogenetici determinanti nell’insorgenza dell’OSCC.
Dal punto di vista diagnostico, una delle maggiori difficoltà che si riscontrano nella pratica
clinica è rappresentata dalla diagnosi differenziale tra la lesione iniziale, a carattere benigno, e la
forma carcinomatosa maligna. [19]
Per questo motivo è opportuno sottolineare come soltanto un’attenta e dettagliata valutazione
clinica della lesione unitamente ad un accurato esame anatomo-istopatologico della stessa, consentano
di pervenire ad un’esatta diagnosi e di valutare così quale sia il reale potenziale di sviluppo e diffusione
di tale patologia neoplastica nella specie ovina.
Bibliografia
1.
Wilcock BP, 1993: The eye and ear, pp. 512-522. In: Jubb KVF, Kennedy PC, Palmer N (Eds), 1993,
Pathology of Domestic Animals, 4th Ed, San Diego, Academic Press.
2.
Russel WO, Wynne ES, Loquvam GS. Studies on ocular squamous cell carcinoma (“cancer eye”). I.
Pathological Anatomy and Historical Review. Cancer 1956; 9: 1-52.
3.
Fischer CA, Lindley DM, Carlton WC, Hecke HV, 2002: Tumors of the cornea and sclera, pp. 149-202. In:
Pfeiffer RL, Simons KB (Eds), 2002, Ocular Tumours in Animals and Humans, Ames, Iowa State Press.
4.
Wilcock B, Rosenbraum PS, Boniuk J, 2002: Tumors of the conjunctiva, pp. 87-125. In: Pfeiffer RL,
Simons KB (Eds), 2002, Ocular Tumours in Animals and Humans, Ames, Iowa State Press.
5.
Steiner PE, Bengston JS: Research and economic aspects of tumors in food-producing animals. Cancer, 4:
pp. 1113-1124, 1951.
6.
Nakamura Y, Mashima Y, Kameyama K, Mukai M, et al: Detection of human papillomavirus infection in
squamous tumours of the conjunctiva and lacrimal sac by immunohistochemistry, in situ hybridisation, and
polymerase chain reaction. British Journal of Ophthalmology, 81: pp. 308-313, 1997.
7.
Taylor RL, Hanks MA. Developmental changes in precursor lesions of bovine ocular carcinoma. Vet Med
Small Anim Clin 1972; 67: 669-671.
8.
Heeney JL, Valli VEO: Bovine ocular squamous cell carcinoma: an epidemiological perspective. Canadian
Journal of Comparative Medicine, 49: p. 21, 1985.
9.
Rebhun WC, 1995: Ocular diseases, pp. 443-468. In: Rebhun WC, 1995, Diseases of Dairy Cattle,
Philadelphia, Lippincott Williams & Wilkins.
10. Mendez A, Perez J, Ruiz-Villamor E, Garcia R, Martin MP, Mozos E: Clinicopathological study of an
outbreak of squamous cell carcinoma in sheep. Veterinary Record, 141 (23): pp. 597-600, 1997.
11. Marà M, Di Guardo G, Venuti A, Marruchella G, et al: Spontaneous ocular squamous cell carcinoma in
twin goats: pathological and biomolecular studies. Journal of Comparative Pathology, 132: pp. 96-100,
2005.
12. Anderson DE: Genetic aspects of cancer with special reference to cancer of the eye in the bovine. Ann N Y
Acad Sci, 108: pp. 948-962, 1963.
13. Anderson DE, Chambers D: Genetic aspects of cancer eye in cattle. Misc Publ Okla Agric Exp Sta No MP,
48: p. 28, 1957.
14. Moore CP, 1990: Diseases of the Eye, pp. 1197-1257. In: Smith BP, 1990, Large Animal Internal Medicine,
St. Louis, C.V. Mosby Company.
15. Blackwell RL, Anderson DE, Knox JH: Age incidence and heritability of cancer eye in Hereford cattle.
Journal of Animal Science, 15: pp. 943-951, 1956.
16. Anderson DE, Skinner PE: Studies on bovine ocular squamous carcinoma (cancer eye). XI. Effects of
sunlights. Journal of Animal Science, 20: pp. 474-477, 1961.
518
17. Anderson DE, Badzioch M: Association between solar radiation and ocular squamous cell carcinoma in
cattle. American Journal of Veterinary Research, 52: pp. 784-788, 1991.
18. Pulley LT, Stannard AA, 1990: Tumors of the Skin and Soft Tissue, pp.23-87. In: Moulton JE (Ed), 1990,
Tumors in Domestic Animals, 3rd Ed, Berkeley, University of California Press.
19. Monlux AW, Anderson WA, Davis CL: The diagnosis of squamous cell carcinoma of the eye (“cancer eye”)
in cattle. American Journal of Veterinary Research, 18: pp. 5-34, 1957.
20. Kircher CH, Garner FM, Robinson FR: X: tumours of the eye and adnexa. Bulletin of the World Health
Organisation, 50: p. 135, 1974.
21. Giaccone P, Portolano B, Todaro M: La pecora “Valle del Belice”. Arti Grafiche Provideo, pp. 15-20,
1988.
22. Mazzullo G, Fasciana G, Marotta S, Marino F, Macrì B: Congenital hypotrichosis in Valle del Belice sheep:
gross and histopathological study of skin lesions. Abstracts 24th ESVP Meeting, Munich, Germany, p. 145,
2007.
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520
SHEEP OF THE ISLAND OF PAG: LAMBING FREQUENCY AND LAMB
BIRTH WEIGHT DURING WINTER-SPRING SEASON
PAŠKA OVCA: UČESTALOST JANJENJA I PORODNA MASA JANJADI
TIJEKOM ZIMSKO-PROLJETNE SEZONE
V. Sušić1, A. Ekert Kabalin1, I. Štoković1, Ž. Pavičić2, B. Mioč3, V. Pavić3, Z. Barać4
1
2
Zavod za stočarstvo, Veterinarski fakultet, Sveučilište u Zagrebu, Hrvatska
Zavod za animalnu higijenu, okoliš i etologiju, Veterinarski fakultet, Sveučilište u Zagrebu, Hrvatska
Zavod za specijalno stočarstvo, Agronomski fakultet, Sveučilište u Zagrebu, Hrvatska
3
4
Hrvatski stočarski centar, Zagreb, Hrvatska
Sažetak
Paška ovca je hrvatska autohtona pasmina. Glavni proizvodni cilj uzgoja paške ovce je mlijeko koje se
prerađuje u cijenjeni tvrdi sir. Meso janjadi je visoke kvalitete i kulinarski specijalitet. Kao u slučaju većine ovaca
u Hrvatskoj, paška ovca se janji sezonski, tijekom zimskih i proljetnih mjeseci. U istraživanju je analizirano
4005 janjenja tijekom četverogodišnjeg razdoblja. U svim godinama janjenja su započela tijekom studenog
i završila slijedeće godinje u svibnju. Najveća dnevna učestalost janjenja bila je tijekom prosinca i siječnja.
Prosječna porodna masa janjadi iznosila je 3,28 ± 0,58 uz statistički značajne (p<0,01) varijacije ovisne o spolu
(muška janjad 3,32 ± 0,58 i ženska janjad 3,25 ± 0,57). Općenito, janjad ojanjena tijekom rane zime imala je
veću porodnu masu u usporedbi s janjadi ojanjenom tijekom kasne zime ili proljeća. Utvrđeni rezultati koristiti
će se za karakterizaciju reprodukcijskih obilježja Paške ovce.
Abstract
Sheep of the Island of Pag is Croatian autochthonous breed. It’s main production goal is milk which is
processed into the well known hard cheese. Lamb meat is, because of high quality, culinary speciality. Like
majority of sheep in Croatia, Sheep of the Island of Pag has seasonal lambing during winter and spring months.
In this investigation we analyzed 4005 lambings during 4 years period. In all years lambings started in November
and finished next year in May. The maximal daily frequency of lambings was in December and January. Average
lamb birth weight was 3.28 ± 0.58, but it varied depending of lamb gender (3.32 ± 0.58 for males and 3.25 ± 0.57
for females, statistically significant difference at level P<0.01). In general, lambs born during early winter days
had higher birth weight in comparison to lambs born in late winter and spring months. The results will used for
the characterisation of reproduction traits of Sheep of the Island Pag.
Uvod
Paška ovca je hrvatska autohtona pasmina. Procijenjena brojnost ove pasmine je oko 30.000
grla, što je čini najbrojnijom otočkom populacijom ovaca u Hrvatskoj. Broj uzgojno valjanih ovaca,
obuhvaćenih uzgojno-selekcijskim radom, tijekom vremenskog razdoblja od 2004. do 2006. godine
prikazan je na tablici 1.
521
Tablica 1 – Pasminska struktura uzgojno valjanih paških ovaca od 2004 do 2006. g (Izvor: HSC, 2007)
Table 1 - Breed structure of Sheep of the Island of Pag under selection control from 2004 to 2006 (Source:
HSC, 2007)
Broj šilježica
Godina /
Broj ovaca /
Ukupno /
Number of ewes
/ Number of
yearlings
Broj ovnova /
Year
Number of rams
Total number
2004.
1207
170
41
1418
2005.
1466
186
43
1695
2006.
1944
411
88
2443
Na otoku Pagu uzgajao se otočni tip pramenke koja je bila osnova za stvaranje izvorne paške
ovce. Mijenjanjem proizvodnih ciljeva u prošlosti mijenjale su se i pasmine kojima se pokušavala
oplemeniti paška ovca. Pretpostavlja se da je uz oplemenjivanje pramenke ovnovima pasmine
Negretti i Bergamo, na formiranje paške ovce znatno utjecala i talijanska pasmina Pentile de Puglia
(Pavić i sur., 2005).
Osnovni proizvodni cilj uzgajivača paške ovce je mlijeko koje otkupljuju tri veće sirane na otoku
Pagu, ili se mlijeko unutar gospodarstava prerađuje u poznati i cijenjeni punomasni tvrdi paški sir.
Posebno mjesto u ponudi mesa na hrvatskom tržištu predstavlja i paška janjetina.
Janjenje paških ovaca, kao i većine ovaca drugih pasmina, događa se sezonski tijekom zimskih
i proljetnih mjeseci. Cilj rada je utvrditi dnevnu učestalost janjenja paških ovaca tijekom sezone, te
postoje li razlike u porodnoj masi janjadi ovisno o razdoblju unutar sezone.
Materijal i metode
Podaci o janjenju uzgojno valjanih paških ovaca sustavno su praćeni u razdoblju od 20. 10. 2004.
do 9.6.2007., te analizirani statističkim računalnim programom Statistica 7.1 (StatSoft Inc., 2005).
Prikupljeni podaci obrađeni su uobičajenim postupcima deskriptivne statistike. Analiza varijance
(uz Unequal n HSD test za post hoc analizu) korištena je da bismo usporedili porodnu masu janjadi
ojanjene tijekom pojedinog mjeseca i godine. Značajnost razlika u tjelesnoj masi pri porodu s
obzirom na spol utvrđena je Studentovim T-testom.
Sezonalnost janjenja tijekom promatranog razdoblja prikazana je na grafu 1. U tablici 2 navedene
su prosječne porodne mase janjadi u pojedinoj godini, kao i prosječna porodna masa muške i ženske
janjadi dok je na tablici 3 i grafu 2 prikazano kretanje porodne mase ovisno o mjesecu janjenja.
522
240
220
200
180
160
140
120
100
80
60
20
0
May-2007
Mar-2007
Apr-2007
Feb-2007
Dec-2006
Jan-2007
Nov-2006
Sep-2006
Oct-2006
Aug-2006
M o n th a n d ye a r o f la m b in g
Jul-2006
May-2006
Jun-2006
Mar-2006
Apr-2006
Feb-2006
Dec-2005
Jan-2006
Nov-2005
Sep-2005
Oct-2005
Aug-2005
Jul-2005
May-2005
Jun-2005
Mar-2005
Apr-2005
Feb-2005
Dec-2004
Jan-2005
Nov-2004
Oct-2004
Number of lambed ewes
40
Graf 1 – Učestalost janjenja u razdoblju od 20. 10. 2004. do 9.6.2007.
Graph 1 - Lambing frequency in period from 20. October 2004. till 9. June 2007.
Tablica 2 - Prosječna porodna masa janjadi uzgojno valjanih paških ovaca
Table 2 - Average lamb birth mass of Sheep of the Island of Pag under selection control
Broj janjadi
/ Number of
lambs
3918
Prosječna porodna masa, kg (arit. sred. ± std. dev.)
Average birth mass, kg (Mean ± SD)
Sva janjad / All lambs
3,28
±
0,58
Muški / Male
3,32
±
0,58
Ženski / Female
3,25a
±
0,57
statistički značajna razlika (p<0,01) u odnosu na porodnu masu muške janjadi / statistically significant difference
(P<0.01) according to the birth mass of male lambs
a
523
Tablica 3 – Kretanje prosječne mase janjadi ovisno o mjesecu janjenja
Table 3 - Kinetics of average lamb birth weight depending on month of lambing
Godina / Year
2004
arit.
sred.
Mean
±
220 3,40
II
±
51
III
±
IV
±
Mjesec
/ Month
I
arit.
sred.
Mean
2005
n*
±
std.
dev.
SD
2006
±
std.
dev.
SD
arit.
sred.
Mean
±
0,52 308 3,33
3,18
±
0,45
63
45
2,86
±
0,56
33
2,62
±
0,39
0,27
28
2,48
2
2,15
n*
2007
±
std.
dev.
SD
arit.
sred.
Mean
±
std.
dev.
SD
±
0,59 420 3,19
±
0,51
2,86
±
0,48
54
2,92
±
0,55
50
2,95
±
0,51
16
2,50
±
0,67
78
2,82
±
0,41
5
2,70
±
0,51
±
0,37
24
2,52
±
0,32
±
0,07
3
2,43
±
0,42
n*
n*
V
±
7
2,31
±
VI
±
1
2,40
±
VII
±
±
±
±
VIII
±
±
±
±
±
IX
1
X
XI
3,50
±
117 3,59
±
±
0,65
1
3,50
±
2
3,30
±
0,28
2
3,60
±
0,57
±
±
60
3,86
±
0,46
40
3,23
±
0,64
±
455 3,49 ± 0,52 726 3,34 ± 0,56 1104 3,28 ± 0,54
±
XII
* broj janjadi ojanjene po pojedinom mjesecu manji je od broja janjenja – iz statističke obrade porodne mase
izuzeta je ona janjad kod koje nije zabilježena porodna masa / number of lambs born in every month is smaller
than number of lambing - lambs without noted birth mass are excluded from statistical analysis
5,0
4,5
4,0
3,5
3,0
2,5
2,0
1,5
0,5
0,0
May-2007
Jun-2007
Apr-2007
Mar-2007
Feb-2007
Dec-2006
Jan-2007
Nov-2006
Sep-2006
V ertic al bars denote 0,95 c onf idenc e interv als
Oct-2006
M je se c - g o d in a / M o n th - ye a r
Jun-2006
May-2006
Mar-2006
Apr-2006
Feb-2006
Dec-2005
Jan-2006
Nov-2005
Oct-2005
May-2005
Jun-2005
Mar-2005
Apr-2005
Feb-2005
Dec-2004
Jan-2005
Nov-2004
Oct-2004
Lamb birth weight (Kg)
Porodna masa janjadi (kg) /
1,0
Graph 2 - Kinetics of average lamb birth weight depending on month of lambing (during period from 20.
October 2004. till 9. July 2007.)
Graf 2 – Kretanje prosječne porodne mase zavisno o mjesecu janjenja (u razdoblju od 20. 10. 2004. do
9.6.2007.)
524
Tijekom promatranih godina prva janjenja na početku sezone bila su zabilježena krajem listopada,
izuzev u 2006. godini krajem rujna. Pri tome početkom i završetkom sezone janjenja smatrali smo
mjesece u kojima je učestalost janjenja bila veća od pet. Iz grafa 1 i tablice 3 uočljivo je da se najveći
broj ovaca (80% i više) ojanjio tijekom prosinca i siječnja.
Prosječna porodna masa janjadi iznosila je 3,28± 0,58 uz statistički značajne (p<0,01) varijacije
ovisne o spolu (muška janjad 3, 32 ± 0,58 i ženska janjad 3,25 ± 0,57). Općenito, janjad ojanjena
tijekom rane zime imala je veću porodnu masu u usporedbi s janjadi ojanjenom tijekom kasne zime
ili proljeća.
Utvrđeni rezultati koristiti će se za karakterizaciju reprodukcijskih obilježja Paške ovce.
Literatura
1.
Ekert Kabalin, Anamaria, I. Štoković (2007): Očuvanje hrvatskih izvornih pasmina. Zbornik 2. kongresa
studenata veterinarske medicine, 13.-16. lipanj 2007, Zagreb.
2.
FAO (2007): The State of the World’s Animal Genetic Resources for Food and Agriculture, edited by
Barbara Rischkowsky & Dafydd Pilling. Rome.
3.
HSC – HRVATSKI STOČARSKI CENTAR (2007): Godišnje izvješće za 2006. godinu, Zagreb.
4.
MZOPU (Ministarstvo zaštite okoliša, prostornog uređenja i graditeljstva) (2007): Internet stranica (http://
www.mzopu.hr/doc/06Poljoprivreda.pdf).
5.
Pavić, Vesna, B. Mioč, Z. Barać, I. Vnučec, V. Sušić, N. Antunac, Dubravka Samardžija (2005): Vanjština
paške ovce. Stočarstvo 59 (2): 83-90.
6.
Sušić, V. (2006): Utjecaj sezone pripusta na plodnost ovaca. Ovčarsko kozarski list 3, 14 - 16.
7.
Sušić, V., A. Ekert Kabalin, I. Štoković, I. Karadjole, T. Balenović (2006): Hrvatske izvorne pasmine
domaćih životinja. IV. simpozij poljoprivrede, veterinarstva, šumarstva i biotehnologije. Zenica, Bosna i
Hercegovina. (pozvano predavanje).
8.
Sušić, V., A. Ekert Kabalin, I. Karadjole, T. Balenović, I. Štoković, Igor (2006): Paška ovca. IV. simpozij
poljoprivrede, veterinarstva, šumarstva i biotehnologije. Zenica, Bosna i Hercegovina. (poster).
9.
Sušić, V., V. Pavić, B. Mioč, I. Štoković, A. Ekert Kabalin (2005): Seasonal variations in lamb birth weight
and mortality. Veterinarski arhiv 75 (5), 375-381.
10. Sušić, V., T. Balenović, I. Martinić, D. Katica, I. Štoković, A. Ekert Kabalin (2001): Hrvatske autohtone
pasmine domaćih životinja. Zbornik - Proceedings “Veterinarski dani 2001.”, Zagreb, 177-186.
11. Sušić, V., K. Mikulec, I. Karadjole, A. Tomašković, D. Križanović, I. Štoković (2000): Effect of milk yield
on seasonal oestrual activity in sheep. Veterinarski arhiv 70 (Suppl.), S231-S237.
525
526
PREVALENCE AND AETHIOLOGY OF COW MASTITIS IN SMALL
DAIRY HOLDINGS IN CROATIA
PREVALENCIJA I ETIOLOGIJA MASTITISA KRAVA U MALIM
OBITELJSKIM GOSPODARSTVIMA
Benić, M1.; Ž. Cvetnić1, M. Lojkić1, B. Habrun1; G. Kompes1; M. Cergolj2, N. Maćešić2
1Hrvatski veterinarski institut, Savska c. 143, 10 000 Zagreb
2
Veterinarski fakultet, Klinika za porodništvo i reprodukciju, Heinzelova 55
SUMMARY
In spite of improved milking practices and widely applicated preventive measures against intramammary
infections, mastitis is still one of the most commonly observed problems in dairy practice. The aim of the
research was to obtain the data concerning the spread of intramammary infections at herd level at point of time
and to compare it with similar investigations in order to improve preventive measures against new intramammary
infections. Therefore during a cross-sectional field investigation 304 cows from 47 holdings were examined.
Average number of lactating cows per holding at the time of visitation was six (from three to 12). Samples were
taken from individual quarters, tested by California mastitis test and microbiologicaly examined in laboratory.
Microbiological examination was performed on esculin blood agar. Isolated bacteria were subcultivated on
differential and selective media. Ststistical analysis was performed using Stata 6.0 statistical package (STATA
Corp.). At least one CMT positive quarter was found in 174 (57%) of cows. Percent of CMT positive quarters
varied from 0 to 60% per holding while 26% of all examined quarters expressed elevated somatic cells.
Percent of CMT positive quarters was not associated with herd size (p›0.05). However rear quarters were more
frequently affected by elevated cell content than front quarters (p‹0,001). Mastitis pathogens were isolated from
264 quarter sample (23.2%). Contagious pathogens dominated among isolated bacteria, especially S. aureus
which was isolated from 138 (11.3%) of all examined quarter samples. Streptococcus agalactiae was isolated
from 21 (1.7%) samples. Environmental bacteria were isolated from 105 (8.6%) of examined quarter samples.
Agreement between CMT and microbiological examination was moderate (Kappa = 0.6).Since the contagious
pathogens dominated among isolated bacteria, results suggest that improvement in udder health status can be
made by proper hygienic treatment of udder at milking time. Zero percent prevalence of elevated SCC and
negative microbiological findings indicate that small dairy holdings are able to maintain low level prevalence
of intramammary infections.
SAŽETAK
Usprkos napretku u tehnologiji mužnje i široko primjenjivanim preventivnim mjerama za sprečavanje
intramamrnih infekcija, mastitis još uvijek predstavlja jedan od najčešćih problema s kojim se susreće primarna
proizvodnja mlijeka. Cilj ovog istraživanja bio je prikupiti podatke o proširenosti intramamarnih infekcija u
malim stadima muznih krava i usporediti ih s rezultatima sličnih istraživanja kako bi se unaprijedile preventivne
mjere protiv novih infekcija mliječne žlijezde.Provedeno terensko istraživanje presjeka populacije obuhvatilo je
304 muzne krave kod 47 vlasnika. Prosječni broj krava u laktaciji u stadu kretao se od tri do 12 (prosječno šest).
Uzorci su uzeti iz svih četvrti krava u laktaciji, testirani mastitis testom i mikrobiološki pretraženi u laboratoriju.
Mikrobiološka pretraga provedena je na krvnom agaru s dodatkom eskulina, a izolirane bakterije subkultivirane
su na selektivnim i diferencijalnim podlogama. Statistička obrada provedena je korištenjem statističkog
programa Stata 6.0 (STATA Corp.).Barem jedna četvrt s povišenim brojem somatskih stanica utvrđena je u 174
krave (57%). Postotak četvrti s povećanim brojem somatskih stanica u stadima se kretao u rasponu od 0 do
60%, odnosno 26% svih pretraženih četvrti imalo je u manjem ili većem stupnju izraženu pozitivnu reakciju.
Prevalencija mastitis test pozitivnih četvrti nije statistički povezana s veličinom stada (p›0.05). Međutim, stražnje
527
četvrti češće su zahvaćene povećanjem broja somatskih stanica nego prednje, a razlika je statistički značajna
(p<0.001). Uzročnici mastitisa izdvojeni su iz 264 uzorka pojedinačnih četvrti vimena (23.2%). U zastupljenosti
prednjačili su kontagiozni uzročnici, pogotovo Staphylococcus aureus koji je izdvojen iz 138 uzoraka (11.3%).
Streptococcus agalactiae izdvojen je iz 21 (1.7%) uzorka. Uvjetno patogeni uzročnici iz okoliša izdvojeni su
iz 105 (8.6%) uzoraka pojedinačnih četvrti. odudarnost rezultata mastitis testa i mikrobiološke pretrage bila je
umjerena (Kappa =0.6).Budući da među izdvojenim uzročnicima prevladavaju kontagiozni uzročnici, rezultati
nagovješćuju da je smanjenje učestalosti infekcija mliječne žlijezde moguće poboljšanjem higijene mužnje.
Činjenica da u nekim stadima nije bilo ni citološki ni mikrobiološki pozitivnih četvrti upućuje na zaključak da
je mala stada moguće držati u stanju slobodnom od mastitisa.
UVOD
Usprkos brojnim profilaktičkim mjerama u suzbijanju mastitisa, infekcije vimena i dalje su jedan
od glavnih uzroka izravnih i neizravnih šteta u mliječnom govedarstvu. U nastanku i održavanju
infekcija vimena ulogu igraju životinja, okoliš i mikrorganizmi kao neposredni uzročnici infekcija.
Činjenica da se promjene u bilo kojem od ova tri segmenta mogu odraziti na pojavnost mastitisa
razlog je potrebi za opsežnim i redovitim praćenjem sastavnica spomenutih elemenata, od nutritivnog
i fiziološkog stanja same životinje, higijene muzača i muzačke opreme do provjere učestalosti i
zastupljenosti pojedinih uzročnika mastitisa na farmi i njihovoj osjetljivosti prema antibioticima.
Proizvodnja mlijeka u Hrvatskoj doživjela je nekoliko značajnih novosti u posljednjih desetak
godina. Te promjene uvelike su oblikovale i još uvijek utječu na pravac razvoja mliječnog govedarstva.
Treba spomenuti stupanje na snagu Pravilnika o kakvoći svježeg sirovog mlijeka koji je obvezao
cjelokupnu mljekarsku industriju na uvođenje i poštivanje higijenskih standarda kroz formiranje
otkupne cijene mlijeka. Pored ovog i drugi zakonski akti uvjetuju preobrazbu mliječnog govedarstva
od malih držatelja prema tržišno orijentiranim proizvođačima mlijeka s većim brojem krava. Usprkos
tome još uvijek veliki udio proizvodnje mlijeka otpada na držatelje s manjim brojem krava.
Neizravne dijagnostičke metode za otkrivanje infekcija vimena temelje se na brojenju
somatskih stanica, mjerenju električne provodljivosti mlijeka ili aktivnosti nekih enzima u sekretu
mliječne žlijezde. Određivanje broja somatskih stanica obavlja se pomoću uređaja za brojenje ili
polukvantitativno mastitis testom. Pouzdana dijagnoza mastitisa postavlja se mikrobiološkom
pretragom uzoraka sekreta sumnjivih četvrti.
MATERIJAL I METODE
U istraživanje je bilo uključeno 47 proizvođača mlijeka s područja Karlovačke i Zagrebačke
županije. Prema dizajnu istraživanje je cross-sectional tipa pri čemu su ukupno pretražene 304 muzne
krave u laktaciji u jednokratnom obilasku. Uzorci su uzeti prije redovite mužnje na sljedeći način:
prvih nekoliko mlazova mlijeka izmuzli smo u posebnu posudu. U pliticu za testiranje namuzli smo
oko 2 ml mlijeka iz svake četvrti. U pliticu smo potom dodali oko 2 ml mastitis reagensa. Kružnim
pokretima izmiješali smo smjesu mlijeka i reagensa i prosudili reakciju. Nakon toga vrhovi sisa
temeljito su izbrisani vatom namočenom u 70%-tni alkohol. S jednim do dva mlaza iz svake četvrti
napunili smo prethodno označene sterilne epruvete. Uzorci su pohranjeni na led i transportirani u
laboratorij. U laboratoriju su do početka pretrage čuvani na +4 ºC, a pretraga je započeta najkasnije
12 sati nakon uzimanja uzoraka. Uzorci su zagrijani na sobnu temperaturu i nacijepljeni na podlogu
eskulin-krvni agar. Nacijepljene podloge inkubirane pri 37 ºC tijekom 18-24 sata. Porasle kolonije
precijepljene su na diferencijalne i selektivne podloge: Baird-Parker agar, podlogu s trostrukim
528
šećerom (TSI), TKT agar, MacConkey agar radi pouzdanije identifikacije. Izdvojeni stafilokoki
podvrgnuti su koagulaza testu.
Dobivene podatke statistički smo obradili u programu Stata 6. 0 (Stata Corp., USA).
REZULTATI
Opis populacije. Broj krava u laktaciji u trenutku posjete iznosio je prosješno šest (od tri do 12).
Pasminski sastav čine uglavnom simentalac i crnošaro govedo. Životinje se drže na vezu u objektima
zatvorenog tipa s ili bez ispusta. Mužnja se obavlja u staji.
Tablica 1. Razdioba populacije i udio četvrti s povećanim brojem somatskih stanica
Broj krava
Broj držatelja
Ukupno krava
3
4
5
6
7
8
9
10
11
12
Ukupno
3
6
10
5
11
5
2
1
2
2
47
9
24
50
30
77
40
18
10
22
24
304
Mikrobiološki nalaz
Negativan
S. aureus
Streptococcus agalactiae
Streptococcus dysgalactiae
Ostali streptokoki i enterokoki
E. coli
Pseudomonas spp
Kvasci
Bakteriostatik
Atrofija četvrti
UKUPNO
CMT pozitivnih četvrti (%)
Prosjek Prednje stražnje
11.1
29.2
27.0
17.5
23.7
33.8
22.2
45
45.5
22.9
26
Tablica 2. Rezultati mikrobioloških pretraga
Broj četvrti
933
138
21
8
69
11
7
10
7
12
1216
0
11.5
12.5
6.7
8.8
14.4
8.3
20
22.7
7.3
10.5
11.1
17.7
14.5
10.8
14.9
19.4
13.9
25
22.7
15.6
15.5
%
76.7
11.3
1.7
0.7
5.7
0.9
0.6
0.8
0.6
1
100
Tablica 3. Podudarnost mastitis testa s rezultatima mikrobiološke pretrage
Rezultat mikrobiološke pretrage
Pozitivan
Negativan
UKUPNO
Mastitis test
Pozitivan
206
58
264
Negativan
123
810
933
UKUPNO
329
868
1197*
*atrofične četvrti i uzorci s antibioticima nisu uzeti u obzir; Kappa = 0.6
529
RASPRAVA
Proizvodnju mlijeka u Republici Hrvatskoj u zadnjih petnaestak godina zahvatilo je niz promjena
koje su imale za posljedicu nestanak velikih farmi u državnom vlasništvu, smanjenje broja malih
držatelja krava, ali u isto vrijeme i pojavu novih farmi s većim brojem muznih krava u privatnom
vlasništvu. Osim toga tijekom procesa prilagodbe smjernicama Europske unije donesen je veći
broj pravnih akata koji izravno ili neizravno reguliraju proizvodnju i stavljanje u promet mlijeka
i mliječnih proizvoda. Svakako treba spomenuti Pravilnik o kakvoći svježeg sirovog mlijeka (NN
102/00) koji omogućuje razvrstavanje mlijeka u razrede kakvoće uzimajući u obzir osim kemijskog
sastava i broj somatskih stanica i broj mikroorganizama. Dok je broj mikroorganizama često
posljedica sekundarne kontaminacije mlijeka, broj somatskih stanica najčešće je odraz zdravstvenog
stanja mliječne žlijezde.
Udio četvrti s povećanim brojem somatskih stanica u odnosu na veličinu stada prikazan je u
tablici 1. Premda postotak četvrti zahvaćenih povećanjem broja somatskih stanica četvrti varira (1145%) razlike nisu statistički značajne (Kruskall-Wallis p= 0.3). U usporedbi s ranijim istraživanjima
postotak mastitis test pozitivnih četvrti je smanjen što je očekivano s obzirom na stupanje na snagu
Pravilnika o kakvoći svježeg sirovog mlijeka.
Stražnje četvrti u većem su postotku reagirale pozitivno mastitis testom nego prednje, vjerojatno
zbog veće izloženosti podražajima. Uočena razlika statistički je značajna (p<0.001).
Iz tablice 2 razvidno je da među uzročnicima mastitisa krava u malim uzgojima dominiraju
kontagiozni uzročnici, poglavito S.aureus, slično kao u drugim istraživanjima u svijetu. U usporedbi
s ranijim istraživanjima u Hrvatskoj, učestalost kontagioznih uzročnika je smanjena, ali je povećana
učestalost uvjetno patogenih uzročnika iz okoliša. No s obzirom na smanjen udio četvrti pozitivnih
mastitis testom, čini se da se inficirane četvrti ranije otkrivaju i liječe.
Vrijednost mastitis testa kao dijagnostičke metode u otkrivanju upala mliječne žlijezde dokazana
je u brojnim radovima. Budući da je razina detekcije somatskih stanica niža od vršnih vrijednosti
broja somatskih stanica propisanih Pravilnikom o kakvoći svježeg sirovog mlijeka preporučljivo je
izvoditi ga u redovitim vremenskim intervalima i za svaku muznu životinju voditi evidenciju. Na taj
način moguće je uočiti pojavu infekcije prije nego li ista ugrozi kakvoću proizvedenog mlijeka.
LITERATURA
Topolko, S., M. Benić (1997): Aktualni problemi i epizootiološko stanje subkliničkih mastitisa u minifarmskoj
proizvodnji mlijeka. Praxis veterinaria, 45 (1-2), 69-77
Pravilnik o kakvoći svježeg sirovog mlijeka (Narodne novine: 102/2000)
Radostits, O.M., D.C. Blood, C.C. Gay: Veterinary Medicine, Eight edition, London, Bailliere Tindal, 1994
Schalm, O.W., E.J. Carrol, N.C. Jain (1971): Bovine mastitis. Lea & Febiger, Philadelphia
National Mastitis Council: Laboratory Handbook on Bovine Mastitis. National Mastitis Council, Inc. 2820
Madison. USA, 1999
530
CASE REPORT OF ANAPLASMOSIS ON A SHEEP FARM IN THE
NEBRODI MOUNTAIN RANGE (SICILY – ITALY - EUROPE)
Di Marco Vincenzo, Russo Miriam, Aronica Vincenzo, Fiasconaro Michele, Scimeca
Salvatore, Alongi Angelina, Torina Alessandra
Istituto Zooprofilattico Sperimentale della Sicilia
Species of the genus Anaplasma (Rickettsiales: Anaplasmataceae), are obligate intracellular
aetiological agents of tick-borne diseases of mammalian hosts (Dumler et al., 2001) and includes
the causative agents of anaplasmosis of ruminants. The disease in sheep and goats is generally
symptom free and the etiological agent in most cases is Anaplasma ovis. This genus also includes A.
phagocytophilum (previously recognized as E. equi, E. phagocytophila and the human granulocytic
ehrlichiosis (HGE) agent), which infects a wide range of hosts including humans and wild and
domesticated animals, and causes human, canine and equine granulocytic anaplasmosis and tick
fever of ruminants (TBF) (Dumler et al., 2001). A.ovis is transmitted by Rhipicephalus bursa and
most likely other ticks (Friedhoff, 1997). Mammalian or tick hosts with persistent infection serve
as reservoirs of the pathogen in nature (Kocan et al., 2004). Often clinical findings are absent or
scarce. Although anaplasmosis is more frequently associated with haemolytic anaemia in goats, A.
ovis can also cause disease in sheep, particularly in animals exposed to stress or other predisposing
factors (Splitter et al,1956 and Friedhoff, 1997). TBF is rarely fatal, unless there are complications
from other infections. In November 2006, on a sheep and goat farm (300 goats and 220 sheep) in
the province of Messina (Sicily – Italy), 20 sheep deaths were reported. A further 19 animals were
found to be weak, with oedema under the lower jaw and top of the chest, reduced sensory perception,
anorexia, fever and extensive cutaneous tick infestation. The pathological findings were liver damage,
a slightly increased and “soft” spleen (which contained serous and haemorrhagic effusions). There
was evidence of multiple parasitism: tapeworm, roundworm, Lancet liver fluke and Echinococcosis.
All the animals in the flock were poorly nourished. The purpose of this document is to describe this
epidemic focus of sheep anaplasmosis.
Materials and methods
Samples were taken from the livestock farm as follows: whole blood and serum samples were
taken from 155 sheep and 46 goats and 71 ticks were collected. The blood was collected in test tubes
without anticoagulant and in test tubes with EDTA kept at a temperature of 4° C until they reached
the laboratory. The ticks were kept in 70% ethanol at room temperature. Tests were carried out on
the samples according to the following protocols:1) Identification of the ticks according to Manilla’s
morphological keys; 2) Whole blood: DNA extraction and subsequent specific PCR; full blood count
with the Heco-vet SE device. The 95% confidence intervals for preponderance were calculated using
the WinEpiscope 2.0 programme; 3) Serum samples: serologic tests: (cELISA, VMRD, Pullman, WA
99163). An antibody examination for A. phagocytophilum was carried out by slide for IFI (Fuller
Laboratories, Fullerton, CA, USA ) and FITC-conjugated Anti goat IgG (Sigma, St. Louis, MO,
USA) and FITC-conjugated Anti Sheep IgG (Sigma, St. Louis, MO, USA).
531
Results
The blood tests on all sheep samples showed an average level of GR of 6.19 (DS=2.4), GB of
12.97(DS=4.0) haemoglobin 7.9(DS=2.44), hematocrit 21.3(DS=8.4). On the other hand, in the 19
animals with symptoms the average GR was 4.18(ds=1.71), the GB 12.37(ds=2.25),the haemoglobin
4.91(ds=1.86), hematocrit 14.80(ds=5.57). The prevalence of Anaplasma in sheep serum was 98%
(CI 95%= 96.80-99.20%), whilst for Anaplasma phagocytophilum the prevalence was 16.7% (CI
95%=13.51-19.89). In goats the prevalence of Anaplasma was 87% (CI 95%= 78.06-95.94), whilst
for Anaplasma phagocytophilum the prevalence was 78.2%(CI95%=67.22-89.18) On the same
livestock farm, the prevalence of a positive PCR test for Anaplasma sp was for sheep 38%(CI
95%= 33.85-42.15), for Anaplasma ovis it was 20%(CI 95%=16.58-23.42), and for Anaplasma
phagocytophilum 12.9%CI 95% = 10.03-15.77). For the goats on the other hand we found the
following PCRs prevalences: Anaplasma sp. 58.6%(CI 95%= 45.5-71.7), Anaplasma ovis 52.1%(CI
95%=38.82-65.38), Anaplasma phagocytophilum. =0%. 17 ticks belonged to Riphicephalus spp, 32
to Hemaphisalis spp and 22 to Dermacentor spp.
Conclusions
Analysis of the observed haematological parameters, serum prevalence and PCR positivity
confirm the clinical diagnosis of an anaplasmosis focus. The blood tests confirm that all the animals
tested had severe anaemia and it was more severe in the animals showing symptoms when samples
were taken. The interpretation of diagnostic tests leads us to think that the range of disease symptoms
in this livestock farm supports the theory of Anaplasma sp. infection. The high preponderance of
serum abnormality indicates that sheep anaplasmosis is endemic on the livestock farm and rife
infection is supported by the high positivity found with the PCR. Unlike the situation in cattle
infected with A.marginale (who stay lifetime carriers of the infection in a latent form, with fresh
clinical outbreaks whenever the animal is stressed or sick), there is not similar latent state for A. ovis
or for A. phagocytophilum. Finding parasites in the blood of sheep and goats, therefore, indicates
active infection. The severe manifestation of disease in these animals may have been caused by
multiple parasitism and by their generally poor state of health. All the ticks examined belong to the
species which, according to numerous bibliographical references, are considered to be Anaplasma sp.
vectors. Last but not least, it is interesting to look at the differences between sheep and goats. Goats
have always been thought to be more resistant to infection than sheep – this is not only linked to
their genetic make-up, but also due to the fact that goats are rarely treated for parasites and therefore
they undergo natural selection. During the epidemic the goats examined, although they were less in
number than the sheep, gave an interesting outcome. More than 50% were PCR positive for A. ovis
and this result would lead us to believe that the goats are asymptomatic reservoirs for the pathogen
in the area.
532
THEILERIA EQUI IN OVINE: A CASES REPORT
Caracappa Santo, Torina Alessandra, Nicosia Silviane, D’Agostino Rosalia, Russo
Miriam, Alongi Angelina, Di Marco Vincenzo
Istituto Zooprofilattico Sperimentale della Sicilia, via G. Marinuzzi 3, 90129 Palermo.
Abstract
Sicilian kind of breeding, especially for small ruminant is characterized by mixed animal species. Ticks
can bite in their life cycle different host that can became asinthomatic reservoir of the TBD pathogens. In
the present work we describe the accidental discovery of Theileria spp different from to most common ovine
Theileria (B.ovis, T.hirci, T.motasi) in sheep and we intend to demonstrate that piroplasmid infections may
reveal unexpected (but probably not infrequent) in unusual hosts.
Introduction
Piroplasms are tick-transmitted parasitic protozoa divided into the genera Theileria and Babesia.
They are the causative agents of theileriosis and babesiosis, respectively. Piroplasms of both genera
are highly pathogenic for domestic animals. The agents of tick borne disease in ovine are B. ovis, B.
motasi and Theileria hirci. Previous works from other authors (Habela et al, 1990) pointing out that
B. ovis is the most frequent piroplasmid present in sheep and goats in Mediterranean regions. B. ovis
is transmitted by Rhipicephalus bursa, R. turanicus, Hyalomma anatolicum excavatum (Friedhoff
et al, 1997). The acute form has characteristic clinical signs, as jaundice and haemoglobinuria; subacute or chronic form can only be diagnosed by demonstration of the parasite in the blood (smear,
PCR, RLB). Indigenous sheep in enzootic areas usually acquire a mild infection and are immune
thereafter. Sicilian kind of breeding, especially for small ruminant is characterised by mixed animal
species. Ticks can bite in their life cycle different host that can became asinthomatic reservoir of the
TBD pathogens. Molecular diagnosis methods has led to the discovery of some new piroplasmids in
the lapse of just a few years (H. americanum, described by Vincent-Johnson et al., 1997; Theileria
annae by Zahler et al., 2000a).
In the present work we describe the accidental discovery of Theileria spp different from to most
common ovine Theileria (B.ovis, B.motasi, T.hirci,) in sheep and we intend to demonstrate that
piroplasmid infections may reveal unexpected (but probably not infrequent) in unusual hosts.
Sampled were collected in Sicilian sheep breeding in Messina province. DNA extracted (N=100)
positive by Reverse Line Blot hybridization for probe Theileria/Babesia catch all, Theileria sp1 china,
Theileria sp2 china and negative for T. buffely, T. annulata, T. velifera, T. taurotragi, T. mutans, T.
lestoquardi, T. ovis, B. bovis, B. bigemina, B. crassa, B. motasi, B. ovis, B. major, B. divergens, T. hirci,
B.sp1 (Turchey),B.sp2 (Lintan). (Gubbels et al. 1999) were tested by T. equi specific PCR (Badgar
Battsetseg et al, 2002). For sequencing purposes, amplified products were separated in agarose gels
and visualized with ethidium bromide. Bands of interest were isolated with the Wizard SV Gel and
PCR Clean-up purification System (Promega Corporation, Madison, WI, USA). Fragments obtained
by PCR Theileria equi were sequenced using primers EMA5 (5’-TCGACTTCCAGTTGGAGTCC3’) and EMA6 (5’-AGCTCGACCCACTTATCACC -3’) for identification of isolates through the
533
sequence of merozoite antigen (published by Kakuda T, Sugimoto C and Onuma M). Sequencing
was carried out by Macrogen.Inc automated sequencer.
Results
Thirty sheep were infected with T.equi as demonstrated by PCR and two samples were select
for sequencing to ascertain if the infection was present in the samples. The obtained sequences were
compared with those in GeneBank using Blast software and then analyzed in silico using Bioedit
v.7.0.9.0. The results showed a high degree of identity (99.5%) with the Merozoite antigen of T. equi
isolate D-31 (GeneBank accession number AB015220).
Discussions
The degree of identity between the sequences obtained and the GeneBank data shows that there
is a high probability that in the ovine samples tested was present the T. equi DNA.
Our study would show the existence, among the sheep population tested, of a piroplasm not
common for small ruminants. T.equi has never been described before as a parasite of sheep. Animals
positive for T.equi PCR analyses were clinically asymptomatic, which might be indicative of a nopathogenicity in that host. Theileria equi is a common protozoan disease of horses in Sicily and
infected horses become carriers of T. equi and potential disseminators of the parasite. Trans-ovarian
transmission in ticks for Theileria spp has been never described in previous reports, only trans-stadial
transmission can occur. Due to this reason the interspecies transmission between equine and ovine
is possible throughout the bite of the same tick in its different biological stages; ticks that acquire T.
equi from infected horses could successfully transmit the parasite to naive sheep. In fact there are in
Sicily many farms where cohabit different animal species, ovine, equine, bovine, dogs; we suppose
that domestic animal species may play a role as asymptomatic reservoir for Tick Borne pathogens.
References
Badgar Battsetseg, Susana Lucero, Xuenan Xuan, Florencia G. Claveria, Noboru Inoue, Andy Alhassan,
Tsutomo Kanno, Ikuo Igarashi, Hideyuki Nagasawa, Takeshi Mikami, Kosa Fujisaki, 2002. Detection
of natural infection of Boophilus microplus with Babesia equi and Babesia caballi in Brazilian horses using
nested polymerase chain reaction. Veterinary Parasitology 107: 351-357.
Friedhoff KT, 1997. Tick-borne diseases of sheep and goats caused by Babesia, Theileria or Anaplasma spp.
Parassitologia; 39(2):99-109.
J.M. Gubbels, A.P.de Vos, M.van der Weide, J. Viseras, L.M.Schouls, E.de Vries, F.Jongejan, 1999.
Simultaneos detection of bovine Theileria and Babesia species by Reverse Line Blot Hybridization. Journal
of Clinical Microbiology, 1782-1789.
Habela M, Reina D, Nieto C, Navarrete I. 1990Isolation and identification of Babesia ovis in Extremadura
(Spain). Vet Parasitol; 35 (3): 233-8.
Vincent-Johnson, N.A., Macintire, D.K., Lindsay, D.S., Lenz, S.D., Baneth, G., Shkap, V. and Blagburn,
B.L., 1997. A new Hepatozoon species from dogs: description of the causative agent of canine hepatozoonosis
in North America. J. Parasitol. 83, pp. 1165–1172.
Zahler, M., Rinder, H., Schein, E. and Gothe, R., 2000. Detection of a new pathogenic Babesia microti-like
species in dogs. Vet. Parasitol. 89, pp. 241–248.
534
ANIMAL IDENTIFICATION: CONDITIONS OF THE INDIVIDUAL
IDENTIFICATION OF BOVINE ANIMALES IN ASTURIAS (SPAIN)
Barriada Alvarez M.1, Martínez López L. M.2
1
2
Dirección General de Ganadería y Agroalimentación. Consejería de Medio Ambiente y Desarrollo Rural.
Principado de Asturias. Asturias. Spain
Dirección General de Informática. Consejería de Administsraciones Públicas y Portavoz del Gobierno. Principado de Asturias. Asturias. Spain
SUMMARY
This paper aims to present some data on the status of the identification of cattle in Asturias, a small
autonomous community placed in the north of Spain. The Identification of cattle is based on several elements
including the ear tags, that every bovine animals should keep throughout its life, with the same identification
code. Another element is simogan, the database containing the identification and movement of bovine animals
and that, on the one hand, must be able to control the information that is provided in order to maintain their
reliability, and, on the other hand, has to provide data that allow ensure the traceability of production, such as
information relating to the ear tags replacement in the animals.In this paper we have checked the data pertaining
to the replacement of the ear tags in the animals of our holdings, as one of the parameters to assess the quality of
information stored in terms of traceability. We have controlled cattle censuses, births, and requests for duplicate
ear tags, in the period of time between the years 2004 2007. The results show that while from 2004 to 2007 in
the region there has been a decline of close to 14%, of the census and the number of births of bovine animals,
the number of duplicates of ear tags that were requested has experienced a sharp increase, about 78%. This
significant, and not expected, increase in the number of requested duplicates of ear tags, leads us to analyze
data in search of some factor that allows explain it, and if possible, seek solutions. So we have analysed the
relationship with the type of holding, age of the animals, and the provenance of ear tags.
RESUMEN
Este trabajo pretende presentar algunos datos sobre la situación de la identificación del ganado bovino
en Asturias, una pequeña Comunidad Autónoma situada al norte de España. La identificación del bovino está
basada en varios elementos entre los que se encuentran las marcas auriculares o crotales, que todas las reses han
de mantener puestas a lo largo de toda su vida y con el mismo código de identificación. Otro de los elementos
es simogan, la base de datos que recoge la identificación y los movimientos de los bovinos y que, por una
parte, ha de ser capaz de controlar la información que se le facilita a fin de mantener su fiabilidad, y que, por
otra parte, ha de suministrar datos que permitan garantizar la trazabilidad de la producción, como es el caso
de la información referida a las recrotalizaciones de los animales. En este trabajo se han revisado los datos
referidos a las recrotalizaciones de los animales de nuestras explotaciones, como uno de los parámetros para
valorar la calidad de la información almacenada, en cuanto a la trazabilidad. Para ello se han controlado los
censos de ganado bovino, los nacimientos, y las solicitudes de duplicados de marcas auriculares, en el período
de tiempo comprendido entre los años 2004 a 2007. Los resultados obtenidos muestran que si bien desde el año
2004 a 2007 en la región se ha producido una disminución, próxima al 14%, del censo y del nº de nacimientos
de bovinos, el nº de duplicados de crotales solicitados experimentó un fuerte incremento, del 78,37%. Este
considerable, y no esperado, incremento del número de duplicados solicitados, nos lleva a analizar los datos en
busca de algún factor que permita explicarlo y, si es posible, buscar soluciones. Así, se buscó la relación con el
tipo de explotación, la edad de los animales, y la procedencia de los crotales.
535
INTRODUCCCIÓN
Los consumidores valoran cada vez más la calidad y la seguridad de los alimentos que
consumen.
En el campo de la producción animal, y de modo especial en la producción de bovino, este hecho
unido a las diferentes crisis alimentarias y de sanidad animal que hemos presenciado en los últimos
años, han llevado a una revisión de la normativa relacionada con el control de los alimentos. Así, la
nueva normativa que se elaboró, nació bajo la máxima de buscar la seguridad alimentaria desde la
granja hasta la mesa.
En este contexto, la identificación de los animales constituye uno de los puntos de partida para
que sea viable mantener la trazabilidad de la producción, a fin de asegurar la calidad alimentaria.
Particularizando para el ganado bovino, su identificación está basada en varios elementos entre
los que se encuentran las marcas auriculares que se les colocan a los animales después de nacer y
que han de mantener durante toda su vida, y las bases de datos, que gestionadas por las autoridades
competentes, recogen esa información referida a la identificación de las reses.
Con este sistema de identificación del bovino mediante marcas auriculares, las sustituciones de
dichas marcas, en casos de pérdida, es uno de los puntos que se han de controlar de manera efectiva,
para poder cumplir los requisitos relativos a garantizar la trazabilidad de los alimentos.
OBJETIVO
Este trabajo presenta datos referidos a la identificación del ganado bovino en Asturias, una
pequeña Comunidad Autónoma situada al norte de España.
El fin es valorar la información que existe, en nuestra base de datos de identificación y registro
de ganado bovino simogan, sobre las sustituciones que se hacen de las marcas auriculares cuando se
pierden. Las nuevas marcas han de ser duplicados de las originales, manteniendo el mismo código
de identificación.
Por una parte, se pretende estimar qué nivel de reposición de marcas auriculares tenemos
anualmente (nº de crotales duplicados solicitados un año respecto al censo de ganado bovino ese
año). Y por otra parte, se valora la evolución de ese nivel de reposición a lo largo de varios años y su
relación con factores como la edad de los animales y el tipo de animal de producción.
DATOS
Se analizan los datos de simogan, relativos a número de bovinos, número de nacimientos y
número de crotales solicitados duplicados para esos bovinos, localizados en las explotaciones
asturianas durante los años 2004, 2005, 2006 y 2007.
En el cálculo de la cantidad de crotales duplicados solicitados, además de considerar el nº total
de peticiones, se diferenciaron las solicitudes en que se pedía un único ejemplar de la marca auricular
(duplicados simples) de aquellas en las que se pedían los dos ejemplares de la marca (duplicados
dobles), por las repercusiones que este hecho tiene en el control de esos animales.
También se realizan cálculos para determinar la distribución del número de duplicados solicitados
según
la edad de los animales, considerando 4 grupos: animales de 1 año de edad o menos, animales
con edad comprendida entre 1 y 3 años, animales con edad comprendida entre 3 y 6 años, y animales
con edad superior a 6 años.
536
el tipo de animal de producción, considerando a este efecto la raza del animal y obteniéndose
datos para las cuatro razas que representan aproximadamente el 99 % del censo: frisona, asturiana de
los valles (AV), asturiana de la montaña (AM) y animales mestizos.
RESULTADOS
En la tabla 1 se presentan los datos referidos al número de bovinos localizados en las explotaciones
asturianas, número de nacimientos y número de duplicados solicitados.
TABLA 1. Número de reses, nacimientos y crotales duplicados solicitados para ganado bovino en las explotaciones asturianas, correspondientes a los años 2004 a 2007
Censo bovino
Nº Nacimientos
Nº duplicados simples (DS)
Nº duplicados dobles (DD)
Ratio DS / Censo bovino
Ratio DD / Censo bovino
Ratio Nº total D / Censo bovino
2004
452.556
215.545
25.216
2.264
0,055
0,005
0,060
2005
424.392
203.438
30.684
2.342
0,072
0,005
0,078
2006
386.913
192.546
35.876
2.190
0,093
0,006
0,098
2007
386.954
186.405
44.979
2.145
0,116
0,005
0,121
Se observa que, desde el año 2004, si bien ha ido disminuyendo el censo de ganado bovino
en nuestras explotaciones, llegando a ser en el año 2007 un 14% menor que en el 2004, ha ido
aumentado el número de las solicitudes de crotales duplicados simples, llegando a ser en el año 2007
un 78,73 % mayor que en 2004. No se ha observado una tendencia clara en el caso de las solicitudes
de duplicados dobles, con incrementos y descensos que oscilaron del 3 al 8 %.
Así con estos datos, se ve que el porcentaje de reposición de las marcas auriculares ha ido en
aumento todos los años, pasando de valores de 6,07 % en el año 2004 a 12,18 % en el 2007 (Gráfico
1), lo que supuso un aumento del 71,48 % en el nº total de crotales solicitados.
Ratio nº de
duplicados/ censo
bovino
GRAFICO 1. Evolución desde el año 2004 a 2007 en la reposición de
las marcas auriculares del bovino en la comunidad de Asturias
0,14
0,12
0,1
0,08
0,06
0,04
0,02
0
2004
2005
2006
Años
2007
Ratio DS /
Censo bovino
Ratio DD /
Censo bovino
Ratio N □total D
/Censo bovino
En la tabla 2 se presentan los datos del número de crotales duplicados que se solicitaron en esos
años, teniendo en cuenta la edad de los animales.
537
TABLA 2. Crotales duplicados solicitados, durante los años 2004 a 2007, teniendo en cuenta la edad.
menos de 1 año
de 1 a 3 años
de 3 a 6 años
más de 6
2004
Simples
Dobles
4.307
610
4.526
284
9.886
791
6.497
579
2005
Simples
Dobles
4.050
441
4.456
226
9.771
610
2006
Simples
Dobles
4.823
353
4.093
133
10.228
480
2007
Simples
Dobles
5.934
333
5.159
158
10.667
381
12.407
16.732
23.219
1.065
1.224
1.282
Se puede ver que para el grupo de animales con edad superior a 6 años se ha producido un
incremento continuado e importante del número de solicitudes registradas. Este aumento se detecta
tanto para las peticiones de duplicados simples como de duplicados dobles. Al comparar los datos
de los años 2005, 2006 y 2007, respecto a los de 2004, las peticiones aumentaron más del doble,
llegando a ser 3,5 veces superiores las peticiones de duplicados simples, en el año 2007 (Gráfico 2).
Nª de crotales
duplicados
GRAFICO 2. Evolucion desde el año 2004 a 2007 del número de
solicitudes de crotales duplicados simples en función de la edad de
los animales
25000
20000
15000
10000
5000
0
2004
2005
2006
2007
menos de 1a□o
de 1a 3 a□os
de 3 a 6 a□os
m疽de 6 a□os
Edad de los animale s
Cuando analizamos la información del número de crotales duplicados pedidos, en función de la
raza, el resultado constata que el incremento continuado, desde el año 2004 al 2007, en la cantidad
de solicitudes registradas para los animales de más de 6 años de edad, se produce para todas las razas
(Gráfico 3).
GRAFICO 3. Evolución desde el año 2004 al 2007 del nº de solicitudes de
duplicados simples en función de la raza para animales mayores de 6 años de edad
10000
8000
6000
4000
2000
0
2004
2005
2006
MESTIZO
AM
FRISONA
AV
2007
R aza
538
Al revisar los datos que tenemos de los otros 3 grupos de edad, no se aprecia en ninguna de las
razas, variaciones interanuales importantes en la cantidad de duplicados pedidos. Paralelamente,
durante los cuatro años, se ve que el rango de edad para el que se registra el mayor y el menor
número de solicitudes no varía dentro de cada una de las razas, de modo que parece que pueda existir
una referencia para determinar en qué edades puede ser más necesario, en cada raza, el control de la
reposición de marcas auriculares (Gráfico 4).
GRAFICO 4. Evolución desde el año 2004 al 2007 del numero de solicitudes de crotales
duplicados en función de la raza y la edad
10000
9000
8000
7000
6000
5000 N □C R OT A LES
4000
3000
2000
1000
0
2004
2005
2006
2007
ED A D
Así, en los animales de conjunto mestizo ese mayor nº de solicitudes se centra en animales
de menos de 1 año y de más de 6 años de edad; en los bovinos de raza asturiana de la montaña se
realizan más peticiones de duplicados para los animales de más de 6 años; en la raza frisona destaca
el bajo nº de solicitudes para animales de menos de 1 año y, en comparación con las otras razas, un
alto número para los animales de 1 a 3 y de 3 a 6 años; finalmente en la raza asturiana de valles la
mayor cantidad de solicitudes corresponden a los animales de los grupos de menos de 1 y de más de
6 años de edad.
DISCUSIÓN
De acuerdo con los resultados expuestos, en el año 2004 la reposición de las marcas auriculares
en la cabaña bovina de nuestra comunidad fue del 6%, cifra que coincide con los porcentajes de
pérdidas anuales que se citan en la bibliografía (Ghirardi et al., 2004; Caja et. al, 2005) para este tipo
de crotales convencionales de poliuretano.
En cuanto a los porcentajes de reposición que se obtienen para los siguientes años, resultan muy
superiores a esa cifra del 6%. Aquí se ha de tener en cuenta que entre los resultados de este trabajo
se señala un incremento muy importante del número de crotales duplicados solicitados al aumentar
la edad de los animales, especialmente para animales de más de 6 años de edad, y por tanto cabe
suponer que se trata de animales que portan esas marcas de identificación durante un período de
tiempo superior a los 7 años que Ghirardi et al., citan que duro la experiencia de su trabajo.
539
Si a su vez consideramos que esa relación entre el incremento en el nº de solicitudes de crotales
duplicados y la mayor edad de los animales, se mantiene independientemente de la raza de bovino
que consideremos, y por extensión del sistema de producción, nos lleva a pensar en que el factor que
está influyendo es la duración o vida media de la marca auricular.
En relación con la duración y resistencia de los crotales únicamente se han encontrado
referencias de los fabricantes a la duración del marcado de los caracteres que llevan inscritos, citando
10 años como mínimo, y a la resistencia a que las dos piezas que componen el crotal sean separadas,
soportando fuerzas de más de 40 kg.
Respecto a los rangos de edad en los que el número de solicitudes de duplicados es mayor para
cada raza, se podrían relacionar, con un mayor censo de animales de esas edades y a la vez con los
sistemas de producción en que se les mantiene:
ü orientaciones productivas de carne, en sistemas semiextensivos e intensivos, para los bovinos
de raza asturiana de valles y conjunto mestizo (con reproductoras y cebo de reses jóvenes), y
en sistemas más extensivos, para los bovinos de raza asturiana de montaña (reproductoras y
producción de carne de buey)
ü y orientaciones productivas de leche para los bovinos de raza frisona, con reproductoras
jóvenes.
Como comentario final, señalar que no se pudo disponer del censo de animales de cada año
desglosado por raza y edad para poder hacer una valoración objetiva de esa relación censo- sistema
de producción - nº de solicitudes.
AGRADECIMIENTOS
Al personal de la empresa “Atos Origin” y del Servicio de Producción y Bienestar Animal
(Consejería de Medio Ambiente y Desarrollo Rural) por su colaboración.
REFERENCIAS
Ghirardi J.J., Caja G., Conill C., Hernández-Jover M. & Garín D. 2004. J. Anim. Sci.
82 (Supl. 1):351 (Abstr.).
C. Saa, M.J. Milán, G. Caja, J.J. Ghirardi, O. San Miguel, A. Ruiz y M.J. Lueso. 2005. ITEA Prod. Animal,
(volumen extra) 26: 318-320
Información técnica de fabricantes de crotales.
540

Posters Posteri - Università di Bologna

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