THE INDIAN JOURNAL OF ANIMAL SCIENCES Previous Issue : Vol. 80, No.2, pp. 89–188

Vol. 80, No.3 March 2010

CONTENTS

ANIMAL HEALTH Molecular typing of field isolate of Salmonella by using random amplified polymorphic DNA (RAPD) 191 Anand Kumar and Mumtesh Kumar Saxena Occurrence of gastrointestinal parasites in domestic yaks in Sikkim 195 H Rahman, P Pal and S Bandyopadhyay Evaluation of methanolic extract of Allium sativum and Saussurea costus in yaks with infectious 199 keratoconjunctivitis Samiran Bandyopadhyay, T K Biswas, D Sasmal, I Samanta and M K Ghosh Ultrasound guided biopsy and fine needle aspiration biopsy of splenic and prostatic affections in dogs 203 S K Mahajan, S S Singh, J Mohindroo, N S Saini, N Singh and N K Sood Effect of granulosa cells monolayer and oviductal epithelial cells co-culture on cleavage rate and 209 embryo development of in-vitro fertilized goat oocytes K P Singh, Atul Saxena, S D Kharche and Praneeta Singh Changes in endometrial histology following enrofloxacin therapy in crossbred cows suffering from endometritis 213 J K Prasad, M S Saxena, S K Rastogi and J P Korde Serum lipid profile during lactation in buffalo 217 P M Tripathi, S D Ingole, B T Deshmukh, A S Nagvekar and S V Bharucha Clinico-haematobiochemical profile in chronic anaemic crossbred cattle 220 R K Bhardwaj, C S Randhawa, S S Randhawa And P S Dhaliwal

Short Communications Isolation and molecular characterization of avian pathogenic Escherichia coli and Pseudomonas 225 aeruginosa from mortality in ducks in Kashmir, S D Qureshi, S A Wani, I Hussain, M T Banday, I A Mir, S Farooq and M A Bhat Estimations of blood plasma metabolites following melatonin implants treatment for initiation 229 of ovarian cyclicity in true anestrus buffalo heifers Jagir Singh, S P S Ghuman, D Dadarwal, M Honparkhe, G S Dhaliwal and A K Jain Factors influencing gestation length of Thoroughbred mares in India 232 Sumeet Sharma and G S Dhaliwal

ANIMAL PRODUCTION MHC-DRB exon-2 (BuLA-DRB3) polymorphism in Banni breed of Indian buffalo 234 Jyotsna Dhingra Behl, Rahul Behl and N K Verma Role of additive and multiplicative age correction factors in sire evaluation of Hariana cattle 239 D S Dalal and A S Khana Prediction of lactation milk yield from test day records in Murrah buffaloes 244 D Chakraborth, S S Dhaka, B L Pander, A S Yadav, S Singh and P K Malik Genetic analysis of lactation traits in Jamunapari goats 246 R Roy and Ajoy Mandal Effect of naturally fermented rice straw based diet on the performance of buffalo calves 249 M Wadhwa, K Kaur and M P S Bakshi Effect of feeding raw or water soaked rapeseed cake on carcass characteristics and meat quality in kids 253 M Palanivel, K Sharma, Narayan Dutta and S K Mendiratta Micro-minerals status in goats of different age in semi-arid region of India 258 Neeru Bhooshan, Puneet Kumar and M C Yadav Effect of concentrate levels on the production performance of Angora rabbits 262 R S Bhatt, Davendra Kumar, R B Shrama and K S Risam Short Communications Estimation of genetic parameters in Sahiwal cattle using single and multi-trait restricted maximum 266 likelihood method S Banik and R S Gandhi Sire evaluation using single and multiple trait animal models in Sahiwal cattle 269 S Banik and R S Gandhi Effect of seasonal variation on primary physiological responses of yak 271 G Krishnan, K P Ramesha, G Kandeepan, V S Chouhan and S Jayakumar Price : Rs 125.00 per copy 636 (3) CODEN : IJLAA4 80 (3) : 189–272 (2010) II ISSN 0367–8318 MARCH 2010 The Indian Journal of Animal Sciences

INDIAN COUNCIL OF AGRICULTURAL RESEARCH NEW

3 Indian J Anim Sci Vol. 80 No. 3 pp. 189–272 March 2010 VOL.80, NO. 3 VOL.80, NO. THE INDIAN JOURNAL OF ANIMAL SCIENCES VOL.80, NO. 3 NO. VOL.80, THE INDIAN JOURNAL OF ANIMAL SCIENCES MARCH 2010 R.N. 14357/57 Postal Regd. No. DL(C)–12/1146/10–12 Price : Rs 125.00 per copy

Highlights of Forthcoming Issue (April 2010)

• Molecular detection of Staphylococcus aureus mastitis in crossbred cows based on genus specific gap gene and species specific aroA gene PCR assay • Uterine torsion in bovines: a review • Insulin-like growth factor-I and -II in buffalo ovary: mRNA expressions and partial sequences • Effect of Bt cotton plants on oxidative stress in sheep • Effect of age and reproductive state on phosphatase enzymes and steroid hormones profile in Indian goats • Biochemical and enzymatic changes in downer cow syndrome • Therapeutic and residual efficacy analysis of some anti-tick compounds against natural Boophilus microplus infestation in crossbred cattle • Inventorization of Gaushala resources and their use in breed improvement and conservation programmes • Environmental and genetic effects on growth traits of Chokla sheep • On-farm evaluation of urea molasses multi-nutrient blocks enriched with minerals in goats • Carcass and meat quality characteristics of designated indigenous sheep breeds of India • Identification of single nucleotide variations in the genes related to reproduction in riverine Buffalo (Bubalus bubalis) • Influence of genetic and non genetic factors on growth profile of Bharat Merino sheep in semi arid region of • Housing and feeding managemental practices for goat followed in South Gujarat • Activity of extracts of Synzium aromaticum against microbes of veterinary importance

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• All disputes are subject to the exclusive jurisdiction of competent courts and forums in Delhi/New Delhi only • The Council does not assume any responsibility for opinions offered by the authors in the articles and no material in any form can be reproduced without permission of the Council • The Council is not responsible for any delay, whatsoever, in publication/delivery of the periodicals to the subscribers due to unforeseen circumstances or postal delay • Readers are recommended to make appropriate enquiries before sending money, incurring expenses or entering into commitments in relation to any advertisement appearing in this publication. The Council does not vouch for any claims made by the advertisers of products and services. The publisher and the editor of the publication shall not be held liable for any consequences in the event of such claims not being honoured by the advertisers. Indian Journal of Animal Sciences 80 (3): 191–94, March 2010 Molecular typing of field isolate of Salmonella by using random amplified polymorphic DNA (RAPD)

ANAND KUMAR1 and MUMTESH KUMAR SAXENA2

Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, Uttarakhand 263 145 India

Received: 7 May 2007; Accepted: 18 September 2009

ABSTRACT In present study molecular typing of poultry isolates was carried out by RAPD-PCR by 5 reported primers. All used primers for 28 isolates were generated 359 loci in total. None of these loci showed common banding pattern. All 28 isolates were differentiated in 28 different molecular types. Our present study reveals that RAPD-PCR can be used for differentiation of field isolates of Salmonella. RAPD may be proving as efficient tool for molecular epidemiological studies of Salmonella.

Key words: Molecular typing, RAPD, Salmonella

Salmonella has long been recognized as important MATERIALS AND METHODS zoonotic pathogen of global significance. As per OIE report The 28 isolates belonging to 4 serovars were obtained (2004) there are 2541 seroypes of Salmonellae and many of from Animal Biotechnology Centre of the University. The these serotypes are well documented human pathogens genomic DNA of 28 isolates was isolated by C-TAB method (Kwang et al. 1996) but only 2 serovars Salmonella (Wilson 1987). Five primers were used for RAPD-PCR with Enteritidis and Salmonella Typhimurium, account for about conditions according to the given references. All reagents half of all human infections. Interest in Salmonella has used in PCR were procured commercially. heightened in recent years due to the devastating effect of PCR reaction conditions for Primer R1 and R2: The PCR Salmonella in poultry industry and the globalization of trade. mixture of 25μl contained 200μM οφ each dNTPs, 40 pmol Hence, a more rapid method of identification and primer, 10 × PCR buffer, and 20ng DNA template with differentiation of an organism (Salmonella) is the pre- MgCl2 concentration optimized at 2.5 mM and U/μl of Taq requisite for eradication of disease condition. Differentiations DNA polymerase. It was carried out in a thermal cycler of Salmonella isolates are equally important in order to programmed for 30 cycles composed of one step of prevent vaccination failure, indiscriminate use of antibiotic denaturation for 1 min at 94°C, one step of annealing for 1 and molecular epidemiological studies. Though several min at 36°C followed by one step of synthesis for 2 min at conventional and molecular biological methods have been used for strain differentiation of Salmonella (Threllfall et al. 1994, Bianca et al. 2003 and Franandez et al. 2003) but Primer Sequence Reference randonm amplified polymorphic DNA (RAPD) is found to R1 OPE19- 5’-ACG GCG TAT G-3’ Welington et al. (2004) be the most convenient and rapid method (Welington et al. R2 OPX09- 5’-GGTY CTG Welington et al. (2004) 2004). Tarai region of India is a prosperous region in terms GTT G-3’ of poultry but Salmonella is a major problem in this area. In R3 784- 5’-GCG GAA TGA G-3’ Bianca et al. (1990) this study efficiency of RAPD was evaluated to differentiate R4 797- 5’-AGC GTC ACT C-3’ Bianca et al. (1990) poultry isolates of Salmonella isolated from tarai region of R5 P1254 -5’-CCG CAG CCA A-3’ Hilton et al. (1997) India. 72°C. Each of sample amplified thrice to avoid possibility of artifacts. Present address: 1Research Scholar (e mail: PCR reaction conditions for primer R3: The PCR mixture [email protected]), 2Assistant Professor, Animal of 25μl contained 200μM of each dNTPs, 40 pmol primer, Biotechnology Centre, Department of Veterinary Biochemistry, 10 × PCR buffer, and 20ng DNA template with MgCl2 College of Veterinary and Animal Sciences. concentration optimized at 2.5 mM and 3 U/μl of Taq DNA 3 192 KUMAR AND SAXENA [Indian Journal of Animal Sciences 80 (3) polymerase. It was carried out in a thermal cycler scorable band profile with primer R1. The isolate ML42 (S. programmed for 40 cycles composed of initial denaturation Heidelberg) was the one that has not shown any band profile at 94°C for 5 min followed by one step of denaturation for 1 (Fig. 1). The size of amplicon produced by this primer ranges min at 94°C, one step of annealing for 1 min at 40°C followed from 201 base pairs to 2055 base pairs. The maximum size by one step of synthesis for 1 min at 72°C and a final of the band was produced by isolate ML30 (S. Heidelberg) extension step at 72°C for 5 min. Each sample was amplified and minimum size of band was produced by ML7, ML8, thrice to avoid possibility of artifacts. PCR reaction conditions for primer R4 and R5: The PCR Table 1. Description of isolates of Salmonella mixture of 25μl contained 200μM of each dNTPs, 40 pmol S no. Isolate no. Serovars primer, 10 × PCR buffer, and 20ng DNA template with MgCl2 concentration optimized at 3.5 mM and 3 U/μl of Taq DNA 1ML1 S. Galiema polymerase. It was carried out in a thermal cycler 2ML2 S. Galiema programmed for 40 cycles composed of one-step of 3ML4 S. Typhimurium denaturation for 1 min at 94°C, one step of annealing for 1 4ML5 S. Typhimurium min at 36°C followed by one step of synthesis for 1 min at 5ML6 S. Typhimurium 72°C. Each sample was amplified thrice to avoid possibility 6ML7 S. Heidelberg of artifacts. 7ML8 S. Heidelberg PCR amplified product (10 μ litre) was mixed with 2μl 8ML9 S. Virchow 9ML10 S. Virchow of 6 × loading dye and loaded on 1.6% agarose gel with 10 ML S. Typhimurium 1%TBe buffer. The samples were run at 5 volt/cm of gel for 11 11 ML12 S. Virchow 3 h. The gels were visualized by UV tran illuminator and 12 ML13 S. Galiema photographed by gel documentation system. Molecular 13 ML14 S. Ttyphimurium weight of each band was calculated by comparing with 14 ML15 S. Heidelberg standard molecular weight marker 100 bp ladder plus. 15 ML19 S. Heidelberg Combined molecular typing and dendogram construction: 16 ML21 S. Typhimurium Bands produced from all 5 primers were arranged in 17 ML25 S. Virchow decreasing order. If band of respective size was present isolate 18 ML26 S. Typhimurium 19 ML27 S. Galiema was allotted “1” if band was not present isolate was allotted 20 ML S. Typhimurium “0”. 01 profile was used in Treecon software for construction 29 21 ML30 S Heidelberg of phylogenetic tree. 22 ML31 S. Virchow 23 ML S. Virchow RESULTS AND DISCUSSION 32 24 ML33 S. Virchow Primers (5) were used for Salmonella serovar 25 ML34 S. Heidelberg differentiation by RAPD method. All 5 were able to produce 26 ML35 S. Virchow scorable amplicon. 27 ML37 S. Typhimurium Out of 28 isolates, 27 isolates were able to produce 28 ML42 S. Heidelberg

Fig 1. RAPD profiles of Salmonella isolate of poultry using primer R1. M- Marker (100 bp leader), 1-ML1, 2- ML2, 3- ML4, 4- ML5, 5- ML6, ML7, 7- ML8, 8- ML9, 9-ML10, 10- ML11, 11- ML12, 12- ML13, 13- ML14, 14- ML15, 15- ML19, 16- ML21, 17- ML25, 18- ML26, 19- ML27, 20- ML29, 21- ML30, 22- ML31, 23- ML32, 24- ML33, 25- ML34, 26- ML35, 27- ML37, 28- ML42, M- Marker (100 bp leader). 4 March 2010] MOLECULAR TYPING OF FIELD ISOLATES OF SALMONELLA 193

Fig 2. RAPD profiles of Salmonella isolate of poultry using primer R2. M- Marker (100 bp leader), 1-ML1, 2- ML2, 3- ML4, 4- ML5, 5- ML6, ML7, 7- ML8, 8- ML9, 9-ML10, 10- ML11, 11- ML12, 12- ML13, 13- ML14, 14- ML15, 15- ML19, 16- ML21, 17- ML25, 18- ML26, 19- ML27, 20- ML29, 21- ML30, 22- ML31, 23- ML32, 24- ML33, 25- ML34, 26- ML35, 27- ML37, 28- ML42, M- Marker (100 bp leader).

Fig 3. RAPD profiles of Salmonella isolate of poultry using primer R3. M- Marker (100 bp leader), 1-ML1, 2- ML2, 3- ML4, 4- ML5, 5- ML6, ML7, 7- ML8, 8- ML9, 9-ML10, 10- ML11, 11- ML12, 12- ML13, 13- ML14, 14- ML15, 15- ML19, 16- ML21, 17- ML25, 18- ML26, 19- ML27, 20- ML29, 21- ML30, 22- ML31, 23- ML32, 24- ML33, 25- ML34, 26- ML35, 27- ML37, 28- ML42, M- Marker (100 bp leader). and ML15 isolates (S. Heidelberg) (Table 1). Though isolates i.e. ML26 and ML29 (S. Typhimurium) and ML31 amplicon were obtained in 27 isolates but no serovar or genus (S. Virchow) have not given any band profile. The size of specific band could be observed. The isolate ML14 (S. amplicon ranged from 273 base pairs to 1772 base pairs. Typhimurium) produced most distinguishable profile as it Further this primer failed to show any genus specific and produced 2 bands of size 289 and 201 base pairs. serovar specific scorable profile. Rare profiles were observed All 28 isolates were able to produce scorable band profile in isolates ML14 and ML25, where each isolate was able to with primer R2. The size of amplicon ranges from 249 base produce a single band (452 and 917 bp respectively). This pairs to 1870 base pairs (Fig. 2). Although this primer failed could be an isolate specific locus, the prime R3 also showed to produce any serovar specific scorable band profile yet it good discriminating power by producing 25 different scorable was having great discriminating ability as it produced 28 profiles. With prime R4 and R5 amplicons were produced in different scorable band profiles but the common profile was 28 isolates. Both of the primers were having high not observed between different serovars. Common bands of discriminative value but none of the primer could produce a 835 and 971 base pairs were observed in 12 isolates and 11 serovar or genus specific profiles. Common band was shared isolates, respectively, and these bands were present in all 4 in between the serovars. serovars, which indicate that common loci have shared by 4 Dendrogram (Fig. 4) was constructed using Treecon different serovars. software. In dendrogram five major clusters were identified The primer R3 was tested for 28 isolates of which 25 but strains were not clustered on the basis of serovar as isolates produced observable band profile (Fig. 3) where 3 common clusted was shared between the 2 different serovars. 5 194 KUMAR AND SAXENA [Indian Journal of Animal Sciences 80 (3)

Isolate-19: et al. (2004). In our study all 24 isolates were differentiated Isolate-15: by RAPD based combined molecular typing. On the basis of Isolate-10: our study and findings of earlier workers we can conclude Isolate-21: that RAPD is an efficient tool for molecular typing of Isolate-4: Salmonella. Isolate-11: Isolate-14: REFERENCES Isolate-26: Isolate-25: Bianca R, Quintaes C, Leal M F, Reis I, Fonseca and Ernesto H. Isolate-13: 2002. Conventional and molecular typing of Salmonella Isolate-34: Typhi strains from Brazil. Rev. Inst. Med. trop. S. Paulo 44 Isolate-25: (6): 1–11. Isolate-35: Fernandez J, Fernandez J, Fica A, Ebensperger G, Calfullan H, Prat S, Fernandez A, Alexandre M, Heitmann I. 2003. Analysis of Isolate-29: molecular epidemiology of Salmonella enterica serotype Isolate-30: Enteritidis isolates by pulse field gel electrophoresis and Isolate-31: bacteriophage typing. Journal of Clinical Microbiology 24: 56– Isolate-33: 68. Isolate-32: Hilton A C, Banks J G and Penn C W. 1997. Optimization of RAPD Isolate-42: for fingerprinting Salmonella. Letters in Applied Microbiology Isolate-37: 24: 243–48. Isolate-6: Kwang J, Littledike E T and Keen J E. 1996. Use of the polymerase Isolate-5: chain reaction for Salmonella detection. Letters in Applied Isolate-8: Microbiology 22: 46–51. Isolate-2: Mather K O D, Morris J G and Gotuzzo E. 1996. Molecular Isolate-12: technique in the study of Salmonella Typhi in epidemiological Isolate-9: studies in endemic area: comparison with vi phage typing. Isolate-7: American Journal of Medical Hygiene 35: 831–35. Isolate-1: Threlfall E J, Torren E and Ward L R. 1994. Insertion of sequence IS200 Fingerprinting of Salmonella Typhi an assessment by Fig 4. Dendrogram constructed by combined molecular typing. pulse field electrophoresis. Epidemiology and Infections 112: 253–61. The isolate No-7 was the most distantly located in Welington Luiz Araujo, Derlene Attili de Angellis and Joao Lucio dendrogram, hence having maximum genetic variation. Azevedo. 2004. Direct RAPD evaluation of without RAPD has been used for differentiation of Salmonella field conventional DNA extraction. Braz. Arch. Boil. Technol 47 (3): isolates. Welsh and Mccllean (1990) used this technique first 1–8. Welsh J and Mcclellan M. 1990. Fingerprinting of genome using time for differentiation of Salmonella and from that day PCR with arbitrary primers. Nucleic Acid Research 28: 2755– onwards this technique was frequently used for strain 61. differentiation. Hilton et al. (1997) found it more useful than Wilson K. 1987. Preparation of genomic DNA from bacteria. phage typing in differentiation of Salmonella Enteritidis. Current Protocols in Molecular Biology. Unit 2. 4. 4. Wiley, Direct RAPD for strain differentiation was used by Welington New York.

6 Indian Journal of Animal Sciences 80 (3): 195–98, March 2010 Occurrence of gastrointestinal parasites in domestic yaks in Sikkim

H RAHMAN1, P PAL2 and S BANDYOPADHYAY3

ICAR Research Complex for NEH Region, Sikkim Centre, Gangtok, Sikkim 737102 India

Received: 22 May 2009; Accepted: 26 October 2009

ABSTRACT Occurrence of gastrointestinal parasites in domesticated yaks of North and East districts of Sikkim was studied during the year 2001 to 2008. Of the 4,792 animals examined, 991 (20.68%) were found positive for different gastrointestinal parasites. The overall occurrence of different parasites recorded was strongyles (17.36%), Strongyloides spp. (4.34%), coccidia (4.55%), Toxocara spp. (2.25%), Trichuris spp. (2.88%), amphistomes (0.52%) and Moniezia spp. (3.07%). The infestation was more in Sub-Alpine low humid area (29.34%) as compared to Alpine dry zone ((19.0%) and Cold Desert zone (10.05%). The faecal egg counts (eggs per gram, epg) of nematodes ranged from 100 to 2 900, with higher loads during rainy and post-rainy seasons. The infection was higher in calf (1–5 months) and the parasites mostly recorded were Haemonchus spp., Toxocara spp. and Eimeria spp. The seasonal distribution of parasitism indicated a higher percentage of infestation in autumn (26.02%) and summer (21.57%) as compared with spring (20.65%) and winter (13.73%). On coproculture of positive samples, the nematode infestations in order of prevalence were Haemonchus, Bunostomum, Oesophagostomum and Nematodirus spp.

Key words: Gastrointestinal parasites, Prevalence, Sikkim, Yaks

Yak (Poephagus grunniens), native of Tibet, is reared Census 2003) and they are reared in the Northeastern ranges (3000–6700 masl) in West Kameng and Tawang districts of bordering Tibet and Bhutan and the Western region bordering Arunachal Pradesh; West, North and East districts of Sikkim; Nepal. Three kinds of yaks are described in Gazettier of Lahul, Spiti and Kinaur districts of Himachal Pradesh and Sikkim, Lho-guag, larged size yak is found in the Western Ladakh of Jammu and Kashmir (Pal and Barari 1994) and is Sikkim; Bho-gyag (smaller in size than Lho-guag); and the known for its ability to withstand low temperature, third kind called A-gu are polled yaks seen in high altitude surefootedness and capability to thrive on coarse fodder at areas of North and Eastern ranges of Sikkim (Katiyar and higher altitude where no other ruminant can survive (Katiyar Sinha 1982). and Sinha 1982). The yak is the only species, which produces Yaks are maintained mainly on grazing. They graze during milk, meat, hair fibre and provides transport (camel of snow) the day time and sit down at intervals for rumination. for trade and agricultural production, serves as financial asset Migratory system of grazing is in practice. In winter, yaks and security for investment and family ceremonies. This are taken to a lower altitude grazing land (<3000 masl), which prized animal is considered to be an efficient converter of is shared by other domesticated animals. Probably due to forest biomass into valued beef (Rahman et al. 2007). Yak is this reason, yaks become exposed to many of the diseases exceptionally a hardy animal and can withstand severe winter particularly those affecting cattle. The occurrence of many with snowy environment. Yak farming has been recognized of the common diseases of cattle has been reported in yaks as an important source of livelihood for various pastoral tribes (Lensch 1996). However, the information in yaks with of Gaddis in Himachal Pradesh, Gujjars and Backarheals in particular reference to health is very scarce. There are 2 Jammu and Kashmir (Jithendran and Bhat 1996) and Rai, limiting factors for this, firstly, the remoteness and Lepchas and Limbus in Sikkim of Eastern most Himalayan inaccessibility of yak tracts and secondly, the migratory region of Indian Subcontinent. system of yak husbandry. Due to these limiting factors, In Sikkim, yak population is around 6 521 (Livestock mortality and morbidity data on yak diseases could not be recorded and the light of the modern health management practices has not reached to most of the yaks herdsmen. Present address: 1Joint Director, 2Research Associate, 3Senior Gastrointestinal parasitism in livestock is common and Scientist, Veterinary Parasitology, IVRI Eastern Regional Station, economic losses due to parasites are enormous, mainly due 37 Belghachia Road, Kolkata, West Bengal 700 033 India. to morbidity and mortality. The present study was undertaken 7 196 RAHMAN ET AL. [Indian Journal of Animal Sciences 80 (3) to ascertain the occurrence of gastrointestinal (GI) parasitic Tabe 1. Influence of altitude on the occurrence of gastrointestinal infestation in yaks in Sikkim as monitored by faecal egg parasites in yaks counts. Agroclimatic zone No. of animals No. of animals MATERIALS AND METHODS (Altitude) examined found positive (%)

Sikkim covers a total land area of 7,096 sq. km. between Sub-Alpine low humid zone 1077 316 (29.34) 27°46 – 28°7N and 88° – 88.5°E and has an altitude range of (2 200–4 285 m amsl) 300–7500 m amsl. Alpine dry zone 3367 640 (19.0) The investigation was carried out between April 2001 and (4 285 – 6 667 m amsl) March 2008 on randomly selected yaks of different ages and Dry High cold Desert zone 348 35 (10.05) both sexes in East and North Hill districts of Sikkim. (7 6667 m amsl) Total 4792 991 (20.68) The study was conducted in 11 villages including Gurudongmar (7 238 m asl) at North Sikkim (latitude 28°5 N; longitude 28°4 E; altitude 3 200–6 670 masl) and low rate infestation with gastrointestinal parasites in yaks Gnathang (5 258 masl) at East Sikkim where temperature recorded in the cold desert area might be due to uncondusive varies from subzero to minus 20°C during winter and spring agroclimatic conditions prevailing in these selected areas of and then rises slowly from June to November (summer and study, which does not favour the propagation of the parasitic autumn). The rainfall normally begins in mid April and ova leading to unavailability of infective larvae on the grazing extends up to October with heaviest spell from June to ground. The overall occurrence of different parasites recorded August. In the Alpine pasture snowfall usually occurs is presented in Table 2. The most commonly occurring between mid October to early April with heavy spell from parasites in the region were Strongyle (17.36%) followed December to March. by Eimeria spp (4.55%), Strongyloides spp (4.34%), Faecal samples from 4 792 animals were collected Moniezia (3.07%), Trichuris spp. (2.88%), Amphistomes randomly from 11 villages and brought to the laboratory and (0.52%) and Dicrocoelium spp (0.25%). Among strongyles, stored at 4° C until analysed. Faecal samples were subjected Haemonchus spp. was most prevalent parasite than other to qualitative and quantitative examination for nemaodes, Toxocara spp (2.88%). No relationship could be gastrointestinal parasites during 7 years from 2001 to 2008. observed between the presence of a particular species and The animals were of various ages and belonged to individual the time of year or sex of the host. The higher rate of farmers or Private/Government farm. Most of the farmers infestation could be possibly due to conducive agroclimatic sell their animals during the local festival called Lossong, so conditions, constant exposure of infestation and availability the exact age, lactation and other history of the most of the of infective stage larvae on the grazing ground by animals. subjects were not known. The faecal egg counts (eggs per The present findings are in agreement with those reported gram of faeces, epg) of nematode eggs was determined by by Katiyar et al. (1981), Ansari et al. (1989), Ranga Rao et Modified McMaster technique (MAFF 1984), while fluke al. (1994) and found 3 species of trematodes and cestodes (amphistome and Dicrocoelium) egg counting was done as respectively, and 10 species of nematodes and reported per Soulsby (1982). Representative number of faecal samples infection in 75% yaks with predominant infection of were pooled in equal quantities and used for coproculture at Haemonchus contortus and other strongyles and 20°C. The infective larvae were harvested and used for larval amphistomes from Sikkim. The comparatively less identification. Table 2. Overall prevalence of gastrointestinal parasites recorded RESULTS AND DISCUSSION in yaks in Sikkim (N= 4,792) The present study revealed that out of 4 792 animals tested from 3 agroclimatic zones of Sikkim, 991 (20.68%) were Parasite Infected animals (%)* positive for various intestinal parasites either singly or in Nematodes Strongyles 832 (17.36) mixed infestation (Table 1). The occurrence of Strongyloides spp. 208 (4.34) gastrointestinal parasites was higher (29.34%) among Toxocara spp. 108 (2.25) animals reared at a lower altitude Sub - Alpine low humid Trichuris spp. 138 (2.88) zone (3 000 m amsl) than those reared at higher altitude Cestodes Moniezia spp. 147 (3.07) Alpine dry humid zone (19%, 4 286 m masl) and lowest Flukes Amphistomes 25 (0.52) incidence (10.05%) were noticed in Dry high cold desert Dicrocoelium spp. 12 (0.25) zone in North District of Sikkim (above 6 667 m amsl). Protozoa Eimeria spp. 218 (4.55) Total 991(20.68) Statistical analysis indicated a significant difference (P<0.05) in the occurrence of intestinal parasitism among animals • Total numbers and overall percentage of an animals exceed reared at different agroclimatic zones (Table 1). An overall expected values because of multiple gastrointestinal parasitism. 8 March 2010] OCCURRENCE OF GASTROINTESTINAL PARASITES IN YAKS 197

Table 3. Occurrence of gastrointestinal parasites in yaks – Table 5. Influence of age on the occurrence of gastrointestinal season-wise parasitism in yaks

Season Animals Animals Animal group Animals Animal examined infected (%) (age wise) examined infected (%)

Spring (March – May) 736 152 (20.65) Group A (1–5 months) 804 256 (31.84) Summer (June – August) 1386 299 (21.57) Group B (6 months –2 years) 1 506 286 (19.0) Autumn (September- November) 1410 367 (26.02) Winter (December- February) 1260 173 (13.73) Group C (above 2 years) 2 482 449 (18.09) Total 4792 991 (20.68) Total 4 792 991 (20.68) prevalence of amphistome and Dicrocoelium spp. ova in the in strongyle epg could be attributed to a more favourable present study was probably due to less availability of the temperature and humidity for the development and survival intermediate host. Haemonchus spp. emerged as the most of the preparasitic stages leading to increased availability of prevalent species in the region with Bunostomum, infective larvae on the pasture during the subsequent months. Oesophagostomum and Nematodirus also being common in The study also indicated that, under normal conditions, the yaks. animals harboured a worm burden without any clinical signs, The observations on monthly seasonal fluctuation of but the worm burden reached a threshold pathogenic level in prevalence (per cent positive samples) and mean monthly the monsoon and postmonsoon seasons (Jithendran and Bhatt faecal egg counts are summerised in Tables 3 and 4. Out of 1999). The egg count data revealed that September to October 991 positive samples subjected to faecal egg counts for were the months with the highest risk of GI parasites and gastrointestinal parasites, the count ranged from 100–2 900 pasture contaminations. epg with higher prevalence (28.09%) and the overall mean The influence of age on the prevalence of GI parasites in epg (160.56) were recorded during rainy and post rainy yaks revealed that the infestation was higher (31.84%) in season. The occurrence of helminths specially strongyles was yaks at 1–5 months of age followed by in animals of 6 months high throughout the period of study with a peak during to 2 years of age (19.0%) and in those of above 2 years of September to November. With the onset of winter season age (18.09%, Table 5). from December onwards, the mean total egg count fell to a The coprocultural studies showed that yaks harboured low level, steadily falling to a minimum of 42.08 mean epg some highly pathogenic nematode species of which strongyle in January after that it rose slowly with the increase in was maximum (Table 6). The most predominant nematode temperature and rainfall. The prevalence of gastrointestinal species were Haemonchus (37.5%), Bunostomum (14.06%), parasites did not show a marked seasonal variation in the Oesophagostomum (12.5%) and Nematodirus species present study. However, the occurrence of parasites was (7.81%). The present findings are in agreement with those minimum (13.73%) during winter (December-February) and of Katiyar et al.(1981) and Ansari et al. (1989). higher levels were recorded during autumn (26.02%) The data generated in this study indicated that under followed by summer (21.57%) and spring (20.65%). The rise prevailing agroclimatic conditions of Sikkim, India, the occurrence of gastrointestinal parasites is slightly high among Table 4. Average monthly prevalence and EPG of gastrointestinal yaks. However, the EPG estimated in the present study parasites in yaks indicated the level of subclinical infestations in the host Month No No. infected Mean Range of examined (%) epg mode Table 6. Incidence of gastrointestinal nematode infection through the faecal larval culture in yaks (N= 64) January 382 55 (14.39) 42.08 100–500 February 257 56 (9.98) 45.416 100–700 Species identified Per cent larval March 87 9 (12.31) 52.214 100–300 composition (%) April 324 52 (16.04) 58.993 100–400 May 325 91 (28.01) 88.324 100–700 Haemonchus spp.* 24 (37.5) June 516 86 (16.66) 115.678 200–700 Bunostomum spp. 9 (14.06) July 292 76 (26.02) 57.353 100–2200 Oesophagostomum spp. 8 (12.5) August 578 137 (23.7) 152.62 100–1300 Nematodirus spp. 5 (7.81) September 372 76 (26.03) 124.846 100–1800 Other strongyle spp. 18 (28.16) October 591 166 (28.09) 160.56 100–2900 *The species were confirmed on the basis of morphomatric November 447 125 (27.96) 65.42 100–900 characteristics of released larvae (L ) after coproculture, and December 621 62 (10.08) 60.253 100–300 3 percentage of bursate (strongyle) worms were calculated out of Total 4792 991 (20.68) 111.75 100–2900 total strongyle reported in the host species. 9 198 RAHMAN ET AL. [Indian Journal of Animal Sciences 80 (3) species and may initially be of help for planning Jithendran K P and Bhatt T K. 1999. Epidemiology of parasitoses chemotherapeutic and prophylactic strategies for yaks from in dairy animals in the North West humid region of India with the area studied and also for the regions with similar climatic particular reference to gastrointestinal nematodes. Tropical conditions. Further, investigations on epizootiology of Animal Health and Production 31: 205–14. Katiyar R D, Srivastava V K, Khera R C and Sinha S B. 1981. gastrointestinal parasites in yaks in high alpine region, larval Incidence of helminthes in domesticated yaks (Bos poephagus) bionomics and pasture burden are required for better in Sikkim. Livestock Research 1: 115–8. understanding of GI parasitism in the region. Katiyar R D and Sinha S B. 1982. Yak breeding and their health problems in Sikkim. Livestock Research 2: 1–9. ACKNOWLEDGEMENTS Lench J. 1996. Crankheiten: Der yak (Bos grunniens) in Authors thank the Indian Council of Agricultural Zentralasien. pp. 237–46. (Eds) Jurgen Lensch, Peter Schley and Rong – Chang Zhang. Duncker & Humblot– Research, New Delhi, for providing financial support for the Berlin. present study under the project of All India Network MAFF. 1984. Manual of Veterinary Investigation. Vol.2 (Reference Programme on Gastrointestinal Parasitism (AINP on GIP). Book 390, HMSO, London), 161–87. Techniques. HMSO. Thanks are also expressed to the Director and Joint Director Pal R N and Barari S R. 1994. Yak Production and Health Advances of this institute, Gangtok for providing the necessary in Veterinary Research and their impact on Animal Health and facilities. Production. , India. pp. 21–27. Ranga Rao G S C, Sharma R L and Hemapresanth. 1994. Parasitic REFERENCES infections of Indian yak Bos (Poephagus) grunniens- an overview. Veterinary Parasitology 53: 75–82. Ansari M Z, Rai M K and Chauhan H V S. 1989. Pathology of Rahman H, Bhattacharya M, Rajkhowa J, Soud N, Nandankar U liver, lung and caecum of yaks (Bos poephagus) infected with and Mukerjee S. 2007. Seroprevalence of brucellosis in yaks Dicrocoelium, Fasciola, Echinococcus and Trichuris. Indian (Poephagus grunniens) in India. Indian Journal of Animal Journal of Animal Sciences 59: 552–54. Sciences 77: 4–7. Jithendran K P and Bhatt T K. 1996. Prevalence of Dicrocoeliasis Souls by E J L. 1982. Helminths, Arthropods and Protozoa of in sheep and goats in Himachal Pradesh, India. Veterinary Domesticated Animals. 7th edn, 809 pp. ELBS and Baillire, Parasitology 61: 265–71 Tindall, London.

10 Indian Journal of Animal Sciences 80 (3): 199–202, March 2010 Evaluation of methanolic extract of Allium sativum and Saussurea costus in yaks with infectious keratoconjunctivitis

SAMIRAN BANDYOPADHYAY1, T K BISWAS2, D SASMAL3, I SAMANTA4 and M K GHOSH5

National Research Centre on Yak, ICAR, Dirang, West Kameng, Arunachal Pradesh 790 101 India

Received: 21 May 2009; Accepted: 26 October 2009

ABSTRACT The present study was undertaken to evaluate the efficacy of crude methanolic extract of fresh bulb of Allium sativum (G-MeOH), and roots of Saussurea costus (SC-MeOH) in yaks (Poephagus grunniens) with infectious keratoconjunctivitis. Bactericidal activity of G-MeOH and SC-MeOH was evaluated against Moraxella bovis and Neisseria spp isolated from eyes of yaks and yak hybrid with infectious keratoconjunctivitis and flies (Musca domestica). Following single disc diffusion assay, 5 resistant isolates of both bacteria were included for further study. Single disc diffusion and broth dilution method were used to determine the inhibitory effect of G-MeOH and SC-MeOH. Both the extracts displayed dose dependent antibacterial effect against all the isolates. Thereafter, the diluted plant derived products were subjected to clinical trials. Yaks (18) with infectious keratoconjunctivitis were included for clinical trial. Following application of the extracts, 8 yaks out of 12 (5 cured by G-MeOH, 3 cured by SC-MeOH) were completely cured within 7 days. However, 6 yaks where no extract was applied did not show any recovery. G-MeOH (83.33%: 5/6) exhibited the most prominent clinical efficacy followed by SC-MeOH (50%: 3/6). The results supported the traditional wisdom of herbal remedy use and suggested a potential clinical testing for G-MeOH and SC-MeOH on infectious keratoconjunctivitis.

Key words: Antibacterial activity, Costus, Garlic, Keratoconjunctivitis, Moraxella bovis, Neisseria spp, Yak.

Yak (Poephagus grunniens) is a unique bovine species of resistance (Radostits et al. 2000). A safe, cost-effective, high economic importance in high hill and snow bound areas alternative therapeutic strategy against these microbial of India, China, Mongolia, Bhutan, Nepal and Russia pathogens especially from herbal origin is the need of the hour. (Bandyopadhyay and Bhattacharya 2008). Apart from typical Garlic (Allium sativum) has been documented for its inherent problems of yak husbandry several diseases were antibacterial properties against various microbial pathogens also found responsible for diminishing yak population of India (Ahshan et al. 1996, Ankari and Mirelman 1999). Saussurea (Bandyopadhyay et al. 2008a, Bandyopadhyay et al. 2008b). costus, commonly known as ‘costus’, is usually found in Infectious keratoconjunctivitis (pink eye) is the most moist shady areas in brich forests of East Asia. The root of common ocular problem encountered in Indian yaks the plant is reported to be used as tonic, stimulant and (Bandyopadhyay et al. 2007a, Bandyopadhyay et al. 2007b, antiseptic in traditional Chinese and Tibetan medicine (Yeung Bandyopadhyay et al. 2008 c). Moraxella (Branhamella) bovis 1985, Tsarong 1994). The present investigation describes was reported to be the main causative organism in association about the effect of methanol extract of A. sativum (G-MeOH) with Neisseria, Chlamydia and Mycoplasma (Radostits et al. and S. costus (SC-MeOH) on the growth and viability of 2000). The conventional antibiotic therapy against these Moraxella bovis and Neisseria spp in vitro. It also elaborates pathogens is very expensive for the poor tribal farmers. the clinical efficacy of these extracts in the yaks with Moreover, the pathogens are not always responsive to infectious keratoconjunctivitis. conventional antibiotics due to emergence of multiple drug MATERIALS AND METHODS Collection, culture and maintenance of bacterial isolates: 1 Present address: PhD Scholar, Department of Clinical Moraxella bovis, Neisseria spp were isolated from the eyes Veterinary Medicine, Ethics and Jurisprudence e-mail: of yaks and yak hybrids with clinical manifestation of [email protected], 2Scientist, 3Subject Matter Specialist, 5Sr Scientist. infectious keratoconjunctivitis and flies (Musca domestica) 4Assistant Professor, Department of Veterinary Microbiology, abundant in the regions. The samples were cultured in nutrient West Bengal University of Animal & Fishery Sciences, 37, K B broth and subsequently in trypticase soy agar and blood agar Sarani, Kolkata – 700 037. at 37°C overnight. The isolated colonies were identified by 11 200 BANDYOPADHYAY ET AL. [Indian Journal of Animal Sciences 80 (3) colony structure, haemolysis, bipolar staining and standard For each concentration of A. sativum and S. costus, equal biochemical tests (Bandyopadhyay et al. 2008c). amount of DMSO was added to each tube to exclude out the In vitro antimicrobial drug sensitivity profile of the bactericidal effect of the vehicle, if any. isolates: The bacterial isolates recovered from yak were Determination of antibacterial effect of methanolic extract subjected to in vitro drug sensitivity assay following the (MeOH) using single disc diffusion method: Further, standard single disc diffusion method (Bauer et al. 1966). The disc diffusion method was employed with little modification. antibiotics used in the assay were cifrofloxacin (10 μg), Briefly, sterile discs were impregnated with each of the colistin (10 μg), co-trimoxazole (25 μg), gentamicin (10 μg), extract (200 mg/mL) to get the desired concentration. The nitrofurantoin (300 μg), streptomycin (10 μg), tetracycline discs were dried under laminar flow cabinet overnight. (30 μg), ampicilin (10 μg), cephotaxime (30 μg), cephalexin Following spreading the bacterial inoculums (Resistant (30 μg), chloramphenicol (30 μg), nalidixic acid (30 μg), strains of M. bovis and Neisseria spp as stated above) evenly furazolidone (50 μg), norfloxacins (10 μg) and over the surface of Muller Hinton agar plates, the oxytetracycline (30 μg). For each bacterium 5 resistant strains impregnated discs were positioned and incubated at 37°C were included for further in vitro study. for 24 h. Sterile disc was used as negative control. The Plant materials and extract preparations: Fresh bulbs of antibacterial activity was interpreted from the size of the A. sativum were collected from the local market and fresh diameter of zone inhibition measured to the nearest roots of S. costus were collected from Tawang Botanical millimeter (mm) as observed from clear zones surrounding Nursery, Tawang, Arunachal Pradesh, India. The bulbs and the discs. roots were shade dried and finely chopped. The chopped Dilution of the stock solution for in vivo trial: The stock materials were suspended in petroleum ether at 4°C overnight solution of 200 mg/ml of A. sativum (G-MeOH), and S. costus to remove the lipid substances. The supernatant was discarded (SC-MeOH) was diluted further in sterile double distilled and residue was again dried at room temperature. The dried water at various concentrations (1–50 mg/ml). The diluted residue was further suspended in methanol under refrigeration solutions were applied on the eyes of the swiss albino mice overnight. The supernatant was collected and filtered through thrice daily consecutively for 7 days to observe for adverse Whatmann No. 1 filter paper. The filtrate was dried in effect, if any. The aliquots were preserved at –20°C for further rotational vacuum concentrator to evaporate the methanol. use. The stock solution was prepared by dissolving the extract in Clinical trial in yaks with keratoconjunctivitis: Yaks (18) dimethyl sulfoxide (DMSO) at 200 mg/mL. Each time a fresh from the farm of the institute with overt and typical clinical stock solution was prepared for the assay of A. sativum (G- manifestation of infectious keratoconjunctivitis like unilateral MeOH) and S. costus (SC-MeOH). or bilateral conjunctivitis, keratitis, profuse ocular discharge, Determination of antibacterial effect of methanolic extract photophobia and corneal opacity were included for the study. (MeOH): Sterile test tubes containing 10 mL of trypticase Bacterial culture isolated from ocular swabs was maintained soy broth were supplemented with different concentrations on nutrient agar slant/trypticase soy agar slant and were of G-MeOH and SC-MeOH (ranging from 0.00–12.0 mg/ conventionally identified as described earlier. The yaks (18) mL for G-MeOH and 0.00–30 mg/mL for SC-MeOH). The were divided in 3 groups with 6 animals each. Two groups tubes were inoculated separately with 5 multi-drug resistant were tested with G-MeOH and SC-MeOH. In the infected strains of M. bovis and Neisseria spp at a concentration of eyes, the extracts were administered @ 0.5 ml/eye 4-times 1.8 ×106/mL, 2.5 ×106/mL respectively. The initial OD daily (at the concentration of 30 mg/mL), for 7 days. Another (optical density) was observed at 650 nm. Following group was kept as vehicle control where 0.5 ml of 0.5% incubation of the organism for 24 h again the OD650 was DMSO (v/v) in water was applied. observed. The growth and viability of the organisms was Statistical analysis: The results were expressed as the calculated in terms of turbidity using UV spectrophotometer. mean±SE of the results of 5 isolates in duplicate. MIC50 and The difference between final OD650 and the initial OD650 MIC80 were determined by fit spline/lowness using Graph- (specific OD650) is interpreted as the growth of the bacteria. pad prism software version 4.0. The viability of bacteria at any specific concentration was RESULTS AND DISCUSSION calculated as the percentage of specific OD650 at that concentration versus the specific OD650 of the tube without Infectious keratoconjunctivitis associated with Moraxella A. sativum and S. costus supplementation (negative control). bovis, Neisseria spp. was found wide spread among the yaks The calculation can be shown as follows in India. The problem increased substantially during autumn and spring. This was probably due to abundance of flies Specific OD650 (final OD650 –initial OD 650 ) (Musca domestica) and increased intensity of sunlight during of any concentration of MeOH×100 Percent viable = these seasons. Flies and intense UV irradiation were reported Specific OD650 (final OD650 –initial OD 650 ) as important predisposing factors for keratoconjunctivitis of the negative control (MeOH) apart from microorganisms (Akerstedt and Hofshagen 2004). 12 March 2010] EFFECT OF ALLIUM AND SAUSSUREA ON YAKS WITH INFECTIOUS KERATOCONJUNCTIVITIS 201

Large hair growth around the eyes is also an important included for this study. The effect of G-MeOH and SC-MeOH contributing factor due to constant irritation (Bandyopadhyay on the bacterial viability is depicted in Tables 1 and 2 et al. 2007a). The wide spread presence of the disease can respectively. G-MeOH and SC-MeOH at a concentration of be attributed to the way yak husbandry is practised in India. 7.57 mg/mL and 7.516 mg/mL, respectively, caused a marked Yaks are generally reared under transhumance system of decline (80%) of the viability of the resistant strains of M. migration and during lean season they come down to lower bovis (Table 1). Strains of Neisseria were also sensitive at altitude in search of feed. During this period, they share 6.14 mg/mL and 28.996 mg/mL concentration of G-MeOH common grazing land, falls and streams with other domestic and SC-MeOH respectively. The disc diffusion assay revealed and wild animals like hill cattle and mithun (Bandyopadhyay (Table 2) comparable zones of inhibition with G-MeOH (10– et al. 2007a, Bandyopadhyay et al. 2008b, Bandyopadhyay 15 mg/disc) and SC-MeOH (30 mg/disc) against Neisseria et al. 2008e). Moreover, yaks are kept in herds of 50–200 spp and M. bovis at 24 h of incubation. G-MeOH and SC- animals. Thus increased contact between animals provides MeOH reduced the growth of M. bovis and Neisseria spp an easy avenue for the transmission of pathogen conspicuously. Control discs had no observable effect on the (Bandyopadhyay et al. 2008b, e). bacterial growth. It is clear that the extracts suppressed the M. bovis and Neisseria sp were resistant to colistin, co- growth of multi-drug resistant isolates of M. bovis and trimoxazole, gentamicin, nitrofurantoin, streptomycin, Neisseria spp, recovered from the eyes of yaks and yak hybrid ampicilin, cephalexin, nalidixic acid, furazolidone. Till date, with infectious keratoconjunctivitis or pink eye, in a very limited literature is available on the multi-drug concentration-dependent manner using broth dilution resistance profile of M. bovis and Neisseria sp. However, method. Similar profile was also observed in disc diffusion the increased use of antibiotics both as a growth promoter assay. This may be due to the fact that G-MeOH and SC- and in disease management exposes these pathogens to low MeOH mediate its antibacterial property in different and sub-lethal concentrations of these agents and indirectly mechanisms than the antibiotics. helps them to evolve novel resistance mechanism (Williams G-MeOH and SC-MeOH at the concentration of 30 mg/ et al. 1989). Overall antibiogram profile of this study reflects mL was subjected to in vivo trial. This concentration was that this resistance feature is remarkably increased when the more than the MIC80 value of both herbal extract tested isolates were taken from the hosts (yaks and hybrids) rather against all the isolates. Moreover, above this concentration than from carriers (flies). This may be due to extensive and swiss albino mice started showing adverse reaction like indiscriminate use of antibiotics in these animals. The restlessness, irritation etc. when applied on their eye. For exploration of muti-drug resistant pathogens in the present clinical trial 18 yaks with overt clinical and microbiological study is quite alarming and strongly indicates towards the evidence of infectious keratoconjunctivitis were included for judicious use of antimicrobials in clinical application. the study. M. bovis and Neisseria spp were detected in the To study the antibacterial efficacy of A. sativum (G- conjunctival swab of all the 18 yaks with infectious MeOH) and S. costus (SC-MeOH), strains of M. bovis and keratoconjunctivitis. Twelve yaks (6 in each group) were Neisseria spp were selected on the basis of their antibiogram tested with extracts of G-MeOH and SC-MeOH, @ 0.5 ml/ profile. Isolates resistant to more than 5 antibiotics were eye/4 times daily (at the concentration of 30 mg/mL) for

Table 1. MIC50 and MIC80 of methanolic extract of A. sativum (G-MeOH) and S. costus (SC-MeOH) against 5 different multidrug resistant isolates of Moraxella bovis and Neisseria spp (data shown as mean ± SEM; n = 5 different isolates in duplicate)

Organism G-MeOH SC-MeOH 5 mg/disc 10 mg/disc 15 mg/disc 10 mg/disc 20 mg/disc 30 mg/disc mm mm mm mm mm mm

Neisseria 17.6 ± 0.59 24.2 ± 0.21 27.8 ± 0.79 13.4 ± 0.51 17.8 ± 0.62 21.9 ± 0.17 M. bovis 13.8 ± 0.35 20.9 ± 0.67 28.9 ± 0.36 18.3 ± 0.71 21.8 ± 0.53 29.6 ± 0.42

Table 2. Evaluation of G-MeOH and SC-MeOH against M. bovis and Neisseria spp by disc diffusion method (data shown as mean ± SEM; n = 5 different isolates duplicate)

Organism G-MeOH SC-MeOH

MIC50(mg/mL) MIC80(mg/mL) MIC50(mg/mL) MIC80(mg/mL) Neisseria 2.63 ± 0.65 6.14 ± 0.55 12.201 ± 0.92 28.996 ± 0.69 M. bovis 3.96 ± 0.42 7.57 ± 0.38 4.513 ± 0.71 7.516 ± 0.57

13 202 BANDYOPADHYAY ET AL. [Indian Journal of Animal Sciences 80 (3) seven days. Eight yaks, 5 by G-MeOH and 3 by SC-MeOH K P and Bhattacharya M. 2007a. Common Health Ailments of were completely cured. G-MeOH (83.33%: 5/6) exhibited Yaks (Poephagus grunniens L.). Technical bulletin, NRC-Y the most prominent clinical efficacy followed by SC-MeOH publication, Dirang, AP. (50%: 3/6).The effectiveness of extracts was noticed from Bandyopadhyay S, Ghosh M K and Rajkhowa J. 2007b. Unilateral trummatic keratoconjunctivitis with corneal opacity in a Dzomo- the third day onwards. However, no clinical recovery was a case report. Indian Veterinary Journal 84: 296–97. observed in the yaks with vehicle control up to 7 days. Garlic Bandyopadhyay S and Bhattacharya M. 2008. Scope constraints is active against wide range of gram positive and gram- and strategies of yak husbandry in poverty alleviation and negative bacteria even against antibiotic resistant strains development of hilly regions. Compendium of National Seminar (Ashan et al. 1996, Ankari and Mirelman 1999). However, on “Integrated farming systems relevant to NE regions”. the response may vary depending upon the bacterial species. February 07–08, 2008, at College of Veterinary Science and The proposed active compound of garlic is allicin or di-allyl- Animal Husbandry, Central Agricultural University, Selesih, thiosulphinic acid, which is released from a precursor when Aizwal, Mizoram, pp 82–85. the garlic bulbs are crushed (Sasmal et al. 2005). The root of Bandyopadhyay S, Sasmal D, Ghosh M K, Borah B, Bora M and Biswas T K. 2008a. Collibacillosis in new born yak calves and costus is commonly used as herb and Ayurvedic medicine antibiogram profile of rectal flora isolated from the same. Indian where it is used mainly as tonic, stimulant and antiseptic Journal of Animal Sciences 78: 1101–02. (Yeung 1985, Tsarong 1994). The root is also used as Bandyopadhyay S, Sasmal D, Dutta T K, Ghosh M K, Sarkar M, anodyne, antibacterial, antispasmodic, aphrodisiac, Sasmal N K and Bhattacharya M. 2008b. Seroprevalence of carminative, skin stimulant, stomachic, tonic and vermifuge brucellosis in yaks (Poephagus grunniens) in India and (Yeung 1985). The antimicrobial effect could be attributed evaluation of protective immunity to S19 vaccine. Tropical to different kinds of flavonoids and sesquiterpene lactone Animal Health Production (in press; DOI 10.1007/s11250–008– derivatives like anthocyanins, ellagitannins, and 9228–0) proanthocyanidin, which were reported to be present in this Bandyopadhyay S, Sasmal D, Ghosh M K, Bora M and Bhattacharyya M. 2008c. Antibacterial efficacy of neem plant. (Azadirachta indica) against Morexella bovis and Neisseria. This preliminary study is able to open the floodgate of Indian Journal of Animal Sciences 78: 1105–07. new knowledge as the literature on the treatment of Bandyopadhyay S, Sasmal D, Bora M, Ghosh M K, Sarkar M. keratoconjunctivitis by some herbal compound is not 2008d. Antibacterial profile of Rubus idaeus (raspberry): available. It can be concluded from the results that G-MeOH An in vitro study. Indian Journal of Animal Sciences 78: and SC-MeOH supported the traditional wisdom of herbal 1247–49. remedy use and suggested a potential clinical testing by Bandyopadhyay S, Chakrabarty D, Sarkar T, Pal B, Sasmal D, improving the product formulation with greater efficacy and Biswas T K, Ghosh M K and Sarkar M. 2008e. A serological duration of action would be of interest and awaits further survey of bovine herpesvirus type –1 antibodies in yaks (Poephagus grunniens). Review Scientific Technique Office investigation. International Epizootics (in communication). ACKNOWLEDGEMENT Bauer A W, Kirby W M M, Shemis J C and Truck M. 1966. Antibiotic susceptibility testing by a standard simple The author is thankful to the Director of NRC-Yak for disc method. American Journal of Clinical Pathology 45: 493– providing necessary facilities. 96. Radostits O M, Gay C C, Blood D C and Hinchcliff K W. 2000. REFERENCES Veterinary Medicine, A textbook of the Diseases of Cattle, Horses, Sheep, Pigs and Goats. 9th edn, WB Saunders Company Ahsan M, Azad Chowdbury A K, Islam S N and Ahmed Z U. 1996. Ltd. Garlic extract and allicin: Broad spectrum antibacterial agents Sasmal D, Babu Surendra C and Abraham T J. 2005. Effect of effective against multiple drug resistant strains of Shigella garlic (Allium sativum) extract on the growth and disease dysenteriae type 1 and Shigella flexneri, Enterotoxigenic resistance of Carrassius auratus (Linnaeus, 1758). Indian Escherichia coli and Vibrio cholerae. Phytotherapy Research Journal of Fishes 52: 207–14. 10: 329–31. Tsarong T J. 1994. Tibetan Medicinal Plants. Tibetan Medical Akerstedt J and Hofshagen M. 2004. Bacteriological investigation Publications, India. of infectious keratoconjunctivitis in Norwegian sheep. Acta Yeung Him C. 1985. Handbook of Chinese Herbs and Formulas. Veterinariae Scandinavia 45: 19–26. Institute of Chinese Medicine, Los Angeles. Ankari S and Mirelman D. 1999. Antimicrobial properties of allicin Williams J D, Moosdeen F, Teoh-Chan C H, Lim V K E and from garlic. Microbes Infection 1: 125–29. Jayanetra P. 1989. Surveillance of antibiotic resistance in South Bandyopadhyay S, Saravanan BC, Ghosh M K, Sarkar M, Ramesha East Asia. Euroepan Journal of Epidemiology 5: 207–13.

14 Indian Journal of Animal Sciences 80 (3): 203–208, March 2010 Ultrasound guided biopsy and fine needle aspiration biopsy of splenic and prostatic affections in dogs

S K MAHAJAN1, S S SINGH2, J MOHINDROO3, N S SAINI4, N SINGH5 and N K SOOD6

Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab 141 004 India

Received: 2 April 2008; Accepted: 26 October 2009

ABSTRACT The clinical study was conducted on 20 clinical cases of dogs for various abdominal disorders involving the spleen and prostate, which included USGCB in 4 cases of spleen and 1 case of prostate and USG-GNAB in 7 cases of spleen and 8 cases of prostate. The technique of ultrasound guided tissue core biopsy (USGCB) and ultrasound guided fine needle aspiration biopsy (USG-FNAB) was found to be accurate without any complications for spleen and prostate. Ultrasonography helped characterization of splenic parenchyma for change in size and echotexture. Hemangiosarcoma, hemosiderosis and chronic splenitis were the conditions which were diagnosed with USGCB. The USG-FNAB helped diagnosing purulent splenitis, reactive hyperplasia and plasmacytoma of the spleen. USG-FNAB of the prostate helped diagnosing benign prostatic hyperplasia, prostatitis, prostatic abscess and prostatic adenocarcinoma. Radiography and ultrasonography was effective for assessing enlargement and change in echotexture of the prostate. USGCB and USG- FNAB were found reliable tools for the diagnosis of splenic and prostatic affections in dogs.

Key words: Biopsy, Dogs, Prostate, Spleen, Ultrasound

Different workers have conducted studies on ultrasound- small animal teaching hospital, with varying symptoms. The guided biopsy of spleen (Leveille et al. 1993, Bonfanti et al. clinical signs in cases of splenic affections varied from fever, 2004) and ultrasound guided fine needle aspiration of prostate abdominal pain, anorexia, reduced general activity and (Nyland et al. 2002, Powe et al. 2004). Ling (1995) has abdominal distension. In cases of prostatic affections the reported that ultrasound guided fine needle aspiration of the clinical signs ranged from anorexia, blood in urine, prostate improves the quality of diagnosis by more accurately urolithiasis, constipation, pasty faeces and hindlimb identifying and sampling diseased areas of tissue. Fine needle weakness. The animals were selected as having splenic or biopsy is a widely accepted diagnostic technique that can be prostatic affections on the basis of clinical, hemato- used to establish the diffuse lesions of spleen (O’Keefe and biochemical, radiographical and ultrasonographical findings. Couto 1987). The aim of the study was to assess the efficacy All the animals were subjected to systematic evaluation for and diagnostic value of ultrasound guided fine needle diagnosis of the diseased conditions. Hematological and aspiration biopsy (FNAB) in cases of prostatic affections and biochemical parameters including serum alkaline both ultrasound guided FNAB and ultrasound guided and phosphatase (ALP), alanine aminotransferase (ALT), alanine core biopsy (USGCB) in cases of splenic affections in dogs. aminoaspartate (AST), blood urea nitrogen (BUN), creatinine, total leucocyte count (TLC) (×103 /μl), differential MATERIALS AND METHODS leucocyte count (DLC) (%), Hb (g/dl) were determined The study was conducted on 20 clinical cases of dogs of depending upon the case. Ultrasound-guided core biopsy varying breeds, of either sex, aged 6 months to 14 years and (USGCB) was collected in 4 cases of spleen and 1 case of body weight ranging from 6 to 48 kg presented at the institute prostate with a Bard’s disposable biopsy gun using the free hand technique. Ultrasound guided fine needle aspiration biopsy was collected in 7 cases of spleen and 8 cases of Present address: 1Associate Professor (e mail: skmahajan73 prostate using 20–22 gauge needle attached to a 10 ml syringe @yahoo.co.in), 3, 4Associate Professor, Department of Veterinary Surgery and Radiology, 2Dean, College of Veterinary Science, using the free hand technique. Survey abdominal radiography 5Associate Professor (Surgery), Department of Veterinary and and ultrasonography was performed in all the cases. Animal Husbandry Extension, 6Professor, Department of Veterinary Prothrombin time was estimated in some critical cases where Pathology. USGCB was carried out. In the study ultrasonography was 15 204 MAHAJAN ET AL. [Indian Journal of Animal Sciences 80 (3) carried out using a concept/MCV veterinary ultrasound the technique for splenic biopsies was 90%. Sufficient scanner, with 3.5 MHz micro convex and 7.5 MHz linear histopathological/cytological samples of good diagnostic array transducer. The USGCB was carried out in dorsal quality were obtained in the present study. However, De recumbency in non-anesthetized animals. In 2 cases local Rycke et al. (1999) reported that difficulty experienced in infiltration anesthesia (lignocaine HCl 2%) was injected at differentiating hematoma from hemangioma or the site where patients were non-cooperative during the hemangiosarcoma on the basis of a cytological sample limits procedure. The instrument used for all biopsies was the diagnostic value of this procedure. No complication was disposable biopsy instrument, which was resterilized and found in the present study. The animals generally had reused. The size of the needle was 18 g × 16 cm in length. neutrophilic leucocytosis with left shift. The ALP values were The length of sample notch was 1.8 cm and depth of the markedly elevated. PT was 8.5 sec in 1 case, which was penetration was 22 mm. Two examiners performed the within normal range. No major complications were associated procedure, one handling the ultrasound transducer and other with this procedure in the present study, which was in operating the biopsy gun. The organ was localized concurrence with Leveille et al. (1993) who also found no ultrasonographically, the overlying skin aseptically prepared, complications with ultrasound guided biopsy or fine needle the needle was pierced at an angle from ultrasound transducer aspiration of the spleen in dogs and cats. Radiography in the through the skin, as the organ was being visualized on the present study showed there was a big mass in the abdomen monitor. Biopsy of each organ was made after seeing the in most of the cases. However, Couto and Hammer (2002) image of both the organ and the needle tip. One to two reported that the splenic neoplastic masses lead to lumpy attempts were made to collect the biopsy. After removal of margins with multiple lobulations. But no such specific the biopsy instrument, a post biopsy scan was done to check radiographic findings were seen in the present multiple the haemorrhage. The tissue samples thus obtained were lobulations. But no such specific radiographic findings were immediately stored in 10% buffered formalin for seen in the present study. Pechman Jr. (1998) also reported histopathological analysis. In spleen, the samples were taken that evaluation of the splenic size radiographically was purely from the animals, which were having significant radiographic subjective and might not be a reliable criterion. In all cases and ultrasonographic findings indicative of any alteration in the spleen was found enlarged ultrasonographically. O’Brien size or structure of the splenic parenchyma. The ultrasound (1978) reported that the neoplasia, inflammation, vascular guided fine needle aspiration biopsy (USG-FNAB) was done stasis, torsion and trauma usually cause diffuse splenic in spleen and prostate using a 20–22 g needle attached to a enlargement. 5–10 ml syringe. For aspiration, after placing the transducer Out of 4 animals in which ultrasound-guided core biopsy on the affected organ, the needle was advanced slightly was done, the spleen was found to be neoplastic in 2 animals. oblique to the long axis of transducer through the skin. When The clinical signs in these cases ranged from fever, pain, the needle was seen in middle of the lesion, moderate and anorexia, reduced general activity and pain. In 1 case there rapidly repeated suction (approximately 3–6 times) was was neutrophilic leucocytosis (85% neutrophilia) and in other applied to the syringe plunger while the needle was moved case there was leucopenia with eosinophilia. The ALP values within the lesion. After complete release of the suction, the were found markedly elevated in both the cases and mass syringe and attached needle were removed from the was palpable in the mid abdomen. The increase in ALP values abdominal cavity. Double samples were obtained in all the 8-times greater than the upper limit of the normal range and cases. Smears were made from tissue samples and submitted are usually indicative of the neoplastic conditions in dogs for cytological evaluation. (Bush 2002). The organ of the abdominal mass could not be ascertained by the abdominal palpation. Survey radiographs RESULTS AND DISCUSSION revealed a round radiodense mass visible at mid ventral Spleen (N=11): The mean age of dogs suffering from abdomen in the first case (Fig. 1) while in second case the various splenic affections was 6.60 years (range 1–10 years). spleen was enlarged and pushed caudally along with liver Various physiological parameters i.e. temperature, pulse rate enlargement. Ultrasonography of the spleen showed that in and respiratory rate of dogs suffering from splenic affections the first case there was a circumscribed hypoechoic cavity were statistically nonsignificant (P<0.05). Based on clinical, in splenic parenchyma within splenic capsule (Fig. 2) and in hematological, radiographic, ultrasonographic and the second case spleen appeared to have a mixed echotexture histopathological findings animals having splenic disease and circumscribed anechoic areas. Partington and Biller were broadly divided into 2 categories—neoplastic in origin (1996) reported that in hemangiosarcoma, the spleen is and non-neoplastic in origin. In the present study all the enlarged and contains multiple masses, which often protrude samples contained sufficient splenic tissue for from the splenic surface or may appear adjacent to or near histopathological/cytological analysis which suggested 100% spleen. These masses have a mixture appearance. accuracy of the technique. Similar findings were shown by Ultrasonographic findings may not be definitive for the De Rycke et al. (1999), who reported that the accuracy of histopathologic cell type, but focal neoplasms as 16 March 2010] ULTRASOUND GUIDED BIOPSY IN DOGS 205

1. 2.

3. 4.

5. 6.

7. 8. Figs 1–8. 1. Songram showing circumscribed hypoechoic cavity (F) apparent in the splenic parenchyma.2. Histopathology of the spleen showing haemangiosarcoma of the spleen. Tumour cells are forming cord like arrangements and at times forming vascular lumen (HE × 200) 3. Sonogram showing mass in spleen having mixed echotexture in the caudal half. 4. Histopathology of the spleen showing hemosiderosis and depletion of lymphoid tissue in the spleen (HE × 150). 5. Lateral pneumocystogram showing a soft tissue mass (P) evident at the neck of urinary bladder pushing it cranially indicating prostatic enlargement along with dilated prostatic urethera (PU). 6. Sonogram showing an enlarged prostate (P) at the neck of UB. 7. Sonogram showing a hyperechoic mass (P) evident at the neck of UB in prostatic region having cavitations. 8. Cytology of prostate showing adenocarcinoma characterized by pleomorphism, hyperchromatic nuclei, altered C/N ratio and nucleoli in some cells. 17 206 MAHAJAN ET AL. [Indian Journal of Animal Sciences 80 (3) leiomyosarcoma, fibrosarcomas, anaplastic sarcomas, plasma echotexture (Fig. 6). In 1 case the spleen was hyperechoic cell and mast cell tumors were reported to be homogenously with the congested areas. The enlarged spleen was wider, hypoechoic (Feeny et al. 1984). Histopathology of the biopsy thicker and was extending caudally up to the pelvic inlet and sample showed hemangiosarcoma in both the cases on the right side. Nyland and Hager (1985) also reported characterized by cells forming cord like arrangements and that diffused splenomegaly could be due to active or passive at time forming vascular lumen (Fig. 3). The condition was congestion, vascular compromise and diffuse infiltration. confirmed at surgery histopathologically. Splenectomy was Partington and Biller (1996) also reported that diffuse splenic done under general anaesthesia. The animals were disease presents uniformly enlarged spleen with normal or premedicated with butorphenol (0.2 mg/kg body weight) + decreased echogenecity and a normal or inhomegeneous acepromazine (0.05 mg/kg body weight) + glycopyrrolate echotexture. The USG-FNAB findings in the study ranged (0.01 mg/kg body weight) by intra-muscular route followed from purulent splenitis (N=2) and reactive hyperplasia by induction with thiopentone @ 10–15 mg/kg body weight characterized by various sizes of lymphoid and plasma cells intra-venously and maintained with halothane (1.5–2%). (N=2) (Fig. 7). The USG-FNAB and ultrasonographic After splenectomy both these animals improved but the findings in the study in non-neoplastic affections were usually animals died 4 and 6 months after surgery. associated with reactive spleen, which might be due to some The diseased spleen was non-neoplastic in 2 animals. The other infections or inflammatory disease in the body. In the clinical signs in these animals were fever (104° F and 106° study history of ticks, fever, enlargement of lymph nodes F), anorexia, and abdominal distension from 15–20 days. could be linked to some systemic or hemoprotozoan disease, Neutrophilic leucocytosis (82% neutrophilia) was seen in which might have lead to splenomegaly. one case. The ALP (mean 641.5 ± 29.5) values were elevated The spleen was found neoplastic in 1 animal on USG- in both whereas the ALT (225 U/L) and AST (208 U/L) values FNAB. The animal, concomittantly had diffused enlargement were elevated in 1 case. A soft tissue mass was palpable in of all lymph nodes, mixed mammary tumor and moggoted mid-abdomen in both the cases, but organ of origin of the wound. Radiograph of the animal revealed a soft tissue abdominal mass could not be ascertained by abdominal density in the caudoventral abdomen in the region of spleen palpation. The ultrasonographic findings showed (Fig. 8). Ultrasonography revealed an enlarged and irregular enlargement of spleen with mixed echotexture in the caudal spleen with well-defined hyperechoic mass within its half in 1 case (Fig. 4) and an enlarged spleen with normal parenchyma (Fig. 9). The USG-FNAB of the lesion revealed echotexture in other case. The enlarged spleen was extending plasmacytoma indicated by proliferation of plasma cells. up to the urinary bladder and also on the right side of the Partington and Biller (1996) reported that plasma cell tumors abdomen. The biopsy findings showed that there was are frequently more homogeneously hypoechoic than hemosiderosis and marked depletion of spleen in 1 case (Fig. fibrosarcomas and anaplastic sarcomas, but the 5) and chronic splenitis in the other case. Both the conditions ultrasonographic appearance of primary and metastatic were confirmed histopathologically at surgery. Splenectomy splenic tumor is not specific for cell type. The owner denied was done in both the animals under general anaesthesia. The surgery and animal died one month after the diagnosis of animal with chronic splenitis died during surgery while the this condition. other animal improved after splenectomy. In the study, USG-FNAB was effective in the diagnosis In 7 cases, where the USG-FNAB was done, the spleen of the splenic diseased conditions in dogs. The technique was non-neoplastic in 6 animals. The term fine needle biopsy was effective for the diagnostic of neoplastic and non- is reserved for needles smaller than 20 gauge. Tissue core neoplastic (reactive and inflammatory) conditions of the biopsies using larger (usually 14 to 18 gauge) automatic spleen. needles, yield tissue plugs for histopathologic examination Prostate: In 1 case USGCB and in 8 cases USG-FNAB (Siatoh 1984). The clinical signs in these cases ranged from of the prostatic affections was done depending upon the case. fever (103–107° F), anorexia, swelling of lymph nodes, tick No complications were recorded during the study. The USG- infestation and conjunctivitis. The clinical signs in splenic FNAB was preferred over USGCB in the present body as it affections in the study were similar as seen by Bhadwal was cheaper, easier, less labour intensive and because of the (1997). Generally, the animals were anemic and the blood thin monolayer obtained with cytological smears, it is picture revealed neutrophilic leucocytosis. The changes in possible better assess cell detail and the presence of blood values as anemia and neutrophilic leucocytosis in the etiological agents (Powe et al. 2004). Prostatic disorders were study might be related to the concurrent disease condition. more common in middle-aged and older intact male dogs in No significant radiographic findings were seen, except in 1 the study. Olsen et al. (1987) reported similar observations. case where an elevated trachea was evident in the cranial Majority of affected animals in the study were more than 4 mediastinum suggesting a mediastinal mass and base of the years of age. Lamb (1990) also opined that BPH occurred in heart could not be visualised. Ultrasonography of the cases 50% dogs over 4 years and 90% over 5 years and might revealed spleen was diffusely enlarged with the uniform produce a diffused mild increase in the echogenicity. 18 March 2010] ULTRASOUND GUIDED BIOPSY IN DOGS 207

Statistically nonsignificant (P<0.05) difference was recorded USG-FNAB showed degenerated neutrophils indicating among various physiological parameters (temperatue, heart sepsis. The animal, however, died on the subsequent day and rate and pulse rate) of dogs suffering from prostatic the condition was confirmed on post-mortem. affections. Mean age of dogs with prostatic affections was In prostatic adenocarcinoma, ultrasonography showed that 7.77 years (4–12 years) with a mean body weight of 24.80 a hyperechoic mass was evident at the neck of urinary bladder kg (15–44 kg). The animals generally had neutrophilic in the region of prostate having cavitations (Fig. 15). Barsanti leucocytosis with left shift. The ALT, AST, and ALP values and Finco (1979) reported that prostatic neoplasia might be were generally within normal range. The animals had various one of the causes of asymmetric enlargement of the prostate. affections as benign prostatic hyperplasia (N=5), prostatitis, Sonographically prostatic neoplasia reportedly produced prostatic abscess or prostatic adenocarcinoma (N=1 each) symmetric and asymmetric prostatomegaly of mixed, non- diagnosed on USG-FNAB. Similarly Thrall et al. (1985) had uniform echogenicity. Pathy echogenic areas coalescing classified the prostatic disease as hyperplasia, cyst, together and cavitations, though infrequent were reported inflammation, primary and metastatic neoplasia and (Feeney et al. 1985, Feeney et al. 1987). USG-FNAB of the squamous metaplasia. The clinical signs in these cases ranged lesion showed prostatic adenocarcinoma characterized by from anorexia, blood in urine, and history of urolithiasis, pleomorphism, hyperchromatic nuclei, altered C/N ratio and constipation, pasty faeces and hindlimb weakness. Similar nucleoli in some cells (Fig. 16). In prostatitis, radiograph clinical signs were reported by Singh (2006). The majority revealed radiopaque calculi in the prostatic urethra along with of cases showed mild neutrophilic leucocytosis. The survey the enlarged prostate (Fig. 17), which was pushing the urinary and contrast radiography (pneumocystography and/or barium bladder cranially. Ultrasonography showed that prostate was enema) of the caudal abdomen showed that there was a enlarged with mixed echotexture. One of the common causes symmetric increase in the soft tissue density caudal to the of the prostatic enlargement is reported to be prostatitis, neck of the urinary bladder with cranially displaced and which is usually bacterial (Barsanti and Crowell 1980, distended urinary bladder (Fig. 10). Our radiographic findings Lattimer 1998). Green and Homco (1996) reported that acute were concurrent with the findings of Lattimer (1998) who inflammation is marked by an enlarged and symmetric reported that with the uniform prostatomegaly, urinary prostate gland. The USG-FNAB of the case showed bladder was displaced cranially along the floor of abdomen inflammation of the ducts indicating prostatitis. with dorsal displacement of colon. Cartee and Rowles (1984) The animal in which the ultrasound guided core biopsy stated that benign prostatic hyperplasia (BPH) was was done had a history of constipation and swelling in the characterized by symmetric and enlarged prostate, with firm perineal region since 4–5 days. Plain radiograph of the caudal smooth margins and increased overall echogenicity. In the abdomen revealed soft tissue density in the perineal region. study similar sonographic findings were seen in case of BPH, Ultrasonography revealed that the swelling was due to an which showed that the prostate was enlarged with normal or enlarged prostate (84.6 mm dia) with multiple areas of generalized increase in echogenicity (Fig. 11). The prostatic hyperechogenicity in parenchyma. Histopathology of the urethra appeared to be dilated in most of the cases. The USG- prostatic tissue revealed benign prostatic hyperplasia FNAB of the cases suffering from benign prostatic characterized by formation of the papilliform projections in hyperplasia was characterized by a large number of intact the lumen. and degenerated prostate epithelial cells (Fig. 12). Open The USG-FNAB of the prostate proved to be an effective castration was done in all the animals suffering from BPH technique for obtaining sufficient cytological samples from under general anaesthesia. Same anaesthetic protocol as for prostate for diagnosis of the condition. The technique was splenectomy was used. The animals generally recovered well effective for the diagnosis of various prostatic conditions after surgery. Singh (2006) also reported similar findings after such as BPH, prostatitis, abscess and adenocarcinoma. castration in prostatomegaly due to BPH. In animal with REFERENCES prostatic abscess, radiograph of the case showed a soft tissue mass in the prostatic region (Fig. 13). The sonography of the Barsanti J A and Crowell W. 1980. Renal amyloidosis. Current case showed that the prostate was enlarged with Veterinary Therapy VII. pp. 1063–66. (Ed.) Kirk R W. W.B. circumference 209.5 mm having multiple hypoechoic Saunders Co., Philadelphia. cavitations with echogenic particles suggestive of prostatic Barsanti J A and Finco D R. 1979. Canine bacterial prostatitis. abscess (Fig. 14). 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20 Indian Journal of Animal Sciences 80 (3): 209–212, March 2010 Effect of granulosa cells monolayer and oviductal epithelial cells co-culture on cleavage rate and embryo development of in-vitro fertilized goat oocytes

K P SINGH1, ATUL SAXENA2, S D KHARCHE3 and PRANEETA SINGH4

U.P.Pt.Deen Dayal Upadhyaya Pashu-Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan, , Uttar Pradesh 281 122 India

Received: 20 April 2009; Accepted: 4 November 2009

ABSTRACT The study was undertaken to estimate the effect of granulosa cells monolayer and oviductal epithelial cells co-culture on cleavage rate and embryo development of in-vitro matured and fertilized prepubertal and pubertal goat oocytes. The oocytes were matured in maturation media (TCM–199) supplemented with either different supplements, viz. 20% estrus goat serum (EGS), 20% new calf serum (NCS), or 10% new calf serum (NCS) plus hormone (FSH + LH + E2) or 20% caprine follicular fluid (CFF). After 27 hours (h), the matured oocytes were picked up and denuded by treating with 0.1% hyaluronidase and by repeated pipetting through a fine bore pipette. The denuded oocytes were transferred in fert-TALP medium (fertilization medium). The capacitated sperm having concentration of 1×106 live sperm/ml were used for in- vitro fertilization. Oocytes were co-incubated with capacitated spermatozoa for 18 h at 38.5±1°C in an atmosphere of 5% CO2 in humidified air. The fertilized oocytes culture in EDM over their respective cell culture i.e. granulosa cells monolayer and oviductal epithelial cells were obtained for further cleavage rate and embryo development (2 celled, 4 celled, 8 celled, and >8 celled) under steriozoom microscope at every 24 h with 50% replacement of pre-equilibrated EDM media up to 7 days. Forty-eight hours post insemination the cleavage rates with granulosa cells monolayer and oviductal epithelial cells co-culture were 40.78 and 45.93% and 47.36 and 43.49% for prepubertal and pubertal goat oocytes respectively. The embryo development up to the 2 cell, 4 cell, 8 cell, and >8 cell with granulosa cells monolayer and oviductal epithelial cells co- culture were 65.32, 22.58, 6.45, 5.64 and 63.92, 27.26, 6.96, 1.89% and 50.92, 30.55, 12.03, 6.48 and 40.78, 22.36, 7.23, 6.57%, respectively, for prepubertal and pubertal goat oocytes. In conclusion, the present study indicate that both granulosa cell monolayer and oviductal epithelial cells co-culture behaved similarly for cleavage rate and embryo development of in-vitro matured and fertilized prepubertal and pubertal goat oocytes.

Key words: Embryo development, Goat, Granulosa cells monolayer, In-vitro maturation, In-vitro fertilization, Ovaries, Oviductal epithelial cells

In-vitro embryo production provide an excellent source of early stage embryo. It could provide embryonic growth of embryos to carry out basic research on developmental factor or remove toxic substances from the culture medium or physiology, farm animal breeding and for commercial both. In goats, granulosa cells monolayer or oviductal application of the emerging biotechniques like embryo epithelial cells have been used in IVM-IVF to improve sexing, sperm injection, nuclear transfer, cloning and embryonic development program. The oviductal cells have transgenic (Nandi et al. 2003). been defined as crucial factor for better cleavage and further Somatic cells are repeatedly used to improve in-vitro culture development of IVF embryos in cattle (Xu et al. 1987), goat (Watson et al. 1994) and buffaloes (Madan et al. 1994). Present address: 1Assistant Professor, Teaching Veterinary The present study was aimed to evaluate the effect of Clinical Complex, College of Veterinary Sciences and Animal granulosa cells monolayer and oviductal epithelial cells co- Husbandry, Narendra Deva University of Agriculture and culture on cleavage rate and developmental competence of Technology, Kumarganj, Faizabad (Email: drkpsvet in-vitro fertilized goat oocytes. @rediffmail.com). 2 Associate Professor, Department of Obstetrics and MATERIALS AND METHODS Gynaecology, 3Senior Scientist, Division of PR and SM, CIRG, Makhdoom, Farah, Mathura 281 122. Collection and processing of abattoir ovaries: Goat 4Veterinary Officer, Barun, Faizabad. ovaries (prepubertal and pubertal stage) collected from local 21 210 SINGH ET AL. [Indian Journal of Animal Sciences 80 (3) abattoir in normal saline solution (NSS), having 100 IU/ml streptomycin) kept at 4 ºC. In laboratory the oviducts were penicillin and 100 microgram/ml streptomycin sulphate at cleared from ligaments, connective tissue and adjoining blood 37°C were brought to the laboratory and washed with vessels and 2 cm of the ampullary and isthmic region excised. 1physiological normal saline solution and DPBS (Dulbecco’s The trimmed oviducts were then subjected to washing at least phosphate buffer saline, PH 7.3–7.4) before subjecting to for 3-times with sterile physiological saline solution the oocytes recovery. supplemented with antibiotics. Oocytes maturation: The oocytes were recovered by After washing with physiological saline solution, the puncturing the visible surface of atretic follicle (1–5 mm oviducts were washed 3-times with Dulbecco’ s phosphate diameter) with an 18–gauge needle into a 35 mm sterile buffer saline solution (DPBS) kept at 37°C. The oviduct was petridish containing oocytes collection media (OCM). placed in a petri-dish and with the help of a pair of forceps Oocytes were graded as per Kharche et al. (2008). its lumen was squeezed, which yield in thick fluid containing The selected cumulus oocyte complexes (COCs) were oviductal epithelial cells. The collected oviductal epithelial washed 10–20 times in 100 μl drop of oocyte handling media cells were mixed with oocyte collection media (OCM) and (OHM) followed by 8–10 washing in 100 μl drop of were subsequently passed through a syringe fitted with 26 maturation medium (MM) which had been preequilibrated gauge needle. The process resulted in collection of in a CO2 incubator at 38.5±1°C for 2 h. About 10–15 cumulus homogenized epithelial cells. oocyte complexes (COCs) were placed in micro drop of The homogenized oviductal epithelial cells were 50 μl of the maturation medium (TCM–199, M–7528) transferred to a centrifuge tube containing 5 ml of oocyte containing either 20% concentration of EGS or 20% NCS or collection media (OCM) and were subjected to centrifugation 10% NCS plus hormone (FSH @ 1 microgram/ml + LH @ at 200 rpm for 5 min. After centrifugation, the supernatant 100 IU/ml + E2 @ 1 microgram/ml) or 20% CFF which had was discarded with the help of a pipette without disturbing been equilibrated at 38.5°C in a CO2 incubator. The oocytes the oviductal epithelial cells pellet. The process of washing in the 50 μl droplets were covered with sterile mineral oil of oviductal epithelium cell pellets was repeated, which was and cultured in humidified atmosphere of 5% CO2 in air at followed by washing in maturation media (100 μl) (pre- 38.5±1 °C for 27 h in a CO2 incubator. The maturation of equilibrated at 38.5±1°C for 2 h in a CO2 incubator) for 2–3 oocytes was confirmed by cumulus expansion and presence times. of first polar body after 27 h of incubation with maturation The oviduct cell either in 100 or 200 μl droplets and media. covered with sterile mineral oil were cultured in a humidified The in-vitro capacitation and in-vitro fertilization of in- atmosphere of 5% CO2 in air at 38.5±1°C for 24 h in a CO2 vitro matured oocytes were done as per the procedure incubator. At the time of collection, oviductal cells were described by Singh et al. (2009). devoid of any cilliary (hair like projection on epithelium) Preparation of granulosa cells monolayer: Masses of goat movement but it appeared after 3–4 h of culture. After 20 h cumulus and granulosa cells were isolated in a petri-dish of culture, live cells were seen in rotatory motion. As expected following recovery of cumulus oocytes complexes (COCs) in the middle of drop cells showed restricted movements and washed 4–6 times in 100 μl drops of oocytes handling while those on the periphery were highly motile. After 3 days medium (OHM). Thereafter, 2–3 washings were given in 100 of culture most of the cell formed vesicles attaining oval or μl drops of maturation medium (MM) which had been pre- round shape and become more transparent resembling equilibrated in a CO2 incubator at 38.5±1°C for 2 h. After blastocyst. washing, packed cumulus cells were placed in a micro drops In-vitro culture: Eighteen hours post insemination, oocytes of 100 μl of the maturation medium (TCM–199, M–7528) were washed in a culture media (embryo development containing 20% concentration of EGS, which had been medium, EDM) 8–10 times to separate adhering sperm cell. equilibrated at 38.5±1°C in a CO2 incubator. The packed The oocytes were finally transferred either on monolayers cumulus cells in the 100 μl droplets were covered with sterile (equilibrated with EDM under oil for 18 h) or 50 μl drop of mineral oil and cultured in humidified atmosphere of 5% EDM co-incubated with oviductal epithelial cells under CO2 in air at 38.5±1°C in a CO2 incubator. After 48 h the sterile mineral oil (equilibrated for 2 h at 38.5±1°C in a CO2 expended cumulus masses forming monolayers were used incubator) for 48 h in a drop. They were cultured at 38.5±1°C for co-culture of presumptive zygote and early 2 cell embryos. at 5% CO2 in a CO2 incubator. Preparation of oviductal epithelial cells: Goat oviducts Assessment of cleavage rate and embryo development: were collected from slaughtered animal within an hour of Forty-eight hours post insemination, cleavage rate was their slaughter. Only those genitalia were selected where the evaluated by counting the number of cleaved >2 cell embryos/ ovaries contain either corpus haemorrhagicum or corpus number of oocytes used for fertilization. After evaluation of luteum. The genitalia were brought to the laboratory in a cleavage rate, embryo development was seen thermos flask containing physiological saline solution morphologically every 24 h under stereozoom microscope fortified with antibiotic (100 IU penicillin and 100 μg/ml and classified as 2 cell, 4 cell, 8 cell and >8 cell embryos 22 March 2010] EFFECT OF CO-CULTURE ON EMBRYO DEVELOPMENT 211 depending on a possible in-vitro cellular block. After every Improvement in the in-vitro fertilization and culture in farm 24 h, 50% of the media was replaced with a fresh pre- animals by incorporating granulosa cell monolayer co-culture equilibrated EDM media. could be due to the additional support in nutritional The difference between cleavage rates and embryo requirement or generation of specific signals to the development stages were analyzed by the chi square analysis developing oocytes (Staigmiller and Moor 1984). This (Sendecor and Cochran 1989). support can be further enhanced by granulosa cell monolayer as it produced growth factors (Mulheron and Schomberg RESULTS AND DISCUSSION 1992, Satoh et al. 1994). Statistical analysis (χ2 = 1.73) did not reveal any Several investigators reported the superiority of co-culture significant differences between the cleavage rates and with oviductal epithelial cells compared to culture without embryonic development (2 to >8 cell stage) (χ2 =3.37) of cells for goat (Sakkas et al. 1989, Prichard et al. 1991), cow two different groups of oocytes which were co-cultured with (Eyestone et al. 1989, Nagao et al.1994) and sheep (Gandolfi granulosa cell monolayer (Table 1). et al. 1987) embryo development. On the other hand it was Statistical analysis between prepubertal and pubertal also observed that cumulus and oviductal cells did not support cleaved oocytes, which was co-cultured with oviductal embryonic development (Wiemer et al. 1991, Shamsuddin epithelial cells also did not reveal any significant difference et al. 1993). (χ2=0.74) between the cleavage rates and embryonic In goat, Keskintepe et al. (1994) obtained higher number development (2 to >8 cell stage) (χ2 = 0.78) of 2 different of embryos reaching the morula stage with cumulus cells groups of collected oocytes (Table 2). than with oviductal epithelial cells using an atmosphere of In the present study 2 different co-culture systems i.e. 90% of N2 for embryo co-culture. However, Bavister (1988) granulosa cell monolayer and oviductal epithelial cells were opined that the role of oviductal tissue to supports early used for enhancing cleavage rate and embryo development development in vitro is not well known, but it seems likely of in-vitro matured prepubertal and pubertal goat oocytes. that these cells produce material beneficial to the young Several workers have used granulosa cell to induce nuclear embryos, remove substances with negative effects from the and cytoplasmic maturation and suggest that these cells media and/or reduced the oxygen concentration or both. enhance the rate of fertilization and subsequent Izquierdo et al. (1999) carried out study for cleavage and developmental capacity of the oocytes in bovine and caprine further embryo development of prepubertal goat oocytes. (Lu et al. 1988, Sharma et al. 2001, Teotia et al. 2001). After 24 h of co-culture in TCM–199 with bovine and caprine

Table 1. Effect of granulosa cell monolayer co-culture on cleavage rate and embryo development of in-vitro fertilized goat oocytes

Types of ovaries Total Total Recov- Oocytes Cleaved Unclea- Cleavage 2 cell 4 cell 8 cell >8 cell number of number ered used for oocytes ved rate (%) (%) (%) (%) (%) replicates of ovaries oocytes fertilization oocytes

Prepubertal 19 185 384 304 124 180 40.78 65.32 22.58 6.45 5.64 (81/124) (28/124) (8/124) (7/124) Pubertal 19 272 419 344 158 186 45.93 63.92 (27.26) 6.96 1.89 (101/158) (43/158) (11/158) (3/158)

*Nonsignificant (χ2 = 1.73) difference between the cleavage rate of 2 different groups of oocytes; *nonsignificant (χ2 =3.37) difference between the embryonic development stage (2 to >8 cell stage) of 2 different groups of cleaved oocytes.

Table 2. Effect of oviductal epithelial cell co-culture on cleavage rate and embryo development of in-vitro fertilized goat oocytes

Types of ovaries Total Total Recov- Oocytes Cleaved Unclea- Cleavage 2 cell 4 cell 8 cell >8 cell number of number ered used for oocytes ved rate (%) (%) (%) (%) (%) replicates of ovaries oocytes fertilization oocytes

Prepubertal 15 146 303 228 108 120 47.36 50.92 30.55 12.03 6.48 (55/108) (33/108) (13/108) (7/108) Pubertal 15 203 314 269 117 152 43.49 40.78 22.36 7.23 6.57 (62/117) (34/117) (11/117) (10/117)

* Nonsignificant (χ2 = 0.74) difference between the cleavage rate of 2 different groups of oocytes; *nonsignificant (χ2 =0.78) difference between the embryonic development stage (2 to >8 cell stage) of 2 different groups of cleaved oocytes. 23 212 SINGH ET AL. [Indian Journal of Animal Sciences 80 (3) oviduct epithelial and cumulus cells and following 48 h of oocytes and co-cultured with cumulus and oviductal cells. post insemination the cleavage rates in different culture Theriogenology 42: 591–600. systems ie caprine cumulus cells, bovine cumulus cells, Mulheron G W and Schomberg D W. 1992. Effects of caprine oviductal epithelial cells, bovine oviductal epithelial diethylstilbestrol on rat granulosa cells and thecal/intestinal cell transforming growth factor beta 2 mRNA expression in-vitro: cells and M–199 supplemented with 20% EGS were 42.6, analysis by reverse transcription polymerase chain reaction. 41.2, 41.5, 45.2 and 42.4%, respectively. In present study Biology of Reproduction 46: 546. we have used caprine oviductal epithelial cells and granulosa Nagao Y, Saeki K, Hoshi M and Kinuma H. 1994. Effects of oxygen cell monolayer (caprine) co-cultures for cleavage and further concentration and oviductal epithelial tissue on the development embryo development of prepubertal and pubertal goat of in vitro matured and fertilized bovine oocytes cultured in oocytes. A cleavage rate of 47.36 and 40.78% for caprine protein free medium. Theriogenology 41: 681–7. oviductal epithelial cells and granulosa cell monolayer Nandi S, Ravindranath B M, Gupta P S P, Raghu H M and Verma P for prepubertal goat oocytes were obtained. On the other S. 2003. Developmental competence and post-thaw survivability hand the cleavage rates for pubertal goat oocytes of buffalo embryos produced in-vitro: effect of growth factors in oocyte maturation medium and of embryo culture systems. were observed as 43.49 and 45.93% for caprine oviductal Theriogenology 60: 1621–31. epithelial cells and caprine granulosa cell monolayer. The Prichard J F, Pool S H, Blakewood E G, Menezo Y and Godke R A. present results are somewhat comparable with the above 1991. Culture of early stage caprine embryos using goat group of workers. oviductal cell monolayers. Theriogenology 35: 259. Various workers reported that the beneficial effect of the Sakkas D, Batt P A and Cameron A W N. 1989. Development of oviductal epithelial cells does not depend on the species from perimplantation goat (Capra hircus) embryos in vivo and in- which the cells are taken since oviductal epithelial cells from vitro. Journal of Reproduction and Fertility 87: 359–65. cattle and buffalo are equally competent for development of Satoh T, Kobayashi K, Yamashita S, Kikuchi M and Hashi H. 1994. goat embryo (Betteridge 1995, Yadav et al. 1998). Tissue inhibitor of metalloproteinase (TIMP-I) produced by granulosa and oviductal cells enhance in-vitro development of In conclusion, the present study indicates that both bovine embryos. Biology of Reproduction 50: 835–44. granulosa cell monolayer and oviductal epithelial cells co- Sharma G T, Teotia A and Majumdar A C. 2001. Meiotic culture behaved similarly for cleavage rate and embryo competence of caprine oocytes during IVM on granulosa cell development of in-vitro matured and fertilized prepubertal monolayers developed from small and large follicles in and pubertal goat oocytes. comparison to granulosa cell culture. Asian-Australian Journal of Animal Science 4: 777–84. REFERENCES Singh K P, Saxena A, Kharche S D and Singh P. 2009. 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24 Indian Journal of Animal Sciences 80 (3): 213–216, March 2010 Changes in endometrial histology following enrofloxacin therapy in crossbred cows suffering from endometritis*

J K PRASAD1, M S SAXENA2, S K RASTOGI3 and J P KORDE4

Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, Uttarakhand 263 145 India

Received: 19 January 2009; Accepted: 18 September 2009

ABSTRACT The histo-pathological changes in endometrium following enrofloxacin therapy were studied in crossbred cows suffering from endometritis. Twelve cows (group A) were treated with intra-uterine enrofloxacin (1 000 mg in 30 ml distilled water) for 3 consecutive days, and 12 cows (group B) were infused with 20 ml of PBS in an identical schedule. Endometrial biopsies were collected prior to treatment and at 72 h after treatment from all the cows for histo-pathological studies. Enrofloxacin treatment of this study caused the histopathological changes in endometritis cows during post– treatment period. The effects were evident by an additional increase of 25% cows showing denuded epithelium at 72 h post-treatment. Further, a poor recovery of cases with pseudostratification with reduced cellular infiltration following antibiotic treatment indicated an adverse effect of enrofloxacin on uterine defense mechanism. On the other hand, infusion of PBS did not cause any such changes in endometrial epithelium of cows under control group. It, however, reduced the number of cases with denuded epithelium with an appreciable increase in the infiltration of mononuclear and PMN cells. It is concluded that intra-uterine infusion of enrofloxacin may cause changes in endometrial histology and have some adverse effect on uterine defense mechanism in the cows suffering from endometritis.

Key words: Cows, Endometritis, Endometrium, Enrofloxacin, Histopathology

Endometritis is a superficial inflammation of the Antibiotics are very commonly used in the treatment of endometrium only, extending no deeper than the stratum endometritis in bovines. Tetracycline among those is reported spongiosum. Histologically, endometritis is characterized by to be effective for treatment of most of the mixed bacterial some disruption of surface epithelium, infiltration of infections of postpartum uterus (Olson et al. 1986). However, inflammatory cells, vascular congestion, stromal edema and it causes irritation to the endometrium, might induce by varying degrees of lymphocyte and plasma cell endometrial inflammation and reduced phagocytosis (Singh accumulation in the superficial layers (McEntee 1983, De- et al. 1997). Recent reports indicated that enrofloxacin is Bois and Manspeaker 1986). The detrimental effect of non-irritant in nature and proved to be good choice for intra- endometritis on subsequent fertility is well known. The uterine therapy in repeat breeder in bovines (Markandeya et incidence of the condition varies between 8.5 to 56.6% in al. 1999, Rane et al. 2003). But studies pertaining to its effect repeat breeder bovines (Rahumathulla et al. 1986, Maneta on endometrial histology and uterine defense mechanism et al. 1990, Singh and Sinha 1991, Mohanty et al. 1992, (UDM) has not been studied to the best of our knowledge. Sawamukai et al. 1994). Causes of endometritis are Therefore, in this study, we have examined the changes in multifactorial in nature. Bacterial endometritis occurs more endometrial histology and UDM after enrofloxacin treatment often than others with an incidence ranges from 77 to 100% in cows suffering from endometritis. amongst the repeat breeding cows (Easley et al. 1951, MATERIALS AND METHODS Dholakia et al. 1987, Shukla 1988, Singh et al. 1989, Dohmen et al. 1996). Crossbred cows (24) with endometritis were selected on the basis of per-rectal examination followed by appearance * Part of Ph.D. work of first author. Present address: 1Associate Professor, 2Formerly Professor and of cervical mucus and white side test. Cows were divided Head, 3Professor and Head, Department of Animal Physiology. randomly into 2 groups. In group A cows (12) were infused 4Associate Professor, Department of Animal Physiology, JNKV, with enrofloxacin @ 1 000 mg, intrauterine consecutively Rewa, Madhya Pradesh. for 3 days, and group B 12 cows served as untreated control 25 214 PARSAD ET AL. [Indian Journal of Animal Sciences 80 (3) in which PBS (20 ml) was infused intrauterine once. enrofloxacin on histopathological changes of endometrial Endometrial biopsy samples were collected from all cows epithelium have been studied with respect to denudation of for histopathological studies at 0 h (before treatment) and epithelium, pseudostratification and cellular infiltration. 72 h (after treatment). The biopsy technique consisted of Overall, the histopathological changes were recorded in 5 collecting a representative piece of endometrium aseptically cows prior to treatment, which increased by 25% to 8 using Nielson’s uterine biopsy punch from either of the horns, following enrofloxacin treatment. But intra-uterine infusion 2 to 3 inches deep from the uterine body. of PBS did not alter the changes in terms of population of cows affected at pre-treatment and post-treatment period in RESULTS AND DISCUSSION this study. All the experimental cows of this study were positive for Denuded endometrial epithelium was observed in 2 cows white side test and majority of them had purulent or at pre-treatment, which increased to 5 cows at the end of mucopurulent discharge prior to the assignment of the antibiotic treatment (Fig. 1). Further, cellular infiltration was treatment. After enrofloxacin treatment, about 91.7% cows reduced at the end of enrofloxacin treatment probably showed clear cervical mucus during post-treatment heat while because of the marked inhibition/destruction of the only 25% cows in control group showed clear cervical phagocytic activity following antibiotic therapy (Jayappa discharge. The response in relation to recovery obtained after and Loken 1983, Paisley et al. 1986). While in control antibiotic therapy in our study supports the previous findings group, denuded epithelium was found in 3 cows (Saini et al. 1999, Rane et al. 2003). Although, a larger pre-treatment, to 1 at post-PBS treatment with an appreciable population (91.7%) of cows showed clear mucus discharge increase in the infiltration of mononuclear and PMN cells in but only about 75% cows showed negative white side test 2 affected cows. following intrauterine infusion of enrofloxacin. The In the present study, the changes associated with discrepancy in recovery rate by visual observation and white endometrial epithelium were observed mostly in mild side test might be due to changes of mild endometritis with endometritis cases. The natural uterine defense remains less number of leucocytes causing positive white side test affected with antibiotic therapy demonstrating its harmful but unable to reveal visible changes in cervico-vaginal impacts as previously documented (Hoedemaker 1998). response. Endometrial glands: In this study, histopathological changes in endometrial glands were recorded in cows of both Changes in histopathology of endometrium enrofloxacin (2/12; 16.7%) and control groups prior to the Various histopathological changes were observed in the treatment. The changes like glandular hyperplasia (1/12; endometrium of cows prior to and following antibiotic 8.3%) and atrophied endometrial glands (1/12; 8.3%) were treatment in this study. The changes were recorded in the recorded in enrofloxacin group while glandular hyperplasia endometrial epithelium and glands, cellular infiltration of (1/12,8.3%), atrophy of enmetrial glands (1/12; 8.3%), stratum compactum and spongiosum with lymphoid nodule denuded and detached epithelial cells (1/12; 8.3%) as well formation and hyaline degeneration etc. as cellular infiltration of endometrium (2/12; 16.7%) were Endometrial epithelium:The effects of intra-uterine noticed in untreated cows. One cow in enrofloxacin group,

Fig 1. Denuded epithelium, infiltration of mononuclear cells, Fig 2. Extreme glandular-cystic dilatation with necrosis of hemorrhage in stromal tissue and hyperplasia of endometrial gland. stroma. H & E stain. 400 ×. H & E stain. 400 ×. 26 March 2010] CHANGE IN ENDOMETRIAL HISTOLOGY IN CROSSBRED COWS 215 however, developed into cystic dilatation of endometrial glands after 72 h of treatment (Fig. 2). This is possibly because of repeated intervention of endometrium by intrauterine infusion of antibiotic leading to chronic inflammatory conditions i.e chronic endometritis. The said cow finally did not conceive and remained non-pregnant. Cellular infiltration: Prior to the treatment, the infiltration of mononuclear cells (MNCs) was observed in 25% (3/12) cows in both the groups. But after 72 h of enrofloxacin infusion, all the 3 cows recovered. However, infiltration of a large number of MNCs was still noticed in majority of the untreated cows (2/3) after 72 h, probably they turned into sub-acute stage. In the present study, enrofloxacin infusion induced necrotic changes in the endometrial stroma in few cows after 72 h of treatment. Besides, distorted endometrial stroma was also recorded in some enrofloxacin treated cows. Some of Fig 4. Glandular hyperplasia with hyalinization of stromal tissue the untreated cows also showed loss of stromal tissue in this at 72 h in control group, H & E stain. 400 ×. study, about 16.6% of which did not conceive. Lymphoid nodule formation: Lymphoid follicles in the endometrium are not usual. Most probably they represent showed lymphoid nodules at 72 h (Fig. 3), which was merely an exaggerated but physiological reaction of a local although absent at 0 h. Low grade of infections for a lymphatic tissue. These follicles act as a mechanism of prolonged period of time probably elicit lymphoproliferative defense against noxious agents, not only exogenous but reactions causing lymphoid nodule formation in our study. endogenous as well (Dallenbach-Hellweg 1981). It is Hyaline degeneration: Hyaline degeneration generally considered that low grade of infections may elicit appears in sub-acute and chronic type of endometritis. Such lymphoproliferative reactions with formation of lymphoid degenerative changes were recorded only in one untreated nodules and follicles to meet the demand of differentiation cow in this study, which was characterized by heavily into other cell types i.e. plasma cells and multipotential stem infiltrated mononuclear cells in the stroma that did not cells. These cells also supply nutrients to other cells and to subside later on (Fig.4). participate in the immune response. It is concluded that intra-uterine infusion of enrofloxacin In the present study, lymphoid follicles were observed in may cause changes in endometrial histology and have some 2 cows in enrofloxacin group prior to the initiation of the adverse effect on uterine defense mechanism in the cows treatment. However, in untreated control group, only 1 cow suffering from endometritis. Future studies are, therefore, warranted to confirm such adverse effect involving a larger population of animals. ACKNOWLEDGEMENTS Authors are thankful to Dean, College of Veterinary and Animal Sciences, and Director, Experiment Station, Pantnagar for providing all the necessary facilities during the course of study.

REFERENCES

Dallenbach-Hellweg G. 1981. Histopathology of Endometrium. 3rd edn, pp. 30–31. Springer-Verlag Berlin Heidelgerg New York. DeBois C H W and Manspeaker J E. 1986. Endometrial biopsy of the bovine. Current Therapy in Theriogenology. (Ed.) Morrow D A. 2nd edn, pp. 424–26. Philadelphia, W.B. Saunders Company. Dholakia P M, Shah N M, Purohit J H and Kher N N. 1987. Bacteriological studies on nonspecific genital infection and its Fig 3. Denuded / desquamated epithelial cells in the lumen and antibiotic spectra in the repeat breeders. Indian Veterinary lymphoid nodule formation in the periglandular area and infiltration Journal 64: 637–40. of mononuclear cells H & E stain. 400 ×. Dohmen M J W, Husjenicza Gy, Fodor M, Kulcsar M, Vamos M, 27 216 PARSAD ET AL. [Indian Journal of Animal Sciences 80 (3)

Porkolab L, Szilagyi N and Lohuis J A C M. 1996. Bacteriology buffaloes. Cherion 15: 78–89. (fide Veterinary Bulletin 1987, and fertility in healthy postpartum cows and cows with acute 57: 993.) endometritis. XIX World Buiatrics Congress. Edinburgh, 2–12 Rane R S, Jadhav R H, Mazkori R J and Swami S S. 2003. Efficacy July, 1: 238–40. of intrauterine enrogil in the treatment of repeat breeding in the Easley H T, Leonard R H and Trotter D H. 1951. Bacteriological, buffaloes. Indian Veterinary Journal 80: 169–72. pathological and clinical studies of reproductive tract of the Saini P S, Nanda A S, Grewal A S and Singh J. 1999. Uterine defense Hereford cows and bacteriological studies of Hereford bull modulation for the treatment of repeat breeding due to infectious semen. Preliminary report. North America Veterinarian 32: 258– endometritis in bovines. Indian Journal of Animal Sciences 69: 66. (fide Singh, 1979.) 307–09. Hoedmaker M. 1998. Pospartal pathological vaginal discharge: to Sawamuki Y, Itho J and Togashi R. 1994. Relationship between treat or not to treat? Reproduction in Domestic Animals 33: 141– bacteriological and histopathological finding and fertility in 6. cows with clinical endometritis. Proceedings of XVIII World Hussain A M. 1989. Bovine Uterine Defense Mechanisms: A Buiatrics Congress. XXVI Congress of the Italian Association Review. Journal of Veterinary Medical Services B 36: 641–51. of Buiatrics, Aug 29–Sep 2, Bologna, Italy. pp. 305–8. Jayappa H G and Loken K I. 1983. Effect of antimicrobial agents Shukla S P. 1988. ‘Studies on biochemical changes in the uterine and corticosteroids on bovine polymorphonuclear leucocyte fluid in relation to bacteriology and histopathology of uterus in chemotaxis. American Journal of Veterinary Research 44: 2155– repeat breeding cattle.’ Ph.D. Thesis, Punjab Agriculture 59. University, Ludhiana. Korudzhiiski N, Tasankova S, Bonovska M and Kaflov K. 1988. Singh B and Sinha A K. 1991. Reproductive problems in crossbred Effect of some antibiotics on non-specific defense mechanisms cattle. National Symposium on Recent Biotechnological of cows and its significance in development of chronic Advances in Animal Reproduction and 9th National Convention endometritis. Veterinary Bulletin 59: 1062. of ISSAR, HAU, Hisar. 6–8th Feb. 1991 Maneta M, Elezov G, Angelov A and Stoev S. 1990. Clinical and Singh B, AminuDeen, Gupta A K and Srivastava A. 1997. morphological changes in reproductive organs of infertile cows Therapeutic trials on infertile cows. Indian Veterinary Journal due to chronic inflammatory process. Veterinary Sbirka 88: 60– 74: 265–6. 3 (fide Veterinary Bulletin 1991 61: 29–30.) Singh J, Nanda A S, Dhaliwal G S and Pangaonkar G R. 2003. Markandeya N M, Bhikane A V and Deshpande A R. 1999. Clinical Treatment of bacterial endometritis in crossbred cows using response of enrofloxacin in infections repeat breeder cows and intrauterine oyster-glycogen, a non-specific immunomodulator. buffaloes. National symposium on biotechniques in optimizing Indian Journal of Animal Sciences 73: 844–7. fertility in farm animals: 49. (fide Rane et al. 2003) Singh K C P, Pandey J N, Sinha M N, Prasad C B, Prasad C R and McEntee K. 1983. The female genital system. Pathology of Singh S S. 1989. Studies on genital microflora of repeat breeder Domestic Animals. (Eds) Jubb K V F, Kennedy P C and Palmer cows. Indian Journal of Veterinary Medicine 13: 61–3. N, Academic Press, Orlando, FL. Studer E and Morrow D A. 1978. Postpartum evaluation of bovine Mohanty B C, Mohanty B N, Ray S K H and Mohanty D N. 1992. reproductive potential; comparison of findings from genital tract, Clinical and therapeutic studies of bovine endometritis. Indian examination per-rectum, uterine culture and endometrial biopsy. Veterinary Journal 69: 379–80. Journal of American Veterinary Medical Association 172: 1966– Paisley L G, Mickelsen W D and Anderson P B. 1986. Mechanisms 72. and therapy for retained fetal membranes and uterine infections Zerbe H, Schuberth H J, Hoedemaker M, Grunert E and Leibald of cows: A review. Theriogenology 25: 353–81. W. 1996. A new model system for endometritis basic concepts Rahumathulla P S, Rajasundram R C and Gajendran K. 1986. and characterization of phenotypic and functional properties of Incidence of various reproductive disorders among cattle and bovine uterine neutrophils. Theriogenology 46: 1339–56.

28 Indian Journal of Animal Sciences 80 (3): 217–219, March 2010 Serum lipid profile during lactation in buffalo

P M TRIPATHI1, S D INGOLE2, B T DESHMUKH3, A S NAGVEKAR4 and S V BHARUCHA5

Maharashtra Animal and Fishery Sciences University, Nagpur, Maharashtra 400 016 India

Received: 20 November 2008; Accepted: 6 November 2009

ABSTRACT Serum lipid profile was studied during gestation in 18 lactating Murrah buffaloes. The animals were grouped as early, mid and late lactation. The differences amongst 3 stages of lactation in all the lipid components studied were statistically highly significant. The serum total cholesterol, HDL cholesterol, triglycerides, phospholipids increased from early to mid stage of lactation and then decreased from mid to late stage of lactation, while LDL cholesterol increased with advancing stage of lactation. This increase may be related to the effect of estrogen on carbohydrate metabolism, which in turn causes increases in production of cholesterol in endocrine gland tissue from acetate and also due to demand of udder for fatty acid synthesis for milk fat. However, serum NEFA differed significantly with decreased level during mid lactation and this decrease from early to mid lactation is attributed to high energetic requirement during first stage of lactation that cannot be supported by dietary intake and thus buffalo must utilize body fat as source of energy. The correlation between serum lipid profile with milk fat per cent was nonsignificant with each other during all stages of lactation.

Key words: Buffaloes, Lactation, Serum lipids

The blood lipids play an important role in milk fat experimental animals in early lactation group were synthesis in dairy animals (Bauman and Davis 1974). inseminated, and those in mid and late stages of lactation Evidence for the utilization of blood lipids for milk fat were pregnant in early to mid stages of gestation. The synthesis comes from arterio venous differences. The buffaloes were maintained under standard managemental and triglycerides, phospholipids, cholesterol-esters and protein nutritional conditions. The average maximum temperature, bound unesterified fatty acids are the major blood sources minimum temperature and relative humidity recorded during of milk fatty acids. Thus the role of blood lipids in milk fat the period of study were 23.60±0.68 and 16.70 ±0.39 °C and synthesis is quite evident. But scanty information is available 95±1.29% respectively. on the blood lipids during lactation in buffaloes. Hence it Jugular blood samples (8–10 ml) were collected was decided to study lipid profile during lactation in aseptically in morning hours between 8 AM and 10 AM. Clear buffaloes. serum was separated by centrifugation. The blood samples were collected for 6 consecutive days and milk samples were MATERIALS AND METHODS collected in plastic bottles from all the buffaloes on days 1, Apparently healthy lactating Murrah buffaloes (18) in 3 and 5 during the experimental periods, for determination fourth to sixth lactation selected from ShriWarana Sahakari of fat content. The serum samples were stored at –20° C Dudh Utpadak and Prakriya Sangh Ltd, Warana Nagar, until used for biochemical analysis. Kolhapur, were divided into 3 groups, 6 in each, based on Estimation of serum total cholesterol, triglycerides, HDL their stage of lactation: early (days 51–65), mid (days 125– cholesterol and LDL cholesterol were done by using an 160) and late (days 198–262). At the start of the experiment, autoanalyser, phospholipids by using spectrophotometer and the average ages of experimental animals was 6 to 8 years NEFA by copper soap formation method (Falholt et al. 1973). and were yielding on average 6.6 to 12.5 kg milk. The Fat content in milk was estimated using fat analyzer on fresh milk samples. The data were analysed as per Snedecor and Cochran (1998). Present address: 1Senior Research Fellow, RBR Unit, Anand Agricultural University, Anand. RESULTS AND DISCUSSION 2Associate Professor, 3Professor, 4, 5Assistant Professor, Department of Physiology and Biochemistry, Bombay Veterinary Serum total cholesterol: The highest value of serum total College, Parel, Mumbai 400 012. cholesterol was recorded during mid lactation, least during 29 218 TRIPATHI ET AL. [Indian Journal of Animal Sciences 80 (3)

Table 1. Serum lipid profile during lactation in buffaloes

Stage of Serum total Serum HDL Serum LDL Serum Serum Serum non- lactation cholesterol cholesterol cholesterol triglycerides phospholipids esterified fatty (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) acids (μmol/litre)

Early lactation 154.19c±1.13 80.29c±1.14 70.55b±0.29 16.66c±0.57 139.12c±1.60 454.33a±21.01 Mid lactation 198.73a±0.68 112.03a±0.94 80.96a±0.46 28.40a±0.42 196.98a±1.39 417.41b±19.39 Late lactation 173.97b±1.76 87.98b±2.35 81.65ac±1.66 21.80b±0.46 186.72b±3.11 460.15ac±23.53

Mean with atleast one common superscript do not differ significantly (P<0.05). early lactation and intermediate during late lactation lactation and intermediary during mid lactation. The increase (Table 1). The differences in serum total cholesterol in serum LDL cholesterol concentration amongst the early concentration amongst 3 stages of lactation were significant and mid, and in early and late stages of lactation were (P<0.01). Our results are in agreement with those obtained statistically highly (P<0.01) significant. During late lactation by Polat et al. (2002) and Grasso et al. (2004) who found the the levels were highest as compared to early and mid same trend as increased levels from early to mid lactation lactation. Similar trend was observed by Basoglu et al. (1998) due to high energy requirement of animal during first stage during lactation but the values were lower than the present of lactation. However decreased levels were reported by study. However the lower levels of LDL cholesterol during Stanley et al. (1991), Singh et al. (2003) and Jagatheesan et early lactation are due to increased LDL catabolism or the al. (2005). Higher values were reported by Setty and Razdan reduced transformation of VLDL into LDL (Sevinc et al. (1966) since the gonadal steroid which have correlation with 2003) cholesterol metabolism, are produced in much greater amount Triglycerides: The serum triglycerides exhibited the during these stages. Prakash and Tandon (1979) found the identical trend to that of serum total cholesterol and HDL increased level of serum cholesterol and were due to the cholesterol. The lowest serum triglycerides concentration was mechanism by which estrogen affected the complex recorded during early lactation followed by increase during interrelationship of pituitary-thyroid-adrenal function. The mid stages of lactation and then decreased during late estrogen had an effect on the carbohydrate metabolism that lactation. The differences in serum triglycerides in turn caused increased production of cholesterol in concentrations amongst 3 stages of lactation were statistically endocrine gland tissue from acetate. significant (P<0.01). The observation of the present study HDL cholesterol: The serum HDL cholesterol followed are in accordance with Varman and Schultz (1968) and the similar trend to that of serum total cholesterol. The Basoglu et al. (1998) who observed similar trend during early differences in serum HDL cholesterol concentration amongst and mid lactation. Sevinc et al. (2003) reported high 3 stages of lactation were statistically significant (P<0.01). triglycerides levels during 1 - 6 weeks of lactation as During early lactation the levels were lower than mid compared to present study and according to them low lactation and late lactation. Basoglu et al. (1998) also found triglycerides can be thought to be caused by an influx of free HDL cholesterol activity significantly high in late lactation fatty acids from adipose tissue near the time of parturition than early lactation and prepartum. The decreased level of and a low out put of lipoprotein by liver. The rapid increase HDL cholesterol during early and late lactation may be related in the triglycerides during lactation may be attributed to to the lower cholesterol level during same period as HDL increase demand of the udder for fatty acid synthesis for milk consists of about 60% cholesterol (Sevinc et al. 2003). fat, and also to lowest level of circulatory estrogen and LDL cholesterol: Serum LDL cholesterol concentration thyroxin profile which influence the lipid metabolism gradually increased from early to late lactation. The highest (Tainturier et al. 1984). values were recorded during late lactation, least during early Phospholipids: The serum phospholipids exhibited the

Table 2. Correlation of fat content in milk with lipid components in serum in buffaloes

Stage of Serum lipid components lactation Total HDL LDL Triglycerides Phospholipids Nonesterfied cholesterol cholesterol cholesterol fatty acids

Early 0.16NS 0.22 NS –0.516 NS –0.099 NS 0.592 NS 0.106 NS Mid –0.018 NS –0.103 NS 0.396 NS –0.579 NS –0.206 NS –0.45 NS Late 0.22 NS 0.378 NS –0.594 NS 0.235 NS –0.556 NS 0.617 NS

30 March 2010] SERUM LIPID PROFILE IN BUFFALOES 219 identical trend to that of serum total cholesterol and HDL Bauman D E and Davis C L. 1974. Biosynthesis of Milk Fat in cholesterol. The lowest serum phospholipids concentration Lactation. A Comprehensive Treatise.Vol.II. (Eds) Larson B L was recorded during early lactation followed by increases and Smith. Academic Press INC (London) Ltd. 31–75. during mid stages of lactation and then decreased during late Falholt K, Lund B and Falholt W. 1973. Lipids and lipoproteins. Practical Clinical Biochemistry. Vol I. (Eds) Varley H, lactation. The differences in serum phospholipids Gowenlock A H and Bell M. William Heinemann Medical concentrations amongst 3 stages of lactation were statistically Bookd Ltd., London. highly (P<0.01) significant. The observations of the present Grasso F, Terzano G M, Napolitane F, Rosa G D and Tripalidi C. study are in accordance with Varman and Schultz (1968) who 2004. Influence of housing conditions and calving distance on observed the same increasing trend from early lactation to blood metabolites in water buffalo cows. Italian Journal of late lactation. The highest concentration of phospholipids Animal Sciences 3: 275–82. during mid and late lactation could be due to increased Hayashi T, Shah J and Kumagai H. 2005. Dairy production and demand of nutrients for milk fat synthesis during lactation. nutritional status of lactating buffalo and cattle in small scale Non esterified fatty acids: There was a significant (P<0.01) farms in Terai. Nepal Livestock Research for Rural Development 17 (6). difference in the serum non-esterified fatty acids Jagatheesan P N R, Selvaraju M, Kumar V R S and Chandrahason concentrations in the early lactation and mid lactation and in C. 2005. Effect of advanced pregnancy and early lactation on the early lactation and late lactation. The result revealed that blood biochemical constituents in Murrah buffaloes. Indian the levels decreased from early lactation to mid lactation Veterinary Journal 82 (4): 401–03 and again increased during late lactation. These results are Polat U, Cetin M and Yalcin A. 2002. The relations between some in agreement with those obtained by Grasso et al. (2004) biochemical blood parameters and milk yield during various who observed the same trend as decreased levels from early lactation stages in high yielding dairy cows. Uludag University to mid lactation and this decreased level during mid lactation Journal Faculty of Veterinary Medicine 21: 65–9. may be attributed to high energetic requirement during first Prakash B S and Tandon R N. 1979. A note on the effect of late pregnancy and early lactation on blood serum cholesterol and stage of lactation that cannot be supported by dietary intake. total lipids on Holstein × Tharparkar first lactation cows. Indian As a result the buffalo must utilize body fat as a source of Journal of Animal Sciences 49 (4): 308–09. energy. But this result was not in agreement with Varman Setty S V S and Razdan M N. 1966. Studies on the chemical and Schultz (1968) who observed the increased level of free composition of blood in dairy cattle. Indian Journal of Dairy fatty acids during early lactation as compared to late lactation. Science 19: 5–59 They postulated that this unusual observation at this particular Sevinc M, Basoglu A and Guzelbektas H. 2003. Lipid and time of lactation (early lactation and under certain lipoprotein levels in dairy cows with fatty liver. Turkish Journal circumstances) may be due to greatly enhanced mobilization of Veterinary Animal Sciences 27: 295–9. of the depot fat and also a possible change in the ability of Singh M, Tiwari D P, Kumar A and Kumar M. 2003. Effect of feeding transgenic cotton seed vis-a-vis non transgenic cotton the mammary gland secretary tissue to remove and seed on haematobiochemical constituents in lactating Murrah metabolize higher amounts of plasma free fatty acids. buffaloes. Australian Journal of Animal Science 16 (12): 1732– However, the low levels were observed by Hayashi et al. 37. (2005) and high level were observed by Vanzquez anon et Snedecor G W and Cochran W G. 1998. Statistical Methods. 8th al. (1994) during early pregnancy. edn. Oxford and IBH Publishing Company, New Delhi. The correlation (Table 2) between serum total cholesterol, Stanley T A, Cochram R C, Harmon D L and Vanzant E S. 1991. HDL cholesterol, LDL cholesterol, triglycerides, Periparturient changes in intake, rumen capacity and selected phospholipids and NEFA levels with milk fat during early, blood metabolites in beef cows. Cattlemens Day 57–59 mid and late lactation was nonsignificant. Tainturier D, Braun J P, Rico A G and Thouvenot J P. 1984. Variations in blood composition in dairy cows during pregnancy REFERENCES and after calving. Indian Veterinary Journal 37: 129–31 Varman P N and Schultz L H. 1968. Blood lipids of cows at different Basoglu A M, Sevinc M, O k M and Gokcen M. 1998. Pre- and stages of lactation. Journal of Dairy Science 51: 1971–74. post parturient concentrations of lipid, lipoprotein, insulin and Vazquez anon M, Bertics S, Luck M and Grummer R R. 1994. glucose in normal dairy cows. Tropical Journal of Veterinary Peripartum liver triglyceride and plasma metabolites in dairy Animal Sciences 22: 141–44. cows. Journal of Dairy Science 77: 1521–23.

31 Indian Journal of Animal Sciences 80 (3): 220–224, March 2010 Clinico-haematobiochemical profile in chronic anemic crossbred cattle

R K BHARDWAJ1, C S RANDHAWA2, S S RANDHAWA3 and P S DHALIWAL4

Guru Angad Dev University of Veterinary and Animal Sciences, Ludhiana, Punjab 141004 India

Received: 20 April 2009; Accepted: 23 September 2009

ABSTRACT Crossbred cows (250) of 3–9 years of age, were screened for gastrointestinal parasites and blood protozoan/rickettesia and acid-fast bacilli (AFB) in faeces. Anemic, apparently healthy crossbred cows (43), negative for gastrointestinal parasites, AFB in faecal smears and protozoa/rickettesia in blood smear were finally sampled to establish the etiology of chronic anemia in crossbred cows. The blood samples were analyzed for haematological parameters, viz. Hb, PCV, TEC, red cell indices, red cell morphology and regenerative response in blood smear. Bone marrow aspirate examination was done to calculate M:E ratio and to assess iron stores in ten anemic cows to see regenerative response. Blood was also analyzed for biochemical parameters, viz. iron biochemistry, total bilirubin, TPP, albumin, globulin, A:G ratio, fibrinogen, PUN and TPP:Fib. ratio. Anemia was mild in 55.8%, moderate in 30.3% and severe in 13.9% of crossbred cows. Normal plasma iron and normal TIBC was observed in 55.9% of anemic cows. Hypoproteinemia and hypoalbuminemia was detected in only 4.65% cows, low A:G ratio in 16.3% and low TPP: Fib. ratio in 11.6% of anemic cows. Plasma urea nitrogen was significantly low in anemic cows. Mean M:E ratio was low (0.6±0.05) in anemic as compared to non-anemic (1.02±0.03) cows suggestive of regenerative anemia . Bone marrow stores were adequate in 7 and low in 3 anemic cows. Considering morphology of red cell, iron biochemistry, A:G and TPP:Fib. ratio, tentative diagnostic possibilities of idiopathic chronic anemia was evolved as anemia of chronic inflammatory diseases in 30.2%, early iron deficiency in 16.3%, iron deficiency in 9.3%, nutritional factors other than iron in 16.3% and etiology was obscure in 9.3% of chronically anemic cows. Trypanosoma evansi infection was etiology of chronic anemia in 18.6% cows.

Key words: Bone marrow aspirate, Cattle, Chronic anemia, Fibrinogen, Hemogram

Anemia in cattle is of great clinical importance because study on clinico-haematobiochemical profile in chronic of the direct and indirect economic losses. Direct losses are anemic crossbred cattle of Punjab. due to mortality and morbidity and indirect losses are in the MATERIALS AND METHODS form of reduced production and physical performance. Baseline surveys in Punjab showed that anemia is widely Crossbred cows (250) with mean age range of 3–9 years, prevalent in crossbred cattle compared to buffaloes and were screened from rural dairy units of Punjab. Cows having incidence of chronic anemia varied from 30 and 25% (Singh (Hb<8 gdl–1 and PCV<24%) were categorized as anemic. 1999), 26.4 and 13.2% (Singh 2002) and 30 and 25% These anemic cows were examined on 3 occasions for (Heigopal 2003) on hemoglobin and packed cell volume basis gastrointestinal (GIT) parasites and AFB in faeces and blood respectively. Though, the anemic cows were apparently smears for protozoan/rickettsia, viz. Babesia spp or Theileria healthy, subclinical effects are expected because of spp or Anaplasma spp infection. Cows (43) found negative physiological adjustments in cardiovascular system (Jain for GIT parasites and blood protozoa/rickettsia were finally 1986). Sarkar et al. (1996) reported infertility in the form of taken up to determine the etiology of chronic anemia. Thirty- increased services per conception and delayed postpartum five non-anemic cows (Hb>8 gdl–1 and PCV>24%) were ovarian activity in crossbred anemic cows and high mortality selected as healthy control. History about the feeding regime, rate of 36.5% in anemic crossbred cattle due to debility and weight loss, and consistency of faeces, urine discoloration, weakness followed by pneumonia in 25% of anemic any previous illness or any underlying disease was recorded. crossbred cattle (Chavai et al. 2001). The present article is a Anemic animals were examined for general body condition, color of conjunctival mucous membrane and heart rate. Present address: 1Assistant Professor, 2,3,4Professor, Division The blood samples were collected from each cow (15–20 of Clinical Veterinary Medicine Ethics and Jurisprudence. ml) in heparinised and (2–3 ml) in disodium EDTA mineral 32 March 2010] CLINICO-HAEMATOBIOCHEMICAL PROFILE IN CHRONIC ANEMIC CATTLE 221 free screw capped glass vials. The blood samples were Table 2. Classification of anemia on basis of hemoglobin in examined for Hb, PCV and TEC within 5–10 h of collection anemic cows (mean±SE) (Jain 1986). Reticulocyte response was observed by new methylene blue staining. Ziehl’s Neelson staining of faecal Parameter Mild anemia Moderate Severe (7–8 gdl–1) anemia anemia smears was done for demonstration of acid-fast bacilli (Carter (n=24) (6–7 g dl–1) (<6 gdl–1) 1975). Bone marrow aspiration, from dorsal end of 9 to 11th (n=13) (n=6) ribs was collected aseptically using Klima needle from 10 anemic, and 5 healthy cows. Bone marrow aspirate smears Hb (gdl–1) 7.65±0.06a 6.46±0.08b 5.23±0.15c were prepared, air-dried and fixed (Freeman 2000). Bone PCV (%) 22.6±0.35a 19.9±0.62b 18.3±1.30c 6 –1 a 16ab b marrow aspirate smears were stained by giemsa and TEC (10 ml ) 5.36±0.13 4.56±0. 4.11±0.23 a a a heamatoxylin and eosin stain, for myeloid to erythroid ratio MCV (fl) 42.8±1.06 44.0±1.52 43.4±0.37 MCH (pg) 14.4±0.30a 14.38±0.52a 12.8±0.50a (M:E) (Bruce and Blue 2000). Bone marrow iron stores were MCHC (%) 33.9±0.63a 32.8±1.03a 29.0±1.34a evaluated by staining of bone marrow smears with Prussian blue stain (Luna 1968). Simultaneously blood samples were collected from healthy and anemia cows for TLC and DLC. anemia was a usual feature of non-regenerative anemia of Plasma was analyzed for total plasma Fe and total iron inflammatory diseases, chronic infections, gastrointestinal binding capacity (Caraway 1962) and per cent transferrin worms, ectoparasites infestation or infection of saturation was calculated. Total plasma proteins and albumin Eperythrozoon wenyoni (Morris 2001). Mean value of TEC were estimated by biuret method and BCG method was significantly lower compared to non-anemic cattle. Total respectively. Globulins were determined by taking the erythrocyte count was less than 5×106 ml–1 in 53.5% of difference in total plasma proteins and albumin and A/G ratio anemic cows. Low erythrocyte count with hemoglobinemia, was derived. Plasma fibrinogen was estimated using heat hyperbilirubinemia, hemoglobinuria and hyperbilirubinuria total precipitation refractometer method (Jain 1986). was a feature of hemolytic anemia (Jain 1986). Concentration of plasma total bilirubin (Malloy and Evelyn), Mean values of MCV, MCH and MCHC were significantly urea nitrogen (Ritcher and Laponite) and total plasma lower in anemic cattle compared to non-anemic cattle. cholesterol were estimated by commercial kits. Univariate However, mean values of red cell indices were similar in analysis was done to compare for multiple comparison mildly, moderately and severely anemic cows (Table 3). between groups to find out significant difference between Anemia on the basis of red cell indices was normocytic- them, using SPSS12.0 Software. normochromic in 70%, normocytic-hypochromic in 13.9% and microcytic-normochromic in 16.1% of anemic cows. RESULTS AND DISCUSSION Freshly prepared new methylene blue (NMB) stained smears Mean values of red cell parameters of non-anemic and also did not reveal reticulocytosis in any of the anemic cows. anemic crossbred cows are presented in Table 1. Anemia was Peripheral blood smear examination of anemic cows revealed mild, moderate and severe in 55.8, 13.9 and 30.3% of normocytic-normochromic, macrocytic-hypochromic, crossbred cows respectively (Table 2). Considering PCV microcytic-hypochromic and normocytic-hypochromic values, anemia was mild, moderate and severe in 55.8, 27.9% anemia in 64%, 23%, 7% and 6%, respectively in anemic and 16.3% of crossbred cows (Table 3). Mild to moderate cows. Thus anemia was characterized as non-regenerative

Table 1. Red cell parameters of non-anemic and anemic cows Table 3. Classification of anemia on basis of packed cell volume (mean±SE) in anemic cows (mean±SE)

Parameter Non-anemic (n=35) Anemic (n=43) Parameter Mild anemia Moderate Severe (23–24%) anemia anemia Hb (gdl–1) 10.6±0.21a 6.82±0.16b (n=24) (19–22%) (<19%) (9.10–14.6) (4.40–8.70) (n=12) (n=7) PCV (%) 33.6±0.76a 20.6±0.54b –1 a b c (28.0–48.0) (8.00–28.0) Hb (gdl ) 7.45±0.12 6.78±0.22 5.55±0.22 a b c TEC (106 ml–1) 6.63±0.19a 4.86±0.13b PCV (%) 22.6±0.17 20.4±0.19 16.5±0.57 6 –1 a ab a (5.00–9.84) (2.05–6.85) TEC (10 ml ) 5.39±0.13 4.64±0.14 4.03±0.18 a a a MCV (fl) 50.9±0.92a 42.8±0.82b MCV (fl) 42.4±0.99 44.3±1.12 40.2±1.32 a a a (38.3–60.6) (24.9–54.4) MCH (pg) 13.9±0.32 14.7±0.58 13.8±0.44 a a a MCH (pg) 16.2±0.34a 14.2±0.27b MCHC (%) 33.0±0.64 33.2±1.08 33.7±1.74 (12.4–20.1) (10.4–18.5) Means with different superscripts in row differ significantly (P MCHC (%) 32.3±0.72a 33.5±0.64b < 0.05). Figures in parentheses show range. (n) refers to number of (24.8–34.9) (24.2–37.5) cows. 33 222 BHARDWAJ ET AL. [Indian Journal of Animal Sciences 80 (3) on the basis of erythrocytic indices. Whitney (1999) hemolysis or rapid removal of red cells from the circulation. emphasized that erythrocytic morphology noted on careful Other possible cause in 3 of 10 cows with relative low bone examination of blood films, must always be considered in marrow iron stores, low plasma Fe and high TIBC was likely conjunction of the calculated red cell indices. Calculated to be secondary iron deficiency, might be due to chronic erythrocytic indices are also less useful in cattle because of intermittent blood loss. higher degree of anisocytosis and thereby resulting in Mean value of plasma Fe concentration and per cent unreliability of their reference range (Weiss and Perman transferrin saturation in anemic cows were significantly lower 1992). Underlying cause of normocytic- normochromic than non-anemic cattle (Table 4). Mean values of TIBC of anemia might be early iron deficiency, hypoproliferative anemic cows were significantly higher as compared to non- anemia due to selective depression of erythropoiesis as in anemic cows. Low plasma Fe concentration (<100 mgdl–1) chronic infections, uremia and hypothyroidism due to was observed in 39.5% (17/43) of anemic cows. Normal flourosis. Macrocytic-hypochromic anemia in cattle was a plasma Fe and normal TIBC; low-normal plasma Fe and feature in remission anemia. Normocytic-hypochromic and normal TIBC; low plasma Fe and high TIBC and normal microcytic-hypochromic anemia resulted as a consequence plasma Fe and high TIBC were detected in 55.9% (24/43), of early iron deficiency. Microcytic-hypochromic anemia was 20.9% (9/43), 18.6% (8/43) and 4.65% (2/43) of anemic purely a feature of iron or copper deficiency (Jain 1986). cows. Low plasma Fe, high TIBC and low PTS in 18.6% of Erythrocytic indices might vary in cattle due to anisocytosis, anemic cows thus suggested iron deficiency as the possible which was also observed in blood smear of non-anemic cows cause of anemia. Primary iron deprivation in adult cows was in this study. A proportion of cows with normocytic- unlikely as: the soil iron content is many times higher than normochromic anemia on the basis of calculated red cell the plants iron content, physiological losses from animal body indices, revealed macrocytic-hypochromic anemia on are minimal and fodder is contaminated with soil and dust examination of blood smears. Thus microscopic erythrocyte (Underwood and Suttle 1999). Secondary iron deficiency due morphology examination revealed that macrocytosis was to chronic blood loss was thus considered as the cause of probably indicating regenerative bone marrow response. Normocytic-normochromic anemia in ruminants cannot be Table 4. Comparative biochemical parameters of healthy and always categorized into regenerative or non-regenerative by AFB shedding cows simply examining peripheral blood smears (Cole et al. 1997) Name of parameter Healthy AFB shedding and bone marrow examination becomes essential for its cows (N=35) cows (N=45) differentiation. Also there was discrepancy in the morphological classification of anemia as determined from Plasma iron (mgdl–1) 156.1±7.08a 110.8±6.70b calculated red cell indices and from peripheral blood smear (92.5–267) (65.6–233.4) a b examination. Therefore bone marrow aspirate examination Total iron binding 433.5±18.4 573.6±42.0 –1 was done in 10 anemic cows to confirm whether anemia was capacity (mgdl ) (240–680) (252.0–1156.2) Per cent transferrin 39.4±2.71a 30.3±3.10b regenerative or non regenerative and also to access iron status saturation (%) (13.9–76.2) (5.84–92.7) for the differentiation of iron deficiency and inflammatory Protein (gdl–1–-) 8.25±0.09a 8.08±0.17a diseases anemia. (7.36–9.16) (5.69–11.2) Mean myeloid to erythroid ratio (M:E ratio) was 0.60±0.05 Albumin (gdl–1) 3.19±0.03a 3.05±0.06a in anemic cows showed predominance of erythroid over (2.80–3.72) (1.90–4.00) myeloid cells and 1.02±0.03 in non-anemic cows showed Globulin (gdl–1) 5.02±0.10a 5.01±0.16a comparable number of myeloid and erythroid cells. Prussian (3.84–6.03) (2.90–7.41) a a blue stained smears showed normal hemosiderin (+1 to +2) A:G ratio 0.64±0.17 0.62±0.02 in 7 of 10 and low hemosiderin (+0.5 to +0.75) in remaining (0.48–0.91) (0.32–0.97) Fibrinogen (mgdl–1) 328.5±18.6a 377.7±20.0a 3 anemic cows. The M:E ratio of anemic cows was thus below (200–600) (100–700) 1.0 and was also lower than non-anemic cows (1.02), these TPP: fibrinogen ratio 28.1±1.71a 25.8±2.02a observations indicated that anemia was regenerative in (13.3–45.0) (11.8–74.8) anemic cows. It may be used to conclude that normocytic- Total plasma bilirubin 0.41±0.05a 0.35±0.20a normochromic anemia in crossbred cows was not always non- (mgdl–1) (0.09–1.20) (0.03–1.34) regenerative and bone marrow examination was essential for Plasma urea nitrogen 17.8±0.67a 11.5±0.90b their differentiation. As from the bone marrow examination, (mgdl–1) (11.6–26.7) (8.03–20.4) a a it was inferred that anemia was regenerative with adequate Total plasma cholesterol 113.8±6.66 101.3±6.68 –1 iron stores in more number of cows (7/10) and bone marrow (mgdl ) (59.7–210) (39.50–210) regeneration with low iron store in 3 of 10 of anemic cows. Means with different superscripts in a row differ significantly It was finally theorized that regenerative idiopathic anemia (P < 0.05); figures in parentheses show range. (n) refers to number in 7 of 10 cows was probably either from slow degree of of cows. 34 March 2010] CLINICO-HAEMATOBIOCHEMICAL PROFILE IN CHRONIC ANEMIC CATTLE 223 iron deficiency anemia. Although the blood loss was not inflammation. A ratio of less than 15:1 indicated a relative established routinely during this study on anemia, however increase in fibrinogen over plasma proteins and ratio below the intermittent urinary blood loss and occult fecal blood 10:1 indicated a marked increase in fibrinogen (Jain 1986). loss was not monitored. Hypoferremia was a feature of iron Thus low TPP:Fib. ratio also supported anemia of chronic deficiency due to increase iron loss, chronic blood loss, inflammatory diseases in 5 cows. Usually the elevation of decreased iron intake or intestinal absorption and acute or plasma fibrinogen leveled off within several days after the chronic inflammation (Stockham and Scott 2002). Low active inflammation had stopped and thereafter elevation of plasma Fe concentration, high or normal TIBC and low per plasma gamma globulins occurred in chronic inflammatory cent transferrin saturation was a common finding in iron disease (Benjamin 1997). Commonly plasma fibrinogen was deficiency anemia (Jain 1986). Early iron deficiency might more in inflammatory, suppurative and neoplastic diseases. accompany normal plasma iron and high TIBC (Jain 1986 Thus low TPP:Fib. probably indicative of anemia of chronic and Stockham and Scott 2002). inflammatory diseases in anemic cows. Mean plasma total Normal plasma iron and high TIBC in 4.65% cows was bilirubin of anemic and non-anemic cows was comparable considered to be due to early iron deficiency. Anemia of (Table 4). However plasma bilirubin of cows infected with chronic or non-infectious inflammatory conditions caused a T. evansi varied from 0.75 – 1.25 mgdl–1 and was high in 1 fall in TIBC, transferrin saturation and normal to decreased cow only (1.25 mgdl–1). Plasma bilirubin concentration plasma Fe concentration (Mc gillivary et al. 1985), whereas decreased to 0.60±0.09 mgdl–1 following treatment. Icterus anemia of organ dysfunction or bone marrow dyscrasia did in absence of hemoglobinuria might occur if significant and not affect iron metabolism (Cole et al. 1997). Normal to persistent erythrocyte destruction occurred (Jain 1986). Thus, decreased plasma Fe and TIBC concentrations was a feature the mean and range of plasma bilirubin concentration of anemia of chronic infection and inflammatory diseases suggested that persistent and remarkable extravascular or (Jain 1986, Smith 1997). Normal or low-normal plasma Fe intravascular hemolysis was probably not undergoing in the and normal TIBC in 76.7% cows could be attributed to chronically anemic crossbred cows in the present study. anemia of inflammatory diseases. Mean plasma urea nitrogen (PUN) concentration of Mean concentrations of TPP, albumin, globulins and A:G anemic cattle was lower (P<0.05) than non-anemic cows ratio of anemic cows were not significantly different from (Table 4).The concentration of PUN had been demonstrated non-anemic cows (Table 4). However, the range of plasma to be directly proportional to daily protein intake (Payne et proteins was broader in anemic than of non-anemic cows. al. 1974, Whitlock et al. 1976, Pelletier 1985). A study by Frank hypoproteinemia (TPP<6.5 gdl–1) and Manston et al. 1975) recorded a concentration of 7.5 mg/dl in hypoalbuminemia (albumin<2.4 gdl–1) was recorded in cows maintained on low protein diet, associated with low Hb 4.65% (2/45) of anemic cows. Anemia was normocytic- and PCV values. Concentration of PUN was less than 7.5 normochromic in one cow with very low albumin mgdl–1 in 18 of 43 anemic cows. It indicated that anemic cows concentration (1.9 gdl–1). Total globulins were higher in 7 of were probably on low protein diet at time of sampling. Low 43 anemic cows. Elevated levels of globulins occurred Hb had also been recorded by Manston et al. (1975) in animals because of benign plasma cell proliferation in response to on low protein diet. persistent antigenic stimulation as in chronic infections, Cows that had low TPP:Fib. or low A:G ratio and normal chronic inflammatory diseases or immune mediated diseases plasma Fe and TIBC were attributed to anemia of (Jain 1986). Albumin/globulin ratio (A:G) was below-normal inflammatory diseases or chronic infection. Cows with range in 16.3% (7/43) cows. Therefore chronic inflammatory normocytic-normochromic anemia, low iron and high TIBC anemia was suspected in 16.3% of cows. and normal TPP: Fib. or A:G ratio was incriminated to early Mean concentration of plasma fibrinogen in non-anemic iron deficiency. Cows with normocytic-normochromic and anemic cows were within normal range of 100–700 mg/dl anemia with normal plasma Fe and TIBC and high TPP: Fib. as suggested by Jain (1986) and Radostits et al. (2000). The or A:G ratio was considered due to nutritional factors other difference in the ratio of total plasma proteins to fibrinogen than iron. Anemic cows showing normocytic-normochromic (TPP: Fib.) between non-anemic and anemic group was also anemia accompanied by low normal or normal plasma Fe, nonsignificant, because of extensive variability of the normal or high TIBC, high TPP:Fib. and A:G ratio were leukocyte response in chronic inflammation. Estimation of unusual findings where tentative etiology could not be fibrinogen was a useful adjunct to leukogram for the established. Anemia in other 35 crossbred cows when the determination of inflammatory response. Though plasma biochemical and hematological changes were considered for fibrinogen concentration was within the physiological range, all the cows was idiopathic as clinical, hematological and low TPP: Fib. ratio (15:1) was present in 5 of 43 anemic biochemical changes failed to confirm the exact cause. cows. Plasma fibrinogen is acute phase proteins, the levels Considering morphology of red cell, iron biochemistry, A:G of which are not affected by age, sex, exercise, hemorrhage and TPP:Fib. ratio, tentative diagnostic possibilities of and repeated bleeding, however elevation suggest idiopathic anemia was evolved as anemia of chronic 35 224 BHARDWAJ ET AL. [Indian Journal of Animal Sciences 80 (3) inflammatory diseases in 30.23%, early iron deficiency in Manston R, Russell A M, Dew S M and Payne J M. 1975. The 13.95%, iron deficiency in 11.76%, nutritional factors other influence of dietary protein upon blood composition in dairy than iron in 16.27% and etiology was obscure in 6.97% of cows. Veterinary Record 96: 497–502. chronically anemic cows. Trypanosoma evansi infection was Morris D D and Johnston. 2001. Alterations in blood proteins. Large Animal Internal Medicine. pp 429–32. (ed) Smith B P. Mosby etiology of chronic anemia in 18.6% cows. Inc. Missouri, USA. The present study concluded that anemia was mild Payne J M, Rowlands G T, Manston R, Dew S M and Parker W A. to moderate in nature and of normocytic- normochromic 1974. A statistical appraisal of the results of the metabolic profile and regenerative type in chronic anemic cows. Etiology of test on 191 herds in the BVA/ADAS, Joint exercise in animal anemia was evolved as anemia of chronic inflammatory husbandry and productivity. British Veterinary Journal 130: 34– diseases in 30.2%, early iron deficiency in 16.3%, iron 40. deficiency in 9.3%, nutritional factors other than iron in Pelletier G, Tremblay A V and Heelie P. 1985. Factors affecting the 16.3% and etiology was obscure in 9.3% of chronically metabolic profile of dairy cows. Canadian Veterinary Journal anemic cows. The study also concluded that Trypanosoma 26: 36–37. Radostits O M, Gay C C, Blood D C and Hinchcliff K W. 2000. evansi infection should be ruled out as an etiology of chronic Veterinary Medicine: A Textbook of Diseases of cattle, sheep, anemia in cows. pig, goat and horses. pp 414–16. WB Saunders, Harcourt REFERENCES Publishers Ltd., London. Ritcher H and Laponite Y. 1962. New reagent for use in Benjamin M M. 1997. Outlines of Veterinary Clinical Pathology. determination of blood urea nitrogen with special reference to 3rd edn. Kalyani Publishers, New Delhi. manual analysis. Clinical Chemistry 8: 335. Bruce D C and Blue J T. 2000. Approaches to evaluation of bone Singh C. 1999. ‘Epidemiological, immunopathological and marrow function. Feldman B F, Zinkl J G and Jain N C. Schalm’s therapeutic studies in dairy animals with reference to copper Veterinary Haematology. 5th edn, pp 33. Lippincot Williams and iodine.’ Ph. D. Dissertation, Punjab Agricultural University, and Wilkns. Philadelphia, USA. Ludhiana, India. Caraway. 1962. Clinical diagnostic specificity of laboratory tests. Singh S T. 2002. ‘Epidemiological and therapeutic studies on American Journal of Clinical Pathology 37: 445. calcium and phosphorus deficiency in dairy animals.’ M. V. Sc. Chavai B R, Ulmek B R, Asawalw S P and Deokar D K. 2001. Thesis, Punjab Agricultural University, Ludhiana, India. Studies on mortality pattern in crossbred cattle. Indian Journal Smith J F. 1997. Iron metabolism and its diseases. Clinical of Animal Production and Management 15: 82–83. Biochemistry of Domestic Animals. pp 223–39. (Eds) Kaneko J Cole D J, Roussel A J and Whitney M S. 1997. Interpreting a bovine J, Harvey J W and Bruss M L. Academic Press, New York. CBC: Collecting a sample and evaluating the erythron. Stockham S L and Scott M A. 2002. Fundamentals of Veterinary Veterinary Medicine 5: 460–68. Clinical Pathology. pp 86–148. Iowa state press, 2121 state Coles E H. 1980. Veterinary Clinical Pathology. W.B. Sunders, Avenue America, Iowa. Philadelphia pp. 106. Underwood E J and Suttle N F. 1999. The Mineral Nutrition of Freeman P K. 2000. Bone marrow evaluation. Feldman B F, Zinkl Livestock. 3rd edn, pp 375–96. CAB international 1999, Walling J G and Jain N C Schalm’s Veterinary Haematology 5th edn. pp Ford, Oxon, and U.K. 29–32. Weiss D J and Perman V. 1992. Assessment of the hematopoietic Heigopal. 2003. ‘Pathophysiology and treatment of chronic anemia system in ruminants. Veterinary Clinics of North America 8: in crossbred cattle.’ M.V.Sc. Thesis, Punjab Agricultural 411–28. University, Ludhiana, India. Whitlock R H, Manston R, Rowlands J, Little W and Payne J M. Jain N C. 1986. Schalm’s Veterinary Haematology. 6th edn, pp 31– 1976. Anaemia in dairy cattle: its incidence and relationship to 65, 566, 610–11 Lea and Febiger, Philadelphia. the metabolic profile. Proceedings of Third International Luna L G. 1968. Manual of histologic methods of the armed forces Conference on Production Diseases in Farm Animals. institute of pathology. 3rd edn, Mc Graw-Hill, New York. Wageningem the Netherlands, pp 61–63. Malloy and Evelyn. 1937. Microanalysis in Medical Biochemistry Whitney M S. 1999. Hematology of Food Animals. Current 4th edn. J and A Churchill Ltd., 10W Gloucester Palace, London Veterinary Therapy III Food Animal Practice. pp 482–90. (Eds) W I. Howard J L and Smith R A. W B Saunders, Philadelphia, USA.

36 Short Communications Indian Journal of Animal Sciences 80 (3): 225–228, March 2010 Isolation and molecular characterization of avian pathogenic Escherichia coli and Pseudomonas aeruginosa from mortality in ducks in Kashmir, India

S D QURESHI1, S A WANI2, I HUSSAIN3, M T BANDAY4, I A MIR5, S FAROOQ6 and M A BHAT7

Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shuhama (Alusteng), Srinagar, Kashmir 190 006 India

Received: 9 June 2009; Accepted: 18 September 2009

Key words: APEC, Escherichia coli, Exotoxin A iss, Pseudomonas aeruginosa

A group of Escherichia coli (E. coli) collectively termed diarrhoea. Necropsy revealed echymotic and petechial as avian pathogenic E. coli (APEC) causes colibacillosis in haemorrhages on heart and liver, respectively. Besides, spleen poultry and other avian species. APECs most likely enter and kidneys were swollen. and colonize the avian respiratory tract by inhalation of faecal Isolation of bacteria: Heart blood, pieces of liver, lung dust, leading to localized infections such as septicaemia, and intestine of 6 duck carcases referred to this Division were cellulitis, air sacculitis and pneumonia (Dho-Moulin and plated onto nutrient agar, blood agar and MacConkey agar, Fairbrother 1999). A variety of virulence attributes of APEC and incubated aerobically at 37°C for 24 h. The well- that include increased serum survival (iss), presence of type separated colonies were picked up onto nutrient agar slants 1 fimbriae (fimC), temperature sensitive haemagglutinin as pure culture and subjected to standard morphological, (tsh), haemolysin (hlyE), S-fimbriae (sfa) and colicin V biochemical tests (Holt et al. 1994) to ascertain their identity. (cvaC) are implicated in promoting these extraintestinal The isolates were identified as E. coli and Ps. aeruginosa. diseases in avian species (McPeake et al. 2005). Screening for presence of virus: Besides, morbid material Pseudomonas aeruginosa (Ps. aeruginosa) a gram was inoculated into Vero cell monolayer to look for presence negative bacterium is implicated in various infections of virus permissible to Vero cells. The liver and lung tissues including lung infections in man and animals. Ps. aeruginosa of dead ducks were homogenised with homogenizer and produces an extracellular toxin called exotoxin A, which is centrifuged at 10 000 × g. Supernatant was filtered through encoded by exoA gene (Khan and Cerniglia 1994) and is 0.2μm syringe filter and used for inoculation on vero cell 10 000 times more toxic to mice than Pseudomonas monolayer for detection and isolation of virus. A vero cell endotoxin (Iglewski and Kabat 1975). Present work describes line was grown in a 12–well cell culture plate in a CO2 the isolation and molecular characterization of APEC and incubator at 37°C under 5% CO2 tension and 200 μl of tissue Ps. aeruginosa associated with heavy mortality in ducks in filtrate was inoculated into duplicate wells. Additional two an organised farm in Kashmir valley. wells without any inoculums served as negative control. The An outbreak of disease occurred in ducks (Khaki plate was incubated as before. Cells were examined for Campbell layer type) at Duck Farm, Faculty of Veterinary cytopathic effects (CPE) after every 24 h up to 72 h using an Sciences and Animal Husbandry, SKUAST-K, Shuhama inverted microscope. (Alusteng), Srinagar, in December 2007. The ducks were Screening of iss, sfa, cvaC, tsh, hlyE and fimC virulence aged between 6 and 10 weeks. Out of 250 ducks, 199 died genes of APEC and exoA gene of Ps. aeruginosa: For during the outbreak. The affected ducks showed paralytic detection of various virulence specific genes bacterial DNA signs and were unable to stand. Some of the ducks exhibited was extracted (Wani et al. 2006). The iss, sfa, cvaC, fimC, lameness. Most of the ducks suffered from greenish watery hlyE and tsh genes of APEC were detected as per McPeake et al. (2005). APEC strain belonging to serogroup O12 was used as positive control for iss gene. The exoA gene of Ps. Present address: 1, 3Assistant Professor-cum-Jr. Scientist. aeruginosa was detected as per the protocol and primers 2Professor and Head. 4Associate Professor-cum-Senior Scientist, Division of Livestock Production and Management; 5–6MVSc described by Xu et al. (2004). Amplified PCR products were Students; 7PhD Student; Bacteriology Laboratory Division of analysed by gel electrophoresis in 1.5–2% agarose gel Veterinary Microbiology and Immunology, Faculty of Veterinary containing ethidium bromide (0.5μg/ml). The products were Sciences & Animal Husbandry. visualised with UV illumination. 37 226 QURESHI ET AL. [Indian Journal of Animal Sciences 80 (3)

Vero cytotoxicity assay: The Ps. aeruginosa was inoculated and was associated with a 20–fold increase in complement into 5 ml of Luria-Bertani (LB) broth and incubated overnight resistance and a 100–fold increase in virulence toward 1– at 37°C in the shaking incubator. The overnight culture was day-old chicks (Chuba et al. 1989). An association between centrifuged at 10 000 × g. Cell free supernatant was filtered pathogenicity and serum resistance has been previously through 0.2μm syringe filter and used for cytotoxic assay. demonstrated by Knobl et al. (2001). The iss gene was also Sixty microlitre of cell free filtrate (CFF) was inoculated demonstrated among the isolates from cellulitis and into duplicate wells of vero cell monolayer and incubated as colisepticaemia in chickens (Jeffrey et al. 2002). Tivendale described above. Equal volume of sterile LB broth instead et al. (2003) detected the iss gene in 3 highly virulent strains of CFF served as negative control. The plate was incubated of APEC, and suggested that it is required in association with and observed for CPE as before. iucA (aerobactin operon) gene for the highest level of Serogrouping of APEC was carried from National virulence. Salmonella and Escherichia centre, C.R.I., Kasauli The Ps. aeruginosa isolate revealed the presence of exoA (Himachal Pradesh). gene as it yielded a product of 396 bp (Fig.2) by PCR. It is a In vitro antibiotic susceptibility testing: The in vitro specific marker of Ps. aeruginosa (Xu et al. 2004), which antibiotic sensitivity tests of the E. coli and Ps. aeruginosa codes for exotoxin A. The role of exotoxin A in the mortality isolates were carried out by disc diffusion technique as of experimentally infected mice has been demonstrated described by Bauer et al. (1966). (Manafi et al. 2009). Woods et al. (1982) assessed the role Present study records the investigation of heavy mortality of Ps. aeruginosa exoA in chronic lung infection in the rat in an organised duck farm in Kashmir valley. Two types of model, and demonstrated that active toxin A is required for organisms were isolated from all the 6 samples subjected to maximum virulence. They observed that chronic bacteriological examination. One isolate was identified as inflammatory changes could be induced only with toxigenic APEC and the another one as Ps. aeruginosa. The APEC strain. It appears that the virulence potential of exotoxin A isolate was untypeable (UT) and possessed iss gene as it may vary with the site and tissue involved (Xu et al. 2004). produced an amplicon of 323 bp (Fig.1) by PCR but did not Cell free filtrate of Ps. aeruginosa produced prominent CPE harbour sfa, cvaC, tsh, hlyE or fimC virulence genes. There on vero cells after 48 h. No CPE was observed on vero cell seems to be no report available about the isolation of APEC monolayer, inoculated with tissue filtrate, even after 72 h of with iss gene from ducks. However, there are reports of the inoculation. The CPE on vero cells due to CFF of Ps. isolation of APEC strains harbouring iss gene from outbreaks aeruginosa may be due to its exotoxin A. However, this needs of heavy mortality in poultry (McPeake et al. 2005, to be investigated further. Absence of CPE in vero cells after Rodriguez-Siek et al. 2005). The iss gene was first identified inoculation of filtered tissue homogenate was suggestive of in a human septicaemic E. coli isolate (Binns et al. 1982) absence of virus permissible to vero cells. Determination of in vitro antimicrobial susceptibility of the bacterial isolates is prerequisite for judicious use of M123 1M2

400 323 bp 500 bp 300 396 bp 400 bp 200

100 200 bp

Fig 1. Detection of iss gene in APEC isolate by PCR. Lane M: Fig 2. Detection of exoA gene in Pseudomonas aeruginosa by 100 bp DNA ladder, Lane 1: APEC isolate positive for iss gene, PCR: Lane 1: Pseudomonas aeruginosa isolate positive for exoA Lane 2: positive control, Lane 3 negative control. gene. Lane M: 100 bp DNA ladder Lane 2: negative control. 38 March 2010] APEC AND PSEUDOMONAS AERUGINOSA FROM DUCK MORTALITY 227 antimicrobials for effective treatment and to avoid the of complement resistance conferred on Escherichia coli by the development of drug resistance. The APEC isolate was plasmid genes traT of R100 and iss of colV, I-K94. Infection sensitive to gentamicin and ampicillin/cloxacillin while Ps. and Immunity 35: 654–59. aeruginosa was sensitive to gentamicin, ciprofloxacin, Chuba P, Leon M, Banerjee A and Palchaudhuri S. 1989. Cloning and DNA sequence of plasmid determinant iss, coding for enrofloxacin and ampicillin/cloxacillin. These are the increased serum survival and surface exclusion, which has antibiotics less abused in poultry industry. Both isolates were homology with lambda DNA. Molecular and General Genetics resistant to tetracycline, norfloxacin, cephalexin, nalidixic 216: 287–92. acid and doxycycline. In addition the APEC was also resistant Dho-Moulin M and Fairbrother J M. 1999. Avian pathogenic to ciprofloxacin and enrofloxacin. This multi drug resistance Escherichia coli (APEC). Veterinary Research 30: 299–316. of the isolates might be due to indiscriminate use of Holt J G, Krieg N R, Sneath P H A, Staley J T and Williams S T. antibiotics in clinical practice. These resistance levels are 1994. Bergey’s Manual of Determinative Bacteriology. 9th edn, comparable to those previously reported for APEC by other pp. 787. Williams and Wilkins, Baltimore, USA. investigators (Saha et al. 2007) and for Ps. aeruginosa by Iglewski B H and Kabat D. 1975. NAD-dependent inhibition of protein synthesis by Pseudomonas aeruginosa toxin. Sekiguchi et al. (2007). Antibiotic therapy with gentamicin Proceedings of National Academy of Sciences, USA, Vol. 2, and ampicillin/cloxacillin checked the mortality of ducks at pp. 2284–2288. the farm. Jeffrey J S, Nolan L K, Tonooka K H, Wolfe S, Giddings C W, Keeping in view the symptoms and lesions of the affected Horne S M, Foley S L, Lynne A M, Ebert J O, Elijah L M, birds and the virulence attributes of the isolates, it appears Bjorklund G, Pfaff-McDonough S J, Singer R S and Doetkott that APEC and Ps. aeruginosa isolates could have been the C. 2002. Virulence factors of Escherichia coli from cellulitis or cause of heavy mortality in ducks. colisepticemia lesions in chickens. Avian Diseases 46: 48–52. Khan A A and Cerniglia C E. 1994. Detection of Pseudomonas SUMMARY aeruginosa from clinical and environmental samples by amplification of the exotoxin A gene using PCR. Applied Present study records the investigation of heavy mortality Environmental Microbiology 60: 3739–45. in an organised duck farm in Kashmir valley. Bacteriological Knobl T, Baccaro M R, Moreno A M, Gomes T A T, Vieira M A M, examination of the morbid material revealed the presence of Ferreira C S A and Ferreira A J P. 2001. Virulence properties of avian pathogenic Escherichia coli (APEC) and Pseudomonas Escherichia coli isolated from ostriches with respiratory disease. aeruginosa (Ps. aeruginosa). The APEC isolate carried iss Veterinary Microbiology 83: 71–80. virulence gene but lacked sfa, cvaC, tsh, hlyE or fimC Manafi A, Kohanteb J, Mehrabani D, Japoni A, Amini M, virulence genes. The Ps. aeruginosa revealed the presence Naghmachi M, Zaghi AM and Khalili N. 2009. Active of exoA virulence gene. The cell free filtrate of Ps. aeruginosa immunization using exotoxin A confers protection against produced prominent cytopathic effect (CPE) on vero cells Pseudomonas aeruginosa infection in a mouse burn model. BMC Microbiology 9: 23 (Available online: http:// after 48 h. The tissue filtrate from morbid material did not www.biomedcentral.com/1471–2180/9/23). produce CPE on Vero cells. Both the isolates were resistant McPeake S J W, Smyth J A and Ball H J. 2005. Characterization of to tetracycline, norfloxacin, cephalexin, nalidixic acid and avian pathogenic Escherichia coli (APEC) associated with doxycycline but sensitive to gentamicin and ampicillin/ colisepticaemia compared to faecal isolates from healthy birds. cloxacillin. Treatment of the affected flock with gentamicin Veterinary Microbiology 110: 245–53. and ampicillin/cloxacillin was effective to check the mortality Rodriguez-Siek K E, Giddings C W, Doetkott C, Johnson T J and at duck farm. This appears to be first report of its kind from Nolan L K. 2005. Characterizing the APEC pathotype. India. Veterinary Research 36: 241–56. Saha T, Guha C, Biswas U, Chakraborty D, Chakaborty G C and ACKNOWLEDGEMENT Sadhukhan T K. 2007. Escherichia coli isolates from respiratory disease of broiler birds. Indian Veterinary Journal 84: 915–17. The authors are grateful to Professor Anwar Alam, Vice- Sekiguchi J I, Asagi T, Akiyama T M, Kasai A, Mizuguchi Y, Araake Chancellor, SKUAST-Kashmir, for providing facilities. M, Fujino T, Kikuchi H, Sasaki S, Watari H, Kojima T, Miki H, Thanks to Director, National Salmonella and Escherichia Kanemitsu K, Kunishima H, Kikuchi Y, Kaku M, Yoshikura H, Centre, Central Research Institute, Kasauli 173204 Kuratsuji T and Kirikae T. 2007. Outbreaks of Multidrug- (Himachal Pradesh) for serogrouping of the APEC isolate. Resistant Pseudomonas aeruginosa in Community Hospitals The Vero cell line used in this study was kindly supplied by in Japan. 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Escherichia coli in Kashmir, India. FEMS Microbiol Lett 261, Xu J, Moore J E, Murphy P G, Millar B C and Elborn J S. 2004. 238–44. Early detection of Pseudomonas aeruginosa–comparison of Woods D E, Cryz S J, Friedmass R L and Iglewski B H. 1982. conventional versus molecular (PCR) detection directly from Contribution of toxin A and elastase to virulence of Ps. adult patients with cystic fibrosis (CF). Annals of Clinical aeruginosa in chronic lung infections of rats. Infection and Microbiology and Antimicrobials. (Available online:http:// Immunity 37: 1223–28. www.ann-clinmicrob.com/content/3/1/21).

40 Indian Journal of Animal Sciences 80 (3): 229–231, March 2010 Estimations of blood plasma metabolites following melatonin implants treatment for initiation of ovarian cyclicity in true anestrus buffalo heifers

JAGIR SINGH1, S P S GHUMAN2, D DADARWAL3, M HONPARKHE4, G S DHALIWAL5 and A K JAIN6

Guru Angad Dev Veterinary and Animal Science University, Ludhiana, Punjab 141 004 India

Received: 13 October 2007; Accepted: 18 September 2009

Key words: Anestrus, Blood metabolite, Buffalo, Estrus melatonin

Poor breeding efficiency in buffalo heifers due to delayed Taking day 0 as day of implant insertion, blood samples initiation of ovarian cyclicity remains one of the persisting from treatment group heifers were collected at –30, –15, +15, problems in buffalo dairying (Singh et al. 2000). Melatonin, +30 days. Similar blood sampling schedule was followed a pineal gland hormone, is implicated in the sequence of for control group heifers. Blood samples were collected from events leading to the onset of puberty in cow heifers jugular vein in heparinised vials. Plasma was separated (Tortonese and Inskeep 1992). Recently, melatonin implants following centrifugation (1500×g for 10 min) and then stored treatment was successfully exploited for initiating ovarian at –20°C for later analyses. Plasma progesterone was assayed cyclicity in true anestrus buffalo heifers (Ghuman et al. with a solid-phase radioimmunoassay (Kamboj and Prakash 2007). However, the precise mechanisms involved remain 1993) using antisera raised in our laboratory (Ghuman et al. unknown. Alterations in melatonin release with change in 2009). Sensitivity of the assay was 0.1 ng/ml; intra- and inter- photoperiod are associated with changes in lactation body assay variation coefficients were 5.5% and 9.5%, growth and composition in cattle (Dahl et al. 2002). Because respectively. Plasma glucose, total proteins, albumin, urea, of the interaction of melatonin with various endocrine creatinine, lipid profile (total cholesterol, triglycerides-TG, systems (Zieba et al. 2007), it was thought that melatonin total lipids, high-density lipids-HDL, low density lipids-LDL, treatment may initiate ovarian cyclicity in true anestrus very low density lipids-VLDL), aspartate amino transferase buffalo heifers through its influence on body metabolism (AST), alaline amino transferase (ALT), lactate (Darul and Kruczynska 2004). Hence, this study was planned dehydrogenase (LDH), calcium (Ca) and phosphorus (P) to investigate blood plasma metabolic alterations following were determined colorimetrically with auto analyzer using melatonin implants treatment in true anestrus buffalo heifers. commercially available kits. This study was conducted on clinically healthy true Data of pre- or post-treatment days of various blood anestrus (no corpus luteum or utero-ovarian pathology) plasma metabolites was pooled and mean (±SEM) were buffalo heifers (control, 5; treatment, 5) raised under uniform calculated for control and treatment group buffalo heifers. feeding, managemental and environmental conditions at Differences between pre- and post-treatment values in control University Dairy Farm, Ludhiana. Each treatment group and treatment group were examined post-hoc using Student’s heifer was inserted (sub-cutaneously at the base of ear) with paired t-test (two tails; Dytham 1999). Probabilities of <0.05 8 absorbable melatonin ear implants (16–19.8 mg melatonin were considered significant. Statistical procedures were per implant) with the help of an implanter (Ghuman et al. performed in Minitab release 14.2 statistical software. 2007). All the animals were offered ad lib. feeds and fodders In the present study, all the true anestrus buffalo heifers twice daily while fresh drinking water throughout the day of treatment group ovulated within 1 month following and night. To detect the onset of ovarian cyecliciky, if any in melatonin implant insertion; however none of the control control and treatments group heifers, transrectal group heifers ovulated during the study period. These results ultrasonography was carried out at weekly interval for 1 further validate our previous findings (Ghuman et al. 2007). month before and 1 month after treatment. Control and treatment group data of pre- and post-treatment blood plasma metabolites are presented in Table 1. To the Present address: 1Gynaecologist ([email protected]), 2Associate Professor, 3,4Assistant Gynaecologist, Department of best of our knowledge, this is the first study in buffalo heifers Animal Reproduction, Gynaecology and Obstetrics; 5Professor, aimed at delineating the mechanisms behind melatonin- Department of Veterinary Clinical Services Complex; 6Professor, induced onset of ovarian cyclicity, therefore references of Department of Animal Breeding and Genetics. this species are not available of the comparison of results of 41 230 SINGH ET AL. [Indian Journal of Animal Sciences 80 (3)

Table 1. Melatonin treatment-induced metabolic alterations in true anestrus buffalo heifers (mean±SEM)

Control (n=5) Treatments (n=5) Plasma metabolites Pre-treatment Post- treatment Pre-treatment Post- treatment

Progesterone (ng/ml) 0.25±0.03 0.32±0.06 0.30±0.05 0.81±0.19* Glucose (mg/dl) 58.1±1.1 59.1±2.7 59.1±2.1 62.1±2.8 Total proteins (g/dl) 7.7±0.2 7.1±0.3 7.4±0.2 7.0±0.4 Albumin (g/dl) 3.0±0.3 3.0±0.4 3.0±0.1 2.7±0.2* A/G ratio 0.7±0.02 0.7±0.02 0.7±0.01 0.6±0.02* Urea (mg/dl) 35.0±1.0 37.2±1.1 37.0±1.0 27.3±1.2 Creatinine (mg/dl) 1.8±0.4 1.9±0.4 1.8±0.3 2.0±0.4 total cholesterol (mg/dl) 59.5±2.0 58.4±3.0 57.7±20 50.5±3.8 Triglycerides (mg/dl) 39.6±4.2 39.4±1.9 40.6±3.2 40.6±2.2 Total lipids (mg/dl) 195.5±9.1 191.1±15 196.6±9.2 182.2±10.0 HDL (mg/dl) 39.7±1.5 38.1±2.0 38.8±1.4 35.1±2.8 LDL (mg/dl) 11.7±1.3 10.0±1.7 10.8±1.4 7.4±1.7 VLDL (mg/dl) 8.1±0.5 8.10±0.5 8.1±0.6 8.2±0.5 AST (U/L) 148.7±7.4 149.5±51.0 150.8±8.5 158.3±21.0 ALT (U/L) 60.6±2.0 58.4±4.3 62.6±3.0 56.4±4.6 LDH (U/L) 1655±99 1607±96 1715±74 1437±206 Calcium (mg/dl) 10.0±0.2 9.7±0.1 10.1±0.2 8.7±0.3 Phosphorus (mg/dl) 7.1±0.3 7.1±0.4 7.1±0.4 7.2±0.5

*P<0.5; Significantly different from pre-treatment in respective groups. blood metabolic alterations following prolonged melatonin Furthermore, nonsignificantly low plasma urea seen in this implant treatment. study during post-treatment period in melatonin implanted Plasma progesterone concentration is an indicator for heifers could be an indicator of low dietary protein supply in status of ovarian cyclicity. An animal is considered anestrus ruminants (Schroder et al. 2003). It remains to be further when plasma progesterone concentrations persist <0.50 ng/ investigated that how the observed alterations in protein ml (Ghuman et al. 2007). In the present study, plasma metabolism in heifers following melatonin treatment are progesterone concentrations were consistently <0.50 ng/ml associated with onset of ovarian cyclicity in these animals. in controls and during pre-treatment period in melatonin Expression of estrus is more likely in dairy animals with group, whereas the presence of plasma progesterone >0.50 higher plasma cholesterol (Westwood et al. 2002). In this ng/ml during post-treatment period (P<0.05) suggested the study, following melatonin treatment, no alterations were onset of ovarian cyclicity in treated buffalo heifers. observed in lipid profile of buffalo heifers suggesting non- Plasma glucose is a metabolic signal providing significant role of lipids in melatonin treatment-induced onset information for the central control of GnRH release (Foster of ovarian cyclicity. and Nagatani 1999). High plasma glucose was previously Deranged enzymatic actions affect normal reproductive reported in cycling dairy cattle compared to anestrus (Singh behaviour of animal (Fischbach 2000). Similar to control and Singh 2006). In this study, similar to control group, no group heifers, there were no post-treatment significant alteration was observed in the plasma glucose following alterations in plasma concentrations of ADT, ALT and LDH. melatonin implants treatment in buffalo heifers, though Moreover, there were no alterations in plasma concentrations treatment group buffaloes exhibited onset of ovarian cyclicity. of Ca and P following melatonin implants treatment though This suggests that plasma glucose was not the metabolic initiation of ovarian cyclicity has significant positive factor responsible for initiation of ovarian cyclicity following correlations with plasma Ca and P (Shah et al. 2003). melatonin treatment. In conclusion, results suggest that majority of the observed Optimum total serum proteins are essential for the blood plasma metabolites were not altered following expression of estrus (Tandle et al. 1998). In treatment group melatonin implants treatment of true anestrus buffalo heifers, heifers, post-treatment plasma albumin and albumin/globulin though this treatment was able to induce ovarian cyclicity in (A/G) ratio was significantly (P<0.05) low compared to their all the buffalo heifers as suggested by plasma progesterone pre-treatment concentrations. These results are in contrast concentrations. Thus, melatonin treatment induces ovarian to previous findings where higher A/G ratio was seen in cyclicity in true anestrus buffaloes through mechanisms other cycling heifers (Singh and Singh 2006). Low protein uptake than involving alterations in blood metabolites. Further is associated with low A/G ratio (Fischbach 2000). studies investigating underlying reproductive endocrine 42 March 2010] MELATONIN TREATMENT AND BLOOD METABOLITES IN BUFFALOES 231 mechanisms involved in melatonin-induced onset of ovarian Ghuman S P S. Dadarwal D, Honparkhe M, Jagir Singh and cyclicity in buffalo heifers are needed. Dhaliwal G S. 2009. Production of polyclonal antiserum against progesterone for radioimmunoassay. Indian Veterinary Journal SUMMARY 86: 909–11. Aim of the study was to investigate alterations in blood Ghuman S P S, Honparkhe M, Dadarwal D, Jagir Singh, Dhaliwal G S and Jain A K. 2007. Melatonin implant treatment for plasma metabolites following melatonin implants treatment initiation of ovarian cyclicity in anestrus buffaloes. Proceedings for the induction of ovarian cyclicity in true anestrus buffalo of XXIII ISSAR conference held at Orissa University of heifers. True anestrus buffalo heifers (10) were equally Agriculture and Technology, Bhubaneshwar. Dec 7–9, 2007. divided into control and treatment groups. Each treatment pp 36. group buffalo was inserted with 8 absorbable melatonin ear Kamboj M and Prakash B S. 1993. Relationship of progesterone in implants (16–19.8 mg melatonin/implant). Taking day 0 as plasma and whole milk of buffaloes during cyclicity and early day of implant insertion, blood samples were collected on – pregnancy. Tropical Animal Health Production 25: 185–92. 30, –15, +15, +30 days. Similar blood sampling schedule Schroder B, Schoneberger M, Rodehutscord M, Pfeffer E and was followed for control group. Plasma samples were Breves G. 2003. Dietary protein reduction in sheep and goats: different effects on L-alanine and L-leucine transport across analyzed for progesterone, glucose, total proteins, albumin, the brush-border membrane of jejuna enterocytes. Journal of urea, creatinine, lipid profile, AST, ALT, LDH, Ca and P. Comparative Physiology (B) 173: 511–18. Melatonin implants were able to induce ovarian cyclicity in Shah R G, Dhami A J, Vadodaria V P, Kharadi V B , Desai P M and all the treatment group buffalo heifers. Following melatonin Kavani F S. 2003. Effect of GnRH treatment on biochemical implants treatment, significant alterations were observed in and mineral profiles in postpartum Surti buffaloes. Indian plasma progesterone, plasma albumin and albumin/globulin Journal of Animal Sciences 73: 1324–28. (A/G) ratio in buffalo heifers; however majority of blood Singh A S and Singh O N K. 2006. Blood biochemical and enzyme plasma metabolites remains unchanged. This suggested that profile in estrus and anestrus heifers. Indian Veterinary Journal melatonin implants treatment-induced modulations in blood 83: 726–29. Singh J, Nanda A S and Adams G P. 2000. The reproductive pattern plasma metabolites may not have a major role in initiation and efficiency of female buffaloes. Animal Reproduction Science of ovarian cyclicity in true anestrus buffalo heifers. 60–61: 593–604. REFERENCES Tandle M K, Biradar U S, Amanullah Md, Honnappogal S S, Kartikesh S M, Sonwane S D and Jagjiwanram. 1998. Blood Dahl G E, Auchtung T L and Kendall P E. 2002. Photoperiodic biochemical profiles in cyclic and anestrus Deoni cows. Indian effects on endocrine and immune function in cattle. Journal of Dairy Science 51: 66–68. Reproduction Supplement 59: 191–201. Tortonese D J and Inskeep E K. 1992. Effects of melatonin treatment Darul K and Kruczynska H. 2004. Effect of melatonin on on the attainment of puberty in heifers. Journal of Animal biochemical variables of the blood in dairy cows. Acta Science 70: 2822–27. Veterinaria Hungarica 52: 361–70. Westwood C T, Lean I J and Garvin J K. 2000. Factors influencing Dytham C. 1999. Choosing and Using Statistics, A Biologoist’s fertility of Holstein dairy cows: A multivariate analysis. Journal Guide. Vol. 2. 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43 Indian Journal of Animal Sciences 80 (3): 232–233, March 2010 Factors influencing gestation length of Thoroughbred mares in India

SUMEET SHARMA1 and G S DHALIWAL2

Punjab Agricultural University, Ludhiana, Punjab 141 004 India

Received: 21 July 2009; Accepted: 30 September 2009

Key words: Foal gender, Gestation length, Mare, Thoroughbred

Gestation length in the mare is highly variable, and the software (SPSS 12.00). Results were expressed as variability in physical signs of imminent parturition makes mean±sem. Chi squared test was used to analyse the ratio of predicting foaling particularly difficult. To date, any detailed colt to filly foals. Statistical significance was considered at reference on the gestation length of Thoroughbred mares in P<0.05. India is obscure. Additionally, the gestation length is also The mean gestation length for 422 Thoroughbred foals reported to be influenced by climate (Astudillo et al. 1960) was 343.13 ± 0.39 days with a range of 319–378 days. The and management therefore the reported findings from other mean gestation length obtained in this study is consistent countries might not hold true for Indian Thoroughbreds. with the observation (344.1±0.49 days) of Davis Morel et Therefore this study aims to assess the effect of various al. (2002) but comparatively longer than recorded (335.15 factors on gestation length of Thoroughbred mares in India. days) by Singhvi (1987) in Thoroughbreds. The mean Reproductive records for 422 Thoroughbred mares those gestational length for all foals falls within previously reported were foaled (1998-2005) at 8 stud farms of Punjab were ranges of 310-388 days (Immegart 1997, Davies Morel et analysed. On all the surveyed stud farms ultrasonographic al. 2002). The calculation of gestation length (ovulation to examination and/or transrectal palpation of ovaries were foaling) plus the use of large number of single breed performed daily or every other day to confirm ovulation (day pregnancies, allows this work to be considered more accurate 0). The mares were grouped according to their age (3-7,8- and reliable than previous work. 12,13-17,>18 years) and reproductive status, viz. maiden, There was a nonsignificant (P>0.05) difference in the barren, lactating, rested and aborted (Sharma et al. 2009). percentage of colt (46.92%) and filly (53.08%) foals born. Univariate analysis of variance was used to examine the effect Further, year of foaling and reproductive status of dam did of age of mare, reproductive status, foal gender, stud farm, not significantly affect gestation length. No effect of mare year and month of foaling on gestation length, using statistical age on gestation length in all the stud farms surveyed may

Table 1. Gestation length of Thoroughbred foals in different months of foaling

Month of Mean gestation length, n Mean gestation length, n Mean gestation length, n foaling all pregnancies (days) filly pregnancies (days) colt pregnancies (days) ± s.e.m. ± s.e.m. ± s.e.m.

January 335.79a±1.29 29 334.60±1.73 15 337.07±1.95 14 February 340.21b±0.81 80 338.79±0.99 42 341.79±1.27 38 March 344.02c±0.57 141 342.55±0.80 75 345.70±0.78 66 April 346.08c±0.77 124 344.27±0.80 67 348.21±1.33 57 May 342.75bc±1.33 36 341.16±1.80 19 344.53±1.95 17 June 340.33b±1.37 12 339.17±1.54 6 341.50±2.32 6 Total 343.13±0.39 422 341.62±0.48 224 344.83±0.61 198

Within column mean gestation lengths with different superscripts differ significantly (P<0.05)

Present address: Rural Veterinary Officer, Civil Veterinary Hospital, Mahatam Nagar, Fazilka, Punjab 152123. (e mail: well be accounted for by the close proximity of the stud farms [email protected]) and their similar management regimes. 2Professor, Department of Teaching Veterinary Clinical Services Pregnancy was significantly longer when mares gave birth Complex GADVASU, Ludhiana 141 004. to colts rather than fillies (344.83±0.61 vs. 341.62±0.48 days, 44 March 2010] FACTORS AFFECTING GESTATION LENGTH IN MARES 233

P<0.001; Table 1), consistent with Pe'rez et al. (2003). This REFERENCES may be due to difference to androgen action and to sex- Astudillo C R, Hajek G E and Diaz O H. 1960. Influencia de algunos chromosome-linked effects (Monteiro et al. 1998). factores climaticos sobre la duracion de la gestacian de carrera: The shortest gestation lengths were in January estudiio preliminary (the influence of some climatic factors on (335.79±1.29) and longest (346.08 ± 0.77) in April, is in pregnancy duration in Thoroughbred mares: preliminary accordance with Davies morel et al. (2002). As climate in account). Zoolatria 2 (35): 37–38 (Animal Breeding Abstracts particular the photoperiod is thought to affect the foaling 30, 2348). time (Astudillo et al. 1960) therefore, significant difference Davies Morel M C G, Newcombe J R and Holland S J. 2002. Factors in gestation length was expected between the months in this affecting gestation length in the Thoroughbred mare. Animal study (Table 1). It has previously been suggested that nature Reproduction Science 74: 175–85. Evans W J and Torbeck R L. 1998. Breeding Management and attempts to bring the timing of parturition back to the ideal, Foal Development. Equine Research Incorporated Texas, pp. early spring and that this may be achieved by shorter or longer 700. gestation lengths in late born and early born foals, Immegart H M. 1997. Abnormalities of pregnancy. Current Therapy respectively (Evans and Torbeck 1998). Previous work, in Large Animal Theriogenology. (Ed.) Yougquist R S, pp. 113– therefore, agrees with the decline in gestation length in later 129, Saunders, Philadelphia, USA. months as seen in the current study, but not the short gestation Monteiro V, Derom C, Vlietinck R, Kohn N, Lesser M and lengths in January. No ready explanation for this difference Gregerson P K. 1998. Commitment to X-inactivation precedes is found. the twinning event in monochronionic MZ twins. American In conclusion, true gestation length for Thoroughbred foals Journal of Human Genetics 63: 339–46. Pe'rez C C, Rod'riguez I, Mota J, Dorado J, Hidalgo M, Felipe M born in India is 343.13 ± 0.39 days. The mares carrying colt and Sanz J. 2003. Gestation length in Carthusian Spanishbred foals due to be born in mid season are likely to have the mares. Livestock Production Science 82: 181–87. longer gestation lengths. Sharma S, Davies Morel M C G, Dhaliwal G S and Dadarwal D. 2009. The pattern of embryonic fixation and its relationship to SUMMARY pregnancy loss in Thoroughbred mares. Reproduction in Factors affecting gestation length of thoroughbred mares Domestic Animals (doi: 10.1111/j. 1439-0531.2009.01523.x.) Singhvi N M. 1987. Gestation in equines and factors affecting it. in India, were studied. Reproductive records of 422 mares Journal of Remount Veterinary Corps 26: 6–11. that foaled in 8 stud farms of Punjab were collected. True Winter G H Z, Rubin M I B, De La Corte F D and Silva C A M. gestation length for mare was 343.13 ± 0.38 days in India. 2007. Gestational length and first postpartum ovulation of The mares carrying colt foal due to born in mid season are Criollo mares on a stud farm in Southern Brazil. Journal of likely to have longer gestation length. Equine Veterinary Science 27: 53–34.

45 Indian Journal of Animal Sciences 80 (3): 234–238, March 2010 MHC-DRB Exon-2 (BuLA- DRB3) polymorphism in banni breed of Indian buffalo

JYOTSNA DHINGRA BEHL1, RAHUL BEHL2 and N K VERMA3

National Bureau of Animal Genetic Resources, P. O. Box 129, GT Bypass Road, Karnal, Haryana 132 001 India

Received: 15 March 2009; Accepted: 3 October: 2009

ABSTRACT Polymorphism in the DRB3- exon 2 of the BoLA (BuLA) gene of the Banni buffalo breed was studied by the technique of PCR-RFLP. The PCR product (304 bp) was digested with three enzymes RsaI, BstYI and HaeIII. The digestion products were separated on native PAGE gels which were silver stained. The results obtained showed presence of 6 different BuLA-DRB3.2 alleles viz. BuLA –DRB3.2* 19, *20, *36, *41, *abd and *gbb.

Key words: PCR-RFLP, DRB 3-exon2, BuLA-DRB3.2*

Buffaloes play an important role in agricultural economy Complex Class II exon-2 (DRB3 exon 2 (DRB3.2)) locus of with their contribution to milk and meat production and draft the Banni breed of the buffaloes by PCR-RFLP. power. They contribute about 55 per cent of total milk Major Histocompatibility Complex (MHC) class II loci production of the world. Buffaloes are able to survive well encode cell-surface glycoproteins that are composed of α, - under tough, harsh tropical climatic conditions. They are and ß- chains on the antigen-presenting cells that function to higher producers of milk even under rough climatic bind and present foreign peptides to the immune system conditions. (Klein 1986). The major histocompatibility complex of cattle Among buffaloes, the Banni breed of Indian buffaloes is is referred to as BoLA (Bovine Lymphocyte Antigen). The distributed throughout the Kutchh region of Gujarat and in corresponding gene in buffaloes is referred to as BuLA parts of Banaskantha and Patan districts of North Gujarat. (Aravindakshan et al. 2000). The genes that encode the MHC These buffaloes are prevalent in Khavda, Nakhatrana, Haji protein receptors are the most polymorphic loci of all the Peer, Bhrindiara, Hodka, Zarpara villages. Banni is riverine nuclear-encoding genes in vertebrate species (Hughes and type of buffalo and is usually black coloured with a few (about Hughes, 1995). These molecules bind foreign antigens and 5%) brown also, which are reasonably good yielders. ‘The present them to specifically stimulated helper T lymphocytes average milk yield is 12.2 to 17.1 1. Body size is medium to (Allen et al. 1987). The high degree of polymorphism present large. Morphologically these buffaloes have distinct curling in the MHC Class II region is considered responsible for of horn which is aligned vertically to the body and curls differences among individuals in the immune response to inwardly. The animals are wedge shaped with heavy backs infectious diseases (Brown et al. 1993). Wenink et al. (1998) and are maintained under (almost) zero input system. Banni studied genetic diversity present in the major area has 44 villages spread out in 840 sq. miles and has a histocompatibility complex by PCR-RFLP, in the African total of approximately 35,000 to 40, 000 buffaloes. The area buffalo, that had experienced bottlenecks, thereby resulting is characterized by high salinity, no arable farming, little grass in genetic losses in the population. It was observed that in and plenty (excess) of Israeli babul. These animals live under spite of the historically known bottlenecks in the population, such tough environmental conditions. They are strongly the MHC-DRB3 gene exhibited surprisingly high level of contributing in the Banni area and the farmers in the tract allelic diversity. For each population sample a deficiency in are generally dependent on Banni buffaloes (Annual Report, the expected number of heterozygous animals was found. NBAGR, 2004-2005). These results contrasted with the data observed for MHC The present study was taken up to study the extent of genes in larger populations such as humans where a genetic diversity present at the Major Histocompatibility significant homozygote deficit has been earlier reported (Black and Salzano, 1981: Boyce et al. 1996; Markow et al. Present address: National Bureauof Animal Genetic Resources, 1993). The excess of heterozygosity has been interpreted as P.O. Box 129, GT Bypass Road, Karnal 132 001. Haryana India a consequence of overdominance, because heterozygous 46 March 2010] MHC-DRB EXON-2 POLYMORPHISM IN BANNI BREED OF INDIAN BUFFALO 235 individuals are able to recognize a broader spectrum of Digestions were performed in 200μl PCR tubes, using a foreign antigens, increasing their relative fitness when thermal cycler. After heat denaturation of the enzymes at compared with homozygotes (Hedrick et al. 1991; Hughes 65°C (Rsa I) or at 85°C (BstYI, HaeIII) for 30min the and Nei, 1989). restriction fragments were resolved by gel electrophoresis Several studies have been carried out to study in 12% polyacrylamide at 200V for 4–5 h. A 50-base pair polymorphism (genetic diversity) present at the BoLA- DNA ladder in some gels and a 20-bp DNA ladder in other DRB3.2 locus in case of cattle (Behl et al. 2007, Nassiry et gels were used as DNA size markers. The fragments were al. 2008). However, there are only few studies on the visualized by silver staining of the gels following the silver polymorphism present at this locus in case of buffaloes staining procedure for ion-denaturing PAGE gels (Sambrook (Aravindakshan et al. 2000, Sena et al. 2003, De et al. 2002, et al. 2001). Pipalia et al. 2004, Singh et al. 2004). There has been no report so far on the polymorphism of the MHC class II exon Population genetic analysis 2 of the Banni buffaloes. The POPGENE version 1.32 program was used to estimate observed and expected heterozygosities of the populations MATERIALS AND METHODS studied (Yeh et al. 1999). DNA isolation from blood RESULTS AND DISCUSSION Blood was collected from 31 animals of the Banni animals from their breeding tract. It was collected from animals Polymerase chain reaction residing in the Khavda, Luna, Haji Peer and Nakhtrana blocks A PCR product of size 304 bp was obtained by using the in the Kutchh area of Gujarat. Genomic DNA was isolated primers specific for the corresponding region in cattle. The from blood by digestion with Proteinase K and extraction size of the PCR product was the same in Banni buffalo as with the phenol: chloroform extraction procedure and that obtained for cattle. This indicated that there is some precipitation with ethanol. The working DNA concentration degree of conservation of the DNA sequences at the DRB3 was adjusted to 50–100 ng/μl for being used in the locus in Banni buffalo and cattle. Similar results were polymerase chain reaction. obtained by Aravindakshan et al. (2000) when the polymorphism of the DRB3 locus was studied in case of the Polymerase chain reaction Murrah and Surti breeds of Indian buffaloes. Buffalo DRB3.2 gene was amplified by the polymerase chain reaction (PCR) by using oligonucleotide primers viz., Digestion of the PCR product with the RsaI, BstYI and HaeIII LA3l, 5' GATGGATCCTCTCTCTGCAG CACATTTC- CT- restriction digestion enzymes 3' and LA32, 5'., CTTGAATTCGCGCT CACCTCGCC- Digestion of the 304 bp PCR product of the DRB3.2 locus GCTG-3' as described by Sigurdardottir et al. (1991), for with RsaI in the present resulted in digestion patterns ‘1’ the PCR amplification of the BoLA-DRB3 exon 2 in cattle. (237, 67), ‘s’ (144, 93, 67), ‘a’ (81, 33, 30, 39, 54, 67), ‘g’ The reactions were carried out in a final volume of 25μ1. (144, 39, 121), and ‘b’ (114, 30, 39, 54, 67) in Banni buffaloes. Each 25μl PCR reaction contained 50–100 ng of genomic When the PCR product was digested with BstYl enzyme only DNA, 2.5 μl of l0X PCR reaction buffer (I0mM Tris-pH- one pattern was observed viz. ‘b’ (304) as described by 9.0, 50mM KCl, 1.5 mM MgCl2 and 0.1% gelatin), 100 μM Gelhaus et al. (1995). This pattern corresponds to the 304 bp of each of the dNTPs, 5 pmole of each oligonucleotide primer PCR product wherein there is no restriction site for the mentioned above and 1 unit of the Taq DNA polymerase. A enzyme BstYl. Digestion of the PCR product with Hae III negative control to which no genomic DNA was added, was resulted in digestion patterns ‘a’ (170, 52, 82), ‘b’ (222, 82) also taken, to rule out any non-specific amplification of the and ‘d’ L 93, 29, 82). gene. The PCR was carried out in PCR Thermal Cycler. The As per the nomenclature followed by Van Eijk et al. (1992) thermal cycling profile followed was: initial denaturation for for the BoLA alleles, a cattle obtained by PCR-RFLP using 2 min. at 94°C followed by 30 cycles of 45 s at 92°C, 45s at the enzymes RsaI, BstYI and Hae III; the corresponding 66°C and 45 sec at 72°C. Final extension was for 10 min. at BuLA-DRB3 alleles observed to be present in Banni 72°C. buffaloes in the present study were BuLA-DRB 3.2 *19, *20, *36, *41, *abd and *gbb. Thus a total of 6 BuLA alleles of Restriction digestion of the PCR product (PCR-RFLP) the DRB3 locus were found to be present in the Banni animals The PCR amplified products from the previous step were taken up or the study. digested separately with the restriction endonucleases Rsal, The RsaI enzyme exhibited highest degree of BstY1 and Haelll (New England Biolabs). For restriction polymorphism as compared to other two enzymes viz. BstYl endonuclease digestion 10 μl of the PCR products were and HaeIII used in the study since the number of restriction digested for 3 hours at 37°C with 5 units of Rsa1 and Hae III digestion patterns obtained when the PCR product was and at 50°C with 5 units of BstY1 in a total volume of 15 μl digested with RsaI was maximum (5 digestion patterns) as 47 236 DHINGRA ET AL. [Indian Journal of Animal Sciences 80 (3)

Table 1. Different digestion patterns and the corresponding ‘b’ , ‘d’, ‘e’ and ‘i’ as compared to the three digestion patterns alleles of the DRB-3 exon 2 locus in the Banni buffalo breed obtained upon digestion with the Hae III enzyme in the Banni buffaloes viz. ‘a’, ‘b’ and ‘d’ in the present study. Also BuLa-DRB 3.2 Digestion pattern Allelic digestion of the PCR product with the Rsa I enzyme in the allele type Frequency RsaI BstYI HaeIII Murrah animals in the study carried out by Aravindakshan *19 b b 0.1774 et al. (2000) resulted in 10 different restriction digestion *20 I b b 0.0968 patterns while in Surti buffaloes digestion of the amplified *36 I b a 0.1129 product with RsaI resulted in 12 different RsaI enzyme *41 a b a 0.5161 digestion patterns. The present study showed the occurrence *abd a b d 0.0645 of 5 patterns when digestion was carried out by the enzyme *gbb g b b 0.0323 RsaI. Sena et al. (2003) carried out a study on the polymorphism in the MHC-DRA and- DRB alleles of Bubalus bubalis. The compared to the number of patterns obtained when digestion study was carried out on 19 Murrah and 20 Jafarabadi river was carried out with BstYI (1 pattern) and Hae III (3 patterns). buffalo animals besides 19 swamp buffalo animals (Carabao The allelic frequencies of the BuLA alleles in the Banni breed) from Brazilian herds and 17 individuals of river animals are given in Table 1. The BuLA alleles *abd and buffalo (Mediterranean breed). Primers LA31 and LA32 as *gbb found in this study on Banni buffaloes are allelic described earlier by Sigurdardottir et al. (1991) were used patterns that have not been reported previously in any of the for PCR amplification. The PCR products were sequenced studies on gene polymorphism of this locus either in cattle ABI Prism 377 DNA sequencer. A total of 6 Bubu-DRB or in buffaloes. Therefore, it could be assumed that these (BuLA-DRB) alleles were found in the Murrah animals and two alleles are putative new alleles of the Bola (BuLA) gene 5 Bubu -DRB (BuLA-DRB) alleles were found in the 20 present in the Banni breed of buffaloes. The DRB3.2 allele Jafarabadi animals. Therefore the number of Bubu -DRB *41 is present at the highest frequency of 0.5161. The alleles (BuLA-DRB) alleles in the Murrah and Jafarabadi animals *19 is present at a frequency of 0.1774, followed by *36 was almost similar to the number of BuLA-DRB alleles which is present at an allelic frequency of 0.1129. The observed to occur in the present study in the Banni breed. corresponding genotypes present in the Banni breed are *41/ *41, *19/*36, *41/*abd, *41/*20, *41/*gbb, *19/*19 and Population genetic analysis *41/*19 in the present study at genotypic frequencies of When population genetic analysis (heterozygosity 0.290, 0.225, 0.129, 0.193, 0.064, 0.032 and 0.064, estimates; observed and expected) of the Banni breeds was respectively. carried out using the POPGENE 1.32 version of the software The results obtained in the present study depict that while program of analysis of different population genetic digestion with RsaI and Hae III enzymes result in patterns 1, parameters it was observed that the value of the observed s, a, g , and ‘b’ and ‘a’ ‘b’ ‘d’, respectively. Digestion of the heterozygosity (Ho= 0.6774) was close to the expected PCR product with the Bst YI enzyme results in only a single heterozygosity (He= 0.6859). Therefore, the observed pattern in all the animals of the Banni buffalo breed viz.’b’. heterozygosity was almost the same as the expected This pattern corresponds to a size of 304 bp thereby indicating heterozygosity. So, the Banni population was almost in the that the DRB3 locus in the Banni buffaloes does not have a Hardy Weinberg equilibrium. It varied only very slightly from restriction digestion site for the enzyme BstYI. This is similar the Hardy- Weinberg equilibrium with the observed to the results obtained in the study carried out on the heterozygosity (Ho= 0.6774) being a little greater than polymorphism of the DRB3 locus in two Indian buffalo the expected heterozygosity (He= 0.6859). Takeshima breeds viz. Surti and Murrah by Aravindakshan et al. (2000) et al. (2003) studied the polymorphism present t the using PCR-RFLP. In both these buffalo breeds the digestion DRB3 locus in four breeds viz. Holstein, Japanese pattern obtained, when the product obtained by amplifying Black, Japanese Shorthorn and Jersey; the observed the DRB3.2 locus, with primers same as those that have been heterozygosity values for these breeds were 92.1, 92.0, 91.3, used in the present study, is digested with the BstYI enzyme 0.5, respectively as against the expected heterozygosity is, ‘b’. This observation showed that the DRB3 locus in case values of 90.1 ± 0.7, 91.2 ± 0.7, 88.7 ± 1.0 and 91.4 ± 0.5, of these two buffalo breeds also does not have a digestion respectively. site for the BstYI enzyme as observed in the case of Banni However, in the present study the population size that has animals studied in the present study. Digestion of the been taken for study is every small. If the sample size amplified product with the Hae III enzyme in the increases, there is quite a probability that the relative polymorphism study carried out by Aravindakshan et al. frequencies of the different alleles, the different genotypes (2000) on Murrah (n=34) and Surti (n= 36) buffalo resulted as well as the frequency of heterozygous individuals versus in the detection of five restriction enzyme patterns viz. ‘a’ homozygous individuals may change. 48 March 2010] MHC-DRB EXON-2 POLYMORPHISM IN BANNI BREED OF INDIAN BUFFALO 237

Certain major histocompatibility complex DRB3 exon-2 clearer picture regarding the distribution of the different alleles have been observed to be related to several diseases alleles of the DRB3.2 region in Banni buffaloes will emerge in cattle. For example, Dietz et al. (1997) detected an when the sample size for the study is increased. Earlier reports association between allele *16 and the risk to acute mastitis, on the study of polymorphism of the DRB3.2 locus in case BoLA DRB3.2 *16 and *22 were associated with a lower of Banni buffaloes are not available. risk of cysteic ovarian disease. BoLA allele *03 was ACKNOWLEDGEMENT associated with a decrease in the probability of occurrence of retained placenta. Lunden et al. (1990) were the first to This work was supported by the Indian Council of evaluate associations between BoLA class II genes and bull Agricultural Research, New Delhi. We are deeply grateful breeding values for diseases including clinical mastitis, to Mr Subhash Chander for collection of blood samples from ketosis, retained placenta, arid milk fever. However, there the breeding tract of the Banni buffalo and for his technical are no reports so far on the association of the buffalo DRB3 assistance. Thanks are due to Mr Sandeep for his exon 2 alleles with disease. unconditional help in the laboratory. Using PCR-RFLP, a total of 6 BuLA DRB3 alleles have been detected in the population size of 31 Banni animals. REFERENCES Three enzymes viz. RsaI, BstYI and HaeIII have been used Allen P M, Babbit B P and Unanue. 1987. T-cell recognition of in this study. MHC DRB Exon2 is one of the most important lysozyme: the biochemical basis of presentation. Immunology genetic systems involved in the generation of an effective Review 98:171–187. immune response. The study gives information on the Annual Report. 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Animal Genetics 26: 147–53. population as compared to the characterization of breed (s) Hedrick P W, Whittam T S and Parham P. 1991. Heterozygosity at using microsatellite markers which are neutral markers and individual amino acid sites: Extremely high levels for HLA-A do not give any information on occurrence of selection forces and HLA-B genes. Proceedings of National Academy of Science if any on a population. Also association with the trait of 88: 5897–5901. interest (in this case, resistance or susceptibility of disease) Hughes A L and Nei M. 1989. Nucleotide substitution at major histocompatibility complex class II loci: evidence for can directly be related if the MHC DRB exon 2 gene is overdominant selection. Proceedings of National Academy of characterized, in comparison to the microsatellites which can Science, USA 86: 958–62. be associated with the desired trait only if they are closely Hughes A L and Hughes M K. 1995. 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50 Indian Journal of Animal Sciences 80 (3): 239–243, March 2010 Role of additive and multiplicative age correction factors in sire evaluation of Hariana cattle

D S DALAL1 and A S KHANNA2

CCS Haryana Agricultural University, Hisar, Haryana 125 004 India

Received: 10 November 2008; Accepted: 5 October 2009

ABSTRACT Data on 491 first lactation records of Hariana cows sired by 41 sires maintained from 1995-2006 at 5 farms, viz. Germ Plasm Unit, CCSHAU, Hisar; Government Livestock Farm, Hisar; Kurukshetra Gaushala, Hisar; Gaushala Bhiwani and Gaushala Jind under ICAR project entitled, “Genetic Improvement of Indigenous Breeds of Cattle (Hariana Unit)” were analyzed for developing and evaluating the efficiency of additive and multiplicative age correction factors. Age at first calving was classified into 11 classes with an interval of 120 days between classes. The model used for least squares analysis and for deriving the age correction factors included herd, period of calving, season of calving and age groups as fixed effects and lactation length as a covariate. Both the age correction factors were equally effective on the basis of mean, F-ratio and R2 value, while multiplicative age correction factors were more effective in retaining similar coefficient of variation (CV) for all subclasses of age groups as that of uncorrected data and reducing the residual variance as compared to uncorrected and additively corrected data. Out of 41 sires; 19 sires were having positive estimated breeding value (EBV’s) from uncorrected, additively (ACD), and multiplicatively corrected data (MCD) with percent range of superiority over herd average as 0.96–37.30, 1.49–38.77 and 0.99–38.53, respectively. These findings indicated that ACD and MCD discriminated among the sires to the maximum extent as compared to uncorrected data. The rank correlations among EBV’s of sires estimated from uncorrected, ACD and MCD were very high (0.98) revealing that both additive and multiplicative correction factors were equally effective in evaluating the sires. As far as comparison among both the age correction factors is concerned, multiplicative age correction factors were more effective in cow evaluation, whereas additive age correction factors seemed to be more effective for sire evaluation.

Key words: Additive correction factors, Cattle, Multiplicative correction factors

The age at first calving varies from cow to cow as this for age at first calving. Therefore, this investigation was trait is considered to be governed more by environmental undertaken to develop additive and multiplicative age factors i.e. feeding and managemental practices. Apart from correction factors for cow and sire evaluation in Hariana genetic influence, cow’s milk yield is affected by age at cattle. calving. Therefore, the milk production record of a dairy MATERIALS AND METHODS animal has to be pre-corrected for age at calving to make a more reliable and less biased comparison among cows. Data on 491 first lactation milk yield of Hariana cattle, Mostly, sires are evaluated on the basis of first lactation milk progeny of 41 sires; maintained at 5 farms, viz. Germplasm yield of their daughters; so, there is a need to correct the first Centre, CCSHAU, Hisar; Government Livestock Farm, lactation milk yield of cows for age at first calving to have Hisar; Gaushalas, Hisar; Bhiwani and Jind from 1995–2006 more accurate estimate of breeding values of sires (Gandhi were analysed. Only those cows completing a lactation length 2000, Kumar and Singh 2003, Dawande et al. 2007). of atleast 100 days were included in the study. The whole Influence of age at first calving on milk yield is more duration of 12 years was divided into 3 periods of 4 year pronounced even that of sires (Miller 1973, Kumar and each. The year was divided into 4 seasons namely April to Gandhi 1995). Hence, this becomes more pertinent to correct June (summer); July to September (rainy); October to the first lactation milk yield of daughters of different sires November (autumn) and December to March (winter). Age at first calving was classified into 11 classes with an interval Present address: 1Professor, 2Professor and Head, Department of 120 days between classes, starting from < 1200 days to > of Animal Breeding. 2281 days. 51 240 DALAL AND KHANNA [Indian Journal of Animal Sciences 80 (3)

The least squares analysis of variance technique (Harvey of calving as fixed effects and sire within set as random effect. 1987) was used to study the effect of age at first calving and The estimated breeding value of sires was obtained by other factors on milk yield. A model incorporating herd, multiplying the BLUP values by 2. period of calving, season of calving and age groups as fixed The spearman’s rank correlations (Steel and Torrie 1980) effects and lactation length as covariate was used. Least were calculated among the ranking of sires by different squares constants and least squares means obtained from the methods. above model were used to derive the additive and RESULTS AND DISCUSSION multiplicative age correction factors for milk yield as follows: Additive correction factors (ACF): Additive correction The least squares analysis of variance revealed that age factors were obtained by substracting least squares constant groups had significant (P<0.05) effect on first lactation milk of the base class from the least squares constant of the yield. The coefficient of determination (R2) value for age respective class. The class with largest number of group was 3.79%, hence there is need to correct the milk observations (1561–1680 days) was considered as the base yield for age. The least squares constants and means along class. with additive and multiplicative age correction factor for Multiplicative correction factors (MCF): Multiplicative various age classes are given in Table 1. The highest least correction factors were derived from the least squares means squares constants (60.11±29.37 kg) and means (962.16±30.76 using the following expression as used by Miller et al. (1966) kg) were observed for 1921–2040 days age class. The least and Gacula et al. (1968). squares constants and mean for the base class (1561–1680 MCF= LSMb/LSMi days) was–31.35±23.75 kg and 870.70±23.47 kg, LSMb is the least squares means for the base class. respectively. The least squares constants and means did not LSMi is the least squares means for the ith age class. show regular trend for the different age groups. Similar trends The first lactation milk records were corrected by using were also observed by Gandhi and Gurnani (1997) and Kumar age correction factors derived above. Additive correction and Singh (2003) in Karan Fries cattle and Dawande et al. factors were subtracted from the observed milk yield whereas, (2007) in Holstein Friesian halfbreds. On the other hand, multiplicative correction factors were multiplied with the Gandhi and Singh (1999) and Gandhi (2000) observed an observed milk yield to obtain age corrected milk yield. increasing trend in least squares constants and means with To compare the effectiveness of ACF and MCF the same increase in age in Karan Fries cattle. model incorporating the same effects was fitted again on The additive correction factors ranged between –18.93 additively (ACD) and multiplicatively corrected data (MCD) kg for 1321–1440 days age class and 91.45 kg for 1921– and these correction factors were compared using criterion 2040 days age class. Multiplicative factors ranged between of mean, coefficient of variation (CV), F-ratio of age groups, 0.90 for 1921–2040 days age class to 1.02 for 1321–1440 residual variance, and R2 value. days age class. Age correction factors also did not show any The best linear unbiased prediction (BLUP) procedure regular trend. described by Henderson (1973) was used to estimate the breeding value of sires from uncorrected, ACD and MCD. Effectiveness of correction factors The mixed model used for estimation of EBV’s of sires The overall population means for uncorrected, additively comprising set of sires, herd, period of calving and season corrected data (ACD) and multiplicatively corrected data

Table 1. Least squares constants (LSC), means (LSM) and additive and multiplicative age correction factors for first lactation milk yield in Hariana cattle

AFC (days) No of observatoins LSC±SE LSM±SE ACF MCF up to 1200 (1) 30 0.20±38.82 902.25±40.83 31.55 0.9650 1201–1320 (2) 27 2.59±38.29 904.64±40.80 33.94 0.9625 1321–1440 (3) 52 –50.28±29.56 851.77±29.91 –18.93 1.0222 1441–1560 (4) 77 –25.49±24.13 873.56±24.67 2.86 0.9967 1561–1680 (5) 87 –31.35±23.75 870.70±23.47 0.00 1.0000 1681–1800 (6) 63 1.55±26.14 903.59±27.15 32.90 0.9636 1801–1920 (7) 55 18.53±27.33 920.58±28.54 49.88 0.9458 1921–2040 (8) 46 60.11±29.37 962.16±30.76 91.46 0.9049 2041–2160 (9) 21 37.98±43.29 940.03±46.88 69.33 0.9262 2161–2280 (10) 11 –24.83±58.02 877.22±64.26 6.52 0.9926 >2280 (11) 22 13.99±42.29 916.04±45.10 45.34 0.9505

Figures in parentheses are age groups. 52 March 2010] ADDITIVE AND MULTIPLICATIVE AGE CORRECTION FACTORS 241

Table 2. Mean (kg) and coefficient of variation (%) of first lactation milk yield for uncorrected, additively (ACD) and multiplicatively corrected data (MCD) in different age groups

Age No of Uncorrected ACD MCD groups observations Mean CV Mean CV Mean CV

1 30 902.25 24.79 870.25 25.69 876.44 25.03 2 27 904.64 23.44 870.64 24.35 869.89 23.91 3 52 851.77 25.32 869.77 24.80 869.02 24.35 4 77 873.56 24.78 870.56 24.86 874.39 24.29 –5 87 870.70 25.14 870.70 25.14 872.52 24.61 6 63 903.59 23.84 870.60 24.75 866.43 24.40 7 55 920.58 22.99 870.58 24.31 872.65 23.79 8 46 962.16 21.68 870.16 23.98 857.88 23.86 9 21 940.03 22.85 870.03 24.69 871.75 24.18 10 11 877.21 24.30 870.21 24.49 868.70 24.07 11 22 916.04 23.09 870.03 24.31 867.99 23.91 Overall 491 902.05 32.53 870.32 33.70 869.78 33.09

(MCD) were 902.05, 870.32 and 869.78 kg, respectively, were in consonance to the findings of Kumar and Gandhi with 21.83, 22.47 and 22.07% CV for each set of data (Table (1995), Gandhi and Gurnani (1997), Gandhi (2000) and 2). The overall mean for ACD was slightly higher than the Kumar and Singh (2003). Dawande et al. (2007) also reported mean from MCD. The milk yield corrected for age at first around zero F-value for effect of age groups on milk yield. calving by ACF were similar in age classes, similarly, milk The residual variance of MCD was lesser than the residual yield corrected by MCF was more or less compatible to each variance from uncorrected and additively corrected data. It other with the difference of 18.56 kg between the maximum revealed that MCFs were more efficient as compared to (<1200 days) and minimum (1921–2040 days) age class. ACFs. These results were in agreement with the findings of Since ACF retained constant least squares means, it could Chauhan (1988), Saxena et al. (1991), Gandhi and Gurnami be regarded as the most efficient method. Similar results were (1997) and Gandhi (2000). However, Kumar and Gandhi also reported by Chauhan (1988) and Saxena et al. (1991) (1995) found that ACFs were more effective as compared to and Malik (1995). The CV of ACD and MCD were similar MCFs in terms of reducing residual variance in Murrah to that of uncorrected milk yield for all age groups as well as buffaloes. The coefficient of determination (R2 value) of for overall age groups. As both the correction factors were fitting the model from ACFs and MCFs factors were 0.5 and effective in retaining overall mean and CV of population 0.8% less that from uncorrected data (64.5%). As both the near to the mean and CV of uncorrected data, it revealed that correction factors were having almost similar coefficient of both the age correction factors were almost equally variation, both seem to be equally effective for correcting appropriate. These finding were in agreement with the the first lactation milk yield. Kumar and Gandhi (1995) and findings of Chauhan (1988), Saxena et al. (1991), Kumar Gandhi (2000) also reported similar findings. The herd and Gandhi (1995), Gandhi and Gurnani (1997) and Gandhi average and range of estimated breeding values (EBVs) for (2000). different sires for all the 3 data sets revealed that about The F-value for the effect of age groups on first lactation 46.34% (19 out of 41) of sires were having positive EBVs milk yield was almost zero for both the corrected data as over herd average (Table 4). The per cent range of EBVs of compared to significant F-value (P<0.05) for uncorrected sires having positive deviation from herd averages from data (Table 3). It indicated that both the correction factors uncorrected, ACD and MCD were 0.96–37.31, 1.49–38.78 were appropriate ones and equally effective in removing the and 0.99–38.53, respectively. The high range of superiority effect of age from first lactation milk yield. These findings in ACD and MCD indicated that both the corrected data sets

2 discriminated among the bulls to the maximum extent as Table 3. F-value, residual variance and R value of first lactation compared to uncorrected data. milk yield for uncorrected, additively (ACD) and multiplicatively corrected data (MCD) in different age groups A simulation study was done to find out the superiority of bulls over the herd average when 5, 10, 15 , 20 and 25% of Uncorected ACD MCD sires were selected (Table 5). When 5% (2 out of 41) of sires were selected the percent ranges of superiority of sires over F-value 1.89 0.00 0.02 herd average was more from additively and multiplicatively Residual variance 40379.06 40379.06 38865.77 2 corrected data. Gandhi (2000) also reported higher range of R value (%) 64.5 64.0 63.7 superiority of ACD and MCD over uncorrected data. On the 53 242 DALAL AND KHANNA [Indian Journal of Animal Sciences 80 (3)

Table 4. Range of breeding values (EBVs) of sires estimated from uncorrected, additively corrected (ACD) and multiplicatively corrected data (MCD)

Data Herd Range of EBVs No of sires Range of EBVs of sires Range of average (kg) having ‘+’ EBVs having positive percent (kg) deviation (kg) superiority

Uncorrected 915.48 655.87 (12)– 19 924.29 (27)– 0.962– 1257.91 (11) 1257.91 (11) 37.305 ACD 894.14 637.86 (12)– 19 907.44 (17)– 1.487– 1240.84 (11) 1240.84 (11) 38.775 MCD 894.32 6944.14 (12)– 19 903.14 (17)– 0.986– 1235.86 (11 1235.86 (11) 38.525

Figures in parentheses indicate number of sires.

Table 5. Range of superiority of sires over herd average for different proportions of sires selected

Data Proportion of sires selected 25% 20% 15% 10% 5%

Uncorrected 976.07–1257.91 1010.59–1257.91 1052.34–1257.91 1103.05–1257.91 1207.23–1257.91 (6.62–37.31) (10.39–37.31) (14.95–37.31) (20.49–37.31) (31.87–37.31) ACD 968.82–1240.84 987.20–1240.84 1036.06–1240.84 1084.18–1240.84 1156.40–1240.84 (8.35–38.78) (10.41–38.78) (15.87–38.78) (21.25–38.78) (29.33–38.78) MCD 970.64–1238.86 979.96–1238.86 1034.52–1238.86 1082.78–1238.86 1149.14–1238.86 (8.53–38.53) (9.58–38.53) (15.68–38.53) (21.07–38.53) (28.49–38.53)

Figures in parentheses indicate superiority over herd average in per cent. other hand, Kumar and Gandhi (1994) reported lower range ACKNOWLEDGMENT of superiority over herd averages from ACD than that from The authors are thankful to the Indian Council of uncorrected data in Murrah buffaloes. Higher per cent ranges Agricultural Research, New Delhi, for providing the grant of superiority from ACD and MCD as compared to for the project. uncorrected data for different proportions selected (10, 15, 20 and 25%) were also observed, indicating that both the REFERENCES correction factors seem to have an edge over uncorrected data. Chauhan V P S. 1988. Additive versus multiplicative pre-corrections The rank correlations between the EBVs of sires estimated of dairy records for environmental effects in sire evaluation. on different data sets were high (0.980). The rank correlation Journal of Dairy Science 71: 195–203. of EBV’s of uncorrected data with ACD and MCD were 0.980 Dawande D, Kuralkar S V and Ali S Z. 2007. Efficiency of different and 0.982 , respectively. The rank correlation of EBVs of age correction factors for milk yield in Holstein Friesian halfbred ACD and MCD was 0.998. These results revealed that cows. Indian Journal of Veterinary Research 1: (Old 16) 1: 1– 8. ranking of sires from uncorrected and both the corrected data Gacula M C Jr., Gaunt S N and Damon R A Jr. 1968. Genetic and sets were almost similar. Kumar and Gandhi (1994) also environmental parameter of milk constituents for five breeds. reported near to unity rank correlations among uncorrected, 1. Effect of herd, year, season and age of cows. Journal of Dairy additively corrected and multiplicatively corrected data sets Science 51: 428–37. in Murrah buffaloes. Gandhi R S. 2000. Role of additive and multiplicative age correction Combining the results from different criteria, it may factors in Karan Fries sire evaluation. Indian Journal of Dairy be concluded that both additive and multiplicative Science 53: 131–36. age correction factors were comparatively more effective Gandhi R S and Gurnani M. 1997. Additive and multiplicative age in sire evaluation than from uncorrected data in correction factors in Sahiwal cattle. Indian Journal of Dairy Science 50: 325–28. Hariana cattle. As far as comparison among both the age Gandhi R S and Singh A. 1999. Effectiveness of additive and correction factors is concerned, multiplicative age correction multiplicative age correction factors in Karan Fries cattle. Indian factors seem to be more effective in cow evaluation, whereas Journal of Animal Sciences 69 (9): 715–17. additive age correction factors were more effective for sire Harvey W R. 1987. Mixed model least-squares and maximum evaluation. likelihood computer programme, January, 1987. PC-1 version. 54 March 2010] ADDITIVE AND MULTIPLICATIVE AGE CORRECTION FACTORS 243

Henderson C R. 1973. Sire evaluation and genetic trends. Buffalo Journal 11: 193–97. Proceedings of the Animal breeding and genetic symposium in Miller P. 1973. A recent study on age adjustment. Indian Journal honour of Dr J L Lush. ASAS and ADSA Chapaign, Illinois, of Dairy Science 56: 952–58. pp. 10–41. Miller R H, Harvey W R, Talbler K A, McDaniel B T and Corley E Kumar P V V and Singh A. 2003. Efficiency of additive and L. 1966. Maximum likelihood estimates of age effects. Indian multiplicative age correction factors for milk yield in Karan Journal of Dairy Science 49: 65–73. Fries cattle. Indian Journal of Dairy Science 56: 320–22. Saxena R K, Raheja K L and Singh A. 1991. Additive and Kumar R and Gandhi R S. 1994. Additive versus multiplicative multiplicative correcton factors for age at calving in Sahiwal. age correction factors for Murrah sire evaluation. Buffalo Indian Journal of Dairy Science 44: 47–53. Journal 10: 205–12. Steel R G D and Torrie J H. 1980. Principles and Procedures of Kumar R and Gandhi R S. 1995. Effectiveness of additive and Statistics: A Biometrical Approach. 2nd edn. McGraw-Hill multiplicative age correction factors in Murrah buffaloes. International Book Co., London.

55 Indian Journal of Animal Sciences 80 (3): 244–245, March 2010 Prediction of lactation milk yield from test day records in Murrah buffaloes

D CHAKRABORTY1, S S DHAKA2, BL PANDER3, A S YADAV3, S SINGH4 and P K MALIK5

CCS Haryana Agricultural University, Hisar, Haryana 125 004 India

Received: 24 December 2008; Accepted: 19 May 2009

ABSTRACT The data pertaining to 326 Murrah buffaloes, pregnancy of 48 bulls calved during period from 1987 to 2006 maintained at the University were considered for the present study. The index involving test day records from first to seventh had an accuracy of selection of 0.593, which is greater than accuracy of direct selection and thereafter, there was trivial increase in accuracy by adding subsequent test day records. Accuracy of bimonthly test day recording is good as accuracy for lactation yield and accuracy for monthly records. The accuracy of selection (rIH) for odd test day records (TD1 + TD2 + TD3 + TD4 + TD5 + TD7 + TD9) was 0.686 and for even test day records (TD2 + TD4 + TD6 + TD8 + TD10) was 0.652. Both the alternate test day records of first lactation had higher accuracy of selection than the accuracy of direct selection (0.566). Therefore, the bimonthly recording of test day could be better alternative for selection of bulls as it saves cost of recording. The coefficient of determination (R2) was maximum (39.89%) for TD8 among all ten prediction equations having only one variable and minimum for TD2 (4.69%). The equation having the combination of test day recording third, seventh, eight, and tenth is recommended for prediction of first lactation milk yield as it has 56.97% value for R2 and after that there was slight increase in the value of coefficient of determination.

Key words: Buffaloes, Genetic improvement, Milk yield, Test day records

For a breeder, it is mandatory to access the rate of changes Determination of accuracy of prediction of phenotypic and occurring in the traits of economic importance over a period breeding value was obtained from test day records. of time to estimate net gain as a result of breeding programme. First test day yield was recorded from the seventh day The selection of sires is mostly based on the complete after calving, and 10 test day milk records were taken at every lactation yield that is predicted from individual test day milk 4 week interval. Multiple regression analysis was used to records taken at monthly intervals (Pander et al. 1992). predict lactation production from test day records. However, Danell (1990) and Machado et al. (2000) reported Test day records were used for genetic prediction of that test day milk yield records in mid lactation have high lactation yield using selection index procedure. genetic and phenotypic correlations with lactation yield, RESULTS AND DISCUSSION thereby, increase the accuracy of sire evaluation. Hence, the investigation was also planned to study the inter-relationship Prediction of breeding value of FLY from test day records: of test day with that of first lactation milk yield to develop Ten independent variables on terms of test day milk records suitable predictors for the prediction of phenotypic value and from TD1 to TD10 were taken for this purpose. Table 1 breeding value of first lactation milk yield. presents the accuracy of selection for the prediction of breeding value of FLY from test day records. The accuracy MATERIALS AND METHODS of direct selection for first lactation yield is 0.566. The index The present study was conducted on data pertaining to 326 involving only first test day had an accuracy of 0.076 and Murrah buffaloes maintained at Buffalo Research Centre the accuracy of selection increased by 264.47% when TD1 (BRC) of the university, Hisar over a period of 20 years from + TD2 were considered. Subsequently, the accuracy of 1987 to 2006, were delineated as summer (March to June), selection increased with the incorporation of later test day monsoon (July to October) and winter (November to February) in the index The index involving test day records from first on the basis of geo-climatic conditions prevailing in the region. to seven had an accuracy of selection 0.593, which is greater Present address: 1Ph. D. Scholar 2, 3 Senior Scientist. than accuracy of direct selection and thereafter, there was 4SMS, KVK Sangaria, RAU, Bikaner, Rajasthan. trivial increase in accuracy by adding subsequent test day 5Assistant Professor, Nivasari Agricultural University, Nivasari, records. Therefore, it is worth to include the test day records Gujarat. up to seventh to get better results for bull selection. The 56 March 2010] PREDICTION OF LACTATION MILK YIELD IN BUFFALOES 245

Table 1. Accuracy and genetic gain in breeding value of FLY from selection on test day records

Traits in index Accuracy of % increase Genetic gain % increase ∆ ∆ selection (rIH) in rIH ( G) in G TD1 0.076 – 1.39 – TD1+TD2 0.277 264.47 18.10 1202.16 TD1+TD2+TD3 0.299 7.94 21.10 16.57 TD1+TD2+TD3+TD4 0.395 32.10 36.85 74.64 TD1+TD2+TD3+TD4+TD5 0.460 16.46 50.08 35.90 TD1+TD2+TD3+TD4+TD5+TD6 0.464 0.87 50.82 1.48 TD1+TD2+TD3+TD4+TD5+TD6+TD7 0.593 27.80 83.22 63.75 TD1+TD2+TD3+TD4+TD5+TD6+TD7+TD8 0.632 6.58 94.44 13.42 TD1+TD2+TD3+TD4+TD5+TD6+TD7+TD8+TD9 0.701 10.92 117.74 24.67 TD1+TD2+TD3+TD4+TD5+TD6+TD7+TD8+TD9+TD10 0.725 3.42 124.18 5.47

Accuracy of direct selection = 0.566 accuracy of prediction of breeding value using the sum of recording third, seventh, eighth and tenth is recommended 10 test day records is 0.725. The result was in close agreement for prediction of first lactation milk yield as it had 56.97% with the findings of Pander et al. (1993). value for R2 and after that there was slight increase in the Recording of daily yield at monthly interval is costly and value of coefficient of determination. Similarly, Saini et al. hence an attempt was made to predict the breeding value of (2005) also reported that prediction equation having 3 FLY from bimonthly recording. Results of bimonthly variables, i.e. first, second and seventh test day milk yield recording (Table 2) indicated that the indexes involving could give adequate accuracy (78.42%) in estimating 300 bimonthly recording had accuracy of 0.62 and 0.652 for odd days milk yield in Rathi cows. However, Appannavar et al. and even test day bimonthly recording, which are greater (1995) reported that when 3 test day yields were incorporated than the accuracy of direct selection (0.566). The increase in the accuracy was 67.43%; when 4 test day yields accuracy of selection over that of accuracy of direct selection were incorporated, the accuracy was 76.01%; when 5 test was 21.20 and 15.19%, respectively, for odd and even test day yields were incorporated the accuracy was 81.64% and day bimonthly recording. Therefore, the bimonthly recording when 6 test day yields were incorporated the accuracy was of test day could be a better alternative for selection of bulls. 85.49%. Similar findings were reported by Pander and Hill (1993) in From the results, it may be inferred that accuracy of Hostein-Friesian cattle. bimonthly test day records is as good as accuracy of lactation Prediction of phenotypic value of FLY from test day yield and accuracy of monthly records and it could be taken records: The prediction equation and their R2 values for the as better alternative for selection of bulls and is cost effective prediction of phenotypic value of lactation milk yield from also. The equation having the combination of test day test day records are presented in Table 3. The coefficient of recording third, seventh, eighth and tenth is recommended determination (R2) for prediction equation for the prediction for prediction of first lactation milk yield. of phenotypic value of lactation milk yield from the test day REFERENCES records involving only TD1 was 4.92%. The coefficient of 2 determination (R ) was the maximum (39.89%) for TD8 Appannavar M M, Kumar S and Shashidaran T. 1995. Test day among all 10 prediction equations having only 1 variable models in predicting 305 days first lactation milk yield in Surti and minimum coefficient of determination (R2) was obtained buffaloes. Indian Journal of Dairy Science 48: 411–12. for TD2 (4.69%). Moreover, several other prediction Danell B. 1990. Genetic aspects of different parts of lactation. equations were also developed by utilizing various Proceeding of 4th World Congress on Genetic to Livestock Production. Edinburgh 14: 114–17. combinations of test day recording. First, third, fourth and 2 Machado S D, Freitas M A R and Gadini C H. 2000. Genetic seventh to tenth was having the maximum (R ) to the tune parameters of test day milk yields of Holstein cows. Animal of 60.78%. The equation having the combination of test day Breeding Abstract 68: 88. Pander B L and Hill W G. 1993. Genetic evaluation of lactation Table 2. Accuracy in breeding value of FLY from selection on yield from test day records on incomplete lactation. Livestock alternate test records Production Science 37: 23–26. Pander B L, Hill W G and Thompson R. 1992. Genetic parameters Traits in index Accuracy of %increase of test day records of British Holstein-Friesian heifers. Animal selection (r ) in r IH IH Production 55: 11–21. TD1+TD2+TD5+TD7+TD9 0.686 21.20 Saini T, Gahlot G C and Kachwaha R N. 2005. Prediction of 300 TD2+TD4+TD6+TD8+TD10 0.652 15.19 days lactation yield on the basis of test day milk yield in Rathi cows. Indian Journal of Animal Sciences 75: 1087–89. 57 Indian Journal of Animal Sciences 80 (3): 246–248, March 2010 Genetic analysis of lactation traits in Jamunapari goats

R ROY1 and AJOY MANDAL2

Central Institute for Research on Goats, Makhdoom, Uttar Pradesh 281 122 India

Received: 2 April 2008; Accepted: 30 August 2009

ABSTRACT Data on 1367 lactations from 571 Jamunapari does, maintained (1985 to 2005) at the Central Institute for Research on Goats, Makhdoom, Mathura, Uttar Pradesh, India, were utilized to study the lactation traits and their genetic parameters. The 90-day, 140-day and total milk yield for this breed averaged 79.89±1.41, 119.17±2.66 and 150.79±3.37 lit., respectively. The average lactation length was 162.01±2.68 days. Significant differences among sires existed within the breed for all the lactation traits. There were significant variations for all lactation traits of does among different kidding years/periods and lactation order. Does kidded from March-April had significantly higher milk yields than the does kidded from October-November. The interaction effect of period and season of kidding also yielded significant variations for all lactation traits. Both the linear and quadratic regressions of days of lactation (milking) showed significant effect on total milk yield. The heritabilities of all lactation traits were moderate in magnitude, ranging from 0.17 to 0.24. Genetic correlations among the lactation traits were medium (0.41±0.32) to high (0.99±0.01) in this study.

Key words: Genetic analysis, Heritability, Jamunapari goats, Lactation traits

Adjustmant of data for non-genetic factors and estimation (MY90D), 140-days milk yield (MY140D), total milk yield of geneic parameters for the various traits are necessary to (TMY) and lactation length (LL) of animals. The data on obtain reliable estimates for important economic traits and 90-days milk yield less than 30 kg or higher than 175 kg and to increase accuracy of selection of breeding animals. The days of milking less than 90 days were excluded from this importance of the various environmental effects on lactation analysis. The data were classified according to period of traits of goats has also been examined in other studies (Kala kidding, season of kidding and parity/lactation order of does. and Prakash 1990, Roy et al. 2001, Kumar et al. 2004, Singh The days of lactation (milking) were considered in the model et al. 2005). The present study was undertaken to identify as linear and quadratic covariate for total milk yield (TMY) various factors, viz. period of kidding, lactation number of of does. dam and season of kidding, influencing the lactation traits, Data were analyzed using the mixed model least-squares and to estimate the genetic and phenotypic parameters of analysis for fitting constants (Harvey 1990) including all these traits in Jamunapari goat. main effects and interaction. The following models were used for different traits: MATERIALS AND METHODS Yijklm= μ+Si+Pj+Lk+Al+ (P×A)jl+ eijklm (for MY90D, Data on 1367 lactations from 571 Jamunapari does, MY140D and LL) 2 maintained at the institute over a period of 21 years (1985 to Yijklm= μ+Si+Pj+Lk+Al+ (P×A)jl + b1 L+b2L +eijklm (for 2005) were utilized for the study. Generally, the animals were TMY only) maintained under semi-intensive feeding systems (Singh et where Yijklm is the record for the mth animal, μ is the overall al. 2008), where they are allowed to graze during the day for mean, Si is the random effect of the ith sire, Pj is the fixed 5 to 6 h and supplemented with some amount of concentrate effect of the jth period of kidding, Lk is the fixed effect of according to age and physiological category of animals and the kth lactation number of doe, Al is the fixed effect of the with seasonally available green and dry fodders. The lactation 1th season of kidding, (P×A)jl is the interaction effect traits analyzed for this study were 90-days milk yield between jth period of kidding and 1th season of kidding, b1 and b2 are the linear and quadratic regression coefficients, Present address: 1Principal Scientist and Head, 2 Senior Scientist, respectively, for the days of lactation of doe, eijklm is the Genetics and Breeding Division (e mail: ajoymandal residual error element with standard assumptions. @rediffmail.com). The genetic parameters of various traits were estimated 58 March 2010] LACTATION TRAITS ANALYSIS IN JAMUNAPARI GOAT 247 by the paternal half-sib method. Duncan’s multiple range kidding had highly significant (P<0.01) effect on all lactation test (DMRT) as described by Kramer (1957) was applied to traits in this study. Similarly the significant effects of period/ compare different sub-groups mean. years of kidding on lactation traits of does for various breeds were reported by Kala and Prakash (1990), Roy et al. (2001), RESULTS AND DISCUSSION Kumar et al. (2004) and Singh et al. (2005). The significant Lactation traits: The overall least-squares means (Table 1) differences in milk yields and lactation length among for lactation traits obtained in this study were well Jamunapari does kidded in different periods/years may be comparable with the earlier findings of Roy et al. (2000, attributed to differences in management, selection of bucks 2001) for this breed. However, Kala and Prakash (1990) and environmental conditions such as ambient temperature, reported lower milk yield at 90-days of lactation in humidity, rainfall etc. Significant (P<0.01) differences in milk Jamunapari does. The random effects of sire had significant yield as well as lactation length existed among does of (P<0.01) influence on all lactation traits under study. Similar different lactation orders. Lower milk yields were observed significant sire effect on milk yield at different milking days in does of first lactation as compared to does of second and and lactation length was observed by Roy et al. (2003) and onward lactations (Table 1). Significant effect of lactation Singh et al. (2005). The significant effect of sire on all these number of does was observed for 90-days/lactation milk yield traits studied indicated that these traits could well be (Kala and Prakash 1990, Singh et al. 2001, Roy et al. 2001) improved by introduction of superior bucks. The period of and lactation length (Roy et al. 2001) in different breeds of

Table 1. Least-square means along with standard errors of different lactation traits in Jamunapari goat

Effects MY90D (lit.) MY140D (lit.) TMY (lit.) LL (days)

Overall mean 79.89±1.41 (1367)* 119.17±2.66 (969) 150.79±3.37 (909) 162.01±2.68 (1295) Period of kidding Pd 1 (1985-87) 80.35±6.11bcd (74) 126.31±12.64bc (25) 156.66±13.58bc (25) 135.33 ±6.17a (73) Pd 2 (1988-90) 73.64±4.70ab (136) 115.53±9.61ab (67) 158.81±10.80bc (65) 137.61±9.90a (125) Pd 3 (1991-93) 75.75±3.55abc (118) 112.58±6.69ab (77) 136.72±7.71ab (73) 172.49±7.58c (106) Pd 4 (1994-96) 85.37±3.18de (171) 121.90±6.45bc (107) 152.64±7.40b (104) 151.68±6.74ab (158) Pd 5 (1997-99) 66.67±3.46a (139) 100.94±7.05a (72) 127.45±8.16a (60) 167.31±7.51bc (114) Pd 6 (2000-02) 80.72±4.40cd (279) 116.92±8.26ab (211) 146.98±9.48ab (194) 183.63±9.33c (271) Pd 7 (2003-05) 96.78±5.36e (450) 140.00±9.65c (410) 176.25±10.91c (388) 186.04±11.31c (448) Lactation number 1 71.10±2.01a (513) 105.23±3.57a (354) 135.00±4.23a (331) 169.18±4.01cd (490) 2 82.07±1.83b (347) 120.45±3.28bc (250) 149.75±3.97b (236) 174.93±3.64d (324) 3 79.92±1.93b (224) 115.40±3.40b (170) 149.24±4.04b (159) 162.94±3.84bc (216) 4 83.46±2.25b (131) 125.44±3.94c (95) 156.77±4.56b (90) 166.33±4.62bcd (121) 5 83.99±2.90b (78) 128.07±5.00c (56) 160.27±5.68b (54) 155.71±5.92ab (74) 6 78.84±3.23b (74) 120.43±5.84bc (44) 153.70±6.67b (39) 142.99±6.66a (70) Season of kidding S-1 (Mar-Apr) 82.63±1.63a (722) 123.50±3.09a (505) 154.50±3.77a (480) 159.16±3.21a (689) S-2 (Oct-Nov) 77.17±1.58b (645) 114.84±3.27b (464) 147.07±3.95b (429) 164.87±3.10a (606) Period season interaction Pd 1×S 1 82.85±6.61bcd (42) 133.62±13.91bc (17) 156.05±14.98cde (17) 152.79±13.81abc (41) Pd 1×S 2 77.84±6.72bcd (32) 119.01±15.24abc (8) 157.28±16.19cde (8) 117.87±13.95a (32) Pd 2×S 1 75.08±5.06abc (98) 117.91±9.83abc (58) 149.80±10.87bcd (57) 153.93±10.69bc (90) Pd 2×S 2 72.21±5.36ab (38) 113.15±12.47abc (9) 167.83±14.08def (8) 121.29±11.45a (35) Pd 3×S 1 72.86±4.27ab (53) 110.23±8.09ab (30) 137.50±9.06abc (30) 161.80±9.25cd (48) Pd 3×S 2 78.64±4.01bcd (65) 114.93±7.36abc (47) 135.93±8.52abc (43) 183.18±8.59de (58) Pd 4×S 1 97.38±4.20ef (47) 141.35±8.71cd (20) 182.42±9.71ef (20) 126.98±9.08ab (40) Pd 4×S 2 73.36±3.15ab (124) 102.45±6.10a (87) 122.87±7.06a (84) 176.38±6.56cde (118) Pd 5×S 1 68.68±4.06ab (72) 101.13±8.49a (32) 129.05±9.70abc (26) 162.66±8.57cd (62) Pd 5×S 2 64.66±3.87a (67) 100.75±7.60a (40) 125.85±8.86ab (34) 171.97±8.80cde (52) Pd 6×S 1 76.29±4.42bcd (180) 108.80±8.33ab (127) 139.46±9.56abcd (113) 170.48±9.34cde (175) Pd 6×S 2 85.17±4.81cd (99) 125.04±8.77bc (84) 154.50±10.01cd (81) 196.77±10.17e (96) Pd 7×S 1 105.26±5.32f (230) 151.46±9.55d (221) 187.24±10.80f (217) 185.47±11.20de (233) Pd 7×S 2 88.30±5.63de (220) 128.55±10.03bc (189) 165.26±11.32def (171) 186.60±11.87de (215)

Means with different superscripts differed significantly (P<0.05) from each other. *Figures in parentheses indicate number of observation. 59 248 ROY AND MANDAL [Indian Journal of Animal Sciences 80 (3) goat. Season of kidding had significant (P<0.05) influence view of very high and positive genetic correlations between on all lactation traits except lactation length (Table 1). MY90D and MY140D and TMY. Significant variations in lactation traits between does kidded ACKNOWLEDGEMENTS in different seasons were also observed by Kala and Prakash (1990) in Jamunapari and Barbari goats; Roy et al. (2001) in The authors are thankful to Dr B U Khan, Ex-Project Jamunapari goat and Singh et al. (2005) in Barbari goat. Coordinator, All-India Co-ordinated Reasearch Project on However, Singh et al. (2001) reported nonsignificant effect Goat Improvement, for his devoted contribution and help of season of kidding on lactation yield in Black Bengal and since the inceptoin of this project. We wish to acknowledge its halfbreds. In this study, does kidded in March-April the contribution of the staff of the Jamunpari Project. The produced significantly more milk than does kidded in support extended by Director, CIRG, in providing facilities October-November, which may be due to the fact that the to carry out this work is also gratefully acknowledged. does that kidded in March-April pass through a period with REFERENCES favorable climate, when good quality pasture was available. This corroborated by the earlier findings of Roy et al. (2001) Harvey W R. 1990. User’s guide for LSMLMW PC-2 version mixed for this breed. The interaction effect between period of model least squares maximum likelihood computer programme. kidding and season of kidding was also significant (P<0.01) Mimeograph Columbue, Oghio. USA. for all lactation traits (Table 1). Days of lactation (milking) Kala S N and Prakash B. 1990. Genetic and phenotypic parameters were statistically significant (P<0.01) as linear and quadratic of milk yield and mik composition in two Indian goat breeds. covariates for TMY in this study. Small Ruminant Research 3: 475–84. Kramer C Y. 1957. Extension of multiple range tests to group correlated adjusted means. Biometrics 13: 13. Genetic and phenotypic parameters Kumar Arun, Tomar A K S and Mehta B S. 2004. Lactational The heritabilities of MY90D , MY140D, TMY and LL of performance of Kutchi goats under semiarid condition animals were 0.24±0.07, 0.19±0.08, 0.21±0.09 and of Rajasthan. Indian Journal of Dairy Science 57 (4): 0.17±0.07, respectively. The moderate heritabilities for milk 285–87. yields were corroborated by findings for other goat breeds Roy R, Rout P K, Singh L B and Khan B U. 2000. Factors affecting (Kala and Prakash 1990, Singh et al. 2004, Kumar et al. milk yield in Jamunapari goats. Indian Journal of Animal 2004). On the contrary, Singh et al. (2005) reported the lower Production 32 (1–4): 6–8. (0.07) and higher (0.63) heritability estimates for 90 days Roy R, Rout P K, Singh L B and Khan B U. 2001. Influence of non-genetic factors on milk yield of Jamunapari goats. and total lactation milk yield in Barbari goats, respectively. International Journal of Animal Sciences 16 (1): 81–83. The moderate heritability estimates for lactation traits in this Singh S K, Rout P K and Khan B U. 2005. Peroformance evaluation study suggest the existence of ample scope for improving and genetic parameters of early life growth, lactational and these traits through selection. The phenotypic and genetic reproduction traits in Barbari does. Indian Journal of Animal correlations of MY90D with MY140D and TMY were Genetics and Breeding 26 (1, 2): 68–75. significantly high (0.86 to 0.99). Similarly, the genetic and Singh D K, Singh C S P, Singh N S and Singh L B. 2000. Genetic the phenotypic correlations of MY140D with TMY were analysis of milk production characteristics of Black Bengal and its halfbreds. Indian Journal of Animal Sciences 70 (11): 167– also higher (rp=0.92, rg=0.94) and significant. The phenotypic and genetic correlations of LL with MY90D, MY140D and 69. Singh D K, Singh L B and Das P. 2004. First lactational traits of TMY ranged from medium to moderately highu (0.30 to Black Bengal goats. Journal of Research 16 (1): 153–58. 0.70). High phenotypic and genetic correlations among Singh M K, Rai B and Sharma N. 2008. Factors affecting lactation traits were also reported by Singh et al. (2005) for survivability of Jamunapari kids under semi-intensive Barbari goats. This result indicated that selection of does on management system. Indian Journal of Animal Sciences 78 (2): the basis of part lactation records is probably as efficient, in 178–82.

60 Indian Journal of Animal Sciences 80 (3): 249–252, March 2010 Effect of naturally fermented rice straw based diet on the performance of buffalo calves

M WADHWA1, K KAUR2 and M P S BAKSHI3

Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana 141 004 India

Received: 10 June 2009; Accepted: 26 October 2009

ABSTRACT The effects of fermented (FRS) and unfermented rice straw (RS) based complete diets on the nutrient utilization and growth performance of 8 male Murrah buffalo calves (114±9 kg BW) was assessed. The animals divided in to 2 equal groups were offered isonitrogenous and isocaloric diet containing either RS supplemented with high protein (CP 21%) concentrate mixture or FRS with low protein (CP 17%) concentrate mixture in 60:40 ratio for 270 days. The DM intake was comparable in both the groups. The digestibility of nutrients especially that of cellulose and hemicellulose improved significantly, but that of CP depressed significantly in calves fed FRS than RS based diet. The significantly low ammoniacal-N in the FRS based diet was partially responsible for considerably higher TCA-N in the rumen liquor. Bacterial and protozoal populations in the rumen of animals fed FRS was higher than those fed RS base diet. The entodinomorphs were more in number compared to holotrichs, in general as well as in FRS fed groups. The N-intake was comparable in both the groups, however, the faecal-N excretion was significantly higher in FRS than RS supplemented group. Reverse but statistically non significant trend was observed in urinary-N excretion, which resulted in higher efficiency of utilization of absorbed-N in FRS than RS fed group. The ME content of the diet containing FRS improved significantly. The favorable microbial population in the rumen, higher digestibility and efficient utilization of nutrients were responsible for considerably higher daily live weight gain in calves fed with FRS than that with RS based diet. It was concluded that naturally fermented of RS with urea could be efficiently utilized in improving performance of calves, besides sparing about 70% of oilseed cake.

Key words: Fermented rice straw, Growth, Nutrient utilization, Rumen microorganisms, Straw

Wheat straw is the basal roughage in the traditional MATERIALS AND METHODS feeding system in the Northern region of India especially Fermentation of rice straw: Naturally fermented rice straw Punjab, Haryana and Uttar Pradesh. Therefore, most of the was prepared by sprinkling urea solution (14.0 kg urea in rice straw produced in these states (24.5 mt/annum; 200 litre water) on 386 kg dried chaffed rice straw (2.5–3.5 Anonymous 2007) is surplus and more than 80% is cm) and stacked for 9 days. The ratio of urea to rice straw burnt, causing environmental pollution. The remaining was kept 3.5: 96.5 and moisture was 40% (Bakshi et al. 1987, rice straw is used either for card board/paper industry, Bakshi and Wadhwa 2001). About 50q dried, chaffed rice as packing/bedding material, mushroom cultivation or straw was treated and stacked. for feeding to livestock by land-less/marginal farmers. Earlier studies revealed that rice straw could be Table 1. Ingredients composition of concentrate mixtures fermented with urea in stacks and bales successfully and fermented rice straw had higher degradability Constituent Conc-1 (HP) Conc-2 (LP) than unfermented rice straw (Kaur et al. 2007 2008). In Maize 30 35 the present study an attempt was made to ferment rice straw Mustard cake 10 10 with urea and to assess its impact on the performance of Deoiled mustard cake 20 05 buffalo calves. Rice bran 15 15 Deoiled rice bran 22 32 Mineral mixture 02 02 1 2 Present address: Senior Biochemist; Reseach Associate; Common salt 01 01 3Senior Nutritionist cum Head (e-mail: [email protected]), Department of Animal Nutrition. HP, High protein; LP, low protein. 61 250 WADHWA ET AL. [Indian Journal of Animal Sciences 80 (3)

Feeding schedule: A 270–day growth trial was conducted total volatile fatty acids (Barnett and Reid 1957). The data on 8 male Murrah buffalo calves (114±8.9 kg BW). The were analyzed by using completely randomized design animals divided into 2 equal groups were offered either 3.00 (Snedecor and Cochran 1994). kg untreated rice straw supplemented with 1.45±0.21 kg high RESULTS AND DISCUSSION protein concentrate mixture with 2 kg green fodder (Table 1) or 3.00 kg naturally fermented straw (FRS) was In the low protein concentrate mixture only 25% of deoiled supplemented with 1.55±0.09 kg low protein concentrate mustard cake was used as compared to that used in high mixture with 2 kg green fodder, depending upon the nutrient protein concentrate mixture (Table 1). The proximate and requirements of calves (NRC 2001). The animals were cell wall constituents of both concentrate mixtures were offered respective diet individually as total mixed ration similar, except their CP (20.9 vs. 17.0% in HP vs LP) content (TMR). The roughage to concentrate ratio in the TMR was (Table 2). The fermented rice straw was enriched with maintained at 60:40 in both the groups. The animals were microbial protein, as during natural fermentation of straw weighed every fortnight for 3 consecutive days and the with urea, there was significantly higher microbial population feeding schedule was adjusted accordingly. A 7–day (bacteria, fungi and antinomycetes) in the stacks (Bakshi et metabolism trial was conducted at the termination of the al. 1986, Gupta et al. 1987, Makhdoomi et al. 1996), resulting growth study to assess nutrient utilization in the two groups. in increased CP (9.9 vs. 4.8%). The hemicellulose in FRS Rumen studies: For conducting rumen studies, 3 rumen reduced (P<0.05), because microbes proliferating in the stack fistulated male buffaloes (464.7±5.1 kg body weight) were utilized part of it, resulting in low NDF content. The NDF offered either 6–7 kg untreated rice straw supplemented with content is negatively correlated to DM intake and digestibility 2.5 kg high protein concentrate mixture and 2 kg fresh of cell wall constituents. available green fodder or 6–7 kg naturally fermented rice The DM intake was higher in FRS than RS fed group, but straw supplemented with 2.5 kg low protein concentrate the differences were statistically nonsignificant (Table 3). mixture and 2 kg available green fodder (to meet vitamin A The digestibility of nutrients especially of cellulose and requirement) as TMR. The animals were adapted on the hemicellulose improved significantly (P<0.05), because of respective diet for 30 days. Thereafter, the samples of rumen the breakdown of the ligno-cellulose bond in RS by ammonia contents were collected from each animal for 3 consecutive released from urea, resulting in availability of free cellulose days at 0, 2, 4, 6, 8 and 10 h after feeding. The samples were and hemicellulose for degradation in the rumen. As observed filtered through 4-layered muslin cloth and preserved with in ammoniated wheat straw (Horten et al. 1982), the few drops of mercuric chloride. digestibility of CP depressed significantly (P<0.05) in FRS Analytical methods: Samples of feed, orts and faeces were than RS fed group, due to poor digestion and absorption of pooled for each animal separately and thoroughly mixed. microbial cell wall-N and endogenous protein (Dias-Da-Silva These were then ground (1 mm) and analyzed in duplicate and Sundstol 1986). for nitrogen and total ash (AOAC 1995), cellulose (Crampton The effect of feeding FRS on the rumen environment in and Maynard, 1938) and other cell wall constituents buffaloes revealed that total-N and NPN content in the (Robertson and Van Soest 1981). The urine samples were strained rumen liquor were statistically comparable in both analyzed for nitrogen content only. The ME was determined the groups (Table 4). The significantly low (P<0.05) from the apparent digestible OM using the relationship given ammoniacal- N in the FRS based diet was partially by Broster and Oldham (1981). The SRL samples were responsible for considerably higher TCA-N in the rumen analyzed for total-N, ammoniacal-N (AOAC 1995), liquor. trichloroacetic acid precipitable-N (Cline et al. 1958) and The microbial (protozoa and bacteria) populations in the

Table 2. Chemical composition of feedstuffs offered to buffalo calves

Constituent Concentrate Rice straw Green fodder HP LP Untreated Urea treated

Total ash 9.3 8.0 13.2 15.3 8.2 Organic matter 90.7 92.0 86.8 84.7 91.8 Crude protein 20.9 17.0 4.8 9.9 7.7 Neutral detergent fibre 44.5 46.0 74.0 70.0 71.0 Acid detergent fibre 17.5 17.0 48.5 56.0 42.0 Hemicellulose 27.0 29.0 25.5 14.0 29.0 Cellulose 9.0 8.5 36.0 37.0 33.0

HP, High protein; LP, low protein. 62 March 2010] EFFECT OF FERMENTED RICE STRAW BASED DIET ON CALVES 251

Table 3. Effect of naturally fermented rice straw on digestibility Table 6. Effect of naturally fermented rice straw on N-utilization of nutrients in buffalo calves (g/d) in buffalo calves

Parameter RS FRS Pooled SE Parameter RS FRS Pooled SE

Dry matter intake, kg/d 3.7 4.5 0.40 N-intake 65.4 86.2 7.40 Digestibility of nutrients, % Faecal-N 20.9a 47.4b 2.80 Dry matter 42.4 47.5 4.54 N-digested 44.5 38.9 4.97 Organic matter 47.2 54.0 4.27 Crude protein 67.8b 45.0a 1.54 Urinary-N 18.1 11.6 2.46 Neutral detergent fiber 33.1 47.4 5.28 N-Retained 26.5 27.3 3.38 Hemicellulose 44.9a 73.3b 5.29 Cellulose 40.1a 59.8b 4.77 attach fibrous feed and invades and breaks the plant tissue to RS, Untreated rice straw; FRS, fermented rice straw; figures smaller pieces. The cellulase enzyme of Epidinia are usually with different superscripts in a row differ significantly, P<0.05. endo-gluconase type, but they have to depend upon other entodinomorphs for complete hydrolysis. The higher number Table 4. Impact of naturally fermented rice straw on the of Epidinium, Polyplastron, Ophryoscolex and biochemical changes in the rumen, mg % Ostracodinium spp. in the rumen of animals fed FRS, were responsible for higher digestibility of hemicellulose. Parameter RS FRS Pooled SE Feeding FRS did effect N-intake significantly, however, Total-N 90.0 116.7 9.43 the faecal-N excretion was higher (P<0.05) in FRS than in NPN 30.7 29.3 2.85 RS supplemented group (Table 6) because of low CP TCA-N 59.7 85.7 7.61 digestibility in FRS than in RS fed group. Reverse but Ammoniacal-N 13.0a 6.3b 1.49 statistically non significant trend was observed in urinary-N TVFA 8.4 8.8 0.18 excretion, which resulted in higher efficiency of utilization RS, Untreated rice straw; FRS, fermented rice straw; NPN, non- of absorbed-N in FRS than in RS fed group. Animals in both nitrogen protein nitrogen; TVFA- total volatile fatty acids; TCA- the groups were in positive-N balance and the differences N- trichloro acetic acid precipitable nitrogen; figures with different were statistically non-significant. superscripts in a row differ significantly, P<0.05. The lower digestibility of CP on FRS resulted in significantly (P<0.05) lower DCP content of the diet (Fig 1) rumen of animal fed FRS was higher than that in the rumen than that of the RS based diet. The ME content of the diet of animals fed RS. Their number increased 4 h after feeding containing FRS improved (P<0.05) because of the higher in the rumen of animals fed either FRS/RS, though the digestibility of free cellulose and hemicellulose. The calves increase was more pronounced in the rumen of animals fed fed FRS had considerably higher live weight gain than those FRS (Table 5). The entodinomorphs were more in number fed on RS based diet (Table 7), mainly due to favorable as compared to holotrichs, in general as well as in FRS fed microbial population in the rumen, higher digestibility and group of animals. Amongst entodinomorhs; E. efficient utilization of nutrients, confirming to the earlier longinucleatum, E. bursa, and E. caudatum predominated. report on naturally fermented wheat straw (Bakshi and Langar Entodinomorphs ingest plant material to degrade cellulose; 1990, 1994). It was concluded that natural fermentation of this observation clearly justified their higher number in rumen RS with urea could improve the nutritive value and about of animals fed FRS resulting in higher cellulose digestibility. Epidinium spp. are one of the most important protozoa, which RS FRS b Table 5. Impact of naturally fermented rice straw on the 7.1 microbial changes in the rumen 6.0a 5.5b Time, HAF RS FRS 3.6a Protozoa, ×104/ml 0 1.98 3.05 4 3.90 5.12 Bacteria, ×109/ml 0 3.32 8.06 DCP, % ME, MJ/kg DM 4 4.39 10.6 Fig. 1. Natural fermentation of rice straw with urea and nutritive HAF, Hours after feeding; RS, untreated rice straw; FRS, value of diets in buffalo calves. Figures with different superscript fermented rice straw. in a parameter differ significantly <0.05 63 252 WADHWA ET AL. [Indian Journal of Animal Sciences 80 (3)

Table 7. Performance of buffalo calves fed naturally fermented Broster W H and Oldham J D. 1981. Recent Developments in rice straw-based diet Ruminant Nutrition. Pp. 194–99. (Eds) Hariesign W and Cole D J A. Butterworths, London. Parameter RS FRS Pooled SE Cline J H, Hershberger T V and Bantely O G. 1958. Utilization and/or synthesis of valeric acid during digestion of glucose, Initial BW, kg 81.7 80.7 14.05 starch and cellulose by rumen microorganisms in vitro. Journal Final BW, kg 101.7 110.7 15.47 of Animal Sciences 17: 284–92. Gain in BW, kg 20.0 30.0 3.19 Crampton E W and Maynard L A. 1938. The relation of cellulose Gain, g/d 166.7 250.0 26.6 and lignin content to the nutritive value of animal feeds. Journal of Nutrition 15: 383–95. RS, Untreated rice straw; FRS, fermented rice straw. Dia-Da-Silva A A and Sundstol F. 1986. Urea as a source of 70% of deoiled mustard cake could be spared for the feeding ammonia for improving the nutritive value of wheat straw. to other productive animals. Animal Feed Science and Technology 14: 67. Gupta V K, Bakshi M P S and Langar P N. 1987. Microbiological REFERNCES changes during natural fermentation of urea-wheat straw. Biological Wastes 21: 291–99. Anonymous. 2007. Statistical Abstracts of Punjab. Economic Horten G M J, Nicholson H H and Christensen D A. 1982. Advisor to Government of Punjab, Chandigarh. Ammonia and sodium hydroxide treatment of wheat straw in AOAC. 1995. Official Methods of Analysis. 16th edn. Association the diet for fattening steers. Animal Feed Science and of Official Analytical Chemists, Washington, DC. Technology 19: 1. Bakshi M P S and Langar P N. 1990. Effect of feeding naturally Kaur K, Kaur J, Wadhwa M and Bakshi M P S. 2007. Natural fermented wheat straw on the availability of nutrients and fermentation of rice straw in bales and stack and its evaluation growth of buffalo calves. Proceedings National Symposium as feed. Indian Journal of Animal Nutrition 24: 88–91. Buffalo Development for Economic Returns 23–27. Kaur K, Kaur J, Wadhwa M, Balwinder Kumar and Bakshi Bakshi M P S and Langar P N. 1994. Utilization of naturally M P S. 2008. Fermented rice straw as a source of fermented urea-crop residues in buffalo production. Buffalo nutrients for ruminants. Indian Journal of Animal Nutrition Journal 10: 19–27. 25: 195–200. Bakshi M P S and Wadhwa M. 2001. Nutritive evaluation of Makhdoomi A A, Bakshi M P S and Langar P N. 1996. Factors inoculated fermented wheat straw as complete feed for responsible for low crude protein digestibility of naturally buffaloes. Indian Journal of Animal Sciences 71: 710–11. fermented urea-wheat straw. Indian Journal of Animal Sciences Bakshi M P S, Gupta V K and Langar P N. 1986. Fermented straw 66: 203–05. as complete basal ration for ruminants. Agricultural Wastes 16: N R C. 2001. Nutrient Requirements of Dairy Cattle. 7th revised 37–46. edn. National Academy of Sciences. National Research Council, Bakshi M P S, Gupta V K and Langar P N. 1987. Effect of moisture Washington, D.C., USA. level on the chemical composition and nutritive value of Robertson J B and Van Soest P J. 1981. The detergent system of fermented straw. Biological Wastes 21: 283–89. analysis and its application to human foods. The analysis of Barnett A J G and Reid R L. 1957. Studies on the production of Dietary Fibre in Foods. pp. 123–58. (Eds) James WPT and volatile fatty acids from the grass in artificial rumen. 1. Volatile Theander O. Marcel Dekker, Inc., New York. fatty acid production from fresh grass. Journal of Agricultural Snedecor G W and Cochran W G. 1994. Statistical Methods. Oxford Science 46: 315–18. and IBH Publications, New Delhi.

64 Indian Journal of Animal Sciences 80 (3): 253–257, March 2010 Effect of feeding raw or water soaked rapeseed cake on carcass characteristics and meat quality in kids

M PALANIVEL1, K SHARMA2, NARAYAN DUTTA3 and S K MENDIRATTA4

Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh 243 122 India

Received: 28 May 2009; Accepted: 7 November 2009

ABSTRACT Growing male kids (18) of 11.10± 0.77 kg BW, 6 months age were randomly assigned to 3 dietary treatments of 6 each. Groundnut cake of the concentrate mixture of control group (GNC) was replaced by rapeseed-mustard cake (RMC) in group 2 and 3, but the concentrate in group 3 was soaked with water (1:3) overnight and fed as mixed ration (sani) with wheat straw. In other groups, respective concentrate mixtures were offered in the morning and wheat straw was offered ad lib. During an experimental period of 180 days, fortnightly body weight changes of kids were recorded. The glucosinolates (GLS) content of RMC was 149.50 μmol/g DM which was reduced by 31.96% after overnight water soaking. DM intake in group 3 was reduced as compared to other groups. All the kids were slaughtered at the end of the experimental feeding. Preslaughter weight (PSW), carcass yield, carcass length and loin eye area of kids were similar between control and group 2, while all of these were significantly (P<0.05) lower in group 3 than that in control group. While yield of wholesale cuts and, edible and non-edible visceral organs were comparable among groups, weight (g/kg of PSW) of liver and pluck was significantly higher in groups 2 and 3 than in control group. Chemical composition, physico-chemical properties and thiocyanate concentration of Longissimus dorsi (LD) muscle did not differ significantly among groups. Organoleptic scores of pressure cooked LD muscle sample with salt (1.5% w/w) were comparable among the groups; however, tenderness score was slightly poor in groups 2 and 3. It may be concluded that despite reduction in GLS content of RMC based supplement by overnight water soaking, its feeding as mixed ration (sani) to growing kids had no positive effect in terms of meat quality attributes.

Key words: Carcass characteristics, Glucosinolates, Growing kids, Rapeseed-mustard cake, Water soaking

High glucosinolates (GLS) content in the Indian varieties The adverse effects of high GLS content of RMC can be (Brassica juncea) of rapeseed–mustard cake limit its potential reduced or eliminated by plant breeding, proper processing as a protein supplement in the ruminant rations (TERI 2003, or combination of both (Liu et al. 1995). It has been reported Ravichandiran et al. 2008). Upon ingestion, these intact that soaking water of RMC is a cheap and conventional thioglucoside compounds are enzymatically (endogenous; processing technique resulting in substantial reduction (36%) exogenous) hydrolysed into toxic metabolites such as of GLS content by endogenous enzymatic hydrolysis into thiocyanate, isothiocyanates and nitriles which are deposited volatile metabolites (Tyagi et al. 1997). The present study in large amount in the vital organs and muscles (Pailan and was therefore, planned to evaluate effect of feeding raw or Singhal 2004) in addition to exerting adverse effect on DM water soaked RMC based diet on the carcass characteristics intake, growth performance and subsequently carcass yield and meat quality attributes in growing kids. of young ruminants. In a recent study, the kids fed high MATERIALS AND METHODS GLS (6.2%) mustard cake based diet produced low dressed carcass weight (400 to 421 g/kg) as compared to control (461 Experimental animals and diets: Eighteen intact male kids g/kg) which could be attributed to lower PSW as the result (9 Jamunapari and 9 Barbari; 3 each per treatment) of about of low DM intake by kids fed high GLS mustard cake based 6 months old with an average body weight of 11.10 ± 0.77 diet (Pailan and Singhal 2004). kg, were randomly allocated to 3 dietary treatments, viz. Control, group 2 and group 3 containing groundnut cake as Present address: 1Indira Street, Kangikoil Road, Erode, Tamil major protein source, 100% replacement of groundnut cake Nadu 638052. with RMC, and overnight water soaked RMC concentrate 2Formerly Head, 3 Senior Scientist, Division of Animal mixture fed as mixed ration (sani), respectively. All the kids Nutrition, 4 Senior Scientist, LPT Division. were dewormed prior to experimental feeding and reared 65 254 PALANIVEL ET AL. [Indian Journal of Animal Sciences 80 (3) under uniform managerial conditions throughout the of vital organs and LD muscle samples was determined using experimental period of 180 days. tissue extract (Bowler 1944). Feeding procedure: The kids were housed in a well- Organoleptic evaluation: Pooled LD muscle samples from ventilated shed having a provision made for individual each treatment group were pressure cooked with salt (15% feeding. They were fed for maintenance and an expected daily w/w) and subjected to organoleptic evaluation. The sensory weight gain of 50 g/d as per the stipulated nutrients attributes, viz. appearance, flavour/taste, texture/tenderness, requirement (Kearl 1982). The kids of control and group 2 juiciness and overall acceptability of cooked meat samples were offered measured quantities of respective concentrate were assessed by 8 semi-trained panelists, using an 8 point mixture in the morning at 10.00 AM. Wheat straw was offered hedonic scale as described by Piggott (1984). ad lib. after ensuring complete consumption of the The data were subjected to one way analysis of variance concentrate. About 200g green fodder (oats/maize) was as per Snedecor and Cochran (1989). Differences among the provided to each kids for their vitamin A requirement. The means were ranked using Duncan's multiple range test body weights of all the kids were recorded fortnightly in the (Duncan, 1955). morning, before feeding and watering and amount of RESULTS AND DISCUSSION concentrate mixture offered to each animal was revised accordingly. The animals were provided fresh and clean tap Chemical composition: The chemical composition of water free choice twice daily. wheat straw offered as basal feed was within the normal range Carcass studies: After 180 days of experimental feeding, reported earlier (Sharma et al. 2004, Dey and Sehgal 2005). all kids were fasted overnight with free access to water and The concentrate mixtures were isonitrogenous and slaughtered by standard procedures in the experimental comparable with respect to other parameters related to their abattoir of the Institute after recording pre slaughter weight proximate composition and fibre fractions (Table 1). Total (PSW). The carcass length (cm) was measured (Yeates et al. GLS content (149.50μmol/g DM) of rapeseed-mustard cake 1975). Weights of carcass (before and after evisceration), was high and comparable to the values reported for Indian gastro-intestinal tract (full and empty), vital organs, viz. varieties of RMC (Banday and Verma 2003; TERI 2003). thyroid, liver, kidneys, lungs with trachea, heart and spleen Overnight water soaking of RMC reduced GLS content by were recorded. The carcass was divided into 7 primal 31.96%. Similar reduction in GLS content of water soaked wholesale cuts as per ISI (1963). Loin eye area (cm2) was RMC was reported by Singhal and Senani (1991) and Tyagi determined by tracing on the cut section of longissimus dorsi et al. (1997) following the water soaking of mustard cake (LD) muscle between the 10th and 11th rib using a butter for a period of 12 h. paper. Weight of edible and non- edible offals were recorded Voluntary feed intake: Intake (g/d) of concentrate moiety and expressed as g/kg PSW. The LD muscle from left side was lower (P<0.01) in RMC fed groups compared to control of the carcass from each animal in respective treatment was (Table 2), which could be attributed to the bitter taste and pooled and deep frozen for organoleptic evaluation later. The pungent odour of GLS metabolites in the diet (Bell 1984, LD muscle from the other side of carcass was individually Tripathi and Mishra 2007, Ravichandran et al. 2008). Wheat preserved by deep freezing (–20°C) until further analysis. Table 1. Ingredients and chemical composition of feeds Samples of kidney, liver, lung and thyroid gland were offered to kids collected from each carcass and preserved for analysis of thiocyanate concentration. Attributes Concentrate mixture Wheat straw Chemical analysis: The samples of feed offered and Control Group 2 Group 3 residues left were analysed for proximate principles (AOAC 2000) and fibre fractions (Van Soest et al. 1991). The total Ingredients composition (% as such basis) GLS content (μmol/g DM) in the extract of raw and overnight Maize 30.00 29.00 29.00 – water soaked (1:3 w/v) and sun-dried RMC was analysed GNC 30.00 – – – using HPLC following desulphoglucosinolates method (Sang RMC – 43.00 43.00 – and Truscott 1984). The gradient of acetonitrile and water Wheat bran 37.00 25.00 25.00 – Mineral mixture 2.00 2.00 2.00 – was kept at a flow rate of 4ml/min by using the wavelength Salt 1.00 1.00 1.00 – at 226nm. Chemical composition (% DM) Representative samples of thawed and minced LD muscle OM 91.95 91.15 91.15 91.20 were analysed for chemical composition (AOAC 2000), pH CP 22.70 22.90 22.90 3.68 value using digital pH meter, shear force value (SFV) using EE 2.18 2.11 2.11 0.76 Warner-Bratzler shear force apparatus (Chrystall et al. 1973) Total Ash 7.45 8.04 8.04 8.15 water holding capacity (WHC) by centrifugation technique NDF 35.31 37.43 37.43 80.10 (Wardhw et al. 1973) and muscle pigments by acid haematin ADF 13.37 13.04 13.04 50.74 method (Hornsey 1956). Thiocyanate concentration (μg/g) GLS (μmol/g DM) – 64.28 43.74 – 66 March 2010] EFFECT OF RAPESEED-MUSTARD CAKE ON KIDS 255 straw intake (g/d) was significant (P<0.05) lower in RMC Table 3. Yield of visceral organs and edible: non-edible ratio in g sani primarily due to low DM intake from moistened straw rowing kids fed raw or water soaked RMC moiety as reported by Peacock (1996) who suggested that DM intake have shown to be lower in goats fed high Aattributes Treatments SEM moistened feed and fodders. Control Group 2 Group 3 Slaughter and carcass characteristics: The slaughter Yield of visceral organs (g/kg of PSW) weight (PSW and EBW) of kids fed group 2 diet was Pluck* 35.73b 40.81a 40.53a 1.42 camparable to control or group 3, while it was lower (P<0.05) Liver* 17.42b 19.82a 19.57a 0.67 in group 3 than control group (Table 2) due to lower DM Heart 4.41 4.20 4.52 0.22 intake. The carcass weights (kg) taken after skinning and Lung with trachea 14.05 16.82 16.52 1.27 evisceration respectively, were comparable between control Kidney 5.17 5.23 5.05 0.21 and group 2 treatments, while these were lower (P<0.05) in Testes 7.24 7.31 6.52 0.44 group 3. Dressed carcass weight of kids was similar between Head 79.63 79.63 84.62 1.42 control and group 2 (462.8 vs 461.8 g/kg of PSW) and these Feet 32.91 30.72 35.83 1.23 GIT (Ful) 316.42 306.58 319.42 6.45 values were comparable to values reported earlier in Barbari GIT (Empty) 83.72 86.51 86.03 4.83 kids (Sebsibe and Mathur 2000). However, dressed carcass Spleen 1.34 1.42 1.56 0.11 weight was lower (P<0.01) in group 3 which could be Blood 34.79 29.83 31.07 1.43 attributed to lower PSW as dressed carcass weight is reported Skin 87.32 85.51 75.61 3.94 to be directly related to PSW (Sen et al. 2004). Thyroid 0.070 0.072 0.073 0.002 The carcass length (cm) and loin eye area (cm2) were Edible: non-edible ratio 2.51 2.79 2.48 0.09 significantly (P<0.05) lower in group 3 than in control group, abMean values bearing different superscripts within a row differ though both parameters in group 2 and control were significantly; *(P<0.05), ** (P<0.01). comparable. The lowest loin eye area was (4.77 cm2) in group 3 and highest 6.24 cm2 in control. These observations are in further observed that animals with heavier carcass weight agreement with these of Pal Agnihotri (1999) in goats. They within each weight category having higher loin eye area Table 2. DM intake, carcass characteristics and yield of resulted in better dressed carcass weight. The yield of wholesale cuts in growing kids fed raw or water soaked RMC wholesale cuts (g/kg of carcass weight) was comparable among dietary treatments. Leg contributed maximum to hot Attributes Treatments SEM carcass followed by shoulder, breast and shank, irrespective Control Group 2 Group 3 of the dietary treatments as recorded in Barbari male kids (Agnihotri and Pal 1997). DM intake (g/d) Yield of visceral organs: The weight of visceral organs, a b b Concentrate** 192.46 166.17 159.04 4.07 thyroid and GIT (g/kg of PSW), and edible:non edible ratio Wheat straw* 136.98a 142.66a 111.66b 4.60 did not differ significantly (P>0.05) among the treatments Total DM* 329.44a 308.83a 270.69b 9.00 Carcass characteristics (Table 3). However, weight of pluck (liver, lungs with trachea Pre slaughter weight 15.10a 14.27ab 11.34b 0.71 and heart) was higher (P<0.05) in RMC fed groups due to (kg)* liver enlargement (P<0.05). The present results are in Carcass weight after 6.99a 6.59a 4.69b 0.37 agreement with, earlier reports in different species fed with skinning (kg)* RMC (Papas et al. 1979, Bourdon and Aumaitre 1990). Carcass weight after 6.12a 6.02a 4.04b 0.36 Higher GLS content of RMC based diets increased the liver evisceration (kg)* size since liver is the major organ detoxifying the GLS a a b Dressed carcass weight** 462.8 461.8 413.4 96.1 derivatives (Mandiki et al. 2002). (g/kg of PSW) Chemical composition and physico-chemical properties Carcass length (cm)* 52.80a 51.50ab 45.33b 1.44 Loin eye area (cm2)* 6.24a 5.78ab 4.77b 0.24 of meat: The chemical composition of LD muscle was Yield of whole sale cuts (g/kg of dressed carcass weight) comparable (P>0.05) among the treatments (Table 4) and Legs 329.93 317.46 324.61 6.21 the mean moisture, protein, fat and ash content of LD muscle Loin 71.87 66.24 69.52 2.07 were similar to the reported values in Barbari kids (Agnihotri Rack 85.52 94.38 82.89 2.83 and Pal 1997). Physicochemical properties of LD muscle Flank 30.33 27.11 33.31 0.91 did not vary among the treatments. However, shear force Breast & Shank 199.93 197.04 200.03 2.66 value (SFV) of LD muscle (<5.5kg/cm2 ) in RMC fed groups Shoulder 205.21 218.27 213.07 3.43 was usually considered as objectionably tough (Shackelfold Neck 81.92 75.53 74.02 2.62 et al. 1991). The variation in SFV may be attributed to the abMean values bearing different superscripts within a row differ differences in post-mortem carcass treatment, age, live weight significantly; *(P<0.05), ** (P<0.01). and the types of muscles used (Dhanda et al. 1999) 67 256 PALANIVEL ET AL. [Indian Journal of Animal Sciences 80 (3)

Table 4. Chemical composition, physico-chemical and sensory differ significantly among treatments. Thyroid and kidney attributes of meat and thiocyanate concentration of vital organs have the trend to accumulate thiocyanate, and kidneys can in growing kids fed raw or water soaked RMC be considered as a way of thiocyanate elimination. It may be concluded that groundnut cake can be replaced Aattributes Treatments SEM with RMC in the diet of the growing kids without adverse Control Group 2 Group 3 impact on the carcass yield and meat quality attributes. Chemical composition (% on fresh basis) Despite reduction in the substantial amount of GLS content Moisture 75.22 75.25 77.01 0.62 of RMC by overnight water soaking, its feeding as mixed Protein 20.95 20.91 19.51 0.50 ration (sani) to growing kids had no positive effect in terms Fat 2.81 2.78 2.25 0.14 of meat quality attributes; rather PSW and subsequently Total ash 0.88 1.04 1.06 0.16 carcass yield of kids were reduced due to low DM intake Physico-chemical properties from high moistened straw moiety. pH 5.51 5.13 5.22 0.03 SFV (kg/cm2) 4.79 6.25 5.92 0.32 ACKNOWLEDGEMENT WHC (ml/100 g) 11.99 9.99 10.55 1.02 Pigments (ppm) 93.30 71.74 82.31 6.07 The authors are thankful to Director, IVRI, Izatnagar, for Sensory attributes necessary facilities provided to complete this study Appearance 7.17 6.79 6.80 0.14 successfully in time. Financial assistance provided by ICAR, Flavour/taste 7.09 6.78 6.79 0.12 New Delhi, is duly acknowledged. 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69 Indian Journal of Animal Sciences 80 (3): 258–261, March 2010 Micro-minerals status in goats of different age in semi-arid region of India*

NEERU BHOOSHAN1, PUNEET KUMAR2 and M C YADAV3

Central Institute for Research on Goats, Makhdoom, Uttar Pradesh 281 122 India

Received: 20 April 2008; Accepted: 24 November 2009

ABSTRACT The Present study was conducted to evaluate and compare blood zinc (Zn), copper (Cu) and cobalt (Co) status of healthy female goats (210: 105 of Barbari breed and 105 of Jamunapari breed) of different ages, managed under semi- intensive system at the institute farm. Blood Zn and Cu concentrations were significantly influenced by the age of goats, while blood Co concentration was significantly affected by breed of goats. In Barbari and Jamunapari goats, Zn level was 5.74 ± 0.73 and 4.26 ± 0.69 ppm, respectively, at birth which further increased to 6.03 ± 0.73 and 4.94 ± 0.69 ppm during 1 month of age. Thereafter it decreased significantly with the advancement of age up to 9–10 months of age in Barbari goats. Zn level was significantly low at pubertal age than pre-pubertal age in Barbari goats, while in Jamunapari goats, Zn level was not different in pre-pubertal, pubertal and post-pubertal ages. In these goats, Cu concentration was low at birth which increased with the advancement of age. While blood Cu concentration was not different at pre- pubertal, pubertal and post-pubertal ages. Blood Co concentration did not change with the advancement of age. Barbari goats have significantly higher blood Co concentration than Jamunapari goats

Key words: Age, Cobalt, Copper, Goat, Zinc

Zinc (Zn), copper (Cu) and cobalt (Co) are micro-minerals evaluate and compare micro-minerals status of two breeds essential in multiple enzyme systems. Early deficiency of of goats of different age groups on the basis of mineral zinc reduces feed intake, growth rate and feed efficiency concentrations in blood, so as to form the basis for their (McDowell 2003); and cobalt deficiency impairs energy and optimum growth and fertility. protein metabolism and then growth and development of the MATERIALS AND METHODS deficient animal (Kadim et al. 2006). However, goats are considered as being more resistant to low levels of dietary Animals and feeding: Healthy indigenous female goats cobalt (Mburu et al. 1993). (210: 105 Barbari and 105 Jamunapari goats) of different Mineral concentrations in goat blood are different from age groups (Table 1), maintained under semi-intensive system those of other ruminants such as cattle and sheep (Haenlein of management at the institute farm, were used in this study. 1980), and there is a need to more fully understand its micro- Feeding was done according to NRC (1981). Newly born mineral requirements. Breed, age, productivity, physiological kids were fed with mother’s milk for first 15 days with bottle. state of animal, mineral intake, chemical form of elements Weaning was done at 3 months of age. After weaning, and interrelationships with other nutrients, affect mineral experimental goats were allowed 4–6 h grazing and were requirements and status (NRC 1985, Khan et al. 2007). Young stall-fed with dry roughage ad lib. For Barbari goats, 200 g animals absorb minerals more efficiently than older animals to 350 g/animal/day pelleted concentrate mixture with 13% (McDowell 2003). The objective of the present work was to and 69% digestible crude protein (DCP) and total digestible nutrients (TDN) respectively was given from 2 to 3 months *Part of Ph.D. thesis, Submitted to Dr B.R. Ambedkar of age to 9 to 10 months. An additional 50 g feed per animal University, . was given to Jamunapari goats. The adult goats above 1 year 1 Present address: Scientific Officer, U P Council of Agricultural of age were given 400 g concentrate mixture daily. Drinking Research, 8th Floor, Kisan Mandi Bhawan, Vibhuti Khand, Gomti water was given ad lib. The animals were dewormed regularly Nagar, , Uttar Pradesh 226 010 e-mail address: [email protected] as per standard health practices. 2Principal Scientist, Division of Physiology. Analysis of micro-minerals: Blood (5 ml) was collected 3Principal, Narain College, , Firozabad, Uttar in nitric acid washed heparinized vials from jugular vein at Pradesh. days 0 (birth), 30, 90, 180, 270–300, 330–360 besides pre- 70 March 2010] MICRO-MINERALS STATUS IN GOATS OF DIFFERENT AGE IN SEMI ARID REGION OF INDIA 259 pubertal, pubertal and post pubertal (one week after estrus) further increased to 6.03 ± 0.73 and 4.94 ± 0.69 ppm (Table ages for estimation of Zn, Cu and Co. Blood samples were 1) during 1 month of age. Thereafter it decreased significantly digested as per AOAC (1984). Blood Zn, Cu and Co were (P<0.05) with the advancement of age up to 9–10 months of estimated in digested samples using flame atomic absorption age in Barbari goats. Similarly, plasma Zn concentration spectrophotometer. Element specific hollow cathode lamps decreased significantly with increase in age in Nubian goats were used and analytical quality was maintained by repeated (Ahmed et al. 2001) and in calves (Kincacid and Hodgson analysis of reference samples. Eight working standards were 1989). In Barbari goats, 11–12 months of age, Zn level prepared freshly from stock (Naresh 1997). increased significantly (P<0.05) up to 5.18 ± 0.68. Similarly, Analysis of data: Data obtained was analyzed by using in cattle, calves did not carry higher concentration of total mixed model (MIXMDL PC-2) program with a least square body Zn than did mature animals (Akan et al. 1991). In technique for fitting non-orthogonal data and maximum Jamunapari goats, Zn level also increased at 11–12 months likelihood computer program developed by Harvey (1990). of age though not significantly. In Barbari goats, the Zn level Duncan’s multiple range test (DMRT) modified by Kramer was significantly (P<0.05) low at pubertal age than pre- (1957) was used for pair-wise comparison among least square pubertal age. While in Jamunapari goats, the Zn level was means for age within breed, effect to find out any significant not different in pre-pubertal, pubertal and post-pubertal age. difference among them. Correlation Coefficient (R) was Similarly, no significant difference between the age groups carried our in pooled manner by using standard method was found in Assami goats (Bhattacharyya et al. 1995) and described by Snedecor and Cochran (1994). in Kivircik lambs (Akdogan et al. 2000). In this study, blood Cu concentration was affected RESULTS AND DISCUSSION significantly by age (P<0.01) but there was no effect of breed In this study, the overall means of blood zinc (ppm), and interaction between breed and age on Cu concentration. copper (ppm) and cobalt (ppm) for Barbari and Jamunapari In present investigation, blood copper concentration goats irrespective of age were 5.44 ± 0.21 and 5.14 ± 0.20 increased with the advancement of age which may be due to ppm, 0.92 ± 0.03 and 0.92 ± 0.02 ppm and 0.34 ± 0.02 and increasing physiological demands of growth. In Barbari and 0.26 ± 0.02 ppm, respectively (Table 1). In sheep, the blood Jamunapari goats, blood copper concentration was low (Table plasma concentration of Zn, Cu and Co was 8–12 ppm, 0.7– 1) at birth which increased with the advancement of age and 1.3 ppm and 0.1–0.3 ppm respectively (Radostits et al. 2000). attained highest level at 11 to 12 months of age. The copper Similarly, in adult lactating healthy cow, the Zn, Cu and Co concentrations are related to age in sheep (Church 1993), in concentration was 8.46 ± 1.10 ppm, 0.62 ppm and 0.40 ± beef and dairy calves (Puschner et al. 2004) and in Sudanese 0.03 ppm respectively (Naresh 1997). camels (Camelus dromedarius) (Mohamed 2004). Ahmed Least square analysis of variance indicated that goat blood et al. (2001) showed that an association exists between age Zn concentration was significantly (P<0.01) affected by age and physiological status of dairy Nubian goats, pregnancy, (Table 2) but not with breed. Concentrations of Zn fluctuate lactation and concentration of copper and zinc. Plasma copper with age, stress, infections and feed restriction (Kincaid levels increased significantly in adult compared to young 1999). In Barbari and Jamunapari goats at birth, the Zn levels animals. The increase in Cu level with age could be associated was 5.74 ± 0.73 and 4.26 ± 0.69 ppm, respectively, which with higher concentrations of circulating oestrogens in the

Table 1. Least square mean±SE of zinc (ppm), copper (ppm) and cobalt (ppm) at various ages for Barbari and Jamunapari goats

Age (days) n Zinc (ppm) Copper (ppm) Cobalt (ppm) Barbari Jamunapari Barbari Jamunapari Barbari Jamunapari

Birth 8 5.74abc±0.73 4.26±0.69 0.76±0.1 0.52c±0.08 0.41ab±0.06 0.27±0.07 30 8 6.03abc±0.73 4.94±0.69 0.87±0.10 1.01ab±0.08 0.34ab±0.06 0.27±0.07 90 15 4.51c±0.53 4.67±0.50 0.91±0.07 1.02ab±0.06 0.39ab±0.04 0.21±0.05 180 6 4.29c±0.84 4.89±0.79 0.77±0.12 0.90ab±0.10 0.39ab±0.07 0.26±0.09 270–300 21 4.82c±0.45 4.91±0.42 0.96±0.06 1.03ab±0.05 0.24b±0.05 0.27±0.05 330–360 9 5.18abc±0.68 5.93±0.65 1.08±0.09 1.08a±0.08 0.25ab±0.06 0.26±0.07 Pre-pubertal 14 6.65ab±0.55 5.40±0.65 0.97±0.07 0.85b±0.06 0.22b±0.05 0.23±0.06 Pubertal 12 4.84bc±0.59 5.63±0.56 1.02±0.08 0.92ab±0.07 0.68ab±0.05 0.29±0.06 Post-pubertal 12 6.88a±0.59 5.64±0.56 0.96±0.08 0.93ab±0.07 0.44a±0.05 0.28±0.06 Overall 105 5.44±0.21 5.14±0.20 0.92±0.03 0.92±0.02 0.34±0.02 0.26±0.02

Means marked with different a,b,c (superscript) in a column between ages indicate DMRT significance (P<0.05); n=denotes the no. of observations for each age group of each breed. 71 260 BHOOSHAN ET AL. [Indian Journal of Animal Sciences 80 (3) mature animals as a consequence of oestrous cycle (Desai et effects of serum IgG and trace elements Copper and Zinc on al. 1978) and probably for normal functioning of endocrine the developments of Kivircik lambs following colostrums intake. glands during puberty (Pathak et al. 1986). In these goats, Veteriner-Fakultesi Dergisi Istanbul 26: 475–478. blood copper concentration was not different at pre-pubertal, AOAC. 1984. Official Methods of Chemical Analysis. Association of Official Analytic Chemists. pp 444–76. Virginia. pubertal and post pubertal ages. Contrary to this, blood copper Bhattacharyya B N, Talukdar S C, Baruah R N, Baruah K K Sr, concentration was significantly higher on the day of oestrous Baruah K K Jr and Baruah A. 1995. Studies on circulatory levels than during the other stages of reproduction in Assami goats of trace mineral at different reproductive status in goat. Indian (Bhattacharyya et al. 1995) and in nulliparous heifers (Small Journal of Animal Reproduction 16: 96–98. et al. 1997). Church C D. 1993. El ruminates: fisiologia digestive ynutricion. In present study, blood Co concentrations was significantly pp. 397–408. (Ed.) Acribia S A Zaragoza, Espana. (P<0.01) affected by breed but not with age. Barbari goats Desai M C, Thakkar T P, Ami D R and Janakiraman K. 1978. A have significantly (P<0.01) higher blood Co concentration note on serum copper levels in relation to reproductive than Jamunapari goats. Zadjali et al. (2004) also reported performance in Surti buffaloes. Indian Journal of Animal Sciences 47: 398–409. that there are likely genetic differences between breeds of Ferguson E G W, Mitchell G B and Mac Pherson A. 1988. Cobalt the same species. Co plays a more important role in early deficiency and ostertagia circumcincta infection in the lambs. growth and development (Kadim et al. 2006). In Barbari and Veterinary Record 124: 20. Jamunapari goats, Co concentration did not change with Haenlein G F W. 1980. Mineral nutrition of goats. Journal of Dairy advancement of age. Contrary to this, kids in the age group Science 30: 1729–42. 1–3 months showed significantly (P<0.05) lower levels of Harvey W R. 1990. User’s Guide for MIXMDL PC-2 version Mixed Model Least–Squares and Maximum Likelihood Computer serum vitamin B12 than older animals (Zadjali et al. 2004). Robertson (1971) suggested that need of the young animal Program (Mimeo). Ohio State University, Columbus, OH. for serum vitamin B is greater than that of adults because Kadim I T, Mahgoub O, AI-Ajmi D, Al-Habsi K R and Johnson E 12 H. 2006. Comparative effects of low levels of dietary cobalt of their higher metabolic rate. 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73 Indian Journal of Animal Sciences 80 (3): 262–265, March 2010 Effect of concentrate levels on the production performance of Angora rabbits

R S BHATT1, DAVENDRA KUMAR2, R B SHRAMA3 and K S RISAM4

Central Sheep and Wool Research Institute, Rajasthan, Avikanagar 304 501 India

Received: 31 December 2007; Accepted: 15 November 2009

ABSTRACT

Experiment was conducted on adult male German Angora rabbits (54) with 18 rabbits in each group. Rabbits in T1 were given 140 g pellet feed whereas in T2 group 110 and in T3 80 g pellet per day was given. The grass in all the groups was given ad lib. Other management practices were kept common in all the groups. Feed intake and body weight was monitored fortnightly. Rabbits were weighed and sheared after 75 days interval and the experiment was conducted for 375 days up to four consecutive shearing. Wool production of five consecutive shearing was recorded. Initial body weights of rabbits were 3.13, 3.12 and 3.04 kg in T1, T2 and T3 groups, which were 3.28, 3.03 and 3.07 kg, respectively, in these groups by the end of fifth shearing. Concentrate level did not reveal any marked effect on the body weight. The annual wool yield was 803 g, 772.4 g and 767.4 g in T1, T2 and T3 groups with significant differences among treatments, although there were no differences between T2 and T3 groups. The significant differences were recorded for daily dry matter intake between groups with the respective value of 157.5 g, 133.1 g and 115.1 g in T1, T2 and T3 groups. Significant differences were recorded for digestibility of dry matter, crude fibre and ether extract digestibility. The crude fibre digestibility decreased whereas ether extract digestibility increased with the lowering of concentrate feeding. Total mortality was two rabbit each in T1 and T2 and five in T3 groups. Dry matter required for producing 100 g wool was 7.35 kg in T1, 6.46 kg in T2 and 5.62 kg in T3 group. From this experiment it is concluded that feeding of 140 g of pellet feed per day is higher and 110 g of pellet per day by virtue of almost similar wool production and mortality is appropriate in adult rabbit for economical Angora rabbit production.

Key words: Angora rabbit, Balance, Digestibility, Pellet, Wool production

Fulfilling the nutritional needs is a prerequisite for MATERIALS AND METHODS harnessing the full genetic potential of the Angora rabbits. Adult Angora male rabbits of German Angora breeds (54) Hence both feed quantity and quality has importance in feed were divided into 3 equal groups with 18 animals in each management techniques for achieving higher wool yield. As group. They were offered 140 g (T ), 110 g (T ) or 80 g (T ) feed cost accounts for more than 60 per cent of total recurring 1 2 3 concentrate pellet feed along with ad lib. green grass/day. cost a small saving may make the Angora farming more Green grass fed was a mixture of all the seasonal grasses successful. The disadvantage with ad lib feeding as compared and consisted of mixture of Festuca arundinacea, Lolium to rationed feeding is that it not only increases the feeding perene, Trofolium repens, Paspalum sp., Puereria cost but also causes unnecessary fattening which hamper thunbergiana, Panicum sp. Themeda sp. and Setaria sp reproduction. The aim of developing feeding schedule is to mainly. The ingredient composition of pelleted concentrate meet the nutrient requirement of different physiological of mixture was groundnut cake 15, mustard cake 5, sunflower processes without significantly altering the body cake 5, soyaflakes 5, maize 25, wheat bran 15, deoiled rice composition. Maertens (1992) optimized the restricted bran 15, fish meal 1.5 and common salt 0.5 parts. Rabbits quantity of concentrate to the adult males and non breeding were offered concentrate with a standardized scoop daily and females as 35 g/day/kg live weight. However for Angora their dry matter intake was recorded once per week. The rabbit such information is not available. With this aim the experiment was conducted for 375 days. The animals were present experiment was planned on adult Angora rabbits to weighed and sheared before start of experiment. Thereafter optimize the concentrate feeding. the animals were weighed and sheared at the interval of 75 days. The wool samples were taken from the dorsal surface Present address: 1–3Senior Scientist, Division of Nutrition, of each rabbit for quality evaluation. After second shearing 4Extension Director, Sher-e-Kashmir University of Agriculture and a digestibility trial of 5 days duration was conducted on four Technology, Jammu, R S Pura, Jammu and Kashmir. representative rabbits from each group. During the trial the 74 March 2010] EFFECT OF CONCENTRATE LEVELS ON PRODUCTION PERFORMANCE OF ANGORA RABBITS 263 total concentrate and grass intake were recorded, faeces climates. The extent to which this effect is dependent on voided out were collected and weighed daily. Urine samples temperature alone or on other environmental components, were collected, measured and samples were taken for nitrogen such as day length is unknown. The body weight of rabbits estimation. Fresh faecal samples were collected for crude during second, third, fourth, and fifth shearing did not change protein and dry matter estimation. The feed, grass and faeces much within the group and remained statistically similar were analyzed for proximate principles (AOAC 1990) and among groups. Increased amount of nutrients in terms of fibre fractions (Goering and Van Soest 1984). Wool samples higher concentrate in T1 and T2 as compared to T3 did not collected were studied for staple length (cm) with scale after result higher body weight in adult Angora rabbits. These fixing sample on the board. The dry matter required for results are in contrast to grower broiler rabbits where ad lib. producing 100 g wool was calculated for studying concentrate fed rabbits yielded significantly (Pd<0.05) higher comparative economics in two regimes. The purchased cost body weight than 80 and 50 g concentrate fed groups (Bhatt of concentrate and the prevalent cost of roughage and fodder et al. 2005).In growing Angora rabbits the higher concentrate were used for calculating feed cost. The data were analyzed feeding was reported good for growth as compared to statistically (Snedecor and Cochran 1994). restricted feeding (Singh et al. 2004). There was no treatment difference in wool yield in rabbits RESULTS AND DISCUSSION during first, second, third, fourth, and fifth shearing between Composition of experimental diets (Table 1) revealed different groups. However, the wool yield in T1 was higher 22.92% crude protein, 7.05% crude fibre, 3.37% ether extract, than T2 which was higher than T3 during all the five shearings. 7.93% total ash, 58.73% nitrogen free extract, 18.40% acid When the wool yield of all the shearings was added together detergent fibre and 13.20% cellulose. Roughage fed was a to calculate the annual yield the differences between groups mixture of cultivated and seasonal grasses and consisted of widened and as a result the differences in annual yield were 10.50% crude protein, 21.05% crude fibre, 2.40% ether statistically significant (P<0.05). The wool yield during extract, 10.30% total ash, 55.75% nitrogen free extract, second shearing was reduced in all the treatments which also 36.06% acid detergent fibre and 31.93% cellulose. The higher correlated well with the reduced body weight of group during content of protein and lower content of crude fibre in this period. This is due to the summer stress due to higher concentrate was kept to compensate the low value of protein temperature during this period. Seasonal influences have been and higher of crude fibre in roughage so that the rabbit as a Table 2. Body weight, wool yield and plane of nutrition whole get the nutrient as per NRC (1977) in the complete in rabbits ration. The composition of concentrate and roughage is as per Bhatt et al. (2005) with slight variations which were due Parameters T1 T2 T3 Standard to compositional variations of feed ingredients. Error The initial body weight of rabbits in different groups were 3.06, 3.05 and 3.08 kg in T , T and T groups, respectively, Body weight (kg) 1 2 3 Initial 3.06 3.05 3.08 0.05 and the differences between groups were statistically 1st Shearing 2.98 2.91 2.84 0.06 nonsignificant (Table 2). Body weight decreased in all the 2nd Shearing 3.04 3.08 3.19 0.26 groups during first shearing and was due to summer stress. 3rd Shearing 3.07 3.00 3.09 0.18 Similar summer stress was also reported earlier in Angora 4th Shearing 3.37 3.13 3.15 0.27 (Bhatt et al. 1999) and broiler rabbits (Stephen et al. 1979) 5th Shearing 3.28 3.03 3.07 0.29 stating that higher environmental temperature resulted in Wool yield (g) lower wool yield in Angora rabbits and is related to higher 1st Shearing 161.4 159.2 154.6 6.7 fibre density and longer fibre in colder compared to warmer 2nd Shearing 136.6 141.3 129.3 5.0 3rd Shearing 155.3 146.9 145.3 4.8 4th Shearing 187.7 165.0 177.4 7.2 Table 1. Chemical composition of concentrate and roughage 5th Shearing 162.0 160.0 160.8 5.9 (% on dry matter basis) Av. wool yield/shearing 160.6 154.5 153.5 4.8 Annual wool yield (g) 803.0a 772.4b 767.4b 6.4 Nutrients Concentrate Roughage Wool yield/kgW0.75 68.2 67.3 66.2 3.2 Dry matter 87.59 90.57 Mortality (No.) 2 2 5 a b b Crude protein 22.92 10.50 Staple length (cm) 5.40 5.20 5.09 0.07 Crude fibre 7.05 21.05 Plane of nutrition (g dry matter intake/d) Ether extract 3.37 2.40 Concentrate intake 118.1 94.9 70.3 1.4 Total ash 7.93 10.30 Roughage intake 39.4 38.2 44.8 3.6 Nitrogen free extract 58.73 55.75 Total 157.5 133.1 115.1 2.5 Acid detergent fibre 18.40 36.06 Values with different superscripts in a row differ significantly Cellulose 13.20 31.93 (P<0.05). 75 264 BHATT ET AL. [Indian Journal of Animal Sciences 80 (3)

Table 3. Digestibility of nutrients, nitrogen balance and dry in different groups indicating that different amount of matter intake in different treatments concentrate did not affect the roughage intake. Similar results were reported by Bhatt et al. (2005) in broiler weaners. Parameters T1 T2 T3 Standard Significant (Pd<0.05) differences were observed in total dry Error matter intake of rabbits in different groups with highest value Digestibility coefficient (%) (157.5 g) in T1, followed by (133.1 g) T2 and T3 (115.1 g) DM 64.8a 69.8b 66.4ab 1.5 groups. The notable difference in total dry matter intake in CP 73.6 78.3 77.1 1.5 different groups is due to the variable levels of concentrate CF 25.1a 39.7b 44.0b 4.4 offered in different groups .As per Shemin et al. (1991) the a b b EE 68.9 85.6 85.4 3.2 daily crude protein requirement for Angora with this NFE 73.1 75.0 70.2 1.3 production potential is between 25 and 26 g /day. In T regime ADF 39.0 40.8 43.5 1.7 1 the daily total (concentrate+ roughage) supplied is 31.20 g Cellulose 45.0 46.3 50.7 2.7 Balance of nitrogen whereas in T2 it is 25.8 g which is well within the requirement. Daily intake (g) 5.03a 4.13b 3.42c 0.10 In T3 the total daily protein supplied is 20.8 g which is below Daily outgo the requirement. Through faeces (g) 1.32a 0.89b 0.78c 0.05 Digestibility coefficient data (Table 3) revealed Through urine (g) 2.19 2.31 2.24 0.22 nonsignificant differences for dry matter, crude protein, Balance (g) 1.52a 0.92b 0.59c 0.12 nitrogen free extract, acid detergent fibre and cellulose a b c Balance (% of intake) 30.7 22.0 14.3 1.86 digestibility. Significant differences exist for crude fibre and Dry matter consumed/ 100g wool produced ether extract digestibility. Crude fibre digestibility was Concentrate 5.51 4.61 3.43 highest (44.0%) in T group, followed by T (39.7%) and T Roughage 1.84 1.85 2.19 3 2 1 Total 7.35 6.46 5.62 (25.1%) group. Xiccato and Cinetto (1988) also reported low fibre digestibility at high nutritive level and vice-versa. In Values with different superscripts in a row differ significantly agreement to these observations Bhatt et al. (2004) also (P<0.05). reported increased fibre and ether extract digestibility with low concentrate level as compared to ad lib concentrate fed recorded to be significant for wool production in Angora group. Low crude fibre digestibility at high concentrate rabbits. Winter harvests are reported to be more productive feeding is due to the fact that when available rabbits derive than spring which were higher than summer (Charlet-Lery most of its energy from non-fibre carbohydrates and pass et al. 1985). Similar results were reported by Bhatt et al. the fibre as unutilized from the gastro intestinal tract. EE (1999) in Angora rabbits. Annual wool yield of rabbits in T1, digestibility was 68.9% in T1 which significantly (Pd<0.05) T2 and T3 groups was 803.0 g, 772.4 g and 767.4 g increased to 85.6% in T2 and 85.4% in T3 group .The respectively. The wool yield in T1 group was significantly digestibility of EE is related to digestibility of CF in the diet, (Pd<0.05) higher than T2 and T3 groups. Wool yield in T2 and is due to the fact that the major part of the plant lipid is and T3 groups was almost similar indicating no effect of always associated with cell walls in non-added fat diets nutrition on wool yield in these two feeding regimes. In (Xiccato, 1998). Significant (P<0.05) differences were contrast Bhatt et al. (2005) reported nonsignificant observed for the nitrogen balance in different groups. The differences in weight gain in broiler rabbits at 80 g and ad daily nitrogen balance was 1.52 g in T1, which decreased to lib. (113.3±11.3 g) concentrate level and significant 0.92 g in T2 and 0.59 g in T3. Similar values were reported differences (Pd<0.05) at 50 and 80 g of concentrate level. In by Singh et al. (1985) and Lall et al. (1984) for Angora rabbits agreement to our observations Singh et al. (2004) reported fed pellets plus roughage and different protein levels significant effect of concentrate level on wool yield at second respectively. shearing in Angora weaners. Among wool attributes staple Calculated dry matter used for producing 100 g wool was length in T1 group was significantly (Pd<0.05) higher than 7.35 kg in T1, 6.46 kg in T2 and 5.62 kg in T3 group with the T2 and T3 groups. The differences between T2 and T3 were respective concentrate intake value of 5.51 kg in T3, 4.61 kg non significant which correlated well with the wool yield in in T2 and 3.43 kg in T1 group indicating the T3 group as these respective groups. From these findings it is revealed most economical. But higher mortality in this group is most that higher nutrients in the form of concentrate stimulated important factor to be considered before concluding. In the staple length which increased the wool yield in the agreement to our observations Schlolaut and Lange (1983) respective groups. reported lower wool yield (188 g) and feed/kg of wool Plane of nutrition revealed higher concentrate in T1, produced (60.9 kg feed) in rationed rabbit as compared to followed by T2 which was followed by T3 group as the rabbits (203 g wool and 80.9 kg feed) ad lib. fed rabbits. In another were offered variable amounts of concentrate. The non experiment Schlolaut and Lange (1983) reported 79% wool significant differences were observed in the roughage intake production in 80 g concentrate+ ad lib roughage as compared 76 March 2010] EFFECT OF CONCENTRATE LEVELS ON PRODUCTION PERFORMANCE OF ANGORA RABBITS 265 to ad lib. concentrate+ roughage feeding regime. He further production. 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Shemin L, Li Z, Zhuang X, W C Qiao Yongzhong and Dezhi Y. ACKNOWLEDGEMENT 1991. Studies on the Nutrient requirements of Angora rabbits. The first author is thankful to Director, Central Sheep and Digestible energy, crude protein, methionine and Lysine. Journal of Applied Rabbit Research 14: 260–65. Wool Research Institute for consistent encouragement and Singh B, Goel G C and Negi S S. 1985. Effect on wool production providing necessary facilities for conducting this experiment. of supplementing maggar (Dendrocalamus hamiltoni) and khirk REFERENCES (Celtis australis) leaves ad libitum to a concentrate diet of Angora rabbits. Journal of Applied Rabbit Research 8: 87–89. AOAC. 1990. Official Methods of Analysis. 15th edn, Vol.2. Singh Umesh, Sharma S R, Bhatt R S, Risam K S, Davendra Kumar, Association of Official Analytical Chemists, Washington DC. Sawal R K and N Swain. 2006. Effect of feeding on the Bhatt R S, Sawal R K and Mahajan A. 1999. 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77 Short Communications Indian Journal of Animal Sciences 80 (3): 266–268, March 2010 Estimation of genetic parameters in Sahiwal cattle using single and multi-trait restricted maximum likelihood method

S BANIK1 and R S GANDHI2

National Dairy Research Institute, Karnal, Haryana 132 001 India

Received: 11 August 2008; Accepted: 17 October 2009

Key words: Cattle, DFREML, Genetic correlation, Heritability, Multi trait, Single trait

The present study was conducted to estimate variance and yijk =×bi + Z uj + eijk [1] covariance components using single and multiple traits Where, yijk was the kth observation of jth random effect of procedures and to compute heritabilities, genetic and ith fixed effect, bi was the observation vector of the fixed phenotypic correlations among different first lactation effect (e.g. season, period etc.), uj was the additive genetic production and reproduction traits in Sahiwal cattle. vector of random effect (e.g. animal effect),×was design Data: The first lactation records of 1367 Sahiwal cows, matrix/incident matrix of fixed effect (b), Z was design matrix/ sired by 81 sires, each having at least five daughters, calved incident matrix of random effect (u) and eijk was a vector of over a period of 37 years (1966 to 2002), maintained at residual errors with E (y) = Xb; and E (u) = 0, E (e) = 0. National Dairy Research Institute, Karnal and Government The following were the assumptions of the model: Livestock Farm, Chak Ganjaria, Lucknow, were used for estimation of genetic parameters of Sahiwal population. The Var (u) = G traits considered were age at first calving (AFC), first Var (e) = R lactation total milk yield (FLMY), first lactation 305 days and Cov (u, e/) = 0 or less milk yield (FL305DMY), first lactation length (FLL), So that, V(y) = V = ZGZ/+ R, where Z was the incidence first dry period (FDP), first service period (FSP) and first matrix pertaining to u. calving interval (FCI). Animals having abnormal lactations The additive genetic value of all the animals was estimated and lesser than 300 kg milk production in a lactation were as described by Grasser et al. (1987) using the convergence excluded from the study. The year was classified into 4 criteria. Estimates of the heritability, genetic and phenotypic seasons, viz. December - March (winter), April - June correlations among different traits under single and multi (summer), July -September (rainy) and October - November trait procedures were obtained from converged values of sire (autumn). The various seasons were formed on the basis of and residual variance-covariance components by half sib temperature and relative humidity prevailing during the correlation method. various months of the year. The data were classified in 12 Estimates of heritability, genetic and phenotypic periods on the basis of period of birth/calving. correlations among different traits obtained from single trait Model and methods: All the variance and covariance and multi traits analysis are given in Table 1. Heritability components for seven traits were estimated by using single estimates of AFC, FDP, FSP and FCI obtained from single and multiple trait derivative free restricted maximum trait analysis were higher than those obtained from multi likelihood procedures (DFREML) program of Meyer (1998). traits analysis. However, heritability estimates of FLMY, The same model was applied for each trait in single and as FL305DMY and FLL were higher in multi trait analysis than well as in multi trait procedures. The effects of farm, season single trait analysis. Estimates of genetic and phenotypic of calving and period of calving were taken as fixed effects correlations (absolute values) were generally smaller under and sires were taken as random effect in each model. The multi trait analysis. Much higher differences were observed single trait model was: in genetic correlations of FLL with FDP, FSP and FCI. The heritability estimates of AFC from single and multiple traits Present address: 1Senior Scientist, NRC on Pig, Asom (e-mail: estimated under the present investigation were lower than [email protected]). the estimate of heritability of this trait reported by Kumbhare 2Principal Scientist and Head Dairy Cattle Breeding Division, and Gandhi (2007). e-mail: [email protected] For estimation of heritability by multi trait model, one of 78 March 2010] ESTIMATION OF GENETIC PARAMETERS IN SAHIWAL CATTLE 267

Table 1. Estimates of heritability (diagonal), genetic (above diagonal) and phenotypic (below diagonal) correlations among single and multi trait analysis

AFC FLMY FL305DMY FLL FDP FSP FCI

Single trait model AFC 0.193 0.034 -0.029 0.115 0.596 0.546 0.269 FLMY 0.004 0.102 NE 0.387 NE NE NE FL305DMY 0.006 0.894 0.222 0.188 NE -0.889 -0.918 FLL 0.001 0.789 0.547 0.089 0.751 0.622 0.560 FDP 0.008 -0.221 -0.260 -0.188 0.023 NE NE FSP 0.062 0.269 0.029 0.459 0.751 0.138 NE FCI -0.012 0.278 0.046 0.462 0.754 0.989 0.134 7- traits model AFC 0.143 0.000 0.000 0.002 0.009 0.001 0.003 FLMY -0.001 0.290 0.004 0.029 -0.016 0.013 0.017 FL305DMY 0.000 0.002 0.315 0.009 -0.003 0.008 0.007 FLL -0.002 0.013 0.008 0.138 -0.008 0.011 0.006 FDP 0.003 -0.002 -0.004 -0.008 0.013 0.024 0.008 FSP -0.001 0.004 0.006 0.006 0.007 0.098 0.019 FCI -0.001 0.004 0.005 0.003 0.004 0.005 0.039

AFC, Age at first calving; FLMY, first lactation total milk yield, FL305DMY, first lactation 305 days or less milk yield; FLL, first lactation length; FDP, first dry period; FSP, first service period; FCI, first calving interval; NE, not estimable. the milk production traits (first lactation milk yield) was taken The results indicated that estimates of genetic and into consideration. The combinations of different traits under phenotypic correlations not only changed in magnitude, but multi trait analysis were based on importance of different also in sign depending upon analysis (single or multi trait) production and reproduction traits simultaneously. The as well as the number and type of traits included in multi- estimates of heritability under 2–trait, 3 trait, 4–trait, 5–trait trait analysis. Similar findings were reported by Raheja et and 6– trait analysis were presented in table 2. The al. (2000) while comparing the correlations between the sire comparison of estimates of heritability of different traits by proofs and the variance covariance values obtained from multi trait model having all the seven traits are presented in single and multi trait analysis in Murrah buffalos. The multi Table 1. trait analysis used full information on all the traits including

Table 2. Estimates of heritability of different traits by multi trait analysis

AFC FLMY FLL FDP FSP FCI

2-Traits analysis 1) AFC and FLMY 0.500 0.348 2) AFC and FLL 0.261 0.037 3) AFC and FCI 0.942 0.308 4) FLMY and FLL 0.338 0.068 5) FLMY and FCI 0.342 0.340 6) FLL and FCI 0.131 0.884 3-Traits analysis 7) AFC, FLMY and FLL 0.500 0.372 0.128 8) AFC, FLMY and FCI 0.500 0.375 0.057 9) FLMY, FLL and FCI 0.357 0.135 0.040 4-Traits analysis 10) AFC, FLMY, FLL and FSP 0.114 0.382 0.118 0.042 11) AFC, FLMY, FLL and FCI 0.446 0.383 0.094 0.044 5-Traits analysis 12) AFC, FLMY, FLL, FSP and FCI 0.150 0.386 0.142 0.096 0.041 6-Traits analysis 13) AFC, FLMY, FLL, FDP, FSP and FCI 0.157 0.386 0.129 0.008 0.094 0.041

AFC, Age at first calving; FLMY, first lactation total milk yield; FLL, first lactation length; FDP, first dry period; FSP, first service period; FCI, first calving interval. 79 268 BANIK AND GANDHI [Indian Journal of Animal Sciences 80 (3) complete information of inter-correlations among all yield, first lactation 305 days or less milk yield, first lactation the traits. On the contrary, only pair wise correlations length, first dry period, first service period and first calving (simple correlations) without including the inter-correlations interval using single and multi trait models (2–, 3–, 4–, 5–, among the traits were used to estimate the parameters 6– and 7– traits). The estimates of heritability for first in single trait analysis. So, the multi trait analysis was lactation total milk yield, first lactation 305 days or less milk much desired as the pair wise correlations were yield and first lactation length were higher from 7–trait model highly affected by choice of pair in the analysis. Thus, the as compared to single trait model. However, multi trait still parameters estimated on simple pair wise correlations may gave higher estimates of heritability for most of the traits include the biases attributed to choice of pair, while in multi when considered in different combinations. trait analysis the inter correlations among all the traits can give bias free estimates of all the parameters. Therefore, it REFERENCES was suggested that multi trait analysis might be used to Banik S and Gandhi R S. 2006. Animal model versus conventional remove the selection biases, which occur due to correlated methods of sire evaluation in Sahiwal cattle. Asian Australasian traits. This study supported the findings of Pollak et al. Journal of Animal Science 19: 1225–28. (1984), Raheja et al. (2000), Banik and Gandhi (2006) and Graser H U, Smith S P and Tier B. 1987. A derivative-free approach Kumar (2007). for estimating variance components in animal models by Differences in heritability estimates (Table 2) of all the restricted maximum likelihood. Journal of Animal Science 64: traits from multi trait model suggested influence of other 1362–70. correlated traits on the estimation of genetic and phenotypic Kumar A. 2007. ‘Genetic analysis of stayability in Sahiwal cattle.’ parameters of a trait in a population. The inclusion of different Ph. D Thesis, NDRI Deemed University, Karnal. Kumbhare S and Gandhi R S. 2007. Heritability estimates of first production and reproduction traits in multiple trait model lactation traits from time series adjustments in Karan Fries cattle. affected the estimates of genetic parameters of all the traits. Indian Journal of Dairy Science 60: 274–77. The estimates of genetic and phenotypic parameters obtained Meyer K. 1998. DFREML (Derivative Free Restricted Maximum from multi trait analysis further revealed the dependence Likelihood) Program. Version 3.0ß. User Notes. University of upon the number and type of traits included in the model. New England, Armidale, NSW 2351, Australia. So, it was suggested that under a genetic improvement Pollak E J, Vander Warf and Quaas R L. 1984. Selection bias and programme of a breed, the goal oriented multi trait model multiple trait evaluation. Journal of Dairy Science 67: 1590– should be used for the selection of animals for the overall 99. adaptive value instead of a single trait model. Raheja K L, Vinayak A K and Kalra S. 2000. Genetic and phenotypic parameters estimated from single and multi-trait SUMMARY restricted maximum likelihood procedure. Indian Journal of Animal Sciences 70: 497–500. Data on first lactation records of 1367 Sahiwal cows were Schaeffer L R and Wilton J W. 1987. Comparison of single and used to obtain variance and covariance components among multiple trait beef sire evaluation. Canadian Journal of Animal seven traits, viz. age at first calving, first lactation total milk Science 61: 565–72.

80 Indian Journal of Animal Sciences 80 (3): 269–270, March 2010 Sire evaluation using single and multiple trait animal models in Sahiwal cattle

S BANIK1 and R S GANDHI2

National Dairy Research Institute, Karnal 132 001 Haryana

Received: 10 February 2009; Accepted: 18 September 2009

Key words: Animal model, Breeding value, First lactation traits, Milk yield, Sahiwal cattle, Sire evaluation

The effectiveness of sire evaluation is the backbone of lowest error variance from fitting a model. any breed improvement programme as the contribution of Derivative free restricted maximum likelihood sire path is higher than the dam path for the overall genetic (DFREML) method gave the average breeding value (BV) improvement for a trait (Robertson and Randel, 1954). In as 1503.99 kg for the evaluation of sires. Forty (49.38%) out addition, very intense selection can be practised in case of of 81 sires showed higher values than the average for BV. males, as few males are required for breeding purpose. Hence, The highest and the lowest breeding values were estimated one of the main criteria for enhancing the genetic potential as 1911.74 kg and 856.12 kg, respectively. In this method, of progenies in a herd is to use proven sires to transmit three sires showed BV over and above 20% as compared to superior genetic potential for higher milk production. The overall average BV. Out of 81 sires, 7, 17 and 28 sires were derivative free restricted maximum likelihood (DFREML) having breeding values 15, 10 and 5% higher than the overall method as described by Meyer (1989) has become a useful average value. In multi trait DFREML method, the average method for single/multi trait sire evaluation with the breeding value of sires for 305 MY, AFC, FSP and FCI were advancement of computational facilities. Therefore, the 1503.99 kg, 1216.92 days, 175.63 days and 413.24 days, present investigation was undertaken to estimate breeding respectively. Out of 81 sires, 39 and 42 sires were having value of Sahiwal sires by this latest method of sire evaluation breeding value below and above the average for 305 MY. using single and multiple traits. The corresponding figures for AFC, FSP and FCI were 46 Data on 1367 first lactation records of Sahiwal cows, and 35, 43 and 38 and 43 and 38, respectively. progenies of 81 sires, spread over a period of 37 years (1966– The breeding value of sires for 305 MY ranged from 2002), maintained at the National Dairy Research Institute, 1912.64 to 846.89 kg from multi trait model. These estimates Karnal and Government Livestock Farm, Chak Ganjaria, were 27.17% higher and 43.69% lower than the average Lucknow were analyzed. Sires having five or more progenies breeding value. The difference between these two extremes were evaluated on the basis of first lactation 305 days or less was 1065.75 kg. There were three sires whose breeding value milk yield by single and multiple trait derivative free was 20% and higher than the average value. Eight, 17 and maximum likelihood (DFREML) and their effectiveness were 28 sires showed 15, 10 and 5%, respectively, higher values compared. DFREML version 3.0-ß computer package as than the average breeding value. The lowest estimate for AFC suggested by Meyer (1998) was used. For both the models was 1066.84 days, which was 12.33% lower than the average of DFREML, season and period of calving were used as fixed breeding value. Whereas, the highest estimate was 1594.87 effects, age at first calving was used as covariable and sires days, which was 31.06% higher than the average breeding were considered as random effect. For multi trait evaluation value. The difference in these values was 528.03 days. FSP of breeding value of sires, a combination of four traits namely, showed a lowest value of 121.15 days, which was 31.02% 305 days or less milk yield (305 MY), age at first calving lower than average breeding value. While the highest value (AFC), first service period (FSP) and first calving interval was observed as 228.13 days, which was 29.89% higher than (FCI) were incorporated in the model. The effectiveness of the average breeding value for the trait. The difference in different sire evaluation methods was judged using the the range of breeding value for first service period was 106.98 following criteria viz. within sire variance or error variance days. The lowest breeding value of sires (386.12 days) and rank correlations. The most efficient method had the estimated for FCI was 6.56% lower than average breeding Present address: Senior Scientist, NRC Pig, Gowhati. e-mail: value. However, the highest breeding value was observed as [email protected] Corresponding Author: Principal 440.55 days, which was 6.61% higher than the average Scientist & Head, Dairy Cattle Breeding Division, National Dairy breeding value of sires for this trait. The difference in the Research Institute, Karnal, Haryana. 132001. range of breeding value for FSP was 54.43 days. 81 270 BANIK AND GANDHI [Indian Journal of Animal Sciences 80 (3)

Table 1. Rank correlations among breeding values of sires SUMMARY estimated from single/multiple traits by DFREML method Sire evaluation was carried by derivative free restricted 305 AFC FSP FCI maximum likelihood (DFREML) method for single (305 days milk yield) and multiple traits (305 day milk yield, age at 305 DMY 1.000 0.634 0.0050 0.1024 first calving, first service period and first calving interval) AFC 1.000 0.1239 0.0555 using 1367 first lactation records on daughters of 81 sires, FSP 1.000 0.8846** having 5 or more progenies of Sahiwal cattle. The highest FCU 1.000 and lowest overall average breeding value of sires for first lactation 305 days or less milk yield was obtained by single **singnificant at 1% level trait model ranged from 1911.74 to 856.12 kg with an average of 1503.99 kg. The corresponding figures from multi trait Within sire variance or error variance for first lactation model were 1912.64 to 846.89 kg with an average of 1503.91 305 days or less milk yield by multi trait DFREML was kg. The average breeding values of sires from multi trait 2 smaller (190580 kg ) than the single trait DFREML (191111 model for age at first calving, first service period and first 2 kg ); therefore, it was concluded that efficiency of multi trait calving interval were 1216.92 days, 175.63 days and 413.24 DFREML was slightly higher than the single trait model. days, respectively. On comparing the breeding value of 305 The relative efficiency (%) of single trait DFREML in days or less milk yield from a single trait model with the comparison to multi trait DFREML was 99.72%. These multi trait model, it was observed that the breeding value findings revealed that single trait DFREML method was and the ranks of sires do not differ significantly from both almost equally effective as compared to multi trait model the models. The rank correlations amongst breeding value for this set of data. of sires for various traits from multi trait model were low Comparing the breeding value of 305 days or less milk and non-significant except for first calving interval with first yield from a single trait model with the multi trait model, it service period. was observed that, the breeding value and the ranks of sires do not differ significantly for both the models. Rank REFERENCES: correlation between the breeding value of 305 days or less milk yield of single trait REML and multi trait REML was Gaur G K, Tripathi V N, Mukherjee S and Choudhary V K. 2001 . Efficiency of sire evaluation proceduresin Frieswal cattle. Indian very high approaching unity as ranking of sires by both these Journal of Veterinary Research 10: 1–6. methods was similar for all most all the sires. On the other Jain A and Sadana D K. 2000. Sire evaluation using animal model hand, the rank correlations among the estimates of breeding and conventional methods in Murrah buffaloes. Asian value of sires from multi trait DFREML method for different Australasian. Journal of Animal Sciences 13: 1196–1200. traits were non-significant (Table 1 ). This indicated that Kumar A. 2007. ‘Genetic analysis of stayability in Sahiwal cattle.’ ranking of sires on the basis of age at first calving, first service Ph. D Thesis NDRI Deemed University. Karnal, India. period and first calving interval was almost entirely different Meyer K. 1989. Restricted Maximum Likelihood to estimate than that from first lactation 305 days or less milk yield. variance components for animal models with several random However, the rank correlation of first service period with effects using a derivative-free algorithm. Genetic Selection Evolution 21: 317–40. first calving interval was very high (0.8846) and significant Meyer K. 1998. DFREML (Derivative Free Restricted Maximum (P<0.01). These results indicated that ranking of sires for Likelihood) Programme. Version 3.0ß. User notes. University first service period and first calving interval was similar to of New England, Armidale, .NSW 2351 , Australia. the extent of 88%. Mukherjee S. 2005. ‘Genetic evaluation of Frieswal cattle.’ Ph D The results of the present study were comparable fairly Thesis, NDRI Deemed University. Karnal, India. well with those reported by Raheja et al. (2000), Jain and Pundir R K, Malik R P S and Prakash B. 2004. Comparison of Sadana (2000), Gaur et al. (2001), Pundir et al. (2004), different sire evaluation methods in Sahiwal cattle. Indian Mukherjee et al. (2005) and Kumar (2007). Journal of Animal Sciences 74: 229–31 . It can be concluded that both the single and multi trait Raheja K L, Vinayak A K and Kalra S. 2000. Genetic and phenotypic parameters estimated from single and multi-trait models were almost equally effective in estimating breeding restricted maximum likelihood procedure. Indian Journal of value of sires for milk yield in Sahiwal cattle. However, multi Animal Sciences 70: 497–500. trait model seems to have an edge over the single trait model Robertson A and Randel J M. 1954. The performance of heifers in terms of accuracy of evaluation of Sahiwal sires for this got by artificial insemination. Journal of Agricultural Sciences set of data. 44: 184–92.

82 Indian Journal of Animal Sciences 80 (3): 271–272, March 2010 Effect of seasonal variations on primary physiological responses of yak

G KRISHNAN1, K P RAMESHA2, G KANDEEPAN1, V S CHOUHAN1 and S JAYAKUMAR1

National Research Centre on Yak (ICAR), Dirang, Arunachal Pradesh, India 790 101

Received: 26 May 2008; Accepted: 16 September 2009

Key words: Pulse rate, Rectal temperature, Respiration rate, Summer, Winter

Yak, a multipurpose domesticated animal of high altitude results are in agreement with the findings of Sarkar et al. mountainous terrain, has many characteristics which are (2000) and Krishnan et al. (2008) in yaks. The rectal regarded as adaptation to extreme cold and high solar temperature of yaks was within the expected values during radiations (Cai and Gerald 2003). Body temperature, pulse winter season at an altitude of 2750 m above msl with average rate and respiration rate are three body responses considered ambient temperature of 4.83±0.19ºC. During summer, the as an index to the climatic conditions, shelter stresses and observed rectal temperature was significantly (P<0.05) higher comforts (Blackshaw and Blackshaw 1994). Respiration rate than winter. Prolonged exposure of yaks to increasing is the most consistent of all the physiological responses ambient temperature and relative humidity indicated poor studied, which is highly influenced by solar radiations. There compensatory heat loss mechanism of yaks to excessive heat is paucity of information on the effect of environmental load. It could be due to lesser number of functional sweat conditions of yak. Hence, the present study was designed to glands present in yak. The upper limit of ambient temperature determine the effect of seasonal variations on primary at which yaks could maintain a stable body temperature is physiological responses in different age groups of yak. 10°C (Zhang 2000). If the ambient temperature exceeds The present study was carried out at the National Research beyond 10°C, rectal temperature started to rise from the Centre on Yak, located at 2750 m above mean sea level (msl) expected value (99.68ºF). The rectal temperature of yak on 91°4’ East longitude and 27° North latitude in calves were significantly (P<0.05) lower during winter and Nyukmadung area of West Kameng district of Arunachal higher during summer than adult yaks which might be Pradesh, India. Healthy yaks (30) of different age groups attributed to higher thermal adaptability of adult yaks. and body weight were utilized in the present study. The Respiration rate: Respiration rate (Table 1) was animals were divided into 3 groups, viz. calves (8–12 months significantly (P<0.05) higher during summer than winter in with body weight of 67.94±5.09 kg), adult bulls (3–5 years all 3 groups of yak. Respiration rate of yak calves were with body weight of 365.25±17.33 kg) and lactating yak cows significantly (P<0.05) lower than adult bulls and lactating (5–7 years with body weight of 265±10 kg), with ten animals yak cows during winter but it was significantly (P<0.05) in each group. They were maintained under semi-extensive higher during summer. Present results corroborated with the system of management. The primary physiological responses findings of West (2003) in dairy cattle and Krishnan et al. of selected animals were recorded according to Sarkar et al. (2009) in buffaloes. When yaks were exposed to fluctuating (2000) at fortnightly intervals during winter and summer. ambient temperature, the respiration rate increased The average ambient temperature and relative humidity were significantly (P<0.05) in response to heat stress during 4.83±0.19ºC and 76.54±1.32% during winter and summer. The rise in respiration rate precedes rectal 14.07±0.10ºC and 89.61±1.04% during summer. temperature and similar results were reported by Blackshaw Rectal temperature: The results of average physiological and Blackshaw (1994) in cattle. An increase in respiration responses are presented in Table 1. The rectal temperature rate is an important thermoregulatory mechanism to heat was significantly (P<0.05) higher during summer than winter stress and aids in heat dissipation through evaporative cooling in all 3 groups of yak. The rectal temperature of yak calves (West 2003, Krishnan et al. 2009). When the ambient was significantly (P<0.05) lower than adult bulls and lactating temperature is higher, the peripheral warm receptors are yak cows during winter. But it was significantly (P<0.05) activated and they send neuronal signals to the warm higher during summer compared to other groups. Present receptors located in the anterior hypothalamus which stimulates increase in respiration rate to dissipate heat from Present address: 1Scientist (e mail: [email protected]), the body. 2Senior Scientist. Pulse rate: Pulse rate (Table 1) was significantly (P<0.05) 83 272 KRISHNAN ET AL. [Indian Journal of Animal Sciences 80 (3)

Table 1. Average physiological responses of yak calves, adult bulls and lactating yak cows during winter and summer seasons (mean ± SE)

Physiological responses Group of animals Winter Summer

Rectal temperature (°F) Calves 100.01±0.01Aa 101.24±0.10Ba Adult bulls 100.19±0.02Ab 100.95±0.08Bb Lactating yak cows 100.12±0.02Ab 101.00±0.08Bb Respiration rate (Breaths/min) Calves 19.63±0.11Aa 29.26±0.75Ba Adult bulls 21.11±0.15Ab 27.08±0.57Bb Lactating yak cows 20.92±0.10Ab 27.23±0.60Bb Pulse rate (Beats/min) Calves 58.41±0.13Aa 67.52±0.68Ba Adult bulls 60.29±0.17Ab 64.91±0.54Bb Lactating yak cows 59.70±0.14Ab 66.34±0.55Bb

The means bearing different superscripts of capital letters are significantly (P< 0.05) different in same row. The means bearing different superscripts of small letters are significantly (P< 0.05) different in the same column. higher during summer than winter among different groups than bulls and lactating yaks during summer and lower of yaks. The pulse rate of yak calves was significantly during winter. It is concluded from the physiological (P<0.05) lower than adult bulls and lactating yak cows during responses that yaks generally experience heat stress during winter but it was significantly (P<0.05) higher during summer but not cold stress during winter at 2750 m above summer. The present results are in agreement with Collier et mean sea level. al. (2006) in cattle and Krishnan et al. (2009) in buffaloes. Pulse rate increased significantly (P<0.05) with increase in REFERENCES body temperature during summer. The increase in pulse rate Blackshaw J K and Blackshaw A W. 1994. Heat stress in cattle and indicated cardiac ability to fulfill increased demand of gas the effect of shade on production and behaviour: A review. transport. This functional ability served as a heat loss Australian of Journal of Experimental Agriculture 34: 285–95. mechanism by increasing blood flow to peripheral organs Cai L and Gerald W. 2003. The Yak. 2nd edn. Pub. FAO regional and skin taking away heat from core of the body. office for Asia and the Pacific. It is concluded that at an altitude of 2750 m above msl, Collier R J, Dahl G E and VanBaale M J. 2006. Major advances yaks generally experience heat stress during summer when associated with environmental effects on dairy cattle. Journal ambient temperature exceeded 10ºC and did not experience of Dairy Science 89: 1244–53. cold stress with an average ambient temperature of Krishnan G, Sarkar M, Ramesha K P, Ghosh M K and Kataktalware M A. 2008. Physiological responses of yaks (Poephagus 4.83±0.19ºC during winter. grunniens L.) during winter season. Indian Veterinary Journal SUMMARY (In press). Krishnan G, Singh G and Shukla D C. 2009. Effect of electrolyte A study was conducted to investigate effect of seasonal supplementation on physiological responses in heat stressed variations on different age groups of yaks. Thirty healthy male buffalo calves. Indian Journal of Animal Sciences 79 (1): yaks were divided into three groups viz., calves, adult bulls 34–37. and lactating yak cows. They were maintained under semi- Sarkar M, Das B C, Das D N, Mandal D B and Chatterjee A. 2000. extensive system of management. The primary physiological Physiological responses of yak under different environments. responses viz. rectal temperature, respiration rate and pulse Proceedings of the Third International Congress on Yak. Lhasa, P.R. China. pp. 380–87. rate were recorded at fortnightly intervals during summer West J W. 2003. Effects of heat-stress on production in dairy cattle. and winter in all the three groups. The physiological responses Journal of Dairy Science 86: 2131–44. were significantly (P<0.05) higher during summer Zhang R C. 2000. Effect of environment and management on yak than winter in all the three groups. The physiological reproduction. Pub. International Veterinary information Service, responses of calves were significantly (P<0.05) higher Ithaca, New York, USA.

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