International Buffalo Information Center (IBIC) BUFFALO BULLETIN ISSN : 0125-6726

Aims

IBIC is a specialized information center on water buffalo. Established in 1981 by Kasetsart University (Thailand) with an initial fi nancial support from the International Development Research Center (IDRC) of Canada. IBIC aims at being the buffalo information center of buffalo research community through out the world.

Main Objectives

1. To be world source on buffalo information 2. To provide literature search and photocopy services 3. To disseminate information in newsletter 4. To publish occasional publications such as an inventory of ongoing research projects

Buffalo Bulletin is published quarterly in March, June, September and December. Contributions on any aspect of research or development, progress reports of projects and news on buffalo will be considered for publication in the bulletin. Manuscripts must be written in English and follow the instruction for authors which describe at inside of the back cover.

Publisher International Buffalo Information Center, Offi ce of the University Library, Kasetsart University

Online availible http://ibic.lib.ku.ac.th/e-Bulletin

Advisory Board Prof. Dr. Charan Chantalakhana Thailand Prof. Dr. John Lindsay Falvey Faculty of Veterinary and Agricultural Science, University of Melbourne, Australia Prof. Dr. Metha Wanapat Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Thailand Mr. Antonio Borghese International Buffalo Federation, Italy Dr. Aree Thunkijjanukij International Buffalo Information Center, Offi ce of the University Library, Kasetsart University, Thailand Miss Wanphen Srijankul International Buffalo Information Center, Offi ce of the University Library, Kasetsart University, Thailand

Editorial Member Dr. Pakapan Skunmun Thailand Dr. Kalaya Bunyanuwat Department of Livestock Development, Thailand Prof. Dr. Federico Infascelli Department of Veterinary Medicine and Animal Science, University of Naples Federico II, Italy Dr. Rafat Al Jassim School of Agriculture and Food Sciences, Faculty of Science, The University of Queensland, Australia Prof. Dr. Nguyen Van Thu Department of Animal Sciences, Faculty of Agriculture and Applied Biology, Can Tho University, Vietnam Prof. K. Sarjan Rao Department of Livestock Production and Management, College of Veterinary Science, India Prof. Dr. Masroor Ellahi Babar Virtual University of Pakistan, Pakistan Asst. Prof. Dr. Asif Nadeem Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Pakistan Prof. Dr. Raul Franzolin Departamento de Zootecnia, Universidade de São Paulo, Brazil

Editor Dr. Sunpetch Sophon Faculty of Veterinary Medicine, Mahanakorn of Technology, Thailand

Journal Manager Mr. Chalermdej Taterian International Buffalo Information Center, Offi ce of the University Library, Kasetsart University, Thailand

Assistant Journal Manager Miss Jirawadee Wiratto International Buffalo Information Center, Offi ce of the University Library, Kasetsart University, Thailand

BUFFALO BULLEITN IBIC, KASETSART UNIVERSITY, P.O. BOX 1084, BANGKOK 10903, THAILAND E-mail : [email protected] Tel : 66-2-9428616 ext. 344 Fax : 66-2-9406688 Buffalo Bulletin (September 2014) Vol.33 No.3

CONTENTS

Page

Case Report Therapeutic management of concurrent vitamin A and zinc defi ciency in buffalo calves- Case report S. Beigh , J.S. Soodan, A. Tiko and H. Tantary...... 244

Successful management of a compound fracture in a buffalo using a fabricated polyvinylchloride splint in a fi eld setting A. Velavan, S. Sivaraman and K. Krishnakumar...... 246

Original Article Prevalence of gastrointestinal parasites in buffaloes (Bubalus bubalis) in and around Tirupati, India C. Sreedevi and Md. Hafeez...... 251

Haemato-biochemical effects of oral sub-chronic cypermethrin toxicosis in buffalo calves Shabir Ahmad Dar, Bibhuti Ranjan and Rajdeep Kaur...... 256

Incidence of parthenogenetic development of buffalo (Bubalus bubalis) oocytes Ch. Srinivasa Prasad, A. Palanisamy, S. Satheshkumar, V.S. Gomathy and S. Rangasamy...... 267

Genetic diversity and conservation of animal genetic resources in Iraqi buffalo using microsatellite markers Talib Ahmed Jaayid and Maytham Abdul Kadhim Dragh...... 271

Cryopreservation of endometrial stromal cells of buffalo (Bubalus bubalis) Chethan Sharma G., S.K. Singh, Jessihun Nongsiej, H.B. Rakesh, R.P. Singh and S.K. Agarwal...... 277

Adoption level of buffalo farming practices in the arid zone of Rajasthan M.L. Meena and Dheeraj Singh...... 283 Buffalo Bulletin (September 2014) Vol.33 No.3

CONTENTS

Page

Original Article

Milk production and reproduction performance of Murrah buffaloes of Tamil Nadu, India A.K. Thiruvenkadan, S. Panneerselvam, N. Murali, S. Selvam and V. Ramesh Saravanakumar...... 291

Effect of genetic and non-genetic factors on morphometric traits of buffaloes Shashi Shankar, Dhirendra Kumar and K.G. Mandal...... 301

Observations on buffalo sarcoptic mange in Jammu, India T. Nazir, R. Katoch, R. Godara, Anish Yadav and Vijay Pandey...... 308

Buffalo milk marketing by the Gujjars (Tribals) in the Jammu and Kashmir state in India Tarunvir Singh and Sudhakar Dwivedi...... 316

Prevalence of ticks in buffaloes in the Upper Sindh Pakistan Majid Hussain Soomro, Shahida Parveen Soomro, Muhammad Bachal Bhutto, Zeeshan Akbar, Muhammad Yaqoob and Abdullah G. Arijo...... 323

Comparison of blood acid base gas parameters in venous and arterial blood of healthy buffaloes S.A. Hussain and S.K. Uppal...... 328

Prevalence of Salmonella and Escherichia coli associated with diarrhea in buffalo and cow calves M. Anwarullah, J.A. Khan, M.S. Khan, K. Ashraf and M. Avais...... 332

Distribution of Salmonella species in buffaloes in some middle governorates of Iraq Afaf Abdulrahman Yousif and Ali, D.M. Al-Hashimi...... 337 Case Report Buffalo Bulletin (September 2014) Vol.33 No.3

THERAPEUTIC MANAGEMENT OF CONCURRENT VITAMIN A AND ZINC DEFICIENCY IN BUFFALO CALVES- CASE REPORT

S. Beigh, J.S. Soodan*, A. Tiko and H. Tantary

ABSTRACT donot respond to the light. The meance refl ex was almost absent but palperal and corneal refl ex Vitamin A defi ciency is either due to an was present; however, animal was not apparently absolute defi ciency of vitamin A or its precursor blind. The clinical examination of skin revealed carotene in the diet or secondary in which the dietary dermatophytosis with rough dry hair coat, heavy supply of vitamin A or its precursor is adequate deposition of scales, with alopecia and wrinkling but their digestion, absorption or metabolism is at the neck and head. Plucked hairs and scrabbed interfered with to produce defi ciency at the tissue scales were examined for fungal agent by direct level. Secondary vitamin A defi ciency may occur microscopy in 10% KOH and lactophenol. Samples in case of chronic diseases of liver or intestine or were inoculated on mycobiotic agar. The plates were due to defi ciency of zinc (Radostits et al., 2000). incubated at 28°C for 2-6 weeks and examined for Absorption, metabolism, hepatic release, transport, colony formation. Culture examination revealed T. and tissue utilization of vitamin A may depend verrucosum as the usual cause of dermatophytosis. on adequate zinc status while severe vitamin A Blood samples were taken from all the animals defi ciency may reduce absorption and lymphatic in heparinised vials for the estimation of vitamin transport of zinc by altering synthesis of a zinc- A and serum zinc concentration. Vitamin A and dependent binding protein (Bendich, 1993). serum zinc concentration were 7.12 ± 0.34 μg/dl (reference values 15.4-32.3) and 5.12 ± 0.26 μg/L Keyword: buffalo calves, vitamin A, zinc, (reference values 6-12). On the basis of history, therapeutic management clinical examination and decreased serum zinc and vitamin A concentration present, the animals were diagnosed to be suffering from combined vitamin CASE HISTORY AND OBSERVATION A and zinc defi ciency.

Five buffalo calves of 1-2 years of age were referred to the Veterinary Clinics and TREATMENT AND DISCUSSION Teaching Hospital of SKUAST-Jammu with the history of sudden inappetance, reduced weight and The calves were administrated orally with heavy lacrymation. Clinical examination revealed zinc sulphate at the rate of 1 gm per week for 45 slight clouding of the cornea, dilated pupils which days with vitamin A 300,00 IU and Belamyla 3

Division of Veterinary Clinical Medicine and Jurisprudence, Faculty of Veterinary Sciences and Animal Husbandry-R.S.Pura-181102, SKUAST- Jammu, India, *E-mail: [email protected]

244 Buffalo Bulletin (September 2014) Vol.33 No.3

ml intramuscularly, as reported by Anand et al. often results in increased prevalence of infectious (2005). The affected parts were topically treated diseases like dermatophytosis in the present zinc oxide ointment once daily for three weeks. case. Therefore, supplementation of zinc along Marked improvement were reported in terms of with vitamin A in vitamin A defi cient animals is disappearance of skin lesions and convulsions, and benefi cial . the appetite returned to normal, but the clouding of eyes remained unaffected. All other buffalo owners were advised to supplement vitamin A and zinc to REFERENCES the animals. Vitamin A has numerous functions such as maintenance of epithelial cells, vision, immune Anand, K.J., C. L. Srinivas, Dananjay Sing, Harsha cell function and gene regulation (Chew, 1987). Kumar and S.M. Dhoolapas. 2005. Zinc Absorption, metabolism, hepatic release, transport, defi ciency in two calves. Indian Vet. J., 85: and tissue utilization of vitamin A depends on the 768-769. adequate level of serum zinc. Defi ciency of zinc Bendich, A. 1993. Physiological role of antioxidant predisposes the animal for vitamin A defi ciency in the immune system. J. Dairy Sci., 76: (Serdar et al., 2009). Zinc plays a regulatory role 2789-2794. on vitamin A transport mediated through protein Chew, B.P. 1983. Vitamin A and β-carotene in host synthesis. Zinc defi ciency depresses the synthesis defence. J. Dairy Sci., 70: 2732. of a.. A brand of Sarabhai Zydus, Ahmedabad retinol Christian, P. and K.P. West. 1998. Interaction binding protein (RBP) in the liver and leads to lower between zinc and vitamin A: an update. Am. concentrations of RBP in the plasma (Christian J. Clin. Nutr., 68: 435-441. et al., 1998). Vitamin A and zinc also interact Ewans, P. and Halliwell B. 2001. Micronutrients: through the ubiquitous, oxidative conversion of Oxidant/antioxidant status. Brit. J. Nutr., retinol to retinaldehyde (retinal), a critical step in 85: 57. the metabolic pathway of vitamin A that is well- Naresh, R., S.K. Dwivedi, D. Swarup and S. Dey. described in the visual cycle in the retina of the 2001. Zinc, copper, and cobalt concentrations eye and requires the action of a zinc dependent in blood during infl ammation of mammary retinol dehydrogenase enzyme (Christian et al., gland in dairy cows. Asian Austral. J. Anim., 1998). Zinc is also essential components of the 14: 564. body’s antioxidant defense that play an important Radostits, O.M., C.C. Gay, D.C. Blood and K.W. role in the prevention of free radical-induced Hinchcliff. 2000. Veterinary Medicine: A damage to tissues for maintenance of health and Textbook of Diseases of Cattle, Sheep, Pigs, production (Ewans et al., 2001). Susceptibility to Goats and Horses, 9th ed. WB Saunders, infl ammatory and infectious conditions increases Harcourt Publishers Ltd., London. during zinc defi ciency (Naresh et al., 2001). Along Serdar, P. and K. Funda. 2009. Serum zinc and with zinc, vitamin A is also an important defense vitamin A concentrations in calves with booster. Vitamin A increases disease resistance and dermatophytosis. Kafkas Üniv. Vet. Fak. has stimulatory effects on cell-mediated immunity Derg., 15: 1. (Bendich, 1993). Defi ciency of vitamin A and zinc

245 Case Report Buffalo Bulletin (September 2014) Vol.33 No.3

SUCCESSFUL MANAGEMENT OF A COMPOUND FRACTURE IN A BUFFALO USING A FABRICATED POLYVINYLCHLORIDE SPLINT IN A FIELD SETTING

A. Velavan*, S. Sivaraman and K. Krishnakumar

ABSTRACT a challenging type to treat. In buffaloes repair of fractured long bones is not commonly reported due A six-year-old female Murrah buffalo was to their heavy body weight (Turner, 1984; Ayaz, brought with the complaint of hind limb fracture. 2000). Prognosis in adults is guarded and mainly Clinical examination revealed compound fracture limited by the weight of the animal and degree of of a metatarsal bone with an open wound on the the contamination of the wound (Mulon, 2010). medial aspect. The fracture was stabilized using So, management of this type of fracture is not a fabricated PVC splint external coaptation. The much explored. In this paper a modifi ed method of daily dressing of wound was carried out using 5% utilizing a PVC (polyvinylchloride) splint for the povidone iodine through the window provided in successful management of a compound fracture of the PVC splint. Inj. streptopenicillin 5 gm was given the metatarsal bone in a buffalo is described. i/m daily for 10 days. The animal started partial weight bearing on the fractured limb on day 40. On day 90, the animal was bearing its complete weight HISTORY AND CLINICAL SIGNS on the fractured limb. There was no complication reported thereafter. A six-year-old female buffalo weighing 450 kilograms was reported to the Teaching Veterinary Keywords: compound fracture, metatarsal bone, Clinical Complex, Veterinary College and buffalo, fabricated, PVC splint Research Institute, Orathanadu with the complaint of breaking a hind limb while jumping across a feed manger. The animal was already treated by a INTRODUCTION local vet using plaster of paris for a week. Clinical examination of the animal revealed that it was a In farm animals, limb fractures are compound fracture of right metatarsal bone (Figure common and occur subsequent to trauma during 1). The open wound was located on the medial handling or dystocia (Anderson and Jean, 2008). aspect of metatarsal bone (Figure 2). Fractured Limb fractures are classifi ed depending on the bone fragment was visible through the wound site. anatomical location, presence of external wound, Pus discharge was noticed at the fractured site. extent of bone damage and direction of the fracture line. Among these types, the compound fracture is Department of Teaching Veterinary Clinical Complex, Veterinary College and Research Institute, Orathanadu, Thanjavur-614625, India, *E-mail: [email protected]

246 Buffalo Bulletin (September 2014) Vol.33 No.3

RESULTS AND DISCUSSION amount of soft tissues covering over the metatarsal and metacarpal bones, these fractures are frequently The wound at the medial site was dressed converted into compound fractures resulting from using 5% povidone iodine and Ringers lactate the penetration of the bone within (Ayaz, 2000). solution and bandaged. Cotton padding was Due to the lack of satisfactory immobilizing devices provided from stifl e to fetlock joint except at the with open dressing facilities, compound fractures wound area. The fracture was stabilized using a usually do not respond to the treatment and develop fabricated PVC splint. Fabrication of PVC splint such complications that only amputation will save was done as per the following procedure. A 10 the animal (Nayak and Samantara, 2010). Since, mm thickness, 4” diameter PVC pipe was cut at the compound fracture needs daily dressing of the 75 cm length. Then the PVC pipe was divided wound, external skeletal fi xation is the right choice. into two halves and a 5 cm rectangular piece was Economic considerations and non-availability of removed from the medial side of the splint. This orthopaedic implants for large animals at the fi eld window provision was done for the daily dressing level makes compound fractures non-treatable. of the wound. Then the splint was heated at fi re Dealing with the compound fracture will always and fabricated according to the shape of the hind remain a challenge to the clinician (Mulon, 2011). limb from stifl e to hoof. The fabricated PVC splint Moreover, radiological assessment is almost always is shown in Figure 3. Over the cotton padding impracticable at the fi eld level, for there is no x-ray the fabricated PVC splint was applied. Fractured infrastructure facility. fragments were aligned anatomically and adhesive Considering all these factors, we fabricated tape was applied over the fabricated PVC splint and the PVC splint with window provision for daily bandaged. The bandage was covered with parceling dressing of wound. Common antibiotics used in tape to keep the bandage dry despite urine spillage. orthopaedic infection are penicillin, cephalosporin, Daily wound dressing was carried out using 5% fl uoroquinolone and trimethoprim-sulpha as it has povidone iodine and bandaged for 40 days. Inj. been established they reach a tissue concentration streptopenicillin 5 gm i/m was given for 10 days. above MIC in bones. The control of the infection

Supplements like Inj.vitamin AD3E 10 ml i/m is the main target to reach the ultimate goal of and Inj.phosphorus 10 ml i/m were administered fracture healing (Mulon, 2010). In this present once weekly for 4 weeks . Oral calcium 60 ml case I organisms were identifi ed in the wound and was advised for 40 days. Animal was kept under inj. streptopenicillin was used. Administration of complete rest. On day 40, the wound had healed antioxidant vitamins A, E and C could accelerate completely (Figure 4). The animal started mild bone healing after long bone fi xative surgery weight bearing from day 20. Partial weight bearing (Sandukji et al., 2011). In this present case apart was observed on day 40. Complete weight bearing from calcium and phosphorus supplementation, of the fractured limb was noticed on day 90.The administration of vitamins AD3E would help in fabricated PVC splint removed on day 90. earlier bone healing. So, compound fracture in The compound fracture is one type of buffaloes can be successfully treated using the fracture wherein there is communication between above protocols. the fracture site and outer skin wound. Due to limited

247 Buffalo Bulletin (September 2014) Vol.33 No.3

Figure 1. Buffalo lying down with fractured hind limb before treatment.

Figure 2. Fractured hind limb (white arrow) indicates wound at the medial side of fractured area.

248 Buffalo Bulletin (September 2014) Vol.33 No.3

Figure 3. Fabricated PVC splint (white arrow indicates window provision for daily wound dressing).

Figure 4. Status of the animal after 40 days of treatment with partial weight bearing.

249 Buffalo Bulletin (September 2014) Vol.33 No.3

REFERENCES

Anderson, D.E. and G. St. Jean. 2008. Management of fractures in fi eld settings. Vet. Clin. North Am. Food Anim. Pract., 24: 567-582. Ayaz, M.M. 2000. Successful repair of metacarpal fracture in a buffalo using a novel gadget. Pak. Vet. J., 20: 49-50. Mulon, P.Y. 2011. Management of open fractures in cattle. www.acvs.org/symposium/ proceedings 2011/ data/papers/222/pdf: 672-673. Nayak, S. and S. Samantara. 2010. Management of open facture and dislocation in bovines without apmuation. www.vetscan.co.in., 5: 68. Sandukji, A., H. Al-Sawaf, A. Mohamadin, Y. Alrashidi and S.A. Sheweita. 2011. Oxidative stress and bone markers in plasma of patients with long-bone fi xative surgery: role of antioxidents. Hum. Exp. Toxicol., 30: 435-442. Turner, A.S. 1984. Large animal orthopaedics, p. 816-821. In Jennings, Jr. P.B. (ed). The Practice of Large Animal Surgery, Vol 2. W.B. Saunders Co, Philadelphia. USA.

250 Original Article Buffalo Bulletin (September 2014) Vol.33 No.3

PREVALENCE OF GASTROINTESTINAL PARASITES IN BUFFALOES (BUBALUS BUBALIS) IN AND AROUND TIRUPATI, INDIA

C. Sreedevi1 and Md. Hafeez2

ABSTRACT gastrointestinal parasitic infections in buffaloes in and around Tirupati. The prevalence of gastro-intestinal parasites of buffaloes was studied in and around Keywords: gastrointestinal parasites, buffalo, Tirupati of Andra Pradesh for a period of one prevalence, age, season year through coprological examination. A total of 694 buffaloes were examined; among them, 279 (40.20%) buffaloes were found infected with INTRODUCTION one or more species of gastro-intestinal parasites. Ten species of gastro-intestinal parasites were Bubalus bubalis (buffalo) is one of the identifi ed; of them, seven species were helminthes, most important species of domestic livestock as a namely, Amphistome, Fasciola, Strongyles, source of dairy, meat, manure and drought power Strongyloides, Toxocara, Trichuris, Moniezia spp. and plays an important role in Indian rural economy. and two species were protozoa, namely, Buxtonella, In India, the majority of small and marginal farmers Eimeria and Entamoeba spp. Amphistomes were are more dependent on buffaloes than cattle for their predominant (15.42%) followed by Strongyles livelihood as they also serve as an insurance against (6.19%). Among the gastro-intestinal parasites the risk of crop failure due to natural calamities mixed infection was common (3.17%). In this (Dhanda, 2004). The Food and Agriculture study, prevalence of parasites in relation to age and Organization (FAO, 2000) has termed the buffalo seasonal dynamics was also studied. Signifi cantly as an important but ‘an asset undervalued’. Buffalo (p<0.05) higher prevalence of gastro-intestinal diseases have been considered as one of the major parasites was observed in the rainy season constraints for the development of the industry in the followed by the summer and winter seasons. In developing countries causing substantial economic the age groups, adults (above 1 year) were most loss to poor subsistence farmers (Jithendran and (p<0.05) susceptible to gastro-intestinal parasites. Bhat, 1999). The parasitic diseases, gastro-intestinal Based on the results on the prevalence of gastro- helminthiasis, coccidiosis, fasciolosis and mange are intestinal parasites in buffaloes it is obligatory to not less important in buffaloes than other infectious follow integrated strategies and measures to control diseases (Griffi ths, 1974). Epidemiological survey

1Department of Veterinary Parasitology, NTR College of Veterinary Science, Gannavaram. Andhra Pradesh, India, E-mail: [email protected] 2Department of Veterinary Parasitology, College of Veterinary Science, Sri Venkateswara Veterinary University, Tirupati, Andhra Pradesh, India

251 Buffalo Bulletin (September 2014) Vol.33 No.3 of parasitic infection is an important aid to combat RESULTS AND DISCUSSION infections more effectively and in controlling economic losses by adopting effective control The prevalence of various gastrointestinal measures. The incidence of G.I. parasites in cattle parasitic infections in buffaloes is summarized and buffaloes from different parts of India has been in Table 1. Faecal examination of 694 buffaloes published from time to time for this purpose (Sanyal revealed 40.20 percent incidence of parasitic et al., 1992; Pal et al., 2001; Muraleedharan, 2005; infections. Jagannath et al. (1988) and Hirani Ashutosh Wadhwa et al., 2011.). The present study et al. (1999) also reported 42.12 percent and was undertaken to study the prevalence of common 38.86 percent of incidence of gastrointestinal G. I. parasites in and round Tirupathi, Andhra helminthiasis in buffaloes in Karnataka and Gujarat, Pradesh, India. respectively. Whereas Muraleedharan (2005) and Ashutosh Wadhwa et al. (2011) recorded the lower percentages of 20.45 percent in Karnataka MATERIALS AND METHODS and 13 percent in Rajastan, respectively. Slightly higher prevalence was observed by Anish yadav et Six hundredninety-four buffaloes from six al. (2004) and Mamun et al. (2011) who recorded surrounding villages and organized dairy farms 60.51% and 61.02% in Jammu and Bangladesh. located in and around Tirupati were randomly Low and high prevalence of gastrointestinal selected for the study. The faecal samples were helminthiasis from different parts of India could collected as per standard procedure throughout be due to the deworming of buffaloes and the the year for a one-year period for observing the managemental practices followed in the particular G. I. parasites in different seasons viz. summer area. A signifi cant (P<0.05) relationship between (March-June), monsoon or rainy (July-October) age group and incidence of parasitism was observed and winter (November-February). The faecal in the present study. The results indicated higher samples were examined by direct microscopic prevalence rate in adults than young buffaloes method and concentration methods (sedimentation (Anish yadav et al., 2004); this could be due to and fl oatation) and samples positive for helminth the grazing of adult animals in and around marshy eggs and protozoan cysts/oocysts were specifi cally lands that were infested with infective stages of identifi ed and counted (Soulsby, 1982). Samples parasites. Contrary to this Samanta and Santra positive for coccidian oocysts were preserved in (2009) reported high prevalence rate in the below- 2.5% potassium dichromate solution for species one-year age group. specifi c identifi cation. The quantum of infection Amphistome, Fasciola sp., Strongyles, among the animals was derived in terms of Strongyloides sp., Toxocara sp., Trichuris sp., percentage positive of the total samples examined. Moniezia sp., Buxtonella sp., Eimeria sp., and Data obtained were classifi ed according to age and Entamoeba sp. were recorded in the studied season and were analyzed as per standard statistical geographical area. The helminthic infection was techniques (Snedecor and Cochran, 1980). 30.54 percent followed by protozoa (6.48%). The prevalence of amphistomes recorded was higher (15.42%) than other common parasitic infections.

252 Buffalo Bulletin (September 2014) Vol.33 No.3 Total Total positive samples

Mixed

Total

sp. Entamoeba

sp. Eimeria

sp. Balantidium

Total

sp. Moniezia

sp. Trichuris

sp. Toxocara

sp. Strongyloides

Helminthes Protozoa

Strongyles

Fasciola Amphistome 15.42 2.02 6.19 2.59 1.44 1.58 0.57 30.54 4.89 1.15 0.43 6.48 3.17 40.20 No. of samples examined % 3 yr 87 12 2 3 - - - - 17 5 - - 5 5 27 3 yr 198 45 6 18 9 - 4 1 83 13 - - 13 4 100 3 yr 90 16 2 4 3 - - - 25 4 - - 4 4 32 3 yr 375 73 10 25 12 - 4 1 125 22 - - 22 12 159 Age Total 131Total 15 2 6 1 1 1 1 27 6 2 1 9 8 44 Total 391Total 70 8 28 15 8 7 2 138 21 5 2 28 8 174 Total 172Total 22 4 9 7 1 3 1 47 7 1 - 8 6 61 Total 694Total 107 14 43 18 10 11 4 212 34 8 3 45 22 279 < 1 yr 20 - - 1 1 1 - - 3 - 1 - 1 1 5 < 1 yr 76 1 - - 2 8 - - 11 - 4 1 5 3 19 < 1 yr 19 - - 1 1 1 1 - 4 - 1 - 1 2 7 < 1 yr 115 1 - 2 4 10 1 - 18 - 6 1 7 6 31 1-3 yr 24 3 - 2 - - 1 1 7 1 1 1 3 2 12 1-3 yr 117 24 2 10 4 - 3 1 44 8 1 1 10 1 55 1-3 yr 63 6 2 4 3 - 2 1 18 3 - - 3 1 22 1-3 yr 204 33 4 16 2 - 6 3 69 12 2 2 16 4 89 group Infection total Grand Season Winter Summer Monsoon Table 1. Seasonal and age wise prevalence of gastrointestinal parasites buffaloes. Table

253 Buffalo Bulletin (September 2014) Vol.33 No.3

Similar fi ndings on the prevalence of higher stages on pasture. Sanyal and Singh (1995) also percentages of amphistome infections in bovines indicated an increased parasitic burden in hosts have been recorded from Gujarat, Kashmir and and pastures during the rainy season, based on Bangladesh (Hirani et al., 1999; Anish yadav et al., nationwide survey on parasitic epidemiology in 2004; Mamun et al., 2011). Buffaloes are usually dairy animals in seven different agro climatic zones exposed to a higher risk of infection with snail- of India. With the onset of winter, the infection borne helminthes due to their tendency to seek rate gradually decreased, agreeing with the report rivers, pools or swamps for wallowing (Cockrill, of Samanta and Santra (2009). The results of the 1974). present study revealed that the prevalence of gastro- Mixed infections of Toxocara vitulorum, intestinal parasites in buffaloes is very common Strongyloides papillosus and Eimeria sp. were and quite severe. Keeping in view these factors, observed in the below-one-year age buffaloes strategic treatment and control programme may as pointed out by Bharkad et al. (1999), but ova be formulated to control gastrointestinal parasitic of Amphistomes and cysts of Buxtonella were infections in buffaloes in Tirupati and elsewhere in observed only in animals above one year of age. Andra Pradesh. Mixed infection with ova of Toxocara sp. and Strongyloides sp. could be due to their common prenatal and transmammary route of infection. In ACKNOWLEDGEMENT addition Toxocara vitulorum ova were exclusively identifi ed in below-one-year age buffaloes as this The authors are thankful to the Associate parasite is spontaneously expelled at the age of up Dean, College of Veterinary Science, Tirupati for to 4 months (Usharani Devi et al., 2000). Absence the facilities provided. of toxocarosis in aboveoneyear age animals might be due to the arrested larval development resulting in non-patent infection. The infection rates of REFERENCES Moniezia sp. (0.57%) and Eimeria sp. (1.15%) were similar to the report of Muraleedharan (2005) Anish yadav, J.K. Khajuria and A.K. Raina. 2004. and were quite low compared to the report from Gastrointestinal parasitic infestation profi le Gujarat (Hirani et al., 1999). of bovines at R.S. Pura, Jammu. J. Vet. Seasonal effect on prevalence of G. I. Parasitol., 18(2): 167-169. parasites in the present study revealed that the Ashutosh Wadhwa, R.K., Tanwar, L.D. Singla, S. infection rate was signifi cantly higher (P<0.05) Eda, Naveen Kumar and Yogesh Kumar. during the rainy season (44.50%) followed by the 2011. Prevalence of gastrointestinal summer (35.46%) and winter (33.58%) seasons helminthes in cattle and buffaloes in Bikaner, similar to the observations reported by Mamun et Rajasthan, India. Veterinary World., 4(9): al. (2011). The environmental conditions of this 417-419 region are hot and humid, and thus favourable Bharkad, G.P., P.D. Deshpande and B.W. Narladkar. for the development and survival of preparasitic 1999. Gasrointestinal parasitosis in bovine stages leading to increased availability of infective calves in Mrathwada. J. Vet. Parasitol.,

254 Buffalo Bulletin (September 2014) Vol.33 No.3

13(2): 143-146. Bangladesh. J. Bangladesh Agril. Univ., Cockrill, W.R. 1974. The working buffalo. In 9(1): 103-109. Cockrill, W.R. (ed.) The Husbandry and Muraleedharan, K. 2005. Prevalence of Health of the Domestic Buffalo. Food and gastrointestinal parasites of livestock in Agriculture Organization of the United a central dry zone of Karnataka. J. Vet. Nations, Rome, Italy. Parasitol., 19(1): 31-33. Dhanda, O.P. 2004. Developments in water buffalo Samanta, A. and P.K. Santra. 2009. Prevalence of in Asia and Oceania, p. 17-28. In Proceedings gastrointestinal helminthes in hot and humid of the 7th World Buffalo Congress, Manila, zone of West Bengal. J. Vet. Parasitol., Philippines. 23(1): 73-76. FAO. 2000. Water Buffalo: an Asset Undervalued, Sanyal, P.K., A.J. John and M.R. Knox. 1992. pp.1-6. FAO Regional Offi ce for Asia and Epidemiology of gastrointestinal the Pacifi c, Bangkok, Thailand. nematodiasis in buffalo compared to Griffi ths, R.B. 1974. Parasites and parasitic crossbred cattle in subtropical climate of diseases, p. 236-275. In Cockrill, W.R. (ed.) Western India. Buffalo J., 1: 31-38. The Husbandry and Health of the Domestic Sanyal, P.K. and D.K. Singh. 1995. Parasitic Buffalo. Food and Agriculture Organization gastroenteritis. In Proceedings of the of the United Nations, Rome, Italy. Workshop on Control Strategy Against Hirani, N.D., M.A. Katariya, Abdulla Patel, J.J. Gastrointestinal Parasites in Dairy Animals Hansani, L.G. Kathiria and P.V. Patel. 1999. in India using Urea Molasses Blocks. Prevalence of gastrointestinal parasitic National Dairy Development Board, Anand, infections in cattle and buffaloes of Kheda India. District of Gujarat. J. Vet. Parasitol., 13(2): Snedecor, G.W. and W.G. Cochram. 1980. 147-149. Statistical Methods, 7th ed. The Iowa State Jagannath, M.S., P.E. D’souza and S. Abdul University Press, Ames, Iowa, USA. 593p. Rahman. 1988. Gastrointestinal parasites Soulsby, E.J.L. 1982. Helminths, Arthropod and of cattle and buffaloes in Bangalore and Protozoa of Domesticated Animals, 7th ed. Mysore milk unions. Mysore J. Agric. Sci., Bailliere Tindal and Cassell Ltd., London, 22: 91-96. pp. 35-740. Jithendran, K.P. and T.K. Bhat. 1999. Epidemiology Usharani Devi, H., M. Ansari, S.K. Singh and of parasites in dairy animals in the North K.H. BijubalaDevi. 2000. Prevalence and West Humid Himalayan Region of India epidemiology of Toxocara vitulorum in cow with particular reference to gastrointestinal and buffalo-calves in and around Ranch, nematodes. Trop. Anim. Health Pro., 31(4): Bihar. Indian J. Anim. Sci., 70: 817-819. 205-214. Mamun, M.A.A., N. Begum and M.M.H. Mondal. 2011. A coprological survey of gastro- intestinal parasites of water buffaloes (Bubalus bubalis) in Kurigram district of

255 Original Article Buffalo Bulletin (September 2014) Vol.33 No.3

HAEMATO-BIOCHEMICAL EFFECTS OF ORAL SUB-CHRONIC CYPERMETHRIN TOXICOSIS IN BUFFALO CALVES

Shabir Ahmad Dar, Bibhuti Ranjan and Rajdeep Kaur*

ABSTRACT due to their high effi cacy against target species, their relatively low mammalian toxicity and rapid Cypermethrin, a type II synthetic biodegradability. Synthetic pyrethroid pesticides pyrethroid insecticide, at a dose rate of 0.5 mg/kg/ account for over 30% of the global pesticide use day for 14 consecutive weeks, produced mild signs and these are now being preferentially used in of toxicity in buffalo calves. Repeated exposure to place of organophosphates and organochlorines cypermethrin produced a signifi cant increase in the (El-Tawil and Abdel-Rahman, 2001). plasma levels of lactate dehydrogenase (15.4%), The toxicity of pyrethroid insecticides to gamma-glutamyl transpeptidase (18.7%), aspartate mammalian animals has received much attention aminotransferase (13.5%), blood urea nitrogen in recent years because animals exposed to these (75%) and plasma creatinine (33.9%). It also insecticides exhibit changes in their physiological produced signifi cant decrease in the levels of total activities besides other pathological features. proteins (4.9%), haemoglobin concentration (9.5%), Cypermethrin is a synthetic type II pyrethroid. packed cell volume (8.9%) and total erythrocytic It is widely used as an insecticide in developing count (14.3%), but, there was a signifi cant increase countries for controlling pests and in various in the erythrocyte sedimentation rate (6.6%). agricultural practices (Usmani and Knowles, 2001). Due to the central role played by the liver in the Keywords: buffalo calves, cypermethrin, oxidative detoxifi cation of cypermethrin, there is a tendency stress, insecticide, toxicity for its accumulation and subsequent toxicity to the liver, disrupting the normal hepatic functioning. Low levels of chronic exposure to agricultural INTRODUCTION chemicals may not have clinically recognizable symptoms but could produce subtle cumulative In the present world scenario, insect effects that eventually affect the health of an pest management has gained impedus, leading organism. The potential hazard due to pesticide to constant evolution of pesticide technology residues on the health of livestock is a growing as a result of which newer and safer compounds concern. are being developed for various agricultural and Although extensive research work is being veterinary practices. Pyrethroid pesticides have done on various aspects of synthetic pyrethroids, gained popularity over other conventional pesticides including their metabolism, pharmacological

Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana - 141 004, India, *E-mail: [email protected]

256 Buffalo Bulletin (September 2014) Vol.33 No.3

characteristics, ecotoxicity and detection of residues, (LDH), plasma gamma-glutamyl transpeptidase little attention has been paid to their biochemical (GGT), aspartate aminotransferase (AST), alanine effects in nontarget species. Although some work aminotransferase (ALT), blood urea nitrogen (BUN), on the toxicity of cypermethrin has been done in plasma creatinine and total proteins were estimated different animals, there is limited information using Bayer Autopak kits on a Photometer 5010 available regarding its toxic mechanisms in buffalo (Nicholas Piramal). In addition, the haematological species. So, in order to better understand the parameters like erythrocyte sedimentation rate mechanisms involved in cypermethrin toxicity, it (ESR), packed cell volume (PCV), haemoglobin was thought pertinent to investigate its ability to concentration, total erythrocyte count (TEC), total modulate various biochemical and haematological leucocyte count (TLC), differential leucocyte count parameters in buffalo calves. (DLC), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC) and mean corpuscular haemoglobin (MCH) were MATERIALS AND METHODS estimated by the methods described by Benjamin (1985). Statistical analysis was done using an The experiments were performed on eight SPSS® 16.0 software package. healthy male buffalo calves of 6-12 months of age and weighing between 60-120 kg, procured from the University Dairy Farm and local market. RESULTS The animals were acclimatized in the animal shed of department under uniform conditions for Oral administration of cypermethrin at the 2 weeks prior to the commencement of study. dose rate of 0.5 mg/kg/day for 14 consecutive weeks The animals were dewormed, fed seasonal green resulted in an increase in plasma cypermethrin fodder and wheat straw and had access to water concentration for the initial four weeks, and ad libitum. Permission to conduct the experiment thereafter, the cypermethrin concentration declined was duly obtained from the University Animal slowly (Figure 1). The initial incline could be Ethics Committee. The animals were randomly due to continuous exposure of cypermethrin, divided into two groups of four animals, each. and thereafter, the decline in cypermethrin Animals of Group 1 served as healthy control concentration may be attributed to the induction of whereas Group 2 animals were orally administered the metabolizing enzymes or decrease of plasma cypermethrin at a dose rate of 0.5 mg/kg/day for 14 protein levels as observed in the present study. consecutive weeks. Blood samples were collected Cypermethrin exposure produced in heparinized vials via jugular venipuncture at signifi cant elevation in the enzymatic activity weekly intervals during the treatment period and on of lactate dehydrogenase and gamma-glutamyl 2nd week after the withdrawal of treatment. Levels transpeptidase from the 12th week onwards (Table of cypermethrin were analyzed in the plasma of 1). This elevation in LDH and GGT was observed animals by gas chromatography using an electron to be 16.3% and 23.6%, respectively, after 14 weeks capture detector (Gill et al., 1996). Various of insecticide exposure. Similarly, the enzymic parameters viz. plasma lactate dehydrogenase activity of aspartate aminotransferase increased

257 Buffalo Bulletin (September 2014) Vol.33 No.3

signifi cantly from the 12th week onwards, elevating weeks post treatment. These fi ndings are in league by 14.9% by the end of treatment period (Table with those reported by Padma and Ashok (2010) in 1). On the other hand, no signifi cant changes were rats, Nagarjuna et al. (2008) in rabbits and Khan et observed in the activity of alanine aminotransferase al. (2009) in goats, exposed to pyrethroids. in cypermethrin exposed animals (Table 1). These Diseased or damaged kidneys cause an fi ndings are consistent with those reported by Aslam elevated BUN because the kidneys are less able et al. (2010), Khan et al. (2009) and Remya et al. to clear urea from the bloodstream. Elevated (2010) in poultry, goats and rats, respectively. blood urea is correlated with an increased protein The increase in plasma LDH activity in catabolism in the mammalian body (Murray et present investigation refl ects damage to a range of al., 1990). Increase in plasma creatinine and tissues including skeletal, cardiac muscles, kidney BUN levels probably indicate renal damage, and liver. Although the exact cause of increased which may be attributed to urinary obstruction, GGT level in the present study could not be which potentiates decreased secretion of urea ascertained, yet cholestatic disorders of all species from the body. Decrease in protein levels of are associated with increased GGT activity (Braun plasma are usually seen in chronic liver diseases et al., 1987). Aspartate aminotransferase is a not due to impaired synthesis and in renal diseases an organ specifi c enzyme; however, as the cardiac due to loss of proteins. Hypoproteinemia is seen and skeletal muscles have high concentrations of in infl ammations and is due to increased protein aspartate aminotransferase (Brar et al., 2000), its catabolism during stress. Leakage of proteins is elevation in present investigation suggests marked also observed during glomerulonephritis. The muscular damage. Alanine aminotransferase is decrease in protein levels of plasma in the present employed as a marker of hepatocellular damage in investigation could be attributed to impairment of various species and is usually elevated in disorders protein synthesis following chronic exposure to of infl ammatory, toxic or degenerative origin (Dial cypermethrin. 1995). Leakage of this enzyme into the blood occurs Long term oral exposure to cypermethrin when there is damage to hepatocytes, so its level (0.5 mg/kg; 14 weeks) resulted in signifi cant changes increases in plasma. In large domestic species, the in the blood profi le of exposed animals. This was activity of ALT in the liver is low and hence during manifested by a signifi cant decrease in haemoglobin liver injury, the ALT is not remarkably elevated (18.7%) and packed cell volume (11.7%) by the (Tennant 1997). 14th week of treatment, as depicted in Table 3. The Cypermethrin produced signifi cant levels however returned to normal within 2 weeks increase in the levels of blood urea nitrogen post-treatment. Similarly, a signifi cant decline in and plasma creatinine to the extent of 75% and TEC was observed in insecticide exposed animals 33.9%, respectively by the 12th week of insecticide from the 10th week onwards, declining by 16.9% exposure (Table 2). However, chronic cypermethrin by the 14th week of treatment (Table 3). In contrast, exposure resulted in a signifi cant decline in the there was a signifi cant increase in ESR from the total plasma proteins, to the extent of 4.9% after 8th week onwards reaching a maximum of 8.02% the 14th week of cypermethrin exposure (Table 2). by the 14th week of cypermethrin exposure (Table The levels however returned to normal within 2 3). However, the values returned to normal during

258 Buffalo Bulletin (September 2014) Vol.33 No.3 ranspeptidase, 38.6±3.4 41.0±3.4 44.6±3.9 43.4±6.2 45.2±5.6 42.2±5.2 37.4±5.1 37.2±3.3 a a a a a a a a 34.7±6.0 34.0±4.3 33.0±5.6 ** ** ** 131.2±9.1139.2±7.2 35.8±3.1 135.4±7.2 34.0±6.3 137.0±6.1 37.0±5.7 35.3±5.8 134.8±4.9 35.0±3.1 145.0±7.6 143.0±6.6 141.6±6.8 a a a a a a a a 127.7±6.5 126.3±7.6 127.0±5.8 Aspartate aminotransferase Alanine aminotransferase * * * 16.6±0.816.4±0.8 131.0±6.1 16.6±0.8 125.0±10.7 17.0±0.8 122.7±9.7 124.7±8.2 15.2±0.7 123.7±6.5 17.4±0.7 17.4±0.7 17.8±0.8 a a a a a a a a transpeptidase Gamma-glutamyl 15.0±0.7 15.3±0.8 15.0±0.9 cantly from each other. (*P<0.05 and **P<0.01). cantly from each other. fi * ** ** 1334.4 ± 53.61344.8 ± 64.1 15.0±0.7 1366.4 ± 59.7 15.1±1.0 1370.2 ± 55.9 14.9±1.0 15.4±0.8 1291.4 ± 40.9 15.7±0.9 1410.6 ±45.8 1423.0±56.5 1413.6±51.2 a a a a a a a a Control Treatment Control Treatment Control Treatment Control Treatment 023 1193.4±49.64 1136.2±47.95 1215.6 ± 35.1 1174.6±44.66 1227.0 ± 46.4 15.0±1.1 1203.4±56.67 1235.0 ± 48.4 14.0±0.8 1189.3±56.38 1256.0 ± 55.6 14.4±0.8 12.8±0.8 1216.6±44.29 15.0±0.8 1271.0 ±51.6 14.0±0.8 1235.7±64.6 119.6±6.3 1302.4 ± 52.4 13.8±0.8 1214.0±63.8 15.7±1.0 122.6±6.4 14.8±0.7 15.2±0.7 1256.7±48.9 123.2±8.0 132.4±4.0 14.8±0.8 121.4±6.3 131.0±5.9 16.4±0.7 30.2±1.2 121.4±8.3 133.0±4.9 29.4±2.6 125.0±10.2 31.2±1.6 119.8±6.42 30.0±5.4 124.0±8.8 33.8±4.8 31.0±3.0 133.0±9.2 33.6±3.0 35.0±4.6 39.0±5.1 1222.7±49.5 33.2±2.6 39.2±5.1 33.0±5.3 aspartate aminotransferase and alanine aminotransferase in buffalo calves. aspartate aminotransferase and alanine in buffalo 101213 1236.0±64.5 14 1233.0±53.6 1224.3±64.3 1228.7±57.6 Time Time (weeks) Treatment Lactate dehydrogenase Post treatment Values given are expressed as U/L and represent the Mean ± S.E. of 5 animals unless stated. given are expressed as U/L Values a= Mean ± S.E. of 4 animals. signi with superscript in a given row differs Values Table 1. Effect of repeated oral administration cypermethrin 0.5 mg/kg/day on plasma lactate dehydrogenase, gamma-glutamyl t 1. Effect Table

259 Buffalo Bulletin (September 2014) Vol.33 No.3 * * 6.98±0.11 7.11±0.10 6.91±0.13 6.90±0.25 6.76±0.15 6. 81±0.12 6.75±0.11 6.73±0.13 eins in buffalo calves. eins in buffalo a a a a a a a a 7.03±0.17 7.03±0.15 7.08±0.16 7.08±0.14 ** ** ** ** 1.41±0.11 7.04±0.19 1.27±0.061.30±0.10 7.02±0.22 7.10±0.20 1.27±0.08 7.11±0.09 1.49±0.11 1.45±0.10 1.50±0.12 1.50±0.12 a a a a a a a a 1.15±0.10 1.17±0.09 1.12±0.10 1.21±0.08 1.16±0.10 cantly from each other. (*P<0.05 and **P<0.01). cantly from each other. * ** ** ** ** fi 3.4±0.95.4±1.6 1.16±0.06 1.25±0.08 5.4±0.3 1.19±0.08 7.4±0.5 6.7±0.4 7.2±0.6 7.0±0.5 7.1±1.0 a a a a a a a a 8 4.3±0.3 2 4.0±0.6 9 4.3±1.5 0234 3.2±0.45 4.2±0.86 3.2±0.77 3.6±0.7 3.6±0.5 3.8±0.4 2.8±0.3 3.2±0.5 3.2±0.4 2.5±0.6 3.2±0.4 1.14±0.06 2.6±0.3 1.20±0.07 3.8±0.4 1.13±0.05 1.20±0.05 1.18±0.06 1.13±0.08 1.11±0.06 1.22±0.09 1.24±0.06 7.21±0.18 1.23±0.11 7.11±0.17 1.27±0.12 7.11±0.21 1.23±0.08 7.17±0.19 7.16±0.18 7.06±0.17 7.05±0.15 7.14±0.22 7.07±0.16 7.09±0.18 7.01±0.17 7.08±0.15 14 4.0±0.6 101213 4.7±0.7 4.0±0.6 4.3±0.3 Treatment nitrogen Blood urea Creatinine proteins Total Time (weeks)Time Control Treatment Control Treatment Control Treatment Post-treatment Table 2. Effect of repeated oral administration cypermethrin 0.5 mg/kg/day on blood urea nitrogen, creatinine and total prot 2. Effect Table Values given are expressed as mg/dl and represent the Mean ± S.E. of 5 animals unless stated. Values a= Mean ± S.E. of 4 animals. signi with superscript in a given row differs Values

260 Buffalo Bulletin (September 2014) Vol.33 No.3 * * ** ** 5.11±0.26 4.91±0.26 4.74±0.24 4.67±0.23 4.67±0.18 4.49±0.25 4.31±0.19 4.38±0.21 a a a a a a a a edimentation rate and 5.05±0.21 5.13±0.18 5.00±0.22 5.03±0.19 5.03±0.21 * ** ** ** ** 134.2±1.6135.2±1.4 5.11±0.20 5.09±0.17 133.8±1.4 5.05±0.24 140.0±2.5 137.4±1.7 139.0±1.7 139.8±2.3 139.0±2.2 a a a a a a a a 133.0±2.1 131.3±1.8 133.0±1.7 131.7±2.6 132.0±2.6 131.3±2.9 * ** ** ** ** ** 35.86±0.54 131.0±2.3 37.62±0.64 131.7±1.5 34.31±1.0 34.51±1.03 34.82±0.79 34.38±0.95 34.33±0.87 34.04±0.95 a a a a a a a a cantly from each other. (*P<0.05 and **P<0.01). cantly from each other. fi 37.10±0.62 37.87±1.07 37.37±1.20 37.58±0.95 ** ** ** ** 8.83±0.308.36±0.27 37.03±0.83 8.24±0.31 36.83±0.51 37.40±0.85 8.85±0.27 38.05±1.08 8.15±0.30 8.07±0.22 7.94±0.25 7.90±0.31 a a a a a a a a total erythrocytic count in buffalo calves. total erythrocytic count in buffalo 2 8.79±0.25 89 9.15±0.26 8.86±0.28 023 8.96±0.294 8.94±0.405 9.10±0.31 8.75±0.316 9.12±0.21 38.46±1.19 8.84±0.217 9.03±0.32 38.29±0.78 8.90±0.28 38.55±0.56 9.01±0.24 37.82±0.34 8.90±0.34 38.21±1.12 8.84±0.35 37.69±0.42 8.99±0.30 130.2±1.8 37.87±0.46 8.71±0.31 36.66±0.81 131.0±1.4 37.54±0.43 37.49±0.45 131.8±2.7 36.56±0.68 129.6±1.2 130.0±1.6 36.72±0.60 131.2±1.9 131.6±1.4 5.08±0.29 129.6±2.9 132.2±1.6 5.08±0.13 132.2±1.7 5.21±0.12 5.05±0.21 133.2±1.8 5.18±0.29 5.06±0.23 135.6±2.1 5.22±0.25 5.03±0.16 5.14±0.22 5.10±0.22 5.24±0.25 5.03±0.27 121314 8.87±0.23 8.65±0.28 8.73±0.23 10 8.88±0.25 Treatment Haemoglobin Packed cell volume sedimentation rate Erythrocyte count erythrocytic Total Time (weeks)Time Control Treatment Control Treatment Control Treatment Control Treatment Post-treatment Table 3. Effect of repeated oral administration cypermethrin 0.5 mg/kg/day on haemoglobin, packed cell volume, erythrocyte s 3. Effect Table Values given are expressed as g/dl and represent the Mean ± S.E. of 5 animals unless stated. Values a= Mean ± S.E. of 4 animals. signi with superscript in a given row differs Values

261 Buffalo Bulletin (September 2014) Vol.33 No.3 24.25±0.80 24.02±1.18 23.97±1.25 23.95±1.58 23.51±1.23 23.53±0.37 22.90±1.20 23.59±1.65 e, mean corpuscular a a a a a a a a Mean corpuscular Mean corpuscular haemoglobin concentration 17.42±0.9516.98±0.43 24.72±1.14 17.15±1.00 23.9±0.81 17.14±1.21 23.68±1.07 17.41±0.59 23.94±0.02 17.39±1.78 23.44±1.09 17.01±0.41 23.15±0.19 23.28±0.67 17.61±0.89 23.22±0.53 a a a a a a a a haemoglobin Mean corpuscular Mean corpuscular and represent the Mean ± S.E. of 5 animals unless stated. 3 71.82±3.8470.71±1.88 17.92±0.56 71.53±2.20 17.36±0.59 71.55±0.72 17.55±1.08 74.05±3.34 17.33±0.62 73.92±8.17 17.74±0.87 74.29±3.29 17.19±0.66 17.34±0.53 75.53±7.26 17.67±1.47 a a a a a a a a volume Mean corpuscular Mean corpuscular cantly from each other. (*P<0.05 and **P<0.01). cantly from each other. fi for total erythrocytic count and as mm 3 /mm 9240±640.8 74.11±1.51 9080±336.8 72.38±2.63 9560±613.8 74.93±3.16 11170±592.5 74.25±2.55 6 10760±855.9 72.52±1.81 10560±605.5 72.36±0.79 10930±913.5 75.68±2.10 10580±852.3 75.93±4.74 a a a a a a a a Control Treatment Control Treatment Control Treatment Control Treatment 89 11733±712.7 8850±859.8 2 10625±632.5 023 10590±613.84 10420±671.1 11000±528.0 76.15±2.235 10820±674.1 10700±637.3 74.34±4.55 75.31±3.006 9944±752.3 10760±568.7 73.71±5.027 11680±687.0 17.63±0.90 74.83±3.27 10920±862.0 74.45±3.03 10560±933.1 17.59±0.58 72.52±6.59 10600±911.7 17.29±1.94 73.01±3.14 72.82±3.10 10320±772.9 11700±810.7 17.32±1.56 17.59±0.85 71.63±2.56 73.46±2.12 23.15±0.85 17.66±1.33 72.60±3.49 17.59±0.44 23.35±1.63 17.68±0.93 23.25±1.074 17.27±0.57 17.44±1.12 23.14±2.35 17.33±0.57 23.86±1.52 23.72±1.08 17.21±0.62 23.86±2.13 24.28±0.61 23.56±1.83 23.74±1.14 24.19±0.66 23.71±1.15 haemoglobin and mean corpuscular haemoglobin concentration in buffalo calves. haemoglobin and mean corpuscular concentration in buffalo 101213 8953±535.6 14 12366±856.8 11666±529.2 9266±581.2 Time Time (weeks) Treatment leukocytic count Total Post-treatment a= Mean ± S.E. of 4 animals. signi with superscript in a given row differs Values Table 4. Effect of repeated oral administration cypermethrin 0.5 mg/kg/day on total leukocytic count, mean corpuscular volum 4. Effect Table given are expressed as 10 Values

262 Buffalo Bulletin (September 2014) Vol.33 No.3 hils, lymphocytes, . 24.8±1.727.2±1.6 68.4±2.423.8±4.4 65.8±1.9 4.2±0.625.4±2.9 69.8±4.7 4.2±0.423.2±6.1 2.2±0.4 67.8±2.9 4.6±0.727.4±1.3 2.0±0.3 0.4±0.2 69.2±5.9 4.6±.222.6±1.8 1.4±0.2 0.8±0.4 68.0±1.4 4.4±0.5 0.4±0.2 2.0±0.3 71.0±1.8 2.8±0.526.6±1.1 2.6±0.4 0.2±0.2 4.2±0.6 1.4±0.2 0.6±0.2 67.2±1.5 2.0±0.3 0.4±0.2 4.6±0.4 0.2±0.2 1.6±0.2 0.0±0.0 a a a a a a a a 0.5±0.3 0.5±0.5 0.0±0.0 0.7±0.3 0.0±0.0 0.0±.0.0 0. 7±0.3 a a a a a a a 1. ±0.3 2.0 ±0.3 0.0±0.0 1.8±0.3 1.5±0.3 1.7±0.3 1.7±0.3 2.3±0.0 0.0±0.3 cantly from each other. (*P<0.05 and **P<0.01). cantly from each other. fi a a a a a a a a 5±0.6 Control Treatment 3.5±0.6 3.5±0.3 3.7±0.9 3.3±0.3 6.3±0.9 4.0±1.0 4.3±0.9 a a a a a a a a 65.0±13 61.5±3.3 64.5±2.3 64.3±1.9 72.7±4.3 67.0±2.1 62. 7±3.2 65. 7±2.4 a a a a a a a a NLMEBNLMEB 89 30.0±2.2 32.0±2.1 2 27.7±2.1 023 22.2±7.54 28.2±5.8 69.4±7.95 28.0±7.1 63.2±5.86 27.8±3.2 5.2±0.7 63.4±7.17 34.2±6.1 4.8±0.5 66.0±2.4 2.2±0.6 31.6±3.9 4.6±0.4 59.8±6.2 3.0±0.4 3.8±0.4 32.7±3.2 62.2±4.1 1.0±0.3 2.8±0.6 3.8±1.0 0.8±0.2 1.8±0.4 3.8±0.7 23.2±6.1 1.2±0.4 1.8±0.8 0.6±0.24 20±2. 6 68.6±6.0 1.8±0.6 21.8±2.8 0.4±0.24 22.8±3.7 70.6±1.9 4.8±0.6 0.6±0.24 71.4±1.8 24.8±3.4 70.4±3.4 2.8±0.4 4.8±0.4 26.4±2.3 3.8±0.6 68.8±3.9 4.2±0.4 0.6±0.2 3.6±0.5 66.6±2.7 2.6±0.7 4.0±0.3 2.0±0.3 1.0±0.4 0.4±0.2 4.4±0.6 2.0±0.5 0.6±0.2 2.0±0.4 0.4±0.2 0.6±0.4 1012 30.0±1.0 13 26.3±9.7 14 22.7±3.6 25.7±1.6 Time Time (weeks) Post-treatment Treatment Treatment Values given are expressed as % and represent the Mean ± S.E. of 5 animals unless stated, N, L, M, E, B stads for neutrop Values monocytes, eosinophils and basophils respectively. a= Mean ± S.E. of 4 animals. signi with superscript in a given row differs Values Table 5. Effect of repeated oral administration of cypermethrin 0.5 mg/kg/day on differential leukocyte count in buffalo calves leukocyte count in buffalo of repeated oral administration cypermethrin 0.5 mg/kg/day on differential 5. Effect Table

263 Buffalo Bulletin (September 2014) Vol.33 No.3

Figure 1. Serum cypermethrin concentration in buffalo calves after oral administration of cypermethrin 0.5 mg/kg/day. two weeks post-treatment. However, cypermethrin of erythrocyte destruction. This could probably exposure failed to produce any signifi cant changes be explained by the effect of cypermethrin on in total leukocytic count (TLC) and differential erythropoiesis. The subsequent recovery of leukocytic count (DLC) (Table 4 and Table 5). Table haemoglobin after termination of cypermethrin 4 further illustrates that cypermethrin produced treatment indicates that the damage produced was no signifi cant changes in any of the erythrocytic of reversible nature. Consonantly, the decrease in indices i.e. mean corpuscular haemoglobin, PCV could be attributed to the decrease in TEC. mean corpuscular volume and mean corpuscular The decrease in TEC, Hb concentration and PCV haemoglobin concentration. These observations observed in this study could be the result of disruptive are in agreement with those reported by Remya et action of cypermethrin on the erythropoietic tissue al. (2010) in rats, Summaira et al. (2010) in broiler as a result of which the viability of the cells might chicks, Khan et al. (2009) in goats and Yousef et al. have been affected. (2003) in rabbits intoxicated with cypermethrin. Therefore, on the basis of the observations Signifi cant reduction of haemoglobin of the present investigation, it can be concluded content during the later period of the investigation that chronic exposure to cypermethrin induces could be related to decreased synthesis of red signifi cant biochemical and haematological blood cells in bone marrow (Mandal et al., 1986), alterations in buffalo calves. or reduced biosynthesis of heme in bone marrow (Khan et al., 2009) or as a result of increased rate

264 Buffalo Bulletin (September 2014) Vol.33 No.3

REFERENCES Murray, R.K., D.K. Granner, P.A. Mayes and V.W. Rodwell. 1990. Harpers Biochemistry, 22nd Aslam, Faiza, Khan Ahrar, Khan Muhammad ed. Lange Medical publication, Los Altos, Zargham, Sharaf Summaira, Gul Shafi a California, USA. Tahseen and Saleemi Muhammad Kashif. Nagarjuna, A., S. Prasad, S. Mohyuddin, P. Sekhar 2010. Toxico-pathological changes induced and P. Doss. 2008. Alteration in protein by cypermethrin in broiler chicks: their metabolic profi les in gastrocnemius muscle attenuation with Vitamin E and selenium. tissue of rats during cypermethrin toxicosis. Exp. Toxicol. Pathol., 62(4): 441-450. Journal of the Indian Society of Toxicology, Benjamin, M.M. 1985. Outline of Veterinary 4: 17-21. Clinical Pathology, 3rd ed. Kalyani Publisher, Padma, S. and K.S. Ashok. 2010. Cypermethrin Ludhiana, India. induced biochemical alterations in the blood Brar, R.S., H.S. Sandhu and A. Singh. 2000. of albino rats. Jordan Journal of Biological Veterinary Clinical Diagnosis by Laboratory Sciences, 3(3): 111-114. Methods. Kalyani Publishers, Ludhiana- Remya, R. Nair, Mammen J. Abraham, C.R. New Delhi, India. Lalithakunjamma, N.D. Nair and C.M. Braun, J.P., G. Siest and A.G. Rico 1987. Uses of Aravindakshan. 2010. Haemtological and gamma-glutamyltransferase in experimental biochemical profi le in sub lethal toxicity of toxicology. Adv. Vet. Sci. Comp. Med., 31: cypermethrin in rats. International Journal 151-172. of Biological and Medical Research, 1(4): Dial, S.M. 1995. Clinicopathologic evaluation of 211-214. liver. Vet. Clin. N. Am. Small, 25: 257-273. Summaira, S., A. Khan, M.Z. Khan, F. Aslam, El-Tawil, O.S. and M.S. Abdel-Rahman. 2001. M.K. Saleemi and F. Mahmood. 2010. The role of enzyme induction and Clinico-hematological and micronuclear inhibition on cypermethrin hepatotoxicity. changes induced by cypermethrin in broiler Pharmacol. Res., 44: 33-40. chicks: Their attenuation with vitamin E Gill, U.S., H.M. Schwartz and B. Wheatley. 1996. and selenium. Exp. Toxicol. Pathol., 62: Development of a method for the analysis 333-341. of PCB congeners and organochlorine Tennant, B.C. Hepatic function, p. 327-52. In pesticides in blood/serum. Chemosphere, Kaneko, J.J., J.W. Harvey and M.L. Bruss 32: 1055-1061. (eds.) 1997. Clinical Biochemistry of Khan, Ahrar, Hafi z A.M. Faridi, Muhammad Ali, Domestic Animals, 5th ed. Academic Press, Muhammad Zargham Khan, Muhammad San Diego. Siddique, Iftikhar Hussain and Maqbool Usmani, K. and C. Knowles. 2001. Toxicity Ahmad. 2009. Effects of cypermethrin of pyrethroids and effect of synergists on some clinico-hemato-biochemical and to larval and adult Helicoverpa Zea pathological parameters in male dwarf Spodopterafrugiperda and Agrotis ipsilon goats (Capra hircus). Exp. Toxicol. Pathol., (Lepidoptera: Noctuidae). J. Econ. Entomo., 61(2): 151-160. 94: 868-873.

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Yousef, M.I., F.M. El-Demerdash and K.S. Al- Salhen. 2003. Protective role of isofl avones against the toxic effect of cypermethrin on semen quality and testosterone levels of rabbits. J. Environ. Sci. Heal. B., 38: 463- 478.

266 Original Article Buffalo Bulletin (September 2014) Vol.33 No.3

INCIDENCE OF PARTHENOGENETIC DEVELOPMENT OF BUFFALO (BUBALUS BUBALIS) OOCYTES

Ch. Srinivasa Prasad1, A. Palanisamy2, S. Satheshkumar2, V.S. Gomathy2 and S. Rangasamy3

ABSTRACT rate is much lower in buffaloes, due to their inherent poor developmental competence resulting in poor The incidence of parthenogenetic cleavage and embryo development. Recent reports development of oocytes in routine IVF practices i.e. suggested that in vitro matured buffalo oocytes had without any chemical stimuli, was assessed. After better inherent developmental competence. Oocyte 24 h of maturation while washing and transferring maturation can be defi ned as those events associated to fertilization medium nine (1.39%) oocytes out with the initiation of germinal vesicle breakdown of 646 oocytes were found cleaved. All those (GVBD) and completion of the fi rst meiotic division parthenotes (cleaved oocytes) were transferred to (Leibfried-Rutledge et al., 1987). Maturation allows embryo culture medium instead of fertilization the oocyte to express its developmental potential medium for observing further development. It was after fertilization and is not merely confi ned found that three (33.33%) out of nine were arrested to nuclear events or the ability to be fertilized at the four-cell stage and the remaining (66.67%) (Gordon, 2003). Maturation media supplemented were arrested at the two-cell stage itself. with hormones, serum, growth factors etc. improve the in vitro developmental competence of oocytes. Keywords: buffaloes, Bubalus bubalis, Oocyte maturation triggered by hormone signaling parthenogenesis, oocytes alters membrane permeability, causes ion currents 2+ and increases cytosolic free calcium [Ca ]i. At the end of maturation, the oocyte reaches an adequate INTRODUCTION Ca2+ store.

In buffalo the overall in vitro embryo production effi ciency is lower than in cattle, mainly MATERIALS AND METHODS due to the lower cleavage rate (Gasparrini, 2002). In vitro fertilization procedures in water buffalo were Ovaries from sexually mature buffaloes derived from those in cattle. However, the success (Bubalus bubalis) were collected irrespective of age,

1Department of Veterinary Physiology, Madras Veterinary College, Chennai - 600 007, India, E-mail: [email protected] 2Centralized Embryo Biotechnology Unit, Department of Animal Biotechnology, Madras Veterinary College, Tamilnadu Veterinary and animal Sciences University, Chennai - 600 007, India 3Department of Animal Reproduction, Gynaecology and Obstetrics, Madras Veterinary College, Chennai - 600 007, India

267 Buffalo Bulletin (September 2014) Vol.33 No.3

body condition, stage of oestrous cycle and season percent of oocytes were classifi ed as Grades A, B, from the Chennai Corporation abattoir and utilized C, D and E (expanded oocytes) respectively. After in this study. The ovaries were removed within 24 h of maturation while washing and transferring 30 minutes of slaughter and washed in phosphate to fertilization medium nine (1.39%) oocytes out buffered saline (PBS) supplemented with 50 μg/ml of 646 oocytes were found cleaved. All those gentamicin sulphate to remove blood and extraneous parthenotes (cleaved oocytes) were transferred to material. The washed ovaries were transported at 37oC embryo culture medium instead of fertilization in a thermos fl ask in the same media to the laboratory medium for observing further development. It was within 30 minutes. The extra-ovarian tissues were found that three (33.33%) out of nine were arrested trimmed off and the ovaries were washed with at the four-cell stage and the remaining (66.67%) PBS to remove blood clots and superfi cial bacterial were arrested at the two-cell stage itself. It was also contamination. The washed ovaries were kept in a found that all the parthenotes were derived from sterile beaker containing PBS supplemented with 50 culture grade oocytes (‘A’ and ‘B’ grade oocytes). μg/ml gentamycin until oocyte retrieval by aspiration. Chemical activation of in vitro matured The oocytes were screened under a stereozoom buffalo oocytes successfully supported development microscope, washed thrice in 35 mm petridishes and to the blastocyst stage (Gasparrini, 2003). Sperm graded based on their cumulus mass investment and penetration provides the natural signal for oocyte homogeneity of ooplasm as described by Nandi et al. activation. The sperm-oocyte interaction results (1998). in the release of Ca2+ from intracellular stores, All the oocytes aspirated were subjected in the form of repetitive waves or spikes that are to in vitro maturation in TCM-199 supplemented responsible for meiotic progression and early with FCS and hormones FSH, LH and oestardiol. development (Bootman and Berridge, 1995). All The cumulus oocyte complexes (COCs) were methods of chemical activation mimic this process rinsed three times in maturation medium and were by providing adequate but nontoxic calcium transferred to 100 μl of IVM droplets (15- 20 COCs signals to the MII arrested oocytes with different per droplet). The oocytes were allowed to mature in mechanisms. these droplets at 38.5oC in an atmosphere of 5 percent Ion currents and cytosolic free calcium 2+ CO2 in air for 24 h in a CO2 incubator. Observation ([Ca ]i) elevations are crucial events in triggering for cleavage of oocytes, if any, was made after the complex machinery involved in both gamete 24 h of maturation while washing and transferring maturation and fertilization. Oocyte maturation is to fertilization medium. triggered by hormone signaling which causes ion 2+ currents and [Ca ]i increase. Because extracellular Ca2+ is required for in vitro GVBD (DeFelici and RESULTS AND DISCUSSION Siracusa., 1982) and for fi rst meiotic division (Paleos and Powers, 1981), Ca2+ ion transport through the By aspiration technique, 646 oocytes were plasma membrane seems to play a functional role retrieved from 344 buffalo ovaries, with recovery in maturation. As a consequence of a net uptake rate of 1.87 oocytes per ovary. 10.52 (68), 33.74 of Ca2+ through the plasma membrane, at the end (218), 29.25 (189), 16.40 (106) and 10.06 (65) of maturation, the oocyte reaches an adequate

268 Buffalo Bulletin (September 2014) Vol.33 No.3

Figure 1. Parthenogenetic oocyte (2-cell) with Figure 2. Parthenogenetic oocyte (2-cell) with out intact cumulus mass ( X 200). cumulus mass ( X 200).

Figure 3. Parthenogenetic oocyte (4-cell) ( X 200). Figure 4. Degenerating parthenote (X 200).

Ca2+ store. This storage may occur via either gap vitro maturation might have induced calcium (Ca) junctions or specifi c channels. In the oocyte at oscillations due to altered electrical properties of fertilization also a dramatic ion fl ux occurs together the oocyte plasma membrane as that of chemical with a temporally abrupt and/or spatially confi ned oocyte activation by calcium ionophore resulting 2+ in cleavage, but to a lesser extent indicating that intracytoplasmic calcium [Ca ]i) increase (Boni et al., 2007). Sperm-induced calcium (Ca) oscillations buffalo oocytes had better inherent developmental at the time of fertilization are a required signal for competence and that the poor cleavage and embryo activation in most mammalian oocytes. A clear development following IVF may be due partly to relationship between electrical properties of the the poor quality of frozen/thawed sperm, improper 2+ sperm capacitation and/or fertilization as opined oocyte plasma membrane and [Ca ]i modifi cations has also been recorded following fertilization, as by Mishra et al. (2006). From this study, it was well as following chemical oocyte activation or concluded that cleavage alone is not a reliable after exposure to specifi c Ca2+ releasers (Tosti et indicator of fertilized oocytes because oocytes al., 2002). matured in vitro may undergo parthenogenic Based on the results it was assumed that in development to the four-eight cell stage (Shioya et

269 Buffalo Bulletin (September 2014) Vol.33 No.3

al., 1988), where in vitro developmental block may Leibfried-Rutledge, M.L., E.S. Crister, W.H. or may not occur. Eyestone and D.L. Northey. 1987. Developmental potential of bovine oocytes matured in vivo and in vitro. Biol. Reprod., ACKNOWLEDGEMENT 36: 376-383. Mishra, V., A.K. Misra and R. Sharma. 2006. The work was carried out in a scheme on A comparative study of parthenogenic “Establishment of Buffalo ES-Cell Line” funded activation and in vitro fertilization of by the Department of Biotechnology, Government bubaline oocytes. Anim. Reprod. Sci., of India. The authors are thankful to the funding 103(3-4): 249-259. agency. Nandi, S., M.S. Chauhan and P. Palta. 1998. Infl uence of cumulus cells and sperm concentration on cleavage rate and subsequent embryonic REFERENCES development of buffalo (Bubalus bubalis) oocytes matured and fertilized in vitro. Boni, R., R. Gualtieri, R. Talevi and Elisabetta Theriogenology, 50(8): 1251-1262. Tosti. 2007. Calcium and other ion dynamics Paleos, G.A. and R.D. Powers. 1981.The effect of during gamete maturation and fertilization. calcium on the fi rst meiotic division of the Theriogenology, 68S: S156-S164 mammalian oocyte. J. Exp. Zool., 217: 409- Bootman, M.D. and M.J. Berridge. 1995. The 416. elemental principles of calcium signaling. Shioya, Y., M. Kuwayama, M. Fukushima, S. Cell, 83: 675-678. Iwasaki and A. Hanada. 1988. In vitro DeFelici, M. and G. Siracusa. 1982. Survival of fertilization and cleavage capability isolated, fully grown mouse ovarian oocytes of bovine follicular oocytes classifi ed is strictly dependent on external Ca2+. Dev. by cumulus cells and matured in vitro. Biol., 92: 539-543 Theriogenology, 30(3): 489-496. Gasparrini, B. 2002. In vitro embryo production Tosti, E., R. Boni and A. Cuomo. 2002. Fertilization in buffalo species: state of the art. and activation currents in bovine oocytes. Theriogenology, 57(1): 237-256 Reproduction, 124: 835-846. Gasparrini, B., V. Caracciolo di Brienza, L. Boccia, R. Di Palo and G. Neglia Discizia. 2003. The parthenogenetic development of buffalo (Bubalus bubalis) oocytes after chemical stimulation. In Proceedings of 15th Congress A.S.P.A., Parma, Iyaly. Ital. J. Anim. Sci., 2(Suppl. 1): 139-141. Gordon, I. 2003. Laboratory production of cattle embryos. Biotechnology in Agriculture No.27 , 2nd ed. p. 119.

270 Original Article Buffalo Bulletin (September 2014) Vol.33 No.3

GENETIC DIVERSITY AND CONSERVATION OF ANIMAL GENETIC RESOURCES IN IRAQI BUFFALO USING MICROSATELLITE MARKERS

Talib Ahmed Jaayid1 and Maytham Abdul Kadhim Dragh2

ABSTRACT INTRODUCTION

In our study, conducted in Iraq and The buffalo contributes effectively in the Huazhong University, China, divided Iraq into three agricultural economy and food security in the main regions: a southern area including Basrah, countries of the Indian Subcontinent and South East Missan, and Dhi-Qar, a middle area including Al- Asia, through meat, milk, leather and labor. It is Qadisiyah, Babil, Karbala and Baghdad, and a well known that the buffalo was domesticated very northern area including Diyala, Kirkuk and Mosul. early in history, but when and where is unknown The aim of the study was to measure the genetic (Cockrill, 1974). The water buffalo emerged in East diversity, polymorphism and heterozygosity in Asia (Potts 1996) and mainland South East Asia. It Iraqi buffaloes using microsatellite techniques. spread north and west to China and to the Indian Sixty-nine blood samples were collected from subcontinent (Lau, 1998). The buffalo has been unrelated animals. Six microsatellite markers were present in the valley of the Indus River in the Indian used (ETH125, CSSM060, BM1706, ETH02, subcontinent since about 4000-5000 years ago, but ETH225 and INRA005). The polymerase chain there are areas of independent domestication of reaction (PCR) was done using specifi c bovine water buffalo in Mesopotamia and China earlier primers and a genetic analyzer (ABI-3730). Our than this-about 2500-7000 BC (Haynes et al., 1991; results revealed that all the six markers amplifi ed Payne, 1991 and Bradley, 2006). Macgregor (1939) the DNA. The marker INRA005 did not show high classifi ed buffalo into two types according to formal polymorphism; it only revealed three alleles (137- criteria and behavior: the river buffalo in the Indian 141 bp). The marker ETH152 showed the highest subcontinent and westerly to the Balkan region and level of polymorphism; it has sixteen alleles ranged the swamp buffalo in Southeast Asia, India, Nepal between 192-217 bp. This study showed that there and northeast to the valley of the Yangtze River in are three main clusters: the fi rst one included Basrah, China. Barker et al. (1997) was the fi rst to analyze Baghdad and Al-Qadisiyah, the second consisted the genetic diversity of buffalo breeds in South of Kirkuk and Missan, while the third consisted of Asia using protein coding spatial microsatellites. Babil and Mosul. They noted a clear distinction between these two types: river and marsh buffaloes, the latter being Keywords: Iraqi buffalo, microsatellites, PCR, raised mainly in the swamps of Southeast Asia, genetic diversity and this distinctiojn was confi rmed later by Zhang

1Production Department, College of Agriculture, Basrah University, Iraq, E-mail: [email protected] 2Department of Animal Production, College of Agriculture, Basrah University, Basrah, Iraq

271 Buffalo Bulletin (September 2014) Vol.33 No.3 et al. (2007). Today, almost all breeds have been MATERIALS AND METHODS described according to geographical location and genetic phenomena. Using the new biotechnology Blood samples: Blood samples were in national research centers, local breeds have collected from 96 individuals from the three main been identifi ed in China, India and Pakistan, the regions in Iraq (approximate 24 from the south Philippines, and Vietnam.In China, four types of and 24 individuals from the north of the country buffaloes were identifi ed in accordance with the and 48 from the middle region of Iraq) all the geographical distribution (Wenping et al., 1998). blood samples were from unrelated animals. Six Using molecular data, Zhang et al., 2007 classifi ed microsatellite markers recommended by FAO and Chinese buffalo breeds into four groupsthough ISAG for domestic buffalo diversity studies were these are not commensurate with the classifi cation used (Hoffmann et al., 2004). Forward primers proposed by Wenping (1998). Therefore, there is a were end-labeled with fl uorescent dyes (6-FAM, need to determine the breeds genetically in order TET and HEX). Genotypes for each marker were to obtain correspondence between geographic and determined using ABI 3730 DNA Sequencer genetic classifi cations of the Chinese breeds and (Applied Biosystems) with the internal size the Indian (Kumar et al., 2006). The discovery of standard Gene-Scan (Applied Biosystems). the existence of genetic differences at the level of DNA can bring about a revolutionary strengthening Data analysis of programs of genetic improvement of this Allele frequency, the number of alleles per animal. Current DNA technology can cover most locus, observed heterozygosity (HO) and expected of the requirements for this purpose. Several recent heterozygosity (HE) were calculated using studies have been publishedon the domestic buffalo microsatellite toolkit. To remove the bias of sample using microsatellite markers. These include studies size, a corrected mean number of alleles (MNA) of the swamp buffalo in south-east Asia (Barker et value for each locus was obtained by computing al., 1997), in India (Kumar, 2006), and in China the means of 96 random samples of 24 individuals (Zhang, 2007) and of the river buffalo in Greece for each population except buffaloes of the and Italy (Moioli, 2001). While many researchers middle area, which was 48 individuals. Wilcoxon have investigated genetic polymorphism in the signed rank tests were conducted to examine the buffalo, in Iraq, this issue has not been studied signifi cant differences on MNA and HE between enough. The present study is to fi ll the gap in this each pair of populations. Exact tests for deviations important aspect and to throw light on the genetic from Hardy-Weinberg equilibrium (HWE) were situation in Iraq. Further scientifi c objectives are performed for each population–locus combination development of biodiversity restoration approaches using GENEPOP version 3.4 with the P-values in agricultural biodiversity; managing agricultural obtained by the Markov Chain randomization biodiversity to improve productivity and conserve test. FST values per pair of populations (Weir & diversity and making the fi rst stage of animal Cockerham, 1984) were computed and tested genetic resources conservation. using the FSTAT program (Goudet, 2002). Two approaches were employed to investigate the genetic relationships among the populations. First,

272 Buffalo Bulletin (September 2014) Vol.33 No.3

Nei’s DAgenetic distances (Nei et al., 1983) were 0.869 in the ETH152 marker, while the observed calculated and then used to construct the neighbor- heterozygosity varied from 0.145 in the INRA005 joining tree using MEGA software. Secondly, marker to 0.916 in the CSSM060 marker. Most principal component analysis (PCA) with gene loci showed heterozygosity rates above 0.5, which frequency was conducted. means these markers are highly informative markers (Table 2). Relationships among populations: RESULTS A neighbor-joining tree on the basis of genetic distances was constructed (Figure 1), and for the Genetic variability: In all 70 alleles were fi rst time in Iraq, three main clusters have been detected across the six microsatellite loci .The detected in this genetic tree map. The fi rst of these total number of alleles per locus (TNA) varied clusters includes Basrah, Baghdad, and Qadisseah from three (INRA005) to 16 (ETH152). The MNA provinces, the second consisted of the buffalo of across thesix loci in Iraqi indigenous buffaloes was both Kirkuk and Missan provinces, while the third 5.98. The results of the present study showed that cluster included both Babil and Mosul provinces. all the loci used in this study amplifi ed the DNA The least genetic distance was between the buffalo of the Iraqi buffalo and showed a high level of of Baghdad and Qadisseah provinces, which was polymorphism except INRA005 (Table 1). 0.083, while the highest distance was between the Heterozygosity: From the results of the buffalo of Mosul and Babil and between the buffalo present study, all the six loci showed variable rate of Mosul and Kirkuk provences, which was about of heterozygosity The expected heterozygosity 0.458. ranged from 0.134 in the INRA005 marker to

POP 1

POP 5

POP 6

POP 3

POP 7

POP 2

POP 4

POP 8

      Figure 1. Genetic linkage map of the Iraqi buffalo.

273 Buffalo Bulletin (September 2014) Vol.33 No.3

Table 1. Polymorphism of 6 microsatellite loci in 3 Iraqi indigenous buffalo populations.

Total number of alleles (TNA) Mean number of Locus Southern area Middle area Northern area alleles (MNA) ETH152 8 9 7.5 8.16 CSSM060 6.5 7.5 6.5 6.83 BM1706 6.5 8 8.5 7.66 ETH02 6.5 6 6.5 6.33 ETH225 4.5 4.75 4.5 4.58 INRA005 2.5 2 2.5 2.33 Mean 5.75 6.20 6 5.98

Table 2. Mean of heterozygosity of six loci in the Iraqi buffaloes.

Locus Ho and He * Southern area Meddle area Northern area Ho 0.875 0.729 0.75 ETH152 He 0.865 0.865 0.869 Ho 0.916 0.833 0.791 CSSM060 He 0.782 0.827 0.824 Ho 0.75 0.770 0.791 ETH02 He 0.740 0.753 0.766 Ho 0.75 0.791 0.708 BM1706 He 0.786 0.833 0.085 Ho 0.625 0.770 0.75 ETH225 He 0.577 0.628 0.693 Ho 0.208 0.145 0.458 INRA005 He 0.197 0.134 0.365 *HO: Observed heterozygosity, HE: Expected Heterozygosity.

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DISCUSSION CONCLUSION

Most of the microsatellite loci used here We have presented the fi rst study of were highly informative in the Iraqi buffalo. In the genetic diversity of Iraqi domestic buffalo using present study, a high level of genetic variability microsatellite markers recommended by FAO and was revealed in Iraqi buffalo. The mean expected ISAG. Results indicate distinct genetic variation, heterozygosity of eight populations varied between high levels of genetic differentiation and genetic 0.197 in the INRA005 to 0.865 in the ETH152 loci. structure with three major clusters. These fi ndings The mean observed heterozygosity was 0.208 in could provide an objective basis for classifi cation the INRA005 and 0.916 in the CSSM060 loci. Our and conservation of indigenous buffalo resources. results were supported by many studies conducted in other countries, all of which showed the weakness of the INRA005 marker to the degree of ACKNOWLEDGEMENTS the absence of polymorphism like in Iranian buffalo (Mirhoseinei et al., 2005) or weak polymorphism The authors are thankful to Professor Dr. in the Egyptian buffalo (Zeinab, 2005), with some Chao from Huazhong University in China for exceptions as in the French buffalo which showed scientifi c support for our work. more than three allelesand in the Italian buffalo, in which it reached six alleles (Ciampolini et al., 1995). These differences may be due to the differences in the REFERENCES breed or cluster included in the study. Among the six microsatellite loci used in Barker, J.S.F., S. Moore, S. Hetzel, D. Evan, S.G. this study, the ETH152 marker showed the presence Tan and K. Byrne. 1997. Genetic diversity of 16 alleles, and this can be considered the highest of Asian water buffalo (Bubalus bubalis) number of alleles of this marker that has ever microsatellite variation and comparison reported. In a study on microsatellite markers in with protein-coding loci. Anim. Genet., 28: the Indian buffalo, this marker showed only three 103-115. alleles (Kale, 2010), while in the Iranian buffalo Bradley, D.G. 2006. Genetic documentation of it has between fi ve and sevenalleles only (Seyed animal domestication in: “Documenting et al., 2005). In Egyptian buffalo the number of Domestication: New genetic and alleles for this marker was eight (Zeinab, 2005). archaeological paradigms” (Eds. Zeder, M. All of these studies and many other studies did not A.), University of California Press, USA, p: reach the number in the present study-the marker 273-354. ETH152 had 16 alleles only in Iraqi buffalo. This Ciampolini, R., K. Moazami-Goudarz, D. Vaimman, fact supports our theory that the Iraqi buffalo C. Dillman, E. Mottanti and D. Cianci. originated in Iraq and was not imported from 1995. Individual multilocus genotypes using India. microsatellite polymorphisms to permit the analysis of the genetic variability within and

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between Italian beef cattle breeds. J. Anim. cytochrome b sequence variation. Anim. Sci., 73: 3259-3268. Genet., 29: 253-264. Cockrill, W.R. 1974. The Husbandry and Health Macgregor, R. 1939. The domestics buffalo. Thesis of the Domestic Buffalo. FAO., UN. Rome, presented to the Royal College of Veterinary Italy, 1974. Surgeons (quoted by I.L.Masonin Cockrill Goudet, J. 2002. FSTAT (version 2.9.3.2): A (1974: 1-47). program to estimate and test gene diversities Moioli, B., A. Georgoudis, F. Napolitano, G. Catillo, and fi xation indices. F-statistics. J. Hered., S. Lucioli, C.H. Ligda and J. Boyazoglu. 86: 485-486, URL: http://www 2.unil.ch/ 2001. Genetic diversity between Italian and ize/softwares/fsata.html. Greek buffalo populations. Animal Genetic Haynes, C.D., M.G. Ridpath and M.A.J. Williams. Resources Information, 29: 31-40. 1991. Monsoonal Australia: Landscape, Nei, F., A. Tajima and Y. Tateno. 1983. Accuracy of Ecology and Man in Northern Lowlands. estimated phylogenetic trees from molecular Taylor and Francis Publications, London, data. II. Gene frequency data, J. Mol. Evol., England: 176. 19: 153-170. Hoffmann, D., P.A. Marsan, J.S.F. Barker, E.G. Payne, W.J.A. 1991. Domestication: A forward Step Cothram, O. Hanotte, I. Lenstra, D. Amilan, in civilization, p. 51- 71. In Hickman, C.G. S. Weigend and H. Simianer. 2004. New (ed.) Cattle Genetic Resources. Elsevier MoDAD marker sets to be used in diversity Health Sciences, New York, USA. studies for the major farm animal species Potts, D.T. 1996. Mesopotamia Civilization. Recommendation of a joint ISAG/FAO Cornell University Press, p. 258. working group. In Proceedings of the Seyed, Z.M., M.F.V. Seyed and G. Behzad. 2005. 29th International Conference on Animal Survey of effi ciency of six microsatellite Genetics, Tokyo, Japan. p. 107. loci in Iranian indigenous cattle and Kale, D.S., D.N. Rank, C.G. Joshi, B.R. Yadav, P.G. buffalo populations. Iranian Journal of Korin, K.M. Thakkar, T.C. Tolenkhoomba Biotechnology, 3(1): 41-47. and P. Solanki. 2010. Genetic diversity Weir, J. and C.C. Cockerham. 1984. Estimating among Indian gir, deon and Kankrej cattle F-statistics for the analysis of population breeds based on microsatellite marker. structure. Evolution, 38: 1358-1370. Indian J. Biotechnol., 9: 126-130. Wenping, H. 1998. Genetic diversity of Chinese Kumar, S., J. Gupta, N. Kumar, K. Dikshit, N. water buffalo. Buffalo Bull., 17(2): 30-34. Navani, P. Jain and M. Nagarajan. 2006. Zeinab, G.A. 2005. Genetic variation in native Genetic variation and relationships among cattle. M.Sc. Thesis in Zoology, Faculty eight Indian riverine buffalo breeds. Mol. Science, Al-Azhar University, Egypt. Ecol., 15: 593-600. Zhang, Y., D. Sun, Y. Yu and Y. Zhang. 2007. Genetic Lau, C.H., R.D. Drinkwater, K. Yusoff, S.G., Tan, diversity and differentiation of Chinese D.J. Hetzel and J.S. Barker. 1998. Genetic domestic buffalo based on 30 microsatellite diversity of Asian water buffalo (Bubalus markers. Anim. Genet., 38: 6-17. bubalis): mitochondrial DNA D-loop and

276 Original Article Buffalo Bulletin (September 2014) Vol.33 No.3

CRYOPRESERVATION OF ENDOMETRIAL STROMAL CELLS OF BUFFALO (BUBALUS BUBALIS)

Chethan Sharma G., S.K. Singh*, Jessihun Nongsiej, H.B. Rakesh, R.P. Singh and S.K. Agarwal

ABSTRACT cryopreserved without any major impairment in their morphological and functional integrity. The aim of the present experiment was to cryopreserve buffalo endometrial stromal cells Keywords: cryopreservation, buffalo, stromal and to study their morphological and physiological cells, culture, prostaglandins characteristics. Stromal cells isolated by enzymatic digestion from the buffalo uterus of day 1-5 of the estrous cycle, were cultured and frozen at INTRODUCTION -80oC using DMSO as a cryoprotectant. Frozen o cells were thawed at 37 C and recultured. The Prostaglandins (PGE2 & PGF2α) are culture media was collected when both unfrozen important regulators of reproductive events viz., (control) and frozen cells reached confl uence for luteolysis, ovulation, implantation and parturition estimation of PGE2 and PGF2α using enzyme- in farm animals. PGF2α plays a major role in linked immunosorbent assay. Frozen stored buffalo regulation of cyclicity, on the other hand, PGE2 endometrial stromal cells immediately after thawing exerts action opposite to PGF2α and favours appeared as single or in small clumps, glistening establishment of pregnancy by its luteoprotective and rounded which changed their morphology from action. The endometrium is a complex tissue rounded to fl at spindle shaped immediately 24 h containing mainly epithelial and stromal cells. after seeding retaining their normal morphological Both types of cells produce isoform of PG, but characteristics. The viability of frozen thawed have different morphological and physiological stromal cells at the time of seeding estimated by properties (Fortier et al., 1988; Asselin et al., Trypan blue dye exclusion was 63.3%. There was 1997). Endometrial epithelial cells mainly secrete no signifi cant difference in the basal production of PGF2α whereas stromal cells are the principal

PGE2 and PGF2α by the frozen-thawed and unfrozen source of PGE2. Identifi cation of a particular cell buffalo stromal cells (289.38 ± 4.39, 3.28 ± 0.16 type responsible for observed effect and interaction pg/μg protein vs. 305.97 ± 3.20, 4.26 ± 0.17 pg/μg between different cell types is diffi cult to study with protein, respectively) on day 7 of culture indicating mixed cell culture. An in vitro system for stromal no adverse effect of cryopreservation on their cell culture, therefore, constitutes a good model to functional characteristics. The results demonstrated study regulation of prostaglandin synthesis (Parent that buffalo endometrial stromal cells can be et al., 2003; Guzeloglu et al., 2004), which avoids

Division of Animal Reproduction, Indian Veterinary Research Institute, Izatnagar, Bareilly (U.P.), 243 122, India *E-mail: [email protected]

277 Buffalo Bulletin (September 2014) Vol.33 No.3

animal handling, ethics problems and variability of antibiotics and transported to the laboratory on ice results too. Recently, stromal cell culture (Gupta et immediately after slaughter. White side test (Popov, al., 2010) has also been established in buffalo in 1969) and uterine cytology (Kasimanickam et al., our laboratory, as a model to study the regulation 2004) were done to rule out infection. and modulation of PGF2α and PGE2. Buffalo endometrial stromal cells were To study the regulatory mechanism isolated by enzymatic digestion as per Gupta et involved in PG synthesis related to either luteolysis al. (2010) with suitable modifi cations. Briefl y, the or establishment of pregnancy, there is a need to myometrial layers were dissected and the horn was cryopreserve uterine endometrial cells in order everted to expose the uterine epithelium and placed to conduct in vitro experiment at any point of in a beaker containing 0.3% trypsin III in HBSS time. Cryopreservation has most commonly been and digested for 2 h at 5% CO2, 95% humidifi ed used for the long term storage of biological cells. air at 37°C. After incubation the digested horn Freezing and storage of endometrial cells allows was further placed for second digestion containing accessibility of cells to conduct experiment at any 0.064% trypsin III and collagenase type II and point of time, reduces repeated procurement of 0.032% DNase I for 1 h. All scrapings and washings genital tracts for isolation of cells and reduction in were combined with the digested cells and fetal variability of results as well. Cryopreservation of calf serum was added to a fi nal concentration of cells derived from reproductive tracts e.g. bovine 10% to block the action of trypsin followed by endometrial epithelial and stromal cells (Murakami centrifugation at 1200 rpm for 10 minutes. The et al., 2003), bovine endothelial cells from the pellets were pooled and washed thrice with HBSS corpus luteum (Acosta et al., 2007), porcine and fi nally suspended in 1 ml RPMI-1640 medium endometrial epithelial cells (Kim et al., 2010) and with 10% FCS, 50 μg/ml gentamicin and 0.25 equine endometrial epithelial and stromal cells μg/ml Amphotericin-B. Cells were counted using (Szostek et al., 2012) have been established. Till haemocytometer and viability was determined by date, information on cryopreservation of stromal trypan blue dye exclusion test (Freshney, 2000). cells in buffalo is lacking. The present experiment, Cell concentration was adjusted to 6.25X105 cells/ therefore, was designed with the objective to study ml in RPMI-1640 media plated on 6 well plates the effect of cryopreservation on stromal cell and cultured at 37°C in an atmosphere of 5% CO2, morphology and physiology. 95% humidifi ed air for 18 h to allow attachment of stromal cells. Media were changed every 2 days until monolayer. Further, stromal cells were MATERIALS AND METHODS subjected to indirect immuno-fl uorescent staining for specifi c marker i.e. fi bronectin (stromal Isolation of endometrial stromal cells cells). Cells stained positive giving green colored Buffalo uteri along with intact ovaries fl uorescence indicating presence of specifi c stromal having corpus hemmorrhagicum (approximately cells (Figure 1-D). day 1-5 of the estrous cycle) apparently free of Cells were harvested upon reaching infection were procured from the local abattoir, monolayer using 0.05% trypsin and 0.02% EDTA placed in sterile HBSS supplemented with for 5-10 minutes at 37oC and washed two times

278 Buffalo Bulletin (September 2014) Vol.33 No.3

with HBSS. Final cell pellet was suspended in unfrozen cells. chilled freezing media (70% culture media + 10% DMSO + 20% serum) to adjust cell concentration at the density of 1X106 cells/ml after counting and RESULTS AND DISCUSSION viability assessment. One ml of cell suspension was dispensed into 2 ml cryovial and subjected to slow Frozen stored buffalo endometrial stromal freezing in cryo 1oC freezing container (isopropanol cells immediately after thawing appeared as single bath) and kept at -80oC in a deep freezer. Cells were or in small clumps, glistening and rounded which thawed by plunging cryovials into a water bath changed their morphology from rounded to fl at maintained at 37oC. Contents were centrifuged and spindle shaped immediately 24 h after seeding the cell pellet was suspended in RPMI 1640 media retaining their normal morphological characteristics with 10% FCS supplemented with antibiotics and and reached confl uence or monolayer by day 7 counted using haemocytometer, the quantitative culture (Figure 1-A,B,C). The viability of frozen survivability of the cells was determined by thawed stromal cells at the time of seeding estimated staining the frozen thawed cells with 0.4%Trypan by Trypan blue dye exclusion was 63.3%. The blue (Freshney, 2000). Growth characteristics of loss of viability may be due to stress of freezing- the frozen thawed stromal cells were evaluated by thawing process causing injury to the plasma re-culturing the cells in RPMI-1640 at a density of membrane, a primary target of cryo-damage (Gao 5 1X10 viable cells/ml in 6 well plate. Media were and Crister, 2000). Production of PGE2 is a useful changed every 2 days till confl uence/ monolayer. parameter for determining the functional status of

The supernatant were collected after bovine endometrial stromal cells. PGE2 has been reaching monolayer on day 7 for PGE2 and PGF2α reported as one of the principal prostaglandin estimation using an ELISA Kit (Neogen, U.S.A). secreted by endometrial stromal cells in cattle and Cells were harvested using 0.05% trypsin and buffalo (Parent et al., 2003; Gupta et al., 2010). 0.02% EDTA for 5-10 minutes at 37oC. The cell There was no signifi cant difference in the basal pellets were dissolved with 0.3 M NaOH and 1% production of PGE2 and PGF2α by the frozen- sodium lauryl sulfate and the protein concentration thawed and unfrozen buffalo stromal cells (289.38 of cell lysates was determined by the Bradford ± 4.39, 3.28 ± 0.16 pg/μg protein vs. 305.97 ± method (Bradford, 1976) using BSA as standard 3.20, 4.26 ± 0.17 pg/μg protein, respectively) on

(Banglore Genei, Banglore). Total PGE2 and PGF2α day 7 of culture indicating no adverse effect of concentration per well were expressed as picograms cryopreservation on their functional characteristics. per microgram of total cellular protein. No difference in the production of PGE2 and PGF2α by frozen thawed stromal cells as compared to Statistical analysis unfrozen cells indicated resistance of stromal cells The data are shown as mean ± SEM of to the freezing and thawing process (Murakami values obtained in three separate experiments, each et al., 2003). The results also corroborated with performed in triplicate. The independent ‘t’ test the fi nding of Szostek et al. (2012) in equines. was used to show the statistical signifi cance among In the present study successful cryopreservation basal production of PGE2 and PGF2α in frozen and of buffalo endometrial stromal cells without any

279 Buffalo Bulletin (September 2014) Vol.33 No.3

Table 1. Basal prostaglandin production (pg/μg protein) by frozen thawed buffalo endometrial stromal cells.

Groups PGE2 PGF2α Frozen stromal cells 289.38 ± 4.39 3.28 ± 0.16 Unfrozen stromal cells (Control) 305.97 ± 3.20 4.26 ± 0.17 ‘t’ value 3.05 NS 4.06 NS

(A) (B)

(C) (D)

Figure 1. Frozen-thawed buffalo endometrial stromal cell immediately after seeding (A), 24 h after seeding (B), at confl uence/ monolayer (C) Immunofl uorescent staining of buffalo endometrial stromal cells (stained positive with anti-fi bronectin) (D).

280 Buffalo Bulletin (September 2014) Vol.33 No.3

major impairment in morphological and functional Freshney, R.I. 2000. Culture of Animal Cells: A integrity was achieved and may serve as useful Manual of Basic Technique, 4th ed. Wiley- model to study cell biology and physiology of the Liss Inc., New York, USA. p. 361. endometrium particularly in buffalos. Gao, D. and J.K. Crister. 2000. Mechanism of cryoinjury in living cells, ILAR J., 41: 187- 196. ACKNOWLEDGEMENTS Gupta, C., S.K. Singh, R.K. Baithalu, K.R. Anuj, A. Saxena, R.P. Singh and S.K. Agarwal, The project is funded by the National Fund 2010. Morphological and functional for Basic and Strategic Research in Agricultural characterization of endometrial stromal Sciences (NFBSRA) under the Indian Council cells in buffalo, p. 119. In International of Agricultural Research, New Delhi, India. Symposium on “Biotechnologies for The authors thank the Director, IVRI, Izatnagar, Optimization of Reproductive Effi ciency of Bareilly, UP for necessary facilities to carry out Farm and Companion Animals to Improve this research work. Global Food Security & Human Health” and XXVI Annual Convention of ISSAR, G.B.P.U.A. & T., Pantnagar. REFERENCES Guzeloglu, A., F. Michel and W.W. Thatcher. 2004. Differential effects of interferon-τ on the Acosta, T.J., S. Yoshioka, J. Komiyama, S.H. Lee, prostaglandin synthetic pathway in bovine A.T. Grazul-Bilska and D.J. Skarzynski. endometrial cells treated with phorbol ester. J. 2007. Effects of storage and passage of bovine Dairy Sci., 87: 2032-2041. luteal endothelial cells on endothelin-1 and Kasimanickama, K., T.F. Duffi elda, R.A. Fosterb,

prostaglandin F2α production. J. Reprod C.J. Gartleya, K.E. Lesliea, J.S. Waltonc and Dev., 53: 473-480. W.H. Johnsona. 2004. Endometrial cytology Asselin, E., P. Drolet and M.A. Fortier. 1997. and ultrasonography for the detection of Cellular mechanisms involved during subclinical endometritis in postpartum dairy

oxytocin induced prostaglandin F2α production cows. Theriogenology, 62: 9-23. in endometrial epithelial cells in vitro: Role of Kim, T.S., E.H. Kwon, S.U. Kim, H.S. Choi, H.J. cyclooxygenase-2. Endocinology, 138(11): Lim and S.B. Park. 2010. Activation and 4798-4805. expression of urokinase-type plasminogen Bradford, M.M. 1976. A rapid and sensitive method activator are modulated by freezing/thawing for quantitation of microgram quantities of process through activation of redox signal protein utilizing the principles of protein- pathway in primary porcine endometrial dye binding. Anal. Biochem., 72: 248-254. cells. Cryobiology, 60: 211-216. Fortier, M.A., L.A. Guibault and F. Grasso. 1988. Murakami, S., M. Shibaya, K. Takeuchi, D.J. Specifi c properties of epithelial and stromal Skarzynski and K. Okudu. 2003. A passage cells from the endometrium of cows. J. and storage system for isolated bovine Reprod. Fertil., 83: 239-248. endometrial epithelial and stromal cells. J.

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Reprod Develop., 49(6): 531-538. Parent, J., C. Villenenve, A.P. Alexenko, A.D. Ealy and M.A. Fortier. 2003. Infl uence of different isoforms of recombinant trophoblastic interferons on prostaglandin production in cultured bovine endometrial cells. Biol. Reprod., 68(3): 1035-1043. Popov, Y.N. 1969. Diagnosis of occult endometritis in cows (using white side test on cervical mucus). Veterinariya Moscow, 4: 85-87. Szostek, A.Z., M.J. Siemieniuch, A.M. Galvao, K. Lukasik, D. Zieba, G.M. Ferreira- Dias and Skarzynski. 2012. Effects of cell storage and passage on basal and oxytocin- regulated prostaglandin secretion by equine endometrial epithelial and stromal cells. Theriogenology, 77: 1698-1708.

282 Original Article Buffalo Bulletin (September 2014) Vol.33 No.3

ADOPTION LEVEL OF BUFFALO FARMING PRACTICES IN THE ARID ZONE OF RAJASTHAN

M.L. Meena and Dheeraj Singh

ABSTRACT knowledge, non-availability of inputs, lack of time and lack of veterinary services. This study was conducted in Pali district. This district was purposively selected for the study Keywords: adoption, buffalo farming, technology, as the fi rst phase of the project was in operation Rajasthan only in this district. The sample size was limited to 200 participants and 100 non-participants considering the time and other resources available INTRODUCTION for the study. A sample of 10 participant farmers were selected by adopting simple random sampling The buffalo (Bubalus bubalis) holds the technique from each of the selected 20 villages. greatest promise for food security and sustainable Likewise, 100 non-participant farmers were development in the 21st century as these animals selected from six villages of the non-project area form an integral part of the typical farming having similar socio-economic status. Thus, in all system in India. In India, the buffalo has been the 300 farmers constituted the sample of the study. It backbone of the rural economy. It is the mainstay could be concluded that 47.50 and 42.50 percent in the production of butter and ghee. Not only of the participant farmers were in the medium and this, the buffalo is also considered more useful for high levels of adoption of recommended practices, reasons of higher fat content in milk, its ability respectively, whereas 54.00 and 45.00 percent of to utilize agricultural byproducts more effi ciently the non-participant farmers in the control villages and requirement for fewer kilo calories to produce were in the medium and low levels of adoption, 1 kg milk. The buffalo has been the friend of small respectively. The extent of adoption of participant farmers, often their largest capital asset. It is a fi t farmers was strikingly more than that of the non- companion for Indian farmers, who grow older with participant farmers and there existed a signifi cant their buffaloes developing a high degree of mutual difference between the two groups. In the case of the understanding. As with many of the live stock non-participant farmers, the majority of them were spices maintained by rural families, there is a socio- non-adopters of various recommended practices cultural linkage with buffaloes also. Livestock play except in practices such as period of grazing and a pivotal role in the country’s rural economy. It feeding of tree leaves in summer. The reasons given contributed nearly 16 percent of total income from by the majority of the participant farmers for non- agriculture in 1970-71, which increased to over adoption of recommended practices were lack of 25 percent in 2009-10. This will continue to be

Krishi Vigyan Kendra, CAZRI, Pali-Marwar, Rajasthan-306 401, India, *E-mail: [email protected]

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so in the coming period due to various economic alleviation efforts. factors like increase in the population, urbanization In order to improve the socio-economic and per capita income growth. The role of livestock status of these farmers by helping them cross is much pronounced in the arid zone of Rajasthan, over the poverty line, a novel project, namely the which is prone to drought and resultant crop failure. Farming System’s Research for Improved Buffalo At the time of crop failure, the livestock sector acts Production (FSRIBP). Central Arid Zone Research as a cushion which the farmers can fall upon. As Institute, aided by FSRIBP, was conceived and per the 2007 Livestock Census, Rajasthan had 10.9 launched in the arid zone of Rajasthan in the year million cattle, 10.4 million buffalo, 10.0 million of 2007. A systematic evaluation on scientifi c lines buffalo and 16.8 million goats. The buffalo is one to understand the impact of the project in changing of the major livestock species contributing to the the knowledge and behavioural aspects of target livelihood security of farmers in Rajasthan. It can farmers is essential because this would form a basis thrive in adverse climatic conditions using sparse for extending this project to the other target areas. vegetation. The major buffalo breeds are Murrah, With this in mind, the present study was undertaken Surti, Nili Ravi, Jafravadi and Mehsana. Buffaloes with the following objectives: are mainly reared by socially and economically 1. To measures the extent of adoption of backward people of the society for whom they act recommended buffalo farming practices among as a store of wealth of high liquidity, a resource participant and non-participant farmers. for meeting the household nutritional security 2. To ascertain the reasons for non-adoption and a source of regular income. The farmers rear of recommended practices. buffalo mainly in an extensive management system using traditional management practices relying on common pastureland for grazing. Various METERIALS AND METHODS improved buffalo management technologies were developed at the Central Arid Zone Research Raipur panchayat samiti of Pali district was Institute, Jodhpur to cater the needs of buffalo purposively selected for the study as the fi rst phase farmers. These techno;ogies pertained to the broad of the project was in operation only in this district. areas of breed management, nutrition, health care, The sample size was limited to 200 participants reproduction and product management. Adoption and100 non-participants considering the time and of improved management practices is expected to other resources available for the study. A sample of increase the income of farmers. The performance 10 participants farmers were selected by adopting and contribution of livestock in recent decades simple random sampling technique from each of the towards agricultural production is phenomenal. selected 20 villages. Likewise, 100 non-participants Livestock not only provide livelihood but also farmers were selected from two villages of non- offer employment opportunities to the poorer and project area having similar socio-economic status. weaker sections of the society. Indian planners and Thus, in all 300 farmers constituted the sample of policy makers realized the need to recognize and the study (Sharma, 2005). promote small scale livestock production, which Rogers (1983) defi ned adoption as the is thought to act as a key component of poverty decision to make full use of an innovation as the

284 Buffalo Bulletin (September 2014) Vol.33 No.3

best course of action available. However, adoption RESULTS AND DISCUSSION of improved management practices for buffaloes in the fi eld is low due to various reasons. Considering The fi ndings on the presented and discussed the importance of buffaloes in the livelihood of in terms of extent and practice wise adoption. rural poor in marginalized environments such as Rajasthan, it is imperative to enhance the adoption Extent of adoption of recommended practices of improved management technologies of buffalo (overall adoption) farming. The term adoption in this study means the The data presented in Table 1 revealed that use of recommended practices by the respondents. about 42.50 and 47.50 percent of the participants In consultation with the FSRIBP and Livestock were high and medium adopters, respectively, Development Project fi eld functionaries, fi fteen whereas only 01.00 and 54.00 percent of the non- skill oriented technologies/practices in buffalo participants were in these categories. The remaining farming recommended through the programme were 10.00 percent of the project farmers and 45.00 identifi ed for study of adoption. The recommended percent of the non-project farmers were in the low practices were described to respondents and they level of adoption category. This indicates that the were requested to indicate their extent of adoption extent of adoption of practices by benefi ciaries was during the previous years. The respondents who higher in the target area than in the control area. The had adopted the recommended practice were given reason is that the project has created a considerable two scores and for those not adopted were given level of technological impact on the participant zero score. The extent of adoption of recommended farmers due to the utilization of services such as practices for each respondent farmer was measured veterinary and extension services rendered by the by using an adoption quotient developed by project offi cials. Besides this, increased awareness Sengupta (1967) and adopted by Swaminathan through mass media exposure and its utilization (1986). Based on the adoption quotient, the and through contact with extension agents would respondents were classifi ed into three categories have certainly increased the level of adoption using mean and standard deviation. along with knowledge gained regarding buffalo husbandry practices. The non-availability of these services and facilities in the control area would be

Table 1. Distribution of buffalo farmers according to their level of adoption behavior. (N = 300) Adopters (N=200) Non-adopters (N=100) Total (N=300) S. No. Level of adoption F% F %F% 1. Low -SD 20 10.00 45 45.00 65 21.66 2. Medium ±SD 95 47.50 54 54.00 149 49.67 3. High +SD 85 42.50 01 01.00 86 28.67 Overall 200 100 100 100 120 100 F = Frequency; % = Percentage

285 Buffalo Bulletin (September 2014) Vol.33 No.3

the main reason for the considerable percentage 6 months of age and isolation of sick animals to of low adoption. The fi ndings are in line with prevent the spread of diseases. the fi ndings of Mohan et al. (2005), Suresh et al. In the case of non-participant farmers, (2008) and Meena et al. (2007). Thus, the buffalo the period of grazing and the feeding of tree farmers who were selected as the benefi ciaries of leaves such as neem (Azadirachta indica A Juss.), the project had increased their level of adoption to khejari(Prosopis cinerria), babool(Acacia nilotica) a moderately high level, indicating a considerable and jharberi pala (Ziziphus mauritiana) spices amount of technological impact on them. were the practices adopted by 84.00 and 52.00 percent of farmers, while the majority of them were Practice wise adoption of recommended non-adopters of almost all the remaining practices practices in buffalo rearing. The reasons given were lack Fifteen practices in buffalo rearing as of knowledge and non-availability of suitable recommended by Farming System’s Research inputs. The main reason that the majority of the for Improved Buffalo Production (FSRIBP) were participant farmers were in the adopter category considered for assessing the adoption by the when compared to non-participants might be the respondent farmers. The data generated in this thrust of the project offi cials to improve buffalo aspect were analyzed and are presented in Table 2. husbandry as a viable enterprise in the project area. It can be seen from the table that of the 15 practices The fi ndings are line with those of Kumaravel and recommended by FSRIBP in buffalo rearing, the Krishnaraj (2005) and Meena (2005). practices namely selection and purchase of quality buffalo (77.50 percent), provision of shelter for Reasons for non-adoption of recommended animals (56.00 percent), feeding of colostrums to practices by participants farmers new born calf (80.00 percent), period of grazing The reasons for non-adoption of various (97.05 percent), feeding of crushed prosopis pods recommended practices were collected, tabulated and tamarind seeds (75.00 percent), feeding of and are presented in Table 3. From the table it tree leaves in summer (83.00 percent), deworming can be observed that with regards to practice of (73.00 percent), vaccination of buffalo (71.00 selection and purchase of quality animals, the percent) and marketing of calf at 15 months of high cost of animals (67.70 percent) and the non- age (67.05 percent) were adopted by the majority availability of good breeds (32.30 percent) were of the participant farmers (Tajane et al., 2005). the reasons given by the farmers for non-adoption The reason for the maximum level of adoption of of the recommended practices. The farmers stated these technologies/ practices might be the farmers’ that they were unable to spend money on such conviction they had gained in these areas by way quality breed of buffalo. of reduction in mortality and enhanced production With respect to provision of shelter for level. The adoption level seemed to be poor in animals, the participant farmers felt that for a few the areas of upgrading, ligation and disinfections animals the overhang in the house was enough of naval cord, practice of weaning at 2 months of (22.70 percent) and some of them opined that age, ectoparasiticide application for eradication of construction of separate shed for providing shelter ticks and lice, castration of male buffalo calves at to the animals (77.30 percent) was a costly affair.

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Table 2. Practice wise adoption of recommended technologies practices in buffalo.

Project area (N=200) Non-project area (N=100) Non- S. No. Technologies Adopter Adopter Non-adopter adopter No. % No. % No. % No. % Selection and purchase of 1 155 77.5 45 22.5 08 8.00 92 92.00 quality buffalo breed Provision of shelter for 2 112 56.0 88 44.0 40 40.00 60 60.00 buffalo 3 Upgrading 16 08.0 184 92.0 05 5.00 95 95.00 4 Colostrums feeding 175 87.5 25 12.5 15 15.00 85 85.00 Ligation and disinfection of 5 72 36.0 128 64.4 00 0.00 100 100 naval 6 Practice of weaning 13 06.5 187 93.5 00 0.00 100 100 7 Period of grazing 195 97.5 05 02.5 84 84.00 16 16.00 Feeding of crushed 8 prosopis pods and tamarind 150 75.0 50 25.0 14 14.00 86 86.00 seeds Feeding of tree leaves in 9 166 83.0 34 17.0 52 52.00 48 48.00 summer 10 Deworming 146 73.0 54 27.0 07 7.00 93 93.00 Ecto-parasiticide 11 54 27.0 146 73.3 03 3.00 97 97.00 application 12 Castration of lamb 13 06.5 187 95.5 00 0.00 100 100 13 Vaccination of buffalo 142 71.0 58 29.0 02 2.00 98 98.00 14 Isolation of sick buffalo 20 10.0 180 90.0 00 0.00 100 100 Marketing of ram cat 16 15 135 67.5 65 32.5 33 33.00 67 67.00 months of age N=Number of respondents, % = Percent

287 Buffalo Bulletin (September 2014) Vol.33 No.3

Table 3. Reasons for non-adoption of recommended buffalo rearing practices (Farmers).

S. Recommended Non No. Reasons for non-adoption No. practices adopter (Percent) Selection and purchase a. High cost 30 ( 66.7) 1 45 of quality animals b. Non-availability of breeds 15 (33.3) Provision of shelter for a. Over hang in the house suffi cient 20 (22.7) 2 88 animals b. Shed construction is costly 68 (77.3) a. Non-availability of quality calves 18 (9.8) 3 Upgrading 184 b. lack of knowledge 166 (90.2) a. Colostrum feeding will induce 17 (68.0) 4 Colostrum feeding 25 diarrhea 8 (32.0) b. Lack of time Ligation and disinfection a. Not necessary 111 (86.7) 5 128 of naval cord b. Lack of knowledge 17 (13.3) a. Lack of knowledge 142 (75.9) 6 Practice of weaning 187 b. lambs will feel lonely 45 (24.1) 7 Period of grazing 05 a. Lack of knowledge 7 (100.0) Feeding of crushed 8 drosopis pods and 50 a. Lack of knowledge 50 (100.0) tamarind seeds Feeding of tree leaves in a. Lack of knowledge 16 (47.1) 9 34 summer b. Time consuming 18 (52.9) a. Lack of knowledge 48 (88.9) 10 Deworming 54 b. Inadequacy of dewormers 06 (11.1) a. Costly method 39 (26.7) Ecto-parsiticide 11 146 b. Lack of knowledge 63 (43.2) application c. Time consuming 44 (30.2) a. Lack of veterinary services 24 (12.8) 12 Castration of lamb 187 b. Lack of knowledge 163 (87.2) a. Buffalo go down in condition 05 (08.6) 13 Vaccination of buffalo 58 b. Failure of vaccination 27 (46.6) c. Lack of knowledge 26 (44.8) a. Non-availability of isolation space 33 (18.3) 14 Isolation of sick animals 180 b. Lack of knowledge 147 (81.7) Marketing of calf at 16 15 65 a. Lack of knowledge 65 (100.0) months of age

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With regards to upgrading, lack of knowledge With regard to ectoparasiticide application, a large about the scientifi c breeding practices (90.20 proportion (43.20 percent) of respondents cited lack percent) and non-availability of quality calves of knowledge about the practice as their reason for (09.80 percent) were the reasons mentioned by their non-adoption. The remaining 30.20 and 26.70 most of the non-adopters. In the case of colostrums percent of non-adopters felt that the recommended feeding the farmers felt that feeding of colostrums practice was time consuming and was a costly immediately after birth would induce diarrhea and method, respectively. cause the animal to go down in condition (68.70 Lack of knowledge was the reason given by percent). The one third of the non-adopters of the majority (87.20 percent) of non-adopters with this practice cited the non-availability of time as respect to the practice of castration of male calves, the reason for non-adoption as they were engaged followed by lack of veterinary services (12.80 in other activities. With regards to the practice of percent) in their villages to perform this operation. ligation and disinfection of naval cord, the non- With respect to the vaccination of buffalo 46.60 adopters felt that the mother itself would lick the percent of non-adopters gave bitter experience umbilical cord and it would heal automatically and of failure of vaccination leading to mortality of hence there was no need to cut and ligate the cord. buffalo as their major reason, followed by lack of A few of the non-adopters said lack of knowledge knowledge (44.80 percent) and the wrong notion was their reason for not adopting this practice. of the condition of animals would deteriorate if In the case of the practice of weaning, vaccinated (8.60 percent). With regard to isolation lack of knowledge was cited by the majority of of sick animals, lack of knowledge (81.70 percent) the respondent farmers. Some of the farmers also and non-availability of isolation space (18.30 opined that weaned animals would feel lonely and percent) were the reasons cited for non-adoption. isolated. As far as period of grazing is concerned In the case of marketing of male calves at 15 very few gave lack of knowledge as the reason for months of age, the non-adopters said that lack of non-adoption of following the recommended time knowledge about organized marketing was their of grazing. In the case of feeding of crushed prosopis reason for non-adoption. pods and tamarind seed, lack of knowledge was the reason given by nearly half of the total participant farmers. With regards to the feeding of tree leaves CONCLUSION in summer, some of the participant farmers stated they lacked knowledge in the feeding of suitable About 47.50 and 42.50 percent of the types of leaves, while the remainder felt that this participant farmers were in the medium and high practice was a time consuming affair. levels of adoption of recommended practices, In the case of deworming, lack of respectively, whereas 54.00 and 45.00 percent knowledge was given by the majority of the non- of non- participant farmers in the control adopters (88.90 percent) while the remaining villages were in the medium and low levels of 11.10 percent of them stated that inadequacy of adoption, respectively. The extent of adoption of dewormers with the local veterinarian and link participant farmers was strikingly more than that worker couples was their reason for non-adoption. of the non-participant farmers and there existed a

289 Buffalo Bulletin (September 2014) Vol.33 No.3

signifi cant difference between the two groups. The Mohan, B., R.L. Sagar, K. Singh, P. Tripathi and recommended practices in buffalo rearing, namely V.S. Vihan. 2007. Present status of goat selection and purchase of quality buffalo, provision rearing under rural condition. Indian of shelter for animals, feeding of colostrums for Research Journal of Extension Education, new born calf, period of grazing of buffalo, feeding 7(1): 43-45. of crushed prosopis pods and tamarind seeds, Rogers, E.M. 1983. Diffusion of Innovations. The feeding of tree leaves in summer, deworming, Free Press, New York: 132. vaccination and marketing of calf at 15 months of Sengupta, H. 2007. Adoption scale to measures age were adopted by majority of the participant livestock production practices in arid zone farmers. Most of the participant farmers had poor of Rajsthan (Unpublished), RAU, Bikaner, adoption in areas such as upgrading of animals, Campus: SKN, COA, Jobner. ligation and disinfection of naval cord, practice of Sharma, N.K. 2006. Construction of adoption weaning, ectoparasiticide application, castration of scale about improved buffalo production male calves and isolation of sick animals. In the technology in Ago-climatic Zone IIIb of case of non-participant farmers, the majority of Rajasthan. Rajasthan Journal of Extension them were non-adopters of various recommended Education, 16(2): 23-26. practices except practices such as period of grazing Suresh, A., D.C. Gupta and J.S. Mann. 2008. and feeding of tree leaves in summer. The majority Adoption of improved management of the participant farmers said that the reason for practices of buffalo in the semi arid region their non-adoption of recommended practices were of Rajasthan. Indian Journal of Extension lack of knowledge, non-availability of inputs, lack Education, 44(1&2): 100-105. of time and lack of veterinary services. Swaminathan, M.S. 1986. Agricultural planning- improving the livestock in rural areas. Indian Farmers Times, 6(11): 32. REFERENCES Tajane, S.B., P.J. Rajkamal and Veeranna. 2005. Work environment of the veterinary Kumaravel, P. and R. Krishnaraj. 2005. Adoption surgeons. Indian Journal of Extension behaviour of livestock farmers. Indian Education, 41(1&2): 99-101. Journal of Extension Education, 41(1&2): 117-121. Meena, M.L. 2005. Problems and prospects of improved buffalo production in the fl ood prone eastern plain zone of Rajasthan. Ph.D. Thesis, RAU, Bikaner Campus: Jobner. Meena, M.L., N.K. Sharma and A. Dudi. 2007. Buffalo keepers’ adoption about improved buffalo production practices. Indian Research Journal of Extension Education, 7(2&3): 65-68.

290 Original Article Buffalo Bulletin (September 2014) Vol.33 No.3

MILK PRODUCTION AND REPRODUCTION PERFORMANCE OF MURRAH BUFFALOES OF TAMIL NADU, INDIA

A.K. Thiruvenkadan*, S. Panneerselvam, N. Murali, S. Selvam and V. Ramesh Saravanakumar

ABSTRACT as service period, calving interval and dry period were slightly higher than those observed in the The data pertaining to variable production home tract of the Murrah buffalo and hence better and reproduction traits of Murrah buffaloes (1980 breeding management and introduction of genetic lactation records of 698 Murrah buffaloes) were evaluation programmes are needed for genetic collected from the Central Cattle Breeding Farm, improvement of these traits. Alamadhi, Tamil Nadu, India. The overall least- squares means (± SE) for peak yield, days to Keywords: coastal region, genetic parameters, attain peak yield, 305-day milk yield, lactation Murrah buffaloes, production, reproduction length, lactation milk yield, service period, calving interval and dry period were 8.87 ± 0.05 kg, 53.4 ± 0.8 days, 1804.9 ± 14.7 kg, 297.8 ± 1.9 days, INTRODUCTION 1855.6 ± 16.1 kg, 225.0 ± 5.5, 532.8 ± 5.5 and 230.2 ± 4.9 days, respectively. Period of calving According to the 2003 livestock census, had a highly signifi cant (P<0.01) effect on all the India possesses 185.2 million cattle and 97.9 traits studied except days to attain peak yield, million buffaloes, which is about 13.7 percent where it had only a signifi cant (P<0.05) effect. of the total cattle and 57.5 percent of the total Season of calving had a signifi cant (P<0.05) effect buffalo population of the world. The dairy industry on peak yield and lactation milk yield and a highly in India has made signifi cant progress in the last signifi cant (P<0.01) effect on days to attain peak few decades. Today, India is the largest producer yield, 305-day milk yield, milk yield per day of of milk in the world. The milk production in the lactation and all the reproduction traits studied. The country has risen to about 88.1 million tonnes in lowest calving interval was observed in south-west 2003-2004 from 17.0 million tonnes in 1950-51. At monsoon calvers, and they differed signifi cantly present, India’s contribution to the total world milk (P<0.05) from winter and summer calvers. Parity production is about 14.3 percent and the national had a highly signifi cant effect (P<0.01) on all the per capita milk availability is 231 g/day. In India, traits studied. Pairwise comparison revealed that although the proportion of buffaloes to cattle is 1:2, the lactation milk yield was lowest in fi rst parity the buffaloes contribute 56.86 percent of the total and differed signifi cantly (P<0.05) from other milk obtained from cattle and buffaloes. Tamil Nadu parities. In general, the reproduction traits such with 9.14 million cattle and 1.66 million buffaloes

Department of Animal Genetics and Breeding, Veterinary College and Research Institute, Orathanadu, Tamil Nadu, 614 625, India, *E-mail: [email protected]

291 Buffalo Bulletin (September 2014) Vol.33 No.3

produces an estimated quantity of 4.75 million buffaloes in the coastal regions of Tamil Nadu to tonnes of milk (Report, 2006). The Murrah breed is suggest suitable managemental practices, selection the fi nest genetic material of milk producing buffalo and breeding strategies for genetic improvement not only in India but also probably in the world. of Murrah buffaloes under hot and humid climatic This breed has been used extensively throughout conditions of India. the country to upgrade the non-descript buffalo stock to improve milk production. The breeding policy of Tamil Nadu state is to use Murrah or Surti MATERIALS AND METHODS as the breeds of choice to improve non-descript buffaloes. As a result of these measures, the Murrah The study was based on the data pertaining and graded Murrah population have been increased to the Murrah buffaloes born and bred at the Central over the years. Although the water buffalo in the Cattle Breeding Farm, Alamadhi, Chennai, Tamil tropics out-produces other domestic animals, Nadu, India from 1979 to 2006 (28 years). This commercial milk production is adversely affected farm is located approximately at 13o N latitude and by a large number of detractive factors such as 80o E longitude at an altitude of about 20 metres late age at fi rst calving, seasonality of oestrus, and above mean sea level. The climate is generally hot, long calving interval and dry period. Therefore, humid and tropical in nature. The mean annual it is necessary to evaluate the relative importance maximum and minimum temperatures were 33oC of various fi xed environmental and physiological and 24.7oC, respectively, and the mean relative effects in infl uencing the milk production and humidity ranged between 69.2 and 76.2%. The reproduction traits in order to devise appropriate Murrah buffaloes were housed in permanent sheds feeding and other managemental practices. From with open type ventilation and maintained under the literature reviewed, it was found that the bulk stall-fed conditions. Roughage in the farm of green of scientifi c information on buffaloes has come fodder and paddy straw was provided. In addition, from the analysis of records made available from concentrate mixture was provided to all age groups institutional and government farms in northern as per the standard requirements. Cows were hand- India (Sethi and Khatkar, 1997; Dass and Sadana, milked twice daily in the morning and evening. 2000; Gogoi et al., 2002; Kundu et al., 2003a,b; Data on production and reproduction performance Yadav et al., 2007). Such information from the of Murrah buffaloes (1980 lactation records form southern peninsular region, especially under hot 698 Murrah cows) were extracted from History and humid coastal regions of Tamil Nadu, is scanty. and Pedigree Sheets. The traits studied were peak The home tract of Murrah buffaloes is a hot and dry yield, days to attain peak yield, 305-day milk yield, climatic region in the north-western part of India. lactation length, lactation milk yield, milk yield per Breeding these buffaloes in the southern peninsular day of lactation, service period, calving interval, region of India, which is hot and humid, may affect dry period and number of services per conception. their performance and adaptability. Hence, the Period and season were the fi xed environmental present study has been made to understand the effects considered for all the traits studied. As the performance and the infl uence of various non- calvings were less in a year, year-season analysis genetic factors affecting economic traits of Murrah was not done. To utilise all available data the entire

292 Buffalo Bulletin (September 2014) Vol.33 No.3

duration was divided into seven periods each to 2006), where the difference was not signifi cant. with an interval of fi ve years assuming that there There was a steep increase in peak yield, 305-day would not be any major managemental changes milk yield and lactation milk yield from the second within a period. Further each calendar year was to the sixth periods and then a slight decline in sub-divided into four seasons, viz. winter (January period 7 (2003 to 2006); the values observed in and February), Summer (March to May), south- period 2 differed signifi cantly (P<0.05) from the west monsoon (June to September) and north-east rest of the periods. monsoon (October to December). LSMLMW and Season of calving had no effect on lactation MIXMDL PC-2 VERSION computer programme length; however, it had a signifi cant (P<0.05) effect of Harvey (1990) was used to study the effect of on peak yield and lactation milk yield and a highly various non-genetic factors and the means were signifi cant (P<0.01) effect on days to attain peak compared using Duncan’s multiple range test. The yield, 305-day milk yield and milk yield per day model used for analysis was Yijkl = μ + Pi + Sj + Ok + of lactation. The peak yield of Murrah buffaloes th th th eijkl, where, Yijkl = the l observation in i period, j calving in the south-west monsoon season was season and kth parity, μ= overall mean when equal the highest and it differed signifi cantly (P<0.05) th subclass frequencies exist, Pi = effect of i period from that of north-east monsoon calvers. However, th (i =1 to 7), Sj = effect of j season (j =1 to 4), Ok there was no signifi cant difference among the other th = effect of k parity (k=1 to 6) and eijkl = random seasons. On the other hand, the lowest 305-day 2 errors NID (0, σ e). milk yield, lactation milk yield and milk yield per day of lactation were observed in cows calving in the north-east monsoon season. Winter and RESULTS AND DISCUSSION summer calvers had higher 305-day milk yield and lactation milk yield than the monsoon calvers. The Production Traits yields observed among winter, summer and south- Least-squares means (±SE) of different west monsoon calvers were not signifi cant but they production traits are presented in Table 1. Period differed signifi cantly (P<0.05) from north-east of calving had a highly signifi cant (P<0.01) effect monsoon calvers. on the variation of peak yield, 305-day milk yield, Parity had a highly signifi cant (P<0.01) lactation length, lactation milk yield and milk yield effect on all the traits studied. First lactation peak per day of lactation and had only a signifi cant yield was signifi cantly (P<0.05) lower than the (P< 0.05) effect on days to attain peak yield. The rest. The yield increased from fi rst to fourth parity highest peak yield, 305-day milk yield and lactation and from there on it started declining. Whereas, milk yield were observed in period 6 (1999 to the days to attain peak yield decreased from fi rst 2002) and they differed signifi cantly from rest of to fi fth parity followed by a moderate increase in the periods, except with periods 5 and 7, where sixth parity. The days to attain peak yield in fi rst the differences were not signifi cant. The lowest lactation was signifi cantly (P<0.05) higher (60.9 lactation length was observed in period 3 (1987 to days) than the rest. The 305-day milk yield and 90) and this period differed signifi cantly (P<0.05) lactation milk yield increased up to third parity, from the rest of the periods except period 7 (2003 was maintained at fourth parity, and declined

293 Buffalo Bulletin (September 2014) Vol.33 No.3 a c c c a a c c b b b b b b b c bc Mil yield per day of per 6.66 ± 0.11 5.38 ± 0.06 6.43 ± 0.07 6.55 ± 0.09 6.66 ± 0.08 5.99 ± 0.04 5.38 ± 0.05 6.45 ± 0.07 6.50 ± 0.08 5.74 ± 0.08 5.72 ± 0.06 6.19 ± 0.07 6.34 ± 0.09 6.14 ± 0.05 6.22 ± 0.06 6.09 ± 0.09 6.33 ± 0.09 lactation (kg) a a a c c a a e b b b b bc cd de bc ab cantly (P>0.05). cantly fi (kg) Lactation milk yield 1706.2 ± 35.8 1629.9 ± 25.8 1658.5 ± 25.6 2030.3 ± 39.6 2055.6 ± 34.1 1687.6 ± 24.8 1967.3 ± 30.4 1925.2 ± 32.7 1983.2 ± 47.4 1894.5 ± 25.7 1966.5 ± 35.5 1853.7 ± 42.4 1763.8 ± 38.5 a c a b b b b b b ab bc bd abd Lactation 301.8 ± 3.7293.3 ± 5.0 1888.6 ± 31.4 1882.4 ± 41.8 298.8 ± 2.6 1849.6 ± 21.8 297.4 ± 2.3 1801.6 ± 19.4 285.1 ± 3.0 310.4 ± 2.9 284.3 ± 4.6 295.6 ± 4.2 300.2 ± 3.1 298.3 ± 3.9 307.2 ± 4.7 305.8 ± 4.1 301.1 ± 3.1 292.5 ± 5.6 302.3 ± 3.6 299.0 ± 4.2 289.8 ± 5.0 length (days) a c a a c c b d b b b d d b ab cd cd yield (kg) 305-day milk 1584.2 ± 23.6 1872.9 ± 29.8 1733.8 ± 17.7 1619.7 ± 22.6 1832.8 ± 23.5 1823.4 ± 38.7 1670.2 ± 32.6 1974.1 ± 31.1 1839.0 ± 28.6 1853.8 ± 38.1 1793.1 ± 19.8 1913.9 ± 27.7 1910.4 ± 32.3 1729.1 ± 35.1 1632.0 ± 23.4 1947.2 ± 36.1 1953.7 ± 43.2 c a a a a a a b b b b ad bc ab ab abc bcd (days) 57.2 ± 1.9 48.3 ± 0.9 54.0 ± 1.3 51.1 ± 1.5 50.6 ± 1.7 50.0 ± 2.1 53.5 ± 1.9 53.0 ± 1.5 57.9 ± 2.0 54.1 ± 1.1 60.9 ± 1.2 peak yield 50.7 ± 1.7 54.9 ± 1.3 52.0 ± 1.3 51.4 ± 1.7 52.2 ± 1.6 55.0 ± 2.3 Days to attain a a a b b b b d b b c e ce ce ab ab cd ** * ** ***** ** * ** ** ** ** ** ** ** ** ** ** (kg) Peak yield 724 9.03 ± 0.07 830 8.79 ± 0.06 ) 1980 8.87 ± 0.05 53.4 ± 0.8 1804.9 ± 14.7 297.8 ± 1.9 1855.6 ± 16.1 6.16 ± 0.04 μ Effect n Least-squares means (± SE) for different milk production traits of Murrah buffaloes Least-squares means (± SE) for different

(1979-82) (1983-86) (1987-90) (1991-94) (1995-98) 268 (1999-2002) 491 8.47 ± 0.12 (2003-2006) 430 7.96 ± 0.09 265 8.30 ± 0.08 171 236 9.16 ± 0.11 9.47 ± 0.13 119 9.52 ± 0.11 9.18 ± 0.16 1 2 3 4 5 6 7 Overall mean ( P Period of calving P P P P P P Season of calving (Jan.-Feb.) Winter Summer (Mar.-May) 276South-west monsoon (Jun.- Sep.) 8.81 ± 0.10 150North-east monsoon 8.84 ± 0.14 (Oct.- Dec.) Parity FirstSecondThirdFourthFifthSixth and above 645 457 7.73 ± 0.08 8.85 ± 0.09 311 193 224 9.37 ± 0.10 8.74 ± 0.13 9.54 ± 0.12 150 8.98 ± 0.14 Table 1. Table signi n= Number of observations. * P<0.05, ** P<0.01.Means bearing same superscript within classes do not differ

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thereafter. Pairwise comparison revealed that the et al. (2004). Differences in the estimates might be 305-day and lactation milk yields observed in fi rst due to sampling errors, genetic constitution of the parity differed signifi cantly (P<0.05) from other herds, agroclimatic variations and managemental parities. Similar to days to attain peak yield, the conditions. In general, the performance in terms of lactation length decreased with the advancement of the fi rst lactation milk yield of Murrah buffaloes parity. There was an initial sharp reduction (3.09 at the Central Cattle Breeding Farm, Alamadhi percent) in lactation length in the second parity is quite similar (Sethi and Khatkar, 1997; Kumar followed by gradual decline later. The reduction in et al., 2002) to those herds in Haryana indicating lactation length between fi rst and second parities that there might not be any appreciable genotype x was statistically signifi cant (P<0.05). The milk environment interaction. yield per day of lactation increased linearly with the advancement of parity up to 4th parity and then Reproduction Traits declined; the values observed in fi rst parity differed Least-squares means (±SE) of different signifi cantly (P<0.05) from the rest of the parities. reproduction traits are set out in Table 2. Analyses The average peak yield obtained in the of variance revealed that the period of calving present investigation was higher than the value infl uenced service period, calving interval, dry reported by a few earlier workers (Rao and Rao, period and number of services per conception in a 1994; Kundu et al., 2003b). However, Chhikara highly signifi cant (P<0.01) manner although there et al. (1998) and Suresh et al. (2004) reported was no consistent trend over the periods. Period 3 higher values for Murrah buffaloes than those (1987 to 90) had the lowest service period, calving observed in the present study. The days to reach interval and number of services per conception, peak yield in the present study was substantially and these differed signifi cantly (P<0.05) from other higher than those reported for Murrah buffaloes periods except period 1(1979 to 1982). Whereas, the at different places in India (Kundu et al., 2003b; lowest and the highest dry periods were observed Suresh et al., 2004). Lactation milk yield of cattle in period 1 and period 5, respectively, and the and buffaloes up to 305-days of lactation is the dry period observed in period 5 was signifi cantly criterion most commonly used for the selection of (P<0.05) different from periods 1, 3 and 4. dairy animals and a study of the performance of this Season of calving also infl uenced all the trait is of paramount importance for carrying out reproduction traits in a highly signifi cant manner selection. The overall 305-day milk yield obtained (P<0.01). Murrah buffalo heifers freshening in the for Murrah buffaloes in this investigation was south-west monsoon had signifi cantly (P<0.05) comparable to the value reported by Ulaganathan et shorter service period, calving interval, dry period al. (1983) and higher than the values reported by and number of services per conception than those other researchers (Kandasamy, 1987; Suresh et al., calving in other seasons. The highest service 2004). The overall least-squares means of lactation period, calving interval and number of services per milk yield obtained was higher than those observed conception were observed in winter season calvers. by Ulaganathan et al. (1984) and Patnaik (1988) in The service period and calving interval observed in the same herd and lower than the present estimate south-west monsoon calvers differed signifi cantly was also reported by Kandasamy (1987) and Suresh (P<0.05) from other seasons except with north-east

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Table 2. Least-squares means (± SE) for different reproduction traits of Murrah buffaloes.

Number of Service period Calving Dry period Effect N services per (days) interval (days) (days) conception Overall mean (μ) 1550 225.0 ± 5.5 532.8 ± 5.5 230.2 ± 4.9 2.31 ± 0.05 Period of calving ** ** ** ** af a a ad P1 (1979-82) 220 182.8 ± 11.4 488.8 ± 11.4 181.6 ± 10.2 1.92 ± 0.11 be be bc b P2 (1983-86) 423 241.3 ± 8.1 548.2 ± 8.2 242.6 ± 7.3 2.45 ± 0.08 a a a ad P3 (1987-90) 316 176.1 ± 8.5 481.9 ± 8.5 191.5 ± 7.6 1.87 ± 0.08 b b b b P4 (1991-94) 210 218.9 ± 10.4 527.7 ± 10.4 228.6 ± 9.3 2.29 ± 0.10 cd cd c c P5 (1995-98) 114 272.8 ± 14.0 580.8 ± 14.0 268.7 ± 12.5 2.82 ± 0.13 cde cde bc bc P6 (1999-2002) 199 252.9 ± 10.7 559.7 ± 10.7 253.3 ± 9.6 2.55 ± 0.10 bdef bd bc bcd P7 (2003-2006) 68 230.5 ± 17.6 542.4 ± 17.7 245.1 ± 15.8 2.30 ± 0.16 Season of calving ** ** ** ** Winter (Jan.-Feb.) 211 246.8 ± 10.3b 554.4 ± 10.3b 248.0 ± 9.2c 2.56 ± 0.10c Summer (Mar.-May) 109 245.2 ± 14.0b 553.4 ± 14.0b 254.1 ± 12.5c 2.44 ± 0.13bc South-west monsoon 587 197.5 ± 7.2a 505.5 ± 7.2a 201.5 ± 6.4a 2.02 ± 0.07a (Jun.- Sep.) North-east monsoon 643 210.6 ± 6.4a 517.9 ± 6.5a 217.1 ± 5.8b 2.24 ± 0.06b (Oct.- Dec.) Parity ** ** ** ** First 513 280.1 ± 8.0c 586.6 ± 8.1c 274.1 ± 7.2c 2.82 ± 0.08c Second 367 237.1 ± 8.3b 544.9 ± 8.3b 240.3 ± 7.4b 2.40 ± 0.08b Third 259 218.4 ± 9.6ab 526.4 ± 9.6ab 220.9 ± 8.6ab 2.24 ± 0.09ab Fourth 175 205.9 ± 11.6a 512.7 ± 11.6a 207.9 ± 10.4a 2.19 ± 0.11ab Fifth 118 187.4 ± 13.7a 495.5 ± 13.7a 201.2 ± 12.2a 2.00 ± 0.13a Sixth and above 118 221.2 ± 14.0ab 530.8 ± 14.1ab 236.8 ± 12.6ab 2.24 ± 0.13ab n= Number of observations. * P<0.05, ** P<0.01. Means bearing same superscript within classes do not differ signifi cantly (P>0.05).

296 Buffalo Bulletin (September 2014) Vol.33 No.3

monsoon calvers. On the other hand, the dry period buffaloes maintained at the National Dairy Research and number of services per conception observed Institute, Karnal and the Buffalo Research Centre, in south-west monsoon season calvers differed Hisar in India. signifi cantly (P<0.05) from winter, summer and The main factor controlling variations in north-east monsoon season calvers. The differences the calving interval is the service period, which of service period among parities were highly in turn depends on post-partum oestrus days and signifi cant (P<0.01). The service period decreased number of services per conception. In addition, with the order of lactation up to fi fth parity and the many other additional factors have been implicated lowest value of 187.4 ± 13.7 days was observed in lengthened calving intervals, such as embryonic at this parity. The reduction in service period in mortality, high milk production, seasonal and the second and third parities was rather sharp and environmental factors, age of cow and sire used for further decline was gradual. In general, pairwise service. The coeffi cient of variation obtained for comparison revealed that the mean service periods service period (67.3 percent) in the present study of fi rst and second parities differed signifi cantly indicates that the herd was more heterogeneous for (P<0.05) from other parities as well as between this trait. This strongly suggests better opportunities them. Similarly the Murrah buffaloes calving for for improvement through good breeding practices. the fi rst time had the longest calving interval and Hence, every effort should be made to reduce the dry period, which declined thereafter. The calving service period suffi ciently to reduce the calving interval and dry period observed in the fi rst parity interval. was signifi cantly (P<0.05) different from second The highly signifi cant infl uence of period of and later parities. In addition, the mean calving calving observed in the present study on different intervals and dry periods observed between the production and reproduction traits was supported second and fourth and fi fth parities differed by similar fi ndings on Murrah buffaloes maintained signifi cantly (P<0.05). The decline from third at different places in India (Kandasamy, 1987; to fi fth parities was gradual and the differences Sethi and Khatkar, 1997; Suresh et al., 2004; between means were not signifi cant. Yadav et al., 2007). The difference in performance The mean service period, calving interval of the animals among different periods might be and dry period of Murrah buffaloes observed were attributed to differences in management practices, in agreement with some research reports on Murrah sires used for breeding, environmental conditions buffaloes (Kandasamy, 1987; Patnaik, 1988; Kundu such as ambient temperature, humidity, rainfall et al., 2003b). However, studies on these traits in etc., and variations in feed and fodder availability. the Murrah breeding tract reported (Chhikara et al., The signifi cant to highly signifi cant effect 1995a; Dass and Sadana, 2000; Banik and Tomer, of season of calving on different production traits 2003) much lower values than those observed in the corroborated with the fi ndings of the earlier workers herd under study. The mean number of services per (Chhikara et al., 1998; Dass and Sadana, 2000) and conception observed (2.31 ± 0.05) was also much indicated that there was a pronounced seasonal higher than that reported by Kumar et al. (2003). infl uence on the traits under study. The buffaloes However, Dutt and Yadav (1988) and Chhikara et calving in the winter season had longer lactation al. (1995b) found comparable estimates for Murrah length and higher lactation milk yield than those

297 Buffalo Bulletin (September 2014) Vol.33 No.3

calving in the rainy seasons. This confi rms the load on conception rate should be implemented to fi ndings in most of the earlier reports on Murrah reduce number of services per conception. This in buffaloes (Rao and Rao, 1994; Dass and Sadana, turn will have a positive effect on service period 2000; Gogoi et al., 2002). The higher lactation and calving interval. milk yield in the winter and summer seasons might The signifi cant infl uence of parity on be due to the fact that the buffaloes calving in different production and reproduction traits is those periods were found to have less gestational in accordance with the results obtained by other stress due to longer service period and delayed researchers (Dass and Sadana, 2000; Kundu et conception and also that during their descending al., 2003b) on Murrah buffaloes. That the highest stage of lactation there was an abundant availability 305-day and lactation milk yields were obtained in of fodders, since it falls on monsoon seasons. The the third parity indicates that lactational maturity lowest milk yield in monsoon calvers might be due is attained in the third lactation. These dindings to the fact that they suffered from hot humid stress aresimilar to the reports of Ulaganathan et al. and non-availability of quality fodder during the (1983) and Kandasamy (1987). The signifi cant major part of the lactation period. infl uence of calving sequence on service period The signifi cant effect of season of calving on and calving interval and longer fi rst calving interval service period and calving interval is in agreement than the rest found in the present study concurred with fi ndings reported in the literature for Murrah with the other reports on Murrah buffaloes buffaloes (Chhikara et al., 1995b; Kumar et al., (Kandasamy, 1987; Dass and Sadana, 2000; Kundu 2003; Suresh et al., 2004). It is generally observed et al., 2003a; Suresh et al., 2004). The reduction that buffalo cows are seasonally polyoestrus during in service period and calving interval over parities October to February and they breed regularly during might be due to differences in age of the animals these periods. This might be one of the reasons for and periodic culling of buffalo cows with longer shorter service period during monsoon seasons. calving intervals. The other plausible reason is that The higher number of services per following the irst two calvings, the physiological conception in winter calvers observed in this study rhythm may be maintained (i.e. reduced postpartum might be due to the fact that those animals calving oestrus days and better conception) which results in in winter exhibit post-partum heat in summer shorter inter-calving periods in pluriparous buffalo months and hence there would be reduction in cows. Similarly the highest dry period in the fi rst conception rate. Conception rate is related to parity and signifi cant reduction in later parities oestrous behaviour, time of oestrus detection and might be due to the reduction of calving interval. insemination and site of semen deposition. Among The slight increase in reproduction traits from the the different factors, accurate detection of oestrus fi fth to sixth and above parities might be due to the is of paramount importance in any reproductive lumping of all later records with the sixth parity. management programmes; however, this is diffi cult in buffaloes during the summer season, since most of them exhibit silent oestrus. Thus CONCLUSIONS accurate detection of oestrus and managemental interventions to ameliorate the effects of heat The study revealed that non-genetic factors

298 Buffalo Bulletin (September 2014) Vol.33 No.3

such as period and season of calving had highly Dairying, Foods and Home Sci., 14: 29-32. signifi cant effect on all the traits studied. In general, Chhikara, S.K., N. Singh, S.S. Dhaka and R.S. Yadav. the milk production performance of the farmbred 1995b. Factors infl uencing reproduction Murrah buffaloes at the Central Cattle Breeding characters of Murrah buffaloes. Indian J. Farm, Alamadhi was comparable to that of animals Anim. Res., 29: 22-26. maintained in other government and institutional Dass, G. and D.K. Sadana. 2000. Factors affecting herds in India. However, the comparatively lower some economic traits in Murrah buffaloes. performance of Murrah buffaloes with respect Indian J. Anim. Res., 34:43-45. to fi tness traits indicates less adaptability of the Dutt, G. and M.C. Yadav. 1988. Factors affecting breed to the hot and humid coastal region. Since number of services per conception and temporary environmental factors play a major role calving interval in Murrah buffaloes. Asian on these fi tness traits, better breeding management J. Dairy Res., 7: 168-170. is needed for improvement. In addition, multi- Gogoi, P.K., D. Das, R.N. Goswami, N. Nahardeka trait evaluation with a combination of production and G.C. Das. 2002. Studies on age at and reproduction traits may be carried out for fi rst calving in Murrah and Surti buffaloes simultaneous improvement of production and maintained in Assam. Indian Vet. J., 79: 854- reproduction performances of Murrah buffaloes 855. in the herd. It is therefore imperative to emphasize Harvey, W.R. 1990. User’s Guide for LSMLMW improvements in husbandry practices and and MIXMDL PC-2 Version. Mixed model introduction of genetic evaluation programmes at least-squares and maximum likelihood the same time. computer program. Ohio State University, Columbus, Ohio, USA. Kandasamy, N. 1987. Genetic Analysis of Milk REFERENCES Records in Farmbred Murrah Buffaloes of Tamil Nadu. Ph.D. Thesis, Tamil Nadu Banik, S. and S.S. Tomar. 2003. Total determination Agricultural University, Coimbatore, India. of calving interval by path analysis in Kumar, A., Z.S. Rana, S.S. Dhaka, D.S. Dalal, Murrah buffaloes. Indian J. Anim. Res., 37: S. Singh and R.K. Sethi. 2002. Test day 19-23. yield and lactation parameters of Murrah Chhikara, S.K., N. Singh and S.S. Dhaka. 1998. buffaloes. In Proceedings of the 7th World Effect of some non-genetic factors on peak Congress on Genetics Applied to Livestock yield and days to attain peak yield in Murrah Production, Montpellier, France. buffaloes. In Proceedings of the 6th World Kumar, D., H. Singh, D. Kumar and C.V. Singh. Congress on Genetics Applied to Livestock 2003. Non-genetic factors affecting some Production, Armidale, Australia, 24: 481- production and reproduction traits in Murrah 484. buffaloes. Indian J. Anim. Res., 37: 24-27. Chhikara, S.K., N. Singh, S.S. Dhaka, R.S. Yadav. Kundu, S., R.S. Pandey and S.K. Singh. 2003a. Non- 1995a. Effect of certain factors on calving genetic factors affecting some reproductive interval of Murrah buffaloes. Journal traits in Murrah buffaloes. Indian J. Anim.

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Sci., 73: 928-929. Kundu, S., R.S. Pandey and S.K. Singh. 2003b. Studies on some persistency traits in Murrah buffaloes. Indian J. Anim. Res., 37: 28-31. Patnaik, A.K.C. 1988. Body weight and its effect on fi rst lactation yield in Murrah. M.V.Sc. Thesis, Tamil Nadu Agricultural University, Coimbatore, India. Rao, A.V.N. and H.R.M. Rao. 1994. Peak yield as related to lactation traits in Murrah buffaloes. Indian Vet. J., 71:1207-1209. Report. 2006. Basic Animal Husbandry Statistics. AHS Series-10. Government of India. Department of Animal Husbandry, Dairying and Fisheries, New Delhi. pp. ix+162+14. Sethi, R.K. and M.S. Khatkar. 1997. Genetic parameters of production traits in Murrah buffaloes. Indian J. Anim. Sci., 67: 141- 142. Suresh, R., D.K. Bidarkar, B. Ramesh Gupta, B. Sudhakarrao and K. Sudhakar. 2004. Production and reproduction performance of Murrah buffaloes. Indian J. Anim. Sci., 74: 854-857. Ulaganathan, V., B. Seetharamasingh and V.K. Sinha. 1984. Factors affecting fi rst lactation characteristics of Murrah buffaloes, p. 4. In Proceedings of the Seminar on Buffalo Breeding. Madras, Tamil Nadu, India. Ulaganathan, V., K. Venkatarajulu and S. Balachandran. 1983. Estimation of breeding values in Murrah buffaloes by maximum likelihood method with adjustment for season of calvings. Tropical Vet. Anim. Sci. Res., 1: 33-36. Yadav, B.S., M.C. Yadav, A. Singh and F.H. Khan. 2007. Study of economic traits in Murrah buffaloes. Buffalo Bull., 26, 10-14.

300 Original Article Buffalo Bulletin (September 2014) Vol.33 No.3

EFFECT OF GENETIC AND NON-GENETIC FACTORS ON MORPHOMETRIC TRAITS OF BUFFALOES

Shashi Shankar 1, Dhirendra Kumar2 and K.G. Mandal3

ABSTRACT order of lactation had the lowest magnitudes of all the morphometric traits and signifi cantly (P<0.05) The present experiment was conducted increased upto third parity indicating that the on 60 randomly selected dairy units consisting of skeletal maturity of the buffaloes might be attained 116 graded Murrah, 70 Diara type and 121 non- at the age of 3rd parity. descript type buffalo cows utilizing the procedures of stratifi ed random sampling with proportional Keywords: morphometric traits, buffaloes, genetic allocation (Snedecor and Cochran, 1967) in and factors, non-genetic factors around Patna (Bihar). The aim of this investigation was to study the effect of genetic and non- genetic factors on various morphometric traits of INTRODUCTION buffaloes under farmer’s management system. The morphometric traits included in the study were Cattle and buffaloes are the main milk height at wither (haw, cm), body length (bl, cm) producing animals in India. The buffalo forms the and chest girth (cg, cm) as well as body weight of backbone of India’s dairy industry and is rightly the adult buffalo cows. The genetic factors were considered as the bearer cheque of the rural the three different genetic groups of buffaloes folk. Although being less than one third of the viz. graded Murrah, Diara and non-descript types total bovine population, the buffalo contributes prevalent in Bihar (India), whereas the non-genetic more than 50% of the total milk production in factors included in the study were location of the country. Although small buffalo herds are herd, farming system and sequence of lactation. distributed throughout the length and breadth of The farming system and the order of lactation had the state, the area under South and North gangetic signifi cant (P<0.05) infl uence on HAW, BL and CG plains of Bihar is densely populated with clusters whereas location of animals did not infl uence these of buffaloes. The majority of them are of non- traits. The animals managed under mixed farming descript type, some are graded Murrah and the rest system had signifi cantly (P<0.05) higher estimates of the population is phenotypically homogenous of morphometric traits than those maintained in the in certain distinguishable characteristics. These units involved dairying alone. The animals of fi rst

1Mahatma Gandhi Veterinary College (M.G.V.C.), Bharatpur, Rajasthan 321 001, India 2Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu (SKUAST-J), Jammu, India, E-mail: [email protected] 3Department of Animal Genetics and Breeding, Bihar Veterinary College, Patna-14, India

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phenotypically homogenous buffaloes are almost to door survey method. Altogether 920 buffaloes true to the breed and have acquired full adoption to consisting of 331 graded Murrah, 221 Diara and the socio-agro-climatic and ecological conditions 368 non-descript buffaloes were enumerated from of Gangetic plains, particularly in the “Tal and 145 dairy units located in and around Patna. Diara” area of the rivers Ganga, Gandak and Sone Out of 145 enumerated units, only 120 units in the division of Patna, Bhagalpur and Magadh. were respondent units which provided relevant These true breeding buffalo populations are information. Out of total 120 units, 50% i.e. 60 particularly called as “Diara” by the farmers. It dairy units, consisting of 430 buffaloes of different is pertinent to mention here that the true breeding genetic groups, were randomly selected utilizing buffalo population (about 1.3 million of the total the procedure of stratifi ed random sampling with buffalo population of the state) locally known as proportional allocation (Snedecor and Chochran, Diara/Deshila which contribute about 32% of the 1967). Data were recorded from buffaloes of State Milk Pool, is almost untouched, so far as defi ned genetic groups, which had completed at their breeding and development programmes are least one calving interval. The morphometric traits concerned, and hence the present study has been included in the study were height at wither (haw, undertaken. cm), body length (bl, cm) and chest girth (cg, cm) as well as body weight of the adult buffalo cows. The data were classifi ed on the basis of genetic MATERIALS AND METHODS group of the buffaloes, location of herd (Zone), farming system and lactation order to study the Buffaloes consisting of three genetic effect of various genetic and non-genetic factors groups namely graded Murrah, Diara and non- on the morphometric traits. descript types maintained in private dairy units at The experimental animals under study farmer’s door located in a radius of 15 km in and were classifi ed under three genetic groups: (i) around Patna were the experimental animals for the graded Murrah, (ii) Diara type (iii) non-descript present study. The whole area under study was type. divided into three distinct zones, which were as Performance records of the buffaloes were follows: classifi ed into four groups on the basis of sequence Zone – I North West Patna of lactation: 1st parity, 2nd parity, 3rd parity, 4th Zone – II South West Patna parity. The enumerated dairy units were grouped Zone – III East Patna according to the farming system adopted by the farmers which were as follows: The primary survey was conducted in i) Mixed farming (animal husbandry private dairy units popularly known as Khatals integrated with agriculture) located in a radius of 15 km in and around Patna. ii) Only animal husbandry. Those Khatals which consisted of at least two To quantify the variation due to various or more buffaloes consisting of graded Murrah, genetic and non-genetic factors on different Diara or non-descript buffaloes either alone or in economic traits, the data were subjected to least combination were enumerated, utilizing a door squares analysis (Harvey, 1966) for which the

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following mathematical model was utilized. estimate of height of wither for Diara buffaloes

YiJKlm = μ + Gi + Zj + Fk + Pl + eijklm reported by Singh et al. (2006) was in consonance with the result of the present study. Thus it may where, Yijklm = The value of mth individual under be concluded that graded Murrah, Diara and non- ith genetic group, Jth location, Kth farming system descript buffaloes in and around Patna (Bihar) were and lth parity. observed to be smaller and lighter than Murrah μ = The overall population mean. but heavier than Mehsana, Surti, Bhadawari and th Gi = The effect of i genetic group Nagpuri buffaloes. (i = 1,2,3). As evident from Table 1, the average height th Zj = The effect of j location of herd at wither was highest (130.456±0.183 cm) in the (j = 1,2,3). animals located in southwest Patna of the study th Fk = The effect of K farming system area followed by northwest (130.180±0.177 cm.) (K = 1,2). and east Patna (130.109±0.198 cm.). Farming system had a signifi cant (p<0.01) th Pl = The effect of l parity (l = 1,2,3,4). infl uence on height at wither. The animals reared

eijklm = The random error associated with under a mixed farming system integrated with individual which is randomly and agriculture had the higher estimate (130.513±0.157 independently distributed with mean cm) for HAW than those managed in the units 2 zero and variance σe exclusively involved in dairying by 0.53 cm (Table 1). The difference was statistically signifi cant The statistical signifi cance of various fi xed (p<0.05). Signifi cantly higher estimates of HAW effect was tested by the F test whereas DMRT, as in all the age groups for animals maintained in modifi ed by Kramer (1957), was applied to carry livestock research stations than those maintained out the pair wise comparisons among least squares under farmer’s management systems as observed means at the 0.05 and 0.01 levels of probability. by Singh (1995a) were in consonance with the fi ndings of the present study. Signifi cant (p<0.05) difference for HAW between animals managed RESULTS AND DISCUSSION under different farming systems might be attributed to the fact that animals managed under better The average height at wither of graded management systems may have better growth. Murrah was estimated to be 132.113±0.187 cm., The average HAW was found to be which differed signifi cantly (p<0.01) from Diara increased signifi cantly (p<0.05) and steadily and non-descript buffaloes by 2.295 cm and 3.299 from fi rst (124.391±0.197 cm) to third lactation cm, respectively. The mean values obtained in (133.952±0.213 cm) by 9.561 cm followed by slight the present investigation for all the three genetic decrease in fourth lactation. However, the difference groups viz. graded Murrah, Diara and non-descript in HAW between third and fourth parity was not types, were lower than those reported elsewhere statistically signifi cant. The signifi cant (p<0.01) for Murrah (Jawarkar and johar, 1975; Sreedharan, infl uence of parity on height at wither observed in 1976 and Manik and Iqbalnath, 1981).The average the present investigation was in accordance with

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the fi ndings of many workers (Jawarkar and Johar, a signifi cant (p<0.01) infl uence on body length. 1975; Saini and Gill, 1986; Jogi and Patel, 1990; The animals managed in the units integrated with Singh et al., 1995. agriculture farming had signifi cantly (p<0.05) higher mean body length (137.003±0.197 cm) in Body length comparison to those involved in the dairying alone Overall least squares mean for body by 1.151 cm (Table 1). Differences in body length length pooled over three different genetic groups were also observed by Singh et al. (1995a) in all of buffaloes viz. Diara, graded Murrah and non- the age groups of Mehsana buffaloes managed in descript types included in this study, was reckoned LRS herd from those managed in milk union sheds to be 136.478±0.138 cm (Table 1). under farmers management systems which supports Genetic group had a highly signifi cant the fi nding of the present study. (p<0.01) effect on body length. As revealed from The lactation order had highly signifi cant Table 1, the graded Murrah had the longest body (p<0.01) effect on body length. The lowest average length (138.598±0.235 cm) followed by Diara body length pooled overall the three genetic groups (135.507±0.232 cm), and non-descript had the viz. graded Murrah, Diara and non-descript types, shortest body length (135.328±0.231 cm). The was estimated to be 130.947±0.247 cm in the fi rst average body length of graded Murrah differed parity and then increased signifi cantly (p<0.05) signifi cantly (p<0.05) from Diara and non-descript over the lactations. The highest average body types. The average estimates for body length in all length (140.007±0.268 cm) was observed in third the three genetic groups obtained in the present parity, which was signifi cantly (p<0.05) longer than study were lower than the body length of Murrah the average body length observed during fi rst and buffaloes as reported elsewhere by Jawarkar and second parity by 4.279 and 9.06 cm, respectively. Johar (1975), Sreedharan (1976) and Manik and The average body lengths during third and fourth Iqbalnath (1981). The signifi cant (p<0.05) effect order of lactations were also signifi cantly (p<0.05) of genetic group on body length reported by Sinha longer than in second parity by 4.781 and 4.505 (2006) and Singh et al. (2006) was in agreement cm, respectively. However, though the body length with the fi ndings of the present study. in fourth parity was little bit shorter than in third As evident from Table 1, the average body parity, the two did not differ signifi cantly. length was found to be highest (136.796±0.230 cm) Signifi cant infl uence of parity on body in the animals located in southwest Patna of the length as observed in the present study has also study area followed by northwest (136.384±0.22 been reported in Murrah buffaloes by Jawarkar cm) and lowest in east Patna (136.253±0.249 cm). and Johar (1975), Saini and Gill (1986), Jogi and The least squares analysis of variance revealed Patel (1990) and in Mehsana buffaloes by Singh et non-signifi cant effect of zone on body length. The al. (1995) and Singh et al. (2000), who stated that non-signifi cant effect of location on body length as body length in buffaloes increased signifi cantly up observed in the fi ndings of the present study is in to third parity. agreement with the results reported by Singh et al. (1995) in Mehsana buffaloes. Chest girth The farming system was found to have The overall least squares mean for chest

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girth pooled over the three different genetic groups under the farmers’ management system. of buffaloes, viz. Diara, graded Murrah and non- The order of lactation had highly signifi cant descript, included in this study was estimated to be (p<0.01) infl uence on chest girth in buffaloes in and 195.692±0.384 cm (Table 1). around Patna. The average chest girth pooled over Genetic group had highly signifi cant all the three genetic groups viz. graded Murrah, (p<0.01) infl uence on chest girth.. As evident Diara and non-descript buffaloes was found to be from Table 1 the graded Murrah had the largest the lowest (186.467±0.688 cm) at fi rst parity. chest girth (199.153±0.653 cm) followed by non- The average estimates of chest girth in descript (196.640±0.6421 cm) and Diara buffaloes second, third and fourth parities were observed to (191.282±0.646 cm). The chest girth in Diara increase signifi cantly (p<0.05) over the average buffaloes was found to be signifi cantly (p<0.05) chest girth at fi rst parity by 7.85, 15.362 and lower than the graded Murrah and non-descript 13.687 cm, respectively. A signifi cant effect of buffaloes by 7.871 and 5.358 cm, respectively. parity on chest girth is also reported by Jawarkar The fi ndings of the present study were and Johar1975), Saini and Gill (1986), Jogi and not in agreement the fi ndings of Jawarkar and Patel (1990) in Murrah and (Singh et al. (1995) and Johar (1975), Sreedharan (1976) and Manik and Singh et al. (2000) in Mehsana buffaloes. Iqbalnath (1981) who reported higher estimates of chest girth in Murrah buffaloes. As evident from Table 1, the highest average SUMMARY chest girth was found to be 196.953±0.641 cm in the animals located in southwest Patna of the study Graded Murrah had signifi cantly (P<0.05) area followed by northwest Patna (195.257±0.619 higher estimates of all the morphometric traits cm) and lowest in east Patna (194.866±0.693 cm). (HAW, BL and CG) than the Diara and non-descript The least squares analysis of variance revealed non- types, whereas, Diara buffaloes had superiority signifi cant effect of zone on chest girth. The non- over non-descript types for height at wither and signifi cant effect of location on chest girth reported chest girth. The average estimates of height at by Singh et al. (1995a) in Mehsana buffaloes is in wither, body length and chest girth of graded agreement with the results of the present study. Murrah were 132.113+0.187, 138.598+0.235 and The farming system was found to have 199.153+0.0653 cm respectively. Therefore, the highly signifi cant (p<0.01) infl uence on chest size of Diara buffaloes was in between Graded girth. The animals managed in the units integrated Murrah and Non-descript types. The farming with agriculture farming had signifi cantly (p<0.05) system and the order of lactation had signifi cant higher average chest girth (197.584±0.549 cm) in (P<0.05) infl uence on HAW, BL and CG whereas comparison to those involved in dairying alone location of animals did not infl uence these traits. by 3.785 cm (Table 1). Similar fi ndings have also The animals managed under mixed farming system been reported by Singh et al. (1995) who observed had signifi cantly (P<0.05) higher estimates of signifi cant difference in heart girth among all the morphometric traits, than those maintained in the age groups of Mehsana buffaloes between LRS units involved dairying alone. herd and milk union sheds where animals were

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Table 1. Least squares means±SE and CV% of morphometric traits of buffaloes in and around Patna.

Morphometric traits (cm) Particulars HAW (Mean±S.E.) B.L. (Mean±S.E.) C.G. (Mean±S.E.) 130.248±0.110 136.478±0.138 195.692±0.384 Overall Mean (μ) (1.47) (1.77) (3.43) Factors Genetic group 132.113a±0.187 138.598a±0.235 199.153a±0.653 Graded Murrah (1.41) (1.69) (3.27) 129.818b±0.185 135.507b±0.232 191.282b±0.646 Diara (1.42) (1.71) (3.41) 128.814c±0.184 135.328b±0.231 196.640c±0.642 Non-descript (1.42) (1.70) (3.34) Location 130.180±0.177 136.384±0.222 195.257±0.619 1. Northwest Patna (1.44) (1.72) (3.35) 130.456±0.183 136.796±0.230 196.953±0.641 2. Southwest Patna (1.44) (1.73) (3.35) 130.109±0.198 136.253±0.249 194.866±0.693 3. East Patna (1.44) (1.72) (3.35) Farming System 1. Animal Husbandry 129.983a ±0.149 135.952b±0.187 193.800a±0.520 alone (1.45) (1.73) 130.513b±0.157 137.003a±0.197 197.584b±0.549 2. Mixed Farming (1.46) (1.74) Lactation order 124.391a ±0.197 130.947a±0.247 186.467a±0.688 1st (1.49) (1.77 (3.48) 128.907b ±0.188 135.226b±0.236 194.317b±0.657 2nd (1.42) (1.70) (3.31) 133.952c ±0.213 140.007c±0.268 201.829c±0.745 3rd (1.38) (1.66) (3.31) 133.743c±0.273 139.731c±0.343 200.154c±0.955 4th (1.38) (1.66) (3.23)

Means with different superscripts (column-wise) differed signifi cantly (P<0.05) Values in parentheses are CV%

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CONCLUSION different physical characteristics in Murrah type heifers and dry buffaloes. Indian J. The animals of fi rst lactation had the lowest Anim. Prod. Mgmt., 3(4): 193-199. magnitudes of all the morphometric traits, and these Singh, D.V., R.K. Pundir, G. Sahana, A.S. Dave magnitudes signifi cantly (P<0.05) increased upto and A.E. Nivsarkar. 2000. Characterization third parity indicating that the skeletal maturity of Mehsana buffaloes. NBAGR Research of the buffaloes might be attained at the age of 3rd Bulletin, 8: 20-27. parity. On the basis of the fi ndings of the present Singh, D.V., V.N. Tripathi and A.S. Dave. 1995. study it can be concluded that the Diara buffaloes Studies on morphological traits in Mehsana were signifi cantly different from Graded Murrah buffalo cows. Indian J. Dairy Sci., 48(1): and non-descript types in respect to morphometric 33-38. traits;therefore, work of a similar type may be Singh, D.V., V.N. Tripathi and A.S. Dave. 1995a. repeated in the entire Tal and Diara areas of the Body measurements in female Mehsana river Ganges, Gandak and Sone pertaining to Bihar buffaloes. Indian J. Dairy Sci., 72: 1282- to identify and enumerate the number of Diara 1285. buffaloes so that a suitable breeding plan can be Singh, S.R., K.G. Mandal, P.K. Singh, R. Kumar chalked out for improvement of Diara buffaloes and R.N. Singh. 2006. Phenotypic parameter and to improve the livelihood of the dairy farmers of true breeding buffalo population in Tal in the state. and Diara areas in Bihar. In Proceedings of National Symposium on Conservation and Improvement of Animal Genetic Resources REFERENCES under Low Input System : Challenges and Strategies, NBAGR, Karnal, Haryana, Harvey, W.R. 1966. Least Squares Analysis of Data India. with Unequal Subclass Number. United Sinha, R.K. 2006. Characterization of buffalo States Department of Agriculture (USDA). genetic resources in Tal and Diara areas in Jawarkar, K.V. and K.S. Johar. 1975. A study on and around Barh (Patna). M.V. Sc. Thesis, some of the body measurements on Murrah RAU, Pusa (Samastipur), Bihar, India. buffaloes. Indian J. Dairy Sci., 28(1): 54- Snedecor, G.W. and W.G. Cochran. 1967. Statistical 56. Methods, 6th ed. Iowa State Univ. Press, Jogi, R.V.K. and D. Patel, U.G. 1990. Various body Ames, USA. measurements and their correlation with Sreedharan, S. 1976. Inheritance of body size milk yield and fat percentage. II Studies on measures in relation to production Surti buffaloes. Buffalo Bull., 9(2): 35-38. effi ciency in buffaloes. Ph.D. Thesis, Punjab Manik, R.S. and Iqbalnath. 1981. Relationship University. of certain body measurements with milk production in Murrah buffaloes. Indian J. Dairy Sci., 34(1): 118-119. Saini, A.L. and R.S. Gill. 1987. Relationship among

307 Original Article Buffalo Bulletin (September 2014) Vol.33 No.3

OBSERVATIONS ON BUFFALO SARCOPTIC MANGE IN JAMMU, INDIA

T. Nazir1, R. Katoch1, R. Godara1,*, Anish Yadav1 and Vijay Pandey2

ABSTRACT INTRODUCTION

Observations on sarcoptic mite infestation Mite infestation, particularly with the in 1440 buffaloes belonging to 72 unorganised obligate, burrowing mite Sarcoptes scabiei, is an dairy farms of Jammu revealed a 3.33% prevalence extremely pruritic, contagious and debilitating rate. Prevalence was signifi cantly (p≤0.05-0.01) disease of buffaloes (Naresh et al., 2005; Chhabra higher during the winter (5.55%) as compared and Pathak, 2011). It is recognized as one of the with the summer (2.91%) and the rainy (1.33%) most serious diseases of dairy animals in the seasons. The disease was recorded in 3.75% and Indian subcontinent (Gill et al., 1989). The disease 2.5% of buffaloes up to 1 year and above 1 year of spreads rapidly in dairy and breeding herds due to age, respectively. Females showed a signifi cantly overcrowding, malnutrition and poor managemental (p<0.05) higher prevalence rate (4.07%) than conditions. Though economic losses must be of a males (1.96%). A strong negative correlation very high magnitude due to hide damage, decreased (r=-0.725) was observed between the prevalence milk and meat production, morbidity and mortality, rate and temperature whereas relative humidity had no accurate fi gures are available. The morbidity rate little or no association (r=0.2). The initial lesions varies from 1.5 to 82%, and reaching up to 100% in were observed on the face and the upper part of severely affected herds, with heavy losses of young neck as papules and exudative crusts, amd then animals (Gill et al., 1989; Patel et al., 2002a). the infestation spread quickly causing generalized Despite this, no epidemiological study vis-à-vis mange. In severe cases, animal had emaciated body the incidence of sarcoptic mange in buffaloes was with no sign of fat deposits and loss of normal skin undertaken previously in the state of Jammu and colour and texture. Infested buffaloes showed a Kashmir of India. The present work was therefore signifi cant (p<0.05) association with inappetance. conducted to determine the prevalence and clinical signs associated with sarcoptic mite infestation Keywords: buffaloes, Sarcoptes scabiei, in buffaloes (Bubalus bubalis) and the effect of prevalence, Jammu climatic factors on the prevalence of sarcoptic

1Division of Veterinary Parasitology, Faculty of Veterinary Sciences and Animal Husbandry, Sher-e-Kashmir University of Agricultural Sciences and Technology, R.S. Pura-181 102, Jammu, India, *E-mail: godara1972@ gmail.com 2Department of Biochemistry, Veterinary College Mathura, Pandit Deen Dayal Upadhaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan, Mathura, India

308 Buffalo Bulletin (September 2014) Vol.33 No.3

mange at Jammu, India. RESULTS

Observations on the prevalence of sarcoptic MATERIALS AND METHODS mange During the year a total of 1440 buffaloes Study area, animals and techniques belonging to 72 unorganised dairy farms of The study was carried out in 1440 Jammu were examined. Out of that total, 3.33% of buffaloes belonging to 72 unorganised dairy farms buffaloes were found to be infested with S. scabiei of Jammu of Jammu and Kashmir state, located var. bubalis (Table 1). The highest prevalence was in north-western region of India, between 74o 50’ found in the month of February (7.5%), followed east longitude and 30o 40’ north latitude. The area by March (6.66%) and December (5.0%). The lies about 332 m above sea level and experiences lowest prevalence (0.83%) was found in the month a subtropical humid climate. The area experiences of May (Figure 1). None of the animals examined four seasons; summer (March to June), rainy (July were found positive during the months of June and to September), post-rainy (October-November) July. Signifi cant association (p≤0.05-0.01) was and winter (December to February). found between the prevalence rate and the seasons Skin scrapings of buffaloes with visible and the highest prevalence rate (5.55%) was dermatitic lesions were collected using the found in the winter season (Table 1). There was method given by Tikaram and Ruprah (1986). a strong negative correlation (r=-0.725, r2=0.525) These samples were brought to the laboratory and between the prevalence rate and the mean monthly examined under stereomicroscope using standard temperature (Figure 2) and a weak positive techniques described elsewhere (Soulsby, 1982). correlation (r=0.2, r2=0.04) was found between The data were recorded according to the age (up to the prevalence rate and mean monthly relative 1 year and above 1 year) and sex (male/ female). humidity (Figure 3). Sarcoptic mange infestation was observed in 3.75% and 2.5% buffaloes up to Statistical analysis 1 year and above 1 year of age, respectively (Table Differences in the prevalence of mite 1). However, the infestation was not signifi cant. between seasons, age groups and sexes were The females had a signifi cantly (p<0.05) higher tested for signifi cance by the chi-square test. The prevalence rate (4.07%) than the males (1.96%) relationship between the monthly variation of (Table 1). the infection index and the one of the climatic parameters: temperature and relative humidity were Clinical observations determined by Pearson correlation analysis using The initial lesions were often localized SPSS 16.0. A p-value of ≤0.05 was considered accompanied by alopecia and were found on the signifi cant. body parts which had thin hair and were prone to scratches/ injuries such as the face and the upper part of the neck. Small papules with intense itching were evidenced as a result of early infl ammatory reactions to the mites’ invasion and injury to the

309 Buffalo Bulletin (September 2014) Vol.33 No.3

Figure 1. The prevalence (%) of sarcoptic mange in buffaloes from January 2010 to December 2010.

Figure 2. Scatter graph showing a strong negative correlation (r=-0.725, r2=0.525) between the prevalence rate (%) of sarcoptic mange and mean monthly temperature (oC).

310 Buffalo Bulletin (September 2014) Vol.33 No.3

Figure 3. Scatter graph showing a weak positive correlation (r=0.2, r2=0.04) between the prevalence rate (%) of sarcoptic mange and mean monthly relative humidity (%).

Figure 4. The prevalence (%) of sarcoptic mange on buffaloes in respect of site locations.

311 Buffalo Bulletin (September 2014) Vol.33 No.3 960 36 3.75 480 12 2.5 q

932 38 4.07 p 0.05-0.01). ≤ Male Female to 1 year Up 1 year Above cantly (p cantly fi on buffaloes in respect of season, sex and age. on buffaloes 88 2 2.27 152 6 3.94 160 6 3.75 80 2 2.5 117 0 0 243 6 2.46 240 6 2.5 120 0 0 144159 5 3 3.47 1.88 216 321 15 11 6.94 3.42 240 320 14 10 5.83 3.12 120 160 6 4 5 2.5 examined infested percent examined infested percent examined infested percent examined infested percent a b b ab Percent Sarcoptes scabiei Sarcoptes No. infested No. 240 8 3.33 examined Total 1440 48 3.33 508 10 1.96 rainy Post- Rainy 360 6 1.66 Winter 360 20 5.55 Season Summer 480 14 2.91 Table 1. The prevalence of 1. Table Figures with different superscripts differ signi superscripts differ Figures with different

312 Buffalo Bulletin (September 2014) Vol.33 No.3

skin, which leads to rubbing of the affected parts The occurrence of sarcoptic mange is with manger or the walls. The infestation spread usually considered to be seasonal and is often quickly from these localized lesions causing restricted to a few months of the year (during winter generalized mange. Although the skin lesions were and early summer months) (Tikaram and Ruprah, found all over the body, the neck (47.5%) and the 1986; El-Khodery et al., 2010). Probably, with face (39.6%) were the most affected parts (Figure increase in the mean temperature during summer 4). However, some animals had the lesions on two months, the mite numbers decrease rapidly due to sites together (Figure 4). The lesions appeared as less active feeding and decreased oviposition by exudative crusts, scales, haemorrhagic and non- the mites (Soulsby, 1982). Further the maximum haemorrhagic fi ssures. A decrease in feed intake survival of both sarcoptic and psoroptic mites and a rapid fall in milk yield in lactating buffaloes has been recorded at 20-27oC rather than at 31- were prominent features of the infestation. In severe 39oC (Maske and Ruprah, 1981). During the hot cases, excess keratinization and thickening of skin summer months, the disease is recognized as were observed, which resulted in corrugations subacute (dormant or quiescent) or chronic with or folds often spread over with a fi ne, chalk-like focal lesions mainly in the groin and submaxillary covering of scurf. Severely infested buffaloes had regions. During this quiescent phase a spontaneous emaciated bodies with no sign of fat deposits. The cure has occurred. Since the more severe lesions characteristic signs of the infestation were loss of may subside, itching and pruritus are relieved and normal skin colour and texture. new hair growth starts. These quiescent lesions are The infested buffaloes showed a signifi cant potential sources of clinical infection and the risk (p<0.05) association with inappetance. Out of 48 of a sudden outbreak of mange within a herd with infested buffaloes, 29 (60.41%) showed inappetance the change of season poses a serious threat. while 19 (39.58%) had normal appetite. The young animals showed a higher infestation rate of S. scabiei than the adults. This could be attributed to soft and tender skin, dense DISCUSSION hair coat, huddling tendency and relatively low level of immunity in young animals (Patel et al., 2003a). Sarcoptic mange is one of the few diseases However, Cernea and Cernea (2006) concluded that seriously threaten the well-being of the water that the mange-mite infestation develops in adult buffalo (Bubalus bubalis). A higher incidence rate animals irrespective of sex with higher prevalence of 27.6% of sarcoptic mange in buffaloes has been of psoroptic mange at 5-13 years of age. Although reported from organised dairy farms of Haryana male and female animals are kept under similar state of India (Tikaram and Ruprah, 1986). El- managemental conditions, a signifi cantly (p<0.05) Khodery et al. (2010) observed Psoroptes and higher infestation rate in females than the males Chorioptes spp. infestation in 16.66% buffaloes suggests that differences in susceptibility between of smallholder farms in the Nile Delta region of sexes may exist. Egypt. As against the above observations, there is In mite infestation, host reactions such as an incidence rate of 3.33% currently recorded in cutaneous infl ammation and intense pruritus are Jammu. associated with response to the mites’ burrowing

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and feeding activities, which usually occur three On the basis of the results of the study, it may weeks after the initial infestation (Colebrook and be concluded that the season and sex are important Wall, 2004; Rahbari et al., 2009). The reaction time and signifi cant predisposing factors for sarcoptic is longer accompanied by severe itching during mange in buffaloes. The application of suitable early stages of infestation when the mite numbers acaricides would help to reduce the infestation and are low. Subsequently, the reaction time becoming to sustain the optimal growth and productivity of much shorter after repeated exposures (Gary buffaloes and minimise the perpetuating fi nancial and Durden, 2009). The induction of cutaneous losses to resource-poor communities in the region. hypersensitivity in response to mite faecal antigens may cause proliferation of mast cells and resultant increase in chymase and tryptase activities (Noviana REFERENCES et al., 2004) as well as decrease in anti-oxidant enzyme activities and trace minerals suggesting Cernea, C. and M. Cernea. 2006. Establishing its association with compromise in anti-oxidant the causal factors in the development of defense and resulting in oxidative stress (Dimri et psoroptic mange in buffalo from S.C.D.C.B. al., 2008); these phenomena are supposed to play Sercaia, Brasov District. Lucrari Stiintifi ce, an important role in development of pathogenicity. 40: 349-354. Itching may develop as a result of histamine that is Chhabra, M.B. and K.L.M. Pathak. 2011. Sarcoptic released from the destroyed cells and that is a potent mange in domestic animals and human pruritogen (Greaves and Wall, 1996). The rubbing scabies in India. J. Vet. Parasitol., 25: 1-10. and scratching of the affected areas in an attempt to Colebrook, E. and R. Wall. 2004. Ectoparasites of alleviate the itchiness, result in the shedding of mites livestock in Europe and the Mediterranean in the surroundings for subsequent transmission to region. Vet. Parasitol., 120: 251-274. other animals. Excessive rubbing and scratching Dimri, U. and M.C. Sharma. 2004b. Effects of leads to excoriation and haemorrhage on the skin sarcoptic mange and its control with oil of surface and even death, particularly in animals Cedrus deodara, Pongamia glabra, Jatropha below one year of age when the lesions are large curcas and benzyl benzoate, both with and (Patel et al., 2002a). without ascorbic acid on growing sheep: The mite infestation is often accompanied assessment of weight gain, liver function, by inappetance, and as a result, loss of weight gain nutrient digestibility, wool production and may occur (Rahbari et al., 2009). El-Khodery et al. meat quality. J. Vet. Med. A, 51: 79-84. (2010) reported a signifi cant (p<0.01) inappetance Dimri, U., S.C. Sharma, D. Swarup, R. Ranjan and in buffaloes infested with Psoroptes spp. mites. M. Kataria. 2008. Alterations in hepatic lipid Inappetance in mite infestation may develop as a peroxidase and antioxidant profi le in Indian result of alteration in hepatic function and structure water buffaloes suffering from sarcoptic and decrease in digestibility of nutrients (Dimri mange. Res. Vet. Sci., 85: 101-105. and Sharma, 2004b). Damage of hepatic tissues in El-Khodery, S.A., S.A. Osman, M. Ishii and M.H. cattle calves infested with Psoroptes ovis has also Al-Gaabary. 2010. Risk factors of infestation been reported (Fisher and Crookshank, 1982). by Psoroptes spp. mites in buffalo (Bubalus

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bubalis) at smallholder farms in the Nile Rahbari, S., S. Nabian and A.R. Bahonar. 2009. Delta region, Egypt. Trop. Anim. Health Some observations on sheep sarcoptic Pro., 42: 275-281. mange in Tehran province, Iran. Trop. Anim. Fisher, W.F. and H.R. Crookshank. 1982. Effects Health Pro., 41: 397-401. of Psoroptes ovis (Acarina: Psoroptidae) on Soulsby, E.J.L. 1982. Helminths, Arthropods and certain biochemical constituents of cattle Protozoa of Domesticated Animals, 7th ed. serum. Vet. Parasitol., 11: 241-251. ELBS and Bailliere Tindall, London, UK. p. Gary, R.M. and Durden. 2009. Medical and 1-809. Veterinary Entomology, 2nd ed. Elsevier Tikaram, S.M. and N.S. Ruprah. 1986. Incidence Publication, Amsterdam. p. 469-470. of sarcoptic mange in buffaloes in India. Gill, B.S., J. Singh, B.S. Gill, A. Singh, S.S. Trop. Anim. Health Prod., 18: 86-90. Khehra, A. Rai and O. Hussain. 1989. Effi cacy of ivermectin against mange and gastrointestinal nematodes of buffalo (Bubalus bubalis). Vet. Parasitol., 31: 141- 147. Greaves, N.W. and P.D. Wall. 1996. Pathophysiology of itching. Lancet, 348: 938-940. Maske, D.K. and N.S. Ruprah. 1981. Note on in vitro survival of psoroptic mange in buffaloes at different temperature and relative humidities. Indian J. Anim. Sci., 51: 563-564. Naresh, R., D. Swaroop, M.C. Sharma and R. Ranjan. 2005. Clinical management of sarcoptic mange in Indian buffalo calves with the botanical ointment. Vet. Rec., 156: 684-685. Noviana, D., D. Harjanti, Y. Otsuka and Y. Horri. 2004. Proliferation of protease-enriched mast cells in sarcoptic skin lesions of racoon dogs. J. Comp. Pathol., 131: 28-37. Patel, J.S., P.R. Patel and H.H. Panchasara. 2002a. Economic losses due to sarcoptic mange in buffalo calves. Vet. Pract., 3: 186-189. Patel, J.S., P.R. Patel, H.H. Panchasara and K.G. Brahmaxtri. 2003a. Epizootiology of sarcoptic mange in buffalo calves. Indian Vet. J., 80: 972-974.

315 Original Article Buffalo Bulletin (September 2014) Vol.33 No.3

BUFFALO MILK MARKETING BY THE GUJJARS (TRIBALS) IN THE JAMMU AND KASHMIR STATE IN INDIA

Tarunvir Singh1 and Sudhakar Dwivedi2

ABSTRACT is actually marketed. Marketed surplus may be less, equal or more than the marketable surplus The present investigation is an attempt to depending upon the external factors operating on study the marketing of buffalo milk produced by a the market economy. well-known pastoral community-the Gujjars of the It is often argued that marketed surplus of a Jammu and Kashmir state. The study was undertaken commodity is a function of the level of production in the Jammu region. About 200 households were and consumption of the commodity. Eventually studied in different districts of the Jammu region. large producers would market more both in Marketed surplus of milk and factors affecting absolute terms as well as in relative terms. But in it, marketing cost, margin and price spread were the case of milk, being a perishable commodity, studied. The Cobb-Douglas production function sometimes there is distress sale, particularly among was used to study the relationships between small and marginal farmers, who will have a cash marketed surplus of milk and factors affecting it, crunch unless adequate market infrastructure is and it was found that milk production and the price provided, and it is often seen that the majority of of milk had a positive and signifi cant impact on large producers sell proportionately less milk than the marketed surplus of milk. The producer’s share what they had produced. Thus, farms, unless they in the consumer’s price was maximum in the fi rst happen to be specializing in dairy farming, do not channel (Producer- Consumer). enter the market in a big way. Because of higher home consumption of milk by large producers, what Keywords: marketed surplus, marketing margin, is just non-marketing level for them is suffi ciently marketing cost, price spread a specialized market level for small or marginal farmers or landless labourers (Singh, 1978). The retention capacity of the producer depends upon INTRODUCTION his fi nancial position, the credit facilities available to him and the price level in the market. The proper The excess of output over sectoral retention incentives and adequate market infrastructure for refers to what is termed as marketable surplus. This the excess production of milk will not only help in is distinct from marketed surplus. which represents increasing milk production, but also increase the only that portion of the marketable surplus which marketed surplus. The marketed surplus is mainly governed by production, domestic consumption, 1Institute of Economic Growth, New Delhi, India, E-mail: [email protected] 2Division of Agricultural Economics and Statistics, Sher-e-Kashmir University of Agricultural Sciences and Technology- Jammu, 180009, India

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price and market infrastructure. based on the classifi cation into three groups on In order to provide the minimum the basis of number of milch buffaloes (cube root nutritional requirements of milk to the majority frequency method) namely small farms (up to 5), of population, the marketed surplus has to be medium farms (6-10) and large farms (10 & above). increased substantially. The disposal of milk in a Primary data on marketing of milk were collected milk producing household consist of liquid milk by the personal interview method with the help of consumption and conversion of milk into various a structured and pre-tested schedule. milk products at the producer household, and The quantity of milk available for sale the balance is available for sale of milk, which depends upon a number of factors like production, constitutes as marketed surplus. price demand, etc. Milk being a perishable cannot The present investigation was an attempt be stored for future date for sale and therefore, to study the marketing aspects of dairying of one of depend upon immediate demand. Although a the well-known pastoral communities-the Gujjars large number of studies have been carried out to of the Jammu and Kashmir state in India. Though develop marketed surplus functions in agricultural information on sociological and anthropological commodities, little empirical evidence is available aspects of these pastoral communities has been on the marketed surplus of milk. Therefore, in this generated in various studies elsewhere, socio- section the empirical nature of relationship between economic studies are scanty. No concerted efforts the marketed surplus of milk and its determinants had heretofore been made to study the economics of was examined. The explanatory variables dairying among the Gujjars, who are well known for considered to develop the marketed surplus function rearing buffaloes only, particularly with reference were total milk production, family size, education 1 to various important economic parameters like level of the head of the family , number of milch marketing of milk. With a view to identifying the animals and price of milk. The education status factors responsible for marketed surplus of milk, (literate or illiterate) of the head of the household marketed surplus functions were fi tted separately was considered as a variable affecting the marketed for different types and categories of milk producers surplus of milk. A critical examination of zero order in the study area. correlation matrix among explanatory variables revealed the absence of multicollinearity problem. The estimated marketed surplus function for milk MATERIALS AND METHODS in respect to Jammu, Udhampur, Rajouri, Poonch and overall are reported in Table 1. Four districts of the Jammu region were The following model was employed to fi nd selected purposively and from each district two the marketed surplus for different households in the blocks were also selected purposively. One village present study. MS = f (X , X , X , X , X ) was selected purposively from each block:that in 1 2 3 4 5 which the population of the Gujjars involved in MS = Marketed surplus of milk per rearing of buffaloes was highest. From each block household per day in kg X = Total milk production per household 25 Gujjar dairy farmers were selected randomly 1 to constitute a total sample size of 200 farmers, in kg

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X2 = Family size of household in Jammu, Udhampur, Rajouri, Poonch districts 2 X3 = Education level of head of household and the overall Jammu region with R values at

X4 = Average price of milk 0.956, 0.925, 0.996, 0.917 and 0.969 respectively,

X5 = Number of milch animals per was statistically signifi cant meaning that about 95 household percent, 92 percent, 99 percent, 92 percent and 96 percent, respectively, of the total variation in milk 1The scale prepared by Trivedi and Pareek production in the respective districts was explained (1963) to measure the education level of rural by the independent or explanatory variables under families was used, the details of which are given consideration. below: The functional analysis revealed that milk Education Weightage production was found signifi cant at the 1 percent Illiterate 0 level of probability in Jammu, Udhampur, Rajouri Can read only 1 and Poonch districts and the overall Jammu region. Can read and write 2 The value of regression coeffi cients for milk Primary 3 production were 1.101, 0.971, 0.924, 1.068 and Middle 4 0.979, respectively. The regression coeffi cient for High School 5 price of milk was signifi cant and positive in Jammu Intermediate and above 6 district at the 5 percent level of signifi cance with the value at 0.193, whereas in the overall Jammu To work out the marketing cost and price region price of milk was found statistically spread during the marketing of milk, the lot method signifi cant at the 5 percent level of signifi cance was used. A specifi c lot or consignment was selected with values at 0.138. The values for family size in and chased through the marketing system until it Rajouri (0.016) and Poonch (0.015) districts, for reached the ultimate consumer. The cost and margin education status in Jammu (0.002), Rajouri (0.001) involved at each stage was assessed (Acharya and Poonch (0.001) and overall (0.083) and for number Aggarwal, 1998). This method is appropriate for of milch animals in Udhampur (0.039), Rajouri such perishable commodities as fruits, vegetables (0.083) and overall (0.059) were, however, positive and milk because the lag between the time the but not signifi cant. commodity enters the marketing system and time The results of marketed surplus function of its fi nal consumption is very small. (Table 1) showed that the values of regression coeffi cients of milk production had a positive and signifi cant impact on the marketed surplus of RESULTS AND DISCUSSION milk in all the four districts and the overall Jammu region indicating thereby that one percent increase Marketed surplus function for buffalo milk in the use of this inputs after keeping the use of all A perusal of Table 1 indicates that the other inputs constant, the marketed surplus could production function for marketed surplus of milk increase by 1.101 percent in the case of Jammu (variables included in the regression model explain district, 0.971 percent in the case of Udhampur more than 91 percent of the total variation for milk) district, 0.924 percent in the case of Rajouri,

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1.068 percent in the case of Poonch district and to be 289, 363, 469 and 335 gm in small, medium, in the case of overall Jammu region, the increase large and overall farms, in Jammu Region. in marketed surplus of milk was 0.979 percent. This shows that in Jammu region overall This clearly implies that as the milk production production of milk was 34.85 litre per household increases, there will be more marketed surplus of per day, in which large farms had the more milk. The quantity of marketed surplus of milk also production per day as compared to other groups, increases with the increase in the price of milk in which was due to the large number of milking the case of Jammu district and the overall Jammu animal kept by large farms in Jammu district. region, where the price of milk shows a positive These fi ndings are supported by the fi ndings of and signifi cant impact on the marketed surplus of Prabhakaran and Ramasamy (1984) and Kumar the milk. In the other districts; the sign for the price (2006). The marketed surplus of milk per day per of milk was however positive but did not show household was found to be 33.29 litres. The overall any signifi cant impact. The other variables like marketed surplus measured in terms of ratio to total family size did not show any signifi cant impact on milk production was 95.53 percent for the overall the marketed surplus. However, their signs were Jammu region, which was highest in large farms. negative in some cases, which clearly indicated that With the increase of herd size, marketed surplus of as the family size increases, the marketed surplus milk also increased, which was observed in all the of milk will decrease. The effect of educational districts of the overall Jammu region. More milk status of the head of the household did not have was retained by small farms and medium farms any signifi cant infl uence on the marketed surplus than large farms due to big family size. The per as most of the heads of household were illiterate. capita consumption of milk in Jammu region was All the variables included in the regression model found to 335 gm per day. explained more than 90 percent of variation. These fi ndings are supported by the fi ndings of Dhaka et Marketing cost and price spread in buffalo milk al. (1998) and Das (2004). marketing The following channels were usually Production, consumption and marketed surplus found for the distribution of milk from producer to of buffalo milk in Jammu region consumer in the Jammu region. Production as well as marketed surplus was Producer - Consumer, highest in large farms followed by medium farms Producer - Vendor - Consumer and small farms (Table 2). On average, the daily Producer - Vendor - Processor (sweet shop) milk production per house hold in different groups In the fi rst channel, either consumers was 21.44, 37.13, 85.21 and 34.85 litres for small, themselves go to take milk from the producers’ medium, large and overall farms, respectively, of houses or producers themselves go to the houses of which 20.25, 35.34, 82.35 and 33.29 litres was sold, consumers to sell the milk. In the second channel, a representing marketed surpluses of 94.45, 95.18, vendor collects milk from the producer and supplies 96.64 and 95.52 percent on small, medium, large it to the consumers. In the third channel, the vendor and overall farm sample households in the Jammu collects the milk from the producers and supplies it region. The per capita milk consumption was found to the processor (sweet shop) for making different

319 Buffalo Bulletin (September 2014) Vol.33 No.3

Table 1. Marketed surplus function for milk for the Jammu region.

Regression Coeffi cients No. of Constant Milk Family Education Price of Districts milch R2 Term production size status milk animals (X1) (X2) (X3) (X4) (X5) 1.101** -0.025 0.002 0.193* -0.073 Jammu -0.845 0.956 (0.049) (0.021) (0.001) (0.091) (0.051) 0.971** -0.013 0.001 0.008 0.039 Udhampur -0.007 0.925 (0.037) (0.014) (0.001) (0.044) (0.040) 0.924** 0.016 0.001 0.023 0.082 Rajouri 0.006 0.996 (0.058) (0.015) (0.001) (0.070) (0.061) 1.068** 0.015 0.001 0.142 -0.057 Poonch -0.675 0.917 (0.075) (0.010) (0.001) (0.089) (0.082) 0.979** -0.029 -0.083 0.138* -0.059 Overall -3.702 0.969 (0.014) (0.039) (0.070) (0.081) (0.120)

Note : Figure in parentheses indicate the standard error of regression coeffi cients. * Signifi cant at 5 percent level. ** Signifi cant at 1 percent level.

Table 2. Production, consumption and marketed surplus of milk in the Jammu region (litres Household/ day).

Avg. no. Milk Marketed Per capita Farm size Milk Milk surplus of milch as % to milk consumption groups production marketed animal production (litres/day) Small 4.27 21.44 20.25 94.44 0.289 Medium 8.26 37.13 35.34 95.18 0.363 Large 20.27 85.21 82.35 96.64 0.469 Overall 7.65 34.85 33.29 95.53 0.335

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milk products. rupee was maximum in fi rst marketing channel due to the absence of any marketing intermediatory. Marketing cost, marketing margin and price Maximum marketing cost was found in third channel spread in marketing of buffalo milk in Jammu whereas minimum marketing cost was found in region the case of the second channel. The marketing Table 3 showing the marketing of milk cost in the fi rst marketing channel in the overall in Jammu region revealed that producer’s share Jammu region was high due to high transportation in the consumer rupee was 93.14 percent, 88.78 cost incurred by the producers in Udhampur and percent and 86.17 percent in the fi rst (Producer- Poonch districts. The maximum marketing margin Consumer), second (Producer -Vendor - Consumer) was found in the third channel. and third (Producer - Vendor - Processor) marketing The marketed surplus function for channels, respectively, whereas marketing cost milk for the Jammu region indicated that milk was 6.86 percent in the case of the fi rst channel, production and the price of milk had a positive and 6.12 percent in the case of the second channel signifi cant impact on the marketed surplus of milk. and 7.45 percent in the case of the third channel, This clearly implies that as the milk production respectively. The marketing margin was found nil increases, there will be more marketed surplus of in the fi rst channel, while it was 5.10 percent and milk and the quantity of marketed surplus will also 6.38 percent for the second and the third channels, increase with the increase in the price of milk. The respectively. production as well as marketed surplus in absolute It was observed in marketing cost and terms was highest in large farms, followed by price spread of the overall Jammu region that 93.14 medium farms and small farms.. The per capita percent of consumer rupee was received by the consumption was found to be higher in large milk producer in fi rst marketing channel (Producer- farms. In estimating the cost of the marketing Consumer), which was the maximum share of the of milk in the overall Jammu region, it was found consumer rupee, while in third marketing channel that the producer’s share in the consumer’s price (Producer-Vendor-Processor), the minimum was maximum in the fi rst channel followed by the producer’s share in consumer rupee was found second and the third. Marketing cost and marketing (86.17%). The producer’s share in the consumer margin was maximum in the third channel.

Table 3. Marketing cost, marketing margin and price spread of milk in the Jammu region (in percentage).

Marketing Channels Particulars Producer to Producer to vender Producer to vender to consumer to consumer processor(sweet shop) Producer’s share 93.14 88.78 86.17 Marketing cost 6.86 6.12 7.45 Marketing margin 0.00 5.10 6.38 Consumer’s price 100.00 100.00 100.00

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REFERENCES

Acharya, S.S. and N.L. Agarwal. 1999. Agricultural Marketing in India. Oxford and IBH Publishing Co. Pvt. Ltd., New Delhi. p. 401. Dhaka, J.P., D.K. Jain, V.K. Kesavan and L. Singh. 1998. A study of production and marketed surplus functions for milk in India. Indo- Swiss Project, National Dairy Research Institute, Karnal. Das, S. 2004. Economic effi ciency of milk production and marketed surplus in rural area of Burdwan district (W.B.). M.Sc. Thesis, NDRI, Karnal, India. Kumar, R. 2006. Economic analysis of dairy farming among nomadic and resettled Van Gujjars in Uttaranchal: A comparative study. Ph.D. Thesis, NDRI, Karnal, India. Prabhakaran, R. and C. Ramasamy. 1984. Infl uence of certain selected factors on marketed surplus of milk. Cheiron, 13(2): 106-108. Singh, K. 1978. Who feeds the milk market? Indian Dairyman, 30(4): 277-280.

322 Original Article Buffalo Bulletin (September 2014) Vol.33 No.3

PREVALENCE OF TICKS IN BUFFALOES IN THE UPPER SINDH PAKISTAN

Majid Hussain Soomro1, Shahida Parveen Soomro2, Muhammad Bachal Bhutto3, Zeeshan Akbar4, Muhammad Yaqoob4 and Abdullah G. Arijo3

ABSTRACT and tactile control of ticks in local breeds of dairy animals in the Sindh province. Tick infestation is still a major economic dilemma for the dairy owners in Pakistan. The Keywords: buffalo, tick, prevalence, Hyalomma, current study reports the prevalence and bionomics Rhipicephalus of tick in the areas of upper Sindh, Pakistan. The study was carried out to identify and to quantify INTRODUCTION variation in the prevalence of bovine tick infestation with respect to host (age and species) Livestock is the key feature of the rural and area studied. Random sampling was used and based economy of Pakistan like other tropical and 1600 samples of Kundi buffaloes from the different sub-tropical countries. Cattle and buffaloes are areas were selected from extensive management the major animals which contribute satisfying systems. Prevalence of bovine tick infestation the ever increasing demand for milk and meat in did not differ signifi cantly (OR = 0.876; p>0.05) the country but the major contribution is fulfi lled by in Kundi (179/800; 24.75%) and Nili-Ravi buffaloes. Indigenous cattle breeds (Sahiwal, Red buffaloes (172/800; 22.3%). Hyalomma was the Sindhi etc.) also shared but these breeds are now major tick species (10.2%; 163/1600), followed by being replaced by crossbred cattle owing to high Rhipicephalus (5.6%; 99/1600). The prevalence of milk production. Although these breeds are resistant ticks in calves (< 1 year) was signifi cantly (p < 0.05) to harsh climatic conditions and resistant to some higher compare to the adult animals (1-2 years and tropical diseases, farmer breed preference is utmost. > 2 year animals). However, the prevalence of tick These crossbred cattle as compared to buffaloes are infestation was not associated (p > 0.05) with the greatly infl uenced by diseases, nutrition and climate location of the district. Moreover, the results of the changes but parasitism is thought to be a major prevalence of the ticks in the studied area provide cause of lower productivity (Sajid et al., 2007). the better understanding for evolving the strategic Parasitism also results in mortality, lowered

1Department of Pathobiology, Faculty of Veterinary Sciences, Bahauddin Zakariya University, Multan 60000, Pakistan, E-mail: [email protected] 2Department of Plant Protection, Sindh Agriculture University, Tandojam, 70060, Pakistan, 3Department of Veterinary Parasitology, Faculty Animal Husbandry and Veterinary Sciences, Sindh Agriculture University, Tandojam 70060, Pakistan 4Faculty of Veterinary and Animal Sciences, Pir Mehr Ali Arid Agriculture University, Rawalpindi 46000, Pakistan

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general health condition, retarded growth, lower and eastern part consists of the Nara Desert. The output of work, and decrease in the production study area lies in the sub-tropical region between of milk and meat. Moreover, fi nancial losses are 27°31’48”N and 68°44’23”E. District Khaipur also signifi cantly associated with nagging irritation has eight sub-divisions (Talukas): Khairpur and depreciation of the value of skins and hides city, Kingri, Gambat, Sobhodero, Kotdeji, Nara, (Chaudhry et al., 2011). Mirwah and Faizgunj. This cross-sectional Pakistan being a tropical and sub-tropical epidemiological survey was conducted at region country, optimal climatic conditions different locations of the district to analyze the are very conducive for raising the buffaloes variation in tick infestation during 2008 and 2009. and these conditions are also favorable for the growth and development of many tick species Sampling of animals (Durrani and Shakoori, 2009). So under these Random sampling was used to select a conditions crossbred cows are considered total of 1600 buffaloes from the study area of unfit for survivability and adaptability. Poor Khairpur District. The samples were collected managemental practices further enhance the severity from small dairy herds having no history of use of the problem (Irshad et al., 2010). Under these poor of acaricide for tick control. About 107 farms and conditions the buffalo is the only animal that survives 1600 animals were selected for sampling of ticks. with optimal production. The buffalo is also prone Animals were categorized on the basis of breed to tick infestation but its adaptability in tropical and (Kundi vs. Nili-Ravi buffaloes) and age (< 1 year sub-tropical conditions is excellent. Previous fi ndings vs. 1-2 years vs. > 2 years). of tick prevalence report that exotic breeds suffer more as compared to local breeds of cattle and Sampling and morphological identifi cation of buffaloes in the climatic conditions of Pakistan.. ticks With regard to the tick prevalence in buffaloes Ticks were collected from body parts there is scanty information available concerning of the infested animals and careful sampling Pakistan (Manan et al., 2007; Sajid et al., 2008, was made using forceps to avoid de-capitulation 2009) and there is no report is available on tick (Solusby, 2006). After collection ticks were kept prevalence from the area of Sindh. Therefore, the in capped bottles properly labeled containing present cross-sectional study was focused on 70% ethyl alcohol for preservation. Morphological prevalence and identifi cation of genera of ticks identifi cation of the ticks was made in a laboratory infesting the buffaloes in the upper areas of Sindh at the Department of Veterinary Parasitology, Sindh province, Pakistan. Agriculture University Tandojam (Soulsby, 1982; Walker et al., 2007).

MATERIALS AND METHODS Statistical analyses Statistical analyses were carried out by The study area included the district of using statistical package for social science (SPSS) Khairpur Mirs of the upper Sindh (Pakistan). It version 11.5. Multiple logistic regression tests were is situated south of the Indus River in the Sindh performed to determine the association between

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the prevalence of ticks and animal breeds, ages, of Sindh Pakistan. study area and tick species. Odds ratio measured The results of host susceptibility for tick the possible association tick prevalence between infestation fi ndings represent that both breeds the factors studied. The signifi cance level was set (Nili-Ravi vs. Kundi) were prone to the infestation at P value less than 0.05. at equal rates. These fi ndings of prevalence were not different from the previous reports in different parts of the country (Manan et al., 2007; Sajid RESULTS et al., 2008). But the fi ndings of the Sajid et al. (2009) in the lower Punjab area do not correspond The results of the present study demonstrate with this current report. This difference might be that the overall prevalence of tick infestation in both due to dry and hot environment of Sindh province breeds was 23% . However, the prevalence of ticks which reduces the activity of ticks. Moreover, did not differ (OR=0.876; p>0.05) signifi cantly the local breeds of cattle (Sahiwal, Red Sindhi) and both breeds were at almost equal risk of tick are resistant due to loose skin coat, and similarly, infestation (Table 1). The study of prevalence of tick their thick skin and wallowing habits prevent the species in bovine infestation found that Hyalomma buffaloes (Nili-ravi, Kundi) from tick infestation (10.5%; 163/1600) was most abundant, followed (Sajid et al., 2009). For the further investigation a by Rhipicephalus (5.6%; 90/1600), Boophilus longitudinal study needs to be done to understand (5.5%; 88/1600) and Ablyomma (2.3%; 36/1600) the frequency of tick infestation during different (Table 1). seasons in the Sindh province climate. In district Khairpur, the current data Data of host age determined that younger showed that the tick prevalence (p>0.05) was found stock were at more tick infestation risk than the to be similar in all Talukas and all were under the adult animals. These fi ndings agrae with the report risk of tick infestation equally. The results of host of Swai et al. (2005) who found that calves are age prevalence showed that a signifi cantly higher more susceptible than adults. But reports that age prevalence of tick infestation was observed in has no signifi cant association with tick infestation calves (p < 0.05) than in adult hosts and that calves of Sajid et al. (2009) and Manan et al. (2007) do were more prone to tick infestation as compared to not agree with our fi ndings . The increased rate of adults (Table 1). infestation in calves than adult might be due to hairy coat of the calves, less attention of the farmer and unhygienic conditions in places where calves DISCUSSION are kept that favor the ticks growth, development and reproduction. Moreover, the thin and easily The issue of tick infestation and prevalence penetrable skin of calves facilitates the easy was reported from all over the world in connection approach to the circulation for the nourishment of with different parts of the Pakistan. So keeping ticks. this in view the present study was designed to This study of the prevalence of ticks investigate the different aspects of prevalence and suggests that there is of a need of a longitudinal study identifi cation of ticks in buffaloes from upper areas to understand comprehensively all epidemiological

325 Buffalo Bulletin (September 2014) Vol.33 No.3

Table 1. Prevalence of ticks in buffaloes in upper area of Sindh Pakistan.

Prevalence Odd Lower Upper Parameter Variable Levels p-value (%) ratios 95% CI 95% CI Host 198/800 Bovine Buffaloes 0.263 0.876 0.696 1.104 species (24.75) 179/800 Cow - - - - (22.38) Age Buffalo < 1 year 182/518 (35.1) 0.425 2.448 1.808 3.315 1-2 years 116/436 (18.0) 0 0.999 0.729 1.37 >2 years 79/336 (18.1) - - - - Origen Khairpur City 44/200 (22.0) 0.73 1.206 0.738 1.971 Kingri 43/200 (21.5)0.78 1.171 0.715 1.917 Gambat 44/200 (22.0)0.78 1.171 0.715 1.917 Sobhodero 63/200 (31.5)0.11 2.073 1.303 3.298 Kotdeji 53/200 (26.5)0.36 1.588 0.987 2.555 Nara 43/200 (21.5)0.78 1.171 0.715 1.917 Mirwah 50/200 (25.0)0.46 1.468 0.909 2.371 Faizganj 37/200 (18.5) - - - - Tick Parasite Amblyomma 36/1600 (2.3) 0.042 0.625 0.284 1.377 Species Boophilus 88/1600 (5.50)0.691 1.13 0.601 2.122 163/1600 Hyalomma 0 0.269 0.156 0.463 (10.2) Rhipicephalus 90/1600 (5.6) - - - -

Value less than P<0.05 shows signifi cant difference.

326 Buffalo Bulletin (September 2014) Vol.33 No.3

aspects of such as frequency, distribution and risk infestation in two districts of lower Punjab, factors associated with tick infestation in the Sindh Pakistan. Preventive Veterinary Medicine, province of Pakistan, in order to design preventive 92: 386-391. medicine programs. Sajid, M.S., Z. Iqbal, M.N. Khan and G. Muhammad. 2008. Point prevalence of hard ticks infesting domestic ruminants ACKNOWLEDGEMENT of lower Punjab, Pakistan. International Journal of Agricultural and Biological This part of study was conducted with Science, 10: 349-351. cooperation the Pak-USAID research project Sajid, M.S., Z. Iqbal, M.N. Khan, G. Muhammad “Building molecular capacity for preventing tick and M.U. Iqbal. 2007. Effect of Hyalomma transmitted diseases in Pakistan”. ticks (Acari: Ixodidae) on milk production of dairy buffaloes (Bos bubalus bubalis) of Punjab (Pakistan). Italian Journal of Animal REFERENCES Sciences, 6: 939-941. Soulsby E.J.L. 1982. Helminthes, Arthropods and Chaudhry, Z.I., A. Saiddain, N. Sabir, N.A. Malik, Protozoa of Domesticated Animals, 7th ed. S. Azeem and A. Sajid. 2006. Prevalence of Bailliere tindall, London, UK. pathological conditions causing skin damage Soulsby, E.J.L. 2006. Helminthes, Arthropods and and consequently reducing its market value Protozoa of Domesticated Animals, 7th ed. in domestic ruminants of Punjab, Pakistan. Elsevier New Delhi, India Veterinary Science Development, 1(4): 19- Walker, A.R., A. Bouattour, J.L. Camicas, A. 23. Estrada-Pena, I.G. Horak, A.A. Latif, R.G. Durrani, A.Z and A.R. Shakoori. 2009. Study on Pegram and P.M. Preston. 2007. Ticks of Ecological Growth Conditions of Cattle Domestic Animals in Africa, Bioscience Hyalomma Ticks in Punjab, Pakistan. Iran. Reports, Edinburgh, UK. J. Parasitol., 4(1): 19-25. Irshad, N., M. Qayyum, M. Hussain and M.Q. Khan. 2010. Prevalence of tick infestation and theileriosis in sheep and goats. Pak. Vet. J., 30(3): 178-180. Manan, A., Z. Khan, B. Ahmed and Abdullah. 2007. Prevalence and identifi cation of Ixodid tick genera in Frontier Region Peshawar. Journal of Agricultural and Biological Science, 2: 21-25. Sajid, M.S., Z. Iqbal, M.N. Khan, G. Muhammad and M.K. Khan. 2009. Prevalence and associated risk factors for bovine tick

327 Original Article Buffalo Bulletin (September 2014) Vol.33 No.3

COMPARISON OF BLOOD ACID BASE GAS PARAMETERS IN VENOUS AND ARTERIAL BLOOD OF HEALTHY BUFFALOES

S.A. Hussain* and S.K. Uppal

ABSTRACT of information for early phase of the disease and they infl uence the development of clinical signs The present study was carried out on ten and therapeutic effectiveness (Brobst 1975). Some clinically healthy adult buffaloes of 4 to 6 years of authors (Brobst, 1975; Singh et al., 1981; Naito age. The parameters of acid-base balance (pH, pCO2, and Murakami, 1982) think that with the exception pO2, HCO3, SBC, ABE, SBE, SAT and O2ct) were of the respiratory components (pCO2 and pO2) measured simultaneously in venous and arterial and the SAT, other components are not different blood. The results from this study revealed that the in venous and arterial blood. Others think that the mean, minimum and maximum values of pH, pO2, venous blood is unsuitable for acid bas gas analysis

ABE, SAT and O2ct were lower in the venous blood (Bergman and Coleman, 1983; Gemelli, 1981). The than in the arterial blood while the values of pCO2 reference values for every parameter help in correct and HCO3 were higher in venous blood than in the interpretation of the results. The reference values arterial blood. Wider differences were observed in in calves are available in the literature. The limited the values of the respiratory parameters of venous overall research of acid-base balance in buffaloes and arterial blood. The differences found in the prompted us for this research, whose purpose was parameters of acid-base balance between both the to describe the blood acid-base gas information for kinds of blood revealed the restricted informative the venous and arterial blood in buffaloes. signifi cance of the venous blood. This calls for the use of arterial blood when it is necessary to assess the acid-base balance. MATEERIALS AND METHODS

Keywords: acid-base balance, arterial blood, The study was carried out on ten clinical buffalo, venous blood healthy buffaloes of age between 4 to 6 years. The selected buffaloes had no history of disease for the current lactation and were clinically healthy at the INTRODUCTION time of sampling. Blood samples were collected in heparinised (1:1000) disposable syringes and The changes in blood acid-base gas status were examined by apparatus ABL- 77 v1.41, are the early manifestation of many diseases in produced by Radiometer- Denmark. Venous blood humans as well as animals. So, they are source samples were collected from the jugular vein and

Department of Veterinary Medicine, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab 141 004 India, *E-mail: [email protected]

328 Buffalo Bulletin (September 2014) Vol.33 No.3

arterial blood samples from auricular artery. The 0.66 mmol/L higher than those in arterial blood. blood acid-base parameters determined were pH, A difference was also found for the ABE, whose partial pressure of carbon dioxide (pCO2), partial average value in the venous blood was 1.03 mmol/L pressure of oxygen (pO2), actual bicarbonate lower than that in the arterial blood. (HCO3), standard bicarbonate (SBC), actual base The analysis of the results for the average, excess (ABE), standard base excess (SBE), oxygen minimal and maximal values of the acid-base saturation (SAT) and oxygen capacity (O2ct). The balance of the venous and arterialized capillary student’s t-test was used to analyze the signifi cance blood revealed that these values of the pH, pO2, of difference between parameters of venous and ABE, SAT and O2ct in the venous blood were arterial blood. The signifi cance level was kept at lower, and for pCO2 and HCO3 were higher than p≤0.05 and p≤0.01. those of the arterial blood. Also the acquired results between the maximal and minimal values of pH,

HCO3 and ABE of the venous and arterial blood

RESULTS AND DISCUSSION were narrower, and the ones for pCO2, pO2, and SAT are substantially wider. Van Sluijs et al. (1983) The mean, minimum and maximum values and Waizenhofer and Mulling (1978) discussed of acid base balance are shown in Table 1. The similar differences between the indexes of the average (7.386±0.023) and the maximal (7.43) venous and arterialized capillary blood of dogs and indexes of pH in the venous blood were over taken calves. According to these authors the values of the with the average (7.463±0.015) and maximal (7.52) indexes of acid-base balance in the arterial blood pH in the arterial blood. The mean pH value in the are closer to the arterialized capillary blood rather venous blood was 0.077 lower than that of the than the venous blood. arterial blood. The difference between the minimal The results of the present study for the venous pH indexes of the two kinds of blood was 0.07 and blood of buffaloes were different that the ones between the maximal pH indexes it was 0.09. of Nangia and Sukhija (1981) and Sobti et al.

The pCO2 in the venous blood was 7.33 (1995) who found higher indexes for pH and lower mmHg higher than that in the arterial blood. for pCO2 and HCO3. The values of pH, pO2 and

The biggest differences were found between the HCO3 of the arterial capillary blood found in this maximal indexes of the venous and arterial blood study were closer to those of Singh et al. (1981) (11 mmHg), the lowest were between their minimal who used arterial blood, rather than Nangia and indexes (4 mmHg). In comparison to pCO2, pO2 in Sukhija (1981) and Sobti et al. (1995) who used the venous was 53.6% to 72.6% lower as compared venous blood. Apart from the bigger differences to that in the arterial blood. Similar substantial of the established values for the respiratory differential rate was found for SAT and O2ct. The indexes (pCO2 and pO2) and SAT between arterial average, minimal and maximal indexes for SAT and venous blood, the values of the metabolic and O2ct in the venous blood were lower than those component (HCO3 and ABE) of the acid-base of the arterial blood. balance and the blood pH of both the blood types

The average values of HCO3 and SBC in the overlapped to a certain degree. For example, the venous blood were respectively 2.17 mmol/L and average and minimal values of HCO3 in the venous

329 Buffalo Bulletin (September 2014) Vol.33 No.3

Table 1. Mean, minimum and maximum values of acid base gas parameters in venous and arterial blood of healthy buffaloes.

Venous blood Arterial blood Mean±S.E Minimum Maximum Mean±S.E Minimum Maximum pH 7.386±0.023 7.35 7.43 7.463±0.015 7.42 7.52

pCO2 (mmHg) 41.33±2.27 35 46 34.0±1.52 31 35

pO2 (mmHg) 49.00±3.26 37 69 86.67±2.64 79 95

HCO3 (mmol/L) 26.20±1.43 23.1 30 24.03±0.66 22.9 25.2

SBC (mmol/L) 26.03±1.89 22.4 28.8 25.37±1.06 23.8 27.4 ABE (mmol/L) 1.10±0.52* -1.9 3.4 2.13±0.54 -0.7 5.4 SBE (mmol/L) 2.13±0.52* -1.7 5.7 0.63±0.48 -0.9 2.4 SAT (%) 80.17±4.23** 71.7 95.2 97.13±3.83 96.2 97.6

O2ct (%) 12.37±1.23** 10.2 14.8 15.50±1.32 10.8 21.6

*Difference signifi cant at p≤0.05; **Difference signifi cant at p≤0.01.

blood overlapped with the maximum and minimum of the organism. Minirva Medica, 72(34): values of the arterial blood and the found borders 2289-2292. for ABE of the venous blood covered most of the Nangia, O.P. and S.S. Sukhija. 1981. A comparative values of arterial blood. Thus, it was inferred that study of acid base components in venous the venous blood of buffaloes could be used for and capillary blood. Indian J. Exp. Biol., assessing pH and HCO3 only. For the respiratory 19(5): 475-476. components (pCO2 and pO2) and SAT the venous Naito, Y. and D. Murakami. 1982. Blood gas blood did not give a clear idea. acid base values in the coccygeal artery of The overall assessment of the venous blood Holstein-Friesian cows. Jpn. J. Vet. Sci., parameters showed that it was not suitable for the 44(5): 777-780. evaluation of acid base balance. Because of this, Singh. J., K. Mirakhur, B. Prasad and R.N. arterial blood is recommended for assessment of Kohli. 1981. Acid base status and gases in acid base balance. cerebrospinal fl uid of healthy calves and buffalo calves. Zbl. Vet. Med. A, 28(2): 131- 134. REFERENCES Van Sluijs, F.J., H.W. de Vries, J.J. De Bruijne and W.E. van den Brom. 1983. Capillary and Bergman, N. and J. Coleman. 1983. Choice of blood venous blood compared with arterial blood for acid-base balance. Lancet, 2: 1135. in measurement of acid base and blood gas Brobst, D. 1975. Evaluation of clinical disorders of status of dog. Am. J. Vet. Res., 44(3): 459- acid-base balance. J. Am. Vet. Med. Assoc., 462. 166(4): 359-364. Sobti, V., S. Singh and B. Prasad. 1995. Effect of Gemelli, A. 1981. Problems of acid-base equilibrium 330 Buffalo Bulletin (September 2014) Vol.33 No.3

storage on acid base status of buffalo bubalis blood. Indian J. Vet. Surg., 6(1): 34-40. Waizenhofer, H. and M. Mulling. 1978. Behavior

of pHact, PO2 and PCO2 in venous, capillary and arterial blood of newborn calves. Berl Munch Tierarztl., 91(9): 173-176.

331 Original Article Buffalo Bulletin (September 2014) Vol.33 No.3

PREVALENCE OF SALMONELLA AND ESCHERICHIA COLI ASSOCIATED WITH DIARRHEA IN BUFFALO AND COW CALVES

M. Anwarullah*, J.A. Khan, M.S. Khan, K. Ashraf and M. Avais

ABSTRACT economically important agents (Acha et al., 2004; House et al., 1978). Salmonella typhimurium is Calf diarrhea caused by Salmonella and a major cause of calf morbidity and mortality Escherichia coli was the particular focus in the in the United States and in Europe (Tsolis et al., current study. A total 150 calves up to 6 months of 1999; Rice et at., 1997). Khan et al. (2009) found age suffering from diarrhea were examined. Fresh the prevalence of calf diarrhea due to Salmonella fecal and blood samples were collected. Fecal was up to 16.3%. Calf diarrhea is a clinical sign samples were cultured on McConkey and on S-S of disease that may have many causes but E. coli agar for isolation of bacteria. Bacterial colonies has been considered as the primary bacterial cause were identifi ed by using an API 20 E Kit. Overall in calves (Yamamoto and Nakazawa, 1997). The prevalence of Salmonella and E. coli was 33.3%. morbidity and mortality rates associated with many Prevalence of Salmonella and E. coli was 18.6% outbreaks of gastrointestinal diseases caused by and 14.6%, respectively. Occurrences of diarrheal Escherichia coli have highlighted the threat that disease due to various risk factors like age, sex these organisms pose to public health (Paton et were recorded for determining the effect of these al., 1998). The prevalence of enterohemorrhagic risk factors. Eschrechia Coli O157:H7 in the feces of dairy calves and feedlot cattle is low. Eschrechia coli Keywords: buffalo, Bubalus bubalis, cow, Bos have been isolated from 0.3 to 2.2% of fecal taurus, Escherichia coli, Salmonella, diarrhea samples collected from healthy calves or cattle in the United States, the United Kingdom, Germany, and Spain (Blanco et al., 1993). INTRODUCTION

Major causes of mortality of neonatal MATERIALS AND METHODS calves were conditions like diarrhea and pneumonia (Khan et al., 2009, Shimizu and Nagatoma 1978). Selection of site: The research was The neonatal calf mortality in the fi rst month of age conducted at different private livestock farms and is more than 80% of the total mortality in calves civil veterinary hospitals of district Jhelum. Total (Jenny et al., 1981). Salmonella and Escherichia duration of the study was of three months. coli are known to be the most common and Animals: A total number of 150 cow and

Department of Clinical Medicine and Surgery, Department of Parasitology, Faculty of Veterinary Science, University of Veterinary and Animal Sciences Lahore-54000 Pakistan, *E-mail: [email protected]

332 Buffalo Bulletin (September 2014) Vol.33 No.3

buffalo calves under six months of age, suffering sas read in millimeters on the left scale after the from diarrhea were studied for determination of time interval of 8-24 h for cow and buffalo calves. Salmonella and E. coli. Seasonal effect: The effect of season, environment, Collection of samples: A fecal sample temperature, rainfall and humidity was observed. was collected directly from the rectum of each of Risk factors: The effects of age, sex, breed, diet, the calves under six months of age. Then the fecal managemental practices and vaccination were also material was shifted to sterilized plastic containers studied. and again those containers were placed in zip bags Statistical analysis: The data collected were to prevent entry of foreign particles. Fecal material tabulated and analyzed statistically by chi-square was kept in contact with ice to avoid further changes test (Steel et al., 1997). in fecal material. Blood: Blood samples from the same individuals were collected from the jugular vein RESULTS after applying disinfectants and immediately shifted into anticoagulant coated vaccutainer tubes. Of the total of 150 cow and buffalo calves Bacterial culture: One gram of fecal under six months of age studied with the signs material was dissolved in 10 ml of autoclaved of diarrhea in district Jehlum, the total number normal saline and cultured on MacConkey agar of E.coli positive samples was 22, and 28 were with the help of safety buds. Then it was incubated Salmonella positive. The prevalence of E. coli was in an incubator for 24 h.The next day the colorless 14.6%, and that of Salmonella was 18.6% The data colonies were again cultured on S-S agar by were analyzed by chi-square test and value of P dissolving those colorless colonies fi rst in 5 ml was greater than 0.05, which means no signifi cant autoclaved normal saline and then culturing on S-S difference was found. The positive samples of agar with safety buds. Salmonella and E. coli in 1-30 day, 31-90 day, 91- The API 20E commercial kit was used for 180 day age groups were 3, 11, 14 for Salmonella the identifi cation of E. coli and Salmonella (Popovic and 2, 6, 14 for E. coli. Thus the prevalence of et al., 2004; API 20E Kit Biomerieux, France). Salmonella and E. coli in 1-30 days, 31-90 days, Blood parameters: Following blood and 91-180 were 2%, 7.33%, 9.33% for Salmonella parameters were compared according to Benjamin and 1.33%, 4%, 9.33% for E. coli, respectively. (1979): (a) Total leukocyte count: Total leukocyte The value of P showed was less than the counts were performed with the help of leukocyte level of signifi cance so there was no difference for diluting pipette. (b) Differential leukocytic the values of different age groups. The prevalence count: The Giemsa stain blood smear method, of positive bacterial samples in the age groups of was preferred for differential leukocyte count. 1-30 days 31-90 days 91-180 days was noted 14%, (c) hemoglobin estimation: Sahli’s method was 23% and 63% for E. coli and 11%, 29% and 60% used to determine the Hb level. (d) Erythrocyte for Salmonella, respectively. The total number of sedimentation rate: Erythrocyte sedimentation positive samples in male calves was 18 and the rate was determined with a Wintrobe hematocrit prevalence was 12%, and in the case of female tube. The upper level of sedimenting erythrocytes calves, the total number of positive samples was

333 Buffalo Bulletin (September 2014) Vol.33 No.3

32 and the prevalence was 21%. values were 13% 9%, 19%, lymphocyte values Out of the total number of 150 diarrheic were 37%, 38%, 59%, monocyte values were 16%, calves, the number of positive samples for cow 8%, 10% for the three age groups, respectively. The calves was 31 and that for buffalo was 19. The effect of season was observed for three months (July, prevalence of Salmonella and E. coli in cow and August, and September). The prevalence rate was buffalo calves was 21% and 12%, respectively. The high in August and least in July. Rainfall increased P value for the test was greater than 0.05 and data the number of bacterial positive samples. showed non-signifi cant. Total numbers of positive samples of Salmonella and E. coli in cow calves was 0, 17, 14 for 1-30 days, 31-90 days, 91-180 DISCUSSION days and for Salmonella were 6, 0, 13 for 1-30 days, 31-90 days, and 91-180 days. The prevalence The prevalence of diarrhea due to of bacteria (Salmonella and E. coli) was 0, 11.33%, bacteria and E. coli in overall calves was 33.3% 9.33% for cow calves and 4%, 0, 8.67% for buffalo in the present study. Although that is very high, a calves for the different age groups. similar prevalence was also seen in Mozambique. The prevalence in males in the 1-30 day In Mozambique, Baule et al. (1995) reported an age group was 0, that in those in the 31-90 day overall prevalence of diarrhoeic calves as high group was 5.33%, and that in those in the 91-180 as 36% but this percentage includes values of daygroup was 7.33%. Similarly in females in the prevalences of diarrhoea from other farms not 1-30 daygroup, the prevalence was 4%; in the 31- included in the present study. This might be due to 90 daygroup, it was 5.33% , and in the 91-180 an outbreak in a particular area in those days or due daygroup, it was 11.33%. The prevalence of E. coli to outbreaks among different farms as sampling was in male calves was 0 %, 6%, 4.66% for the 1-30 done from certain private livestock farms as well. day, 31-90 day, and 91-180 day age groups, and that Among bacteria, enterotoxigenic Escherichia coli for Salmonella was 0%, 0% and 3.33% for the 1-30 and Salmonella are known to be the most common day, 31-90 day, 91-180 day groups. The prevalence and economically most important agents that cause of E. coli in female calves was 2%, 2%, 3% for diarrhea in young calves (House et al., 1978). the 1-30 day, 31-90 day, and 91-180 day, and that The values according to different age for Salmonella was 1%, 3.330% and 5.33% for the groups showed that total leukocytes were greater in 1-30 day, 31-90 day, 91-180 daygroups as shown in age group of 31-90 days, hemoglobin in the younger Figure 1. group of 1-30 days, erythrocyte sedimentation was Blood parameters varied in the 1-30 day, greater in the 91-180 day age group, neutrophils 31-90 day, 91-180 day age groups as shown in were higher in the 31-90 day group, eosinophils, Table 1. Total leukocyte count values in these basophils and lymphocytes were greater in the groups were 9%, 19%, 13% respectively. Similarly, oldest age group (91-180 days) and monocytes hemoglobin values were 14%, 13%, 14% were highest in the youngest age group. In the erythrocyte sedimentation rates were 11%, 8%, current study, the average of the total leukocyte 19%, neutronphil values were 19%, 35%, 26%, count value increased in the cow as compared eosinophil values were 6%, 11%, 19%, basophil to the buffalo calves. Similarly, the average of

334 Buffalo Bulletin (September 2014) Vol.33 No.3

Figure 1. Prevalence of Salmonella and E. coli in calves in different age groups.

Table 1. Average values of blood parameters in cow calves according to different age groups.

Age TLC Hb ESR N E B L M 1-30 days 9% 14% 11% 19% 6% 13% 37% 16% 31-90 days 19% 13% 8% 35 11% 9% 38% 8% 91-180 days 13% 14% 19% 26% 19% 19% 59% 10%

335 Buffalo Bulletin (September 2014) Vol.33 No.3

hemoglobin was higher in the cow calves as Escherichia coli 0157 in cull dairy cows on compared to the buffalo calves, but the averages farm and at slaughter. J. Food Protect., 60: of erythrocyte sedimentation rate and neutrohils 386-387. were higher in the buffalo calves than in the cow Shimizu, T. and H. Nagatoma. 1978. Current status calves. Again. as far as basophiles, eosinophils and of calf diseases in Japan. Bull. Fac. Agri. monocytes are concerned, the average values were Miyazaki Univ., 34: 329-336. greater in the cow calves than in the buffalo calves, Tsolisl, R.M., G. Adams, A. Thomas, Ficht and J. but lymphocytes values were higher in buffalo Andreas. 1999. Contribution of Salmonella calves than in cow calves. typhimurium Virulence Factors to Diarrheal Disease in Calves. Infection and Immunity. 67: 4879-4885. REFERENCES Yamamoto, T. and M. Nakazawa. 1997. Detection and sequences of the enteroaggregative Baule, C., M. Vuuren, J.P. Lowings and S. Bela. Escherichia coli heat- stable enterotoxin 1997. Genetic heterogeneity of bovine 1 gene in enterotoxigenic E. coli strains viral diarrhoea viruses isolated in Southern isolated from piglets and calves with Africa. Virus Res., 52: 205-220. diarrhea. J. Clin. Microbiol., 35: 223-227. Blanco, J., E.A. Gonzalez, S. Garcia, M. Blanco, B. Regueiro and I. Bernardez. 1988. Production of toxins by Escherichia coli strains isolated from calves with diarrhoea in Galicia (North-western Spain). Vet. Microbiol., 18(3-4): 297-311. House, J.A. 1978. Economic impact of Rotavirus and other neonatal agents of animals. J. Am. Vet. Med. Assoc., 173: 573-576. Khan, J.A., M.S. Khan, M.A. Khan, M. Avais, A. Maqbool, M. Salman and Z.U. Rehman. 2009. Epidemiology of major bacterial and viral causes of diarrheoa in buffalo calves in three districts of the Punjab province of Pakistan. J. Zool., Suppl. Ser. (9): 187- 193. Paton, P.C. and A.W. Paton. 1988. Pathogenesis and Diagnosis of Shiga Toxin-Producing Escherichia coli Infections. Clinical Microbiology Reviews, 11: 450-479. Rice, D.H., E.D. Ebel, D.D. Hancock, T.E. Besser, D.E. Herriott and L.V. Carpenter. 1997.

336 Original Article Buffalo Bulletin (September 2014) Vol.33 No.3

DISTRIBUTION OF SALMONELLA SPECIES IN BUFFALOES IN SOME MIDDLE GOVERNORATES OF IRAQ

Afaf Abdulrahman Yousif* and Ali, D.M. Al-Hashimi

ABSTRACT The antibiotic susceptibility pattern of Salmonella against 15 antimicrobial drugs This study was planned to isolate and revealed that all isolates were resistant (100%) type Salmonella from buffalo in three middle to chloramphenicol, suphamethoxazole, governorates in Iraq. One hundred fi fty milk samples erythromycin, cloxacillin, and tetracycline, were collected from 150 lactating buffaloes in the and all isolates were sensitive to amicacin and fi eld, and in the slaughter house (150) fecal samples trimethoprimwhile neomycine, gentamycine, were collected at slaughter time and (900) samples cefi xime, ciprofl oxacin, kanamycin and were collected post slaughtering:150 samples from streptomycin gave intermediate results. each organ: gall bladder, mesenteric lymph nodes, spleen, liver, small intestine and cecum. Keywords: Salmonella, buffaloes, serotyping of Diagnostic study depended upon the Salmonella, Iraq morphological and cultural properties of the isolates on some selective media like Brilliant Green , XLD, SS agar and Hicrome rajhans medium, modifi ed INTRODUCTION (Salmonella agar modifi ed) were used in addition to different biochemical tests, API-20E and KB Buffalo raising is a major contributor to 003 Hi25^TM Enterobacteriaceae Identifi cation the agriculture and livestock industry in many Kit, latex test and serotyping of isolates. Asian countries through the production of good Twenty-two isolates were obtained from the quality meat and farmyard manure (Singh, 2010). fecal samples and organs; these isolates belonged The buffalo is also recognized as the world second to three serotypes (Salmonella anatum (68.18%), most important milk producing species (Bhatti et S.muenchen (18.18%) and S. enteritidis (13.64%), al., 2009). Dairy buffaloes are also called the ‘black while no isolate was obtained from milk samples. gold’ of South Asia, where 95% of the world’s In animals from slaughter houses, the buffalo milk is produced (Javaid et al., 2009) percentage of infection varied in examined organs Therefore, the pathogens either causing disease in and feces: in bile duct and liver, the percentage was buffaloes and their progeny or transmitted through 3.33% each; in mesenteric lymph nodes, 2.67%; their production are important because they affect in spleen and cecum, 2% each; and in feces, milk production and overall livestock production. 1.33%. Salmonellosis is associated with medium to

College of Veterinary Medicine, Department of Internal Medicine, University of Baghdad, Iraq, *E-mail: [email protected]

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severe morbidity and even mortality in farm animals, Brilliant Green agar, Xylose-Lysine Deoxycholate representing a major economic productivity loss agar, Salmonella-Shigella agar and Hicrome in the food and animal industries (Malkawi et al., rajhans modifi ed Salmonella agar and incubated 2004). Salmonella has been widely reported in aerobically at 37oC for 24 h. Salmonella suspected buffaloes (Hassanain et al., 2010; Khan et al., 2009; colonies were identifi ed by Gram staining, in Ribeiro, 2000; Abdulwahid and Raheem 1981), and addition to different biochemical tests: motility, infected animals may shed the organism in their triple sugar iron agar, indole, methyl red and citrate feces without showing any clinical signs of disease utilization tests, API-20E and the use of KB 003 (Fardsanei et al., 2010). Therefore, buffaloes may Hi25^TM-Enterobacteriaceae Identifi cation Kit, carry this organism undetected into an abattoir at which is a standardized colorimetric identifi cation the time of slaughter. system utilizing thirteen conventional biochemical This study aimed at determining the tests and eleven carbohydrate utilization tests distribution of Salmonella in different organs, feces, based on the principle of pH change and substrate and milk samples of buffaloes.in three governorates utilization (Thangamaalr et al., 2009). in Iraq and at determining the sensitivity of the Then, the isolates were grouped by the obtained isolates to different antimicrobials. Wellcolex color latex test for Salmonella (Rohner et al., 1992). Description of kit contents (Figure 1). MATERIALS AND METHODS 1. Latex Reagent 1: one drop of gray-brown suspension of polystyrene latex particles in buffer The study was performed on 300 buffaloes containing 0.05% Bronidox® preservation. The distributed as follows: 150 dairy buffaloes in the latex particles are coated with rabbit antibody with fi eld and 150 buffaloes in slaughter houses. the following specifi city: Information about animals concerning age, Red latex Salmonella group B; Blue latex sex, and any signs of diarrhea were recorded. One S. group C; Green latex; S. group D. hundred fi fty milk samples were collected from 2. Latex Reagent 2: one drop of gray-brown lactating buffaloes in the fi eld , and in the slaughter suspension of polystyrene latex particles in buffer house 150 fecal samples were collected at slaughter containing 0.05% Bronidox® preservation. The time and (900) samples postslaughtering samples latex particles are coated with rabbit antibody with were collected, distributed as follows: 150 samples the following specifi city: of gall bladder, 150 samples of mesenteric lymph Red latex Vi; Blue latex S. group E and G; nodes, 150 samples of spleen, 150 samples of liver, Green latex S. group A. 150 samples of small intestine and 150 samples of 3. Red positive control : Killed bacterial suspension cecum. of organisms with Salmonella group B and Vi The isolation and biochemical identifi cation antigens containing 0.05% Bridox® and 0.5% of Salmonella was carried out according to standard formalin as preservative. laboratory methods (Quinn et al., 2004). Each 4. Blue positive control : Killed bacterial suspension sample was transferred into tetrathionate broth of organisms with Salmonella group C and E for Salmonella enrichment before streaking onto antigens containing 0.05% Bridox® and 0.5%

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Figure 1. Wellcolex color latex test for Salmonella.

***Results of KB003 Hi25 Enterobacteriaceae Identification :

Malonate utilazation

Lactose

Figure 3. Showing results to KB003 Hi25 Enterobacteriaceae.

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Figure 4. Salmonella serogroup : E (blue agglutination- pink background with Reagent 2).

Figure 5. Salmonella serogroup D : (green agglutination-pink background) with reagent 1.

Figure 6. Salmonella serogroup G (blue agglutination-pink background) with reagent 1.

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formalin as preservative. black center, on Brilliant green agar the colonies 5. Green positive control: Killed bacterial appeared gray reddish/pink slightly convex and on suspension of organisms with Salmonella group Hicrome rajhans modifi ed medium the colonies A and D antigens containing 0.05% Bridox® and appeared as pink in color. 0.5% formalin as preservative. The results of biochemical tests by Finally, serotypings of Salmonella isolates Api and KB003 Hi25 Enterobacteriaceae were confi rmed in the Central Public Health Identifi cation system showed that this bacterium Laboratories by using specifi c antisera. was positive for: Lysine utilization, Ornithine Antibiotic susceptibility tests for Salmonella utilization, Nitrate reduction, H2 S production, isolates were performed according to the Kirby Citrate utilization, Methyl red, Arabinose, Xylose, Bauer method (Bauer et al., 1996). Mueller Hinton Rhamnose, Melibiose, Glucose; and negative for: agar was used as growth medium for standard disc ONPG,Urease ,phenylalanine Deamination, Voges diffusion test and growth was spread on plates Proskauer’s, Indole, Malonate utilization, Esculin with the help of a sterilized cotton swab to form hydrolysis, Adonitol, Cellobiose, Saccharose, a smooth bacterial lawn. The discs were placed on Raffi nose, Trehalose, Lactose, Oxidase (Figure 3). to the agar surface using sterile forceps. Each disc The results of serogrouping of Salmonella was gently pressed with the point of sterile forceps isolate according to serogrouping Wellcolex Color to ensure complete contact with the agar surface. Salmonella showed that and 4 isolates gave blue Plates were incubated overnight at 37ºC. agglutination-pink background with reagent 2 [it Characterization of strains as sensitive or resistant belong to group E, (Figure 4)]; 3 isolates gave was based on the size of the inhibition zone around [green agglutination- pink background with reagent the disc compared with the interpretation standards 1 [it belongs to serogroup D, (Figure 5)] and 15 provided by the manufacturers. The antimicrobial isolates gave blue agglutination-pink background drugs used were ampicillin, bacitracin, with reagent 1 [it belong to group G, (Figure chloramphenicol, erythromycin, gentamycin, 6)]. Table 1 showed of Salmonella grouping and kanamycin, novobiocin, penicillin, spectinomycin, serotyping. streptomycin, tetracycline and trimethoprim. Percentage of infection with Salmonella in buffaloes RESULTS Microbiological examination of samples that had been collected from slaughter houses Isolation and identifi cation of Salmonella spp. (organs and feces ) revealed that seven out of 150 Isolation and identifi cation of Salmonella were positive for Salmonella. The milk samples of were confi rmed in different media as showed buffalo in the fi eld revealed negative results so that in Figure 2. The cultural characteristics showed the statistic analysis showed signifi cant differences different colonies, On Salmonella Shigella agar ,the at p>0.05 between animals at the slaughter house colonies appear as small pale, rounded with black and animals in the fi eld. center, on Xylose-Lysine Deoxycholate agar, they showed slightly transparent zone of reddish and Percentage of infection with Salmonella species

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in buffaloes in the slaughter house age groups of buffaloes was found as shown in The results of serotyping of Salmonella spp. Figure 7. from (1050) samples at the slaughter house were that22 isolates of three different serotypes were Distribution of Salmonella species according to recognized according to the Central Public Health sex Laboratories. These serotypes were Salmonella Thirteen isolates from 97 samples were anatum (68.18%), Salmonella muenchen (18.18%) recorded in males and nine isolates from 53were and Salmonella enteritidis (13.64%). The statistical recorded in females. Statistically, there was no analysis showed signifi cant differences at p>0.05 signifi cant difference between males and females between Salmonella anatum and the others . at p ≥ 0.05.

Clinical signs Distribution of Salmonella spp according to Out of 41 animals, three animals showed month of year different clinical signs: diarrhea (1), respiratory Figure 8 shows the distribution of Salmonella signs (1) and more than one symptom (1) were spp. according to month of year. No isolates affected with Salmonella, while four animals out appeared in November and December, one affected of 109 examined that gave positive results for animal (1 isolate) was found in January, but in Salmonella did not show clinical signs (Table 2). In February, three affected animals (12 isolates) were the fi eld, all animals with acute or chronic mastitis found, two affected animals (6 isolates) were found and without clinical signs gave negative results for in March, and one affected animals (3 isolates) was Salmonella infection. found in April.

Percentage of Salmonella isolated from different Results of antimicrobial sensitivity tests samples The antibiotic susceptibility pattern Salmonella was isolated from different of Salmonella against the antimicrobial disc samples with different percentages except samples revealed that all isolates were resistant (100%) of small intestine and milk. The highest percentages to chloramphenicol, suphamethoxazole, appeared in the gall bladder (3.33%) and the liver erythromycin, cloxacillin, and tetracycline. (3.33%) followed by mesenteric lymph nodes Aall isolates were sensitive to amicacin and (2.67%). Statistical analysis showed signifi cant trimethoprim. difference at (p<0.05) (Table 3).

Distribution of Salmonella isolated according to DISCUSSION age According to age of buffaloes at the The results of the present study showed that slaughter house, the results shows that Salmonella Salmonella were isolated at a percentage of 2.33% was highest (6.67%) in the 1.5 - 3 year age group buffaloes, and this is in a agreement with the study and lower (3.23%) in animals more than 3 years of Hassanain 2008 which recorded a percentage old. Statistically no signifi cant difference between 2.16% of Salmonella infection in buffaloes in

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Egypt, and with a study of Al-Nakshabandy In contrast, a study in Pakistan by Iqbal (2001) in buffaloes, which found that the overall et al. (2004) showed isolation of Salmonella at a ratio of infection with Salmonella in Mosul city in percentage of 2.41% in milk samples of lactating Iraq was 2.23%). while Sen et al. (1988) recorded dairy buffaloes . the low percentage 1.60% of Salmonella from The results of present study were in rectal swabs of buffalo in Bangladesh. Phillips et agreement with those of Gunasegaran, 2011 who al. (2008) isolated Salmonella from 1.1% of ground found that Salmonella was resistant to tetracycline buffalo beef samples. and chloramphenicol and sensitive to kanamycin. Other studies have also shown different Also, Singh, et al. (2010) recorded that 46 strains of results, Hassanain et al. (2010) found a high Salmonella enteric.were resistant to streptomycin percentage (11.11%) of Salmonella in buffaloes and kanamycin but disagreed about resistance to in Eygpt. Also Khan et al. (2009) found higher gentamicin and amoxycillin. percentage (16.3%) of Salmonella during study of The present study showed that the diarrhea in buffalo calves in Pakistan . Salmonella anatum and Salmonella enteritidis were Boonmar et al.(2008) isolated Salmonella resistant to seven antimicrobials and Salmonella from fecal samples of buffaloes in Japan at a meunchen was resistant to eight antimicrobials , percentage of 8%. Sharma et al. (1989) found while a study by Boonmar et al. (2008) recorded 16.53% of meat samples were positive to Salmonella that the buffalo isolates were susceptible to most in Indian buffaloes. of antimicrobials (tetracycline, streptomycin, No signifi cant difference between ampicillin, sulfamethoxazole-trimethoprim, Salmonella infection in the three governorates may chloramphenicol, amoxicillin-clavulanic acid and explained by their being in the same geographical nalidixic acid) and all isolates showed sensitivity region and having the same climate. Also. there to cefotaxime, norfl oxacin and ciprofl oxacin. was easy transmission of animals between them. In the present study, some animals showed No study is available concerning the distribution different clinical signs of Salmonellosis, and these of Salmonella in buffaloes at slaughterhouses in all signs were similar to those recorded by Santana et governorates in Iraq. al.(2008) in their study on experimental infection These results resemble those of other with Salmonella dublin in buffalo calves, which studies. In Pakistan, Ali et al.(2008) recorded showed the main clinical signs, i.e., diarrhea, fever, negative results for Salmonella species in 200 respiratory signs and dehydrationwhile other animals mastitis quarters in buffaloes. Also, negative results without clinical signs but gave positive results for for Salmonella in Iraqi buffaloes were recorded by Salmonella infection. This revealed a number of Abdul Razak.(1982). carrier animals without clinical signs, confi rming Our study is compatible with many studies Nabbut and Al-nakhlil (1982), Galland et al.(2000), which showed that Salmonella was not isolated and Radke et al. (2002) who found that animals from milk samples, such as Coroian et al. (2010) in which had recovered from Salmonellosis may Romanian buffaloes ; Khan et al. (2009) in Pakistan continue shedding of Salmonella microorganisms ; Ali et al . (2008) in Pakistan, and Moroni et al. from 2-12 weeks post infection, whereas shedding (2006) in northern Italy. of Salmonella microorganisms from animals from

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Figure 7. Distribution of Salmonella isolated according to age.

Figure 8. The distribution of Salmonella spp. according to month.

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Table 1. Results of Salmonella grouping and serotyping.

Total No. of Salmonella No. of No. isolate Serotyping at center of Salmonella isolates isolates 15 Group E Salmonella anatum (68.18%) 22 3 Group D Salmonella enteritidis (13.64%) 4 Group G Salmonella muenchen (18.18%)

Statistical results showed signifi cant difference at p < o.o5.

Table 2. Distribution of affected animals according to clinical signs before slaughtering.

Clinical signs No. of animals No. of affected animals with Salmonella With clinical signs 41 3 animals (diarrhea(1), respiratory signs (1) and more than one sings (1) Without clinical signs 109 4 animals

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Table 3. Percentage of Salmonella isolated from different samples.

Collection of No. of examined No. of positive No. of Salmonella Sample samples sample sample spp. 4 S. anatum, Gall bladder 150 5 1 S. munchen 3 S. anatum Mesenteric lymph node 150 4 1 S. munchen 2 S. anatum Spleen 150 3 1 S. munchen Organ and feces 3 S. anatum at slaughter Liver 150 4 1 S. munchen house 1 S. enteritidis 2 S. anatum Cecum 150 3 1 S. munchen Small intestine 150 0 0 1 S. anatum Feces 150 2 1 S. enteritidis Total at slaughter house 1050 22 22 Milk 150 0 Field Total 1200 22

Table 4. Antibiotic susceptibility pattern of Salmonella.

Numbers of Salmonella isolates No. Antibiotics used R% I%S% 1 Chloramphenicol 22 100 0 0 0 0 2 Neomycin 2 9.1 15 68.2 5 22.7 3 Suphamethoxazol 22 100 0 0 0 0 4 Gentamicin 21 95.5 0 0 1 4.5 5Cefi xime 18 81.9 4 18.2 0 0 6 Erythromycin 22 100 0 0 0 0 7 Ciprofl oxacin 0 0 1 4.5 21 95.5 8 Kanamycin 1 4.5 11 50 10 45.5 9 Amoxicillin 21 95.5 1 4.5 0 0 10 Streptomycin 0 0 1 4.5 21 95.5 11 Tetracyclin 22 100 0 0 0 0 12 Amicacin 0 0 0 0 22 100 13 Trimethoprim 0 0 0 0 22 100 14 Cloxacillin 22 100 0 0 0 0 15 Nitrofurantoin 1 4.5 2 9.1 18 81.8

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which clinical signs had disappeared may be found Our results also showed that the higher with chronic infection of Salmonellosis. Carrier percentage of Salmonella isolated from organs animals may also be clinical cases if resistance is as compared with feces. The reason for this lowered by environmental stress or inter current might be by that the body does not shed infection. Salmonella microorganisms continuously in feces: In the present study, data demonstrated particularly, carrier animals with Salmonella that 22 Salmonella isolates were isolated from shed microorganisms intermittently. This is in 1050 samples, constituting about 2.09%., and that agreement with the study of Molla et al. (2002) three different species were found. This results is which reported a high percentage of Salmonella consistent with Singh et al. (2010) who recorded isolates (4.2%) in organs (mesenteric lymph node) different species in buffaloes (S. anatum, 13; S. and a low percentage in feces (1.9%).These results weltevreden, 13; S. rostock, 6; S. typhimurium, 5; resemble those of Poernomo et al. (1986) who S. gallinarum, 5; S. stockholm, 1; S. dublin, 1; and found that the percentage of Salmonella isolates S. orion, 2) and with two studies: Hassanain et al. from fecal samples (1.7%) was lower compared (2008) and Hassanain (2010) who found two species with the percentage of Salmonella isolates from in buffaloes (S. enteritidis and S. typhimurium). organs (3.12%). In India, Sharma et al. (1989) recorded Our study revealed a difference in different species in buffaloes: S. anatum, S. the distribution of Salmonella in organs and bareilly, S. stanley, S. Weltevreden, S. newport, S. feces. This indicates that an animal may be a saintpaul, S. typhimurium, S. agona, S. chester, carrier of Salmonella in its organs or actively and S. senftenberq, while Boonmar et al. (2008) excretSalmonella in its feces This is in agreement in Japan found two species only: S. derby and S. with a study of Singh et al. (2010) who mentioned javiana. that if the animal was infected with Salmonella The results of the present study resemble organisms, it may become a clinical case or an a study in Pakistan by Ali et al. (2008) which active or latent carrier . In the active carrier cases, recorded no infection for Salmonella in 200 mastitic the organisms are localized in the intestine and gall quarters in buffaloes, and also a study by Abdul bladder, from whence they are excreted with the Razak (1982) in Iraqi buffaloes which showed no feces, contaminating the environment and posing infection in mastitic milk in buffaloes. Also, many to a threat to susceptible hosts. In latent carrier previous studies reported similar fi ndings. cases, the organisms are localized in the lymph In the present study, the data demonstrated nodes, liver, spleen and tonsils, but the organisms a higher percentage of Salmonella isolates from are not excreted with the feces. the gall bladder and the liver, whereas all of the Salmonella serotypes (S. anatum, S.enteritidis, S.muenchen) were isolated from the gall bladder REFERENCES and the liver at a percentage of 40% from the total of Salmonella isolates in this study followed Abdul Razak, A.W. 1982. Studies of mastitis in by isolates from the mesenteric lymph nodes, the buffaloes. M.V.Sc. Thesis, Univercity of spleen and the cecum. Baghdad.

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Abdul Wahid, N.Y. and K.M. Raheem. 1981. Costa, E.O., F. Garino, Jr. E.T. Watanabe, S.J. Presence of Salmonella in domestic animals Ribeiro, P. Vezon, S.H. Gabaldi, N.R. in Iraq for the ten year 1969-1979, p. 17- Benites, P.S. Baruselli and A. Paske. 1997. 20. In Proceedings of the 1st Scientifi c Evaluation of the CMT positivita and Conference of the General Federation of microbiological status of the mammary Arab Veterinarians, Amman, Jordan. gland over the different lactation phases Ali, G., M. Muhammad, M. Arshad, I. Saqib and in buffalo cows (Bubalus bubalis), p. 631- J. Hassan. 2008. Bacteriology of mastitis 634. In Proceedings of 5th World Buffalo in buffaloes in Tehsil Samundri of district Congress, Caserta, Italy. Faisalabad, Pakistan. Pak. Vet. J., 28(1): 31- Galland, J.C., J.K. House, D.R. Hyatt, L. L. 33. Hawkins, N.V. Anderson, C.K. Irwin and Al-Nakashabandy, A.A. 2001. Some epidemiological B.P. Smeth. 2000. Prevalence of Salmonella and pathological studies on Salmonellosis in beef feeder steers as determination by in buffalo in Mosul province. M.Sc. Thesis, bacterial culture and ELIZA serology. Vet. College of Veterinary Medicine, University Microbiol., 76 : 143-151 . of Mosul, Mosul, Iraq. Gunasegaran, T., X. Rathinam, M. Kasi1, Bauer, A.W., W.M. Kirby, J.C. Sherris and A. Turck. K. Sathasivam, S. Sreenivasan and S. 1966. Antibiotic susceptibility testing by Subramaniam. 2011. Isolation and a standardized single disc method. Am. J. identifi cation of Salmonella from curry Clin. Pathol., 45(5): 493-496. samples and its sensitivity to commercial Bhatti, J.A., M. Younas, M. Abdullah, M.E. Babar antibiotics and aqueous extracts of Camelia and H. Nawaz. 2009. Feed intake, weight sinensis (L.) and Trachyspermum ammi gain and haematology in Nili-Ravi buffalo (L.). Asian Pacifi c Journal of Tropical heifers fed on mott grass and Berseem Biomedicine, 1(14): 266-269. fodder substituted with saltbush (Atriplex Hassanain, N.A. 2008. Detection of antibodies amnicola). Pak. Vet J., 29(3): 133-137. against zoonotic food borne pathogens in Boonmar, S., K. Markvichitrk, S. Chaunchoms, sera of food handlers. Global Veterinarian C. Chanda, A. Bangtrakulnonth, S. 2(5): 265-289. Pornrunangwongs, S. Yamamoto, D. Suzuki, Hassanain, N.A., M.A. Hassanain, R.M. Shaapan, K. Kozawa, H. Kimura and Y. Morita. H.A. Fadaly and A.M. Barakat. 2010. 2008. Salmonella prevalence in slaughtered Diagnosis of Eygyptian bovine meatborne buffaloes and pigs and antimicrobial zoonosis, Journal of American Science, susceptibility of isolates in Vientiane, Lao 6(12): 1526-1533. People’s Democratic Republic . J. Vet. Med. Iqbal, M., Khan, A.M.; Daraz, B. and Siddique, U. Sci., 70(12): 1345-1348. 2004 bacteriology of mastitic milk and in Coroian, A., D.C. Vodnar and M. Trif. 2010. vitro antibiogram of the isolates. Pak. Vet. Study of the main microbiological traits J., 24(4): 161-164. in Romanian buffalo milk. HVM Biofl ux Khan, A.J., M.S. Muhammad, A. Khan, M. Avais, Journal, 2(2): 92-98 . A. Maqbool, M. Salman and Z. Rehman.

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Review pertinent work, cite key references, • Chapter: Author(s) of the chapter. Year. Title of the chapter, explain importance of the research, and state objectives of pages of the chapter. In author(s) or editor(s). Title of the book. your work. Publisher name, Place of publication. Example: Citation in text: Sloss and Dufty. (1980) Materials and Methods. Provide sufficient detail so work Sloss, V. and J.H. Dufty. 1980. Disorders during pregnancy, can be repeated. Describe new methods in detail; accepted p. 88-97. In Sloss, V. and J.H. Dufty (eds.) Handbook of Bovine methods briefly with references. Obstetrics. Williams and Wilkins, Baltimore, U.S.A. Use of trade names. Trade names are to be Sabrani, M., K. Diwyanto and M. Winugroho 1994. A critical avoided in defining products whenever possible. review of buffalo research and development activities in Use of abbreviations and acronyms. At first text Indonesia. Past performanceand future strategies, p. 78-89. In use, define in parentheses. Do not use abbreviations and Proceedings of 1st Asian Buffalo Association Congress, acronyms in titles. Thailand. Results and discussion. Present results concisely using Submission manuscript figures and tables as needed. Do not present the same Submit the following items. information in figures and tables. Discuss principles and Cover letter. Identify the corresponding author and provide his/her relationship, point out exception. Show agreement with full name, address, numbers for telephone and fax, and e-mail address. published research work. The significances of work or Manuscript. In 12 point Times or Times New Roman. Type on one conductions should be presented in the end of discussion. side of A4 paper. Use one inch margins. Number all pages. Send an Tables. Number each table with Arabic numerals. Place a original manuscript and 1 photocopy. Disk. Include an IBM-formatted, 3-1/2" disk or 4-3/4" CD-ROM, descriptive caption at the top of each table. containing the manuscript in Microsoft Word. Figures. (graphs, charts, line drawings, photographs) Mail manuscript to: Number each figure with Arabic numerals under the By post: International Buffalo Information Center illustration. Lettering, data lines and symbols must be Office of University Library sufficiently large so as to be clearly visible when the figure Kasetsart University, is reduced to a size commonly used in the journal. 50 Pahonyothin Road, Chatuchak, References. List only those references cited in the text. Bangkok 10900, Thailand Required format of described below. Tel. 66-2-942-8616 By e-mail: [email protected]

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