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 Kanchana Anuphan International Buffalo Information Center, Offi ce of the University Library, Kasetsart University, Thailand 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 (June 2015) Vol.34 No.2
CONTENTS
Page
Case Report
A rare case of cleft-palate in buffalo fetus delivered through partial fetotomy M.A. Ganie, S.A. Hussain, M. Honparkhe and V. Doudagouddar...... 145
Dystocia due to a bulldog calf in a she buffalo (a case report) A.A. Wani, Tawheed, S. Shafi and R.A. Chowdhary...... 147
Surgical management of massive coccygeal varicosity in a Murrah buffalo V. Sangwan, S.K. Mahajan, A. Kumar, T. Singh and N.S. Saini...... 149
Vaginal prolapse in peri-partum primiparous Murrah buffalo complicated into endometritis and cystitis: A case report Pankaj Kumar, S. Dayal, Ramesh Tiwari, Dipyaman Sengupta, S.K. Barari and A. Dey...... 153
Short Communication
Assessment of blood biochemical profi le and nutritional status of buffaloes under fi eld conditions S.K. Maurya and O.P. Singh...... 161
Review Article
Buffalo production for emerging market as a potential animal protein source for global population M. Wanapat and V. Chanthakhoun...... 169
Original Article
Relationship between udder, skin and milk temperature in lactating Murrah buffaloes during the hot-humid season Bijay Kumar Chaudhari and Mahendra Singh...... 181
Genetic analysis of body weight traits of Surti buffalo G.M. Pandya, C.G. Joshi, D.N. Rank, V.B. Kharadi, B.P. Bramkshtri, P.H. Vataliya, P.M. Desai and J.V. Solanki...... 189 Buffalo Bulletin (June 2015) Vol.34 No.2
CONTENTS
Page
Original Article
Population structure and genetic variability of a closed Jaffarabadi buffalo herd from Brazil Paulo Costa Ferraz, Carlos Henrique Mendes Malhado, Alcides Amorim Ramos, Paulo Luiz Souza Carneiro, José Adrián Carrillo and Ana Claudia Mendes Malhado...... 197
Detection of deltamethrin resistance in buffalo louse, Haematopinus tuberculatus Nirbhay Kumar Singh, Manjurul Haque, Jyoti and Harkirat Singh...... 209
Sub-clinical mastitis in buffaloes: prevalance, isolation and antimicrobial resistance of Staphylococcus aureus K. Nithin Prabhu, Wilfred S. Ruban, G.S. Naveen Kumar, R. Sharada and R.D. Padalkar...... 215
Enteric parasitic infection in diarrhoeic buffalo calves Deepti Naag, Madhu Swamy and A.B. Shrivastav...... 223
Detection of verotoxin producing strain of E. coli in buffalo calves Deepti Naag, Madhu Swamy and A.B. Shrivastav...... 227
Retrospective study of hydatidosis in buffaloes slaughtered in Mirha Exports Private Limited in Punjab, India K. Aarif, B. Suhani, K.N. Mathur, R.L. Sharma, D.M. Makhdoomi, A. Nazir, A. Maria and Mehraj-U-Din...... 231
Induction of estrus in anestrus Murrah buffaloes and programmed breeding M. Thangapandiyan, P. Pothiappan, R.M. Palaniappan, E. Samual Joseph and D. Kathiresan...... 241
In vitro production of buffalo embryos by injection of immobilized and dead spermatozoa K. Tasripoo, K. Srisakwattana, W. Nualchuen and S. Sophon...... 245 Case Report Buffalo Bulletin (June 2015) Vol.34 No.2
A RARE CASE OF CLEFT-PALATE IN BUFFALO FETUS DELIVERED THROUGH PARTIAL FETOTOMY
M.A. Ganie1,*, S.A. Hussain2, M. Honparkhe1 and V. Doudagouddar1
ABSTRACT CASE HISTORY AND OBSERVATIONS
The present case describes the delivery of A full-term buffalo in third parity after a fetus with cleft palate from a dystociac buffalo completion of fi rst stage of labour was presented through partial fetotomy. to the university veterinary clinics. Earlier attempts at fi eld for the delivery of fetus per vaginum were Keywords: cleft palate, cranio-facial defects, not successful. The clinical parameters viz. rectal dystocia temperature, heart rate and respiratory rate were in the normal range. Per vaginal examination revealed a relaxed and wet birth passage without INTRODUCTION any edema. The fetus was in anterior longitudinal presentation and dorso-sacral position. The posture Congenital palate defects have been of the fetus suggested left lateral deviation of the reported in animals especially in dogs (Noden and head with forelimbs extended in the tract. Absence DeLahuanta, 1985; Stanley, 1993). Soft palate cleft of suckling and eye ball refl exes indicated a dead may or may not include the hard palate and may fetus. be unilateral, bilateral or medially located. The soft palate may be hypoplastic usually retaining a medial aspect and described as a pseudo uvula TREATMENT AND DISCUSSION or uvula-like process (Jason and Jonathan, 2004). Hereditary and environmental factors such as Following epidural anaesthesia (6 ml, poisonous plants, some drugs and viruses may 2% lignocaine hydrochloride) and after doing cause these defects (Noden and DeLahuanta, ample lubrication of the birth passage with sodium 1985; Stanley, 1993; Khaksary-Mahabady et al., carboxymethylcellulose gel (carmellose –Na 1%, 2006). The condition is rarely compatible with WDT, Garbsen, Germany) an attempt was made life. The present case describes delivery of buffalo to correct the fetal postural defect of the head, fetus having extensive cleft palate through partial but all the exercise was futile due to slippage of fetotomy. the muzzle from the hands. Thereafter, a decision was taken to perform fetotomy using Thygeson’s 1Department of Veterinary Gynaecology and Obstetrics, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India, E-mail: [email protected] 2Department of Veterinary Medicine, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India
145 Buffalo Bulletin (June 2015) Vol.34 No.2 fetotome loaded with fetotomy wire (Bovivet, REFERENCES Denmark). The right forelimb was amputated to make enough space for the correction of the Batavani, R.A. 2005. Cleft palate in buffalo calf (a deviated head. The fetus was then delivered after case report). J. Fac. Vet. Med. Univ. Tehran., application of moderate traction. The placenta 60(3): 303-304. was completely removed soon after delivery. The Noden, D.M. and A. Delahunta. 1985. Embryology buffalo was discharged on the same day with the of Domestic Animals Developmental routine prescription of antibiotics and supportive Mechanisms and Malformations. Williams therapy. and Wilkins, Philadelphia. p. 351-356. Gross examination revealed a large cleft Stanley, M.D. 1993. Congenital Abnormalities. Vet. on upper jaw extending up to forehead (Figure Clin North Am: Food Anim. Pract., 9. 1) and absence of teeth on the upper jaw. The Jason, F.H. and F.M. Jonathan. 2004. Reconstruction palate works as a barrier between the oropharynx of a bilateral hypoplastic soft palate in a cat. and nasopharynx. A defect that causes a loss of J. Am. Anim. Hosp. Assoc., 40: 86-90. this barrier allows open communication between Khaksary-Mahabady, M., R. Ranjbar, A. Arzi, these two cavities and more commonly occurs A.A. Papahn and H. Najafzadeh. 2006. A in late development (Jason and Jonathan, 2004). comparison study of effects of Echinacea The deformity is most likely a congenital defect extract and levamisole on phenytoin- and is often as a result of failure of the proper induced cleft palate in mice. Regul. Toxicol. mesenchyma to grow laterally to medially. The Pharmacol., 46: 163-166. reason of development of cleft palate in the present case could not be ascertained.
Figure 1. Fetus with cleft palate by partial fetotomy.
146 Case Report Buffalo Bulletin (June 2015) Vol.34 No.2
DYSTOCIA DUE TO A BULLDOG CALF IN A SHE BUFFALO (A CASE REPORT)
A.A. Wani*, Tawheed, S. Shafi and R.A. Chowdhary
ABSTRACT animal was presented with complaint that in spite of consistent straining for the past eight hours since A successful delivery of a bulldog calf the expulsion of the fi rst water bag, there was no (monster) through fetotomy is described. progression to second stage of labour. Obstetrical examination revealed presence of an abnormal Keywords: Bulldog monster, buffalo, congenital, foetus in transverse presentation, with both hind fetotmy limbs and one fore limb in birth canal. Since forced extraction was not possible, fetotomy was performed to deliver the foetus. INTRODUCTION
A monster is a malformed foetus. TREATMENT AND DISCUSSION Monstrosity is a disturbance of the development that involves sexual organs and causes great distortion Since the animal had been straining for of the individual. Monstrosities are associated the previous eight hours and the second stage of with either infectious disease or congenital defects labour had not progressed, per vaginal examination (Arthur et al., 2001) and may or may not interfere revealed dry texture of the foetal parts and birth with birth. It is important to know various types of canal. After the physical examination 5 ml monsters in animals which usually cause dystocia of 2% lignocaine was injected epidurally, to and which cannot be easily removed and so demand reduce the straining. Intramuscular injection of caesarean most of the time. dexamethasone (10 ml) was given to cope with the stress. The birth canal was then lubricated amply by carboxymethylcellulose dissolved in warm CASE HISTORY AND CLINICAL water to facilitate manipulation. Firstly, the whole EXAMINATION of the foetus was repelled into the abdominal cavity to increase space for manipulation, and then An eight-year-old indigenous buffalo with the left hind limb was amputated at the pelvic joint normal gestation period was brought to the Teaching by wire saw using Thygeson’s fetotome. After Veterinary Clinics, Guru Angad Dev Veterinary that, the forelimb was pushed back to extend it and Animal Science University, Ludhiana. The properly and the foetus was rotated to bring it to
Department of Veterinary Gynaecology and Obstetrics, Guru Angad Dev Veterinary and Animal Science University, Ludhiana, Punjab, India, *E-mail: [email protected]
147 Buffalo Bulletin (June 2015) Vol.34 No.2
posterior presentation. After this, the birth passage longer (prognathism). The nose was divided by was further lubricated and a snare was attached to a furrow. The rest of the body organs were fully the right hind limb and an anal hook was fastened developed. A bulldog may be confused with foetal in the anal cavity and traction was applied.With anasarca in which there is accumulation of fl uid in ample traction a male bulldog foetus was removed sub-cutaneous tissue and body cavity (Arthur et successfully after a period of 2 h. The buffalo al., 1989; Roberts, 2004). However in the present was treated with injection amoxicillin cloxacillin case there was no accumulating fl uid and hence combination 5 mg/ kg b.wt, injection meloxicam possibility of anasarca was over-ruled. 0.5 mg/ kg b.wt and supportive therapy for 7 days.
REFERENCES GROSS APPEARANCE OF THE MONSTER Arthur, G.H., D.E. Noakes, H. Pearson and T.J. Parkinson. 2001. Veterinary Reproduction The monster weighed 50 kg and had an and Obstetrics, 8th ed. W.B. Saunders Co. abnormally developed head. The appearance of Ltd., London. 118p. foetus was that of a bulldog with the head lacking Robert, S.J. 2004. Veterinary Obstetrics and distinct eyes and mouth parts (Figures 1 and 2). Genital Diseases, Indian reprints 2004, CBS The tongue of the foetus was protruding, the upper Publishers and Distributors, Delhi-110032. jaw was mal-developed, and the lower jaw was p. 73-74.
Figure 1. Bulldog calf with mal-developed maxilla Figure 2. Whole foetus. and protruding tongue.
148 Case Report Buffalo Bulletin (June 2015) Vol.34 No.2
SURGICAL MANAGEMENT OF MASSIVE COCCYGEAL VARICOSITY IN A MURRAH BUFFALO
V. Sangwan*, S.K. Mahajan, A. Kumar, T. Singh and N.S. Saini
ABSTRACT becomes dilated and tortuous to accommodate the blood volume retaining in it. The incidence The present case report describes successful of varicose veins in animals is low compared to surgical management of massive coccygeal human beings. In cows and buffaloes, lower limb varicosity by tail amputation in a 16 year-old veins like the saphaneous are mostly involved. female buffalo. This condition was 3 years chronic But, radial, cephalic, mammary and scrotal veins and the circumference of the tail had increased to may also show varicosity (Tyagi and Singh 1993; more than double the size of normal (48 cm) but the Rambabu et al., 2009). Although coccygeal length had reduced to half. The skin of the tail was varicosity in buffaloes has been reported, surgical thickened with multiple dry cracks and alopecia. management by tail amputation is not advised Frequent episodes of serosanguinous or blood due to the possibility of extensive haemorrahges discharge from the skin cracks, advanced stage of and subsequent shock (Kulkarni et al., 2005). The pregnancy, diffi culty while defecating, urinating, present case report describes a massive coccygeal sitting and standing were the major concerns. Tail varicosity in a buffalo which was successfully amputation was done from the fi rst inter-coccygeal treated by tail amputation. space under caudal epidural anesthesia. Unusually, both lateral coccygeal veins were markedly dilated but no fresh blood was present, only lengths of CASE HISTORY AND OBSERVATIONS clotted blood were removed. A female buffalo, aged 16 years and Keywords: coccygeal varicosity, buffalo, tail weighing 440 kg was presented to the Teaching amputation, surgery Veterinary Hospital with massive coccygeal varicosity (Figure 1). The history revealed that the condition had started 3 years before. Since then the INTRODUCTION circumference of the tail had gradually increased to more than double the size (48 cm) but the length had Varicosity is a result of valvular defect in reduced to half. The skin of the tail was thickened the superfi cial veins which allows blood to pool with multiple dry cracks and alopecia. Frequent and stretch the veins, further weakening the walls serosanguinous or blood discharge from the skin of the veins. With chronicity, the affected vein cracks was reported by the owner. The buffalo
Department of Veterinary Surgery and Radiology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India, *E-mail: [email protected]
149 Buffalo Bulletin (June 2015) Vol.34 No.2
was in the 10th month of gestation. On clinical lateral coccygeal veins were increased in diameter examination, the buffalo was otherwise healthy and were fi lled with lengths of clotted blood. No but had great diffi culty while defecating, urinating, fresh blood was fl owing in these distended veins. sitting and standing. The weight of the tail also The accumulated clotted blood was squeezed out appeared also pose extra weight on the old animal. from the cranial and lateral sides of sacro-coccygeal Considering all the aspects, surgical amputation of space. The size of the surrounding tissue subsided tail was advised and the owner was informed about following removal of clotted blood. Throughout the surgical risk involved in excessive bleeding and the surgical procedure, no bleeding was observed subsequent consequences. but some precautionary ligatures were applied around the veins to avoid any bleeding. The coccygeal artery on the ventral side was also TREATMENT AND DISCUSSION ligated. A layer of muscle sutures was applied with Polyglactin 910 no. 2 and the skin was closed Surgical amputation was done under using horizontal mattress sutures with braided silk caudal epidural anaesthesia using 2% lignocaine No. 3 (Figure 2). Postoperatively inj. Streptopenicillin hydrochloride in the sacro-coccygeal space. The 5 g twice daily and inj. Meloxicam 15 ml, once daily, skin was too thick and it was diffi cult to feel the joint intranuscularly was administered for 5 and 3 days, space through movement of tail. The amputation respectively. Antiseptic dressing was advised with was done from the most cranial healthy portion of povidone iodine and herbal fl y repellant ointment the tail i.e. at the 1st and 2nd inter-coccygeal joint (Himax; Indian Herbs Company, Saharanpur, space. A careful approach was taken for the major India). Gross examination of the amputated tail blood vessels, but there was no bleeding. Both revealed excessively dilated coccygeal vein and
Figure 1. Photograph showing massive coccygeal varicosity in a Murrah buffalo.
150 Buffalo Bulletin (June 2015) Vol.34 No.2
Figure 2. Photograph of the buffalo after tail amputation.
Figure 3. Photograph showing cross section of amputated tail with dilated coccygeal veins (yellow arrows) and excessive deposition of fi brous tissue (white arrow).
151 Buffalo Bulletin (June 2015) Vol.34 No.2 excessive fi brous tissue deposition on the ventral REFERENCES aspect of the tail (Figure 3). The skin sutures were removed on the 15th post-operative day. Telephonic Rambabu, K., M. Sreenu, R.V. Sureshkumar and follow up revealed that the animal calved normally T.S.C. Rao. 2009. Ultrasonography of the with some assistance and delivered a live calf. udder and teat in buffaloes. Buffalo Bull., Further follow up at 18 months found the animal to 28(1): 5-10. be healthy and doing well. Kulkarni, M.D, A.S. Kadam, A.V. Khanvikar and Varicosity of coccygeal veins in buffalo has O.N. Ladukar. 2005. Vein varicosis in a been reported in literature but surgical treatment was Pandharpuri buffalo-A Case Report. Buffalo not described (Tyagi and Singh, 1993; Kulkarni et Bull., 24(2): 24. al., 2005). In the present study, though the surgical Ramakrishna, O. 1993. Cardiovascular system. In attempt was guarded, it was made considering Tyagi, R.P.S. and J. Singh (eds.) Ruminant owner’s request and on ethical grounds. Chronic Surgery. CBS Publishers, New Delhi, India. retention of blood in the coccygeal veins with 259p. no movement of tail may have led to clotting of blood. Dilated veins, thickened skin and excessive deposition of fi brous tissue might be the cause for massiveness and immobility of the tail. In an adult buffalo, the circumference of a healthy tail at the base usually ranges from 20- 22 cm, but in this case the circumference of the tail at the base was 48cm. For limb varicosities, contrast techniques are advised to fi nd out the exact location of the arteriovenous shunt and attempts can be made to ligate and remove it in early stage, but with chronicity there develops multiple shunts and the ligatures are not helpful as the whole limb is engorged with blood (Tyagi and Singh, 1993). The reason behind the occurrence of coccygeal varicosity only in buffaloes but not in cows, need be explored. The weight and length of the tail along with increased laxity in the sacro-coccygeal joint of buffaloes are two of the hypotheses in the pathogenesis of coccygeal varicosity. These anatomical differences might predispose the buffalo tail to more self trauma than the cow tail. On long term follow up it was observed that once the hanging portion of the tail was amputated the progression of the condition stopped.
152 Case Report Buffalo Bulletin (June 2015) Vol.34 No.2
VAGINAL PROLAPSE IN PERI-PARTUM PRIMIPAROUS MURRAH BUFFALO COMPLICATED INTO ENDOMETRITIS AND CYSTITIS: A CASE REPORT
Pankaj Kumar1,*, S. Dayal, Ramesh Tiwari1, Dipyaman Sengupta2, S.K. Barari and A. Dey
ABSTRACT prolapsed. In such condition, clinician should also look into possibility of urinary bladder involvement Buffaloes are unique in many ways and if left untreated results in delay in recovery due compared to other cattle in many aspects of to persistant straining originating from irritation in animal health. The reproductive performance the urinary bladder. in buffalo is poor due to various diseases of the reproductive system, including genital prolapsed. Keywords: primiparous buffalo, prolapse, Prepartum vaginal prolapse is one of the major endometritis, cystitis reproductive disorders in buffaloes. The presented case report of a primiparous Murrah buffalo which was affected with pre-partum vaginal prolapse INTRODUCTION of low grade intensity which did not respond to conventional therapy instituted and latter after Buffaloes are the backbone of milk assisted parturition complicated into utero-vaginal production in India, whose buffaloes account for prolapse. After manual repositioning, infection more than 55% of the world population. Buffaloes in the uterus resulted in puerperal metritis and are unique in many ways compared to other later urinary bladder infection. The condition cattle in many aspects of animal health (Azawi, was not responding favorably to intrauterine and 2010). The reproductive performance in buffalo systemic medication with suitable antibiotic, anti- is poor due to various diseases of the reproductive infl ammatory and other supportive therapy. It system, including genital prolapse (Rao and resolved only after antibiotic administration both Sreemannarayana, 1983; Akhtar et al., 2012). locally oxytetracycline hydrochloride (500mg tablet Prepartum vaginal prolapse is one of the major Pfi zer® dissolved in 60 ml pyrogen free water) into reproductive disorders in buffaloes (Azawi, 2010). the urinary bladder as well as systemic intravenous It mostly occurs in the seventh month of pregnancy infusion of broad spectrum antibiotic combination or later immediately after parturition (Sah and of Ceftriazone 3000 mg and Tazobactam 375 mg Nakao, 2003). Etiologic factors of prepartum (Vetazo® Zydus Animal Health Limited) for 3 days. vaginal prolapse in buffaloes may be attributed to The present case report signifi es the possibility nutritional imbalance (Kelkar et al., 1989; Ahmed of urinary bladder infection during utero-vaginal et al., 2005), hormonal imbalance (Galhotra et al.,
1Division of Livestock and Fisheries Management, ICAR Research Complex for Eastern Region, ICAR Parisar, Patna, India, *E-mail: [email protected] 2Department of Surgery and Radiology, Bihar Veterinary College, Patna, India 3Department of Animal Reproduction, Gynaecology and Obstetrics, Bihar Veterinary College, Patna, India
153 Buffalo Bulletin (June 2015) Vol.34 No.2
1991), seasonal-managemental factors (Mishra et However, 4 days after parturition, straining resulted al., 1998; Gurcharan et al., 2003; Akhtar et al., 2010) in utero-vaginal prolapse. In time this resulted in and hereditary predisposition (Nanda and Sharma, puerperal metritis and clinical endometeritis with 1982). The hormonal changes that occur during this discharge of cream colour pus from the vagina last trimester of pregnancy, especially the increase on rectal examination and massage of uterus. of estrogen and the production of relaxin, cause a On rectal examination of the reproductive tract, relaxation of the pelvic ligaments and surrounding the uterus was enlarged, fl accid, swollen and soft tissue structures (Wolfe, 2009). Prepartum fl uctuating on palpation. The condition relapsed vaginal prolapse often terminate in uterine even after manual repositioning and necessary prolapse post-partum resulting from the infl uence treatment provided. Severity increased when of parturition. If infection is acquired, metritis or buffalo attempted urination, defecation or lying pyometra may result. The present communication down recumbent. As the duration of the prolapse reports a case of pre-partum vaginal prolapse increased, vascular compromise, trauma and fecal which complicated into uterine prolapse and fi nally contamination resulted in contamination of the terminated in puerperal metritis-endometeritis and vaginal mucosa. The buffalo exhibited discomfort urinary bladder infection. manifest by straining and frequent attempts to urinate and urine fl ow was turbulent (Figure 1 B). Clinical examination revealed a slight tear (Figure CASE HISTORY AND CLINICAL 1 A, marked with an arrow) on the vaginal mucosa OBSERVATIONS near urethral opening with congestion, odema and foul smell. The clinical examination of vital indices A Murrah buffalo aged about 3.5 years, revealed slightly elevated temperature ranging from primaparous and in the last month of gestation, 101.9°F to 103.2°F during the course of the day, was reported initially with the problem intermittent accelerated heart rate and respirations. Fresh urine prolapse of the vagina, with the vagina most sample was examined using reagent strips (Q Dx commonly protruding from between the vulva lips Urine Test 11 Nicholas®) for urinalysis, indicating when the animal was lying down while recumbent. specifi c gravity 1.030, 2+ positive for leukocyte, Vaginal prolapse later became aggravated in proteinuria (1+) and pH (7.5). the course with vaginal mass prolapsed during standing posture while attempting to urinate or defecate or strain. After 20 days, the entire vagina TREATMENTS AND DISCUSSION prolapsed and clinical examination revealed swollen, edematous and congested vaginal mucosa During the fi rst indication of low grade with the visible cervix at the most caudal part of vaginal prolapsed, the buffalo was treated with the prolapse. Animal parturated on day 25 with sodium salt of 4-dimethylamino-2 methylphenyl- mild physical assistance during parturition and phosphinic acid 0.2 gm (Tonophosphan® Vet gave birth to viable male calf weighing 35 kg. Thw Intervet®) injection 15 ml IM for 3 days and placenta (afterbirth) was discharged out without hydroxyprogesterone (Duraprogen® Vetcare®) any physical assistance in due course of time. Injection 2 ml (0.50 gm) by IM route for 3 days.
154 Buffalo Bulletin (June 2015) Vol.34 No.2
The buffalo was also provided with rear elevation daily by IM route for 5 days. Sood et al. (2011) also support using straw bags. The animal showed signs reported use of strepto-penicllin, meloxicam and of improvement but vulvar lip closure was complete. calcium borogluconate for managing postpartum A decrease in progesterone concentrations has been complications in buffalo. After 3 days of parturition, reported (Zicarelli, 2000) in buffaloes suffering the buffalo started showing severe straining and with vaginal prolapse. Successful progesterone this resulted in uterine proplase. Uterine prolapse therapy in pre-partum vaginal prolapse in buffaloes is more frequent in buffaloes in which vaginal (Sah and Nakao, 2003) and cattle (Bhattacharyya prolapse is observed during pregnancy, particularly et al., 2012) has been reported indicative of the beyond the 7th month of gestation. Noakes et al. role of low serum progesterone concentrations (2001b) also observed maximum number of such in causing pre-partum vaginal prolapse. After 20 cases in the last 2 months of gestation. Forced days of treatment prolapse recurred with higher extraction of the fetus has also been incriminated grade, showing prolapse of the vagina even when as an etiological factor (Noakes et al., 2001a). The animal was in standing position and straining was prolapsed mass was fi rst washed with potassium moderate. No treatment was provided due to buffalo permanganate (1:1000) solution and urine was nearing expected date of delivery. Only a rope truss discharged using sterile stainless steel catheter and was applied for physical immobilization around the later the mass was manually repositioned and a vulva. Similar management of prepartum vaginal jute rope truss was applied around the vaginal lip. prolapse using a rope truss has been reported by Considering the possibility of contamination, the Veeraiah and Srinivas (2010). Higher incidence buffalo was treated with a broad spectrum antibiotic of genital prolapse was also observed in buffaloes by the systemic route IM using Enrofl oxacin having more than 8 months of gestation by Patidar 10% (Byrocin®, Bayer HealthCare) for 3 days et al. (2010). High incidence (around 43%) of and provided with an indigenous preparation of prolapse has been reported in buffaloes (Samad et Prolapse-In (Cattle Remedies®) bolus 5 bolus twice al., 1987). The buffalo gave birth to male calf after daily for 3 days orally. Calcium and phosphorus was 25 days. Parturition was non-eventful except for supplemented using intravenous slow infusion of slight manual assistance provided for removal of 450 ml Mifex (Novartis®) once on the assumption the calf. Follow up treatment was provided using that hypocalcium may be one the factor for the Chromostat® (Adrenochrome Monosemicarbazone, prolapse. Hypocalcaemia results in myometrial Life Pharamceuticals) 25 ml IM as haemostatic fatigue and delays cervical involution (Murphy and once, Lixen IU (Cephalexine) 60 ml intrauterine Dobson, 2002). There was appreciable improvement for 3 days, Replanta® powder (Indian Herbs®) initially with decrease in straining and discharge 50gm as commercial indigenous preparation for of the placenta. However, again straining was uterine cleansing twice daily for 3 days, Melonex observed while urinating, urine was disrupted and plus® (meloxicam 5 mg and paracetamol 150 mg the animal tended to remain in urinating posture for per ml, Intas Pharmaceuticals®) as anti-pyretic some time after urination (Figure 1 B). Straining and analgesic 25 ml daily in two divided doses was suspected to result from infection of the uterus for 3 days, strepto-penicllin Dicrysticin® 2.5 gm resulting from contamination of the prolapsed mass (Sharabhia Zydus) given as systemic antibiotic once at the time of uterine prolapse and the systemic
155 Buffalo Bulletin (June 2015) Vol.34 No.2
AB
C D
E F
Figure 1. Buffalo suffering from vaginal prolapse, endometritis and cystitis before treatment (A, B, C and D) and after treatment (E and F).
156 Buffalo Bulletin (June 2015) Vol.34 No.2
antibiotic provided was not able to suppress the per ml, Intas Pharmaceuticals®) 25 ml was also infection. Discharge of pus on rectal examination administered by IM route daily in two divided doses indicated development of clinical endometritis as supportive anti-infl ammatory therapy for 3 days. (Figure 1 B, D). Raman and Bawa, (1977) also The buffalo responded well to this treatment and reported a high prevalence of postpartum infections straining decreased after 2 days of treatment with (38.54%) in buffalo. Puerperal metritis treatment proper urine fl ow and no pus discharge with uterine was attempted by intra-uterine douching with 60 tonicity was observed on rectal examination. ml of 5% Lugol’s iodine on alternate days for 3 There was also improvement in the feed intake of days along with anti-allergic pheniramine maleate the animal and milk production (Figure 1 E, F). (Avilin®, Intervet India) 10 ml by IM route for The animal reportedly showed signs of heat 48 5 days and anti-infl ammatory Melonex plus® days after the last treatment and was successfully (Meloxicam 5mg and Paracetamol 150 mg per ml, inseminated. Intas Pharmaceuticals®) 25 ml daily in two divided doses for 3 days. Lugol’s iodine is most commonly used under fi eld condition to treat endometritis. ACKNOWLEDGMENT Besides the antiseptic activity, Lugol’s iodine causes local irritation of endometrium, releases The authors acknowledge the help provided prostaglandin and regresses ovarian corpus luteum. by Prakash Khatiwada, Technical staff of Animal Postpartum metritis is one of the most important farm in carrying out the treatment and grateful disorders in buffaloes (Azawi et al., 2008), causing to the Head, Division of Livestock and Fisheries high economic losses due to prolonged inter- Management and Director, ICAR RCER, Patna for calving intervals and cost of treatment (Esslemont fi nancial support and guidance. and Peeler, 1993). The severity of condition decreased with thicker consistency and lesser pus discharge of rectal examination. The rope truss REFERENCES was also removed. However straining continued and endometritis persisted resulting in recurrent Ahmed, S.I., L.A. Ahmad, N.A. Lodhi and H.A. prolapse. Suspecting the possibility of urinary tract Samad. 2005. Clinical, haematological and infection based on results of urine examination serum macro mineral contents in buffaloes and clinical symptoms, the buffalo was treated with genital prolapse. Pak. Vet. J., 25: 167- with intra-urinary infusion of oxytetracycline 170. hydrochloride (500 mg tablet Pfi zer® dissolved Akhtar, M.S., L.A. Lodhi, I. Ahmad, Z.I. Qureshi, in 60 ml pyrogen free water) into the urinary and G. Muhammad. 2012. Serum ovarian bladder using sterile A.I. plastic sheet for 3 days steriod hormones and some minerals and systemic intravenous administration of broad concentration in pregnant Nili-Ravi spectrum antibiotic combination of ceftriazone buffaloes with or without pre-partum vaginal 3,000 mg and tazobactam 375 mg (Vetazo® Zydus prolapse. Pak. Vet. J., 32: 265-268. Animal Health Limited) for 3 days. Melonex Akhtar, M.S., L.A. Lodhi, I. Ahmad, Z.I. Qureshi, plus® (meloxicam 5 mg and paracetamol 150 mg and G. Muhammad. 2010. Incidence of
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pre-partum vaginal prolapse in Nili-Ravi Incidence of prolapse of genitalia in Murrah buffalo under two different agro-ecological buffaloes in relation to season, pregnancy, zones of Punjab, Pakistan, p. 785-788. In parity and management. Indian Vet. J., 75: Proceedings of 9th World Buffalo Congress, 254-255. Buenos Aires, Argentina. Murphy, A.M. and H. Dobson. 2002. Predisposition, Azawi, O.I. 2010. Uterine infection in buffalo subsequent fertility and mortality of cows cows: a review. Buffalo Bull., 29: 154-171. with uterine prolapsed. Vet. Rec., 151: 733- Azawi, O.I., S.N. Omran and J.J. Hadad. 2008. 735. A Study of endometritis causing repeat Nanda, A.S. and R.D. Sharma, 1982. Incidence and breeding of cycling Iraqi buffalo cows. etiology of pre-partum prolapse of vagina Reprod. Domest. Anim., 43: 735-743. in buffaloes. Indian J. Dairy Sci., 35: 168- Bhattacharyya, H.K., M.R. Fazili, B.A. Buchoo, 171. and A.H. Akand. 2012. Genital prolapse Noakes, E.D., Parkinson, T.J. and G.C.W. England. in crossbred cows: prevalence, clinical 2001a. Post parturient prolapse of the picture and management by a prolapse in uterus, p. 222-338 In Arthur’s Veterinary crossbred cows: prevalence, clinical picture Reproduction and Obstetrics. 8th ed. and management by a modifi ed Bühner’s Harcourt (India) Pvt. Ltd., New Delhi. technique using infusion (drip) set tubing as Noakes, E.D., T.J. Parkinson and G.C.W. England. suture material. Vet. Arhiv., 82(1): 11-24. 2001b. Prolapse of the vagina and cervix, p. Esslemont, R.J. and E.J. Peeler. 1993. The scope for 145-153 In Arthur’s Veterinary Reproduction raising margins in dairy herds by improving and Obstetrics. 8th ed. Harcourt (India) Pvt. fertility and health. Br. Vet. J., 149: 537- Ltd., New Delhi, India. 547. Patidar, A., S.P. Shukla, S.P. Nema and S.S. Pandey. Galhotra, M.M., G.C. Georgie and V.P. Dixit. 2010. Studies on surveillance of genital 1991. FSH, LH and prolactin in antepartum prolapse in buffaloes (Bubalus bubalis). vaginal prolapse of buffaloes (Bubalus Indian J. Field Vet., 6: 29. bubalis) in relation to cortisol and degree of Raman, S.R.P. and S.J.S. Bawa. 1977. Incidence of stress. Indian Vet. J., 68: 332-335. pre and post partum reproductive disorders Gurcharan, S., S.S. Sidhu and H.K. Verma. 2003. in buffaloes. Haryana Vet., 16: 99-101. Incidence of reproductive disorders of Rao, A.V.N. and O. Sreemannarayana. 1983. buffaloes in different zones of Punjab state. Clinical analysis of reproductive failure Punjab Agri. Uni. J. Res., 40: 79-80. among female buffaloes (Bubalus bubalis) Kelkar, M.A., S.K. Khar and V.M. Mandakhot. under village management in Andhra 1989. Studies on the ante partum prolapse Pradesh. Theriogenology, 18: 403-411. of the vagina in buffaloes and plasma Sah, S.K. and T. Nakao. 2003. Some characteristics trace element concentrations. Archiv. Fur. of vaginal prolapse in Nepali buffaloes. J. Experimentelle Veterinarmedizin, 43: 315- Vet. Med. Sci., 65: 1213-1215. 318. Samad, H.A., C.S. Ali, N.U. Rehman, A. Ahmad Mishra, U.K., R.G. Agrawal and R.K. Pandit. 1998. and N. Ahmad. 1987. Clinical incidence of
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reproductive disorders in buffaloes. Pak Vet. J., 7: 16-19. Sood, P., P. Kumar, A. Sharma, P. Barman and N. Kumar. 2011. Successful management of dystocia due to incomplete abortion in a buffalo. Buffalo Bull., 30: 226-227. Veeraiah, G. and M. Srinivas. 2010. Spontaneous extrusion of the intestines and uterus as a sequelae to vaginal prolapse in a buffalo heifer: A case report. Buffalo Bull., 29: 60- 61, 64. Wolfe, D.F. 2009. Medical and surgical management of vaginal prolapse in cattle. In Proceedings of 81st Annual Western Veterinary Conference, USA. Zicarelli, L. 2000. Considerations about the prophylaxis of the uterine and vaginal prolapse in Italian Mediterranean buffalo cows. Bubalus Bubalis, 3: 71-90.
159 Buffalo Bulletin (June 2015) Vol.34 No.2 Short Communication Buffalo Bulletin (June 2015) Vol.34 No.2
ASSESSMENT OF BLOOD BIOCHEMICAL PROFILE AND NUTRITIONAL STATUS OF BUFFALOES UNDER FIELD CONDITIONS
S.K. Maurya1 and O.P. Singh2
ABSTRACT calcium and phosphorus, whereas their energy requirements are being met under fi eld conditions The present investigation was conducted at the smallholder farmers’ level. to assess the nutritional status and biochemical profi le of buffaloes under fi eld conditions at the Keywords: nutritional status, buffalo, blood farmers’ level. The farmers were divided in to four biochemical profi le categories viz. landless, marginal, small, and large, depending on their land holdings. Measurement of body weight (BW) and body condition scores (BCS) INTRODUCTION along with blood biochemical profi ling for glucose, total protein, albumin, albumin/ globulin (A/G) Assessment of nutritional and health status ratio, cholesterol, serum calcium and phosphorus of animals is invaluable in present-day animal were utilized for nutritional status assessment. husbandry. Body weights and body condition The BW and BCS in the pooled population were scoring are the commonly used methods of 428.16±0.884 and 4.18±0.078, respectively. The assessing nutritional status of animals. But as BW of buffaloes differed signifi cantly among all these methods have drawbacks, the use of blood groups; whereas, the BCS of buffaloes did not metabolites in assessing the nutritional status of differ signifi cantly (P<0.05). The values of blood animals is becoming popular (Ndlovu et al., 2007). glucose, serum total protein, albumin, A/G ratio, Blood metabolite levels indicate the extent of cholesterol, Ca and P in the pooled population were metabolism of energy, protein and other nutrients in 57.66±0.949 mg/dl, 6.85±0.058 g/dl, 3.08±0.039 animals. Blood metabolites can be used objectively, g/dl, 0.83±0.019, 95.27±1.075 mg/dl, 9.42±0.102 reliably and routinely to assess the nutritional mg/dl and 5.15±0.068 mg/dl, respectively. The status of buffaloes. In India, the use of these blood values for blood glucose, total protein, albumin, metabolites is rare due to lack of equipment for A/G ratio and cholesterol were within the normal blood analysis and the high cost of analyzing range, whereas, serum Ca and P were below normal. the blood parameters. Several factors, such as Based on the present observation, it was concluded physiological status of an animal, breed, nutrition, that the buffaloes of this area are defi cient in season and age affect levels of blood metabolites.
1Department of Veterinary, College of Veterinary Science and Animal Husbandry, Narendra Deva University of Agriculture and Technology, Kumarganj, Faizabad, India, E-mail: [email protected] 2Department of Animal Husbandry, College of Veterinary Science and Animal Husbandry, Narendra Deva University of Agriculture and Technology, Kumarganj, Faizabad, India
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Combining body weights, body condition scores and stored at -20oC till further analysis. From the and blood metabolites increases the accuracy of serum samples, glucose, total protein, cholesterol, assessing the nutritional state and welfare of a albumin, calcium and phosphorus were measured buffalo population. Thus the present investigation using commercial kits (Span Diagnostics) as per was done to assess the nutritional status of buffaloes manufacturer’s protocol. of farmers in different landholding categories under Data thus generated were analyzed fi eld conditions. statistically using Graph Pad Prism version 5.00 software.
MATERIALS AND METHODS RESULTS AND DISCUSSION The study was conducted in Sultanpur district of Uttar Pradesh. Farmers were selected The body weight, body condition scores on the basis of a stratifi ed random design for the (BCS) and blood metabolite data of the buffaloes present investigation from the 30 villages of 10 of different categories of farmers are presented in blocks in 5 tehsils. The livestock owners were Table 1. categorized in to four categories depending on The mean and SEM of body weight of their land holdings viz. a.) Landless labourers / buffaloes (kg) in the Landless, Marginal, Small and Farmers (Livestock owners who had no land and Large categories of farmers were 415.97±1.906, whose mode of survival was labour); b.) Marginal 423.20±1.250, 431.87±1.307 and 446.31±2.454, farmers (Livestock owners who had up to 1.0 ha respectively. The overall body weight of the of land); c.) Small farmers (Livestock owners who buffaloes was 428.16±0.884. There was a signifi cant had between 1.0 to 2.0 ha of land) and d.) Large difference (P<0.05) in body weights of among the farmers (Livestock owners who had above 2.0 ha buffaloes of all categories of farmers. of land). The mean and SEM of BCS of the The animals of the above farmers buffaloes of the Landless, Marginal, Small and comprised the subject for the study. A total of 533 Large categories of farmers were 3.83±0.051, lactating buffalo were used for recording of body 4.07±0.032, 4.37±0.216 and 4.46±0.061, weight and body condition scores (BCS). The body respectively. The overall BCS of the buffaloes weights of the individual animals were calculated was 4.18±0.078. The BCS of buffaloes did not by using the Minnesota formula (Verma, 1992). differ signifi cantly (P<0.05) among the different Body condition of the study animals was scored categories of farmers. based on the criteria set by Richards et al. (1986) Similar fi ndings have also been reported and ranged from 1 to 9. by Chantalakhana et al. (1984) who studied the For the analysis of blood metabolites, 30 effect of seasonal fl uctuations and parturition on animals from each category were selected randomly. body weight of swamp buffaloes. They found the Five to ten ml of blood was collected from the jugular body weight of the buffaloes to be within a range vein of each animal. The blood was transported to of 424.6±54.8 to 473.6±43.5. the laboratory in ice, where serum was collected However, higher body weights were
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reported by Jogi and Lakhani (1996); they systems, respectively. They also concluded that reported a mean body weight of 563.87±14.21 and animals that are reared mainly on grazing land 519.39±13.29 in adult male and female buffaloes have in general lower body weight. on a livestock farm at Jabalpur. This difference The mean and SEM of blood glucose (mg/ could be attributed to the fact that Jogi and Lakhani dl) in the buffaloes of Landless, Marginal, Small observed the body weight on an organized farm and Large categories of farmers were 54.99±1.154, and in Murrah buffaloes, which have higher body 55.87±1.302, 58.79±2.066 and 60.98±2.405, weights. respectively. The blood glucose level in the Even lower body weights were reported overall population of buffaloes was 57.66±0.949. by Singh and Tulachan (2001). They reported There was no signifi cant difference (P<0.05) in adult body weights of 209 kg and 398 kg in the blood glucose levels among buffaloes of different smallholder cattle and buffaloes of Uttaranchal. categories of farmers. This may be due to the poor genetic conformation The mean and SEM of serum total protein and nutritional status of animals in the hills. (g/dl) in the buffaloes of Landless, Marginal, Small The present fi ndings concur with the study and Large categories of farmers were 6.51±0.089, of Lanyasunya et al. (2006) who reported that dairy 6.89±0.122, 6.91±0.086 and 7.07±0.098, cows in zero grazing systems recorded higher body respectively. The serum total protein (g/dl) level in weights (480±75 kg) compared to those in free the overall population of buffaloes was 6.85±0.058. (338±39 kg) and semi-zero (397±59 kg) grazing There was a signifi cant difference (P<0.05) in
Table 1. Body weight, BCS and blood biochemical parameters of buffaloes of different groups of farmers.
Parameter/ Landless Marginal Small Large Pooled Animal Body weight 415.97±1.906a 423.20±1.250b 431.87±1.307c 446.31±2.454d 428.16±0.884 (kg) BCS 3.83±0.051 4.07±0.032 4.37±0.216 4.46±0.061 4.18±0.078 Blood Glucose 54.99±1.154 55.87±1.302 58.79±2.066 60.98±2.405 57.66±0.949 (mg/ dl) Total Protein 6.51±0.089a 6.89±0.122 6.91±0.086 7.07±0.098b 6.85±0.058 (g/ dl) Albumin (g/ dl) 2.88±0.094a 3.04±0.053 3.15±0.053 3.27±0.049b 3.08±0.039 Albumin/ 0.81±0.050 0.79±0.034 0.84±0.036 0.87±0.037 0.83±0.019 Globulin Ratio Cholesterol 88.83±1.557a 95.28±2.111a,b 97.10±2.214b 99.86±0.957b 95.27±1.075 (mg/ dl) Calcium (mg/ 9.25±0.228 9.40±0.297 9.48±0.168 9.56±0.077 9.42±0.102 dl) Phosphorus (mg/ dl) 5.01±0.120 5.08±0.088 5.15±0.139 5.35±0.178 5.15±0.068 Values with different superscripts in a row differ signifi cantly: (P<0.05).
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serum total protein levels of buffaloes between the were 9.25±0.228, 9.40±0.297, 9.48±0.168 and Landless and Large categories of farmers. Buffaloes 9.56±0.077, respectively. The serum calcium of the other categories of farmers had no signifi cant level in the overall population of buffaloes was difference (P<0.05) in serum total protein levels. 9.42±0.102. There was no signifi cant difference The mean and SEM of serum albumin (P<0.05) in serum calcium levels of buffaloes levels (g/dl) in the buffaloes of Landless, among the different categories of farmers. Marginal, Small and Large categories of farmers The mean and SEM of serum phosphorus were 2.88±0.094, 3.04±0.053, 3.15±0.053 and levels (mg/dl) in the buffaloes of Landless, 3.27±0.049 respectively. The serum albumin (g/ Marginal, Small and Large categories of farmers dl) level in the overall population of buffaloes was were 5.01±0.120, 5.08±0.088, 5.15±0.139 and 3.08±0.039. There was a signifi cant difference 5.35±0.178, respectively. The serum phosphorus (P<0.05) in serum albumin levels of buffaloes level in the overall population of buffaloes was between Landless and Large categories of farmers. 5.15±0.068. There was no signifi cant difference Buffaloes of the other categories of farmers had no (P<0.05) in serum phosphorus levels among the signifi cant difference (P<0.05) in serum albumin buffaloes of different categories of farmers. levels. The mean values of glucose, protein and The mean and SEM of A/G ratio in the cholesterol were within the normal physiological buffaloes of Landless, Marginal, Small and Large range, whereas the levels of serum calcium categories of farmers were 0.81±0.050, 0.79±0.034, and phosphorus were lower than the normal 0.84±0.036 and 0.87±0.037, respectively. The A/G physiological range (Kaneko et al., 1997). ratio in the overall population of buffaloes was Typically, growth is measured as an 0.83±0.019. There was no signifi cant difference increase in body weight, and it includes not only (P<0.05) in albumin/ globulin ratio among the cell multiplication (hyperplasia) but also cell buffaloes of different categories of farmers. enlargement (hypertrophy) and incorporation of The mean and SEM of serum cholesterol specifi c components from the environment (for levels (mg/dl) in the buffaloes of Landless, example, apatite deposition) (Flier and Maratos- Marginal, Small and Large categories of farmers Flier, 2000). Growth can be monitored by using body were 88.83±1.557, 95.28±2.111, 97.10±2.214 and weights. Body weights are commonly used because 99.86±0.957 respectively. The serum cholesterol measurement is easier and quicker to perform and level in the overall population of buffaloes was does not require much expertise. Body weights are 95.27±1.075. There was a signifi cant difference commonly used for monitoring nutritional status (P<0.05) in serum cholesterol levels of buffaloes and growth of animals (Chimonyo et al., 2000). between Landless and Small and Landless and However, the body weight of an animal per se does Large categories of farmers. Serum cholesterol not refl ect its nutritional status (Oulun, 2005). levels of buffaloes among the other categories Body condition scoring describes the of farmers did not differ signifi cantly (P<0.05). systematic process of assessing the degree of The mean and SEM of serum calcium fatness of an animal (Nicholson and Sayers, 1987). levels (mg/dl) in the buffaloes of Landless, The score refl ects the plane of nutrition on which an Marginal, Small and Large categories of farmers animal has been exposed over a reasonable length
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of time (Stuth et al., 1998). The loin, ribs, tail head, (Reynolds et al., 2003). brisket, fl ank, vulva and/or rectum and udder are the Total protein levels are lower in young important parts of the body used in determining the animals and higher in mature animals whilst score. Physiologically, the proportions of protein albumin levels are lower at birth and then increase and water of the animal’s bodyweight decrease (Doornenbal et al., 1988; Otto et al., 2000). as it gains body condition (NRC, 1996). Several Malnutrition decreases albumin levels. Total authors have documented association between protein and albumin refl ect availability of protein, body condition scoring and fertility (Buckley et al., and their concentration decline in the face of protein 2003) and health (Roche and Berry, 2006). defi ciency. The metabolic profi le test in dairy animals On the basis of above observations, it is used to assess the nutritional status, to predict can be concluded that buffalo of unorganized occurrence of metabolic diseases and to diagnose smallholder farmers of Sultanpur district of Uttar the diseases, and to assess the fertility status of Pradesh are defi cit in the minerals calcium and animals (Ingraham and Kappus, 1988). But the phosphate whereas their energy requirements are success of the metabolic profi le test is limited being met with their diet. I It is also concluded that because several non-dietary factors like herd origin, blood biochemical profi les must be combined with stage of lactation, milk yield and season of the year traditional methods for assessment of nutritional affect the concentration of blood metabolites (Lee status. et al., 1978). Blood metabolite concentrations represent an integrated index of the adequacy of nutrient REFERENCES supply in relation to nutrient utilization of buffaloes (Chester-Jones et al., 1990). They give Buckley, F., K. O’Sullivan, J.F. Mee, R.D. Evans an immediate indication of an animal’s nutritional and P. Dillon. 2003. Relationships among status at that point in time (Pambu-Gollah et al., milk yield, body condition, cow weight, and 2000). In the dairy industry, the use of metabolic reproduction in spring-calving Holstein- profi les for assessing the nutritional and health Fresians. J. Dairy Sci., 86: 2308-2319. status of cows is widespread (Doornenbal et Chantalakhana, C., P. Bunavelchevin and P. Veerasit. al., 1988; Grunwaldt et al., 2005). Use of such 1984. Effect of seasonal fl uctuations and metabolites in the management of fi eld buffalo is parturition on body weight of swamp still uncommon. buffalo. Buffalo Bull., 3(3): 3-7. Blood glucose has a moderate diagnostic Chester-Jones, H., J.P. Fontenot and H.P. Veit. 1990. value in the assessment of nutritional status Physiological and pathological effects of of buffaloes as it varies moderately in blood. feeding high levels of magnesium to steers. Insuffi cient nutrient intake can reduce circulatory J. Anim. Sci., 68: 4400-4413. glucose and cholesterol levels. In conditions of Chimonyo, M., N.T. Kusina, H. Hamudikuwanda undernutrition, the blood levels of propionate and and O. Nyoni. 2000. Reproductive other precursors derived from the diet decrease thus performance and body weight changes in causing a reduction in the rate of glucose synthesis draught cows in a smallholder semi-arid
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farming area of Zimbabwe. Trop. Anim. the nutritional status of beef cattle: current Health Pro., 32(6): 405-415. practices and future prospects. Afr. J. Doornenbal, H., A.K.W. Tong and N.L. Murray. Biotechnol., 6(24): 2727-2734. 1988. Reference values of blood parameters Nicholson, M.J. and A.R. Sayers. 1987. Reliability, in beef cattle of different ages and stages of reproducibility, and sequential use of lactation. Can. J. Vet. Res., 52: 99-105. condition scoring of Bos indicus cattle. Flier, J. and E. Maratos-Flier. 2000. Energy Trop. Anim. Health Pro., 19: 127-135. homeostasis and body weight. Primer. Curr. NRC. 1996. Nutrient Requirements of Dairy Cattle., Biol., 10(6): 215-217. 7th rev. ed. National Research Council. Grunwaldt, E.G., J.C. Guevara, O.R. Estevez, National Academic Press, Washington, DC. A. Vicente, H. Rousselle, N. Alcunten, Otto, F., P. Baggasse, E. Bogin, M. Harun and F. D. Aguerregaray and C.R. Stasi. 2005. Vilela. 2000. Biochemical blood profi le Biochemical and haematological of Angoni cattle in Mozambique. Israel measurements in beef cattle in Mendoza Veterinary Medical Association, 55(3): 1-9. plain rangelands (Agerntina). Trop. Anim. Oulun, Y. 2005. Variation in the blood chemical Health Pro., 37(6): 527-540. constituents of reindeer, signifi cance Ingraham, R.H. and L.C. Kappus. 1988. Metabolic of season, nutrition and other extrinsic profi le testing Veterinary Clinics North and intrinsic factors, Acta Univesitatis America. Food Animal Practice, 4: 391- Ouluensis, Scientiae Rerum Naturalium, A 411. 440. Jogi, S. and G.P. Lakhani. 1996. Study of body Pambu-Gollah, R., P.B. Cronje and N.H. Casey. weights, rate of gain and mortality percentage 2000. An evaluation of the use of blood in Murrah buffalo calves. Buffalo Bull., metabolite concentrations as indicators of 15(3): 51-54. nutritional status in free-ranging indigenous Kaneko, J.J., J.W. Harvey and M.L. Bruss. 1997. goats. S. Afr. J. Anim. Sci., 30(2): 115-120. Clinical Biochemistry of Domestic Animals, Reynolds, C.K., P.C. Aikman, B. Lupoli, D.J. 5th ed. Academic Press, New York, USA. Humphries and D.E. Beever. 2003. Lanyasunya, T.P., W.H. Rong, E.A. Mukisira and Splanchnic metabolism of dairy cows S.A. Abdulrazak. 2006. Performance of during the transition from late gestation dairy cows in different livestock production through early lactation. J. Dairy Sci., 86: systems on smallholder farms in Bahati 1201-1217. Division, Nakuru District, Kenya. Pakistan Richards, M.W., J.C. Spitzer and M.B. Warner. Journal of Nutrition, 5(2): 130-134. 1986. Effect of varying levels of postpartum Lee, A.J., A.R. Twardock, R.A. Bubar, J.E. Hall and nutrition and body condition at calving on C.L. Davis. 1978. Blood metabolic profi les: subsequent reproductive performance in their use and relation to nutritional status of beef cattle. J. Anim. Sci., 62: 300-306. dairy cows. J. Dairy Sci., 61: 1652-1670. Roche, J.R. and D.P. Berry. 2006. Peripaturient Ndlovu, T., M. Chimonyo, A.I. Okoh, V. Muchenje, climatic, animal and management factors K. Dzama and J.G. Raats. 2007. Assessing infl uencing the incidence of milk fever in
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grazing systems. J. Dairy Sci., 89: 2775- 2783. Singh, V.K., P. Singh, A.K. Verma and U.R. Mehra. 2008. On Farm assessment of nutritional status of lactating cattle and buffaloes in urban, peri urban and rural areas of Middle Gangetic Plains. Livestock Research for Rural Development, 20(8), Article #130. Retrieved from http://www.lrrd.org/lrrd/ lrrd20/8/singh20130.htm. Singh, V. and P.M. Tulachan. 2001. Smallholder dairy in Uttaranchal mountains: Performance Indicators. ENVIS Bulletin: Himalayan Ecology and Development, 9(1): 24-26. Stuth, W., P. Dyke, A. Jama and J. Corbett. 1998. The use of NIR/NUBTAL, PHYGROW, and APEX in a meta-modelling environment for an early warning system to monitor livestock nutrition and health. National Workshop on Early Warning System for Monitoring Livestock Nutrition and Health, Addis Ababa, Ethiopia. Verma, D.N. 1992. Cow Keeping and Management. JBD Publishing House, New Delhi, India.
167 Buffalo Bulletin (June 2015) Vol.34 No.2 Review Article Buffalo Bulletin (June 2015) Vol.34 No.2
BUFFALO PRODUCTION FOR EMERGING MARKET AS A POTENTIAL ANIMAL PROTEIN SOURCE FOR GLOBAL POPULATION
M. Wanapat1 and V. Chanthakhoun2
ABSTRACT research and to expand world buffalo production.
The aim of this review paper is to focus Keywords: buffalo production, meat, milk, animal on the world buffalo population trend and the protein, population buffalo’s potential as a protein food source for the increasing world population. The buffalo (Bubalus bubalis) is a ruminant animal that has for a long INTRODUCTION time contributed to integrated farming systems, as a source of draft power, transportation, manure, Currently, livestock systems have both meat, milk and may more. The increase in meat positive and negative effects on the natural consumption is quite dramatic due to the linear resource base, public health, social equity and increase in the world population especially in the economic growth (World Bank, 2009). On the other developing countries. Buffaloes have been raised hand, growth of human population in the world by rural farmers. They are well-adapted to harsh has also been accompanied by unprecedented environments and are capable of utilizing low economic growth that has allowed increases in quality roughages especially agricultural crop- income and purchasing power, and thus, changes residues and by-products. Their potential for meat in food preferences (Cruz, 2010). These recent production using locally available feed resources developments have major impacts on demand for is therefore remarkable. Furthermore, the quality animal derived products meat and milk (Cruz, of buffalo meat has been found high in iron and 2010), while meat consumption in developing conjugated linoleic acid (CLA), which are essential countries has been continuously increasing from for good health. In addition, rumen methane a modest average annual per capita consumption mitigation in buffalo could be manipulated by of 10 kg in the 1960s to 26 kg in 2000 and a feeding feeds which naturally contain plant projected 37 kg around the year 2030 according to secondary compounds (tannins, sapponins) that the FAO. Moreover, livestock products contribute affect rumen microorganisms and feed utilization 17% of the kilocalorie consumption and 33% of and reduce methane, which is a cause of global the protein consumption globally, but there are warming. It is therefore highly recommended for large differences between rich and poor countries those engaged in buffalo production to do more (Rosegrant et al., 2009). While the per capita
1Tropical Feed Resources Research and Development Center (TROFREC), Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand, E-mail: [email protected] 2Faculty of Agriculture and Forestry, Souphanouvong University, Luang Prabang, Laos
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consumption of livestock products is much greater require meat with low fat content, therefore, non- in developed countries, it has fallen slightly over the traditional meat is becoming an important source. last decade; however, substantial growth occurred Among them, buffalo meat has high protein levels, in the developing countries of Asia (FAO, 2009). low fat content and cholesterol compared to beef Demand for livestock products as food will nearly (Murthy and Devadason, 2003). Hence, researchers double in sub-Saharan Africa and South Asia, from are trying to improve buffalo of potential breeds some 200 kcal per person per day in 2000 to around and meat quality for use by humanity for milk, 400 kcal per person per day in 2050. On the other draft power and meat. hand, in most OECD countries that already have According to Heintz (2001) when the meat high calorie intakes of animal products (1000 kcal of young buffalo and young cattle, were compared, per person per day or more), consumption levels it was clearly shown that buffalo meat is indeed will barely change, while levels in South America as good as cattle meat. Results of feeding trials and countries of the former Soviet Union will comparing buffalo and cattle of similar age, feeding increase to OECD levels (Van Vuuren et al., 2009). regimes and carcass treatment clearly demonstrated The agricultural production sector is catering that there is enough scientifi c evidence to show that increasingly to globalized diets. Retailing through buffalo meat were more tender than beef (Neath supermarkets is growing at 20 percent per annum et al., 2007). Moreover, buffalo meat is required in countries such as China, India and Vietnam, and with the increase of population and increased this will continue over the next few decades as requirements of food in developing countries. The urban consumers demand more processed foods, price of buffalo milk is twice that of cows’ milk, thus increasing the role of agribusiness (Rosegrant buffalo skin is used in the leather industry, buffalo et al., 2009). manure is used for fuel in rural areas. Buffalo The world buffalo population is estimated meats have the potential to improve the livelihoods to be approximately 177.247 million, spread of small-holder farmers. Therefore, the objective of through some 42 countries. Of the total, 171 this review paper is to focus on the world buffalo million (97%) buffaloes are found in Asia, while population trend due to the potential capacity of the approximately 5.38 million (3%) are found in the buffalo to produce meat and other by-products to rest of the world (FAO, 2008). In Asia meat from keep up with demand. ruminants constitutes only about 21.0% of the total meat production, and buffalo meat makes up about 11.52% of the total ruminant meat and about 2.7% BUFFALO POPULATION AND of all meat produced in the region (Cruz, 2010). PRODUCTION SYSTEMS The average annual growth rate in production was about 1.3%. Undoubtedly, the majority of The world population of buffalo (Bubalus the world’s buffalo meat is in Asia, representing bubalis) has been estimated at over 140 million 91.89% and amounting to 3.08M tons in 2008 head (FAO, 1991). Of these, 97 percent are found (FAO, 2010). In the past century, buffalo meat was in the Asia and Pacifi c region, mainly in India (75 not accepted by the consumers and there was no million), China (21 million), Pakistan (14 million) market. However, the actual trends in consumption and Thailand (6 million). Moreover, the world
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buffalo population has increased and is estimated to feeding regimes and carcass treatment, clearly be approximately 177.247 million, spread through demonstrated that there is ample scientifi c evidence some 42 countries of which 171 million (97%) of to show that buffalo meat is more tender than beef. them are found in Asia, while approximately 5.38 Anjaneyulu et al. (2007) reported that buffalo million (3%) are found in rest of the world (FAO, meat has been recognized as one of the healthiest 2008). The buffalo population is distributed in major meats for human consumption. It has outstanding buffalo producing countries in Asia; therefore, attributes such as: lower intramuscular fat, lower Asian buffaloes dominate the world population, cholesterol and calories, higher units of essential representing 96.4% of the worldwide population amino acids, higher biological value, and higher of 180.70 million as of 2008 (FAO, 2010). Within mineral content. The quality of buffalo meat has the the Asian region, about 74.8% of buffaloes are been markedly improved with crossbreds, and in in South Asia, 12.8% in East Asia, and only 8.4% Australia, it is hoped to buffalo meat will become are found in South-East Asia. Small proportions of the future standard in the “Tender Buff Program”, the world population are found elsewhere: about which has gained much popularity (Lemcke, 0.626% in America, 0.22% in Europe, and 2.895% 1997). in Africa (Table 1). Compared to beef, buffalo meat contains 1% less intramuscular fat, 92% less saturated fat, 25% fewer calories, 67% less cholesterol, 11 to TREND OF BUFFALO PRODUCTION 30% more protein, and 10% more minerals. The FOR MEAT AND ITS CHEMICAL low cholesterol in buff meat has been emphasized COMPOSITION by Lazar (2001) (Table 2). When swamp buffaloes were fed grass and Khan and Iqbal (2009) reported that about grass with legumes, Polbumrung (2007) found the 88% of world buffalo meat is in Asia and over 21 total fatty acid content was 1,317.2 mg/100 g in million buffaloes slaughtered annually in Asia, the meat of buffalo fed grass+legumes, which were 48% are slaughtered in India and about 18% each higher than that (1,027.2 mg/100 g) of the meat of in Pakistan and China. Pakistan annually produces buffaloes fed grass only by (Table 3). 0.68 m tones of buffalo meat valued at 102 billion Buffalo meat has certain outstanding ($ 1.3 billion). Therefore, in developing countries attributes such as lower intramuscular fat, cholesterol of Asia where meat from ruminants constitutes only and calories, higher units of essential amino acids, about 21.0% of the total meat production, buffalo biological value and iron content (Anjaneyulu et meat is about 11.52% of the total ruminant meat, al., 1990a). Moisture (76.4%), protein (20.4%), and about 2.7% of all meat produced in the region. fat (1.5%), ash (1.0%), water soluble proteins The average annual growth rate in production was (5.1%), salt soluble proteins (7.2%), non-protein about 1.3%. Undoubtedly, the majority of world’s nitrogen (0.37%) and hydroxyproline (0.12%) of buffalo meat is in Asia, representing 91.89% and LD muscle from male buffalo calves were reported amounting to 3.08M tons in 2008 (FAO, 2010). (Anjaneyulu et al., 1985) and also attributed the Neath et al. (2007) reported that feeding low energy value of 6.8 Kcal/g on dry matter basis trials comparing buffalo and cattle of similar age, to the negligible amount of marbling. Buffalo meat
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Table 1. Buffalo populations of major countries, by region.
Region/Country Population Head, % World 180,702,923 100.00% Asia East Asia China 23,271,909 12.879% South East Asia Cambodia 746,207 0.413% Indonesia 2,191,640 1.213% Lao People’s Democratic 1,155,000 0.639% Republic Malaysia 131,000 0.072% Myanmar 2,923,568 1.618% Philippines 3,338,570 1.848% Thailand 1,699,469 0.940% Timor-Leste 110,000 0.061% Vietnam 2,897,700 1.604% South Asia Bangladesh 1,262,000 0.698% India 98,595,000 54.562% Nepal 4,496,507 2.488% Pakistan 29,883,000 16.537% Sri Lanka 318,530 0.176% America Brazil 1,131,986 0.626% Europe Bulgaria 8,968 0.005% Georgia 17,200 0.010% Italy 294,000 0.163% Turkey 84,705 0.047% Africa Egypt 5,231,162 2.895% Source: FAO (2010).
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Table 2. Certain nutritional components of buffalo and cow meat (100 g).
Contents Buffalo Cow Calories (Kcal) 131.00 289.00 Protein (g) 26.83 24.07 Fat (g) 1.80 20.69 Fatty acids Saturated 0.60 8.13 Monosaturated 0.53 9.06 Polysaturated 0.36 0.77 Cholesterol (mg) 61.00 90.00 Minerals (mg) 641.80 583.70 Vitamins (mg) 20.95 18.52 Cholesterol (mg) 61.00 90.00 Minerals (mg) 641.80 583.70 Vitamins (mg) 20.95 18.52 Source: USDA (1996).
Table 3. Effect of feeds (grass and legumes) on buffalo production meat and their chemical composition.
Items Grass+legumes Grass only Color Lightness, L* 35.9 35.5 Redness, a* 14.2 14.9 Yellowness, b* 10.2 9.5 Drip loss, % 5.7 5.9 Chemical composition, % Protein 22.6 22.4 Fat 1.5 1.1 Moisture 74.5 75.8 Chloresterol, mg/100 g 44.6 45.4 Triglyceride, g/100 g 1.8 1.2 Collagen, g/100 g meat Total collagen 1.2 1.3 Soluble collagen 0.3 0.3 Insoluble collagen 0.9 1.0 Shear force value, Newton 47.1 40.3
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Table 3. Effect of feeds (grass and legumes) on buffalo production meat and their chemical composition. (Cont.)
Items Grass+legumes Grass only Sensory evaluation Tenderness 7.6 7.5 Juiciness 7.5 7.4 Flavour 7.1 6.8 Overall acceptability 7.5 7.2 Fatty acids, % Myristic acid (C14:0) 1.9 2.4 Myristoleic acid (C14:1) 0.3 0.3 Pentadecanoic acid (C15:0 0.5 0.5 Palmitic acid (C16:0) 23.5 24.0 Palmitoleic acid (C16:1) 2.2 2.6 Heptadecanoic acid (C17:0) 1.2 1.0 cis-10-Heptadecenoic acid (C17:1) 0.7 0.6 Stearic acid (C18:0) 21.4 21.3 Oleic acid (C18:1n9c) 36.9 37.2 Linoleic acid (C18:2n6) 4.6 3.9 Conjugated linoleic acid (LCA) 0.6 0.7 γ-linoleic acid (C18:3n6) 4.6 3.9 α- linoleic acid (C18:3n3) 2.0 2.1 cis-8, 11, 14-Eicosatrienoic acid 0.2 0.2 (C20:3n6) Arachidonic acid (C20:4n6) 2.2 1.6 Eicosapentaenoic acid (C20:5n3) 1.2 1.2 Docosahexaenoic acid (C22:6n3) 0.3 0.3 Saturated fatty acid (SFA) 48.8 49.4 Mono-unsaturated Fatty Acid (MUFA) 40.1 40.7 Polyunsaturated fatty acids (PUFA) 11.1 9.9 Polyunsaturated fatty acids: Saturated 0.2 0.2 fatty acid (PUFA:SFA) Omega-6 (total n6) 7.0 5.6 Omega-3 (total n3) 3.5 3.6 Omega-6:Omega-3 n6:n3 2.1 1.6 Total fatty acids, mg/100 g meat 1,317.2 1,027.2 Source: Polbumrung (2007)
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Table 4. Fatty acid composition (mg/g fat) of buffalo milk and cheese.
Fatty acid Raw milk Cheese C4:0 24.0±1.2 25.7±2.0 C6:0 19.3±1.1 17.4±2.1 C8:0 14.3±1.4 13.2±1.7 C10:0 31.5±4.1 32.0±2.8 C12:0 32.3±3.2 29.5±1.6 C14:0 68.0±6.9 70.2±3.2 C14:1 5.0±1.50 6.1±0.3 C15:0 19.1±3.6 22.4±2.8 C16:0 319.4±44.1 321.4±12.1 C16:1 10.2±6.7 10.8±2.1 C18:0 111.5±11.3 122.8±9.0 C18:1 (trans 11) 39.5±4.5 38.6±3.4 C18:1 (cis9) 241.6±12.3 251.3±14.3 C18:2 17.1±1.5 16.1±1.8 C18:3 5.1±0.7 5.0±0.4 Trans-10,cis-12 CLA 0.3±0.0 0.4±0.1 cis-9,trans-11 CLA 4.2±0.2 4.8±0.2 CLA/LA 0.266 0.32 SCFA (%) 89.1 88.3 MCFA (%) 463.8 470.4 LCFA (%) 423.3 439.0 Saturated (%) 65.9 65.6 Monounsaturated (%) 31.4 31.8 Polyunsaturated (%) 2.7 2.6 Data are average ± SD. Cheese samples were taken at day 1 after manufacturing. CLA, conjugated linoleic acid; LA, linoleic acids; SCFA, short chain fatty acids (C4-C10); MCFA, medium chain fatty acids (C12- C17); LCFA, long chain fatty acids (C18:0 to C18:3).
Source: Van Nieuwenhove et al. (2004).
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has been recognized as one of the healthiest meats underneath and were regularly pumped out for use for human consumption. India recorded an export on the forage fi elds. The male buffalo calves aged of 0.3 million tons of buffalo meat worth 16, 200 8 to 10 months were purchased from farmers and million (US$ 360 million) in the year 2004-05 quarantined for 15 days. They were then fed on (APEDA, 2005). high energy/high protein diets to gain an additional Since the demand for lean red meat weight of about 120 kg in four months. worldwide has been increasing consistently, buffalo meat is expected to attract consumer preference due to its leanness. According to Borghese (2005), TRENDS OF BUFFALO FOR MILK average per capita demand for meat in developing PRODUCTION AND ITS CHEMICAL countries will be about 25 kg by 2010 and the COMPOSITION buffalo is considered to have strong potential to meet the requirement of increased per capita Recently, buffalo milk production in Asia consumption. represents 96.78% of the total volume of the world’s Ranjhan (2004) has shown us a way buffalo milk of 89.2 million tons. Production in to achieve enhanced yield of hygienic meat South and Southwest Asia, primarily from India and using intensive feeding of buffalo calves under Pakistan, contributed a hefty 93.17% (FAO, 2010). commercial feedlot housing. The facilities included Buffaloes are signifi cant sources of milk in this sub- environmentally controlled animal houses with region contributing as much as 68.35% of the total slatted fl oors where urine and dung are collected milk yield in Pakistan, and 56.85% of the total milk
Table 5. Typical composition of buffalo milk and cow milk.
Traits Cow Buffalo Total solids (%) 13.10 16.30 Fat (%) 4.30 7.90 Protein (%) 3.60 4.20 Lactose (%) 4.80 5.00 Tocopherol (mg/g) 0.31 0.33 Cholesterol (mg/g) 3.14 0.65 Calcium (mg/100 g) 165.00 264.00 Phosphorus (mg/100 g) 213.00 268.00 Magnesium (mg/100 g) 23.00 30.00 Potassium (mg/100 mg) 185.00 107.00 Sodium (mg/100 g) 73.00 65.00 Vitamin A (incl. Carotene) IU. 30.30 33.00 Vitamin C (mg/100 g) 1.90 6.70
Source: Anonymous (1995).
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production in India. Average annual growth rate in the liver, intestines and plasma of mice fed milk or buffalo milk production between the years 1998 to cheese and the concentration of CLA was higher in 2008 was 4.39% in the whole of Asia. Evidently, tissues of mice fed cheese results clearly indicate this growth level can readily be associated with that buffalo milk and cheese are a good source of the consistent good growth in milk production in CLA for human nutrition (Van Nieuwenhove et al., India and Pakistan, with a combined growth rate 2004). of 4.52%. Buffalo milk production in China has Comparative milk composition in the not been as robust as that in the South Asia, with buffalo and the cow is presented in Table 5 and average annual growth of 1.6%. What is notable is shows that buffalo milk is healthy as it is richer the 8.18% average annual growth in SEA, though in in saturated fatty acids. The Indian diet is mainly relative volume, such increases have not been very vegetarian and people relish the hot thick creamy signifi cant. Among the SEA countries, Myanmar milk for their breakfast associated with higher fat registered the highest buffalo milk production, content (Table 5). Swamp buffalo milk has even apparently because it had the highest population higher fat (7.9%), protein (4.2%), calcium (264.0 (about 40,000) of dairy buffalo as of 2000 (Hlaing, mg/100 g) and cholesterol (0.65 mg/g) contents. 2001). Buffalo milk production in the other SEA However, according to Borghese (2010) countries comes mainly from smaller populations milk composition improved in Italy in just a few of introduced riverine buffaloes and the resulting years, with the average protein content moving crossbreds of the dairy breeds with the existing from 4.4 to 4.73% in 2002, to 4.65% in 2003, to swamp buffalo population. In this sub region where 4.66 in 2009 while the fat content moved from farmers tend their animals primarily for work, only 7.3 to 8.3% in 2002, to 8.1% in 2003 and to a small percentage of crossbreds produced are fully 8.24 in 2009 without operating any selection for utilized for milk production. the characters of protein and fat content. These Ruminant meat and milk contain various constraints explain why the percentage of recorded fatty acids, especially conjugated fatty acids (CLA), buffaloes in countries where the buffalo seems to which possess benefi cial biological activities be more important than cattle is so low. The highest in mammals. The cis-9, trans-11 CLA (rumenic proportion of milk recorded buffaloes, in fact, is acid, RA) has been shown to reduce the incidence found in Italy (26%), in Romania it is 19.6%, and in of cancer (Ip et al., 1991). The CLA content of Iran it is 5.7%, while in other countries the recorded meat and milk is strongly linked to the ruminal buffaloes are about 1% of the total dairy females biohydrogenation (BH) of cis-9, cis-12 C18:2 or less, whereas in Bulgaria, the few buffaloes are (linoleic acid, LA) and cis-9, cis-12, cis-15 C18:3 all recorded (Borghese, 2010). Lactation length (linolenic acid, LNA). In Argentina, the buffalo was varies from 180 to 270 days. The latter fi gure is recently introduced as an alternative breed, with a the ideal period but is achieved only in Italy and in milk of good nutritional value and with high protein Romania.In Italy only the mean production is over and fat content (Van Nieuwenhove et al., 2004). 2,200 kg per lactation, while in other countries the The infl uence of buffalo dairy products on lipid production is less than 1900 kg. In Italy, the milk mice tissues was determined as both cis-9,trans-11 production of 46,799 recorded buffaloes (ANASB, and trans-10, cis-12 CLA were incorporated into 2009) was 2,221 kg in 270 days of lactation with
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8.24% fat and 4.66% protein. These buffaloes were consumer demand and market possibilities modern raised in 290 herds with an average of 161.3 head applied technologies for milk and meat processing per farm. and industry could be a substantial basis to increase the availability of quality products make for a positive trend in buffalo development and in higher POTENTIAL EXPORTS OF BUFFALO profi ts for farmers and linked companies (Borghese, MEAT AND MILK PRODUCTS TO 2010). Meat export is desired to effectively utilize VARIOUS MARKETS the available livestock resources and improve returns to the farmers by popularizing buffalo meat Currently, buffalo markets in the world, (Kadeephan et al., 2009). Buffalo meat is the major especially, in Asia, have been increasingly item of Indian animal product export comprising emerging for good quality meat and milk and with 48.76% of the total animal products exported. high acceptability by consumers as shown by the The major destinations of buffalo meat include increase in buffalo meat exports of about 159,703 Malaysia, USA, Jordan, Oman and UAE. Buffalo MT in 1995-96 to as much as 456,907 MT in 2008- meat is exported to the tune of 306,970.81 MT 2009 (APEDA, 2010). Export of buffalo meat from amounting to US$252 million, which is the highest India rose two fold in volume from 234,355M among all animal products that are exported form tons in 2001 to 456,907M tons in 2009, and by India (APEDA, 2008). more than 400% in value from US$243.4M to The share of Indian meat exports in the US$1.043B during the same period. Interestingly, world market is less than 2%. Adequate meat Vietnam, Malaysia and the Philippines received production potential exists in the country to meet about 43.13% of the total Indian buffalo export the domestic demand and to substantially increase 2008-2009; the rest went to 10 countries in the the export. A signifi cant price differential between Middle East (Boghess, 2010). In the Philippines, domestic and export market exists for buffalo meat practically all brands of processed corned beef and it has better prospects for the meat traders. For are derived from imported Indian buffalo meat instance, in Bareilly district, Uttar Pradesh, India, constituting more than 60.0% of the total buffalo buffalo meat is sold by the retailer at the rate of supply in the Philippines (Lanuza, 2009). This 40/kg. Whereas, the same meat used for export has allowed signifi cant growth in the local meat purpose fetches 60/kg at the municipal slaughter processing industry in the country (Boghess, house (Kandeepan et al., 2009). 2010). Intensive feeding of male buffaloes in commercial feedlots for quality meat production CONCLUSIONS started in 1999. Male calves at the age of 8-10 months are purchased from farmers and are fed high Based on this review, it could be concluded protein/high energy diet to put on additional weight that the majority (about 97.1%) of buffalo of 120 kg in 4 months. Murrah yearlings grow by resources are from Asian buffalo. Their potential 0.9 to 1.0 kg/day and would have high dressing for meat and milk production is great. Demand for percentage (Ranjhan, 2004). Buffalo meat and milk buffalo products in the future could be increased
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by human health concerns because they contain Delgado, C., M. Rosegrant, H. Steinfeld, S. Ehui and essential compounds such as high protein, iron and C. Courbois. 1999. Livestock to 2020-The CLA. Promotion and development of smallholders’ Next Food Revolution. Food, Agriculture support for buffalo production using local feed and the Environment Discussion Paper 28. resources should be encouraged and recommended IFPRI/FAO/ILRI. in order to achieve potential production and F.A.O. 1991. Selected Indicators of Food and profi tability. Agriculture Development in Asia Pacifi c Region, 1980-90. FAO Regional Offi ce for Asia and the Pacifi c, Bangkok, Thailand. REFERENCES F.A.O. 2008. Food and Agriculture Organization. Rome Italy. STAT database. www.fao.org. A.N.A.S.B. 2009. Statistical data. Italian Buffalo F.A.O. 2009. Food Outlook: Global Market Breeders’ Association. Analysis. December 2009. p. 42-51. Anonymous. 1995. Indian Dairyman. 47(8): 63- F.A.O. 2010. Production Yearbook. 64. Hlaing, S. 2001. Country report for workshop on Anjaneyulu, A.S.R., S.S. Senger, V. Lakshmanan water buffalo development-Myanmar, p. 65- and D.C. Joshi. 1985. Meat quality of male 69. In Proceedings of Regional Workshop buffalo calves maintained on different levels in Water Buffalo Development, Surin, of protein. Buffalo Bull., 4: 45-50. Thailand. Anjaneyulu, A.S.R., N. Sharma and N. Kondaiah. Heintz, G. 2001. Water buffaloes as meat animals, 1990. Specifi c effect of phosphate on the p. 10-17. In Proceedings of Regional functional properties and yields of buffalo Workshop in Water Buffalo Development, meat patties. Food Chem., 36: 149-159. Surin, Thailand. A.P.E.D.A. 2005. Export statistics for agro and food Ip, C., S.F. Chin, J.A. Scimeca and M.W. Pariza. products India. 2004-05. Processed Food 1991. Mammary cancer prevention by Products Export Development Authority, conjugated dienoic derivatives of linoleic New Delhi, India. acid. Cancer Res., 51(6): 118-116, 124. Borghese, A. 2005. Buffalo production and Lazar, V. 2001. We like buffalo. Meat Proc., 40: research. FAO Ed. REU Technical Series. 80-85. 67: 1-315. Lanuza, M. 2009. An overview of the Philippine Borghese, A. 2010. Development and perspective livestock industry: Looking at the of buffalo and buffalo market in Europe Philippine buffalo (carabao) sub-sector. and Near East, p. 20-31. In Proceedings of In Proceedings of Buffalo Propagation 9th World Buffalo Congress, Buenos Aires, Conference: Conservation and Utilization Argentina. of Buffalo Genetic Resources in Southeast Cruz, L.C. 2010. Recent Developments in the Asia. NLRI, Taiwan. Buffalo Industry of Asia, p. 7-19. In Neath, K.E., A.N. Del Barrio, R.M. Lapitan, J.R. Proceedings of 9th World Buffalo Congress, Herrera, L.C. Cruz, T. Fujihara, S. Muroya, Buenos Aires, Argentina. K. Chikun, M. Hirabayashi and Y. Kanai.
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2007. Difference in tenderness and pH Van Nieuwenhove, C., P.G. Cano, A.P. Chaia and decline between water buffalo and beef S. Gonzalez. 2007. Chemical composition during post mortem aging. Meat Sci., 75: and fatty acid content of buffalo cheese 499-505. from northwest Argentina: Effect on lipid Murthy, T.R.K. and I.P. Devadason. 2003. Buffalo composition of mice tissues. J. Food Lipids, meat and meat products – An overview, p. 14: 232-243. 193-199. In Proceedings of the 4th Asian Van Nieuwenhove, C., S. Gonzalez, A. Perez Chaia Buffalo Congress on Buffalo for Food, and A. Pesce. 2004. Conjugated linoleic Security and Employment. New Delhi, acid in buffalo (Bubalus bubalis) milk from India. Northwest Argentina. Milchwissenschaft, Ranjhan, S.K. 2004. Commercial production 59: 506-508. of buffalo meat with social agenda. In Van Vuuren, D.P., D.P. Vuren van, W.O. Ochola, S. Proceedings of 7th World Buffalo Congress, Riha, M. Giampietro, H. Ginzo, T. Henrichs, Manila, Philippines. 1: 1-7. S. Hussain, K. Kok, M. Makhura, M. Mirza, Rosegrant, M.W., M. Fernandez, A. Sinha, J. Alder, K.P. Palanisama, C.R. Ranganathan, S. Ray, H. Ahammad, C. de Fraiture, B. Eickhout, C. Ringler, A. Rola, H. Westhoek, M. Zurek, J. Fonseca, J. Huang, O. Koyama, A.M. P. Avato, G. Best, R. Birner, K. Cassman, Omezzine, P. Pingali, R. Ramirez, C. Ringler, C. Fraiture, B. de Easterling, J. Idowu, P. S. Robinson, P. Thornton, D. van Vuuren, H. Pongali, S. Rose, P.K. Thornton and S. Yana-Shapiro, K. Ebi, R. Kruska, P. Munjal, Wood. 2009. Outlook on agricultural change C. Narrod, S. Ray, T. Sulser, C. Tamagno, and its drivers, p. 255-305. In McIntyre, M. van Oorschot and T. Zhu. 2009. Looking B.D., H.R. Herren, J. Wakhungu and R.T. into the future for agriculture and AKST Watson (eds.) Agriculture at a crossroads, (Agricultural Knowledge Science and Washington, DC: Island Press. Technology), p. 307-376. In McIntyre, B.D., World Bank. 2009. Minding the stock: bringing H.R. Herren, J. Wakhungu and R.T. Watson public policy to bear on livestock sector (eds.) Agriculture at a Crossroads. development. Report no. 44010-GLB. Rosegrant, M.W., M. Fernandez and A. Sinha. 2009. Washington, DC., USA. Looking into the future for agriculture and AKST (Agric. Knowledge, Sci. Technol.), p. 307-376. In McIntyre, B.D., H.R. Herren, J.W. Wakhungu and R.T. Watson (eds.) Agriculture at a Crossroads: Global Report: International Assessment of Agricultural Knowledge Science and Technology, Island Press, Washington DC., USA. United States Department of Agriculture (U.S.D.A). 1996. Technical Bulletin, Number 8. USDA, New York, USA.
180 Original Article Buffalo Bulletin (June 2015) Vol.34 No.2
RELATIONSHIP BETWEEN UDDER, SKIN AND MILK TEMPERATURE IN LACTATING MURRAH BUFFALOES DURING THE HOT-HUMID SEASON
Bijay Kumar Chaudhari* and Mahendra Singh
ABSTRACT INTRODUCTION
Lactating Murrah buffaloes were selected The buffalo (Bubalus bubalis) has been from the institute herd to investigate the possibility given the name “the black gold of South Asia” of the use of milk temperature as a marker of due to its signifi cant contribution (95%) to milk hot and humid stress during the rainy season. production (Javaid et al., 2009). This species is also Experimental buffaloes were provided with mist known as the world second most important milk and fan facilities while another group of buffaloes producing animal (McDowell et al., 1995; Bhatti et served as control. Udder skin temperature (UST), al., 2009). India has the largest buffalo population skin temperature (ST) and milk temperature (MT) (98 million), which constitute 57% of the total were measured at weekly intervals. The ambient buffalo population in the world. Environmental maximum temperature signifi cantly infl uenced milk factors have a direct effect on the neuroendocrine temperature (P<0.01), skin temperature and udder setup in buffalo and makes them very susceptible to skin temperature in morning and evening intervals. thermal stress owing to low density of sweat glands The changes in milk temperature between animals and scant hair coat (Acharya, 1988; Cockrill, 1993; and between weeks was non-signifi cant. The Pandey and Roy, 1966; Razdan, 1988). Summer availability of mist and fan cooling tended to reduce weather causes stress in lactating buffalo resulting the milk temperature non-signifi cantly in different in depression of milk production (Soch et al., 1997; weeks of the experiment. UST and ST variation Dolejs et al., 2000a), reduction of feed intake (Holter was signifi cant between week and between groups et al., 1996; Holter et al., 1997; Umphrey et al., (P<0.01). The signifi cant changes in ST, UST and 2001) and a deleterious effect on the physiologic non-signifi cant decline in MT indicated that mist status in cows (West, 2003). The effect of heat stress and fan cooling was effective in restoring these on the physiological status of lactating buffaloes variables to normal range in buffaloes. is relatively less studied in comparison with that in lactating cows. It has been found that the upper Keywords: Murrah buffalo, udder skin temperature, limit of the temperature humidity index (THI) at skin temperature, milk temperature, hot and humid which cattle may maintain stable body temperature stress is between 72 and 76 (Igono et al., 1992; Ravagnolo et al., 2000). The body temperature of a buffalo is lower than that of a cow in spite of the fact that
Dairy Cattle Physiology Division, National Dairy Research Institute, Karnal, Haryana, India, *E-mail: [email protected]
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its black skin absorbs more heat due to its having buffaloes were hand milked twice a day in the one-sixth the density of sweat glands of a cow morning (6 am) and in the evening (6 pm). Milk skin (STID, 1981). Rectal and skin temperature temperature was recorded during the milking using fl uctuates much more in buffaloes than in tropical a digital thermometer. The analysis of data was cattle under increased ambient temperature (Koga carried out by three-way ANOVA with interactions. et al., 2004; Aggarwal and Singh, 2008; Marai Mean and standard error was calculated and the and Habeeb, 2010). Milk temperature has been values were tested for signifi cance (Snedecor and reported as a reliable indicator of climatic stress Cochran, 1989). in exotic cows in shaded spray free stalls during the summer (Igono et al., 1985; Igono, 1985) and a positive trend with increasing environmental RESULTS AND DISCUSSION temperature has been reported (Igono et al., 1988); however, information on whether milk temperature The average maximum temperature and udder skin temperature are affected by high during the experiment period of six weeks varied ambient temperature in buffaloes is not available. from 31.50 to 34.10ºC and the THI score ranged The present investigation was undertaken to fi nd between 81.60 and 85.56 (Table 1). The average out the effect of the hot-humid season on skin, THI during the experiment was much higher udder skin and the milk temperature in Murrah than the THI score of 72 (Ravagnolo et al., 2000; buffaloes. Igono et al., 1992). The moderately high ambient temperature and the high humidity resulted in a signifi cantly higher THI score of 85.56 in fi rst MATERIALS AND METHODS week of experiment; THI declined in the second week and was subsequently maintained till the Twelve lactating Murrah buffaloes having end of the experiment. The MT varied signifi cant II or III parity were selected from the livestock herd (P<0.01) between morning and evening intervals of the National Dairy Research Institute, Karnal. in both group of buffaloes (Table 1 and Figure 1). The buffaloes were divided into two groups and However, MT varied non-signifi cantly between had green fodder (maize) available ad lib. while groups, between weeks and between animals. concentrate mixture was offered based on milk However, UST varied signifi cantly between groups yield. A mist and fan facility was provided to the (P<0.01), between intervals (P<0.01) and between experimental group from 10 am to 8:00 pm control weeks (P<0.01). group buffaloes were maintained without mist or The values of UST was signifi cantly lower fan. The temperatures of udder skin (UST), body (P<0.01) in the morning in both the groups and skin temperature (ST) was recorded twice a day in varied between 91.60 and 93.88oF in the morning. the morning (9 am) and in afternoon (3 pm) during A signifi cant increase in the evening UST was the hot and humid season (August-September). observed in all the weeks of the experiment (Figure The temperatures were recorded by an infrared 2). Contrary to this, ST declined signifi cantly thermometer (METRAVI MT-2) at a distance of between morning and evening (P<0.01). A similar about 10 cm away from the site of recording. The pattern of change in ST was found during morning
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Table 1. Mean (±SE) milk, udder skin and skin temperature of control and experiment (mist+fan) Murrah buffaloes during the hot-humid season.
Weeks of Experiment Week I II III IV V VI Avg.Max.Temp.(oC) 34.10 33.5 32.30 33.00 32.10 31.50 Avg. THI M 79.80 77.85 76.84 78.64 78.22 75.60
E 85.56 83.04 82.68 84.69 84.26 81.60
MILK TEMPERATURE(oF) Control 96.08ax 96.91ax 95.66ax 97.00acx 98.00bcx 97.75bcx M ±0.54 ±0.40 ±0.54 ±0.39 ±0.38 ±027 98.31 ay 98.10 ay 99.53 ay 99.31 ay 99.51 ay 99.76ay E ±0.36 ±0.40 ±0.34 ±0.36 ±0.35 ±0.27 Treatment 96.95ax 97.28ax 96.98ax 97.31ax 97.13ax 97.00ax M ±0.51 ±0.44 ±0.62 ±0.48 ±0.38 ±0.37 98.23ay 98.01ax 98.15ay 98.58ay 98.03ax 97.65ax E ±0.47 ±0.41 ±0.49 ±0.44 ±0.41 ±0.40 UDDER SKIN TEMPERATURE(oF) Control 93.88ax 92.23ax 94.18abx 91.60ax 93.22ax 94.63abx M ±0.60 ±0.65 ±0.72 ±0.74 ±0.79 ±0.69 98.35ay 96.46aby 99.86acy 95.46by 96.73aby 97.66aby E ±0.42 ±0.68 ±0.63 ±0.50 ±0.79 ±0.70 Treatment 92.83ax 93.30ax 94.00abx 93.40ax 91.66ax 92.01ax M ±0.59 ±0.62 ±0.59 ±0.48 ±0.72 ±0.69 90.80ax 91.21ax 90.05ay 91.95ax 89.68ax 90.85ax E ±0.71 ±0.61 ±0.52 ±0.65 ±0.61 ±0.65 SKIN TEMPERATURE(oF) Control 94.85ax 92.76ax 93.80bx 94.60ax 93.43ax 94.13ax M ±0.57 ±0.78 ±0.40 ±0.85 ±0.81 ±0.84 98.99ay 97.63ay 101.10ay 99.13by 98.81by 97.96by E ±0.34 ±0.58 ±0.65 ±0.53 ±0.73 ±0.79 Treatment 92.98ax 95.20ax 93.68ax 94.05ax 92.56ax 94.90ax M ±0.78 ±0.65 ±0.59 ±0.45 ±0.55 ±0.87 90.86ax 91.75ax 92.08ax 91.75ax 90.30ax 91.93ax E ±0.65 ±0.71 ±0 .55 ±0.96 ±0.57 ±0.70
Values with different superscript in a row (a, b, c) and in a column(x, y) differ (P<0.05). The values with different superscript a,b in a row and x,y in a column differ (P<0.05).
183 Buffalo Bulletin (June 2015) Vol.34 No.2
Figure 1. Dirunal variation in milk temperature, udder skin temperature and skin temperayure in lactating Murrah buffaloes during the hot humid season.
Figure 2. Overall mean values of milk temperature, udder skin temperature and skin temperature in Murrah buffaloes during the hot humid season.
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and evening recording. ST varied signifi cantly suggesting that cooling the udder would directly between groups (P=0.05) and between weeks cool the internal body temperature. They further (P<0.01). Interaction of week × interval and group observed that current day mean air temperature × interval was signifi cant for ST. The UST was during the hot period had the greatest impact on positively correlated to ST (r=0.568, P<0.01), cow p.m. milk temperature, and minimum air while MT was positively correlated between temperature had the greatest infl uence on a.m. milk morning and evening intervals (P<0.01, r=0.528). temperature. In this study also the milk temperature The correlation between UST with MT, and ST increased in the evening recordings in both groups; with MT was non-signifi cant. however, in the experimental group, the increase in In the present study the signifi cant MT in the evening was non-signifi cant due to effect correlation of the skin temperature with the udder of mist and fan cooling. West et al. (2003) reported skin temperature suggested that high humidity a curvilinear relationship of milk temperature with and high ambient temperature affects the skin dry matter intake and milk yield. The signifi cant temperature in Murrah buffaloes. However, it affects changes in milk temperature in the morning and milk temperature slightly and non-signifi cantly. afternoon also indicated that rhythmicity of UST Therefore, a large number of observations on milk and body temperatures should be considered in temperature could give a clear cut idea of the effect research on the chrono-biology of milk secretion of a hot and humid climate on milk temperature. and mastitis (Bitman et al., 1984). In the present The signifi cant changes in ST and UST was study, none of the buffalo exhibited heat during expected due to the effect of the hot and humid the experimentt as milk temperature also increases climate. The morning and evening variation in ST during oestrus (McArthu et al., 2005). Research on and UST was attributed to the effect of the hot and farm demonstrations show that micro-sprinklers, humid climate as both skin and UST were directly mist, spray jets, fans and ventilation alone or in affected by the hot-humid climate. Milk temperature combination can be effective in relieving heat is solely controlled by internal body temperature stress in farm cows and buffaloes in hot and humid and therefore changes in milk temperature were conditions (Schultz, 1988; Strickland et al., 1989; non-signifi cant. The availability of mist and fan Turner et al., 1989; Anonymous, 2006; Agarwal cooling signifi cantly decreased UST, ST and MT in and Singh, 2008). Our fi ndings also suggest that use experimental buffaloes as reported earlier in cow of mist and fan decreased the skin temperature and and buffaloes (Anonymous, 2006; Gudev et al., udder skin temperature and the milk temperature 2007; Agarwal and Singh, 2008). The fi nding of the in Murrah buffaloes and corroborate the earlier present study is in agreement with the observation report (Agarwal and Singh, 2008, 2010). Further, that milk temperature shows a positive trend with research need be conducted on large number of increasing environmental temperatures (Igono et al., Murrah buffaloes throughout the year covering 1988) and may serve as an indicator of the impact of different seasons on milk temperature association the climatic environment on lactating cattle (Igono with UST and the mastitis. et al., 1988). Bitman et al. (1984) reported a high correlation (R=0.98) between udder temperature and internal body temperature of dairy cows
185 Buffalo Bulletin (June 2015) Vol.34 No.2
REFERENCES Bemabucci, U., B. Ronchi, N. Lacetera and A. Nardone. 2002. Markers of oxidative status Acharya, R.M. 1988. The buffalo: dairy, draught and in plasma and erythrocytes of transition meat animal of Asia, p. 3-17. In Proceedings dairy cows during hot season. J. Dairy Sci., of the 2nd World Buffalo Congress, Vol. 2, 85: 2173-2179. Part I, New Delhi, India. Bhatti, J.A., M. Younas, M. Abdullah, M.E. Babar Aggarwal, A. and M. Singh. 2008. Changes in skin and H. Nawaz. 2009. Feed intake, weight and rectal temperature in lactating buffaloes gain and haematology in Nili-Ravi buffalo provided with showers and wallowing heifers fed on mott grass and Berseem during hot dry season. Trop. Anim. Health fodder substituted with saltbush (Atriplex Prod., 40: 223-228. amnicola). Pak. Vet J., 29(3): 133-137. Al-Ani, F.K. and J.G.E. Vestweber. 1984. Mammary Bitman, J., A. Lefcourt, D.L. Wood and B. Stroud. blood fl ow measurement associated with 1984. Circadian and ultradian temperature development of bovine udder edema. Am. J. rhythms of lactating dairy cows. J. Dairy Vet. Res., 45(2): 339-341. Sci., 67(5): 1014-1023. Al-Ani, F.K., J.G. Vestweber and H.W. Leipold. Blackshaw, J.K. and A.W. Blackshaw. 1994: Heat 1985. Blood fl ow parameters associated stress in cattle and the effect of shade on with udder edema in Jersey cattle affected production and behaviour. Aust. J. Exper. with rectovaginal constriction. Vet. Rec., Agric., 34: 285-295. 116(6): 156-158. Cockrill, W.R. 1993: Developing the water buffalo: Anderson, R.R. 1985. Mammarygland in Lactation, a decade of promise. Buffalo J., 9: 1-11. p. 30-31. In Anderson, R.R., R.J. Collier, R. Cook, N.B., R.L. Mentink, T.B. Bennett and K. Heald, C.W. Jenness, B.L. Larson and H.A. Burg. 2007. The effect of heat stress and Tucker (eds.) The Iowa State University lameness on time budgets of lactating dairy Press, USA. cows. J. Dairy Sci., 90: 1674-1682. Anonymous. 2006. www.uky.edu/Ag/ Dolejs, J., O. Toufar and J. Knizek. 2000a. The AnimalSciences/extension/pubpdfs/aen75. infl uence of a non-homogenous temperature pdf. fi eld on dairy cows (in Czech). Czech J. Armstrong, D. and F. Wiersma. 1986. An update Anim. Sci., 45: 33-36. on cow cooling methods in the west. ASAE FAO. 2002. Water: a precious and fi nite resource. Paper Nº 86-4034. ASAE, St. Joseph, MI, www.fao.org/ag/magazine/0210sp1.htm. USA. Accessed Oct. 3, 2011. Armstrong, D.V. 1994. Heat stress interaction with Gudev, D., S. Popova-Ralcheva, P. Moneva, Y. shade and cooling. J. Dairy Sci., 77: 2044- Aleksiev, T. Peeva, P. Penchev and I. Ilieva. 2050. 2007. Physiological indices in buffaloes Ball, P.J.H., S.V. Morant and E.J. Cant. 1978. exposed to sun. Archiva Zootechnica, 10: Measurement of milk temperature as an aid 1-7. to oestrus detection in dairy cattle. J. Agric. Hahn, G.L. 1985. Management and housing of farm Sci., 91: 593-597. animals in hot environments, p. 151-174.
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In Yousef, M.K. (ed.) Stress Physiology in Igono, M.O., H.D. Johnson, B.J. Steevens, W.A. Livestock, Volume II, Ungulates.CRC Press, Hainen and M.D. Shanklin. 1988. Effect of Boca Raton, FL, USA. season on milk temperature, milk growth Holter, J.B., J.W. West, M.L. McGilliard and A.N. hormone, prolactin, and somatic cell counts Pell. 1996. Predicting ad libitum dry matter of lactating cattle. Int. J. Biometeorol., intake and yields of Jersey cows. J. Dairy 32(3): 194-200. Sci., 79: 912-921. Igono, M.O., H.D. Johnson, B.J. Steevens, Holter, J., J.W. West and M.L. McGilliard. 1997. G.F. Krause and M.D. Shanklin. 1987. Predicting ad libitum dry matter intake and Physiological, productive, and economic yields of Holstein cows. J. Dairy Sci., 80: benefi ts of shade, spray, and fan system 2188-2199. versus shade for Holstein cows during Hurley, W. 2010. Lactation biology website, summer heat. J. Dairy Sci., 70: 1069. University of Illinois, chapter 7: Mammary Javaid, S.B., J.A. Gadahi, M. Khaskeli, M.B. macro structure, dairy cow anatomy. Bhutto, S. Kumbher and A.H. Panhwar http://classes.anci.illinois.edu/ansc438/ 2009. Physical and chemical quality of mamstructure/anatomy-7.html; accessed market milk sold at Tandojam, Pakistan. Nov. 14, 2011. Pak. Vet J., 29(1): 27-31. Igono, M.O., B.J. Steevens and M.D. Shanklin. Koga, A., T. Kuhara and Y. Kanai. 2004. 1985. Spray cooling effects on milk Comparison of body water retention during production, milk and rectal temperature water deprivation between swamp buffaloes of cows during a moderate temperature and Fresian cattle. J. Agr. Sci., 138: 435- summer season. J. Anim. Sci., 68: 979-985. 440. Igono, M.O., G. Bootvedt and T.H. Sanford-Crane. Lin, J.C., B.R. Moss, J.L. Koon, C.A. Flood, R.C. 1992. Environmental profi le and critical Smith III, K.A.Cummins and D.A. Coleman. temperature effects on milk production 1998. Comparison of various fan, sprinkler, of Holstein cows in desert climate. Int. J. and mister systems in reducing heat stress in Biometeorol., 36: 77-87. dairy cows. Appl. Eng. Agric., 14: 177-182. Igono, M.O., H.D. Johnson, B.J. Steevens and Marai, I.F.M. and A.A.M. Habeeb. 2010. Buffalo M.D. Shanklin. 1985. Spray cooling biological functions as affected by heat effects on milk production, milk and rectal stress-a review. Livest. Sci., 127: 89-109. temperature of cows during a moderate McDowell, R.E., J.C. Wilk, S.K. Shah, D.S. Balain temperate summer season. J. Dairy Sci., 68: and G.H. Metyry. 1995. Potential for 979. Commercial Dairying with Buffalo. North Igono, M.O., H.D. Johnson, B.J. Steevens, G.F. Carolina State University, USA. Krause and M.D. Shanklin. 1988. Effect of Mustafa, A. 2001. http://animsci.agrenv.mcgill. season on milk temperature, milk growth ca/courses/460/topics/3/text.pdf, Accessed hormone, prolactin, and somatic cell counts February 23, 2009. of lactating cattle. Int. J. Biometeorol., 32: Pandey, M.D. and A. Roy. 1966. Vitamin A and 194-200. carotene status of domestic ruminants and
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the effects of seasons on vitamin A storage and fan cooling. ASAE Paper Nº 89-4025. in buffaloes. Indian Vet J., 43: 613-621. ASAE, St. Joseph, MI, USA. Peeters, G., A. Houvenaghel, E. Roets, A.M. Umphrey, J.E., B.R. Moss, C.J. Wilcox and Massart-Leen, R. Verbeke, G. Dhondt and F. H.H. Van Horn. 2001. Interrelationship Verschooten. 1979. Electromagnetic blood in lactating Holsteins of rectal and skin fl ow recording and balance of nutrients in temperature, milk yield and composition, the udder of lactating cows. J. Anim Sci., dry matter intake, body weight, and feed 48(5): 1143-1153. effi ciency in summer in Alabama. J. Dairy Ravagnolo, O., I. Misztal and G. Hoogenboom. Sci., 84: 2680-2685. 2000. Genetic component of heat stress Valtorta, S.E., P.E. Leva, M.R. Gallardo and O.E. in dairy cattle, development of heat index Scarpati. 2002. Milk production responses function. J. Dairy Sci., 83: 2120-2125. during heat waves events in Argentina, Roussel, J.D. and J.F. Beatty. 1970. Infl uence p. 98-101. In the 15th Conference on of zone cooling on performance of cows Biometeorology and Aerobiology 16th lactating during stressful summer conditions. International Congress on Biometeorology. J. Dairy Sci., 53: 1085. Kansas City, MO. American Meteorlogical Schultz, T.A. 1988. California dairy corral manger Society, Boston, USA. mister installation. ASAE Paper Nº 88- West, J.W. 2003. Effects of heat-stress on 4056, ASAE, St. Joseph, MI, USA. production in dairy cattle. J. Dairy Sci., 86: Snedecor, G.W. and W.G. Cochran. 1989. Statistical 2131-2144. Methods, 8th ed. Iowa State University Press, West, J.W., B.G. Mullinix and J.K. Bernard. USA. 2003. Effects of hot, humid weather on Soch, M., P. Novák, P. Kratochvíl and J. Travnicek. milktemperature,dry matter intake, and milk 1997. The change of welfare and share of yield of lactating dairy cows. J. Dairy Sci., mechanisms of warmth issues for level 86(1): 232-242. of its total issue from organism of calves Yousef, M.K. 1985. Heat Production: Mechanisms (in Czech), p. 72-73. In Proceedings of and Regulation, in Stress Physiology in International Conference Animal Protection Livestock. Vol. 1. Basic Principles. CRC and Welfare, Part II., Veterinary and Press, Boca Raton, FL. 49p. Pharmaceutical University, Brno, Czech Republic. Strickland, J.T., R.A. Bucklin, R.A. Norstedt, D.K. Beede and D.R. Bray. 1989. Sprinkler and fan cooling system for dairy cows in hot, humid climates. Appl. Eng. Agric., 5: 231- 236. Turner, L.W., J.P. Chastain, R.W. Hemken, R.S. Gates and W.L. Crist. 1989. Reducing heat stress in dairy cows through sprinkler
188 Original Article Buffalo Bulletin (June 2015) Vol.34 No.2
GENETIC ANALYSIS OF BODY WEIGHT TRAITS OF SURTI BUFFALO
G.M. Pandya1, C.G. Joshi2, D.N. Rank2, V.B. Kharadi1, B.P. Bramkshtri1, P.H. Vataliya3, P.M. Desai4 and J.V. Solanki2
ABSTRACT factors under study. Body weights at different ages were found to be moderate to highly heritable and Estimation of genetic parameters for were favorably genetically correlated with each body weight traits is important for understanding other. the genetic basis for growth performance of the animal and for accessing how much improvement Keywords: genetic analysis, body weight, Surti is possible in future generations. The data for the buffalo present study were collected from breeding and body weight records of the Livestock Research Station, Navsari from 1988-2002. The data INTRODUCTION pertaining to body weight of 522 Surti buffaloes for body weight at birth, body weight at 3 months, The Surti is one of the well-defi ned body weight at 6 months and body weight at 12 buffalo breeds of India. The home tract of this months were collected. The records were analyzed breed is Central and South-Western part of Gujarat using least squares analysis procedures. The Harvey state. The breed is generally found in the Middle (1990) programme was used to estimate the genetic Gujarat, the South Gujarat medium rain fall and parameters i.e. heritability, genetic and phenotypic the South Gujarat heavy rain fall agro-climatic correlations by the paternal half sib method. Non- zones of Gujarat state. The breed is known for its genetic factors, such as period of birth, period of sickle shaped horns. The animals of this breed are calving, season of birth, and season of calving were of medium size. incorporated in an appropriate statistical model. Estimation of genetic parameters is
BW0, BW3 and BW6 were signifi cantly affected by important for elucidating the genetic basis of the period of birth and season of birth. The sex of calf trait. Detailed genetic analysis of body weight signifi cantly affected only body weight at birth. traits in Surti buffalo maintained on an organized
BW12 was not affected by any of the non-genetic farm help us in identifying various factors affecting
1Department of Animal Genetics and Breeding, College of Veterinary Science and Animal Husbandry, Navsari Agricultural University, Navsari, India, E-mail: [email protected] 2College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat, India 3College of Veterinary Science and Animal Husbandry, Junagadh Agricultural University, Junagadh, Gujarat, India 4College of Veterinary Science and Animal Husbandry, Navsari Agricultural University, Navsari, India
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the growth of animals. Estimation of genetic Period Year parameters for various body weight traits also helps P1 1988-1990 the breeder in identifi cation of various selection P2 1991-1993 criteria and the planning of breeding programs for P3 1994-1996 genetic improvement in Surti buffalo for growth P4 1997-1998 and indirectly for production traits also. P5 1999-2002 Most of the published research work on Season Duration various aspects of growth trait is available on the S1 (Winter) 16th Oct.-15th Feb. Murrah and Nili-Ravi breeds but the information S2 (Summer) 16th Feb.-15th June on Surti buffaloes is very scanty and needs to be S3 (Monsoon) 16th June-15th Oct. estimated for formulating an effective selection programme. As limited information is available The data in the present study were divided on the growth performance of Surti animals on in to three seasons. Each year was divided into organized farms, the present study was planned three seasons as follows: with the objective to carry out genetic analysis of birth weight and body weight at different ages up Table 1. The distribution of buffaloes according to to 12 months and various genetic and non-genetic period of birth and season of birth for body factors affecting it. weight data.
Season of Period of birth MATERIALS AND METHODS birth P1 P2 P3 P4 P5 S1 S2 S3
The data pertaining to body weight and Total other records of Surti buffaloes were collected from no. of 105 107 115 87 108 186 43 293 animals 1988-2002 for all the farmborn progenies of the Livestock Research Station, Navsari Agricultural University, Navsari. The data pertaining to body weight were collected for different ages i.e. Body METHODOLOGY
Weight at Birth (BW0), Body Weight at 3 months Analysis of genetic parameters (BW3), Body Weight at 6 months (BW6) and Body The records were analyzed using least Weight at 12 (BW12) months. Body weight data for a total 522 (181 males and 341 females) animals square analysis procedures. The Harvey (1990) under 13 sires were collected . programme was used to estimate the genetic The data used in the present study were parameters i.e. heritability (h2), genetic and spread over the years 1988-2002. The entire period phenotypic correlations by paternal half sib method. was divided into fi ve periods as follows: Non-genetic factors such as period of birth, period of calving, season of birth, and season of calving were incorporated in an appropriate statistical model.
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Statistical models used for genetic analysis the present study except for BW6. Rajagopalan and were as follows. Model 1 and Model 2 were run to Nirmalan (1989) reported the average birth weight carry out the genetic analysis of the records. of Surti cross-bred male calves as 28.6 kg. They also reported average body weight at 12 months of Model 1: Fixed model age in cross-bred Surti calves as 169.1 kg.
Yij=μ+Fi+eij The body weights of buffaloes of various
where, Fi represents all fi xed sets of breeds have been reported by different workers. effects Peeva et al. (1994) reported body weights at birth
eij represents unweighted least squares and at different ages in Murrah buffalo which are higher than those reported in the present study. Model 2: Model with one set of cross-classifi ed Such results are expected as the Murrah is a heavier non-interacting random effects breed than the Surti. Na-Chiangmai et al. (1998)
Yij=μ+ai+Fj+eijk analyzed Swamp buffalo body weights at two
where, ai represents the set of random different places in Thailand. The body weights at cross-classifi ed effects birth and at weaning in Swamp buffalo were found
Fj represents all fi xed sets of effects to be higher than those reported in Surti buffalo in
eijk represents unweighted least squares the present study. The average birth weight of Nili- (Harvey, 1990) Ravi buffalo, reported by Naqvi and Shami (1999) is also higher than that in present study. The Surti buffalo, being a moderate size RESULTS AND DISCUSSION breed, has average body weights at birth and other ages that are lower than those reported for Murrah,
The overall means for BW0, BW3, BW6 and Nili-Ravi and Swamp buffaloes.
BW12 were found to be 24.60+0.18, 49.93+0.36, 72.08+0.54, and 129.75+0.94 kg, respectively, Effect of non-genetic factors as presented in Table 2. The least squares means Body weight traits are infl uenced by for BW0, BW3, BW6 and BW12 were found to various genetic and non-genetic factors. Some of be 24.64+0.50, 50.76+0.99, 73.42+1.65, and the important non-genetic factors affecting body 130.50+2.52 kg, respectively are presented in Table weight traits are season of birth, period of birth, 2. sex of calf etc. The least squares means for body Various scientists have studied body weight at different ages with respect to period of weight at different ages in Surti buffalo. Rathi et birth, season of birth and sex are presented in Table al. (1973) found birth weight to be 29.06 kg for 2. The results of analysis of variance for effect of Surti buffaloes, which was higher than the present various genetic and non genetic factors on body fi ndings. Raghvan et al. (1987) analyzed body weights at different ages are presented in Table 3. weight at birth and at 3 and 6 months of ages for Surti buffaloes found them to be 25.66+0.16, Effect of season of birth 56.30+0.33 and 67.73+0.52 kg, respectively. These Effects of season of birth on body weight fi ndings are slightly higher than those reported in in different buffalo breeds have been reported
191 Buffalo Bulletin (June 2015) Vol.34 No.2
Table 2. The least squares mean for body weight traits in Surti buffalo.
BW0 BW3 BW6 BW12 24.60±0.18 49.93±0.36 72.08±0.54 129.75±0.94 Overall (522) (522) (522) (522) 24.64±0.50 50.76±0.99 73.41±1.65 130.50±2.52 μ (522) (522) (522) (522) 22.55a±0.99 50.71b±1.98 73.59b±3.12 128.40a±5.36 Period 1 (105) (105) (105) (105) 22.90a±0.70 52.87c±1.40 76.97c±2.25 131.23a±3.72 Period 2 (107) (107) (107) (107) 26.11c±0.69 50.20b±1.37 70.73a±2.20 129.27a±3.63 Period 3 (115) (115) (115) (115) 24.88b±0.75 46.59a±1.49 69.50a±2.38 135.48b±3.97 Period 4 (87) (87) (87) (87) 26.79c±0.87 53.48c±1.74 76.33c±2.75 128.16a±4.67 Period 5 (108) (108) (108) (108) Season 1 23.85a±0.53 48.32a±1.04 76.33a±2.75 128.53a±2.69 (Winter) (186) (186) (186) (186) Season 2 24.85a±0.76 53.93b±1.50 77.53b±1.73 127.53a±2.69 (Summer) (43) (43) (43) (43) Season 3 25.25b±0.50 50.05a±098 71.18a±1.65 130.77a±2.51 (Monsoon) (293) (293) (293) (293) 24.16a±0.51 50.82a±1.00 73.83a±1.68 130.96a±2.59 Sex F (341) (341) (341) (341) 25.13b±0.56 50.72a±1.09 73.02a±1.81 130.05a±2.84 Sex M (181) (181) (181) (181)
Means with different superscripts differ signifi cantly.
Table 3. The analysis of variance for body wt traits in Surti buffalo.
Source BW0 BW3 BW6 BW12 D.F. MSS D.F. MSS D.F. MSS D.F. MSS Sire 9 42.86** 9 166.28** 9 455.25** 9 1111.93* Period 4 136.32** 4 529.32** 4 833.37** 4 625.53 Season 2 105.35** 2 550.68** 2 697.90** 2 830.59 Sex 1 105.05** 1 0.99 1 72.00 1 93.22 Error 505 15.083 505 61.12 505 144.93 505 462.84 ** = signifi cant (p<0.01). * = signifi cant (p<0.05).
192 Buffalo Bulletin (June 2015) Vol.34 No.2
by many research workers. The present fi ndings body weights at birth and thereafter at different revealed that season had highly signifi cant effects ages.
(P<0.01) at all the ages except for BW12. Calves born in the monsoon season had signifi cantly Effect of period of birth higher birth weights than those in winter and Periods had highly signifi cant effects summer seasons. While at 3 months and 6 months, (P<0.01) on body weight at birth and at 3 and 6 calves born in the summer had signifi cantly higher months. Raghvan et al. (1987) has reported that year body weights. There was no signifi cant difference of birth signifi cantly affects the body weight at all of season observed for body weight at 12 months in ages in Surti buffalo. The effect of year / period of calves. birth have been studied by different workers in the Krishnamoorthy et al. (1979) also studied buffalo breeds of India and other countries. Some
BW0 of calves born in different seasons and found of the important fi ndings include reports by Peeva that season signifi cantly affected the birth weight et al. (1994) in Murrah buffalo and Na-Chiangmai of Surti buffalo. In further analysis, they found that et al. (1998) in Swamp buffalo. These authors have
BW12 was not affected by season, which supports concluded signifi cant effect of year / period on the result of the present study. Rathi et al. (1973) body weight in different buffalo breeds which are have also studied body weight traits at different in agreement of the present fi ndings. ages up to 2 years; they found a signifi cant effect of season of birth on the body weight of the calf. Effect of sex The fi ndings of Raghavan et al. (1987) also suggest The effects of sex was found to be highly a signifi cant effect of season of birth on the body signifi cant (P<0.01) for birth weight while that weight at birth and at 1, 3 and 6 months in Surti for BW3, BW6 and BW12 was found to be non- buffaoes, and the calves born during monsoon signifi cant. season, were signifi cantly heavier at 3 months than Krishnamoorthy et al. (1979) reported that those born in winter. male calves had signifi cantly higher body weight Na-Chiangmai et al. (1998) in Swamp than female calves at all the ages. Raghvan et al. buffalo, Mahdy et al. (1999) in Egyptian buffalo (1987) have also reported that males weighed have also reported signifi cant effect of season on signifi cantly more than females at all ages in Surti
Table 4. Estimates of heritability and genetic and phenotypic correlations among body weight traits.
BW0 BW3 BW6 BW12
BW0 0.188±0.112 1.087±0.149 0.720±0.313 0.637±0.394
BW3 0.388 0.175±0.108 0.899±0.132 0.585±0.384
BW6 0.252 0.724 0.216±0.122 0.736±0.254
BW12 0.209 0.430 0.671 0.144±0.096
h2±SE = along diagonal rG±SE = above diagonal
rP= below diagonal K = 37.59
193 Buffalo Bulletin (June 2015) Vol.34 No.2
buffalo. BW6 and BW12 were found to be 1.087+0.149, 0.720+0313 and 0.637+0.394, respectively. Genetic
Estimation of genetic parameters heritability correlations of BW3 with BW6 and BW12 were estimates found to be 0.899+149, 0.585+0.384, respectively.
Heritability estimates for the body weight A genetic correlation of BW6 with BW12 was found traits at different ages are presented in Table 4. to be 1.736+0.254. The genetic correlations among
Heritabilities of body weight traits at BW0, BW3, different body weight traits were found to be highly
BW6 and BW12 were found to be 0.188+0.112, positive. Phenotypic correlations of BW0 with
0.175+0.108, 0.216+0.122 and 0.144+0.096 BW3, BW6 and BW12 were found to be 0.388, 0.252 respectively. The heritability of BW0, BW3, and and 0.209, respectively. Phenotypic correlation
BW6 was found to be moderate, while that of BW12 of BW3 with BW and BW was found to be 0.671. was found slightly lower than moderate. The phenotypic correlation among different body Different scientifi c workers have estimated weight traits were found to be moderately to highly heritability body weights at different ages in Indian positive. buffaloes. Rathi et al. (1973) have reported that Different scientifi c workers have estimated body weights at different ages, except at birth, are genetic and phenotypic correlation among body highly heritable in the Surti buffalo, while in the weights at different ages in Indian buffaloes. Rathi present study body weight traits have been found to et al. (1973) reveal high genetic and phenotypic be moderately heritable. Tien and Tripathi (1990) correlations among body weights, where the have reported BW0, BW6 and BW36 as highly present study reveals high genetic and moderate to heritable and BW12, BW18, BW24 and BW30 as high phenotypic correlations among body weight moderately heritable in Murrah buffalo, which are traits. high as compare to fi ndings of the present study. Mahdy et al. (1999) in Egyptian buffaloes Mahdy et al. (1999) have found birth weight and and Thevamanoharan et al. (2000) in Swamp weaning weight as low to moderately heritable buffaloes have reported high and positive genetic in Egyptian buffalo while Thevamanoharan et al. and phenotypic correlation between birth and (2000) have found it as highly heritable in Swamp weaning weight which is in agreement of the results buffaloes. of the present fi ndings. As heritabilities in the present study have As the body weights among different been found to be low to moderate, it can be said ages are highly and positively genetically and that environment plays a greater role and the trait phenotypically correlated, they can be effectively can only be slightly improved by selection on the used for selection for improvement in body weight basis of the present fi nding. traits.
Genetic and phenotypic correlations among body weight traits CONCLUSION Genetic and phenotypic correlation among body weights at different ages are presented in From the fi ndings of the above study, it can
Table 4. Genetic correlations of BW0 with BW3, be concluded that Surti buffaloes had lower body
194 Buffalo Bulletin (June 2015) Vol.34 No.2
weights at various stages as compared to Murrah Proceedings of 4th World Buffalo Congress, and Nili-Ravi buffaloes. Furthermore, the body Sao Paulo, Brazil. weight traits except for BW12, were signifi cantly Raghavan, K.C., A.V. Rai and G.R. Lokanath. 1987. affected by period of birth and season of birth. Effect of certain non-genetic factors on body The sex of the animal had only signifi cant effect weights up to six months of age in Surti at birth and not at any other stages. Body weights buffaloes. Mysore Journal of Agricultural at different ages have moderate to high heritability Sciences, 21(2): 205-208. and were favorably genetically associated with Rajagopalan, T.G. and G. Nirmalan. 1989. Pattern each other. of growth of male cross bred Surti buffalo calves. Kerala J. Vet. Sci., 20(2): 42-48. Rathi, S.S., D.S. Balaine and A.S. Kanaujia. 1973. REFERENCES Body weights and their relationship with economic traits in Indian buffaloes. Indian Harvey, W.R. 1990. Users guide for mixed model Journal of Animal Production, 4(1): 1-8. least squares and maximum likelihood Thevamanoharan, K., W. Vandepitte, G. Mohiuddin computer programme PC-2, version, and M. Shafi que. 2000. Genetic, phenotypic USDA-ARS. and residual correlations between various Krishnamoorthy, U., H.K. Balasubramanya, performance traits of Nili-Ravi buffaloes. S. Mallikarjunappa and A.V. Rai. 1979. Buffalo Bull., 19(4): 80-86. Infl uence of season of birth and birth weight Tien, N.Q. and V.N. Tripathi. 1990. Genetic on subsequent body weights in Surthi calves. parameters of body weight at different ages Indian Journal of Dairy Science, 32(1): 20- and fi rst lactation traits in Murrah buffalo 28. heifers. Indian Vet. J., 67(9): 821-825. Mahdy, A.E., O.M. El-Shafi e and M.S. Ayyat. 1999. Genetic study and sire values for some economic traits in Egyptian buffaloes. Alexandria Journal of Agricultural Research, 44(2): 15-35. Na-Chiangmai A., S. Khadee and N. Onwan. 1998. The environmental effects on growth traits of swamp buffaloes. Buffalo-Newsletter. 10: 10-11. Naqvi, A. and S.A. Shami, 1999. Factors affecting birth weight in Nili-Ravi buffalo calves. Pak. Vet. J., 19(3): 119-122. Peeva T., K. Vankov, W.G. Vale, V.H. Barnabe and Mattos-JCA-de. 1994. The effect of some genetic and non-genetic factors on the body weight of buffalo calves, p. 381-383. In
195 Buffalo Bulletin (June 2015) Vol.34 No.2 Original Article Buffalo Bulletin (June 2015) Vol.34 No.2
POPULATION STRUCTURE AND GENETIC VARIABILITY OF A CLOSED JAFFARABADI BUFFALO HERD FROM BRAZIL
Paulo Costa Ferraz1, Carlos Henrique Mendes Malhado2,*, Alcides Amorim Ramos3, Paulo Luiz Souza Carneiro2, José Adrián Carrillo4 and Ana Claudia Mendes Malhado5
ABSTRACT and to reduce the generation interval.
Small closed herds could have signifi cant Keywords: Jaffarabadi buffalo, effective size, negative genetic consequences including small inbreeding, generation interval, pedigree effective size, inbreeding depression and the loss of diversity through genetic drift. Here we evaluate the population structure and genetic variability of INTRODUCTION a closed Jaffarabadi herd through careful analysis of the pedigree. The herd was characterized by a The water buffalo (Bubalus bubalis) was high generation interval (10.17±5.43 years), small originally bred in Asia and has become a widespread effective population size (10) and very low effective dairy animal throughout the world (Borghese, number of ancestors (7) and founders (8). Four 2010). The buffalo has a variety of agricultural ancestors explained 50% of the genetic variability in uses, as a working animal and for the production the population and the most representative ancestor of meat and milk. They are also very well-adapted contributed approximately 33% of the population and productive in low-quality pastures. Buffaloes genetic variability. Estimated inbreeding was of Murrah, Mediterranean, Jabarabadi, and Carabao moderate with a value of 4.22%. However, this may breeds were introduced to Brazil at the beginning of be underestimated because of the shallow pedigree. the 20th century (Cassiano et al., 2003) and recent Based on our results we strongly recommend the estimations suggest that there are currently about introduction of new reproductive individuals, with 1.2 million buffalos distributed across all Brazilian lowest possible average relatedness coeffi cients states (Ibge, 2012). with the herd, to reduce the deleterious effects Studies using historical pedigree records associated with inbreeding and low levels of genetic have the potential to identify the factors that have variability. Moreover, immediate action should be infl uenced the genetic history of a population implemented to increase effective population size (Valera et al., 2005). Moreover, some population 1Universidade Estadual do Sudoeste da Bahia, Praça Primavera, Bairro Primavera, Itapetinga, Bahia, Brazil 2Universidade Estadual do Sudoeste da Bahia, Av: José Moreira Sobrinho, S/N, Jequié, Bahia, Brazil, *E-mail: [email protected] 3Universidade Estadual Paulista, Botucatu, São Paulo, Brazil 4University of Maryland, College Park, Maryland, United States 5Universidade Federal do Alagoas, Maceió, Alagos, Brazil
197 Buffalo Bulletin (June 2015) Vol.34 No.2
parameters are strongly dependent on management 4.8 program (Gutiérrez and Goyache, 2005). and mating systems, having signifi cant impacts on The average generation interval of the genetic variability. In addition to productivity, cattle four gametic pathways was estimated using the breeders are also concerned about the genetic health following criteria: sire-son, sire-daughter, dam-son of their livestock. An assessment of the within- and dam-daughter. The completeness pedigree was population genetic variability and population computed following MacCluer et al. (1983), and structure is necessary for the implementation included the description of the completeness for of effective selection programs and to establish each ancestor in the pedigree until the 4th parental appropriate regimes for managing the genetic generation. The following parameters were stock. In this respect, genealogical assessment is calculated for each individual: an important tool to guide genetic management 1. Number of fully traced generations; de- strategies (Głażewska and Jezierski, 2004). fi ned as the number of generations sepa- In recent years several studies have been rating the offspring from the furthest published on population structure in different generation where the second-generation species (Cervantes et al., 2008a; Ghafouri-Keshi, ancestors were known. Ancestors with no 2010; Malhado et al., 2010; Santana et al., 2012), known parents were considered as found- including buffaloes (Santana et al., 2011; Malhado ers (generation 0) et al., 2012 and Teixeira Neto et al., 2012). However, 2. Maximum number of generations; de- there are no equivalent studies for the Jaffarabadi scribed as the number of generations sepa- breed (in Brazil or other countries). The major rating the individual from its furthest an- objective of the present study was thus to evaluate cestor. the population structure of a closed Jaffarabadi 3. Equivalent complete generations; calcu- buffalo herd employing pedigree analysis. The lated as the sum over all known ancestors n parameters used include effective number of of the terms computed as the sum of ½ , founders, inbreeding, generation interval, effective where n is the number of generations sepa- number of ancestors, average relatedness coeffi cient rating the individual from each known an- (AR), effective population size and completeness cestor (Maignel et al., 1996) of pedigree. This information is valuable for the development of optimal mating strategies in other Genetic history was assessed by calculating buffalo breeds and closed herds. the effective number of founders and the effective number of ancestors. The effective number of
founders (fe) represents the number of animals, MATERIAL AND METHODS which under random mating would produce the same genetic variability as that observed in the studied population. This is computed as where, q Pedigree information was obtained from k 1,272 Jaffarabadi buffaloes born from 1966 to is the probability of gene origin for ancestor k. The effective number of ancestors (f ) 2002 in a closed Brazilian herd. Pedigree analysis a and parameters estimates based on gene origin represents the minimum number of animals (founders probabilities were performed using the ENDOG or non-founders) that are necessary to explain the 198 Buffalo Bulletin (June 2015) Vol.34 No.2
total genetic population diversity. It is calculated in “called realized effective size” by Cervantes et al. a similar way to the effective number of founders:, (2008b). It was computed from , that is obtained where qj is the marginal contribution of ancestor j, by averaging the of the n individuals included in a which represents the genetic contribution made by given reference subpopulation, as . Additionally, a an ancestor that is not explained by another ancestor standard error of was computed from the standard chosen previously. This parameter complements deviation of and the square root of the size (n) of the information offered by the effective number of the reference subpopulation as (Gutierrez et al., founders, by accounting for the losses of genetic 2008). variability produced by the unbalanced use of Secondly, the effective population size was reproductive individuals causing bottlenecks. estimated from the increase in coancestry for all pairs
The founder equivalents (fg) can be defi ned as the of individuals j and k in a reference subpopulation number of founders that would produce the same (Cervantes et al., 2011). This parameter is computed genetic diversity observed in the population, if the as where cjk is the inbreeding value corresponding founders were equally represented and no loss of to an offspring from j and k, and gj and gk are the alleles occurred (Ballou and Lacy, 1995). discrete equivalent generation of individual j and The individual inbreeding coeffi cient (F) was k. Averaging the coancestry increases for all pairs computed following Meuwissen and Luo (1992). of individuals, we can estimate a realized effective The change in inbreeding (∆F) was calculated as population size based on coancestries, as .We also described by Falconer and Mackay and modifi ed computed the standard error of the from the standard by Gutiérrez et al. (2009), using the formula: deviation of these increases in coancestry (and the t−1 ΔFi =1− 1− fi , where Fi is the individual square root of the effective size of the effective inbreeding coeffi cient and t is the equivalent number of paired coancesrtries in the reference sub generation for this individual (Boichard et al., population as. 1997). The expression relating inbreeding in generation t with inbreeding rate proposed by Gutiérrez et al. (2009) is RESULTS AND DISCUSSION
F = 1-(1-∆F)^(t-1) Completeness levels for the whole pedigree were 86.6% 44.7% and 18.2% for the fi rst, second The average relatedness coeffi cient of each and third generations, respectively. Malhado et al. individual is defi ned as the probability that an allele (2012a) reported levels of 76.8% 49.2% and 27.7% randomly chosen from the whole population in the in the same parameters for the Murrah breed. pedigree belongs to a given animal (Gutiérrez and Santana et al. (2011), also studying the Brazilian Goyache, 2005). The average relatedness coeffi cient Murrah breed, calculated the average completeness can thus be interpreted as a representation of the of pedigree for animals born within the last 10 years. animal in the whole pedigree regardless of the They obtained corresponding values of 76.2% knowledge of its pedigree. 66.3% and 57.8%. Likewise, Teixeira Neto et al. The effective size was calculated in two (2012) reported percentages of known pedigree for ways: First, estimate of effective population size, the fi rst, second and third generations as 60.51%
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15.27% and 2.14% in the Mediterranean breed. and ancestor animals contributing to the reference The shallowness of pedigree for Brazilian population (1,059) were 136, 130 and 134, buffaloes can be explained by the relatively recent respectively (Table 1). With much smaller values, foundation of the Brazilian Association of Buffalo effective number of founders (fe) and ancestors (fa) Breeders (ABCB), which occurred in 1966. By were 8 and 7, respectively. Expected increase of contrast, several American and European beef inbreeding caused by the unbalanced contribution cattle associations started the registration earlier of founders was 4.99%. The ratio of effective and consequently its breeds have longer pedigrees number of founders to the number of founders was (Bozzi et al., 2006; Marquéz et al., 2010). 0.058, indicating a high disequilibrium between Generation interval estimates for the four founder contributions. This fact results from gamete pathways were 12.28±6.90 (sire-son), an excessive use of some animals as breeders, 11.55±6.07 (sire-daughter), 8.20±2.63 (dam-son) especially good bulls (as is common practice in and 8.79±4.33 years (dam-daughter), giving an closed herds). According to Gutiérrez et al. (2003) average generation interval of 10.17±5.43 years. the overuse of reproducers also generates loss of Teixeira Neto et al. (2012) reported values of genetic diversity. 8.79±2.29 (sire-son), 9.59±2.80 (sire-daughter), Different authors reported higher values in 7.42±3.04 (dam-son), 7.94±2.7 years (dam- buffaloes from Brazil. Santana et al. (2011) (fe=58, daughter), with an average interval of 8.71±2.85 fa=35, Murrah), Malhado et al. (2012a) (fe=60, years for Mediterranean buffaloes. Santana et al. fa=36, Murrah) and Teixeira Neto et al. (2012)
(2011) also reported generation intervals lower than (fe=71, fa=71, Mediterranean). However, those in our study: 7.38 (sire-son), 7.42 (sire-daughter), authors used data from several buffalo herds in 6.60 (dam-son) and 6.44 (dam-daughter); averaging Brazil. A recent study of a closed population (Zandi
6.89 years across the four pathways. This high sheep) reported higher values for fe (86) and fa (74) generation interval in the Jaffarabadi herd is (Ghafouri-Kesbi et al., 2010). probably a result of using the main breeder (the sire The extent of the genetic bottleneck with the largest number of offspring (271) over a expressed by the fe/fa ratio (effective number of prolonged period of time (from 1975 to 1991). founders/effective number of ancestors) was 1.14, Buffaloes are recognized for their long indicating that the studied Jaffarabadi herd suffered productive and reproductive life. However, the a small bottleneck effect. The fe/fa ratio is caused average interval reported in this study is, in relative by a decrease in the number of breeders in any terms, very high and may result in reduced genetic given generation. Increasing known generations gain per unit of time. Moreover, long breeder will also augment the chance to detect a bottleneck permanence in the herd may increase the chances (Ghafouri-Keshi, 2010). Therefore, in populations of mating between close relatives leading to an with a long historical pedigree, smaller estimates increment in the inbreeding coeffi cient. Therefore, of fa would be expected. the use of young sires is important to reduce the Only fi ve ancestors explained approximately generation interval, mainly sire-son and sire- 56% of the population genetic variability and the daughter intervals. top ancestor contributed approximately 33% of The number of founders, equivalent founders the genetic variability (Table 2). The very small
200 Buffalo Bulletin (June 2015) Vol.34 No.2
Table 1. Parameters characterizing the probability of gene origin in the Jaffarabadi herd.
Size of population 1271 Number of animals in the population reference 1059 Base population (one or more parents unknown) 212 Number of founder animals for the reference 136 population Number of equivalent founder animals for the 130 reference population Number of ancestor animals for the reference 134 population Effective number of founder animal in the reference 8
population (fe) Effective number of ancestor animal in the reference 7
population (fa) Number of ancestors explaining 50% variability 4
Table 2. Description of the ten major ancestors (founders or not) in the Jaffarabadi herd. The table lists the identifi cation number, sire, dam, sex, genetic contribution, the average relatedness coeffi cient (AR) and number of offspring per ancestor.
Year of Contribution AR Ancestral Sire Dam Sex Offspring birth (%) (%) 1239 0 0 M 1967 33.57 28.95 271 1204 0 0 M 1986 7.74 6.80 152 1149 0 0 M 1984 6.52 5.57 25 1205 0 0 M 1993 5.09 4.36 109 1235 0 0 F 1970 3.74 3.25 14 2123 0 0 M 1986 3.51 3.06 69 2192 1162 1148 M 1981 3.33 3.00 40 2126 1231 1233 F 1974 3.02 3.00 14 5610 0 1178 M 1995 2.73 2.40 58 2129 0 0 F 1976 2.31 2.11 14
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number of individuals explaining the variability of Jaffarabadi and Mediterranean). In 1976 there were the Jaffarabadi herd is a consequence of the reduced still only 26 animals registered in the Herd Book effective number of ancestors, in conjunction with of the ABCB. Of these animals, some were used a larger effect of a single animal. Malhado et al. as breeders - a fact which explains why so few (2012a) and Teixeira Neto et al. (2012) observed founders are responsible for such a high proportion 17 and 30 animals explaining 50% of the genetic of the current genetic variability of buffalo in variability in the Murrah and Mediterranean breeds, Brazil. respectively. A recent study of the Murrah breed The average values of inbreeding (F) for the in the Mediterranean (and their crosses in Brazil) whole analyzed pedigree was 4.2% (319 inbreed also reported low genetic variability: the fi rst 20 animals). Lower values of F in buffaloes have ancestors (founders or non-founders) explained been reported by Marcondes et al. (2010) (0.50%), 69.10% of the total genetic variation (Marcondes et Santana et al. (2011) (2.14%), Malhado et al. al., 2010). In a similar study on Murrah buffaloes, (2012a) (1.28%) and Teixeira Neto et al. (2012) 19 animals were responsible for explaining 50% (0.34%). The average values of F for inbreed of the population genetic diversity (Santana et animals was 14.0%, close to that estimated by al., 2011). These fi ndings probably indicate the Teixeira Neto et al. (2012) (16%) in Mediterranean existence of defi ciencies in the Murrah buffalo’s buffaloes. This high value (14.0%) of F indicates breeding plan, such as failures in mating policy, that mating among closely related individuals was intensive use of certain individuals as breeding not being avoided. Indeed, from 1982 to 1990 the animals, and reduced effective population size, average of F for inbreed animals was 25% (Figure among other reasons. 1). A total of 78 matings between half sibs and 67 To our knowledge, all studies that have pairings between parent-offspring were identifi ed analyzed buffalo herds from Brazil have found throughout the study period. In addition, in some that a small number of animals contribute years (e.g.1988 and 1999) most matings were disproportionately to the population genetic inbred (Figure 2). diversity. The low genetic variability of buffalos The increase in inbreeding for each in Brazil can be partly explained by the process of maximum generation (1.21%), complete generation animals’ introduction into the country (Malhado et (5.18%) and equivalent generation (3.57%), and al., 2012a). The fi rst imports of buffaloes occurred the relatively short knowledge of pedigree indicate in 1930, 1952, 1955, 1960 and 1962, and were that the inbreeding value in the population is frequently associated with the importation of underestimated. Recent work on Murrah buffaloes Zebu cattle. However, it is not accurately known demonstrated an increase of approximately 2% how many buffaloes were imported during this for the average F per each complete generation period. In 1966, when the Brazilian Association (Malhado et al., 2012a). More generally, the of Buffalo Breeders (ABCB) was founded, there computation of inbreeding coeffi cients depends were no more than 50 to 60 purebred individuals on the level of pedigree completeness and on the of either the Murrah or the Jaffarabadi breeds. All base year or population used for the calculations other buffalos at this time were some combination (Boichard et al., 1997). Inbreeding levels are of crossbreeding of the three main breeds (Murrah, biased downward when pedigree information is
202 Buffalo Bulletin (June 2015) Vol.34 No.2
Figure 1. Mean value of inbreeding (F), mean value of average relatedness (AR) and average of F for inbreed animals by year of birth.
Figure 2. Inbred animals and total number of animals born by year.
203 Buffalo Bulletin (June 2015) Vol.34 No.2
incomplete, and the Ne is overestimated. using data obtained from several Murrah herds. Inbreeding within a herd deserves attention, Theory suggests that an effective population size mainly due to the possibility of inbreeding of approximately 500 animals is needed to prevent depression. For example, Malhado et al. (2012b) the loss of genetic diversity through genetic drift recently reported deleterious effects of inbreeding and to maintain a suffi ciently fl exible population on growth traits in Mediterranean buffaloes and on (Frankham, 1995). However, an effective milk production in Murrah breed. The relationship population size of at least 50 animals is suffi cient to between inbreeding and depression of traits is not prevent inbreeding depression-the minimum level linear, observing greater depression levels after recommended by the FAO (2007). The effective size 20% of inbreeding. Others studies with buffaloes for the Jaffarabadi breed (10) is much lower than (Khattab et al., 2007, Mirhabibi et al., 2007 and the critical value of 50 for avoiding the deleterious Santata et al., 2011) have also mentioned inbreeding effects of inbreeding or the loss of genetic diversity depression, with potential important economic through genetic drift. Indeed, the loss of genetic implications for the buffalo production system. diversity over 10 generations is approximately 18, The average relatedness coeffi cient (AR) 10, 4, 1.6, and 0.8% when the effective population between individuals was estimated at 12.5% and size is 25, 50, 125, 250, and 500, respectively (FAO the highest individual coeffi cient was 28.95%. 1992). Therefore, actions to increase the effective This is considerably higher than some other recent size are strongly recommended. estimates-3.58% (Santana et al., 2011), 2.05% The ratio was 1.04: close to that expected (=1) (Malhado et al., 2012a) and 0.37% (Teixeira Neto in an idealized population. The comparison between et al., 2012)-and may indicate that inbreeding could and gives valuable information on population increase in the near future if remedial measures structure (Gutierrez et al., 2008) since the two are not implemented. One possible strategy would parameters are assumed to be measures of the same be to use the average relatedness coeffi cient for accumulated drift process, from the foundation of sires’ selection. AR could be used to estimate long- the population to the present time. Because both term inbreeding in a population and to provide values should be asymptotically equivalent in an information to adjust the management practices idealized population, the disagreement between in order to conserve the genetic variability of a them is mainly due to their differential ability to population (Goyache et al., 2010). A very high AR assess the effect of preferential matings. In other indicates that the parents of an individual have close words, the comparison between the two parameters common ancestors, while a low average relatedness could be used to characterize the infl uence of coeffi cient indicates that the animal shares alleles preferential matings in the population. by common descent with only a relatively small percentage of the population. The effective size computed_ via individual CONCLUSION increase in coancestry (Nec) was 10.82±1.29- close to the_ effective size computed by individual The current genetic variability within increase (Ne) in inbreeding (10.40±3.69). Santana the Jaffarabadi buffalo population is very low as et al. (2011) also reported small effective size (40), a consequence of an extremely small number of
204 Buffalo Bulletin (June 2015) Vol.34 No.2
animals contributing to the herd. The observed of buffalo and buffalo market in Europe and levels of inbreeding and the values of the average near east. Rev. Veter., 21: 20-31. relatedness coeffi cients are high and probably Bozzi, R., O. Franci, F. Forabosco, C. Pugliese, A. underestimated due to the limited pedigree Crovettiet and F. Filippini. 2006. Genetic information. variability in three Italian beef cattle breeds Based on these fi ndings, the introduction derived from pedigree information. Ital. J. of new reproductive individuals with the lowest Anim. Sci., 5: 129-137. possible AR coeffi cients is strongly recommend. Cassiano, L.A.P., A.S. Mariante, C. Mcmanus This action will reduce the probability of suffering and J.R.F. Marques. 2003. Caracterização deleterious effects related to inbreeding and fenotípica de raças bubalinas nacionais e do increase future genetic variability in the herd. In tipo Baio. Pesq. Agropec. Bras., 38: 1337- addition, immediate action should be implemented 1342. to increase effective population size and to reduce Cervantes, I., F. Goyache, A. Molina, M. Valera the generation interval. and J.P. Gutierrez. 2008. Application of individual increase in inbreeding to estimate realized effective sizes from real pedigrees. ACKNOWLEDGMENT J. Anim. Breed. Genet., 125: 301-310. Cervantes, I., F. Goyache, A. Molina, M. Valera and The authors thank Dr. Richard Ladle for J.P. Gutierrez. 2011. Estimation of effective the insightful comments and proofreading the population size from the rate of coancestry manuscript. in pedigreed populations. J. Anim. Breed. Genet., 128: 56-63. Frankham, R. 1995. Conservation genetics. An. REFERENCES Rev. Genet., 29: 305-327. FAO. 1992. Monitoring animal genetic resources Ballou, J.D and R.C. Lacy. 1995. Identifying and criteria for prioritization of breeds. The genetically important individuals for Management of Global Animal Genetic management of genetic variation in pedigreed Resources. In Proceedings of an FAO Expert populations, p. 76-111. In Ballou, J.D., M. Consultation, no. 104. J. Hodges, ed. FAO, Gilpin and T.J. Foose (eds.) Population Rome, Italy. Management for Survival and Recovery: FAO. 2007. The State of the World’s Animal Genetic Analytical Methods and Strategies in Resources for Food and Agriculture. Small Population Management. New York: Rischkowsky, B. and D. Pilling (eds.) FAO, Columbia University Press. Rome, Italy. Boichard, D., L. Maignel and E. Verrier. 1997. The Ghafouri-Kesbi, F. 2010. Analysis of genetic value of using probabilities of gene origin to diversity in a close population of Zandi measure genetic variability in a population. sheep using genealogical information. J. Genet. Sel. Evol., 29: 5-23. Genet., 89: 479-483. Borghese, A. 2010. Development and perspective Glazewska, I. and T. Jezierski. 2004. Pedigree
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analysis of Polish Arabian horses based on Maignel, L., D. Boichard and E. Verrier. 1996. founder contributions. Livest. Prod. Sci., Genetic variability of French dairy breeds 90: 293-298. estimated from pedigree information. Goyache, F., I. Fernández, M.A. Espinosa, L. Interbull Bulletin, 14: 49-54. Payeras, L. Pérez-Pardal, J.P. Gutiérrez, L.J. Malhado, C.H.M., P.L.S. Carneiro, A.C.M. Royo and I. Álvarez. 2010. Demographic Malhado, R. Martins Filho, R. Bozzi and and genetic analysis of the Mallorquina R. Ladle. 2010. Genetic improvement and sheep fl ockbook. Itea., 106: 3-14. population structure of the Nelore breed in Gutiérrez, J.P., J. Altarriba, C. Díaz, R. Quintanilla, Northern Brazil. Pesq. Agropec. Bras., 45: J. Cañón and J. Piedrafi ta. 2003. Pedigree 1109-1116. analysis of eight Spanish beef cattle breeds. Malhado, C.H.M., A.C.M. Malhado, P.L.S. Genet. Sel. Evol., 35: 43-63. Carneiro, A.A. Ramos, D.P. Ambrosini Gutierrez, J.P. and F. Goyache. 2005. A note on and A. Pala. 2012. Population structure ENDOG: a computer program for analysing and genetic variability in the Murrah dairy pedigree information. J. Anim. Breed. breed of water buffalo in Brazil accessed Genet., 122: 172-176. via pedigree analysis. Trop. Anim. Health Gutierrez, J.P., I. Cervantes and F. Goyache. Prod., 44(8): 1891-1897. 2009. Improving the estimation of realized Malhado, C.H.M., A.C.M. Malhado, P.L.S. effective population sizes in farm animals. Carneiro, A.A. Ramos, J.A. Carrillo and J. Anim. Breed. Genet., 126: 327-332. A. Pala. 2012 Inbreeding depression on Gutierrez, J.P., I. Cervantes, A. Molina, M. Valera production and reproduction traits of and F. Goyache. 2008. Individual increase buffaloes from Brazil. Anim. Sci. J., 84(4): in inbreeding allows estimating effective 289-295. sizes from pedigrees. Genet. Sel. Evol., 40: Marcondes, C.R., P.A. Vozzi, B.R.N. Cunha, R.B. 359-378. Lôbo, C.V. Araújo and J.R.F. Marques. Instituto Brasileiro de Geografi a e Estatística (IBGE), 2010. Genetic variability in water buffalo 2012. Censo demográfi co [homepage on the from nucleous herd by pedigree analysis. internet]. Instituto Brasileiro de Geografi a Arq. Bras. Med. Vet. Zootec., 62: 706-711. e Estatística, São Paulo, Brasil. Avaliavle Marquez, G.C., S.E. Speidel, R.M. Enns and from url: http://www.ibge.gov.br D.J. Garrick. 2010. Genetic diversity and Khattab, A.S. and A.M. Kawthar. 2007. Inbreeding population structure of American Red and it is effect on some productive and Angus cattle. J. Anim. Sci., 88: 59-68. reproductive traits in a herd of Egyptian Meuwissen, T.H.E. and Z. Luo. 1992. Computing buffaloes. Ital. J. Anim. Sci., 6: 275-278. inbreeding coeffi cients in large populations. MacCluer, J.W., A.J. Boyce, B. Dyke, L.R. Genet. Sel. Evol., 24: 305-313. Weitkamp, D.W. Fenning and C.J. Parsons. Mirhabibi, S., G.H. Manafi azar, S.H. Qaravisi and 1983. Inbreeding and pedigree structure in B. Mahmoodi. 2007. Inbreeding and its Standardbred horses. J. Hered., 74: 394- effect on some productive traits in buffaloes 399. of South Iran. Ital. J. Anim. Scie., 6: 372-
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374. Santana, M.L.Jr., R.R. Aspilcueta-Borquis, A.B. Bignardi, L.G. Albuquerque and H. Tonhati. 2011. Population structure and effects of inbreeding on milk yield and quality of Murrah buffaloes. J. Dairy Sci., 94: 5204- 5211. Santana, M.L.Jr., P.S. Oliveira, J.P. Eler, J.P. Gutierrez and J.B. Ferraz. 2012. Pedigree analysis and inbreeding depression on growth traits in Brazilian Marchigiana and Bonsmara breeds. J. Anim. Sci., 90: 99- 108. Teixeira Neto, M.R., J.F. Cruz, A.A. Ramos, P.L.S. Carneiro, D.M.M.R. Azevêdo, R. Bozzi and C.H.M. Malhado. 2012. Genetic variability in Mediterranean buffalos evaluated by pedigree analysis. Ciência Rural, 42(11). Available at http://dx.doi.org/10.1590/ S0103-84782012001100021 Valera, M., A. Molina, J.P. Gutierrez, J. Gomez and F. Goyache. 2005. Pedigree analysis in the Andalusian horse: population structure, genetic variability and infl uence of the Carthusian strain. Livest. Prod. Sci., 95: 57- 66. Vicente, A.A., N. Carolino and L.T. Gama. 2012. Genetic diversity in the Lusitano horse breed assessed by pedigree analysis. Livest. Sci., 148: 16-25.
207 Buffalo Bulletin (June 2015) Vol.34 No.2 Original Article Buffalo Bulletin (June 2015) Vol.34 No.2
DETECTION OF DELTAMETHRIN RESISTANCE IN BUFFALO LOUSE, HAEMATOPINUS TUBERCULATUS
Nirbhay Kumar Singh*, Manjurul Haque, Jyoti and Harkirat Singh
ABSTRACT INTRODUCTION
Buffalo sucking lice Haematopinus The sucking louse Haematopinus tuberculatus collected from buffaloes of the Tajpur tuberculatus (Burmeister, 1839) Lucas, 1852, is Dairy Complex, Ludhiana, Punjab, India were a harmful ectoparasite found on buffalo (Bubalus tested by in vitro treated surface (contact) bioassay bubalis) and has been reported from various parts of against a range of dilutions of deltamethrin for Asia, Africa, Australia, South America and Europe detection of resistance status. The regression graph (Veneziano et al., 2007). The presence and feeding of probit mortality of lice was plotted against log of lice causes irritation, with the animal reacting by values of progressively increasing concentrations rubbing and scratching, resulting in patchy hair loss, of deltamethrin. From the regression equation sores and untidy appearance. Lice can signifi cantly the LC50, LC95 and LC99.9 values of deltamethrin affect hide and leather quality. Reduced feed intake were calculated as 16.55, 76.66 and 297.18 ppm and weight gain are also common and can have and the resistance factor was calculated as 23.77. a profound impact on productivity of the dairy The indiscriminate and extensive usage of these animals (FAO, 2004). synthetic pyrethroids predominantly deltamethrin Specifi c treatments for lice control are particularly in this part of the country for control uncommon throughout the world; however, products of ectoparasites resulted in the development of aimed at other external parasites (ticks, mites, resistance in lice populations. The implication is that fl ies) have had an effect on buffalo lice populations effective control for the ectoparasites particularly (Levot, 2000). The regularity of some of these lice in dairy animals requires proper control treatments in certain areas has probably reduced management including selection of insecticide lice infestation to sub-clinical levels whereas along with policy for its rotation to increase its indiscriminate use with incorrect concentrations effective life. of insecticides has probably contributed to the development of resistance in these arthropods (FAO, Keywords: deltamethrin, Haematopinus 2004). The synthetic pyrethroids (deltamethrin and tuberculatus, Punjab, resistance cypermethrin) are commercially available in India and at present are the two predominant insecticides used for control of ectoparasites in the country. Large-scale resistance to synthetic pyrethroids has
Department of Veterinary Parasitology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India, *E-mail: [email protected]
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recently been experimentally validated in Indian prepared working dilutions of deltamethrin were isolates of cattle ticks Rhipicephalus (Boophilus) used for layering of clean dry Petri dishes and three microplus (Sharma et al., 2012) and Hyalomma replicates were maintained for each concentration. anatolicum (Shyma et al., 2012). Although dairy Ten live lice were placed into each Petri dish and farmers have reported treatment ineffi ciency of incubated (in darkness) at 34°C and 70%-80% these chemicals in fi eld conditions, data on lice relative humidity (RH) for 24 h. The mortality was resistance to these chemicals are currently not recorded by counting the number of dead lice which available from the country. were immobile and showed signs of desiccation. Keeping in view the requirement of Dose response data were analyzed by probit preserving the life span of costly insecticides, regression (Finney, 1962) and the LC50, LC95 and reluctance of multinationals in funding on LC99.9 values of deltamethrin were determined by insecticide research, and costs involved in applying regression equation analysis to the probit generation and marketing of new group of chemicals transformed data of mortality. Resistance factor for the arthropod control, it is becoming essential to (RF) was worked out by the quotient between develop resistance data for implementation of future LC99.9 of fi eld isolates and LC99.9 of susceptible arthropod control measures. The current study was isolate. Due to absence of a reference susceptible undertaken to generate data on the deltamethrin isolate of H. tuberculatus the LC99.9 of susceptible resistance status of H. tuberculatus collected from was calculated from recommended commercial buffaloes of Ludhiana, Punjab, India. concentration (RCC) of deltamethrin. The RCC
of an insecticide is twice the LC99.9 of susceptible isolates (FAO, 2004) hence, as deltamethrin is used
MATERIALS AND METHODS at 25 ppm the LC99.9 for susceptible isolate as 12.5 ppm was used in the study. Lice were collected by manual picking from the naturally infested buffaloes of the Tajpur Dairy Complex, Ludhiana, Punjab and were RESULTS AND DISCUSSION identifi ed under optical and dissection microscopes based on the keys proposed by Chaudhuri and Lice collected from buffaloes of the Tajpur Kumar (1961). Dairy Complex, Ludhiana, Punjab were identifi ed Technical grade 99.3% pure deltamethrin as Haematopinus tuberculatus. Data on the effects (AccuStandard® Inc. U.S.A) was used to prepare of various concentration of deltamethrin on H. the stock solution in acetone. For the experimental tuberculatus are presented in Table 1. The results bioassay, different working concentrations of of the in vitro study revealed that maximum louse deltamethrin (12.5, 25, 37.5, 50 and 62.5 ppm) responses in terms of mortality were observed within were prepared in distilled water from the stock 1-3 h of exposure. After 6 h of exposure, almost solution and tested against H. tuberculatus. no concentration-dependent change in mortality Laboratory in vitro treated surface was seen following contact with deltamethrin. (contact) bioassay (Levot and Hughes, 1990) was The incubation time is important and an optimum adopted with slight modifi cations. Briefl y, freshly 16 h exposure is recommended, despite highest
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mortality within 2 - 4 h (Levot, 2000). The lice maximum mortality of 96.67% was recorded at were incubated in darkness at 34°C and 70 to 80% 62.5 ppm (Table 1). It was observed that exposure RH to reduce the stress caused by environmental of lice to the concentration at which deltamethrin is conditions as stressed lice may be affected by being widely used (25 ppm) could only achieve 60% lower concentrations and give false susceptibility mortality and even the much higher concentration readings (Levot and Hughes, 1990). of 62.5 ppm failed to produce 100% mortality, The mortality of lice was increased with thus indicating development of resistance against increasing concentrations of deltamethrin and deltamethrin. About 7% of the lice died in the control
Table 1. Effect of different concentrations of deltamethrin on H. tuberculatus.
Concentration Number of lice died Mortality (ppm) N 30 min 1 h 3 h 6 h 12 h 24 h (%) 12.5 30 0 2 8 2 2 0 46.67 25 30 0 4 8 5 1 0 60.0 37.5 30 0 6 12 4 0 0 73.33 50 30 1 5 13 6 0 0 83.33 62.5 30 2 3 18 5 0 0 96.67 Control 30 0 0 0 1 1 0 6.67
H. tuberculatus 8 Probit mortality 7
6
5
Probit mortality 4
3 1.00 1.25 1.50 1.75 2.00 Log conc
Figure 1. Dose mortality curve of H. tuberculatus against deltamethrin.
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group and this may be due to suffocation or some (deltamethrin and cypermethrin) and injection other reason unknown to us. In bioassay, technical (ivermectin) were mainly used for application of grade deltamethrin was selected over commercial insecticides while pour-on (fl umethrin) is used by formulation because commercial products are nearly 15% of the farm owners from Ludhiana prepared with many proprietary ingredients and (Sharma et al., 2012). This indiscriminate and it is diffi cult to assess the responses due to active extensive usage of these synthetic pyrethroids ingredients (Shaw, 1966). particularly deltamethrin resulted in the The regression graph of probit mortality development of resistance in lice populations. of lice plotted against log values of progressively Also, the higher reproductive rate of lice that increasing concentrations of deltamethrin is shown have heritable resistance factors and the resulting in Figure 1. The dotted lines in the regression increase in the proportion of the population of curve represented the 95% confi dence limits. The lice that carry genes for these factors results in slope of mortality was 2.464±0.6783 whereas the the establishment of resistance in the population value of goodness of fi t (R2) was 0.8148. From (FAO, 2004). Further, development of resistance in the regression equation the LC50, LC95 and LC99.9 populations of lice may be rapid for several other values of deltamethrin were calculated as 16.55, reasons: lice are host-specifi c obligate parasites and 76.66 and 297.18 ppm and the RF was 23.77. It there is relatively little immigration from different should be noted, however, that without a reference populations. Hence any resistant genotypes quickly population of pyrethroid naive H. tuberculatus replace the susceptible under the selection pressure it is not possible to unequivocally prove that the imposed by insecticide (Ellse et al., 2012). lice tested have developed resistance. Reference There are two means of pyrethroid populations unfortunately could not be obtained for resistance development in lice, either through single this study due to the scarcity of untreated animals. point mutations in the gene coding for the drug However, there is relatively little target protein (Lee et al., 2000) or by upregulation comparable published data on the effi cacy of of metabolic, monooxygenase enzymes (Scott, deltamethrin against buffalo lice. Previous studies 1999). Therefore, future studies directed against have reported pyrethroid resistance in Australian detection of the mechanism of the resistance fi eld populations of the sheep body louse, Bovicola would be of immense help in development and (Damalinia) ovis (Johnson et al., 1992; Levot et implementation of strategies for effective control al., 1995; Jazayeri, 2004). In similar studies, four for the ectoparasites particularly lice in dairy populations of B. ovis indicated possible resistance animals. to deltamethrin from the United Kingdom (Bates, 2001). The development of resistance against ACKNOWLEDGEMENT deltamethrin in lice from Ludhiana, Punjab may be attributed to the fact that farmers adopted frequent Authors are thankful to Director of treatment of animals with available insecticides Research, Guru Angad Dev Veterinary and Animal without maintaining an optimum dose regime for Sciences University, Ludhiana for providing the control of ectoparasites particularly ticks. Spray facilities to carry out the research work.
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REFERENCES Damalinia ovis (Schrank) (Phthiraptera: Trichodectidae). J. Aust. Entomol. Soc., 29: Bates, P.G. 2001. Sheep chewing lice: an update. 257-259. Proc. Sheep Vet. Soc., 24: 163-168. Levot, G.W. 2000. Resistance and the control of Chaudhuri, R.P. and P. Kumar. 1961. The life lice on humans and production animals. Int. history and habits of the buffalo louse, J. Parasitol., 30: 291-297. Haematopinus tuberculatus (Burmeister) Levot, G.W., P.W. Johnson, P.B. Hughes, K.J. Lucas. Indian J. Vet. Sci., 31: 275-287. Powis, J.C. Boray and K.L. Dawson. Ellse, L., F. Burden and R. Walla. 2012. Pyrethroid 1995. Pyrethroid resistance in Australian tolerance in the chewing louse Bovicola fi eld populations of the sheep body louse, (Werneckiella) ocellatus. Vet. Parasitol., Bovicola (Damalinia) ovis. Med. Vet. 188: 134-139. Entomol., 9: 59-65. FAO. 2004. Resistance Management and Integrated Scott, J.G. 1999. Cytochromes P450 and insecticide Parasite Control in Ruminants, Guidelines. resistance. Insect Biochem. Mol. Biol., 29: Animal Production and Health Division, 757-777. FAO, p. 183-210. Sharma, A.K., R. Kumar, S. Kumar, G. Nagar, N.K. Finney, D.J. 1962. Probit Analysis–A Statistical Singh, S.S. Rawat, M.L. Dhakad, A.K.S. Treatment of the Response Curve. Cambridge Rawat, D.D. Ray and S. Ghosh. 2012. University Press, Cambridge, p. 1-318. Deltamethrin and cypermethrin resistance Jazayeri, J.A. 2004. Biochemical studies on status of Rhipicephalus (Boophilus) sheep body louse Bovicola ovis (Schrank) microplus collected from six agro-climatic (Phthiraptera: Trichodectidae): comparison regions of India. Vet. Parasitol., 188: 337- of pyrethroid and organophosphate resistant 345. and susceptible strains. Pestic. Biochem. Shaw, R.D. 1966. Culture of an organophosphorus Physiol., 80: 41-11. resistant strain of Boophilus microplus Johnson, P.W., J.C. Boray and K.L. Dawson. 1992. (Canestrini) and assessment of its resistance Resistance to synthetic pyrethroid pour-on spectrum. Bull. Entomol. Res., 56: 398- insecticides in strains of the sheep body 405. louse Bovicola (Damalinia) ovis. Aust. Vet. Shyma, K.P., S. Kumar, A.K. Sharma, D.D. Ray and J., 69: 213-217. S. Ghosh. 2012. Acaricide resistance status Lee, S.H., K.S. Yoon, M.S. Williamson, S.J. in Indian isolates of Hyalomma anatolicum. Goodson, M. Takano-Lee, J.D. Edman, Exp. Appl. Acarol., 58(4): 471-481. DOI A.L. Devonshire and J.M., Clark. 2000. 10.1007/s10493-012-9592-3. Molecular analysis of kdr-like resistance Veneziano, V., L. Rinaldi, S. Giannetto and in permethrin-resistant strains of head lice G. Cringoli. 2003. The fi rst record of Pediculus capitis. Pestic. Biochem. Physiol., Haematopinus tuberculatus on Bubalus 66: 130-143. bubalis (water buffalo) in Italy. Bubalus Levot, G.W. and P.B. Hughes. 1990. Laboratory bubalis, 9: 69-75. studies on resistance to cypermethrin in Veneziano, V., M. Santaniello, S. Carbone,
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S. Pennacchio, M.E. Morgoglione, M. Schioppi, R. Condoleo and G. Cringoli. 2007. Lice (Haematopinus tuberculatus) in water buffalo farms from central Italy. Ital. J. Anim. Sci., 6: 926-927.
214 Original Article Buffalo Bulletin (June 2015) Vol.34 No.2
SUB-CLINICAL MASTITIS IN BUFFALOES: PREVALANCE, ISOLATION AND ANTIMICROBIAL RESISTANCE OF STAPHYLOCOCCUS AUREUS
K. Nithin Prabhu1, Wilfred S. Ruban2,*, G.S. Naveen Kumar3, R. Sharada1 and R.D. Padalkar4
ABSTRACT Mastitis prevalence showed signifi cant variation at different Age (p=0.0391), Parity (p=0.0089) This study investigated the status of and Stage of Lactation lactation (p=0.0430). Thus, clinical mastitis among milking buffaloes during mastitis was more at the 4th and 7th lactations. the period September 2010 to August 2011 in The results of antimicrobial susceptibility testing and around Hassan district, Karnataka, India. The revealed that S. aureus was highly susceptible to prevalence of mastitis was assessed by the results chloramphenicol (100%) followed by Enrofl oxacin of bacteriological evaluation of milk samples (97.14%), kanamycin (85.75%), streptomycin collected from clinical and subclinical mastitis (82.85), Cefalexin (74.28) and gentamycin cases. The study was intended to estimate the (65.71%). In contrast, isolates were highly resistant prevalence of mastitis caused by Staphylococcus to tetracyclines (74.28%), penicillin (71.42%) and aureus (S. aureus) and the drug resistance pattern ampicillin (45.71%). In conclusion, this study in collected milk samples. A total of 150 lactating confi rms the importance of S. aureus as a mastitis buffalo cows were tested for mastitis employing causing bacterium and the range of antibiotics to be the California Mastitis Test (CMT) and Electrical used in the control of mastitis. Conductivity (EC). Sixty-eight of the buffalo cows (45.33%) had mastitis, of which 29.41% Keywords: mastitis, buffalo, Staphylococcus (20/68) and 70.58% (48/68) showed clinical and aureus, antimicrobial sensitivity sub clinical mastitis, respectively. A total of 68 (20 from clinical and 48 from subclinical cases) milk samples were collected and cultured for S. aureus INTRODUCTION of which 35 resulted in growth of the bacterium (seven from clinical and 28 from subclinical cases). Buffaloes are the main dairy animal in The Staphylococcus aureus isolates were confi rmed India despite the fact that this species tends to have by conventional biochemical identifi cation. a relatively slow rate of reproduction and more
1Department of Veterinary Microbiology, Veterinary College, Bangalore, India 2Department of Livestock Products Technology, Veterinary College, Karnataka Veterinary Animal and Fisheries Science University, Hassan, Karnataka, India, *E-mail: [email protected] 3Department of Animal Genetics, Veterinary College, Karnataka Veterinary Animal and Fisheries Science University, Hassan, Karnataka, India 4Department of Veterinary Microbiology, Veterinary College, Karnataka Veterinary Animal and Fisheries Science University, Hassan, Karnataka, India
215 Buffalo Bulletin (June 2015) Vol.34 No.2
reproductive problems and mastitis (Ahmed et mortality (Rahman et al., 2010; Sharma and Maiti, al., 2008). Mastitis is the most common infectious 2010). Hence the present study was designed with disease affecting the dairy buffaloes and remains the objective to estimate the prevalence of mastitis the most economically important disease of dairy caused by Staphylococcus aureus in buffaloes and industries around the world (Chrishty et al., 2007). their antimicrobial pattern. Mastitis has two forms. Clinical mastitis usually has all the fi ve cardinal signs of udder infl ammation (redness, heat, swelling, pain and MATERIALS AND METHODS loss of milk production) and hence can be detected without any laboratory test and even by the layman. Study type and study area Whereas the subclinical form of mastitis is hidden The study was conducted in various and needs laboratory aid for diagnosis. Moreover, backyard dairy sectors from various geographic abnormal milk is readily detected in clinical locations in and around Hassan district, Karnataka, mastitis but there is no apparent change in milk in India. A total of 150 lactating cross-bred buffaloes sub-clinical mastitis. were selected from small holder dairy farms having Buffaloes have been reported to be less an average of two to three lactating buffalo cows susceptible to mastitis than cattle (Thapa and each. This study investigated the status of clinical Kaphle, 2002). Sub-clinical mastitis is 3-40 times mastitis among buffalo cows during the period more common than clinical mastitis and causes the September 2010 to August 2011 in and around greatest overall losses in most dairy herds (Singh Hassan district. The prevalence of mastitis was and Bansal, 2004). Subclinical mastitis causes assessed by the results of bacteriological evaluation loss of two-thirds of the total milk production of milk samples collected from clinical and due to affected quarters of animals (Radostits et subclinical mastitis cases. The study was intended al., 2007). These losses might be higher in India to estimate the prevalence of mastitis caused by because of poor management and few prevention Staphylococcus aureus and the drug resistance practices (Kumar et al., 2009) as prevalence of sub- pattern in collected milk samples clinical mastitis is infl uenced by many factors such as husbandry, management, genetics and nutrition Study methodology (Elbers et al., 1998). Data regarding the different potential A wide variety of bacteria can be involved, risk factors (age, parity and lactation stage) but the most common mastitis pathogen is were collected for 150 lactating buffalo cows by Staphylococcus aureus worldwide (Salmon, 2002 interviewing the farm owner. Clinical examination and Taverna et al., 2007). Staphylococcus aureus of the udder was carried out to note any visible is ubiquitous and can colonize the skin as well as signs of mastitis. However, milk samples were the udder. It is capable of causing peracute, acute, screened using the California mastitis test (CMT), subacute, chronic, gangrenous and subclinical electrical conductivity (EC) and bacteriological types of mastitis. The acute form of the disease examination. usually occurs shortly after parturition and tends to produce gangrene of the affected quarters with high
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Collection of milk samples and such milk samples were subjected to cultural Milk samples (n=150) were collected before examinations. milking using standard procedures described by the National Mastitis Council (Oliver et al., 2004). Bacteriological culture Before sample collection, teats were dipped with After initial enrichment in brain heart a premilking teat disinfectant, cleaned thoroughly, infusion broth overnight, 10 micro litre of enriched and dried with individual disposable paper towels, milk samples were streaked onto mannitol salt and teat ends were sanitized with swabs containing agar (MSA) plates, and incubated at 37oC for 70% ethyl alcohol. Milk samples were transported 24 h. The colony morphology and Gram staining on ice until analysis. In the laboratory these milk patterns were recorded and te colonies were samples were subjected for CMT and EC and further streaked onto BHI agar plates to obtain the samples positive by either of the tests were pure colonies and for further identifi cation as considered for bacterial isolation. Stphylococcus aureus based on gram staining, catalase positivity, the coagulase test, the Vogues- California mastitis test (CMT) Proskur test, urease production, the carbohydrate The California mastitis test was conducted fermentation test and other biochemical tests. to diagnose the presence of subclinical mastitis and it was carried out according to procedures given Antimicrobial resistance pattern test by Quinn et al. (1994). A squirt of milk from each The antimicrobial susceptibility test was quarter of the udder was placed in each of four conducted on randomly selected S. aureus isolates shallow cups in the CMT paddle and an equal (n=36) isolated during the study. The isolates were amount of the reagent was added. A gentle circular tested for eight antimicrobials using the Kirby- motion was applied in a horizontal plane. Positive Bauer disk diffusion method (Quinn et al., 1994; samples show gel formation within a few seconds. NCCLS, 1997). The following antimicrobial disks The result was scored based on the gel formation (M/s Hi-media, Mumbai) with their corresponding and categorized as negative if there was no gel concentrations were used: chloramphenicol (C, 30 formation, or positive if there was gel formation μg), enrofl oxacin (Ex, 10 μg), kanamycin (K, 30 ranging from +1 to +3. Further, the positive μg), streptomycine (S, 10 μg), cefalexin (Ce, 5 μg) samples were subjected to isolation and cultural gentamycin (Gn, 10 μg), penicillin G (P, 10 μg), examination. tetracycline (T 10 μg), and amoxacillin (Am, 5 μg). The inhibition zone was reported as the diameter Measurement of electrical conductivity (EC) of the zone surrounding the individual disk in Milk samples procured were subjected tor which bacterial growth was absent. Based on this, the electrical conductivity test using a milk checker the isolates were defi ned as resistant, intermediate (Oriental Instruments, Japan) and the readings and susceptible according to the guidelines of the were taken as per the manufacturer’s instructions. NCCLS (1997). Electrical conductivity of more than 6.5 milli Siemens (mS)/cm was taken as criteria to declare Data analysis the milk/animal as sub clinically mastitic /infected Prevalence was the calculated as the ratio
217 Buffalo Bulletin (June 2015) Vol.34 No.2
of the number of isolates to the number examined. et al., 2012), who reported a prevalence of 55-62% The 95% class interval of prevalence is estimated in buffaloes. This report was also in agreement as where rate is prevalence proportion in decimal with the assertion by Radostits et al. (2007) that, form, n is the population at risk. Proc frequency in most countries and irrespective of the cause, the and Proc logistic of SAS 9.2 program were used to prevalence of mastitis is about 50%. The prevalence fi nd the relationship between antibiotics and levels of subclinical mastitis in this study was in complete of susceptibility, and association of potential risk agreement with the fi ndings of Schultz et al. (1978) factors as age, parity and stage of lactation with who stateed that subclinical mastitis was 3-40 times occurrence of sub clinical mastitis. more common than clinical mastitis and causes the greatest overall losses in most dairy herds. This variation in prevalence between subclinical and RESULTS AND DISCUSSION clinical mastitis may be due to the fact that the defense mechanism of the udder reduces with the Prevalence and bacterial isolation severity of the disease (Sharma, 2010). Of the total 150 lactating buffalo cows All 68 positive samples were cultured examined during the study period, 68 of the total for S. aureus, and of these 35 resulted in growth examined buffalo cows (45.33%) had mastitis of the bacterium (seven from clinical and 28 from as indicated by CMT and EC, of which 29.41% subclinical cases) (Table 1). Many studies from (20/68) and 70.58% (48/68) showed clinical and Asian countries have reported that Staphylococcus sub-clinical mastitis, respectively (Table 1) by aureus is the chief etiological agent of mastitis in CMT and EC. During mastitis, the EC and CMT cattle and buffaloes (Sharma et al., 2004; Abdel- positivity increases signifi cantly due to an infl ux Rady and Sayed, 2009; Rahman et al., 2010; of somatic cells from the blood into the milk and Sharma and Maiti, 2010). release of the intra-cellular ions from the damaged cells, indicating that the milk composition is Associated risk factors adversely affected (Schukken et al., 2003). Logestic regression analysis of the data In the present study the mastitis prevalence revealed that the infl uence of age, parity and of 45.33% was in accordance with many of the stage of lactation on occurrence of sub-clinical earlier workers (Bachaya et al., 2005 and Hameed mastitis was statistically signifi cant with p values
Table 1. Number and percentage of S. aureus isolated from clinical cases and CMT positive subclinical buffalo cows.
No of S. aureus Forms of mastitis No examined Prevalence (%) 95% CI isolated Clinical 20 7 35.00 (14.09-55.90) Subclinical 48 28 58.33 (44.38-72.27) Total 68 35 51.47 (39.59-63.35)
218 Buffalo Bulletin (June 2015) Vol.34 No.2
of 0.0391, 0.0089 and 0.0430 respectively. Odds ampicillin (45.71%). The antimicrobial resistance ratio indicates that the animals in the age group profi les are shown in Table 3. The results of the between 6-8 years, 4th, 5th and 7th lactation, and mid present study were closely related to those reported stage of lactation were more prone for mastitis. by Chanda et al. (1989) and Rehman (1995). Mastitis was least prevalent during second According to the Fisher exact test the relationship lactation and increased with parity. Incidence of between antibiotics and levels of susceptibility infected quarters increases with the age (Sharma was statistically valid (P=2.93X10-62). High et al., 2004; Sharma and Maiti, 2010). Sharma et susceptibility of S. aureus was for chloramphenicol al. (2007) conducted a study on lactating buffaloes (100%) followed by Enrofl oxacin (97.14) and high of different age, parity and stage of lactation resistance of S. aureus was observed for tetracycline and found that higher prevalence of sub-clinical (74.29%) followed by pencillin G (71.43%) (Table mastitis (SCM) in buffaloes was recorded in 5-to- 3). The majority of the S. aureus were resistant 9-year-old animals and in the 3rd and 4th parities. to most commonly used antibiotics such as Older cows (>10 years) are at more risk (44.6%), tetracycline, penicillin and ampicillin. They were particularly for sub-clinical mastitis (38.6%), than found to be highly sensitive to chloramphenicol younger cows (23.6%) in which clinical mastitis and Enrofl oxacin. The development of resistance was predominant (Biffa et al., 2005). As the parity to earlier antibiotics could be due to their increased, there was an increase in the incidence of indiscriminate usage (Mallikarjunaswamy, 1997). mastitis (Lakshmi Kavitha et al., 2009). In previous studies, an association of mastitis with age (Nyman et al., 2007; Hammer et al., 2012) and parity (Parker CONCLUSION et al., 2007; Salem-Bekhit et al., 2010) have been reported. Prevalence, comparative probability of The results of the study revealed the greater than chi-square values, odds ratios and its prevalence of subclinical mastitis is alarming and 95% confi dence Intervals are presented in Table 2. needs to be addressed. Sub-clinical mastitis causes heavy economic losses due to decreased milk Antimicrobial susceptibility yield, increased costs for udder treatment, milk Antimicrobial susceptibility tests were withholding times and discarded milk. In addition, performed on all the 35 isolates of S. aureus obtained Staphylococcus spp. isolates from cases of mastitis from the study. Due to the relatively small size, in buffalo show varying levels of resistance to no separate analysis was undertaken for clinical antibiotics, and caution should be exercised in and subclinical isolates of S. aureus. In this study choosing therapeutics in order to minimize the the results of antimicrobial susceptibility testing risk to public health. From the future study point revealed that S. aureus was highly susceptible to of view it is recommended that regular testing chloramphenicol (100%) followed by Enrofl oxacin for subclinical mastitis be performed to reduce (97.14%), kanamycin (85.75%), streptomycin the economic losses and that the above tested (82.85%), Cefalexin (74.28%) and gentamycin antibiotics be evaluated in vivo. (65.71%). In contrast, isolates were highly resistant to tetracyclines (74.28%), penicillin (71.42%) and
219 Buffalo Bulletin (June 2015) Vol.34 No.2
Table 2. Association of age and lactation number with respect to subclinical mastitis.
Total Factor No. of positives (%) Odds Ratio with 95%CI P>Chisq No Age (years) 3-5 45 9(20) 3.860(0.220-67.609) 0.3552 6-8 54 24(44.44) 8.055(1.541-42.114) 0.0134 >9 31 15(48.39) 1 Parity 2nd 20 2(10) 5.186 (0.263-102.184) 0.2791 3rd 30 8(26.67) 1.306 (0.114-14.920) 0.0298 4th 30 19(63.33) 0.166 (0.043-0.638) 0.0089 5th 19 4(21.05) 1 0.00 6th 19 7(36.84) 3.618 (0.774-16.900) 0.021 7th 12 8(45.65) 1 Stage of lactation <3 months 55 18(32.72) 0.994 (0.345-2.865) 0.2907 3-6 months 46 21(45.65) 0.509 (0.173-1.500) 0.2206 >6 months 29 9(31.03) 1
Table 3. Resistance of S. aureus isolates to different antimicrobials (n=35).
Resistant Intermediate Susceptible Antimicrobials No (%) No (%) No (%) Chloramphenicol 0 (0) 0 (0) 35 (100) Enrofl oxacin 0 (0) 1 (2.86) 34 (97.14) Kanamycin 1 (2.86) 4 (11.43) 30 (85.71) Streptomycin 1 (2.86) 5 (14.29) 29 (82.86) Cefalaxin 2 (5.71) 7 (20.00) 26 (74.29) Gentamycin 3 (8.57) 9 (25.71) 23 (65.71) Penicillin G 25 (71.43) 2 (5.71) 8 (22.86) Tetracycline 26 (74.29) 9 (25.71) 0 (0) Ampicillin 16 (45.71) 12 (34.29) 1 (2.86)
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REFERENCES J. Anim. Plant Sci., 22(3): 375-380. Hammer, J.F, J.M. Morton and K.L. Kerrisk. 2012. Abdel-Rady,A. and M. Sayed. 2009. Epidemiological Quarter-milking-, quarter-, udder- and studies on subclinical mastitis in dairy cows lactation-level risk factors and indicators for in Assiut Governorate. Vet. World, 2: 373- clinical mastitis during lactation in pasture- 380. fed dairy cows managed in an automatic Ahmed, W.M., A.R. Abd El-Hameed and F.M. El milking system. Aust. Vet. J., 90: 167-174. Moghazy. 2008. Some reproductive and Kumar, R, B.R. Yadav and R.S. Singh. 2009, health aspects of female buffaloes in relation Genetic determinants of antibiotic resistance to blood lead concentration. International in Staphylococcus aureus isolates from milk Journal of Dairy Science, 3: 63-70. of mastitic crossbred cattle. Curr. Microbiol., Bachaya, H.A., Z. Iqbal, G.A. Muhammad, Yousaf 60: 379-386. and H.M. Ali. 2005. Sub-clinical mastitis in Kavitha, K.L., K. Rajesh, K. Suresh, K. Satheesh Attock District of Punjab (Pakistan). Pak. and N.S. Sundar. 2009. Buffalo mastitis-risk Vet. J., 25: 134-136. factors. Buffalo Bull., 28(3): 134-137. Biffa, D., E. Debela and F. Beyene. 2005. Prevalence Mallikarjunaswamy, M.C. and G.V.K. Murthy. and risk factors of mastitis in lactating dairy 1997. Antibiogram of bacterial pathogens cows in southern Ethiopia. Int. J. Appl. Res. isolated from bovine subclinical mastitis Vet. M., 3: 189-198. cases. Indian Vet. J., 74: 885-886. Chanda, A., C.R. Roy, P.K. Banerjee and C. Guha. Nyman, A.K, T. Ekman, U. Emanuelson, A.H. 1989. Studies on incidence of bovine Gustafsson, K. Holtenius, K.P. Waller mastitis, its diagnosis, etiology and in vitro and C.H. Sandgren. 2007. Risk factors sensitivity of the isolated pathogens. Indian associated with the incidence of veterinary- Vet. J., 66: 277-282. treated clinical mastitis in Swedish dairy Chrishty, M.A., M. Arshad, M. Avais, S. Hameed herds with a high milk yield and a low and M. Ijaz, 2007. Cross-sectional prevalence of subclinical mastitis. Prev. Vet. epidemiological studies on mastitis in cattle Med., 78: 142-160. and buffaloes of Tehsil Gojra, Pakistan. Oliver, S.P., B.M. Jayarao and R.A Almeida. 2005. Buffalo Bull., 26: 50-55. Food borne pathogens in milk and the dairy Elbers, A.R., J.D. Miltenburg, D. de Lange, farm environment food safety and public A.P. Crauwels, H.W. Barkema and Y.H. health implications. Foodborne Path. Dis., Schukken. 1998. Risk factors for clinical 2(2): 115-129. mastitis in a random sample of dairy herds Parker, K.I., C.W.R. Compton, F.M. Anniss, A. from the southern part of the Netherlands. J. Weir and S. McDougall. 2007. Management Dairy Sci., 81: 420-426. of dairy heifers and its relationships with Hameed, S., M. Arshad, M. Ashraf, M. Avais the incidence of clinical mastitis. N. Z. Vet. and M.A. Shahid. 2012. Cross-sectional J., 55: 206-216. epidemiological studies on mastitis in cattle Quinn, P.J., M.E. Carter, B.K. Markey and and buffaloes of Tehsil Burewala, Pakistan. G.R. Carter. 1994. Clinical Veterinary
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Microbiology. Wolfe Publishing. London, National Mastitis Council, Inc., Washington UK. 648p. DC, USA. Radostits, O.R., D.C. Blood and C.C. Gay. 2007. Sharma, H., S.K. Maiti and K.K. Sharma. 2007. Mastitis Veterinary Medicine: A textbook Prevalence, etiology and antibiogram of of the diseases of cattle, horses, sheep, pigs microorganisms associated with sub-clinical and goats, 9th ed. Bailer tindall, London. p. mastitis in buffaloes in Durg, Chhattisgarh 563-614. state. International Journal of Dairy Rahman, M.M., M.R. Islam, M.B. Uddin and Science, 2: 145-151. M. Aktaruzzaman. 2010. Prevalence of Sharma, N. and S.K. Maiti. 2010. Incidence, subclinical mastitis in dairy cows reared in etiology and antibiogram of sub clinical Sylhet District of Bangladesh. Int. J. Bio. mastitis in cows in Durg, Chhattisgarh. Res., 103: 23-28. Indian J. Vet. Res., 19: 45-54. Rehman, F.U. 1995. Study on Evaluation of Sharma, N., S.K. Maiti and K.M. Koley. 2004. surf fi eld mastitis test for the detection Studies on the incidence of sub clinical of subclinical mastitis in buffaloes and mastitis in buffaloes of Rajnandgaon district cattle, and antibiotic susceptibility of the of Chhattisgarh state. Vet. Pract., 5: 123- pathogens. M.Sc. Thesis, Department of 124. Clinical Medicine and Surgery, University Singh, R.S. and B.K. Bansal. 2004. Variation of Agriculture, Faisalabad, Pakistan. in selected components of milk among Salem-Bekhit, M.M., M.M. Muharram, I.M different milk fractions and it’s relevance to Alhosiny and M.E.S.Y. Hashim. 2010. diagnosis of mastitis in buffaloes. Buffalo Molecular detection of genes encoding Journal, 3: 213-224. virulence determinants in Staphylococcus Taverna, F., A. Negri, R. Piccinini, A. Zecconi, S. aureus strains isolated from bovine mastitis. Nonnis, S. Ronchi and G. Tedeschi. 2007. J. Appl. Sci. Res., 6: 121-128. Characterization of cell wall associated Salmon, S.A. 2002. Use of antimicrobial proteins of S. aureus isolated from bovine susceptibility data to assist in determining mastitis. Vet. Microbiol, 119(2-4): 240-247. the best therapy for clinical mastitis. In Thapa, B.B. and K. Kaphle 2002. Selecting Proceedings NMC 41st Annual Meeting, different drug combinations for control of Orlando, Florida, USA. Bovine clinical mastitis. J. Anim. Vet. Adv., Schukken, Y.H., F.J. Grommers, D. Van de Geer, 1: 18-21. H.N. Erb and A. Brand. 2003. Risk factors for clinical mastitis in herds with a low bulk milk somatic cell count. 2. Risk factors for Escherichia coli and Staphylococcus aureus. J. Dairy Sci., 74: 826-832. Schultz, L.H., R.W. Brown, D.E. Jasper, R.W.M. Berger and R.P. Natzke. 1978. Current Concepts of Bovine Mastitis, 2nd ed. The
222 Original Article Buffalo Bulletin (June 2015) Vol.34 No.2
ENTERIC PARASITIC INFECTION IN DIARRHOEIC BUFFALO CALVES
Deepti Naag, Madhu Swamy* and A.B. Shrivastav
ABSTRACT INTRODUCTION
Diarrhoea is a well-known clinical sign Calves are the livestock industry of the in neonatal animals. It’s etiology is complex future. One of the major causes of neonatal calf involving management, environmental, nutritional mortality and morbidity is diarrhoea. Diarrhoea and physiological variations and various infectious is a well-known clinical sign in neonatal animals. and parasitic pathogens. This study was undertaken It’s etiology is complex involving management, to determine the incidence of parasitic infection in environmental, nutritional and physiological diarrhoeic buffalo calves at the Livestock Farm variations and various infectious and parasitic Adhartal Jabalpur recorded for the period between pathogens. Infections with gastrointestinal parasites June and December 2011. Fecal samples from are among the important factors contributing to diarrhoeic buffalo and cow calves were examined increased calf mortality. Subclinical nematode to ascertain the presence of parasitic infection. parasitic infections cause great economic losses Direct smear, sedimentation and fl oatation methods and these go unnoticed in cattle on rangeland. It were used for detecting parasites in feces. The is also known that calf weight affects survivability overall incidence of enteric parasitic infection as of the calf (primarily in the early days), dam milk determined by faecal examination of diarrhoeic production and later performance (Stronbergh buffalo calves was calculated as 68.75%. The and Averbeck 1999). Growth is strongly affected parasites found comprised Cryptosporidium spp. by the consumption of milk by the calf (Sandoval Eimeria spp. Toxocara spp. and tapeworms. The et al., 2005), because it affects the availability highest incidence observed was Cryptosporidium of nutrients, the development of the digestive spp. (37.70%), followed by Toxocara spp. tract and the appropriate development of the (13.98%), Eimeria spp. (10.20%) and tapeworms immunity system against some diseases such as (8.1%). Maximal parasitic infection was recorded parasitosis (Arrayet et al., 2002; Coverdale et al., in the month of November. 2004). Schottstedt et al. (2005) mentioned that the parasite burden is infl uenced by the milk intake of Keywords: calves, diarrhoea, enteric, parasite the calf. Parasites could be present all year round, causing problems in all stages of calf growth and productivity, decreasing feed intake (Frisch and Vercoe, 1986).
Department of Pathology, College of Veterinary Science and Animal Husbandry, Jabalpur, India, *E-mail: [email protected]
223 Buffalo Bulletin (June 2015) Vol.34 No.2
This study was undertaken to determine buffalo and cow calves from different areas. The the incidence of parasitic infection in diarrhoeic incidence of parasitic infection will defi nitely be buffalo calves at an organized livestock farm governed by the managemental and deworming recorded for a period in which there were three practices followed in different dairy farms. The climatic conditions, that is, hot and dry (average higher incidence of parasitic eggs was found in the ambient temp 42oC), warm and humid (average cool and dry month of November. ambient temp 31oC ) and cool and dry (average The parasitic stages found during the ambient temp 26oC) faecal examination of different animals comprise Cryptosporidium spp., Eimeria spp., Toxocara spp. and tapeworms. The highest incidence observed was MATERIALS AND METHODS Cryptosporidium spp (37.70%). Cryptosporidium occurs in diarrheic calves and more than 10% of Buffalo calves of either sex up to 5 months all the scouring calves excrete cryptosporidium of age were included in the study. Samples were at the same time as rotavirus (Snodgrass et al., collected from 10 diarrhoeic and 10 non-diarrhoeic 1986). Signs are usually unapparent but chronic buffalo calves in each season. Thus, a total 30 diarrhea has been associated with cryptosporidiosis faecal samples were collected from diarrhoeic and especially in neonatal calves. An incidence of 30 from non-diarrhoeic buffalo calves. Diarrhoea 13.98% Toxocara spp. was found in the present was defi ned as an abnormally loose consistency of study. Toxocarosis in buffalo calves is considered as feces, and was classifi ed according to the clinical one of the most common ailments and is responsible signs present in the calves (anorexia, depression, for high mortality .According to Radiostits et al. weakness) and observations including the colour (2000), it is recognized as the number one cause and consistency of feces were also noted. Fecal of calf morbidity and mortality. The workers stated scraps were collected per rectum using sterilized that T. vitulorum larvae are passed in great numbers gloves and samples were placed in polythene in the colostrum 2-5 days after calving, worms are bags which were labeled and sealed properly for matured in the intestine of the calves by 10 days of identifi cation of calves. Direct smear, sedimentation age and eggs are passed by 3 weeks and then the and fl oatation methods were used for detecting adult worms are expelled from the intestine by 5 parasitic stages in feces. month of age, and for this reason, toxocariasis has been considered as calfhood disease. Reports on the incidence of clinical RESULTS AND DISCUSSION bovine calf coccidiosis are very scanty. Radostits et al. (2000) reported 15-20 days prepatent and The incidence of enteric parasitic infection 6-30 days incubation period of Eimeria infection. as determined by faecal examination was calculated 10.20% (10/98), Eimeria spp. stages were observed as 68.75% for diarrhoeic animals and 45% for non- in the present study which is in accordance with diarrhoeic animals.. Variable incidences of parasitic the fi ndings of earlier workers (Shah et al., 1990) infection has been reported by different workers who reported a low incidence (7.77%) of Eimeria (Riberio et al., 2000; Björkman et al., 2003) in spp. infection. However, Priti et al. (2008) reported
224 Buffalo Bulletin (June 2015) Vol.34 No.2
a prevalence of bovine coccidiosis among cattle Garnett, J.W. Oltjen, J. Imhoof, M.E. Kehrli (20.76%) and buffaloes (25%) at Patna (Bihar) Jr and T.W. Graham. 2002. Growth of and its surrounding areas. Thus, the prevalence Holstein calves from birth to 90 days: the of Eimeria infection varies with the geographical infl uence of dietary zinc and BLAD status. location and climatic conditions prevailing in the J. Anim. Sci., 80: 545-552. area. Coverdale, J.A., H.D. Tyler, J.D. Quigley and A low prevalence of 8.1% tapeworm J.A. Brumm. 2004. Effect of various infection was observed in the present study. levels of forage and form of diet on rumen Borthakur and Das (2005) examined faecal development and growth in calves. J. Dairy samples of calves of 240 cattle and 60 buffalo Sci., 87: 2554-2562. for Moniezia spp. infection. The infection rate Frisch, J.E. and J.E. Vercoe. 1986. Utilizing observed by the workers was 5%, 13.75% and 6% genotype x environment interactions for in indigenous, cross-bred cow calves and buffalo improving productivity of cattle in the calves, respectively. tropics. IAEA., 18(3): 57-67. An interesting fi nding was the prescience Sandoval, E., A. Valle, D.Y. Jiménez and O. of parasites in non-diarrhoeic faecal samples also. Márquez. 2005. Evaluación de pesosal This indicates that a subclinical parasitic infection nacery crecimiento en becerros doble of parasites may be prevalent leading to reduced propósito amamantados con vacas nodrizas weight gain. durante la etapa de lactantes. Zootecnia Tropical, 23: 1-16. Schottstedt, T., C. Muri, C. Morel, C. Philipona, CONCLUSION H.M. Hammon and J.W. Blum. 2005. Effects of feeding vitamin A and lactoferrin on In young calves the intestinal parasites epithelium of lymphoid tissues of intestine are usually considered responsible for early calf of neonatal calves. J. Dairy Sci., 88: 1050- mortality. Scientifi c deworming practices are not 1061. properly adopted on many farms. All the animals Stronbergh, B.E. and G.A. Averbeck. 1999. The role present under one shed are not given the dewormers of parasite epidemiology in the management at the same time. It was also observed that most of grazing cattle. International Journal of farmers are not using the recommended dose of a Parasitology, 29: 33-39. de-wormer. Dewormers are usually very expensive and their proper dosing is important not only to obtain maximum effi cacy but also to reduce the treatment cost.
REFERENCES
Arrayet, J.L., A.M. Oberbauer, T.R. Famula, I.
225 Buffalo Bulletin (June 2015) Vol.34 No.2 Original Article Buffalo Bulletin (June 2015) Vol.34 No.2
DETECTION OF VEROTOXIN PRODUCING STRAIN OF E. COLI IN BUFFALO CALVES
Deepti Naag, Madhu Swamy* and A.B. Shrivastav
ABSTRACT port of the test device. The reading was taken after 10 minutes incubation at room temperature. In a Production of verotoxin (Shigatoxin) is positive reaction a distinct red line appears on the most important criteria for the detection of test zone. Out of 32 isolates of E. coli, only 19 enterohaemorrhagic E. coli. There is a paucity of (59.37%) showed a positive reaction for presence data about the prevalence, burden of disease and of verotoxin. complications associated with verocytotoxigenic Escherichia coli (VTEC) in animals in India. The Keywords: calf, diarrhoea, E. coli, verotoxin main reservoir for EHEC is the feces of cattle, sheep and goats. In the present studies the verotoxin rapid test for the detection of verotoxin was performed INTRODUCTION for all the bacterial isolates obtained from faeces of buffalo calves. It is an immunochromatographic Enterotoxigenic Escherichia coli are rapid test utilizing monoclonal antibodies which probably the most common single cause of are labeled by red colored gold particles.The faecal undifferentiated neonatal diarrhea in calves. sample was collected directly from the rectum Escherichia coli have several virulence attributes of each calf by using sterile swabs. The micro- that result in disease in animals. Principally, these organism was cultured on MacConkey agar and promote colonization or adhesion to the mucosa, eosin methylene blue agar medium for preliminary cause metabolic dysfunction or death of enterocytes, characterization. On MacConkey agar lactose affect the local or systemic vasculature or promote fermenting smooth colonies and on EMB agar dark invasion and septicemia. The enteropathogenic centered blue black colonies with metallic shine strains of E. coli which colonize the mucosa of were identifi ed as E. coli. Dacron swabs were taken the small intestine may also secrete cytotoxins from the 32 isolates of E. coli on MacConkey agar (Shigatoxins=verotoxin) that have an effect locally plates obtained from diarrhoeic buffalo calves. or systemically and are categorized as verotoxin The swab was suspended in 0.5 ml distilled water producing (VTEC) or enterohaemorrhagic (EHEC). containing 50 μg/ml polymyxin B to enhance the These are a serious cause of food-borne illness in release of toxin and the mixture was incubated for humans and haemorrhagic enterolitis in calves 30 minutes a 35oC. Using a micropipette 200 μl of (Gyles, 2007). Production of verotoxin is the most the sample was dispensed into the circular sample important criteria for the detection of this group
Department of Pathology, College of Veterinary Science and Animal Husbandry, Jabalpur, India, *E-mail: [email protected]
227 Buffalo Bulletin (June 2015) Vol.34 No.2 of bacteria. There is a paucity of data about the RESULT AND DISCUSSIONS prevalence, burden of disease and complications associated with verocytotoxigenic Escherichia In direct smear examination of rectal swab coli (VTEC) in animals in India (Gopinathan et after staining Gram positive bacilli were seen in few al., 2005). This study was undertaken to detect the cases of diarrhoeic buffalo calves. On MacConkey presence of verotoxin producing Escherichia coli agar lactose fermenting smooth colonies and in feces of buffalo calves. on EMB agar dark centered blue black colonies with metallic shine were identifi ed as E. coli. For epidemiological or clinical purposes E. coli strains MATERIALS AND METHODS are often selected from MacConkey agar plates after presumptive visual identifi cation of lactose The faecal samples were collected directly fermenting pink colonies. Earlier workers (Duffy, from the rectum of each buffalo calf by using 2003; Paul et al., 2010) have also used similar sterile swabs. Direct fecal smears were observed methods for isolation of E coli from faeces of and samples were further processed for bacterial animals. There are no specifi c media recommended culture. The micro-organism was cultured on for isolation of enterotoxigenic E. coli but like MacConkey agar and Eosin methylene blue agar other E. coli strains they can grow on nutrient agar, medium for preliminary characterization. A total of MacConkey lactose agar, and eosin methylene blue 32 isolates of E. coli were obtained. The Duopath (EMB) agar. Blood agar plates can also be used for Verotoxin rapid test was used for identifi cation of isolation of diarrhoeagenic E. coli (Edwards and enterohaemorrhagic E. coli infections. The Duopath Ewing, 1972). Verotoxin test is an immunochromatographic test Swabs were taken from the E. coli culture utilizing monoclonal antibodies which are labeled of MacConkey agar plates obtained from 32 by red-colored gold particles. The test device has a diarrhoeic buffalo calves. Out of these only 19 circular sample port and an oval-shaped test (VT1, (59.37%) showed a positive reaction for presence VT2) and control (C) window. of verotoxin. A number of studies have been The confl uent growth area of the culture conducted to determine the incidence of verotoxic from MacConkey agar plates was swept a few times E. coli in cattle. Khurana and Kumar (2005) using dacron swabs. The swab was suspended in conducted studies to determine the status of dairy 0.5 ml distilled water containing 50μg/ml polymyxin cattle from Hisar and surrounding areas as the B to enhance the release of toxin and the mixture principal reservoir of verotoxic E. coli by faecal was incubated for 30 minutes a 35oC. Using a examination of healthy and diarrhoeic cow and micropipette 200 μl of the sample was dispensed calves, They observed a signifi cant prevalence of into the circular sample port of the test device. The VTEC in faecal samples of both diarrheic as well reading was taken after 10 minutes incubation at as healthy cattle, The workers expressed concern room temperature. In a positive reaction a distinct that healthy animals in close proximity to humans, red line appears on the test zone. manual handling of faeces and poor hygiene at the time of milk production and collection may pose a threat to human health. Rugbjerg et al., (2003) stated
228 Buffalo Bulletin (June 2015) Vol.34 No.2
that among calves aged 1-4 months old, carriage Haryana Veterinarian, 44: 83-85. of VTEC E. coli O157 was reduced if the calf had Paul, S.K., M.S.R. Khan, M.A. Rashid, J. Hassan suckled colostrums from the mother or if the calf and S.M.S. Mahmud. 2010. Isolation and had stayed more than 2 days with the mother after characterization of escherichia coli from calving. Although bovine VTEC belong to a large buffalo calves in some selected areas of number of O serogroups it is not known whether Bangladesh. Bangl. J. Vet. Med., 8(1): 23- all variants of VTEC are equally pathogenic as 26. verotoxin production alone may not be suffi cient Rugbjerg, H., E.M. Nielsen and J.S. Andersen. 2003. for VTEC to cause disease. A factor that may affect Risk factors associated with faecal shedding virulence of VTEC is the ability to cause attaching of verocytotoxin-producing Escherichia coli and effacing (AE) lesions in the intestinal mucosa. O157 in eight known infected Danish dairy Little is known about the putative pathogenic role herds. Prev. Vet. Med., 58: 101-105. of VTEC in cattle. Looking into the present fi nding of a high incidence of VTEC in diarrhoeic calves but negligible incidence of VTEC non-diarrhoeic animals it can be suggested that contrary to the earlier variable reports VTEC may be pathogenic to calves causing diarrhea.
REFERENCES
Duffy, G. 2003. Vero Toxigenic Escherichia coli in animal feces, manures and slurries, J. Appl. Microbiol., 94: 945-1035. Edwards, R. and W.N. Ewing. 1972. Identifi cation of Enterobacteriaceae, 3rd ed. Burgess Publishing Co., Minnesota. Gopinathan, R., G. Kang, G.V. Asokan, N. Arunagirinathan and G. Vasanthana. 2005. Evidence of verocytotoxigenic Escherichiia coli (VTEC) in livestock of Tamil Nadu- an explorative study. Indian J. Anim. Sci., 75(11): 1241-1243. Gyles, C.L. 2007. Shiga toxin-producing Escherichia coli: An overview. J. Anim. Sci. 85: 45-62. Khurana, P. and A. Kumar. 2005. Occurrence of verotoxic E. coli in faeces and milk of cattle.
229 Buffalo Bulletin (June 2015) Vol.34 No.2 Original Article Buffalo Bulletin (June 2015) Vol.34 No.2
RETROSPECTIVE STUDY OF HYDATIDOSIS IN BUFFALOES SLAUGHTERED IN MIRHA EXPORTS PRIVATE LIMITED IN PUNJAB, INDIA
K. Aarif1,*, B. Suhani2, K.N. Mathur3, R.L. Sharma3, D.M. Makhdoomi4, A. Nazir2, A. Maria2 and Mehraj-U-Din5
ABSTRACT INTRODUCTION Hydatidosis, a disease of public health importance, is caused by the metacestodes of tape Hydatidosis is a zoonotic disease of public worm Echinococcus granulosus. An investigation health importance. The tape worm Echinococcus on the prevalence of the disease was undertaken granulosus is found in the small intestine of in buffaloes–calves, heifers and adults–slaughtered carnivores, particularly dogs and the metacestode in Mirha Exports Pvt. Ltd. between March 2010 (hydatid cyst) is found in a wide variety of ungulates to Feb. 2011 revealed the overall prevalence as and man (Varma et al., 1994). The pathogenecity of 50.96%. The group wise prevalence recorded was the cyst depends on the severity of the infection 3.52%, 5.58% and 54% in calves, heifers and adult and the organ in which it is situated. In hydatidosis buffaloes respectively. Females outnumbered the there is development of hydatid cyst in lungs, prevalence in heifers and adult buffaloes as 82.19% liver, brain, spleen and heart. In domestic animals and 84%. The cyst showed higher occurrence in all clinical signs are not commonly seen despite heavy the groups in winter (54.28%), spring (28.14%) infections. Diagnosis of hydatidosis in domestic followed by summer (21.92%) and autumn animals is rarely made at antemortem (Soulsby, (20.92%). Distribution of the hydatid cyst in organs 1982). However, immuno-diagnosis of hydatid showed that the lungs accounted highest followed diseases has been carried out by different workers by liver and spleen. Fertile cysts recoded highest in sheep, camel and cattle (Tassi et al., 1980; Dada than sterile. et al., 1981; Bandyopadhyay and Basu, 1996). Consumption of buffalo meat in India Keywords: buffaloes, Bubalus bubalis, hydatidosis, was around 2 million tons in 2012, as per a GAIN Echinococcus granulosus, cysts, seasons (Global Agricultural Information Network) report. Buffalo meat has doubled in three years between
1Veterinary Surgery and Radiology, Teaching Veterinary Clinical Complex, Apollo College of Veterinary Medicine, Jaipur, India, *E-mail: [email protected] 2,Veterinary Public Health and Epidemiology, Apollo College of Veterinary Medicine, Jaipur, India 3Veterinary Public Health and Epidemology and Veterinary Parasitology, Apollo College of Veterinary Medicine, Jaipur, India 4Teaching Veterinary Clinical Complex, Shere-Kashmir University of Agricultural Sciences and Technology of Kashmir, Jammu and Kashmir, India 5Mirha Exorts Pvt. Ltd., New Delhi, Delhi, India
231 Buffalo Bulletin (June 2015) Vol.34 No.2
2008 and 2011 and is set to scale further heights. out the operations. All the buffaloes come from (Business line, 2012). The estimation of the the different parts of Punjab, viz, Batala, Rajpura, economic importance of hydatidosis varies greatly Ludhiana, Amritsar and Sultanpur. The method of amongst countries and regions. There are reports slaughtering was halal. of hydatidosis in buffaloes slaughtered in different A total of 209,615 buffaloes were regions of India (Khan and Purohit, 2006). The slaughtered from March 2010 through Feb. 2011 contamation rate in North India was reported as 48% (Table 1), which were categorized into three groups for water buffalo (Singh et al., 1998a). Because of (A, B and C) based on the age detected at the ante- extensive distribution throughout the world, it gets mortem examination. such a signifi cant attention that prevention of the Group A consisted of calves (>1yr), disease is one of the dynamic programmes of the accounting for 5,100. (males=4,131 and World Health Organisation (WHO Report, 1982). females=969). Indian subcontinent provides ideal conditions for Group B consisted of heifers (2-3 the establishment, propagation and dissemination yrs), accounting for 7,840. (males=5,644 and of hydatidosis both in man and livestock (Samra et females=2,196). al., 2000 and Gupta et al., 2011). Group C consisted of adult buffaloes Owing to its zoonotic and economic (3-7years), accounting for 196,675 (males= 8,260 importance in India, our study was aimed to and females =188,415). determine the prevalence of hydatid cysts in The organs visualized and palpated buffaloes slaughtered in Mirha Exports Private were lungs, liver, spleen and heart. The cysts Ltd. between March 2010 and Feb 2011. collected were examined in laboratory to ascertain whether fertile or sterile, based on the presence of protoscolices (Soulsby, 1982). The organ wise MATERIALS AND METHODS fertility rate was recorded.
The study was conducted at Mirha Exports Private Ltd. a government-approved slaughter RESULTS AND DISCUSSION house (APEDA No. 125). The slaughter house has a capacity of 700 buffaloes per day. The plant is The 209,615 buffaloes slaughtered from located at SAS-Nagar near Chandigarh, Mohali March 2010 through Feb. 2011 were as depicted India. Mohali has a sub-tropical continental monsoon below in Table 1and were categorized into three climate characterized by a seasonal rhythm, hot groups (A, B and C) based on the age detected at the summers, slightly cold winters, unreliable rainfall ante-mortem examination. The study revealed that and great variation in temperature (-1 to 44oC or 30 the overall prevalence of hydatidosis was 50.96%. to 111oF). In winter, frost sometimes occurs during Earlier, Singh et al. (1998), Khan and Purohit December and January. The average annual rainfall (2006), Verma and Swamy (2009) and Terefe is recorded at 617 ml (24.3 inch). The company (2012) reported overall prevalences of 48%, 34.5%, has seven qualifi ed veterinarians who assist the 23.53% and 40.5% in slaughtered water buffaloes in government appointment Veterinarian in carrying different parts of India. Khan (1996) and Kumar et
232 Buffalo Bulletin (June 2015) Vol.34 No.2
al. (2008) reported a prevalence of 6.52% in buffalo in the spring (30%) and the minimum number of in the north east of India. Pednekar et al. (2009) infections in the winter (24.51%). However, our found a prevalence of 3.8% in buffaloes as against fi ndings are parallel to those of Mohamadin and 5.10% in cattle of Maharashtra region of India. In Abdelgadir (2011) who also documented higher our studies the high prevalence of hydatidosis could prevalence in the winter. The increased incidence of be due to the fact that various sites where buffaloes the disease in the winter may be due to the survival were brought may have had unhygienic conditions, of the cyst in the organs for various days in the cold especially overpopulation by stray dogs. Signifi cant as compared to hot summers. There was also higher variations in the prevalence of hydatidosis has been infl ow of buffaloes for slaughter in the winter as observed in India by Ghourai and Sahai (1989) and compared to other seasons. (Jithendran, 1996) also Irshadullah et al. (1989). reported highest occurrence of hydatidosis in sheep The group wise study showed the and goat as 28.3% in sheep and 19.45% in goats prevalence as 3.52%, 5.58% and 54% in calves, infected in the winter. heifers and adult buffaloes, respectively (Table Distribution of the hydatid cysts in organs 2). Terefe (2012) reported a prevalence of 9.3% showed that lungs accounted (2.17%) followed by in calves. Lahmer et al. (1999) observed a higher liver (1.51%) and spleen (0.04%) in calves (Table prevalence in males (44.8%) than in females 4a). However no cyst was observed in the heart. (25.2%). A prevalence of cysts higher in adults The organ wise fertility showed that 7.04% male than calves was also documented by Gupta et al. cysts were fertile in lungs as compared to 2.94% (2011). The obvious reason for higher prevalence in female cysts. In liver, 54.83% cysts were fertile in advanced age may be due to the various aliments in females whereas 30.95% in males. In heifers of ageing and the chronic nature of the disease. (Table 4b) the lungs showed 1.84% prevalence The prevalence among females was greater (29.92% fertile in females and 64.17% in males) in heifers and adults as 82.19% and 84% as against as compared to liver, 1.31% (24.44% fertile in the calves, where the males showed a higher males and 32.75% in females), spleen 0.21% (40% incidence (63.88%). This is in total agreement with in males and 41.66% females) and heart 0.01%. the fi ndings of Khan and Prohit (2006) and Pour The adult buffaloes (Table 4c) revealed that lungs et al. (2012) who also reported higher incidents constitute 14.96% (0.44% fertile in females and of hydatidosis in female buffaloes slaughtered. 88.23% in males) of hydatid cysts, liver 9.55% The higher prevalence in male calves could be (1.18% fertile in females and 70.66% in males), due to the apathy of the owners who are reluctant spleen 0.01% (69.66% fertile in females and to provide better management to the male calves, 29.26% in males) and heart 0.01% (36.15% fertile owing to their negligible utility. in females and 44.44% in males). The fi ndings are Table 3 revealed that the cysts showed in correlation with Nadery et al. (2011), Pour et higher occurrence in all the groups in the winter al. (2012), who also reported higher prevalence in (54.28%), the spring (28.14%) followed by the lungs as compared to liver. (Singh et al., 1988b) summer (21.92%) and the autumn (20.92%). This reported presence of the cyst as 60% lungs, 32% is contrary to the fi ndings of (Khanmohammad, liver, 4% spleen, 2% kidney, 0.9% heart and 2008) who reported maximum number of infections 0.1% brain. Higher infections of cysts in lungs as
233 Buffalo Bulletin (June 2015) Vol.34 No.2
Table 1. Number of buffaloes slaughtered at Mirha Exports Private Ltd., Punjab.
Calves (1>year) Heifers (2-3 years) Adult (3-7 years) Seasons Months Males Females Males Females Males Females March 644 64 471 181 720 14,810 Spring April 464 71 482 190 545 14,506 May 185 94 467 154 453 12,846 June 154 64 411 121 308 11,766 Summer July 222 43 436 98 427 9,767 August 131 37 489 76 486 8,658 September 265 49 497 167 451 9,845 Autumn October 345 89 412 189 476 12,735 November 436 77 489 204 779 14,634 December 581 78 538 290 785 13,795 Winter January 358 91 454 277 864 14,164 February 346 212 498 249 1,966 50,889 4,131 969 5,644 2,196 8,260 188,415 Total 5,100 7,840 196,675
Table 2. Age-wise prevalence of hydatidosis in respective groups.
Groups Total Positive Male Female Calves 180 115 65 5,100 (1>year) (3.52%) (63.88%) (36.11%) Heifer 438 78 360 7,840 (2-3year) (5.58%) (17.80%) (82.19%) Adult buffaloes 106,208 16,993 89,215 196,675 (3-7years) (54%) (15.99%) (84%)
234 Buffalo Bulletin (June 2015) Vol.34 No.2
Table 3. Seasonal prevalence of Hydatidosis in respective seasons.
Groups Adult buffaloes Seasons Calves (1>year) Heifer (2-3 years) (3-7 years) Total Positive Total Positive Total Positive Spring 1522 456 1945 583 43880 12288 % prevalence (group) 29.96% 29.97% 28.0% % prevalence (season) 28.14% Summer 651 136 1631 342 31412 6911 % prevalence (group) 20.89% 20.96% 22.0% % prevalence (season) 21.92% Autumn 1261 252 1958 391 38920 8173 % prevalence (group) 19.98% 19.96% 20.99% % prevalence (season) 20.92% Winter 1666 643 2306 922 82463 45355 % prevalence (group) 38.59% 39.98% 55% % prevalence (season) 54.28%
Table 4a. In situ involvement of visceral organs and number of cysts recovered with their fertility rate in calves.
Calves (1>year) Organs Total Positive Fertile Sterile Lungs (males) 4,131 71 5 66 Lungs (females) 696 34 1 33 Liver (males) 4,131 42 13 29 Liver (females) 696 31 17 14 Spleen (males) 4,131 02 0 02 Spleen (females) 696 0 0 0 Heart (males) 4,131 0 0 0 Heart (females) 696 0 0 0 Percentage 1.71% (males) 7.04% fertile prevalence in Lungs (males and females with fertility rate) 4.88% (females) 2.94% fertile
Percentage 1.01% (males) 30.95% fertile prevalence in Liver (males and females with fertility rate) 4.45% (females) 54.83% fertile
Percentage 0.04% (males) 0% fertile prevalence in Spleen (males and females with fertility rate) 0% (females) 0% fertile
235 Buffalo Bulletin (June 2015) Vol.34 No.2
Table 4b. In situ involvement of visceral organs and number of cysts recovered with their fertility rate in heifers.
Heifers (2-3 years) Organs Total Positive Fertile Sterile Lungs (males) 5,644 67 43 24 Lungs (females) 2,196 78 21 57 Liver (males) 5,644 45 11 34 Liver (females) 2,196 58 19 39 Spleen (males) 5,644 05 2 03 Spleen (females) 2,196 12 5 7 Heart (males) 5,644 01 0 01 Heart (females) 2,196 0 0 0 Percentage 1.18% (males) 64.17% fertile prevalence in Lungs (males and females with fertility rate) 3.55% (females) 26.92% fertile
Percentage 0.79% (males) 24.44% fertile prevalence in Liver (males and females with fertility rate) 2.64% (females) 32.75% fertile
Percentage 0.08% (males) 40% fertile prevalence in Spleen (males and females with fertility rate) 0.54% (females) 41.66% fertile Percentage prevalence in Heart (males and 0.01% (males) 0% fertile females with fertility rate)
236 Buffalo Bulletin (June 2015) Vol.34 No.2
Table 4c. In situ involvement of visceral organs and number of cysts recovered with their fertility rate in adult buffaloes.
Adult (3-7 years) Organs Total Positive Fertile Sterile Lungs (males) 8,260 612 540 72 Lungs (females) 1,188,415 178,454 789 177,665 Liver (males) 8,260 409 289 120 Liver (females) 1,188,415 113,919 1,345 112,574 Spleen (males) 8,260 41 12 29 Spleen (females) 1,188,415 89 62 27 Heart (males) 8,260 09 04 05 Heart (females) 1,188,415 130 47 83 Percentage 7.40% (males) 88.23% fertile prevalence in Lungs (males and females with fertility rate) 15.% (females) 0.44% fertile
Percentage 4.95% (males) 70.66% fertile prevalence in Liver (males and females with fertility rate) 9.58% (females) 1.18% fertile
Percentage 0.49% (males) 29.26% fertile prevalence in Spleen (males and females with fertility rate) 0.007% (females) 69.66% fertile
Percentage 0.10% (males) 44.44% fertile prevalence in Heart (males and females with fertility rate) 0.01% (females) 36.15% fertile
237 Buffalo Bulletin (June 2015) Vol.34 No.2
43.09%, liver 24.39%, spleen 2.76%, heart 1.65% REFERENCES was reported by Varma and Ahluwalia (1990). Our study is in contrast with Rinaldi et Arbabi, M. and H. Hooshyar. 2006. Survey of al. (2008) who reported only sterile and calcifi ed Echinococcosis and Hydatidosis in Kashan cysts in the lungs and liver of slaughtered buffalo. Region, Central Iran. Iran J. Public Health, Higher prevalence of sterile cysts in buffaloes was 35(1): 75-78. also documented by Varma and Malviya (1988). Bandyopadhyay, S. and A. Basu. 1996. Serological However Arbabi and Hooshyar (2006) found the Survey of hydatid disease in cattle in fertile cysts in lungs of the slaughtered animals. Calcutta, India. Vet. Parasitol., 10(1): 75- The high prevalence of fertile cysts as encountered 78. in the study poses a great public health hazard to Business line. 2012, Times of India 21st June by butchers and meat handlers. Subramani, M.R. Dada, B.J.O., D.S. Adegboye and A.N. Mohamed. 1981. Experience in Northern Nigeria with CONCLUSION counter immunoelectrophoresis, double diffusion and indirect HA teat for diagnosis Higher infections in buffaloes slaughtered of hydatid cyst in camels. J. Helminthol., in the Mirha Exports are due to the overpopulation 55: 197-202. of stray dogs in the areas where the plant gets its Ghourai, S.K. and B.N. Sahai. 1989. Studies on the supply. The lack of unhygienicunhygiene and incidence of hydatid disease in ruminants. appropriate disposal of affected organs at the plant Indian Journal of Animal Health, 28: 39- adds to the worry. So, there is a need of drastic 41. programme of deworming in stray dogs and Gupta, V.K., B. Bist, R.D. Agarewal and P. Gupta. buffaloes in the mandi areas. Proper disposal of the 2011. Buffalo hydatidosis in Agra city of affected organs, the awareness of the butchers and Uttar Pradesh. Vet. Parasitol., 25(1): 88-89. their sero-surveillance is the need of the hour. Irshadullah, M., W.A. Nizami and C.N.I. Maepherson. 1989. Observations on the suitability and importance of the domestic ACKNOWLEDGEMENTS intermediate hosts of Echinococcus granulosus. J. Helminthol., 63: 39-44. The authors are highly thankful to the Jithendran, K.P. 1996. Occurance of hydatidosis Managing Director of the Mirha Exports Pvt. Ltd. and various liverfl uke infections in sheep for facilitating the study. Thanks are due to the and goats in Kangra valley. An Abattoir unlettered butchers whose helped us lot during the study. Vet. Parasitol., 10(1): 63-67. investigation. Khan, N.A. 1996. Prevalence of Echinicoccosis in Buffaloes and applicability of gel diffusion and Immunoelectrophoresis test in diagnosis of disease. M.V.Sc. Thesis, Rajasthan Agriculture University, Bikaner, India.
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Khan, N.A. and S.K. Purohit. 2006. Prevalance of Italy. Zoonoses Public Health, 55(2): 119- Echnicoccosis in bufffaloes. The Scientifi c 123. World Journal, 286357: 5. Samra, M.D., D.K. Deha and M.R. Borkakoty. Khanmohammad., M., S. Gayem and M.Z. Zadeh. 2000. Occurance of hydatidosis and 2008. The prevalance of hydatidosis by porcine cysticercosis in Guwahati city. Vet. sex, season and location in slaughtered Parasitol., 32: 33-34. Buffaloes at Tabriz Abattoir in 2006-2007. Singh, B.P. and D.N. Dhar. 1998. Echinococcus The Internet Journal of Veterinary Medicine, granulosus in animals in Northern India. 4(2): 10.5580/221. Vet. Parasitol., 28(3): 261-266. Kumar, D.D., I. Saidul, B. Manoranjan, A. Saleque, Singh, B.P., D. Sharma and V.K. Srivastava. 1988b. H. Isfaqul and K. Natr. 2008. Bovine Prevalence of hydatid in Buffalo in India. Cysticercosis. Vet. Parasitol., 22(1): 15-35. India Journal Helminthol., 62: 124-126 Lahmer, S., M. Kilani, P.R. Torgerson and M.A. Singh, B.P., V.P. Deorani and V.K. Srivastava. Gemell. 1999. Echinococcus granulosus 1988a. Prevalence of hydatid in buffaloes in larvae in the livers of sheep in Junisia. The India and report of a severe liver infection. effect of host age. Ann. Trop. Med. Parasit., J. Helminthol., 62(2): 124-126. 93: 75-81. Soulsby, E.J.L. 1982. Helminths, Arthropods and Mohamadin, S.A. and A.E. Abdelgadir. 2011. Protozoa of Domesticated Animals, 7th ed. Study on hydatid cyst infection in slaughter 119-127. houses in Khartoum state, Sudan. Archives Tassi, C., S. Dottorini, A.G. Tolu and F. Derosa. of Applied Science Research, 3(6): 18-23. 1980. Diagnosis of hydatid disease in Nadery, B., Y. Mahdi and A.D. Mohammad. sheep by indiresct HA test. Rivista di. 2011. Survey on Hydatid cyst infestation Parassitologia, 41: 61-66. in Sarab city using Epidemological and Terefe, D., K. Kibrusfaw, B. Desta and W. Seroepidemological study. J. Anim. Vet. Anteneh. 2012. Prevalance and fi nancial Adv., 10(16): 2099-2101. loss estimation of hydatidosis of cattle Pednekar, R.P., M.L. Gatne, R.C. Thompson and R.J. slaughtered at Addis Ababa Abattoir Traub. 2009. Molecular and morphological Enterprises. Journal of Veterinary Medicine characterisation of Echinococcus from food and Animal Health, 4(3): 42-47. producing animals in India. Vet. Parasitol., Varma, T.K. and H.C. Malviya. 1988. The 165(1-2): 58-65. incidence of hydatid cysts in slaughtered Pour, A.A., S.H. Hosseini and P. Shayan. 2012. The domestic food animals in Bareilly. Rivista prevalence and fertility of hydatid cysts in iber. Parasit., 49: 45-49. buffaloes from Iran. J. Helminthol., 86(3): Varma, T.K., H.C. Malviya and B.M. Arora. 1994. 373-377. Hydatid cyst from a swamp deer. J. Vet. Rinaldi, L., M.P. Maurelli, F. Capuano, A.G. Parasitol., 8(2): 99-100. Perugini, V. Veneziano and S. Cringoli. 2008. Varma, T.K. and S.S. Ahluwalia. 1990. Prevalence Molecular updates on cystic Echinococcosis of Echinococcus granulosus infection in in cattle and water buffaloes of southern domestic animals of western and central
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Uttar Pradesh. J. Vet. Parasitol., 4(2): 67- 69. Verma, Y. and M. Swamy. 2009. Prevalance and pathology of hydatidosis in buffalo liver. Buffalo Bull., 28(4): 207-211. World Health Organization Report of WHO . 1982. Informal Consulation on research requirements for Hydatidosis. Montreal, Canada, 3rd, WHO CDs VPH 37.
240 Original Article Buffalo Bulletin (June 2015) Vol.34 No.2
INDUCTION OF ESTRUS IN ANESTRUS MURRAH BUFFALOES AND PROGRAMMED BREEDING
M. Thangapandiyan*, P. Pothiappan, R.M. Palaniappan, E. Samual Joseph and D. Kathiresan
ABSTARCT norgestomet ear implant with PMSG successfully induced estrum in Murrah buffaloes and the This study was designed to evaluate the use conception rate of 55 percent is comparable with of norgestomet ear implant and PMSG to induce that of normal cycling buffaloes. ovulatory oestrus in post-partum anestrus Murrah buffaloes. Twenty post-partum Murrah buffalo Keywords: PMSG, norgestomet, anestrus, cows were selected for this study. Rectal palpation conception of ovaries was done twice at a 10-day interval and the buffaloes were confi rmed as anestrus based on the palpation of ovaries. All the twenty buffaloes INTRODUCTION were given 6 mg of norgestomet ear implant and 2 ml of Syncromate-B injection containing 5 mg Buffaloes contribute greatly to the of oetradiol valerate and 3 mg of norgestomet. The economy of the rural masses and to more than implants were removed on day 10 and 500 IU of 50% of the total milk production in India. Further PMSG was administered intramuscularly at the these animals are best suited for our changing time of removal. The response to the treatment was agroclimatic conditions. But the low fertility in 100%. The onset, duration and intensity of estrus buffaloes characterised by prolonged post-partum were assessed. The average time taken for the onset anestrus, continues to be a major constraint to the of estrum was found to be 37+0.433 h (36-40 h) economic production of Murrah buffaloes (Porwal and the average duration of estrum was 20.4+0.674 et al.,1981). True anestrus is the condition in which h (18-24 h). The percentages of animals which both the ovaries are small, smooth, inactive with exhibited intense, normal and weak intensity of the absence of graffi an follice or corpus luteum estrum were 35%, 50% and 15% respectively. The and characterised by cessation of sexual cycle animals were inseminated with frozen semen at 48 and psychic manifestation of estrus (Nayak et and 72 h after the removal of the implants. Seven al., 2009). Higher incidence of anestrus due to animals (35%) conceived with the fi rst artifi cial inactive ovaries in buffaloes than in cows has been insemination and four animals (20%) conceived reported by Tanwar et al. (2003). The dry non- with the second artifi cial insemination. The overall pregnant buffaloes have become a fi nancial burden conception rate over two inseminations was 55%. to the milk man, hence such animals are sold for It is evident from this study that the throw away prices which results in less margin Department of Veterinary Pathology, Madras Veterinary College, Chennai, India, *E-mail: sugigold@gmail. com
241 Buffalo Bulletin (June 2015) Vol.34 No.2
of profi t. Moreover the valuable genetic material treatment with subcutaneous ear implant containing thus goes to slaughter in large numbers especially 6 mg of norgestomet (Intervet, Holland) with around the metropolises like Chennai, Mumbai and intramuscular injection of Synchromate B (Intervet, Calcutta. So there is an urgent need for improving Holland) containing 5 mg estradiol valerate +3 mg the reproductive effi ciency in Murrah buffaloes by norgestomet at the time of inserting the implant. using new reproductive techniques. The answer to On the 10th day the implants were removed and this problem is induction of oestrum in post-partum an injection of 500 IU PMSG (Folligon, Intervet anestrus buffaloes with synthetic progesterone. Pvt. LTD) was given. Estrus detection was carried Rao and Rao (1984) and Kathiresan et al. (1995) out by external observation and by twice daily have tried various hormonal treatment with rectal examination of genitalia. The intensity variable success. The present study was conducted of the induced estrus was assessed based on the on anestrus Murrah buffaloes to see the effi cacy of estrual signs as per Rao and Rao (1984) with slight norgestomet ear implant with PMSG in relation to modifi cation as weak, moderate and intense. The the induction of estrus and conception rate. onset and duration of estrum were recorded.
Breeding and pregnancy diagnosis MATERIALS AND METHODS The buffaloes were inseminated with frozen semen at 48 and 72 h after the removal Experimental animals of the implant. All the animals were kept under The post-partum anestrus Murrah buffaloes observation after insemination and those exhibiting attending the Peripheral Veterinary Hospital, next cyclical estrus were inseminated again at 12 h Madhavaram, Madras Veterinary College and the after the onset of the estrum. Pregnancy diagnosis buffaloes available at the Central Cattle Breeding was done by rectal examination at 45 days after the Farm, Alamadhi, Chennai were used for this study. last artifi cial insemination. The buffaloes were 4-7 years old, apparently healthy, parous and with the history of anestrum for about 3-7 months after calving. The animals RESULTS AND DISCUSSION were fed with adequate green fodder and required concentrates. The anestrus condition was confi rmed Estrus was induced in twenty post-partum by palpation of the ovaries per rectum twice at a 10- anestrus Murrah buffaloes by using the norgestomet day interval. The animals with small, smooth and ear implant system and PMSG. The onset duration inactive ovaries and not showing estrus activity and the intensity, of the induced estrum were were selected as anestrus. A total of 26 animals assessed. All the estrus induced buffaloes were were identifi ed for this study. Six anestrus Murrah inseminated twice at induced estrus and those buffaloes were maintained without treatment as exhibiting estrus again were also inseminated. control. Subsequently pregnancy was assessed at 45 days after the last artifi cial insemination. Induction and detection of estrum All the treated buffaloes (100%) exhibited The buffaloes were given a 10-day estrus signs like vulval edema, vaginal mucous
242 Buffalo Bulletin (June 2015) Vol.34 No.2
membrane congestion, mucus discharge, standing of Rao and Narayana (1983) and Kathiresan et al. to be mounted by herd mates etc. This was in (1995) in anestrus buffaloes. The mean duration accordance with the fi ndings of Nayak et al. (2009) of estrus was 20.40 h; it ranged between 18 and that 100% estrus was achieved in anestrus buffaloes 24 h (Table 1). This is comparable to the fi ndings with Crestar implant with PMSG combination in of Kathiresan et al. (1995) in anoestrus dry and true anestrus buffaloes. The intensity was classifi ed lactating subestrus buffaloes whereas they recorded as intense, moderate and weak oestrus as presented a longer duration of estrum in dry and lactating true in Table 1. Among the treated buffaloes 35%, 50% anestrus buffaloes treated with the SMB system and 15% buffaloes exhibited intense, moderate and PMSG. Kathiresan et al. (1995) have recorded and weak estrus. respectively. None of the animals 28.5, 43.0 and 28.5 and 16.71, 50 and 33.33 intense, in the control group exhibited signs of estrus. normal and weak estrum signs in dry and lactating Comparatively in treated buffaloes, moderate estrus group of true anestrus buffaloes, respectively. The was more prominent (50%) than intense and weak differences might have been due to breed variation estrus. This was contrary to Chede (1990) fi ndings, and differences in the progesterone level at the time who observed only 32.25% intermediate estrus by of giving the norgestomet implant and oestradiol implanting norgestomet for 9 days. level at the time of induced oestrus. The time interval between implant removal The conception rate in SMB induced post- and onset of estrus varied from 36-46 h with the mean partum Murrah buffaloesare shown in Table 2. The of 37.8 h. This was in accordance with the fi ndings conception rate after the fi rst service was 35% and
Table 1. Mean+SE of estrus behavioral pattern in induced estrus in Murrah buffaloes.
S. No. Parameter Mean + SE 1 Onset of estrus (h) 38.80+0.433 (36-46) 2 Duration of estrus (h) 20.40+0.675 (18-24) Intensity of estrus (No. of animals) a. Intense 7 (35%) 3 b. Moderate 10 (50%) c. Weak 3 (15%)
Table 2. Treatment response and conception rate.
S. No. Parameter No. of animals 1 No. of buffaloes treated 20 2 No. of buffaloes exhibited estroul signs and inseminated 20 3 No. of buffaloes pregnant after fi rst artifi cial insemination 7 (35%) 4 No. of buffaloes pregnant after second artifi cial insemination 4 (20%) 5 Overall conception rate 11 (55%)
243 Buffalo Bulletin (June 2015) Vol.34 No.2
that after the second service was 20%. The overall Buffalo Bull., 28(2): 51-54. conception rate was 55%. This was more or less Patel, D.M., N.P. Sarvaiya, A.V. Patel and A.P. in accordance with the conception rate of 57% in Parmar. 2003. Induction of estrus and post-partum anoestrus buffaloes by Rallthra et al. hormonal profi le in buffaloes treated with (1994). But these results were contrary to Patel et norgestomet ear implant. Indian J. Anim. al. (2003) who recorded a high (75%) conception Reprod., 24(1): 67-68. rate in buffaloes with norgestomet ear implant. Porwal, M.L., M.P. Singh and G.W. Kasandikar. 1981. Studies on reproductive performance of Murrah buffaloes. Indian Vet. J., 53: 435- CONCLUSION 437. Rallthra, S., K. Khar and K.P. Singh. 1994. Oestrus It is evident that the 6 mg norgestomet ear induction with synchronization in cows implant for the duration of 10 days and an injection and buffaloes with synthetic progesterones. of 3 mg of norgestomet and 5 mg of oestradiol at Indian. J. Anim. Sci., 64: 1060-1061. the time of implantation in combination with 500 Rao, A.V.N. and S. Narayana. 1983. Induction of IU PMSG at the time of removal successfully ovulatory estrus and fertility in non cycling induced estrum in post-partum anoestrus Murrah buffaloes with norgestomet during low buffaloes. Further the oestrus response was 100% breeding season. Theriogenology, 19: 305- and the treated buffaloes showed different signs of 309. estrum. The overall conception rate was 55% after Rao, A.R. and V.S. Rao. 1984. Improved conception two inseminations. rate in buffaloes after administration of receptal. Indian Vet. J., 61: 813. Tanwar, P.S., N.K. Rakha and J.B. Phogat. 2003. REFERENCES Challenges in buffalo infertility. Intas Polivet, 4(11): 121-127. Chede, S.A. 1990. Pattern of estrus, estrus behaviour and synchronization in buffaloes. Ph.D. Thesis, Punjabrao Krishi Vidyapeeth, Akola. Kathiresan, D., D. Ezhaikial Napolean, J. Antoine, L. Dawson and S.R. Pattabiraman. 1995. Infl uence of ovarian status and lactational stress on the effect of norgestomet, treatment of buffaloes. The Blue Cross Book, 4: 25- 27. Nayak, V., R.G. Agarwal, O.P. Srivastav and M.S. Thakur. 2009. Induction of oestrus in true anestrus buffaloes using Crestar implant alone and in combination with PMSG.
244 Original Article Buffalo Bulletin (June 2015) Vol.34 No.2
IN VITRO PRODUCTION OF BUFFALO EMBRYOS BY INJECTION OF IMMOBILIZED AND DEAD SPERMATOZOA
K. Tasripoo1, K. Srisakwattana1,*, W. Nualchuen1 and S. Sophon2
ABSTRACT those two injected group. In conclusion, this study showed that cleavage rate and later development A preliminary study was conducted of till hatched blastocyst after ICSI with live sperm in vitro production (IVP) of buffalo embryos by (immobilized) were not signifi cantly different from intracytoplasmic spermatozoa injection (ICSI) ICSI with dead sperm. with live (immobilized) sperm and spermatozoa killed (by freezing) and post-additional activation Keywords: injection, immobilized, dead of ICSI-embryos with 5 μM calcium ionophore spermatozoa, buffalo, ICSI (A23187 CaI) and 10 ug/ml cycloheximide (CHX),compared with in vitro fertilization (IVF) without oocyte post-activation. Sham injection is INTRODUCTION aspiration of ooplasm and pushing the ooplasm back into the oocyte. All the oocytes were co- Intracytoplasmic sperm injection (ICSI) cultured with epithelial oviductal cell. There were is a micro-fertilization technique, involving direct no signifi cant differences in cleavage, morula, injection of a single spermatozoon into an oocyte. blastocyst, or hatched blastocyst rate between ICSI bypasses natural selection with spermatozoa treatments with killed sperm and immobilized chosen for injection based on their motility and sperm (77.48%, 81.20%, 48.72%, 39.32% and morphology (Fatehi et al., 2006); zona penetration, 22.22%, respectively vs 81.94%, 83.90%, 55.08%, acrosome reaction and membrane fusion are 42.37% and 27.97%, respectively). In the IVF bypassed (Fujinami et al., 2004a; Ajduk et al., group, the morula, blastocyst and hatched blastocyst 2006). The procedure of intracytoplasmic sperm rates (46.38%, 31.88% and 13.04%, respectively) injection (ICSI) has expanded the possibilities of were not signifi cantly different in the two groups. assisted reproduction technology in animals and However, the cleavage and 8-cell stage rate humans (Galli et al., 2003). ICSI has also been (42.86% and 65.22%) were signifi cantly (p<0.05) demonstrated as a useful way of delivering foreign lower than those two groups. The sham injection DNA into the oocyte for genetic manipulation in plus chemical activation showed a similar cleavage, agriculture (Niemann and Kues, 2000; Houdebine, 8-cell stage, morula and blastocyst rate (84.67%, 2002; Moisyadi et al., 2009). ICSI requires far 74.14%, 50.86% and 31.90%, respectively) to fewer spermatozoa than conventional artifi cial
1Faculty of Veterinary Science, Chulalongkorn University, Henri Dunant Road, Phathumwan, Bangkok, Thailand, *E-mail: [email protected] 2Faculty of Veterinary Medicine, Mahanakorn University of Technology, Nong Chok, Bangkok, Thailand
245 Buffalo Bulletin (June 2015) Vol.34 No.2
insemination (AI) or in vitro fertilization (IVF), and ICSI is necessary to increase cleavage and further is potentially valuable in using very rare or valuable in vitro development as reported by Oikawa et semen samples effi ciently. The spermatozoa in al., 2005. Oocyte activation can be induced by a one straw, divided into hundreds, can be used to variety of physical and chemical agents, including inseminate a large number of oocytes (Horiuchi et an electrical pulse, ionomycin, calcium ionophore al., 2002). ICSI can also be useful in evaluation of A23187 (CaI), 6-dimethylaminopurine (DMAP), oocyte quality because it reduces variation due to cycloheximide (CHX), cytochalasin B (CB) and sperm penetration and allows potential fertilization ethanol (Tian et al., 2006; Oikawa et al., 2005). and embryo development of each one of the MII- Sperm preparation and post-ICSI oocyte activation oocytes injected (Jimenez-Macedo et al., 2007). affect fertilization and the development of ICSI- ICSI is an assisted reproductive technique derived embryos. In buffaloes, the treatment of (ART), allowing us to avoid the problem of poly- oocytes by electric stimulation with CHX and spermy (Garcia-Rosello et al., 2006). Mouse ICSI by ethanol with cycloheximide can activate has been used to overcome male infertility due to parthenogenesis without any deleterious effect on poor sperm motility and abnormal sperm structure. subsequent development of parthenotes (Atabay A direct comparison of ICSI and IVF shows that et al., 2006). Cycloheximide (CHX), a protein there are considerable savings in the number of synthesis inhibitor, has been reported to be oocytes and spermatozoa required to generate an effective in inducting activation when combined embryo (Szczygiel et al., 2002). More importantly, with calcium ionophore. The combined activation ICSI is especially valuable for strains where IVF treatment effectively drives oocytes to resume with fresh spermatozoa produces few or no embryos meiosis (Atabay et al., 2006; Mahmoud et al., (Szczygiel et al., 2002). The availability of ICSI to 2007). Tian et al., (2006) reported that species stabilize yields of in vitro production (IVP) embryos differences exist in oocyte activation after ICSI. was therefore examined using various kinds of At present, research into in vitro production employed frozen semen (Ushijima, 2005). (IVP) in buffalo is still not advanced and there are In cattle the fi rst live offspring were fewer studies compared with cattle. This may be obtained after injection of the dead spermatozoa because most advanced technologies originate frozen without cryoprotectants (Galli et al., 2003). in cattle farming countries and cattle farming Immobilization of a spermatozoon by tail-scoring is much more commercialized. Therefore, most before ICSI can improve formation of the male of the techniques for IVP of buffalo embryos pronucleus and using an appropriate chemical adopt procedures developed originally for cattle pretreatment of spermatozoa is necessary to (Nandi et al., 2002). It is interesting that it is so achieve a higher rate of male pronuclear formation diffi cult to produce a higher number of IVP. Our (Vanderzwalmen et al., 1996; Horiuchi and experience is consistent with several other studies Numabe ,1999; Wei and Fukui, 1999). Fertilization (e.g. Chuangsoongneon and Kamonpatana, 1991; of bovine oocytes by injection of immobilized, Nandi et al., 2002; Kamonpatana, 2003; Atabay et killed spermatozoa and production of normal al., 2004) that found that reproductive performance calves was fi rst reported by Goto et al., 1990. of buffalo is generally inferior to that of cattle. The additional oocyte activation treatment after Live offspring from a variety of species born by
246 Buffalo Bulletin (June 2015) Vol.34 No.2
ICSI techniques have been reported, including MATERIALS AND METHODS humans, and the technique is considered successful (Horiuchi and Numabe, 1999; Ajduk et al., 2006). All chemicals were purchased from Sigma- However, to date there has been no report of Aldrich (St. Louis, USA) unless otherwise stated. buffalo calves produced by ICSI technique. There is also another factor to be considered: a species- In vitro maturation of oocytes specifi c difference for improving the effi ciency Buffalo ovaries were obtained from an of reproductive biotechnologies in mammalian abattoir and transported to our laboratory in 0.9% species (Gasparrini et al., 2004). The evidence normal saline plus antibiotics at 25-30oC. Within reported by Drost (2007) is that fertility in water 3 h of collection, the oocytes were aspirated from buffalo (Bubalus bubalis) is considerably lower follicles with diameter of 3-6 mm through a 21- than in cattle (Bos taulus and Bos indicus) and that gauge needle attached to a 10 ml disposable syringe. poor breeding is attributed to late maturity, poor The oocytes were washed with TCM199 plus 10% estrus expression, prolonged calving intervals, and fetal bovine serum (FBS) (Gibco, Invitrogen). seasonal reproductive patterns (Nandi et al., 2002; A group of 10 cumulus-oocyte complexes with Gasparrini et al., 2004). We should therefore strive compact cumulus cells were placed in a drop (50 to improve the IVP of buffaloes as they remain μl) under mineral oil (Sigma) in a 60 mm X 15 friends to the farmers (Kamonpatana, 1980). mm plastic dish (Nunc) of the medium TCM199 ICSI represents an assisted reproductive supplemented with 10% buffalo follicular fl uid, technology for overcoming inherent reproductive 50 iu /ml human chorionic gonadotrophin (hCG) problems that limit the productivity of buffaloes, (chorulon © Intervet-International BV., Boxmeer, an important species and a fundamental livestock The Netherlands), 0.02 AU/ml follicle stimulating resource in terms of milk, meat, hide, horn and hormone (FSH) and 1 μg/ml estradiol-17ß(E2) draught power for millions of people, particularly and cultured for 19-20 h at 38.5oC in a humidifi ed small scale farmers and for many ecologically atmosphere of 5% CO2. The oocytes were freed disadvantaged agricultural systems (Cockrill, from the cumulus by treatment fi rst with 0.2% 1987; Gasparrini et al., 2004; Suresh et al., hyaluronidase in mPBS supplemented with 1 mg/ 2009). The information in regard to the ICSI with mL PVP (K-30) with gentle pipetting. The oocytes immobilized spermatozoa and dead spermatozoa were washed in TCM199 supplemented with 10% and the technique used for oocyte activation after FBS and 12.5 mM HEPES. The oocytes with ICSI in swamp buffalo (Bubalus bubalis) has been clearly visible fi rst polar body were selected and very limited. transferred into drops (50 μl each) of 12.5 mM Here, we report the in vitro production of HEPES in TCM199 (Gibco) with 10% FBS which buffalo embryos by ICSI with immobilized and had been previously placed under mineral oil. dead spermatozoa and post activation. Preparation of spermatozoa Straws (0.25 ml) of frozen spermatozoa from a buffalo of proven fertility were thawed in a water bath (37oC) for 30 seconds and the
247 Buffalo Bulletin (June 2015) Vol.34 No.2
contents of the straw placed at the bottom of a when the injection pipette, with the inner diameter 1.5 mL-micro-centrifuge tube containing 1 ml 8-10 μm, was introduced. The spermatozoon was of sperm Tyrode’s lactate medium (SPTL) and pushed forward until its head approached the tip incubated for 30 minutes to permit the sperm to of the injection pipette just before the tip of the “swim-up”. The sperm that had swum up into the pipette reached the zona pellucida. The injection top 0.8 ml was then collected and washed in 10 pipette was pushed through the zona pellucida and ml TALP plus 5 mM caffeine by centrifuging at subsequently through the oolema into the cytoplasm 500 g for 5 minutes. After centrifugation the pellet from the 3 o’clock direction (Chung et al., 2000; was resuspended with Tyrode’s albumin lactate Horiuchi et al., 2002). Subsequently, the aspirated pyruvate (TALP) medium and adjusted to the fi nal spermatozoon was expelled into the cytoplasm near concentration of spermatozoa which was 2x106 the holding pipette with a minimum volume of the motile spermatozoa. medium. The pipette was withdrawn gently. Sham injections were performed in a similar manner. Preparation of immobilized sperm Immediately after injection, the injected- One part (5 μl) of the sperm suspension was oocytes were washed four times in TCM199 mixed with three parts of 10% polyvinylpyrrolidone supplemented with 10% FBS and 12 mM HEPES (PVP, Goto et al., 1990) +TCM199+HEPES+ 10% and left at room temperature for 45 minutes in order FBS. A drop (10 μl) of this sperm suspension was to let the cytoplasm return to a normal condition. kept under mineral oil in a 60 mm x 15 mm plastic dish (Nunc). A motile spermatozoa was immobilized In vitro fertilization (IVF) by scoring the sperm tail with the tip of the injection A fi fty microlitre drop of 2x106 /mL of needle against the bottom of the dish immediately spermatozoa in Tyrode’s albumin lactate pyruvate before injection. The immobilized spermatozoa (TALP) medium supplemented with 5 mM was drawn, tail fi rst, into the injection pipette caffeine under mineral oil in 35 mm culture dish was prepared. The oocytes were then placed in Preparation of dead sperm those droplets (10 oocytes / drop) and incubated at o Some spermatozoa, diluted 1:3 with 38.5 C in a humidifi ed atmosphere of 5% CO2 for TCM199+HEPES+ 10% FBS, were killed by 6 h. After washing 4 X with SOFaa plus 1% FBS, freezing at -20oC for 1 h and thawing without any culturing was continued in SOFaa supplemented cryoprotectants at room temperature. A drop (10 μl) with 1% FBS for 2 d at 38.5oC in a humidifi ed of this sperm suspension was kept under mineral atmosphere of 5% CO2 and 5% O2. oil in a 60 mm x 5 mm plastic dish (Nunc). Killed spermatozoa were injected into oocytes within 1 h Activation of oocytes of the initial freeze-thawing. After washing, the sperm-injected oocytes were incubated in TCM199 supplemented with 10% Sperm injection into oocytes FBS +12 mM HEPES for 45 minutes (Goto 1993). An oocyte was positioned on the holding Then the oocytes were activated with 5 μM Calcium pipette (inner diameter 20-30 μm) in such a way that ionophore (A23187) in TCM199 supplemented the fi rst polar body was at the 12 o’clock position with 10% FBS and 12mM HEPES for 5 minutes
248 Buffalo Bulletin (June 2015) Vol.34 No.2
o at 38.5 C under 5% CO2 and followed by washing blastocyst from each treatment are presented as a 4 X in SOFaa supplemented with cycloheximide mean percentage. Comparisons between the groups (CHX) and 10% FBS. The sperm-injected oocytes of treatments were analyzed by Chi square tests. were also incubated in SOFaa supplemented with Signifi cant differences were defi ned as P<0.05. 10 μg/ml CHX and 10% FBS for 5 h. The oocytes were then washed in SOFaa supplemented with 1% FBS and placed in a drop (50 μl) of SOFaa RESULTS supplemented 1% FBS and incubated at 38.5oC under a humidifi ed atmosphere of 5% CO2 and 5% Table 1 summarizes the results when in vitro
O2 for 48 h. Sham injections were performed in the matured oocytes were injected with spermatozoa same manner as the sperm injected group. killed by freezing and thawing or immobilized by scoring the tails. The cleavage rate after ICSI with Collection and establishment of oviductal cell dead sperm (77.48%) was not signifi cantly different monolayer from that of live sperm (81.94%). Oviductal epithelial cells were separated The lowest cleavage rate was found in the from the buffalo oviducts at the slaughterhouse in IVF group (42.86%). The percentage of 8-cell stage animals that had newly formed corpus luteum on embryos after ICSI with dead sperm (81.20%) was their ovaries (Chuangsoongneon and Kamonpatana, not signifi cantly different from that with live sperm 1991; Kamonpatana, 1994). The epithelial cell (83.90%). The signifi cantly lowest (p<0.05) 8-cell monolayer was prepared 1-2 d in TCM199 stage was also found in the IVF group (65.22%). supplemented with 10% FBS. The round oviductal Furthermore, the percentage of morula after ICSI epithelial cells with cilia blowing were introduced with dead sperm (48.72%) and live sperm (55.08%) into a 50 μl drop of SOFaa supplemented with 5% was not signifi cantly different from that of IVF FBS and incubated at 38.5oC under a humidifi ed treatment (46.38%). The blastocyst yield among the atmosphere of 5% CO2 for 15 h before adding the following groups: dead sperm (39.32%), live sperm ICSI-embryo. (42.37%) and IVF (31.88%), was not signifi cantly different. The hatched blastocyst rates were also Embryo culture not different signifi cantly among the treatment The embryos at the 6-8 cell-stage from all with live sperm, dead sperm and IVF groups. The treatments were transferred to a co-culture with hatched blastocyst rates after ICSI with live and oviductal cells in the SOFaa supplemented with dead sperm and of IVF group were not signifi cantly 5% FBS and incubated at 38.5oC in a humidifi ed higher than sham injected plus chemical activation atmosphere of 5% CO2 for 4-5 d. The culture group. However, chromosomal analysis was not medium was changed every 2 days and embryo performed to demonstrate the paternal chromosome development was recorded. defi nitively. From our observation, no cleavage was Statistical Analysis obtained if there were no sperm-injection and The cleavage rates, the 8-cell stage without activation. The proportion of unfertilized embryos, morula, blastocyst rates and hatched and parthenogenesis blastocysts was not determined
249 Buffalo Bulletin (June 2015) Vol.34 No.2 in this study, due to the low number of oocytes DISCUSSION availabile in each batch. The sham injection plus activation also showed similar results of cleavage This study showed that buffalo oocytes till blastocyst rate to those of dead and live sperm matured in vitro can develop apparently normally treatment groups. There was no cleavage of the to the blastocyst stage after being fertilized by sham injected without activation group. injection of immotile and killed spermatozoa. This study showed that, without sperm To our knowledge, this may be the fi rst report of (sham-injection plus activation), the injected oocyte ICSI in buffalo for in vitro embryo production could develop further until reaching the same rate using immobilized and killed spermatozoa. Our of blastocyst stage as those of the IVF group but results showed signifi cantly similar embryonic resulted in a signifi cantly higher percentage of development between using immobilized sperm cleavage than those in the IVF group. and dead sperm for ICSI plus additional activation The highest parthenogenetic development with CaI and CHX. One possible explanation is was found from sham injection plus chemical that the plasma membrane integrity of dead sperm activation treatment, followed by chemical might not have been damaged by our frozen/thaw activation without injection; the least effi cient methodology, therefore it resulted in the same rate treatment was sham injection without activation. of embryonic development found using live sperm.
Table 1. The in vitro development of embryos after ICSI with dead and live spermatozoa.
No. of No. of No. of No. of No. of No. of hatched Group ICSI embryo 8-cell embryo morula blastocyst blastocyst oocyte cleaved (%) (%) (%) (%) (%) Sham injection 137 116 86 59 37 13 with activation ( 84.67 )a (74.14) ab (50.86) a (31.90) a (11.21) a 117 95 57 46 26 Dead 151 (77.48) a (81.20) a (48.72) a (39.32) a (22.22)b 118 99 65 50 33 Live 144 (81.94)ac (83.90)a (55.08)a (42.37)a (27.97)bc 6 9 45 32 22 9 IVF 161 (42.86)b (65.22)b (46.38)a (31.88)a (13.04)d Without 31 26 18 8 injection 54 - (57.41)b (83.87)a (58.06)a (25.81)a with activation Sham injection without 81 - - - - - activation
Values within columns with different letters differ signifi cantly (P<0.05).
250 Buffalo Bulletin (June 2015) Vol.34 No.2
The integrity of the plasma membrane of the sperm bypassed several barriers, because IVF is a natural cell is crucially important for sperm function. fertilization process. In IVF, activation is induced Only a sperm with an intact plasma membrane can by fertilizing spermatozoa which facilitates a more undergo a series of complex changes in the female normal physiological response in the oocyte than reproductive tract and can acquire the ability to when activation is induced by mechanical injection fertilize oocytes (Yanagimachi, 1994; Khalil et al., and chemical activation (Gomez et al., 1998). 2006). Our cleavage, morula and blastocyst yields were Our results clearly showed that the ICSI, higher than those reported by Liang et al. (2011), with dead and live sperm, showed a signifi cantly using Io+CHX for post-activation after ICSI with higher percentage of cleavage and 8-cell stage immobilized sperm. This may indicate that the embryos than those of IVF. This could be inconsistent results could have been attributable to explained partly by the fact that ICSI technique protocol differences. plus additional post- activation affects the capacity It has been reported that the bovine sperm of ICSI-embryos to develop. But in the IVF group, has a more stable nuclear packing than other only sperm activation was performed without species (Martin et al., 2007). It may be possible oocyte activation. Furthermore, the poorer early that buffaloes may differ from bovines because embryonic development following IVF may ICSI by immobilized bovine sperm resulted in a be attributable partly to poor quality of frozen/ higher percentage of embryonic development than thawed sperm, improper sperm capacitation and / that using killed sperm (Horiuchi et al., 2002). or fertilization, improper sperm concentration, and One possible explanation is that the buffalo sperm the condition of the culture media used for IVF nucleus is highly stable and/or no major damage (Mishra et al., 2008; Chankitisakul et al. (2012). resulted in further fertilization process occurred and Even though the IVF group gave a lower cleavage pronuclear formation. However, further research rate than those in dead and live groups, its further is needed to confi rm this. The ultra-structure of development capacity till blastocyst hatching were sperm after freezing (-20oC) is also an interesting not different signifi cantly from those two latter possibility for further investigation. groups. We inferred that activated cleaved ICSI Horiuchi and Numabe (1999) reported that embryos had similar competence to develop to the injection of a motile (live) bull spermatozoon the blastocyst stage compared with cleaved IVF into the ooplasm after immobilizing yields more embryos in our IVC system. The results of this cleaved embryos than the injection of a dead study show that spermatozoa play an important (killed by repeated freezing and thawing) bull role / participate in the activation of oocyte as in spermatozoon, without considering the addition IVF. Chung et al. (2000) reported that injected of oocyte activation. The cleavage and blastocyst spermatozoa contribute to the activation process. yields from immobilized spermatozoa (72% and Our study showed that ICSI with dead 28%, respectively) were signifi cantly higher than and live sperm gave a higher blastocyst rate than those from killed spermatozoa in bovine (28% and those by IVF, but the IVF may give more chance 3%) and activated the ICSI-embryo with ethanol of obtaining embryos of higher competence (Horiuchi et al., 1999). Abnormalities of the and viablility for transfer than by ICSI, which sperm centriole in dead bull spermatozoa killed
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by repeated freezing-thawing may be one of the embryos. Moreover, our results also do not agree reasons for the low rate of cleavage and the behavior with those reported by Fujinami et al. (2004a) that of sperm asters of dead spermatozoa after ICSI is bovine embryonic development by IVF resulted not clear (Horiuchi and Numabe, 1999. Our results in higher cleavage and blastocyst (72% and 24%, contradict results found in bovines by Fujinami respectively) than those by ICSI (immobilized et al. (2004b). They found that the cleavage rate sperm) (51% and 14%, respectively). and developmental rate to blastocyst after ICSI These results highlight the need to take into with live sperm (51% and 12%, respectively) were account species-specifi c differences for improving signifi cantly higher than that after ICSI with killed effi ciency of reproductive biotechnologies in sperm (12% and 0%), respectively. Their results mammalian species (Gasparrini et al., 2004). We indicated that killed sperm do not have suffi cient also agree with Tian et al. (2006), who reported in potential for degradation of maturation promoting porcines that various methods of oocyte activation factor (MPF) activity of oocytes following ICSI and and sperm preparation and post-ICSI oocyte therefore the developmental rate after ICSI with activation signifi cantly affected the developmental killed sperm was lower than ICSI with live sperm capacity of early embryos derived from IVM and Fujinami et al., 2004b. There is another report on ICSI. Furthermore, Tian et al. (2006) and Keefer et bovine that does not agree with our results. Horiuchi al. (1990) reported that species differences exist in et al. (2002) found that spermatozoa “killed” by the oocyte activation after ICSI and bovine oocytes repeated freezing-thawing were far inferior to need artifi cial activation (after ICSI) to improve “immobilized” spermatozoa in their ability to fertilization. Also, technical skill and equipment participate in embryo development. Their blastocyst play very important roles in the success of the rates from ICSI with live and killed spermatozoon ICSI procedure (Suttner et al., 2000). Another were 20.3% and 0.8%, respectively. Post-activation reason for different results may be because the was also important as already reported in bovine; fertilizing ability of each buffalo bull differs in its using ethanol resulted in higher rates of cleavage ability to bind and fertilize oocytes (Abdel Dayem, and blastocyst (74% and 29%, respectively) than 2009). Our results showed that ICSI plus the post without activation (33% and 14%, respectively) activation of ICSI-embryo is a more effi cient and (Horiuchi and Numabe, 1999). However, those effective technique as IVF without post-activation results indicated that individual protocols may for generating buffalo embryos from immobilized need to be established for fertilization by sperm spermatozoa and dead spermatozoa. For blastocyst injection in different species (Keefer et al., 1990). quality comparison among the ICSI-embryos and Our results showed that sham injection IVF-embryos, and the cell numbers need further plus chemical activation and chemical activation investigation. also gave the comparable results of blastocyst In the present study, we found that the rate with dead and live sperm injection and IVF spermatozoa “killed” by freezing-thawing were groups. Therefore, we inferred that activation similar to “immobilized” spermatozoa in their was one of the major factors for parthenogenesis. ability to participate in embryo development. Furthermore, our results showed that sham injection This means that the dead spermatozoa from without chemical activation resulted in no cleaved valuable buffalo still have a chance for fertilizing
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successfully by ICSI. But the usability of dead REFERENCES spermatozoa which are still capable of fertilizing successfully needs further research. Horiuchi Ajduk, A., Y. Yamauchi and M.A. Ward. 2006. et al. (2002) suggested that the immediate Sperm chromatin remodeling after injection of membrane-disrupted spermatozoa intracytoplasmic sperm injection differs is recommended. Although intact sperm plasma from that of in vitro fertilization. Biol. membrane is not essential for successful ICSI, its Reprod. 75: 442-451. presence is important for keeping the sperm nucleus Abdel Dayem, A.M.H., Gh.K. Mahmoud, M.F., physiologically intact (Szczygiel et al., 2002). Our Nawito, M.M. Ayoub and T.H. Scholkamy. results showed that buffalo oocytes can be activated 2009. Fertility evaluation in Egyptian by mechanical stimulus using an injection pipette buffalo bulls using zona pellucida binding and with additional activation treatment after and in vitro fertilization assays. Livestock ICSI. Therefore, ICSI with the appropriate oocyte Sci., 122: 193-198. activation offers an aid to increase in vitro embryo Atabay, E.C., F.P. Atabay, de Vera R.V, F.V. production. Mamuad and L.C. Cruz. 2006. Chemical In conclusion, the present study has and electrical activation of swamp buffalo demonstrated that ICSI with dead and live sperm (Bubalus bubalis) oocytes with or without plus post-activation showed similar cleavage rates cycloheximide treatments. Buffalo Journal, and later development till the blastocyst stage. 22(2): 121-130. Chankitisakul, V., T. Tharasanit, N. Phutikanit, K. Tasripoo, T. Nagai and M. Techakumphu. ACKNOWEDMENTS 2012. Lacking expression of paternally- expressed gene confi rms the failure of Funding was provided by the Thai syngamy after intracytoplasmic sperm government under the Scientifi c Thai –Netherlands injection in swamp buffalo (Bubalus Co-operative Project (Project Code TH/NL 02/51), bubalis). Theriogenology, 77: 1415-1424. Faculty of Veterinary Science, Chulalongkorn Chuangsoongneon, U. and M. Kamonpatana. 1991. University. The authors also thank Dr. Wyn Ellis Oocyte maturation, in vitro fertilization for editing previous drafts of this paper. and culture system for developing preimplantation swamp buffalo embryos using frozen thawed semen. Buffalo Journal, CONFLICTS OF INTEREST 2: 189-198. Chung, J.T., C.L. Keefer and B.R. Downey. 2000. The authors declare that they have no Activation of bovine oocytes following confl icts of interest. intracytoplasmic sperm injection (ICSI). Theriogenology, 53: 1273-1284. Cockrill, W.R. 1987. The buffalo: A world assessment. Buffalo Journal, 3(1): 1-20 Drost, M. 2007. Bubaline versus bovine
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