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 i 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, Ofi 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, Ofi ce of the University Library, Kasetsart University, Thailand Miss WanphenSuraporn SrijankulKongphol International Buffalo Information Center, Ofi 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 .rD hctepnuS nohpoS ytlucaF fo yranireteV ,enicideM nrokanahaM fo Technology, Thailand

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

Assistant Journal Manager Miss Kanchana Anuphan International Buffalo Information Center, Ofi ce of the University Library, Kasetsart University, Thailand

Miss Jirawadee Wiratto International Buffalo Information Center, Ofi 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 (March 2016) Vol.35 No.1

CONTENTS Page Case Report

Reconstruction of lacerated wound on upper lip in a graded Murrah heifer – A case report G. Kamalakar, R. Mahesh and V. Devi Prasad...... 1

Surgical technique for the management of obstructive urolithiasis in a buffalo calf: A case report Md. Moin Ansari...... 5

Ocular squamous cell carcinoma in a buffalo: A case report Deepak Kumar Tiwari, Sandeep Saharan, Satbir Sharma, R.N. Choudhary, Neelesh Sindhu, Vikas Jaglan and Sandeep Potaliya...... 11

Management of irregular sharp molars in a buffalo – A case report P. Ramesh, P. Ravi Kumar, M. Raghunath and P. Vidya Sagar...... 15

Imperforate hymen and subsequent secondary pyometra, pyocervix and pyovagina in Murrah buffalo heifer: A case report Brijesh Kumar, B. Krishnappa, G.K. Das, G. Chetan Sharma, Dushyant Yadav and M.H. Jan...... 19

Monocephalic thoracopagus tetrabrachius tetrapus monster in Murrah buffalo- A case report Vikas Sachan, Brijesh Kumar, Vipin Sonkar and Atul Saxena...... 23

Original Article

Clinico-biochemical studies of ketosis in buffalo (Bubalus bubalis) Gurpreet Bali, Kail Hussain, W.A.A. Razzaque, Utsav Sharma and S.A. Beigh1...... 27

Comparative studies of certain enzyme assay during various reproductive states in buffaloes R. Chaurasia, H.S. Kushwaha, D. Chaurasia, M.K. Gendley, Kiran Kumari, A.K. Santra and B. Shinha...... 33

Buffalo Bulletin (March 2016) Vol.35 No.1

CONTENTS Page Original Article

Cross-sectional survey of helminthiasis in buffaloes at tehsil jatoi and tehsil muzaffar garh, southern Punjab, Pakistan Muhammad Asif Raza, Muhammad Mazhar Ayaz, Muhammad Mudasser Nazir, Muhammad Saleem Akhtar, Mubashir Aziz, Saeed Murtaza and M. Ali Khosa...... 39

Histological study on stromal tissue in mammary gland at lactating, involution and pregnant stage in Murrah buffalo D. Chaurasia, R.S. Dalvi, S.B. Banubakode, N.C. Nandeshwar, R. Churchan, S.P. Ingole1 and B. Sinha1...... 49

Test-day genetic analysis of Murrah buffalo sires for milk production Vijay Kumar and A.K. Chakravarty...... 59

Evaluation of fresh semen quality and predicting the number of frozen semen doses in Jaffrabadi buffalo bull S.N. Ghodasara, P.U. Gajbhiye, A.R. Ahlawat and K.S. Murthy...... 65

Serum concentration of calcium, inorganic phosphorus and magnesium in cyclic, non cyclic and repeat breeder buffaloes Qaisar Shahzad, Muhammad Imran, Hamayun Khan, Armughan Ahmed Wadood, Moazam Ali Khan, Muhammad Binyameen and Amir Ahmad Niazi...... 73

Diagnosis of dermatophilus dermatitis among buffaloes in Kerala P.V. Tresamol, M.R. Saseendranath and K. Vinodkumar...... 77

Management of repeat breeding in buffaloes under ield conditions using hormonal and antibacterial therapies M.G. Butani1, A.J. Dhami, R.G. Shah, N.P. Sarvaiya and Ankita Killedar...... 83 Buffalo Bulletin (March 2016) Vol.35 No.1

CONTENTS Page Original Article

Effect of gonadotropin releasing hormone (GnRH) preparations on induction of estrus and fertility in buffaloes under ield conditions in Haryana Vikash, Meenakshi Virmani, R.K. Malik and Pardeep Sigh...... 93

Farmers’ knowledge on productive and reproductive performances of buffalo under smallholder farming system B.S. Meena, H.C. Verma and Amit Singh...... 101

Standardizing pregnancy rate of Indian Murrah buffaloes for higher milk yield V. Jamuna, A.K. Chakravarty, Vijay Kumar, M.A. Mir and Vikas Vohra...... 109

Sero-prevalence of brucellosis in buffaloes in Gujarat: An on-farm case study B.J. Trangadia and R.M. Patel...... 121

Effect of alfalfa fodder supplementation on enteric methane emission measured by sulfur hexaluoride technique in Murrah buffaloes P.K. Malik and K.K. Singhal...... 125

Histological exploration of graaian and atretic follicles of buffalo ovary: A seasonal study Jasvinder Singh Sasan, Varinder Uppal, Neelam Bansal and Anuradha...... 135 Case Report Buffalo Bulletin (March 2016) Vol.35 No.1

RECONSTRUCTION OF LACERATED WOUND ON UPPER LIP IN A GRADED MURRAH HEIFER – A CASE REPORT

G. Kamalakar*, R. Mahesh and V. Devi Prasad

ABSTRACT often subjected to open wounds on near facial region due to automobile accidents, barbed fencing A 4 year old Graded Murrah Buffalo heifer wires, snake bites, etc. Anterior facial region needs was presented with extensive lacerated wound on special attention in the surgical repair owing to the upper lip and nostril on right side that occurred presence of important structures like buccal nerves, due to an accident. It showed excessive salivation, dental pads, incisors, etc. restlessness, presence of blood clots. The animal was examined thoroughly and prepared for surgery. Reconstructive surgery of upper lip and cheek was CASE HISTORY AND CLINICAL carried out under mental nerve block and linear OBSERVATIONS block using Lignocaine hydrochloride. Good surgical technique and effective post operative A 4 year old Graded Murrah Buffalo heifer management made the case successful recovery. was presented to the clinic with a history of an extensive lacerated wound on face involving right Keywords: graded Murrah buffalo, lacerated side of upper lip and cheek due to an accident wound, lip, cheek, reconstructive surgery occurred yesterday. The animal showed excessive salivation and was unable to take feed and water properly due to the injury. On thorough examination INTRODUCTION the lacerated wound involved right side of lip, cheek and commissures tearing buccinator and A wound is deined as discontinuity levator naso labialis muscles. The injury was of or separation of skin, mucous membrane, or full thickness, with serrated margins and measured any tissue surface. Lacerated wounds occur in 14 cm. Clotted blood was observed on wound animals more commonly due to accidents, while margins with little contamination (F1). crossing fencing wire, injuries by sharp objects like glass pieces. A lacerated wound presents torn and irregular edges (Venugopalan, 2009). These TREATMENT AND DISCUSSION wounds may occur at different places of body like face, inguinal region, abdomen, thorax, legs, etc. The animal was casted in left lateral Because of the nosiposture ruminants are very recumbency and prepared for aseptic surgery. It

Department of Veterinary Surgery and Radiology, College of Veterinary Science, Proddatur, Y.S.R. Kadapa District, Andhra Pradesh, India, *E- mail: [email protected]

1 Buffalo Bulletin (March 2016) Vol.35 No.1

was given mental nerve block and linear iniltration The animal was kept on Intravenous alimentation with 2% Lignocaine hydrochloride along the wound for 3 days in order to restrict the movement of the margins on upper and lower sides. The injury was jaws which otherwise would have impaired wound irrigated with 1:1000 Potassium permanganate healing. The wound healing was observed to have lotion thoroughly to remove debris and clots. To been protracted which was thought to be because make suturing perfect the skin and sub cutis was of the involvement of micro organisms inhabitants separated by under trimming and controlling of the oral cavity. the bleeding points. First the muscle layers were Atropine sulphate was given sub apposed using chromic catgut in continuous manner cutaneously 0.04 mg/kg BW in order to create dry through the centre of the wound. Strepto penicillin mouth condition thus augmenting wound healing. powder was sprinkled on entire wound and skin The wound was found to heal by cicatrisation edges were sutured in horizontal mattress pattern and the wound contraction was minimal. The using 1/0 braided silk. Paste made of Zinc oxide animal was said to have normal prehension and and tincture benzoin was applied on suture line. masticatory habits indicating a complete and Injection Amoxicillin+ Cloxacillin 3 g, injection uneventful recovery. Ketoprofen 2 mg/kg BW, injection Tribivet was The horizontal movement of jaws administered intra muscularly. Owner was advised during mastication and rumination together with to give more of liquid diet. Wound dressing was the presence of normal ruminal fauna and lora done every day with paste of zinc oxide and interfere with normal wound healing. The ruminant tincture iodine. Sutures were removed on 10th post saliva is rich in bicarbonate content thereby further operative day (F2). delaying wound healing. Normally ruminants Lacerated wounds near nostrils are sink their rostral part of the face into the gutters common in working bullocks due to irritation by for drinking soiling the sutured part. Further if the bull nose rope and frequency of nose piercing animals were let outside they ind their way into injuries was 64% (Alam et al., 2010). Here the the pond for wallowing. These factors further pose injury caused by a wheel adjustment guide pointer a challenge before a surgeon for wound healing. of a car. Suturing in these cases was very dificult Hence, the intravenous alimentation used in this because, surgical procedure require mouth to case may be attributed for relatively quick healing. be opened till the suturing was complete as the Chromic catgut was used as it was readily thickness of the wound involved inner surface of available and cheaper. To prevent infection the upper lip and commissures. injection Amoxicillin and to alleviate pain injection A series of opposition sutures were placed Ketoprofen was administered. For better cutaneous using chromic catgut no. 0, joining buccinator, wound healing different authors used various levator naso labialis muscles including fascia agents like ibrin glue (Michel and Harmond, restoring the normal anatomical coniguration of the1990), honey (Bergman et al., 1983), sea buck anterior half of the rostral facial region. Then sub thorn ointment (Gupta, 2002) and obtained better cutaneous sutures were placed using no. 0 chromic granulation tissue formation, reepithelialisation catgut. Cutaneous wound was sutured using no. 1 and other favourable histo-pathological factors. black braided silk in horizontal mattress pattern. In this case paste made of Zinc oxide and tincture

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Figure 1. Photograph showing lacerated wound of lip and cheek. Observe blood clots and contamination.

Figure 2. photograph taken at the time of suture removal. Observe clear apposition.

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benzoin was used on irst day as an anti coagulant 325-333. and from next day onwards combination of zinc Venugopalan, A. 2009. Essentials of Veterinary oxide and tincture iodine was used because of their Surgery, 8th ed. Oxford & IBH Publishing better wound healing and antiseptic properties Co. Pvt. Ltd., New Delhi, India. respectively. Topical application of zinc oxide enhances reepithelialisation of partial thickness wounds in pigs. The cell division in wounds is connected with increased demand for zinc due to its function in enzymes required for cellular replication and zinc found to be slightly mitogenic to epithelial cells (Agren et al., 1991).

REFERENCES

Agren, M.S., M. Chvapil and L. Franzen. 1991. Enhancement of re-epithelialisation with topical zinc oxide in porcine partial- thickness wounds. J. Surg. Res., 50: 101- 105. Alam, M.R., N.G. Gregory, M.S. Uddin, M.A. Jabar, M.S. Choudhury and M.C. Debnath. 2010. Frequency of nose and tail injuries in cattle and water buffalo at livestock markets in Bangladesh. Anim. Welfare, 19: 295-300. Bergman, A., J. Yanai, J. Weiss, D. Bell and M.P. David. 1983. Acceleration of wound healing by topical application of honey. Am. J. Surg., 145(3): 374-376. Gupta, M. 2002. Studies on eficacy of Sea buck thorn (Hippophae rhamnoides) in the healing of infected cutaneous wounds in canines: Thesis submitted to CSKHPKV, Palampur, Haryana. Indian J. Vet. Surg., 23(2): 109. Michel, D. and M.F. Harmand. 1990. Fibrin seal in wound healing: Effect of thrombin and Ca2+ on human skin ibroblasts growth and collagen production. J. Dermatol. Sci., 1(5):

4 Case Report Buffalo Bulletin (March 2016) Vol.35 No.1

SURGICAL TECHNIQUE FOR THE MANAGEMENT OF OBSTRUCTIVE UROLITHIASIS IN A BUFFALO CALF: A CASE REPORT

Md. Moin Ansari

ABSTRACT result in increased concentration of less soluble crystalloids in the urine (Osborne and Kruger, 1984). In the present study a technique of tube A single urolith/calculus is usually responsible cystostomy using Foley’s catheter in terms of for obstruction in cattle, but sheep are normally tolerance by the animal and overall outcome of affected by multiple calculi blocking the urethra the patient suffering from obstructive urolithiasis for several centimeters leading to rupture of urethra is reported and discussed. Foley’s catheter was or urinary bladder. Fatality rate in urolithiasis due blocked permanently when free low of urine was to rupture of the urethra or urinary bladder is very observed through urethra. Post-operatively, the high and so is the economic impact of this disorder calf remained in good health. Foley’s catheter was (Gasthuys et al., 1993; Radostitis et al., 2000). removed by pulling after delating its balloon on Tube cystostomy is a less expensive procedure, 12 days after the free low of urine had established. which can be performed easily, require less time The wound healed uneventful and small opening and preserve breeding ability. However, problem left after removal of Foley’s catheter was dressed of ascending infection, recurrent obstruction and antiseptically until healing. Tube cystostomy and displacement of the tube by the animal may occur oral administration of tablets ammonium chloride (William and White, 1991). The utility of the tube along with cystone were resulted in speedy and cystostomy with medical dissolution of urethral/ uneventful recovery. cystic calculi has been reported (Singh, 2005; Ansari and Moulvi, 2009). The present paper Keywords: Foley’s catheter, tube cystostomy, describes obstructive urolithiasis and its surgical obstructive urolithiasis, buffalo management in a male buffalo calf and puts on record.

INTRODUCTION CASE HISTORY AND OBSERVATIONS Urolithiasis is deined as the formation of uroliths as a consequence of multiple congenital A male buffalo calf of three and half and/ or acquired pathophysiological process that months age presented with the complaint that there

Division of Surgery and Radiology, Faculty of Veterinary Sciences and Animal Husbandry, Sher-e- Kashmir University of Agricultural Sciences and Technology of Kashmir, Shuhama, Srinagar, India, E-mail: [email protected]

5 Buffalo Bulletin (March 2016) Vol.35 No.1

was only few drops of urine passed or none at all parallel to the penis up to the level of preputial for the last 72 h despite making painful attempts oriice and brought up to the site of the incision. A and showing signs of uneasiness. The calf was in K-wire was anchored in the eye of Foley’s catheter poor condition and off-feed. Physical examination and it was inserted into the bladder through its revealed there was marked pitting edema on ventral ventral aspect with a sudden thrust without incising abdominal area and penis could not be extruded the urinary bladder (Figure 3). After insertion of the due to marked subcutaneous edema (Figure 1). catheter its balloon was inlated by infusing 10 ml Percutaneous massage of penis did not provide any of sterile physiological normal saline to prevent it relief except the removal of some minute particle from dislodgement from the bladder and the K-wire through external oriice. Examination of the was pulled out slowly. The laparotomy incision was preputial oriice has disclosed chalky white lakes sutured in standard procedure and the catheter was precipitated of the preputial hairs and resemble ixed to the abdominal wall with simple interrupted calculi in shape. On the basis of clinic-physical sutures along the length of the tube (Figure 4). examinations the animal was diagnosed as suffering The post-operative care included daily dressing from obstructive urolithiasis. Keeping in view the of the skin wound with 0.5% povidone iodine fact of the complete blockage of the urinal passage solution till healing. Antibiotic cover with injection may result in rupture of urethra or the bladder, it Ampicillin plus Cloxacillin at the dose rate of was decided to performed tube cystostomy using 5 mg/kg (AC Vet, Intas Pharma, Ahmedabad) Foley’s catheter to correct the disorder. used twice in a day intramuscularly for 7 days, injection Meloxicam at the dose rate of 0.2 mg/kg (Melonex, Intas Pharma, Ahmedabad) body weight SURGICAL TECHNIQUE intramuscularly once daily for 5 days, injection vitamin A (Intavita, Intas Pharma, Ahmedabad) Paramedian anterior to the brim of the 2 ml intramuscularly on six day interval for three pubis was shaved and prepared for aseptic surgery. time. Tablet ammonium chloride 500 mg/kg body The animal was restrained in right dorsolateral weight and tablet cystone 3 tablets in thrice in a day recumbency. The animal was operated under orally for 10 days were advocated. Foley’s catheter lumbosacral epidural analgesia, induced with 3.5 was blocked permanently when free low of urine ml of 2% lignocaine hydrochloride (Xylocaine, was observed through urethra. Post-operatively, Astra-IDL, Bangluru). This is usually accomplished the calf remained in good health. Foley’s catheter using the “hanging drop” or the “lack of resistance” was removed by pulling after delating its balloon technique during injection (Figure 2). Additional 12 days after the free low of urine had established. local iniltration analgesia at the site operation was The wound healed uneventful and small opening done as and when required. An incision was made left after removal of Foley’s catheter was dressed at the caudal-ventral abdomen, lateral and parallel antiseptically until healing. to the penis. Abdominal muscles were separated by dissection. A Foley’s catheter (no.14) (Uro-cath, Romsons Medicons, Agra) was passed through a subcutaneous tunnel of about 8 inch in length

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Figure 1. A buffalo calf suffering from urolithiasis.

Figure 2. Hanging drop technique during lumbosacral epidural analgesia.

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Figure 3. Placement of Foley’s catheter into the bladder With the help of K-wire.

Figure 4. Fixing of the external part of the Catheter with abdominal wall after completion of tube cystostomy.

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RESULTS AND DISCUSSION REFERENCES

The case was attended instantly without Ansari, M.M. 2005. Surgical management of delay as the complete blockage of urinary passage obstructive urolithiasis in a calf. Vet. World, may result in rupture of urethra or the bladder. 4: 73-75. The Foley’s tube cystostomy was performed in Ansari, M.M. and B.A. Moulvi. 2009. Surgical this as advocated by William and White (1991) in technique and management of obstructive dog and cat. Foley’s catheter was much lexible urolithiasis in a lamb. Intas Polivet, 10: and its inlated balloon or cuff covered the entire 307-309. circumference of the catheter, which prevented Gasthuys, F., M. Steenhaut, A. Dc Moor and leakage of urine. This is in agreement with the Sercuk. 1993. Surgical treatment of urethral indings of Singh (2005). Foley’s catheter was obstruction due to urolithiasis in male well tolerated by the buffalo calf. The surgical cattle: A review of 85 cases. Vet. Rec., 133: maneuvering of the bladder through para-median 522-526. was found easier when the calf was kept in Jones, M.L., R.N. Streeter and C.L. Goad. 2009. dorsolateral recumbency as reported by Ansari Use of dietary cation anion difference for (2005). Dificulty in placing the catheter and control of urolithiasis risk factors in goats. suturing of bladder as observed in other approaches Am. J. Vet. Res., 70: 149-155. (Prasad et al., 1978) could be overcome since Osborne, C.A. and J.M. Kruger. 1984. Initiation the bladder lies very supericially and can be and growth of uroliths. Vet. Clin. N. Am., approached conveniently. The abdominal wound 14: 1400-1404. healed without any complication in 12 days and Prasad, B., S.N. Sharma, J. Singh and R.N. Kohli. calf urinated through the urethra without any 1978. Surgical repair and management of dificulty. Additional combination therapy can be bladder rupture in bullocks. Indian Vet. J., considered more fruitful in combating uraemic 55: 905. toxaemia primarily due to bladder rupture. Radostitis, O.M., D.C. Blood, C.C. Gay and K.W. Ammonium chloride was used for acidiication of Hinchcliff. 2000. Veterinary Medicine: a the urine to induce dissolution of the calculi. The text book of disease of cattle, sheep, pig, eficacy of ammonium chloride in the management goat and horses. Bayllieri Tindall, London. of urolithiasis has been described by others also 1877p. Jones et al. (2009). Cystone tablet by virtue of its Singh, T. 2005. Studies on aetiopathogenesis and marked diuretic action would have contributed surgical management of urolithiasis in to the diuresis. No occurrence of urolithiasis or goats. Ph.D. Thesis, IVRI, Izatnagar, India. other condition leading to retention of the urine William, J.M. and R.A.S. White. 1991. Tube was observed. Author is of the opinion that post- cystostomy in dog and cat. J. Small Anim. operative oral administration of ammonium Pract., 32: 598-602. chloride and cystone tablets might have helped to avoid recurrence. Similar observation has earlier been reported by Ansari (2005) in a cow calf.

9 Buffalo Bulletin (March 2016) Vol.35 No.1 Case Report Buffalo Bulletin (March 2016) Vol.35 No.1

OCULAR SQUAMOUS CELL CARCINOMA IN A BUFFALO: A CASE REPORT

Deepak Kumar Tiwari1, Sandeep Saharan1, Satbir Sharma2, R.N. Choudhary2, Neelesh Sindhu2, Vikas Jaglan1 and Sandeep Potaliya1

ABSTRACT at muco-cutaneous junctions (Goldschmidt and Hendrick, 2002). The malignant tendency of this A four and half year old female murrah disease makes early recognition critical. The buffalo was referred to the Teaching Veterinary etiology of the disease is multifactorial. However, Clinical Complex (TVCC) with the history of prolonged exposure to sunlight (ultraviolet light) growth on the nictitating membrane of the left eye also seems to be a driving force for the disease near the medial canthus since one month. Animal (Anderson and Badzioch, 1991). showed little discomfort due to the growth but have This report communicates a case of ocular normal vision. Neoplastic outgrowth was excised squamous cell carcinoma in a female buffalo, which after ligating its base with 3-0 chromic catgut and was successfully treated by surgical intervention. lavaged with normal saline solution. The animal was recovered uneventfully within 15 days. CASE HISTORY AND CLINICAL Keywords: buffalo, eye, squamous cell carcinoma EXAMINATION

A four and half year old female Murrah INTRODUCTION buffalo was referred to the Teaching Veterinary Clinical Complex, LUVAS, Hisar with the history Squamous cell carcinoma is a tumour of growth on the left eye near the medial canthus of epidermal cells in which the cells show (Figure 1). There was watery discharge from the differentiation to keratinocytes. Squamous cell affected eye since one month. The animal was carcinoma is the most commonly occurring treated with parental administration of antiobiotic neoplasm aflicting the bovine eye (Faziliet al., and topical eye drop since last 15 days but no 2001; Kohlirn and Mashadi, 2008; Sivaseelan et al., signiicant improvement was noticed. 2008). The most common areas affected are limbus Clinical examination revealed a hard (junction of the cornea and the sclera), third eyelid, growth on nictitating membrane near the medial and on the upper and lower eyelid margins primarily canthus of the left eye. The animal had normal vision with mild opacity of cornea. The rectal

1Department of Veterinary Surgery and Radiology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana, India 2Teaching Veterinary Clinical Complex, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana, India

11 Buffalo Bulletin (March 2016) Vol.35 No.1

temperature, heart rate, pulse rate and respiratory 3-0 chromic catgut (Figure 2). The eye was lavaged rate were within the normal physiological limits. with normal saline solution. Blood and serum biochemical values were also Post operatively parental antibiotics within the normal reference range. The surgical Enroloxacin at the rate of 5 mg/kg body weight excision was decided and the site was prepared for and analgesic Meloxicam at the rate of 0.5 mg/kg asepsis. body weight were administered intra muscularly, daily for ive consecutive days. Eye ointment was applied topically in the affected eye (thrice RESULTS AND DISCUSSION per day) for 10 days. The animal was recovered uneventfully within 15 days and no complication Animal was sedated with Injection has been reported since one month after operation. Xylazine at the rate of 0.05 mg/kg body weight On histopathological examination, intravenously and regional anaesthesia was proliferating epithelial cells with concentrating achieved by performing Peterson nerve block using layer of keratin forming cell nest was found. The 10 ml 2% lignocaine hydrochloride. Neoplastic tumourous growth was diagnosed as a squamous outgrowth was excised after ligating its base with cell carcinoma as similar inding reported by Patel

Figure 1. Growth near the medial canthus of the left Figure 2. Excision of neoplastic growth. eye.

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et al. (2009) in buffalo, Al-Asadi (2012) in Iraqi Squamous cell carcinoma of eye in a she dairy cows and Fazili et al. (2001) in jersey cow. buffalo. Tamilnadu Journal of Veterinary and Animal Sciences, 3: 117-118.

CONCLUSION

Early recognition and evaluation of squamous cell carcinoma is necessary and easily be removed successfully without much complication.

REFERENCES

Al-Asadi, R.N. 2012. A survey and treatment of ocular carcinomas in Iraqi dairy Cows from 1987-2012. Kufa J. Vet. Med. Sci., 3(2): 66- 77. Anderson, D.E. and M.S. Badzioch. 1991. Association between solar radiation and ocular squamous cell carcinoma in cattle. Am. J. Vet. Res., 52: 784-788. Fazili, M.R., B.A. Buchoo, M.M. Darzi and S.S. Hussain. 2001. Ocular squamous cell carcinoma in a cow. Indian J. Vet. Surg., 22: 132. Goldschmidt, M.H. and M.J. Hendrick. 2002. Tumors of skin and soft tissues. Meuten, D.J. (ed.) Tumors of Domestic Animals, 4th ed. Iowa State Press. Kohlirn, E.W. and A.G. Mashadi. 2008. Squamous cell carcinoma in an Iranian buffalo. Indian Vet. J., 85: 555-556. Patel, P.B., J.N. Mistry, D.N. Suthar and J.B. Patel. 2009. Surgical Management of ocular squamous cell carcinoma in buffalo calf. Intas Polivet, 10(2): 293-294. Sivaseelan, S., G.A. Balasubranmaniam, P. Srinivasan, P. Balachandran, R. Thangathurai and S. Dharmaceelan. 2008.

13 Buffalo Bulletin (March 2016) Vol.35 No.1 Case Report Buffalo Bulletin (March 2016) Vol.35 No.1

MANAGEMENT OF IRREGULAR SHARP MOLARS IN A BUFFALO – A CASE REPORT

P. Ramesh1,*, P. Ravi Kumar1, M. Raghunath2 and P. Vidya Sagar1

ABSTRACT teeth occurs as quidding, bulging of the cheeks due to impaction of the food material and salivation. A four year old Graded Murrah Buffalo Present report puts on record a case of overgrown was presented with a history of bilateral distension sharp molar teeth and its management in a graded of cheeks, persistent salivation and occasional Murrah buffalo. quidding since one year. Physical examination revealed asymmetrical hard bulge of the both cheeks which was pitting on pressure. Upon oral CASE HISTORY AND CLINICAL examination noticed abnormal accumulation of OBSERVATIONS feed material on either side of the cheeks, ulcerative lesions in oral mucosa and irregular sharp molars. A four year old, graded Murrah buffalo The impacted material from both the sides of buccal was presented to the Teaching Veterinary Clinical cavity was removed and irrigated with potassium Complex with a history of bilateral distension of permanganate solution. The irregular sharp edges cheeks since one year with persistent salivation and of the teeth were rasped by tooth rasp manually to occasional quidding. The animal was anorectic with level the teeth, resulting in complete recovery. decreased water intake since a week. All the vital parameters were within normal range with mild Keywords: Murrah buffalo, sharp molars, tooth dehydration. Physical examination revealed hard rasp, teeth bulge of the both cheeks, which was asymmetrical and pitting on pressure. Needle aspiration revealed presence of INTRODUCTION greenish colour luid resembling rumen luid of neutral pH. Examination of oral cavity revealed Affections of irregular and sharp molar abnormal accumulation of feed material on either teeth is the most common clinical condition in sides of the cheeks (Figure 1), with ulcerative cattle and buffaloes (Tyagi and Singh, 2013). lesions in the oral mucosa and irregular sharp Usually, clinical manifestation of overgrown molar molars. All laboratory indings were within

1Teaching Veterinary Clinical Complex, NTR College of Veterinary Science, Gannavaram, Andhra Pradesh, India, *E-mail: [email protected] 2Department of Veterinary Surgery and Radiology, NTR College of Veterinary Science, Gannavaram, Andhra Pradesh, India

15 Buffalo Bulletin (March 2016) Vol.35 No.1

Figure 1. Photograph showing abnormal Figure 2. Photograph showing tooth rasping of accumulation of feed material in cheeks. overgrown molars.

Figure 3. Photograph showing recovery after treatment.

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normal range. Based on the indings, the case was REFERENCES diagnosed as feed accumulation in the cheeks due to irregular sharp molars. Dollar, John A.W. and J.J. O’Connor. 1958. Dollor’s Vet Surgery: General, Operative and Regional. Bailliere, Tindall and Cox. TREATMENT 1036p. Tyagi, R.P.S. and J. Singh. 2013. Ruminant Surgery. By physical restraint, the mouth was opened CBS Publisher and Distributors Pvt. Ltd. and the impacted food material was removed with a long forceps from both the sides of buccal cavity and irrigation of oral cavity was done with potassium permanganate solution. The irregular sharp edges of the teeth were rasped by irm and controlled strokes of the tooth rasp until a uniform level of the molar teeth was obtained (Figure 2). Dehydration was corrected with injection Dextrose Normal Saline 25 ml/kg body weight intravenously. The animal was supplemented with rumenotorics to improve the feed intake and was advised semi solid diet like gruel and semi cooked cereals until complete recovery. The oral mucosa was painted with boroglycerine paste.

RESULT AND DISCUSSION

After 10 days of the treatment, the animal started taking feed normally and water and made an uneventful recovery (Figure 3). Irregular and sharp molars are the common condition which hampers the mastication and cause simple indigestion in animals. Usually last molars are more predisposed, outer edges of upper molars and inner edges of lower molars become sharp, such animals usually throw feed material and salivation as observed in the present case. (Dollar, 1958). The condition can be diagnosed by detailed oral examination and tooth rasping can effectively reduce the sharp edges.

17 Buffalo Bulletin (March 2016) Vol.35 No.1 Case Report Buffalo Bulletin (March 2016) Vol.35 No.1

IMPERFORATE HYMEN AND SUBSEQUENT SECONDARY PYOMETRA, PYOCERVIX AND PYOVAGINA IN MURRAH BUFFALO HEIFER: A CASE REPORT

Brijesh Kumar*, B. Krishnappa, G.K. Das, G. Chetan Sharma, Dushyant Yadav and M.H. Jan

ABSTRACT secondary pyometra, pyocervix and pyovagina in a buffalo heifer. A rare case of complete persistence of imperforate hymen in murrah buffalo was diagnosed and treated successfully. Animal got permanent CASE HISTORY AND CLINICAL relieve from unwanted symptoms like continuous OBSERVATIONS straining during urination and defecation. Though the animal cycled regularly but failed to conceive A ive year old Murrah buffalo heifer was with follow-up for one year. attended by local practitioner with complaint of intense straining during urination and defecation. Keywords: imperforate hymen, Murrah buffalo, The case was diagnosed as a urinary obstruction and pyometra, pyocervix, pyovagina around three litters non-smelling mucous like luid was evacuated by inserting a medium size canula connected to a rubber tube into buldge luctuating INTRODUCTION portion through rectum. However, animal expressed temporary relief only for few days. After The hymen is formed from the epithelial 48 days, animal developed same symptoms in an lining of the paramesonephric ducts and intense form (Figure 1) and the animal was referred the urogenital sinus at the vestibulovaginal to Referral Veterinary Polyclinic, IVRI, Izatnagar. junction (Roberts, 1971). Canalization of the Detailed anamnesis revealed that the buffalo was hymen is usually complete at birth and leads to bred two times naturally with last mating three communication between the lumen of the caudal months back, during which the male had apparent vagina and vestibule (Roberts, 1971). Congenital dificulty in positioning himself. Further, each imperforate hymen is rare in cattle and similar mating was followed by intense form of straining condition in a buffalo heifer reported by Gupta and lasting few days. Since last mating, female Sharma (1973). The complete blockage results in developed permanent sign of prolong straining accumulation of uterine and cervical secretions during and after defecation. Further, animal had and formation of mucometra, mucocervix and an abnormal low of urine and was also associated mucovagina (Parkinson, 2001).The present report with straining. puts on a record a case of imperforate hymen with Per rectal examination revealed a

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

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Figure 1. Tenesmus during defecation. Figure 2. Visualisation of hymenal membrane cranial to urethral oriice.

Figure 3. Intense low of from genitalia. Figure 4. Apparent decompression of abdomen after evacuation of pus.

voluminous luid illed luctuating mass descending then restrained in lateral recumbency. The hymen into abdomen and it was astonishing to ind a tough membrane was punctured with a trocar guarded membrane obstructing hand to palpate cervix by inger and the hole was dilated to its maximum per vaginally (Figure 2). These indings lead toby digital pressure. Around 4 litres of intense foul diagnose the case as imperforate hymen. smelling pus like luid gush through vagina (Figure 3) and an apparent decompression of abdomen was noticed (Figure 4). Immediate per rectal TREATMENT AND DISCUSSION examination failed to locate cervix and uterine horn. However, per vaginal examination reveals The animal was subjected to epidural tough corrugation all over the wall of uterine horn. anaesthesia with 2% lignocaine hydrochloride and Uterus was lushed with normal saline mixed with

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potassium permagnate (1:1000) and lignocain jelly permanent blocked might have occurred that (Lignocain Hydrochloride 4%) along with pulv. preventing fertilizations. And this may be the Antibiotic 10 gm containing Neosporin Polymyxin reason for the reproductive failure in the present b sulfate, Bacitracin zinc and Neomycin sulphate case. Scanning through literatures revealed no applied locally. Animal was discharged with suficient information to suggest that hymen the prescription of parentrel anti-inlammatory persistence is hereditary. Thus, opens the window Meloxicam 0.05 gm, antibiotic 2.50 gm containing for systematic research on prognostic reproductive Streptomycin Sulphate, Penicillin G Sodium, life of an affected animal and its heritability which Procain penicillin G and intrauterine medication would help the clinicians as well as owners to take with 45 ml 5% Povodone Iodine for ive days. appropriate decision in time. After 45 days, follow-up was made to examine the animal. Cervix and both uterine horn were palpated per-rectally and no reunion of membrane REFERENCES were detected on per-vaginal examination. Animal cycled regularly but could not able to conceive Gupta, S.K. and R.D. Sharma. 1973. Imperforate with follow-up for one year. The animal was culled hymen and persistent hymen in Murrah and failed to follow further. buffalo heifers - a record of three cases. The case, imperforate hymen with Indian Vet. J., 50: 940-943. secondary pyometra, pyocervix and pyovagina Parkinson, T.J. 2001. Infertility in the cow: was diagnosed on the basis of history clinical Structural and functional abnormalities, observation and per-vaginal palpation. The case management and non-speciic infection, p. was earlier mis-diagnosed as urinary obstruction 383-472. In Noakes, D.E., T.J. Parkinson, and insertion of canula into buldge portion of G.C.W. England. (eds.) Arthur’s Veterinary vagina leading to breach in aseptic environment Reproduction and Obstetrics, 8th ed. that might lead to subsequent infection with pus Philadelphia: Saunders, USA. farming bacteria leading to accumulation of pus. Roberts, S.J. 1971. Veterinary Obstetrics and In present case the wall of vagina and uterus Genital Diseases, 2nd ed. CBS Publishers become very thin and dificult to locate the horns and Distributors, Delhi. but per-vaginal examination revealed that the Troiano, R.N. and S.M. McCarthy. 2004. Mullerian inner surface of vagina and uterus was very rough duct anomalies: imaging and clinical issues. and corrugated. This occurs because the normal Radiolog., 233: 19-34. outlow of the uterine secretions is prevented by complete persistency of imperforate hymen leading to accumulation of luid that increases with the age and the cyclic ovarian activity of the female (Troiano and McCarthy, 2004). The duration and volume of luid accumulation could have affected the endometrium via pressure atrophy leading to embryonic loss if fertilization occurred or some

21 Buffalo Bulletin (March 2016) Vol.35 No.1 Case Report Buffalo Bulletin (March 2016) Vol.35 No.1

MONOCEPHALIC THORACOPAGUS TETRABRACHIUS TETRAPUS MONSTER IN MURRAH BUFFALO- A CASE REPORT

Vikas Sachan1, Brijesh Kumar2,*, Vipin Sonkar3 and Atul Saxena1

ABSTRACT in nature (Arthur, 1956) and are the frequent cause of dystocia in cattle and buffalo. Conjoined twins A conjoint monster was delivered by are also known as diplopagus monsters or Siamese caesarean section in a pluriperous murrah buffalo. twins. Structural or numerical duplication during Partially duplicated, two female fetuses joined at the embryonic stage give rise to fetuses whose the thoracic region (Thoracopagus) and having body structures are partially but not completely well developed eight limbs, i.e. four forelimbs duplicated (Roberts, 1971). They are the result (Tetrabrachius) and four hind limbs (Tetrapus) of incomplete division of a fertilized ovum and and both pelvis are separate (Dicaudatus). There show great variation from partial duplication to was clear four nostrils (Tetrarhino) and the post- almost complete separation of two individuals, mortem examination revealed that internal organs joined in just a few places. Dystocia is a common were paired. sequel of monstrosity and most of the cases resolved by caesarean section. In the present study Keywords: conjoined twin, monster, Thoracopagus, a case of monocephalic thoracopagus tetrarhino Tetrabrachius, Tetrapus, Dicaudatus, Tetrarhino tetrabrachius tetrapus dicaudatus monster was relieved by caesarean section.

INTRODUCTION CASE HISTORY AND CLINICAL Monstrosity is a disturbance of the OBSERVATIONS development that involves various organs and systems which can cause great distortion of the A seven year old murrah buffalo presented individual (Vegad, 2007). The incidence of fetal to Teaching Veterinary Clinical complex, Veterinary monsters, though rare, was reported by Khasatiya University (DUVASU) Mathura in recumbent et al., 2009; Jerome et al., 2010; Ravikumar et al., condition with history of full term gestation and 2012 in cows, Dhami et al., 2000; Prasad et al., straining since last two days, water bag ruptured 2006; Sharma et al., 2010 in buffaloes. Conjoined 12 h before and also case was handled by local twins arise from a single ovum and are monozygotic practitioner to relieve the dystocia but failed. The

1Department of Veterinary Obstetrics and Gynaecology, Deen Dayal Upadhyay Veterinary and Animal Sciences University (DUVASU), Mathura, Uttar Pradesh, India 2ICAR RC for NEH Region Sikkim Centre Gangtok, Sikkim, India, *E-mail: [email protected] 3Department of Animal Husbandry, Government of Uttar Pradesh, Uttar Pradesh, India

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clinical parameter such as heart rate 48/minute, TREATMENT AND DISCUSSION respiration rate 30/minute, rectal temperature 101.5oF and animal was lethargy, dull, depressed Attempts were made to relieve dystocia and straining sign was completely ceased. Detail through obstetrical maneuver but futile then it Gynaeco-clinical examination revealed that birth was decided to go for cesarean section to relieve canal was completely impacted with fetal head and the dystocia. The buffalo was stabilize with luid legs and two amputated legs were also palpated. therapy comprises of inj. Dextrose Normal Saline Further detail examination revealed that head and Normal Saline 4 litter each, Ca-borogluconate relatively big and no clear demarcation of thorax 450 ml and antibiotic, antihistaminic and anti- (Figure 1) and palpation of many legs at untoward inlammatory were administered. Lower left lank places conirmed the fetal monstrosity and might laprohystrectomy was made and a full term dead be prime cause for dystocia. female monster was extracted out. The animal

Figure 1. Conjoint Monster with separate pelvis. Figure 2. Four nostrils depression and complete absence of epithelium on pole.

Heart Liver Kidney

Figure 3. A pair of hearts, fused liver and two pairs of kidneys.

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was stood after 6 hrs of cesarean section and took layer arising from single ovum (Kumar and Reddy, the water and walks little and after 24 h animal 2008) that gives rise to monozygotic foetus with showing sign of clear improvement and returning partial duplication of body structures. Simon et to normal and discharge with written prescription al., (2009) stated that conjoined twins were always advising the owner to continue the same treatment genetically identical and shared the same sex. ive more day along with local dressing of surgical Dystocia due to conjoined twin monsters, though site and intrauterine medication. After 12th day of uncommon, have been reported earlier in buffalo operation suture was removed and animal looks (Urankar et al., 1994; Dhami et al., 2000) and in totally recovered. cow (Honnappagol et al., 2005). The present case The monster was a conjoined female twin seemed to be a non-inherited teratogenic defect of with fusion at the thoracic region containing two development as there was no history of monstrosity pairs of fore limbs and posterior regions of both in previous calving. twins were well developed and having separate pelvis with external genitalia and rectum and pair of hind limbs in each but in one pelvis both REFERENCES limbs was in broken condition probably because of previous handling (Figure 1). The heads were Arthur, G.H. 1956. Conjoined and identical twins. fused, lacked distinct eyes, having two ears, four Vet. Rec., 68: 389. clear nostrils (tetrarhino) with two complete jaws. Dhami, A.J., M.T. Panchal and F.S. Kavani. 2000. There was depression and complete absence of Dystocia due to holo acardius acephalic epithelium on pole region (Figure 2).On post- (Asymmetrical conjoined twin) monster in mortem examination, the conjoined twin monster a buffalo. Indian J. Anim. Reprod., 21(2): was found to be attached to the thoracic region and 162-164. encloses a pair of hearts. Other visceral organs like Finberg, H.J. 1994. Ultrasound evaluation in fused liver and two pairs of kidneys, (Figure 3). multiple gestation, p. 121-124. In Callen’s There was well developed urogenital system and Ultrasonography in Obstetrics and small and large intestine were present with separate Gynecology, 3rd ed. Harcourt Publishers. rectums for both fetuses. Honnappagol, S.S., M.H. Tandle and V. Conjoined twins may be caused by number Ramakrishna. 2005. Thoraco- abdomino- of factors such as genetic, environmental, and pygophagus foetal monster in a non-descript infectious agents. Assisted reproductive techniques cow. Indian Vet. J., 82: 441. such as In vitro fertilization (IVF) and Intra Jerome, A., T. Sarath and N. Arunmozhi. 2010. cytoplasmic sperm injection (ICSI) may be a factor Dystocia due to a conjoined twin monster in (Romero et al., 1988). The embryonic disk starts a buffalo. Buffalo Bull., 29: 229-231. to differentiate on the 13th day of conception. If the Khasatiya, C.T., D.M. Patel, D.M. Dabhi and split occurs after day 13, then the twins will share P.P. Choudhary. 2009. Dystocia due to body parts in addition to sharing their chorion and dicephalus dipagus monster in a dangi cow. amnion (Finberg, 1994).This type of foetus is due Indian J. Anim. Reprod., 29: 224-225. to congenital embryonic duplication of germinal Kumar, Y.N. and A.R.M. Reddy. 2008. Syncephalus

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dipus tetrabrachius tetrapus dibrachius monster in ewe. Indian Vet. J., 85: 1335. Prasad, J.K., S. Prasad, A. Kumar and G.K. Singh. 2006. Thoraco-sternopagus monster: a rare case of fetal dystocia in buffalo. Indian J. Anim. Reprod., 27: 122-123. Ravikumar, K., M. Selvaraju and S. Manoharan. 2012. Dystocia Due to “Dicephalus Tetrabrachius Thoracopagus Tetrapus Dicaudatus” monster in a Jersey crossbred cow. Indian Vet. J., 89: 96-98. Robert, S.J. 1971. Veterinary Obstetrics and Genital Diseases, 2nd Indian ed. CBS Publishers, New Delhi, India. Romero, R., G. Pilu and P. Jeanty. 1988. Prenatal Diagnosis of Congenital Anomalies. Norwalk, CT, Appleton and Lange. p. 405- 409. Sharma, A., S. Sharma and N.K. Vasishta. 2010. Diprosopus buffalo neonate: A Case report. Buffalo Bull., 29: 62-64. Simon, M.S., B.J. William and T.A. Kannan 2009. A rare case of conjoined twin monster (Ischiopagus) in a she buffalo. Indian J. Anim. Reprod., 30: 90-91. Urankar, R.M., S.V. Chhonkar and P.M. Gangaprai. 1994. Conjoined twin monstrosity in a buffalo. Indian J. Anim. Reprod., 15(2): 165. Vegad, J.L. 2007. Textbook of Veterinary General Pathology, 2nd ed. International book distribution Company Lucknow, U.P., India.

26 Original Article Buffalo Bulletin (March 2016) Vol.35 No.1

CLINICO-BIOCHEMICAL STUDIES OF KETOSIS IN BUFFALO (BUBALUS BUBALIS)

Gurpreet Bali1, Kail Hussain1,*, W.A.A. Razzaque2, Utsav Sharma2 and S.A. Beigh1

ABSTRACT INTRODUCTION

The study was conducted on 18 clinical Ketosis is a common metabolic disorder cases of ketosis presented in the Teaching frequently observed in dairy cows during the early Veterinary Clinical Complex, Faculty of Veterinary lactation period characterized by increased levels Science and Animal Husbandry (F.V.Sc & A.H), of ketone bodies in the blood, urine, and milk. In R.S. Pura and areas around R.S. Pura Tehsil. buffalo, ketosis remains one of the major diseases Clinical and haemato-biochemical parameters were that decrease the productivity (Ghanem and El- studied in the affected animals. A minor decrease deeb, 2010). Ketosis can be clinical or subclinical in mean body temperature with an increase in depending on the subjectivity of the clinical signs. severity of ketosis was noted. The severe cases had It is generally accepted that clinical ketosis occurs diminished rate of ruminal motility and prominent in ruminants when they are subjected to demands clinical signs were sudden and unexpected drop in on their resources of glucose and glycogen that milk production, depraved appetite, wasting and cannot be met by their digestive and metabolic depression. Biochemical parameters viz. plasma activity. Clinical ketosis has visible clinical glucose, total plasma protein, LDL and HDL- symptoms and typically occurs within the irst six cholesterol, calcium and magnesium decreased to eight weeks post-calving, resulting in anorexia, signiicantly. licking and blindness, hard dry feces, rapid loss of condition, and decreased milk production (Youssef Keywords: ketosis, buffalo, biochemical et al., 2010). In addition, the milk fat yield of parameters ketotic cows is increased due to the availability of beta-Hydroxybutyric acid (BHBA) and fatty acids. Clinical ketosis is easy to diagnose by its clinical

1Division of Veterinary Medicine, Faculty of Veterinary Science and Animal Husbandry, Shere-e- Kashmir University of Agricultural Sciences and Technology of Jammu, R.S. Pura, Jammu (J&K), India, *E-mail: [email protected] 2Division of Veterinary Gynaecology and Obstetrics, Faculty of Veterinary Science and Animal Husbandry, Shere-e-Kashmir University of Agricultural Sciences and Technology of Jammu, R.S. Pura, Jammu (J&K), India

27 Buffalo Bulletin (March 2016) Vol.35 No.1

symptoms. The present study was conducted with sudden decline in milk yield being present to study the clinico-biochemical parameters in in 100 per cent of the cases (Table 1). This was buffaloes with clinical ketosis. followed by selective feeding (79.14%), wasting (49.91%), depression (36.67%), complete anorexia (29.37%), acetone smelling breath (19.35%), dry MATERIALS AND METHODS mucus coated feces (12.90%) and signs of central nervous system involvement (6%) of the cases of The study was undertaken for 8 months bubaline ketosis. The pattern of signs observed in duration. Buffaloes with history of anorexia, hard this study was similar to clinical proile described dry feces, rapid loss of condition, and decreased by other workers (Roy and Ghorui, 2000; Radostits milk production drop in milk production presented et al., 2007). at Teaching Veterinary Clinical Complex of the The present study elucidated that ketosis college, constituted the cases for this study. A total was predominantly accompanied by a drop of of 18 cases (Murrah, Jaffarabadi and few Non- 3.44±0.1 litres milk/animal/day estimating 34.92 descript) were found to be suffering from ketosis on per cent basis. Decline of 25-60 percent in milk diagnosed by history, clinical signs and by urine and production in bovine clinical ketosis has also been milk nitroprusside tests viz. Modiied Rothra’s, Ross placed on record by, Swain and Tripathy (1987) Modiication of Rothra’s test and Multidiagnostic and Mir and Malik (2003). The possible reason for strip reaction (supplied by Siemens, India). Three the decreased milk production could be reduced buffaloes were used as control. Blood samples capacity of the animal to supply the lactogenic from the buffaloes were collected from jugular precursors to mammary gland than the capacity of vein. Plasma was separated by centrifugation at the gland to produce due to homeorhetic drive for 3000 rpm for 15 minutes and was stored at -20oC. production (Lean et al., 1992). Moreover, elevated About 2 ml of blood was collected for glucose blood ketones also result in decreased milk estimation. Blood glucose, total serum protein, production (Andersson and Lundstrom, 1985). albumin, Alanine transaminase( ALT), Aspartate In the present study depressed ruminal motility Aminotransferase (AST), Blood urea nitrogen, was recorded in ketotic buffaloes and this could Createnine, HDL- Cholesterol, LDL-Cholesterol, be attributed to excessive generation of ketone calcium, phosphorus, and magnesium were bodies, as ketones bodies are reported to effect estimated using UV-Spectophometer by employing ruminal motility causing incomplete and depressed standard kits. The statistical analysis was done as ruminal contraction (Lean et al., 1991). In severe per the method described by Snedecor and Cochran cases mean rumen motility was as low as 1.77±0.11 (1994). against 2.17±0.19 per two minutes in mild cases. In the present study a signiicant decrease (P<0.05) in Glucose (36.64 mg/dl), Total protein RESULTS AND DISCUSSION (5.52 g/dl), Albumin(2.03 g/dl) and A:G ratio (0.57) was observed when compared with healthy control The present study revealed varying degrees (Table 2). The decrease of glucose level may occur of frequency of occurrence of symptoms in ketosis in response to intake of low energy diet specially at

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the early stage of lactation when high rate of glucose with control ones could be due to negative energy utilization in the mammary gland is required balance. Similarly, ALT has been found to increase (Nazii et al., 2008). Hypocalcemia can exert an in liver and bile duct malfunctions (Steen et al., additional depressive effect on endogenous glucose 1997). Consequently, in the present study, the high production, hence, aggravating hypoglycemia AST and ALT support the occurrence of hepatic (Schlumbohm and Harmeyer, 2003). Decrease damage in ketotic buffalo. in plasma glucose level in ketosis has also been Signiicantly lower values of Cholesterol were by Youssef et al, (2010 ) in lactating buffaloes. observed in buffaloes 84.9 mg/dl, when compared Since albumin is indicative of the liver’s synthetic with the control group (Table 2). The low cholesterol function (West 1990), the reduction in total observed could be attributed to mild liver asteatosis protein and albumin in our study is an indicator which cause reduction in cholesterol formation in for hepatic injury. In the energy deicient ketotic the liver (Grummer, 1995). However, Anantwar and animals labile pool of body protein also serves as Singh (1993) reported that there was an increase of an important source for energy synthesis of milk cholesterol levels in ketotic animals. The decrease lactose and milk protein (Radostits et al., 2007). of serum cholesterol in ketotic buffaloes is similar This protein catabolism for an increased rate of to clinical conditions caused by liver injuries and gluconeogenesis may be the reason for a reduction fatty liver syndrome in cattle (Marcos et al., 1990). in total plasma protein levels. Similar results have HDL and LDL-cholesterol level (64.90 mg/dl and also been recorded by Youssef et al., (2010). 30.42 mg/dl, respectively) showed a signiicant decrease in ketotic buffalo in comparison to control Buffaloes suffering from ketosis showed groups (Table 2). These results coincide with those a non signiicant increase in blood urinary nitrogen of Turk et al. (2008) and Youssef et al. (2010). (22.27mg/dl) level and createnine (2.18 mg/dl) (Table These results may be attributed to moderate liver 2). The high levels of blood urea results from either steatosis, which causes reduction in cholesterol increased breakdown of tissue or dietary protein level. In contrast to present study, Youssef et al. or impaired excretion. Signiicantly higher average values of AST (144.81 U/L) and ALT (144.8 U/L) (2010) observed there was non-signiicant decrease in LDL-cholesterol levels in ketotic buffalo in were observed in ketotic buffaloes when compared comparison to the normal ones. to healthy control (Table 2). Although AST is non- Signiicantly lower levels of plasma speciic liver enzyme estimation of its activity calcium, phosphorus and magnesium were in dairy cows is most often associated with fatty observed in ketotic buffaloes 8.14 mg/dl, 4.77 mg/dl liver syndrome (Cebra et al., 1997). AST has been and 1.90 mg/dl respectively when compared with the found to increase signiicantly in ketotic cows control group (Table 2). The decrease of phosphorus compared with healthy ones (Youssef et al., 2010). and magnesium level coincided with the indings The iniltration of hepatic cells with fat increases obtained by Ziogas et al. (2007) and Youssef et cell membrane permeability with subsequent al. release of AST enzyme that serves as a good tool (2010). Insuficient phosphorus supply in the diet, prolonged anorexia, and increased urinary for metabolic liver diseases (Karasai and Schefar phosphorus excretion due to hyperparathyroidism 1984). Consequently, in the present study, the could explain presence of hypophosphatemia in this elevated serum AST in ketotic buffalo compared 29 Buffalo Bulletin (March 2016) Vol.35 No.1

Table 1. Important clinical signs observed in clinically ketotic buffaloes (n=18).

Percent S. NO. Clinical Signs animals 1. Nervous signs 6 2. Constipation 12.90 3. Acetone smell in breath 19.35 4. Complete anorexia 29.37 5. Depression 36.67 6. Wasting/ woody appearance 49.91 7. Selective feeding (partial anorexia) 79.14 8. Sudden drop in milk yield 100

Table 2. Plasma biochemical and mineral values in ketotic buffaloes.

Parameters Control Ketotic animals Glucose (mg/dl) 58.4±2.86 36.64±1.19* T Protein (g/dl) 7.44±0.26 5.52±0.13* Albumin (g/dl) 3.13±0.15 2.03±0.05* A:G ratio 0.71±0.01 0.57±0.007* ALT (IU/L) 31±3.1 43.90±3.22* AST (IU/L) 118.2±4.07 144.8±2.19* Cholesterol (mg/dl) 179.11±7.18 84.9 ±3.16* LDL-Cholesterol (mg/dl) 48.4±4.60 30.42±1.05* HDL-Cholesterol (mg/dl) 86.5±2.97 64.90±1.62* Creatinine (mg/dl) 1.71±0.05 2.18±0.05 BUN (mg/dl) 18.3±1.24 22.27±1.10 Ca (mg/dl) 10.8±0.41 8.14±0.09* P (mg/dl) 5.80±0.19 4.77±0.07 Mg (mg/dl) 2.20±0.05 1.90±0.03*

Means marked with asterisk (*) differ signiicantly (p<0.05) from the control group value in a column.

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condition. However, the decrease of magnesium organic nutrient metabolism on feeding the level could be attributed to ketonuria which results transition period dairy cow. J. Anim. Sci., in decrease tubular resorption or to the need of 73: 2820-2833. magnesium that regulate metabolism for milk Karasai, F. and M. Schefar. 1984. Diagnostic secretion. In reference to the fall in calcium levels experiences with metabolic liver diseases of it is suggested that there might be increased loss dairy cows. Monta Fur Veterinar, 39: 181- of base in urine to compensate ketosis induced 186. acidosis. Besides, Cote et al. (1969) indicated that Lean, I.J., R.L. Bruss, R.L. Baldwin and H.F. reduced feed intake may also lead to secondary Troutt. 1992. Bovine ketosis: A review II. hypocalcemia. Biochemistry and prevention. Vet. Bull., 62(1): 1-14 Marcos, E., A. Mazur, P. Carder and Y. Rayssiguier. REFERENCES 1990. Serum apolipoproteins B and A-I and naturally occurring fatty liver in dairy cows. Anantwar, L.G. and B. Singh. 1993. Treatment Lipids, 25: 575-577. of clinical ketosis in buffaloes (Bubalus Mir, A.Q. and H.U. Malik. 2003 Utility of clinical bubalis). Indian Vet. J., 69: 446-448. symptomatology in diagnosis of bovine Andersson, L. and K. Lundstrom. 1985. Effects ketosis under ield conditions. Indian J. Vet. of feeding silage with high butyrie acid Med., 23: 104-105. content on ketone body formation and milk Nazii, S., M. Mohebbi Fani, E. Rowghani and yield in post parturient dairy cows. Zbl. Vet. M.R. Behbood. 2008. Studies on the Med. A., 32: 15-23. relationship between sub-clinical ketosis Cebra, C.K., F.B. Gerry, D.M. Getzy and M.J. and liver injuries within the irst two months Fettman. 1997. Hepatic lipidosis in anorectic, of lactation period in high producing Iranian lactating Holstein cattle: retrospective study Holstein cows. International Journal of of serum biochemical abnormalities. J. Vet. Dairy Science, 3: 29-35. Intern. Med., 4: 231-237. Radostits, O.M., C.C. Gay, D.C. Blood and K.W. Cote, J.M., R.A. Curtis, B.J. McSherry, J.M. Hinchcliff. 2007. Veterinary Medicine: Robertson and D.S. Kronfeld. 1969. A Textbook of the Diseases of Cattle, Bovine ketosis: frequency of clinical Sheep, Pigs, Goats and Horses, 9th ed. WB signs, complications and alterations in Saunders Company, Philadelphia, USA. blood ketones, glucose and free fatty acids. Roy, N. and P. Ghorui. 2000. Spontaneous ketosis Canadian Vet. J., 10: 179-187. in cattle and its therapy. Indian Journal of Ghanem, M.M. and W.M. El-Deeb. 2010. Lecithin Animal Health, 39: 47-50. cholesterol acyltransferase (LCAT) activity Schlumbohm, C. and J. Harmeyer. 2003. as a predictor for ketosis and parturient Hypocalcemia reduces endogenous glucose haemoglobinuria in Egyptian water production in hyperketonemic sheep. J. buffaloes. Res. Vet. Sci., 88(1): 20-25. Dairy Sci., 86(6): 1953-1962. Grummer, R.R. 1995. Impact of changes in Snedecor, G.W. and W.G. Cochran. 1994. Statistical

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Methods, 8th ed. Iowa State University Press, Ames, Iowa, USA. Steen, A., H. Gronstol and P.A. Torjesen. 1997. Glucose and insulin responses to glucagon injection in dairy cows with ketosis and fatty liver. J. Vet. Med. A., 44(1-10): 521- 530. Swain, P.K., and S.B. Tripathy. 1987. Management of ketosis in crossbred cows. Indian J. Vet. Med., 7: 121. Turk, R., D. Juretic, D. Geres, A. Svetina, N. Turk and Z. Flegar-Mestric. 2008. Inluence of oxidative stress and metabolic adaptation on PON1 activity and MDA level in transition dairy cows. Anim. Reprod. Sci., 108(1-2): 98-106. West, H.J. 1990. Effect on liver function of acetonemia and the fat cow syndrome in cattle. Res. Vet. Sci., 48(2): 221-227. Youssef, M.A., S.A. El-Khodery, W.M. El-deeb and W.E. Abou El-Amaiem. 2010. Ketosis in buffalo (Bubalus bubalis): clinical indings and the associated oxidative stress level. Trop. Anim. Health Pro., 42(8): 1771-1777. Ziogas, V., S. Japertas, G. Vorobjovas, J. Kucinskiene and V. Zilaitis. 2007. Morbidity of high yielding cows of ketosis and treatment ketosis with propylene glycol and niacin in Lithuania. Vet. Zootec., 37: 91- 95.

32 Original Article Buffalo Bulletin (March 2016) Vol.35 No.1

COMPARATIVE STUDIES OF CERTAIN ENZYME ASSAY DURING VARIOUS REPRODUCTIVE STATES IN BUFFALOES

R. Chaurasia1, H.S. Kushwaha1, D. Chaurasia2, M.K. Gendley2, Kiran Kumari2, A.K. Santra2 and B. Shinha2

ABSTRACT signiicantly (P<0.01) different among the groups. It was recorded to be highest in repeat breeder Thirty healthy Murrah buffaloes of 5-9 followed by normal cyclic and lowest in anestrus years of age with second to ifth lactation were buffaloes. The serum alanine aminotransferase and randomly chosen from well organized dairies aspartate aminotransferase values were 26.402 and located at Jabalpur to elucidate alteration in 69.124; 32.035 and 104.435; 34.007 and 85.093 concentration of some enzymes viz. serum alkaline U/L in anoestrus , repeat breeder and normal cyclic phosphatase (ALP), acid phosphatase (ACP), groups, respectively. The values of serum alanine alanine transferase (ALT), and aspartate amino aminotransferase and aspartate aminotransferase transferase (AST), in serum of normal cyclic, of repeat breeder was signiicantly (P<0.01) higher repeat breeder and anoestrus Murrah buffaloes. than normal cyclic and lowest in anoestrus group. Gynecological examinations were employed for the diagnosis of reproductive status of animals. Keywords: Serum alkaline phosphatase, Serum The selected 30 Murrah buffaloes were divided acid phosphatase, Alanine amino transferase and into three groups and each group comprised of 10 Aspartate amino transferase animals for the generation of experimental data. Blood samples were collected from animals of different groups on the day of estrus from normal INTRODUCTION cyclic, repeat breeder and anoestrus buffaloes on the same day for gynaeco-clinical examination. In India, estimated populations of buffaloes The serum was prepared following routine are 105.3 million among which female buffaloes procedure. The serum enzymatic activities was consists of 54.5 million (NDDB, 2012). These monitored immediately for serum alkaline and acid animals play an important role in Indian livestock phophatase, alanine amino transferase and aspartate economy. The success of dairy cattle and buffalo amino transferase. The serum alkaline phosphatase economy lies in proper and optimal reproductive and acid phosphatase values were 9.190 and 1.357 rhythm of each individual cow and buffalo in the ; 19.778 and 2.667; 14.064 and 2.100 KA units herd, within normal physiological range (Dhaliwal, /100 ml in anoestrus , repeat breeder and normal 2005). Any deviation or prolongation in the breeding cyclic groups, respectively. The serum alkaline rhythm results in a progressive economic loss due phosphatase and acid phosphatases values were to widening of dry period reduced calvings and

1College of Veterinary Science and Animal Husbandry, Jabalpur (M.P.), India 2College of Veterinary Science and Animal Husbandry, Anjora, Durg (C.G.), India

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lactations during the life span of the animal (Singh cyclic buffaloes. et al., 2006). Barren or infertile buffaloes means a Samples from repeat breeder and anoestrus direct loss in milk production, whereas reduced calf buffaloes were taken on the same day for gynaeco- crops hamper the selection eficiency in long term clinical examination. The serum was prepared dairy herd improvement (Baghel, 2006). About 25 following routine procedure. Separated serum was percent dairy animals. However, repeatability and centrifuged at 3000 rpm for 10 minutes. Serum anoestrus conditions are recognized as the serious was used immediately for monitoring assay of problem in increasing the calving interval, therefore enzyme activities serum alkaline phophatase by subjecting the farmer into heavy economic loss. (ALP) and acid phophatase (ACP), alanine amino In general incidence of anestus has been reported transferase (ALT) and aspartate amino transferase between 9.09-82.50 percent in buffalo (Thakor and AST). Serum alkaline phosphate was measured Patel, 2013). as per Tietz, (1976). Acid phosphatase was Blood proile might be potential in estimated by the calorimetrically method of King characterizing the problem and diagnosing a and Jagatheesan, (1959), alanine aminotransferase deicient condition (Eltohamyet al., 1989; Jain, et and aspartaste aminotransferase as per Henry, al., 2003). The present investigation was designed (1974). The data were analyzed statistically using to study the biochemical changes during different analysis of variance technique (ANOVA) and the reproductive states (Normal cyclic, repeat breeder differences between means were compared using and anoestrus) in Murrah buffaloes. The study was critical difference (Snedecor and Cochran, 1996). aimed to elucidate alteration in concentration of some enzymes in serum of normal cyclic, repeat breeder and anoestrus Murrah buffaloes. RESULTS AND DISCUSSION

Concentration of serum enzymes in MATERIALS AND METHODS different reproductive states in Murrah buffaloes have been given in Table 1. In the present investigation, healthy 1. The serum alkaline phosphatase (ALP) Murrah buffaloes (30) were randomly chosen from activity was signiicantly (P<0.01) increased well organized dairies located at Jabalpur. These in repeat breeder (P<0.01) followed by that in animals were in 5-9 years of age group and within normal cyclic and lowest in anestrous condition second to ifth lactation. These animals were of buffaloes. The trend of higher ALP activity in screened as per the approved technical program. repeat breeder was in agreement with the indings Gynecological examinations were employed for reported by Sharma et al., (1986). They found the diagnosis of reproductive states of animals. higher serum ALP activity in primary infertile The selected 30 Murrah buffaloes were divided Kankrej heifers than normal cyclic heifers. Mehta into three groups and each group comprised of 10 et al. (1989) also recorded similar results on animals for the generation of experimental data. comparison between repeat breeder and normal Blood samples were collected from animals of cyclic cows. Gandotra et al. (1993) reported higher different group on the day of estrus from normal level of ALP activity in repeat breeder cattle and

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buffaloes as compared to normal cyclic cattle and pace of conception while reverse is true in repeat buffaloes. Chandrakar (1999) found serum activity breeder (Sharma et al., 1986). concentration to be signiicantly higher in repeat 2. Serum acid phosphatase (ACP) breeder than normal fertile cows. Yaqub et al. activity showed a similar trend as ALP which was (2013) reported that Aspartate aminotransferase signiicantly (P<0.01) highest in repeat breeder (AST) and Alanine aminotransferase (ALT) followed by reduced in normal cyclic and lowest in concentration non signiicantly luctuated duringanestrous condition. The result correlates with the the estrous cycle in Red Sokoto goats. indings of Gandotra et al. (1993). They recorded In this study ALP activity during anoestrus signiicantly higher values of ACP activity in condition was signiicantly lower than the levels in repeat breeder than normal cyclic buffaloes. normal cyclic Murrah buffaloes. It might be due The result was also similar to Ganguly (2013) to malnutrition.The result can be correlated with who reported that increase in Acid phosphate the indings of Kalnathet al. (2007) who reported concentration decreases with increase in follicular higher Alkaline Phosphatase (ALP) activity during size. It can be inferred that higher concentration of follicular development. However, Derashri et acid phosphate concentration increases follicular al. (1984) reported the ALP levels in anoestrus activity resulting in repeat breeding. On the condition to be higher as compared to the animals contrary, Sharma et al. (1986) reported that mean in oestrus but the difference was non-signiicant. In values of ACP were signiicantly (P<0.01) higher the light of present results it could be hypothesised in normal cyclic Kankrej heifers. Increased activity that decreased level of AKP activity in normal of ACP might be helpful in hydrolysing the organic cyclic as compared to Repeat breeders might be due phosphomonoesters and thus provide energy in the to enhanced folliculogenesis resulting in increased form of phosphates in normal cyclic animals.

Table 1. Concentration of serum enzymes in different reproductive states in murrah buffaloes.

Attributes (KA unit/dl) Normal Cyclic Repeat Breeder Anoestrus Serum alkaline phosphatase 14.064 + 0.58b 19.778+ 0.385c 9.190+ 0.48a (ALP) Serum acid phosphatase 2.100+0.234b 2.667+0.187c 1.357+0.133a (ACP) Serum alanine aminotransferase (ALT) 34.007+1.366c 32.035+1.423b 26.402+0.654a (U/L) Serum aspartate aminotransferase 85.093+ 3.070c 104.435+2.416b 69.124+2.608a (AST) (U/L)

Note: 1. Mean +SE values with different superscripts in row are highly signiicantly different (P<0.01). 2. The enzyme unit (U) is a unit for the amount of a particular enzyme. 3. The enzyme activity unit (KA) is expressed I the term of KA/100 ml.

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The result indicating signiicantly higher signiicantly lower in normal cyclic as compared to ACP activity in normal cyclic as compared to repeat breeders buffaloes, supported the indings of anoestrus buffaloes was in contrary to the indings Pal et al. (1991). They reported that the AST activity of Derashri et al. (1984) who observed ACP in cyclic heifers and cows were comparatively activity level higher in anoestrus surti buffaloes higher and statistically signiicant (P<0.01) than than the normal oestrus buffaloes. The serum ACP the non-cyclic heifers. This enzyme activity was activity level might be inluenced by physiological indicative of increased physiological activity and conditions. Its activity levels may be used as an pathological condition of the tissue. In the present index in assessing the estrogen level in buffaloes. study AST activity was higher in repeat breeder than 3. The serum ALT activity level was normal cyclic buffaloes and differences between signiicantly (P<0.01 increased in normal cyclic, two groups were signiicantly different. This was followed by repeat breeder. The level of ALT was in agreement with Gandotra et al. (1993). They signiicantly (P<0.01) lower in anoestrus buffaloes reported that AST activity level in repeat breeder as compared to other two groups of buffaloes. cows and buffaloes were signiicantly higher than The indings of result were in agreement with the the normal cyclic cows and buffaloes. The possible observation of Derashri et al. (1984). He reported cause of increased AST activity level may be uterine the same trend of ALT activity in oestrus and tissue damage in repeat breeder cows & buffaloes. normal cyclic condition and suggested that possible The present result was in contrary to Sharma et involvement of hormonal levels prevailing during al. (1986) who found AST activity to be higher normal oestrus/normal cyclic helps in regulation of in primary infertile Kankrej heifers as compared ALT activity levels. Sharma et al. (1986) recorded to normal cyclic heifers, however, the difference signiicantly lower activity of ALT in infertile was found to be non signiicant. Sarwaret al. group of heifers than normal group. Pal et al. (1991) (2002) found that AST and ALT were signiicantly found level of ALT activity in the cyclic heifers and higher in endometritis in Nili-Ravi buffalo which cows to be comparatively signiicantly higher than can be correlated with the result of repeat breeder. non cycling ones. In the present study the level of However, result is dissimilar to those of indings ALT was signiicantly lower in repeat breeder than of Yaqub et al. (2013) who reported ALT and AST normal cyclic buffaloes. These indings were again to non signiicantly luctuate during estrus cycle in agreement with Gandotra et al. (1993). They in goat. This might be because goat is a seasonal recorded that the ALT activity level was higher in breeder whereas buffalo is a regular breeder. normal cyclic cattle and buffaloes than the repeat Estrus cycle may produce measurable breeder cattle and buffaloes. Higher level of serum stress during estrus phase resulting in increased ALT activity might be due to increase in metabolic physiological activity which increases the AST activity mediated by physiological activity during activity in blood. But in case of repeat breeder oestrus cycle condition. higher values might be due to tissue damage. 4. The serum AST activity level was signiicantly (P<0.01) increased in repeat breeder compared to normal cyclic and lowest in anestrous group. In the present result AST activity being

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CONCLUSION in normal and repeat breeder rathi cows. Indian J. Anim. Sci., 52: 944-946. Serum alkaline phophatase and acid Ganguly S. 2013. Role of Biochemical factors and phophatase activities in anoestrus group were mineral supplementation in livestock ration signiicantly lower than normal cyclic and repeat for maintenance of their fertility and healthy breeders, Alanine amino transferase and aspartate reproductive status. Res. J. Chem. Sci., 3(6): amino transferase activities in normal cyclic were 102-106. also signiicantly higher than anoestrus buffaloes. Henry, J.B. 1974. Clinical Diagnosis and Management by Laboratory Method. W.B. Saunders & Co. Philadelphia, USA. REFERENCES Jain, A., R.K. Pathak and P.K. Jain. 2003. Effect of mineral supplemention on fertility of Baghel, R.P.S. 2006. Reproductive disorders in crossbred cows. Indian Vet. Med. J., 27: relation to malnutrition in dairy animals, 259-260. p. 94-104. In Souvenir of 22nd Annual Kalnath, G.P. and J.P. Ravindra. 2007. Mineral Convention of ISSAR and National proiles of ovarian antral follicular luid in Symposium. Veterinary College, Mhow, buffaloes during follicular development. India. Indian J. Anim. Res., 41(2): 87-93. Chandrakar, D. 1999. Studies on microbial and King, E.J. and K.A. Jagatheesan. 1959. A method biochemical proile with therapeutic for the determination of tartratelabile, measures in repeat breeder cross-bred prostatic acid phosphatase in serum. J. Clin. cows. M.V.Sc. and A.H. Thesis, I.G.K.V., Pathol., 12(1): 85-89. Raipur, India. Mehta, J.S., J.S. Bhatia, I.S. Kohli, P.K. Pareek, Derashri, H.J., G.A. Prabhu, F.S. Kavani and B.L. Bishnoi and A.K. Gupta. 1989. Studies S.B Kodagali. 1984. Levels of serum on serum alkaline phosphatase and protein phsphatases and reproductive status in surti in various reproductive states in cow. I. J. buffaloes. I. J. A. R., 4: 49-50. A.R., 10: 138-140. Dhaliwal, G.S. 2005. Managing dairy herds for National Dairy Development Board. 2012. NDDB. optimal reproductive eficiency, p. 1-9. In Annual Report, 2011-2012. Souvenir of Advanced Training Course. Pal, S.K., B.N. Mohanty, S.K.H. Ray and D.N. Punjab Agricultural University, Ludhiana, Mohanty. 1991. Studies on serum protein, India. cholesterol and certain enzymes in relation Eltohamy, M.M., M. Younis, H.A. Salem, A. to reproductive status in bovine females. I. Azouz, N. Shawky and A.A. Farahat. 1989. J. A. R., 12: 28-29. Role of some micro and macro-elements Sarwar, A., R.U. Shahid, S. Masood, R. Kausar during repeat breeding in buffaloes. Indian and S.G. Shah. 2002. Serum electeolytes J. Anim. Sci., 59: 1409-1409. and enzymes in endometritic Nili-Ravi Gandotra, V.K., K.R. Choudhary and R.D. Sharma. buffaloes of two age groups and at two 1993. Serum biochemical constituents stages of lactation. Pak. Vet. J., 22(2); 78-81

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Sharma, V.K., G.M. Sidique and V.P. Vadoria. 1986. Levels of serum enzymes in primary infertile and normal cycling Kankrej heifers. I. J. A. R., 7: 36-39. Singh, A.P., R.S. Shah, R.B. Singh, M.H. Akhtar, G.P. Roy, C. Singh and V. Kunj. 2006. Response of mineral mixture, Prajana and GnRH on serum biochemical constituents and conception rate in anoestrus buffaloes. Indian J. Anim. Reprod., 27(1): 51-54. Snedecor, G.W. and W.G. Cochran. 1996. Statistical Methods. IOWA State University Press. Ames, USA. Tietz, N.W. 1976. Fundamentals of Clinical Chemistry. W.B. Sanders and company, Philadelphia, USA. 602p. Thakor, D. and D. Patel. 2013. Incidence of infertility problems in cattle and buffaloes. Dairy Cattle. Available from http:// en.engormix.com/MA-dairycattle/genetic/ articles/incidence-infertility-problems- cattle-t2757/103-p0.htm. Accessed on 15 May 2015. Yaqub, L.S., M.U. Kawu, J.O. Ayo, S.F. Ambali and B. Habibu. 2013. Effect of estrous cycle on serum electrolytes and liver enzymes in Red Sokoto goats. Afr. J. Biochem. Res., 7(9): 174-178.

38 Original Article Buffalo Bulletin (March 2016) Vol.35 No.1

CROSS-SECTIONAL SURVEY OF HELMINTHIASIS IN BUFFALOES AT TEHSIL JATOI AND TEHSIL MUZAFFAR GARH, SOUTHERN PUNJAB, PAKISTAN

Muhammad Asif Raza1, Muhammad Mazhar Ayaz2,*, Muhammad Mudasser Nazir2, Muhammad Saleem Akhtar2, Mubashir Aziz2, Saeed Murtaza2 and M. Ali Khosa2

ABSTRACT world (Ibrahim et al., 1984 and Waler et al., 1987). water buffaloes are considered common host for In the four year study from 2009 to 2013, helminthiasis in tropical and sub-tropical area. a total of 500 faecal samples from buffaloes Helminthiasis inlicts huge economic losses even from different locations of Tehsil Jatoi and Tehsil deaths of infected animals. The economic losses Muzaffar Garh, Southern Punjab were analyzed are in a variety of ways like lowered the fertility, to conirm the presence of gastrointestinal reduction in work capacity, involuntary culling of parasitic infection. The recovered parasites were emaciated animals, reduction in food intake, lesser ive nematodes viz Toxocara vitulorum (16.6%), weight gains, lower milk production, huge treatment Oesophagostomum radiatum (3.2%), Bunostomum costs, and mortality in heavily parasitized animals phlebotomum (1.6%), Cooperia spp. (1.6%), (Lebbie and Irungu, 1994), the other can be reduced Trichostrongylus spp. (0.8%). The two trematodes weight gains and the condemnation of infected were Fasciola hepatica (8.4%), Paramphistomum organs at slaughter (Liu et al., 2009). Prevalence cervi (15%). Age-wise prevalence was 79.5% of Gastrointestinal Tract helminthes in ruminants and 47% in buffalo calf and adult buffalo, while has been reported up to 25 to 92% in different areas sex-wise prevalence was 78.4% and 50.93% in of Pakistan by various workers like Ali et al., 2000; male and female buffalo, respectively. Chi-square Raza et al., 2007; Ijaz et al., 2008; Al-Shaibani et statistical design was applied to data to know the al., 2008; Kakar and Kakar-sulemankhel, 2008; dependence of helminth’s prevalence on sex and Raza et al., 2010; Ayaz et al., 2013 and Iqbal et age of animals. al. (2002) has pointed out various parasitic problems like Facioliasis, hydatidosis, coccidiosis, theileriosis Keywords: buffaloes, helminths, southern Punjab, and babesiosis as the major parasitic problems of Pakistan ruminants in Pakistan in the order of priority. A number of factors that inluence the prevalence INTRODUCTION of helminthes that includes age (Mckenna, 1981), sex (Asanji and Williams, 1987), breed (Miller, Helminths are recognized as a major 1998; Mirza and Razzak, 1998), worm population constraint to livestock production throughout the (Ankers et al., 1997), weather condition (Asanji

1Faculty of Veterinary Medicine, Higher College of Technology, Al-Ain Men’s College, United Arab Emirates 2Faculty of Veterinary Sciences, Bahauddin Zakariya University, Multan, Pakistan, *E-mail: mazharayaz@ bzu.edu.pk

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and Williams, 1987, Mohiuddin et al., 1984) and of Veterinary Sciences, Bahauddin Zakariya husbandry or managemental practices (Tan et University, Multan for identiication of eggs/larvae al., 1996). Some helminths infecting buffaloes or adult helminths. primarily has zoonotic importance such as schistosomiasis, cycstic echinococcosis, and Sample analyses fasciolosis (Liu et al., 2009; Cringoli et al., 2007). Fecal samples were examined for Punjab province in Pakistan has largest population helminthes eggs/larvae/adult by using direct of buffalo so considering the health implications and and indirect techniques (Ayaz, 2010) and for the economic potential of water buffaloes, the issue identiication of certain nematodes, copro-culture/ of investigating parasitic infections of buffaloes is Baermann method (Ayaz, 2010) were performed of relevance (Rinaldi et al., 2007; Veneziano et al., to obtain larval stages. Both eggs and larvae from 2007) for the cross sectional study. copro-culture were identiied by using standard techniques as described by MAFF (1979) and Soulsby (1982). Briely, one gram of fecal sample MATERIALS AND METHODS was mixed well in a drop of water and a relatively homogenous and transparent preparation was The present cross sectional study was obtained and examined under microscope by initiated from January 2009 to June 2013 to placing a drop of suspension on slide with cover determine the point prevalence of Gastrointestinal slip. At least three direct smears were examined tract helminthiasis in the buffalo under ield from each sample. All the samples were also conditions at Tehsil Jatoi and Tehsil Muzaffar examined by using concentration techniques, Garh, Southern Punjab, Pakistan. i.e. loatation and sedimentation. For loatation technique, ive grams of feces was mixed in 50 Study area ml of water and strained through a sieve no. 60 From January 2009 to June 2013, fecal micron mesh to remove the course material. The sample of 500 buffaloes (Between age ranging six mixture was allowed to sediment for half an hour. months to two years and from three years to six The supernatant was discarded and sediment was years and Sex-wise) were brought to the Laboratory mixed with saturated salt solution. The suspension of Veterinary Parasitology, Faculty of Veterinary was centrifuged at 1000 rpm for two minutes. Sciences, Bahauddin Zakariya University, Multan The upper 0.1 ml of centrifuged suspension was for presence of GIT parasites. The study area is transferred to a glass slide and examined under located at latitude 288570 °N to 308460 oN and microscope at 10 X for the presence of helminthes longitude 708300 °E to 718470 °E. eggs. More over a relatively new technique for “a single slide positive sample” was also developed Sample collection as “micro-loatation technique” (Ayaz, 2010). Faecal samples of buffaloes (n=500) were For sedimentation technique to examine heavy randomly collected in sterile polythene bags directly eggs, ive grams of faeces was mixed in 50 ml of (Ayaz, 2010). The faecal samples were brought to water and strained through a sieve mesh no. 60 the Laboratory of Veterinary Parasitology, Faculty micron to remove the course material. The mixture

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was allowed to sediment for half an hour. After RESULTS centrifugation, the supernatant was decanted and washing was continued until supernatant became The present cross sectional study was clear. A drop of 0.1 ml was taken from sediment commenced from January 2009 to June 2013 with the help of Pasteur’s pipette on slide and to determine the prevalence of GIT helminthes was examined under microscope at 10 X for the in buffaloes. Overall Age-wise and sex-wise presence of helminthes eggs. prevalence in buffalo was 60% and 57.8%, respectively. The highest prevalence for nematodes Feco-Copro-Culture (119/500; 23.2%) followed by trematodes (117/500; Feco-Copro-Culture provides an 23.4%) was recorded. A total seven species of environment suitable for hatching and development helminths including ive species of nematodes, of helminths eggs. Samples found positive for i.e. Toxocara vitulorum, Oesophagostomum nematode eggs were broken up inely, using either radiatum, Bunostomum phlebotomum, Cooperia a large pestle and mortar or spatula and were placed spp., Trichostrongylus spp. and two trematodes in a glass jar or petri-dish for incubated at 26°C for species i.e. Fasciola hepatica, Paramphistomum 3-7 days. After incubation, samples were examined cervi were recorded. Among various species of for the presence of larvae and were identiied with helminthes Toxocara vitulorum was the most the help of key by MAFF (1979). prevalent species of helminthes which was followed by Paramphistomum cervi, Fasciola hepatica, Statistical analyses Oesophagostomum radiatum, Bunostomum Data on the prevalence of helminthiasis phlebotomum, Cooperia spp. and Trichostrongylus was analyzed using Chi-square statistical design species respectively. The mixed helminthes and percentage on the basis of sex and age. infections (52/500; 12.4%) was often composed Graphical representation of tabulated data was also of 07 species including Toxocara vitulorum, done. Oesophagostomum radiatum, Bunostomum phlebotomum, Cooperia spp., Trichostrongylus

Table 1. Prevalence of different species of helminths in buffaloes (n=500).

Number of Examined Number of Positive % age GIT Helminths Species Fecal Samples Fecal Samples Infected Toxocara vitulorum 500 83 16.6 Paramphistomum cervi 500 75 15 Fasciola hepatica 500 42 8.4 Oesophagostomum radiatum 500 16 3.2 Bunostomum phlebotomum 500 8 1.6 Cooperia spp. 500 8 1.6 Trichostrongylus spp. 500 4 0.8

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Table 2. Age-wise prevalence of different species of helminths in buffaloes (n=500).

Species of helminths Calf Adult Toxocara vitulorum 60/200; 30% 23/300; 7.67% Paramphistomum cervi 40/200; 20% 35/300;11.67% Fasciola hepatica 16/200; 8% 28/300; 9.33% Oesophagostomum radiatum 4/200; 2% 12/300; 4.00% Bunostomum phlebotomum 4/200; 2% 4/300; 1.33% Cooperia spp. 2/200; 1% 6/300; 2.00% Trichostrongylus spp. 1/200; 0.5% 3/300; 1.00%

Table 3. Sex-wise prevalence of different species of helminths in buffaloes (n=500).

Species of helminth Male Female Toxocara vitulorum 25/125; 20% 58/375; 15.47% Paramphistomum cervi 30/125; 24% 45/375; 12% Fasciola hepatica 13/125; 10.4% 29/375; 7.73% Oesophagostomum radiatum 8/125; 6.4% 8/375; 2.13% Bunostomum phlebotomum 5/125; 4% 3/375; 0.8% Cooperia spp. 2/125; 1.6% 6/375; 1.6% Trichostrongylus spp. 0/125; 0% 4/375; 1.07%

Table 4. Presence of various species of GIT helminths in buffalo.

Species of helminthes Buffaloes Nematodes Toxocara vitulorum + Oesophagostomum radiatum + Bunostomum phlebotomum + Cooperia spp. + Trichostrongylus spp. + Trematodes Fasciola hepatica + Paramphistomum cervi + Cestodes - Total Number of Helminths spp. 7

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Table 5. Age-wise and sex-wise prevalence of GIT helminths in buffalo (n=500).

Age-wise animals Calf Adult Overall prevalence Buffaloes 159/200; 79.5% 141/300; 47.00% 300/500;60% Sex-wise animal Male Female Overall prevalence Buffaloes 98/125; 78.4% 191/375; 50.93% 289/500; 57.8 spp., Fasciola hepatica and Paramphistomum of intermediate host, fauna and lora, frequency cervi. of infection, fecunditity and mal-management practices. In this cross sectional survey, the Age- The prevalence of helminthes was higher wise prevalence of helminthes was higher in in buffalo (Table 2) calves as compared to adult young animals as compared to elder ones, and in buffalo. A total of 12.4% buffalo (62/500) had sex-wise higher in males as compared with the mixed infection comprising 16% (32/200) in calves females. In buffaloes, a total of seven species of and 10% (30/300) in adult buffalo. helminthes including ive nematodes, including Toxocara vitulorum, Oesophagostomum radiatum, In Table 2 and 3 The prevalence of Bunostomum phlebotomum, Cooperia spp., and helminthes was higher in young animals compared Trichostrongylus spp. were found while two with the older ones and the prevalence of helminthes species of trematodes, i.e. Fasciola hepatica, was higher in males compared with the females. Paramphistomum cervi were recorded. The most prevalent nematode recovered in this study area In Table 4 a total of seven species of from buffaloes was T. vitulorum which was reported helminthes (ive nematodes and two trematodes) to be the most frequent occurring nematode in cattle were recorded. and buffaloes by other scientists like El-Maukdad (1979), Iqbal et al. (1984), Mourad et al. (1985), In Table 5 the prevalence of helminthes Anwar et al. (1996) and Motahar et al. (2000). was higher in young animals/ calves (79.5%) The infection was higher in young animals compared with the older ones (47%) while sex-wise as compared to older ones that may be attributed the prevalence of helminthes was higher in males to lesser immunity because of fewer/ maiden (78.4%) as compared with the females (50.93%). exposure to various species of helminthes. It might be interesting that prevalence was higher in males as compared with females. Normally, females DISCUSSION are assumed to be more infected due to stress of pregnancy and parturition due to stall feeding in Helminthiasis being one of the major females around the termination of pregnancy and problems affecting the productivity of buffaloes thus lesser exposure to pasture contamination. Most remains on top of the list. This severity depends of the researchers have observed higher rates of on the prevalence, intensity of infection, presence nematode burden in female hosts as compared with

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Figure 1. Prevalence of different species of helminths in buffaloes.

Figure 2. Prevalence of different species of helminths in buffaloes.

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Figure 3. Age-wise prevalence of different species of helminths in buffaloes.

Figure 4. Sex-wise prevalence of different species of helminths.

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the males like Asanji and Williams, 1987; Pal and Ankers, P., S. Fofana and A.E. Biaye. 1997. Qayyum, 1992; Iqbal et al., 1984; Maqsood et al., Epidemiology of helminths of cattle, 1996; Komoin et al., 1999; Valcarcel and Garcia sheep and goats in Maritime Guinea, Romero, 1999. Guinea. Revue-d’Elevage-et-de-Medecine- In contrast to the current results, Gulland and Veterinaire-des-Pays-Tropicaux, 50: 111- Fox (1992) reported that prevalence and intensity of 116. infection (faecal egg counts) were higher in males Anwar, A.H., C.S. Hayat and M.I. Amir. 1996. than females, except during the lambing periods, and Prevalence of gastro intestinal helminthiasis decreased with increase in age in both sexes. Effect and comparative eficacy of anthelmintics of reproductive cycle has been reported to affect the in parasitized buffalo calves. Pak. Vet. J., worm burdens in animals, which has an important 16: 160-163. epidemiological signiicance as Lyonset al. (1987, Asanji, M.F. and O. Williams. 1987. Variables 1992) reported a progressive increase in the egg per affecting the population dynamics of gram (EPG) and number of helminths in ewes during gastrointestinal helminth parasite of small and after the parturition period. This phenomenon has farm ruminant in Sierra Leone. Bull. Anim. been attributed to a variety of reasons like seasonal Hlth. Prod. Africa., 35: 308-313. changes, host factors, activation of hypobiotic Ayaz, M.M. 2010. The Procedures in Veterinary larvae, parturition stress, poor nutritional status, Protozoology, 1st ed. BBS Life Sciences peri-parturient relaxation in immunity (PPRI) or Pub. Multan. Pakistan. spring rise phenomenon, hormonal changes around Ayaz, M.M, M.A. Raza, S. Murtaza and S. Akhtar. parturition, breed differences etc. etc. In many 2013. Epidemioogical survey of helminthes parts of the world, parturition of grazing animals is of goats in southern Punjab, Pakistan. Trop. synchronized to occur with the favorable climate to Biomed., 30(1): 62-71. pasture growth and also suitable for development Cringoli, G., L. Rinaldi, V. Musella, V. Veneziano, and survival of free-living stages of most helminths M.P. Maurelli, F. Di Pietro, M. Frisiello (Wedderburn, 1970). and S. Di Pietro. 2007. Geo-referencing livestock farms as tool for studying cystic echinococcosis epidemiology in cattle REFERENCES and water buffaloes from southern Italy. Geospatial Health, 2: 105-111. Ali, S., M.Q. Khan, M. Qayyum and M.F.U. Khan. El-Moukdad, A.R. 1979. Helminths of cattle in 2000. Prevalence of gastrointestinal parasites Syria. Angewandte Parasitologie, 20: 11- in sheep and goats maintained at NARC, 16. Islamabad, Pakistan. Pak. Vet. J., 20: 157- Gulland, F.M.D and M. Fox. 1992. Epidemiology 158. of nematode infections of Soay sheep (Ovis Al-Shaibani, I.R.M., M.S. Phulan, A. Arijo and aries L.) on St Kilda. Parasitology, 105(3): T.A. Qureshi. 2008. Epidemiology of ovine 481-492. gastro- intestinal nematodes in Hyderabad Ibrahim, M.A., N. Nwude, R.A. Ogunsusi and Y.O. district, Pakistan. Pak. Vet. J., 28: 125-130. Aliu. 1984. Screening West African plants

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for anthelmintic activity. ILCA Bull., 17: Helminthol. Soc. Washington, 54: 233-236. 19-23. Lyons, E.T., S.C. Tolliver, J.H. Drudge and S. Iqbal, Z., M.N. Khan and A. Qudoos, 2002. Stamprer. 1992. Internal parasites in a Parasitic research on domesticated animals small lock of lambs and ewes during the of Pakistan. University Grants Commission, periparturient in 1987 in Kentucy. Trans. Islamabad-Pakistan. Ky. Acad., 53: 15-18. Iqbal, N.C., M.S. Durrani and A. Tariq. 1984. The MAFF. 1979. Parasitological laboratory techniques, incidence of gastrointestinal parasite in Technical Bulletin No. 18. Ministry of buffalo and cattle of Azad Kashmir. Pak. Agriculture Fisheries and Food Manual Vet. J., 4: 60-61. of Veterinary, Her Majestey’s Stationary Ijaz, M., M.S. Khan, M. Avais, K. Ashraf, M.M. Ofice, London. Ali and Saima. 2008. Infection rate and Maqsood, M., Z. Iqbal and A.H. Chaudhry. 1996. chemotherapy of various helminths in goats Prevalence and intensity of haemonchosis in and around Lahore. Pak. Vet. J., 28: 167- with reference to breed, sex and age of 170. sheep and goats. Pak. Vet. J., 16: 41-43. Kakar, M.N. and J.K. Kakarsulemankhel. 2008. Mckenna, P.B. 1981.The diagnostic value and Prevalence of endo (trematodes) and ecto- interpretation of faecal egg counts in sheep. parasites in cows and buffaloes of Quetta, New Zeal. Vet. J., 29: 129-132. Pakistan. Pak. Vet. J., 28: 34-36. Miller, J.E., M. Bahirathan, S.L. Lemarie, F.G. Komoin, Oka. C., J. Zinsstag, V.S. Pandey, F. Hembry, M.T. Kearney and S.R. Barras. Fofana and A.N. Depo. 1999. Epidemiology 1998. Epidemiology of gastrointestinal of parasites of sheep in the southern forest nematode parasitism in Suffolk and Gulf zone of Coted’Ivoire. Revue-d’Elevage- Coast Native sheep with special emphasis et-de-Medecine-Veterinaire-des-Pays- on relative susceptibility to Haemonchus Tropicaux, 52: 39-46. contortus infection. Vet. Parasitol., 74(1): Lebbie S.H.B., B. Rey and E.K. Irungu. 1994. 55-74. Small ruminant research and development Mirza, M.A. and A. Razzak. 1998. Internal in Africa, p. 1–5. In Proceedings of the parasitism in sheep and goats under Second Biennial Conference of the African extensive grazing system. Pak. Vet. J., Small Ruminant Research Network, ILCA. 18(1): 53-54. Liu, Y., F. Li, W. Liu, R.S. Dai, Y.M. Tan, D.S. He, Mohiuddin, A., M.M. Khan, F.A. Mughal and M.A. R.Q. Lin and X.Q. Zhu. 2009. Prevalence Sheikh. 1984. Taxonomy, incidence and of helminths in water buffaloes in Hunan seasonal variations of helminth parasites of province, China. Trop. Anim. Health Pro., sheep and goats of Sind. Pak J. Zool., 16: 41; 543-546. 25-30. Lyons, E.T., J.H. Drudge and S.C. Tolliver. 1987. Motahar, M.H. 2000. Examination of Epizootology of internal parasites in lambs gastrointestinal helminth in livestock and ewes during periparturient period grazing in grassland of Bangladesh. Korean in Kentucky in 1986. In Proceeding of J. Parasitol., 38: 187-190.

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Pal, R.A. and Qayyum. M. 1992. Breed, age and Schioppi, R. Condoleo and G. Cringoli. sex-wise distribution of gastro-intestinal 2007. Lice (Haematopinus tuberculatus) helminths of sheep and goats in and around in water buffalo farms from central Italy. Rawalpindi region. Pak. Vet. J., 12: 60-63. IJAS., 6: 926-927. Raza, M.A., S. Murtaza, H. Bachaya, A.A. Qayyum Waller, P.J. 1987. Anthelmintic resistance. Vet. and M.A. Zaman. 2010. Point prevalence Parasitol., 72: 391-412. of Toxocara vitulorum in large ruminants Wedderburn, J.F. 1970. Ostertagiasis in adult slaughtered at Multan abattoir. Pak. Vet. J., cattle: a clinical report of outbreak in the 30: 242-244. ield. New Zeal. Vet. J., 18: 168-170. Raza, M.A., Z. Iqbal, A. Jabbar and M. Yaseen. 2007. Point prevalence of gastrointestinal helminthiasis in ruminants in southern Punjab, Pakiatan. J. Helminthol., 81: 323- 328. Rinaldi, L., V. Musella, R. Condoleo, G. Saralli, V. Veneziano, G. Bruni, R.U. Condoleo and G. Cringoli. 2007. Giardia and Cryptosporidium in water buffaloes (Bubalus bubalis). Parasitol. Res., 100: 1113-1118. Roberts, J.A. and S.T. Fernando. 1990. The signiicance of the gastrointestinal parasites of Asian buffalo in Sri Lanka. Vet. Res. Commun., 14: 481-488. Soulsby, E.J.L. 1982. Helminths, Arthropods and Protozoa of Domestic Animals, 7th ed. Baillier Tindall and Cassel Ltd. London. Tan, L.X., X.H. Gong, R.F. Tan, Y.L. Ni, Wang and Z.M. Guo. 1996. The eficacy of an albendazole mediated blocks in controlling sheep nematodes in Xinjiange province. North West China. Vet. Res. Commun., 20: 427-435. Valcarcel, F. and C. Garcia Romero. 1999. Prevalence and seasonal pattern of caprine trichostrongyles in a dry area of central Spain. J. Vet. Med. B., 46(10): 673-681. Veneziano, V., M. Santaniello, S. Carbone, S. Pennacchio, M.E. Morgoglione, M.

48 Original Article Buffalo Bulletin (March 2016) Vol.35 No.1

HISTOLOGICAL STUDY ON STROMAL TISSUE IN MAMMARY GLAND AT LACTATING, INVOLUTION AND PREGNANT STAGE IN MURRAH BUFFALO

D. Chaurasia1,*, R.S. Dalvi2, S.B. Banubakode2, N.C. Nandeshwar2, R. Churchan2, S.P. Ingole1 and B. Sinha1

ABSTRACT INTRODUCTION

The present histological study was Mammary gland stromal tissue undergoes conducted on mammary gland tissue of sixty dramatical histological changes in the various Murrah buffaloes. The samples were categorized stage of lactation under the hormonal inluences. into three stages as lactating, nonlactating The ratio of glandular parenchyma to the stromal nonpregnant (involution stage) and nonlactating tissue is one of the important parameter for pregnant by ascertaining the stage of lactation, selection of cattle as a milch breed. That’s why dry period and pregnancy period. Stroma was histological study of the mammary gland is pre found to be comprised of interalveolar, interlobular requisite. The name “Black Gold” has emerged and interlobar connective tissue. The amount of as synonym for the one very popular breed of stromal tissue varied during different stages of buffaloes i.e. Murrah, which serves as capital lactation. In late pregnant and colostrum stage the reserve or cash crops to rural folk by producing interalveolar connective was minimum and alveoli economic stability, livelihood security and social were almost touching each other. The amount of status (Balbhadra, 2013). During the past one year, stromal tissue increased from colostrum stage a United States dairy irm had purchased Murrah to ten months of lactation. It was maximum in buffaloes, each yielding over 25 kg milk a day, nonlactating nonpregnant stage from one to two at a cost of Rs 2.5 lakh each from Haryana. This months (later stage of involution). Through stomal shows that rich countries will soon switch over to tissue blood and lymph vessels and nerve goes into Murrah husbandry (Sing, 2013). There is paucity of the parenchymal tissue. detail literature of histological study of mammary gland stromal tissue in various stage of lactation Keywords: mammary gland, stromal tissue, in buffalo. So keeping in view the importance of collagen ibers, Murrah buffalo Murrah buffalo in Indian economy, mammary

1Department of Veterinary Anatomy, College of Veterinary Science and Animal Husbandry, Chhattishgarh Kamdhenu Viswavidalaya, Anjora, Durg, Chhattishgarh, India, *E-mail: [email protected] 2Department of Veterinary Anatomy and Histology, Nagpur Veterinary College, Maharashtra Animal and Fishery Sciences University, Nagpur, Maharashtra, India

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gland as important accessory reproductive organ as ibers, elastic ibers and muscle ibers respectively well as scanty information, present experiment was as per the method of Bancroft and Cook (1994). proposed on the mammary gland of Murrah buffalo to study histological changes in stromal tissue of mammary gland. RESULTS AND DISCUSSION

During the present work, the stromal MATERIALS AND METHODS tissue was found to be comprised of interalveolar, interlobular and interlobar connective tissue The present histological study was (Figure 1). The amount of interalveolar connective conducted on mammary gland tissue of sixty tissue varied during different stages of lactation. Murrah buffaloes. The mammary gland samples of The interalveolar connective tissue was dense buffaloes were collected from dairy farms nearby and mainly composed of collagen ibers. Nagpur in Maharashtra and Durg, Rajnandgoan and Blood capillaries were predominantly seen in Raipur District of Chhattisgarh after their natural lactating stage in interalveolar connective tissue death. The samples were ensured for not having any (Figure 2). In colostrum stage, the interalveolar pathological lesions. The samples were categorized connective tissue was very scanty and alveolus into three stages as lactating, nonlactating was almost touching to each other (Figure 2). nonpregnant and nonlactating pregnant by the From colostrum stage onwards, the amount of stage of lactation, dry period and pregnancy period. interalveolar connective tissue was increased Lactating stage was further categorized in ive with the advancement of lactation upto ten month groups as: colostrum stage/ phase, three months of (Figure 2 and 3). Comparatively, more amount of lactation, ive months of lactation, seven months of interalveolar connective tissue was observed in lactation and ten months of lactation. Nonlactating between resting alveoli than active alveoli (Figure nonpregnant stage was categorized in two groups 2 and 4). In nonlactating nonpregnant animals, as: Upto one month and from one to two month. more amount of interalveolar tissue was found Nonlactating pregnant stage was categorized into (Figure 1). In the nonlactating pregnant stage, the three stage as early pregnant stage, mid pregnant interalveolar connective tissue was seen throughout stage and late pregnant stage. the pregnancy (Figure 5). Intalobular duct, blood Mammary tissue of 3-5 mm thickness was and lymph vessels and nerve were present in ixed in 10% neutral buffered formalin ixative interalveolar connective tissue (Figure 6). These for histological and histochemical studies. After inding were in agreement with the Trautmann and ixation tissue were dehydrated in alcohol, cleared Fiebtger (2002) and Riviere (2007) in domestic in benzene and embedded in parafin as per the animals, Sordillo and Nickerson (1988) and method of Drury and Wallington (1980). Three to Bragulla and Konig (2004) in cow, Sulochana et ive micron thick sections were cut and stained in al. (1989) in sheep, Parmar et al. (1985) in goat Haematoxylin and Eosin, Van Gieson’s, Gordon and Nosier (1973) in camel. The blood capillaries and Sweets, Orcein and Masson’s Trichrome stain in the interalveolar connective tissue observed in for histological structure, collagen ibers, reticular mammary gland during lactating stage could be

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Figure 1. Photomicrograph of mammary gland of nonlactating nonpregnant upto one month stage showing interalveolar connective tissue (IAC), interlobular connective tissue (ILC) and interlobar connective tissue (IBC). (Van Gieson’s X 100)

Figure 2. Photomicrograph of mammary gland of colostrum stage of lactation showing active alveoli (A) , interalveolar connective tissue (IAC) and blood capillaries (BC). (Haematoxylin and Eosin X 400)

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Figure 3. Photomicrograph of mammary gland of ten months of lactation showing collagen ibers (CF) in interalveolar connective tissue (IAC), and interlobular connective tissue (ILC). (Van Gieson’s X 100)

Figure 4. Photomicrograph of mammary gland of ten months of lactation showing resting alveoli (RA) and interalveolar connective tissue ( IAC). (Haematoxylin and Eosin X 400).

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Figure 5. Photomicrograph of mammary gland of nonlactating late pregnant stage showing collagen ibers (CF) in interalveolar connective tissue (IAC). (Van Gieson’s X 400)

Figure 6. Photomicrograph of mammary gland of colostrum stage of lactation showing interalveolar connective tissue (IAC), interlobular connective tissue (ILC) and interlobular duct (D). (Van Gieson’s X 100)

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Figure 7. Photomicrograph of mammary gland of ive months of lactation showing fat cells ( FC) in interlobular connective tissue (ILC). (Van Gieson’s X 100)

Figure 8 Photomicrograph of mammary gland of nonlactating nonpregnant one to two month stage showing elastic ibers (EF) in interlobular connective tissu e ( I L C ) a n d i n t e r l o b a r c o n n e c t i v e t i s s u e ( I B C ) . (Orcein X 100)

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Figure 9. Photomicrograph of mammary gland of nonlactating nonpregnant one to two month stage showing interlobar connective tissue (IBC). (Masson ‘s Trichrome X 100) attributed to the higher blood supply demanded by to ten months of lactation. Five to eight times the mammary tissue for the synthesis of milk. increased were noticed in the amount of interlobular Mammary parenchyma was divided into connective tissue from colostrum stage of lactation lobules by the bundles of thick dense connective to involution stage (Figure 1 and 6). Elastic ibers tissue ibers. These ibers bundles were interlobular were observed in interlobular connective tissue in connective tissue present in the form of septae. lactating and nonlactating stages in buffalo (Figure Interlobular artery and vein, lymph vessels, 8). The amount of elastic ibers was comparatively nerves and interlobular ducts were present in the more in the nonlactating nonpregnant stage. interlobular connective tissue. At places, few However, present study did not show reticular fat cells were seen in the interlobular connective ibers in the lactating and nonlactating stages in tissue. However, in some places, fat cells were buffalo. In early pregnant stage lobulations were found to predominate the other connective tissue not distinct. elements (Figure 7). This was in accordance to the In lactating and late pregnant stage the Chaurasia et al. (2012) they found fully developed lobes were not seen in tissue section because of stromal tissue formed chiely of the massive fat large size of alveoli and lobule. The interlobar pad in prenatal period. The most abundant ibers connective tissue was found between lobes were collagen ibers (Figure 1 and 3). The amount (Figure 1 and 9). The interlobar connective tissue of collagen ibers were increased apparently with collagen ibers were more compact and dense advancement of lactation from colostrum stage than interalveolar and interlobular connective.

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It was found that the amount of interlobar REFERENCES connective tissue was increased after weaning during involution stage. Approximately there was Balbhadra, S. 2013. Success stories CIRB achieved two to three fold increase in amount of interlobar signiicant reduction in mortality in Murrah connective from initial one months of involution Buffaloes. ICAR Central Institute for to later period of involution (from one to two Research on buffaloes. www.cirb.res.in/ months). These indings were in agreement with index. the indings of Bloom and Fawcett (1975). They Bancroft, J.D. and H.C. Cook. 1994. Manual opined that after few days of milk cessation, the of Histological Techniques and Their secretion that remains in alveoli and duct, get Diagnostic Application. Churchill absorbed and increases in the activity of lysosomal Livingstone, Edinburgh, London. enzyme leading to degeneration of epithelium. Bloom, W. and D.W. Fawcett. 1975. A Textbook This desquamation of epithelium gradually leads to of Histology, 10th Asian ed., W.B. Saunders collapse of alveoli and get associated with increase Co., Philadelphia, USA. in stromal tissue. The indings of the present study Bragulla, H. and H.E. Konig. 2004. Mammary relect on the active glandular dynamism in the gland, p. 595-603. In Konig, H.E. and terms of alteration in the glandular complex under H.G. Liebich (eds.) Veterinary Anatomy of the inluence of pituitary and gonadal hormones in Domestic Mammals-Textbook and Color tune with the physiological demands and status of Atlas, Stuttgurt, Germany. the animal health. Chaurasia, S., K.M. Panchal, Y.L Vyas and M.C. Desai. 2012. Organogenesis and histogenesis of mammary gland in female ACKNOWLDEGEMENTS goat (Capra hircus). Indian J. Vet. Anat., 24: 67-70. The author (Durga Chaurasia) thank the Drury, R.A.B. and E.A. Wallington. 1980. Former Vice Chancellor (Dr. Hazara) of Indira Carleton’s Histological Technique, 5th ed. Gandhi Krishi Vishwavidyala, Raipur, Dean, Dr. S. Oxford University Press, New York, USA. Jogi for granting me study leave to pursue Ph. D. Nosier, M.B. 1973. Histological structure of the programme from Maharashtra Animal and Fishry mammary glands of the one humped camel Science University. I am thankful to my Husband (Camelus dromedarius). Indian J. Anim. Dr. R. K. Chaurasia for procurement of sample. We Sci., 43: 639-641. thank Dean (Nagpur Veterinary College, Nagpur, Parmar, M.L., R.D. Sinha, G. Prasad and J. Prasad. MAFSU) Dr. B. P. Danndge for providing me all 1985. Histomorphology of lactating and facilities in Nagpur veterinary college to complete nonlactating mammary gland in goat. the research project. Indian J. Dairy Sci., 38: 203-207. Riviere, N.A.M. 2007. Integument, p. 320-349. In Eurell, J.A. and B.L. Frappier (eds.) Textbook of Veterinary Histology, 6th ed. 1st Indian reprint, Blackwell Publishing,

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Philadelphia, USA. Singh. S. 2013. Buffalo gives BMW a run for its money, sold for Rs. 25 lakh . Hindustan Times Rohtak. www.hindustantimes.com Sordillo, L.M. and S.C. Nickerson. 1988. Morphologic changes in the bovine mammary gland during involution and lactogenesis. Am. J. Vet. Res., 49: 1112- 1120. Sulochana, S., M. Hafeezuddin and U.B. Singh. 1989. Histological and histochemical studies of the mammary gland of the Indian goat (Capra hircus). Hy. Agri. Uni. J. Res., 11: 287-291. Trautmann, A. and J. Fiebtger. 2002. Fundamentals of the Histology of Domestic Animals, 1st ed. Indian reprint. Green world Publisher, Lucknow, U.P., India.

57 Buffalo Bulletin (March 2016) Vol.35 No.1 Original Article Buffalo Bulletin (March 2016) Vol.35 No.1

TEST-DAY GENETIC ANALYSIS OF MURRAH BUFFALO SIRES FOR MILK PRODUCTION

Vijay Kumar1 and A.K. Chakravarty2

ABSTRACT INTRODUCTION

The aim of the study was to investigate India is regarded as a treasure house of eficiency of test-day model (TDM) compared to world’s best buffalo germplasm. Buffalo is not lactation model (LM) for genetic evaluation of only a better source of milk but also provides meat Murrah buffalo bulls. Use of TDM instead of LM and works as a draught animal. Indian buffalo is of more interest in genetic evaluation because of contributes 17% of world milk production and 48% variability of lactation days in dairy animals. Data of Asian milk production (Food and Agriculture pertaining to irst lactation monthly test-day milk Organization, 2012). Among the various buffalo yield (FLMTDMY) and irst lactation 305-days breeds available in India, the Murrah buffalo is the or less milk yield (FL305DMY) of 1105 Murrah cynosure for dairy type. Murrah buffalo produces buffaloes during 1993 to 2010 were collected good quantity of milk and it is now well established and adjusted against signiicant environmental that it represents a unique breed in terms of feed inluences. It was found that test-day6 milk conversion ability with low grade feeds, ability yield (FLMTD6MY) had the highest genetic and to sustain under adverse climatic conditions, phenotypic correlation with FL305DMY. An resistance to diseases and production of high value attempt is being made in the present investigation milk containing a higher fat per cent. Keeping the to compare the estimated breeding values (EBVs) importance of buffalo in India, Network Project of Murrah bulls through contemporary comparison on Buffalo Improvement was initiated with the method for FL305DMY and FLMTD6MY. The objective to envisage and undertake progeny testing rank correlations between two traits were highly for improvement of buffalo breeds at various farms statistically signiicant indicating that FLMTD6MY in different parts of the country. equally effective to discriminate amongst sires. In India test bulls are evaluated based on their daughters irst lactation 305-days or less Keywords: Contemporary comparison, lactation milk yield without taking into account variation in model, least-squares, Murrah buffalo, test-day lactation days though the variation in lactation length model is relected in persistency. Genetic evaluation of

1Department of Animal Genetics and Breeding, U.P. Pt. Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan, Mathura, Uttar Pradesh, India, E-mail: [email protected] 2Dairy Cattle Breeding Division, National Dairy Research Institute, Karnal, India

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dairy bulls for milk production based on individual study. The records of the buffaloes with normal monthly test-day yields rather than 305-days or lactation were considered for this study. Data less milk yield has a number of beneits (Jamrozik of buffaloes with a minimum of 500 kg of milk and Schaeffer, 1997). Because of variability of production in at least 100 days of lactation, calving lactation days in dairy animals, the use of test-day and drying under normal physiological conditions models (TDM) instead of lactation model (LM) is were included in the analysis. The buffaloes of more interest in genetic evaluation. First lactation showing abortion, dystocia and other reproductive monthly test-day6 milk yield (FLMTD6MY) had disorders were not included in the study. To ensure the highest genetic and phenotypic correlation with the normal distribution of records, the outliers FL305DMY as obtained by Kumar et al. (2014). So were removed and data within the range of Mean FLMTD6MY of daughter was used in the present ± 3 standard deviation was only considered study. Information on test-day is lacking in Murrah for the study. Hence after standardization and buffaloes and hence, the present study was carried normalization, records of 832 Murrah buffaloes out. were retained for analysis.

Statistical analysis MATERIALS AND METHODS The data were adjusted for signiicant non- genetic factors for buffaloes calved in different Source of Data farms, years and seasons of calving using ixed In the present study, information were linear models. Since the data was non-orthogonal, collected from 7 sets of progeny testing under the least-squares technique suggested by Harvey Network Project on Murrah buffalo Improvement. (1990) was used to estimate the effect of non- In 7 sets of progeny testing 95 (11, 12, 15, 14, 15, genetic factors, and the means were compared 16, and 12) Murrah bulls were evaluated. Lactation using Duncan’s multiple range test (Kramer, 1957). records of 1105 Murrah buffaloes during 1993 to The model considered was as follows: 2010, were collected from the history-cum pedigree sheets and milk yield registers maintained at the Yijkl = m + Pi + Sj + Fk + eijkl National Dairy Research Institute (NDRI), Karnal; th th Central Institute for Research on Buffalo (CIRB), where, Yijkl is the l observation in K farm, Hisar; Guru Angad Dev Veterinary and Animal jth season and ith year of calving; μ the overall mean; th Sciences University (GADVASU), Ludhiana and Pi the ixed effect of i year of calving; Sj the ixed th Choudhary Charan Singh Haryana Agricultural effect of j season of calving; Fk the ixed effect th University (CCSHAU), Hisar. of the k farm; and eijkl, the random error~NID (0, σ2e). Information on Murrah buffalo After adjusting data for signiicant ixed Sires were evaluated on the basis of irst effects, EBVs of Murrah buffalo bulls were lactation 305-days or less milk yield (FL305DMY) estimated for FL305DMY and FLMTD6MY. In 7 and irst lactation monthly test-day 6 milk yield i.e. sets of progeny testing 95 (11, 12, 15, 14, 15, 16 and 155th day milk yield (FLMTD6MY) in the present 12) Murrah bulls were evaluated by contemporary

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comparison (CC) method (Sundaresan et al. 1965) evaluation were highly statistically signiicant as follow: in 5 sets of progeny testing indicating that both traits of sire evaluation were equally effective to where, discriminate amongst sires. I: is the sire index Use of test-day record is easier and H: is the herd average advance than lactation record as in this case only N: is the number of daughters of the sire one or two particular day (test-day) record is D: is the average performance of trait of required instead of taking all day records up to daughters’ of the sire 305-days of lactation. Test-day milk yields offered CD: is the average performance of trait of a better modeling opportunity and more accurate contemporary daughters in genetic evaluation. Also, test-day milk yields in farm should be taken into consideration for The Spearman’s rank correlation method selection of the buffalo for milk yield. The test- (Steel and Torrie, 1960) was used to judge the day models have been suggested as the method of effectiveness of test-day and lactation models choice for the analysis of milk yield traits in order of sire evaluation. To compare two models, to maximize the use of all available information. Spearman’s rank correlations were estimated using This method becomes even more important in ranks of bulls based on EBVs for FLMTD6MY and smaller herd size and without well-established FL305DMY. Two models were compared for the 7 milk recording schemes. In fact, the test-day model sets separately and signiicance of rank correlations appears to be a better alternate of 305-day lactation were tested. model because early selection on the basis of test- days could reduce generation interval. It could economize the genetic evaluation of dairy animals RESULTS AND DISCUSSION and improve accuracy of evaluation. Estimation of breeding value based on test-day milk yield The data were adjusted for signiicant non- would offer a solution to handle complex situation genetic factors. In the present study FLMTDMY like lack of necessary infrastructure for daily milk and FL305DMY were signiicantly affected by recording and hence cost of recording could be farm. EBVs of sires based on FL305DMY and reduced substantially. FLMTD6MY were estimated by CC methods and then sires were ranked subsequently (Table 1 and Table 2). Comparison of two models of sire REFERENCES evaluation was done by comparing the spearman’s rank correlations between ranks of sires based on Food and Agriculture Organization. 2012. Buffalo EBVs for FL305DMY and FLMTD6MY. The production and research. (www.fao.org. rank correlations between corresponding ranks com). (Table 3) based on FL305DMY and FLMTD6MY Harvey, W.R. 1990. Mixed model least squares’ and ranged from 0.566 (in set 2) to 0.882 (in set 1). maximum likelihood computer program, The rank correlations between two traits for sire PC-2 version, Ohio, USA.

61 Buffalo Bulletin (March 2016) Vol.35 No.1 11 1749 3 11 1706 4640 16 11 1555 8 1713 14 1796 1 11 1451 8 1666 9 1922 8 2184 4 11 1165 9 1446 13 1641 5 1836 4 2133 39303949 83966 13 4124 6 4188 1 7 4371 4393 2 1 4395 4523 4619 4 7 3 4637 13 11 1253 6 1153 1 1437 5 1573 12 1717 5 2121 9 3750 9 3924 12 4090 9 4245 10 4506 6 4915 2 11 392 4 93 8 993 10 1319 3 1485 13 1135 9 1419 8 896 3 759 3 1023 5 1341 10 1491 6 1153 1 1727 10 3117 6 1241 5 1131 3 1434 3098 1 761 4 1061 2 1360 4 1524 15 1667 12 1746 12 3125 3127 10 1290 3108 2 829 1 1084 14 1363 12 1536 3206 5 3551 2 1171 3294 7 3638 7 1315 7 1506 2 1749 14 1933 2 2331 5 3462 9 3689 10 1354 4 1538 14 1798 7 1944 10 2363 6 3567 8 3736 12 3865 15 4071 6 4244 3 2028 15 4807 7 Set 1 Rank Set 2 Rank Set 3 Rank Set 4 Rank Set 5 Rank Set 6 Rank Set 7 Rank Table 1. Rank of sires on the of estimated basis breeding values lactation for irst milk or less 305-days yield. Table

62 Buffalo Bulletin (March 2016) Vol.35 No.1 11 2184 6 ** 11 0.811 1717 2 2121 4640 15 ** 0.862 11 ** 4371 1 4523 6 ys or less milk yield and irst lactation monthly test day-6 milk yield. milk day-6 test monthly lactation irst and yield milk less or ys 11 0.478 0.828 1360 5 1524 13 1667 8 1746 12 * 11 1165 3 1446 8 1641 4 1836 5 2133 9 3930 4 4124 39493966 10 12 4188 3 4393 3 4395 4619 6 7 4637 10 0.566 0.578 11 ** Set 1 Set 2 Set 3 Set 4 Set 5 Set 6 Set 7 0.882 1253 3 1153 1 1437 7 1573 3750 10 3924 9 4090 10 4245 12 4506 3 4915 4 11 392896 4 5 93 759 7 6 993 1023 6 2 1319 1341 9 6 1485 1491 15 7 1135 1153 9 1 1419 1727 5 8 3117 6 1241 5 1131 7 1434 12 1555 5 1713 13 1796 1 3098 3 761 4 1061 3206 1 3551 2 1171 13 1451 4 1666 8 1922 3127 10 1290 3108 2 829 9 1084 15 1363 14 1536 10 1706 12 1749 3 329434623567 8 9 7 3638 3689 3736 1 8 12 1315 1354 3865 8 5 14 1506 1538 4071 1 13 2 1749 1798 4244 14 2 9 1933 1944 2028 4 14 16 2331 2363 4807 2 10 7 3125 Set 1 Rank Set 2 Rank Set 3 Rank Set 4 Rank Set 5 Rank Set 6 Rank Set 7 Rank * Signiicant at (P < 0.05) ** Signiicant at (P < 0.01). < 0.05) ** Signiicant at (P * Signiicant at (P Table 2. Rank on the of sires of estimated basis breeding values lactation for irst monthly test d ay-6 milk yield. Table Table 3. Spearman’s rank correlations for the ranks between irst lactation 305-da lactation irst between ranks the for correlations rank Spearman’s 3. Table

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Jamrozik, J. and L.R. Schaeffer. 1997. Estimates of genetic parameters for a test day model with random regressions for yield traits of irst lactation Holsteins. J. Dairy Sci., 8(4): 762-770. Kramer, C.Y. 1957. Extension of multiple range tests to group correlated adjusted means. Biometrics, 13: 13-18. Kumar, V., A.K. Chakravarty, C.S. Patil, J. Valsalan, R.K. Sharma and A. Mahajan. 2014. Genetic Studies on First Lactation Monthly Test Day and 305 Days Milk Yields in Murrah buffaloes. Indian Vet. J. (Accepted). Steel, R.G.D. and J.H. Torrie. 1960. Principles and Procedure of Statistics with Special Reference to the Biological Sciences. McGrew Hill Book Company, New York. 550p. Sundaresan, D., M. Gurnani and S.P Sindhu. 1965. Evaluation of breeding value of bulls used in two herds in India. J. Dairy Sci., 48(11): 1498-1505.

64 Original Article Buffalo Bulletin (March 2016) Vol.35 No.1

EVALUATION OF FRESH SEMEN QUALITY AND PREDICTING THE NUMBER OF FROZEN SEMEN DOSES IN JAFFRABADI BUFFALO BULL

S.N. Ghodasara*, P.U. Gajbhiye, A.R. Ahlawat and K.S. Murthy

ABSTRACT doses, semen parameter

Scientiic information on frozen semen characteristics of Jaffrabadi buffaloes (Bubalis INTRODUCTION bubalis) is scanty and centre to this research is to evaluate frozen semen characteristics in Jaffrabadi Jaffrabadi buffalo is one of the heaviest buffalo bulls. The study included six buffalo bulls buffalo breeds of world, inhabitant of Gir forest with average age 95.83±5.47 months and average area in Saurashtra region of Gujarat, India. These body weight of 834.16±47.63 kg maintained at buffaloes are known on their higher milk fat Cattle Breeding Farm, Junagadh Agricultural per cent (>8%) and larger fat globular size and University, Junagadh. A total of 206 ejaculates were hence the milk is preferred for Ghee and Khoa collected during the period of study. Ejaculates making (Thomas and Sastry, 2005). High quality collected from the six Jaffrabadi buffalo bulls Jaffrabadi frozen semen producing centers in the were clean, dense to very dense (D = 67.2%, DD = region are few and studies on semen characters 32.8%) and milky white (72.5%) to creamy (27.5%) and sexual behavior are scanty. There are many in colour. During this observatory study average agencies/organizations of government and non (Mean±SE) semen parameter like, ejaculatory government organizations working in the ield of volume, mass activity, sperm concentration, initial breed improvement in the Jaffrabadi buffaloes, but progressive sperm motility and total sperm number information need to be assessed on the reproductive per ejaculate were 5.11±0.17 ml, +3.43±0.04, performance, behavior and semen characteristics 838.30±25.74×106/ml, 79.41±0.60% and of Jaffrabadi bull. Changes in the environmental 4053.99±150.56×106 spermatozoa, respectively. condition inluence sperm output, accessory sex Average dilution rate in 0.5 ml medium straw gland secretion and epididymal function, all of (40 million sperm/straw) was found to be ideal which are relected in the ejaculate as volume, sperm at 10.48±0.32 ml. In a year expected number of numbers or sperm motility, morphology, viability ejaculates that could be frozen from the 6 bulls etc. (Koonjaenak et al., 2007). The knowledge of was 34.34±6.43 and correspondingly, the expected sexual behavior and semen evaluation are valuable number of frozen doses produced from bulls could tools to estimate the reproductive eficiency of a be 3546.46±540.30 numbers. breeding bull (Brohi, 1993).

Keywords: Jaffrabadi bulls, ejaculates, frozen

Cattle Breeding Farm, Junagadh Agricultural University, Junagadh, India, *E-mail: [email protected]

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MATERIALS AND METHODS the start of this study, including previous illnesses, mating behavior and libido. The data were compiled Six Jaffrabadi buffalo bulls of Cattle on a total of 206 ejaculates of 6 Jaffrabadi buffalo Breeding Farm, Junagadh Agricultural University, bull during the period from July 2011 to June 2012. Junagadh aged 95.83±5.47 months (mean±SE, Bulls were properly cleaned in perpetual ranges 75 to 108 months) with live weight of area with plenty of clean water and semen was 834.16±47.63 kg (mean±SE, ranges 730-1000 collected using male dummy without giving any kg) with typical breed characters (Figure 1 and 2) false mounting. Samples were collected early in formed the experimental material. Buffalo bulls the morning once a week, using artiicial vagina were kept in individual pens under a loose housing (AV) maintaining inner temperature 40-42oC. system on a concrete loor with the orientation of The temperature of semen processing room was its long axis in the east-west direction. The bulls maintained at 20-25oC during the whole study were fed green fodder such as maize, sorgum, hours. Immediately after collection, each sample sunlower and lucern according to the season and was transferred to laboratory and placed in a availability along with ad lib mature pasture grass water bath at 35oC. Sterilization of all items were hay. Concentrate component of ration comprised maintained before the day of collection and kept in of mixture (50:50) of commercial concentrate an incubator at 45oC. pellet (Amul power dan) and cotton seed cake Ejaculates were collected by AV technique at the rate of 4.5 kg per bull/per day along with by trained person (Figure 3). Two ejaculates mineral mixture powder at the rate of 40 g/bull/day. with a gap of 20 to 30 minutes were collected The bulls were drenched with 10 eggs and 500 ml (Figure 4). Immediately after collection ejaculate edible oil (cotton seed oil) 3 times a month. was evaluated for clarity/cleanliness (1 = clean, A clinical history of each bull was taken at 2 = dirty or contaminated), colour (1 = watery, 2 =

Figure 1. Jaffrabadi buffalo bull. Figure 2. Typical shield like head.

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Figure 3. Semen collection in Jaffrabadi buffalo bull. Figure 4. Thick creamy white semen.

Figure 5. Live and dead count of spermatozoa using eosin and nigrosin staining.

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milky, 3 = creamy), density (0 = thin, D = dense, Table 1. DD = very dense) and Volume (ml, graduated Colour of semen studied was actually the collection tube). Mass activity of spermatozoa was thickness of the semen together with pigment. The recorded by placing a drop of semen on a warm ejaculates collected from the six Jaffrabadi buffalo slide at 100 × magniication under a microscope bulls were clean, dense to very dense (D = 67.2%, with attached stage warmer (temperature set at DD = 32.8%) and milky white (72.5%) to creamy 37oC), camera and LCD screen (0 = no mass (27.5%) in colour (Figure 4). Javed et al. (2000) activity, +1 = slow waves, +2 = quick waves, +3 = reported milky-white caoloured semen in Nili-Ravi very quick waves, +4 = Waves, churning of whirls and in Swamp, buffalo bulls. and eddies) (Nath, 1988). Motility, as a percentage Ejaculate volumes from Jaffrabadi bulls of individually motile spermatozoa, was estimated ranged from 2-12 ml (5.11±0.17 ml), similarly, by examining a drop of diluted fresh semen (with nearly same ejaculatory volume were reported by buffer solution) under a microscope at 200×. Tomar et al. (1966); Shukla and Mishra (2005) Motility percentage was scored on the basis of the in Murrah bulls and Javed et al. (2000) in Nili- percentage of spermatozoa with normal forward Ravi bulls. However, scientists (Bhakat et al., progressive movement, while those showing 2011; Pant et al., 2003; Koonjaenak et al., 2007; circling movements or those oscillating at one Rehman et al., 2012) reported lower ejaculated place were regarded as immotile (Ahmad, 1994). volume as compared to Jaffrabadi in Murrah, Sperm concentration was assessed with Bovine swamp and Kundhi buffalo breeds, respectively. Accucell photometer, (IMV) by diluting 1:100 Differ in the semen volume in various breeds of time neat semen in to Sodium chloride solution buffaloes might be due to differences in genetics, 0.9% W/V. Live and Dead spermatozoa count has reproductive health status of bulls, age of bulls, been carried out using Eosin and Nigrosin staining frequency of collection, pooled volume, nutrition, technique (Figure 5) (Campbell, 1956). season and management (Nazir, 1988; Soderquist, 1992). Variations can also be due to skill of semen collector/attendant and temperature of AV. RESULT AND DISCUSSION Mass activity of experimental Jaffrabadi bulls (range from +2.5 to +4 with a mean of +3.43±0.04) is similar to earlier reports of mass Age of the bulls at the beginning of activity reported by various researcher (Ram, 1988; collection during the study period ranging from 75 Dhami, 1992; Shukla and Mishra, 2005) in Murrah months to 108 months, with a mean of 95.83±5.47 bulls and Rehman et al. (2012) in Kundhi buffalo. months. A total of 217 ejaculates were collected Sperm concentration of six Jaffrabadi during the period of study. The distribution of bulls were ranged from 425 to 2012×106/ml with collections per bull is shown in Table 1. Eleven mean of 838.30±25.74×106/ml. Present indings ejaculates were considered very thin (watery) or are in agreement with the indings of Ram (1988), dirty and were therefore discarded from semen whereas higher concentration of spermatozoa/ml processing and freezing. Semen characteristics of of neat semen were recorded by several workers the remaining 206 ejaculates are summarized in (Prajapati et al., 2000; Pratap et al., 1999; Rehman,

68 Buffalo Bulletin (March 2016) Vol.35 No.1 84.52+1.32 85.94±1.44 85.38±0.42 r of doses frozen/year/bull from Jaffrabadi bulls. frozen/year/bullr of doses from Jaffrabadi 87.09±1.09 1 1 1 1 1 1 - 4535.8 4449.83 4742.4 2538.69 3183.6 1828.46 3546.46±540.30 Laxman Bhagro Moti Nagraj Sundar Raja Average 4.59±0.27 6.89±0.51 4.94±0.24 4.82±0.27 4.20±0.39 4.17±0.36 5.11±0.17 9.15±0.36 7.02±0.52 12±0.56 11.45±0.98 15.16±1.21 12.18±1.07 10.48±0.32 79.43±0.98 75.65±1.92 81.00±1.15 80.43±1.38 83.00±1.10 79.17±1.52 79.41±0.60 57.13±0.86 57.39±0.94 59.26±1.08 59.57±1.11 60.60±0.99 61.94±0.94 58.71±0.42 732.00±29.16 561.37±41.44 960.38±45.02 916.17±78.22 1212.76±96.86 974.00±85.33 838.30±25.74 3348.18±227.45 3314.45±314.13 4817.27±345.80 4394.16±486.14 5025.76±535.66 4069.78±477.79 4053.99±150.56 of Bulls /ml) ) 6 6 Name or Number Number or Name Density (Score: 0-DD) activityMass Initial progressive D-DDsperm motility (%) Sperm concentration 3.39±0.08(10 D-DD 3.30±0.09 D-DD 3.53±0.08 3.48±0.14 D-DD 3.48±0.10 D-DD 3.50±0.12 D-DD 3.43±0.04 - Cleanliness of semen (Score: 1-2) Colour (Score: 1-3) 2-3 2-3 2-3 2-3 2-3 2-3 - Age of bullsAge (Months) of ejaculatesNo. of semen/ Volume 102ejaculate 54 96 46 75 40 108 23 89 25 105 95.83±5.47 18 34.34±6.43 Total sperm number Total (10 % of Live sperm 85.17±0.76 84.76±1.02 85.70±0.99 Average dilution rate/ Average ml of semen ThawPost progressive motility Calculated no. of ejaculate frozen(dose)/ year/bull Table 1. Mean (±SE) of semen parameter/characteristics, dilution rate/ml of semen and Numbe Table

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et al., 2012; Shukla and Mishra, 2005). During the in Nili-Ravi buffalo bulls and Roy (2006) produced study initial progressive sperm motility ranged 5,147.48 doses/year/bull in Murrah bulls which from 65 to 95%, with mean of 79.41±0.60 and is was higher than the estimate for Jaffrabadi bulls in in agreement with the indings of Koonjaenaket al. the present study. (2007) in swamp buffalo. Sahu and Pandit (1997) In vitro semen evaluation parameters and Shukla and Mishra (2005) recorded higher used in the present study are used to determine initial progressive motility in Murrah bulls. Lower fresh semen motility in post-thaw samples. percentage of initial motility than the present Some research workers established a correlation indings in Murrah bulls was also reported bybetween motility and ield fertility; others did Bhakat et al. (2011) and Kumar et al. (1993). The not (Christensen et al., 1999; Tardif et al., 1999). post thaw motility ranged from 45% to 65% with Variations in semen quality parameters recorded in average mean of 58.71±0.42 during the entire study the present investigation, were well supported by period. Percentage of live and dead spermatozoa earlier reports, may be due to individual variations of all the Jaffrabadi bulls was ranged from 75 (Saxena and Tripathi, 1978), ejaculate frequency to 96 percent live spermatozoa with average of (Nath, 1988), differences in age (Bhat et al., 2002), 85.38±0.42 percent live spermatozoa. genetic makeup of the bulls (Tomar et al., 1966), Total sperm number per ejaculate ranged season of study (Tuli, 1984) and agro climatic from 2400 to 10136 million sperm with mean of conditions. 4053.99±150.56×106 spermatozoa. Mean dilution Present study reveled that semen characters rate was found to be 10.48±0.32, with a range of of Jaffrabadi bulls are comparable to Murrah and 4.5 to 25.83 ml. Expected number of ejaculates that there characteristics can be made use of to meet could be frozen from the 6 bulls was 34.34±6.43 the high demand for semen from selected Bulls of (ranging from 18 to 54) and correspondingly, high genetic merit. Harvesting maximum semen expected frozen doses produced from bulls could doses per bull was the other approach to increasing be 3546.46±540.30 (ranged 1828.46 to 4742.4). the number of inseminations possible per bull. Out of six bulls, ejaculates were collected from Information on expected semen doses per bull will three bulls for entire year that produced 4576±67.13 help in planning the functioning of A. I. center at frozen doses/year. Two bulls were used for semen ield level as per the capacity of the semen station collection for only 8 months and one bull was used running at Cattle Breeding Farm. for 5 months. Each semen doses were of 0.5 ml with 40 million sperm concentration per straw. Bhakat et al. (2011) revealed average total sperm output REFERENCE of Murrah buffalo bull was 2,561.05±77.80×106. Average dilution rate was found to be 12.49±0.13. Ahmad, N. 1994. Clinical and experimental Expected number of ejaculates that could be frozen studies of reproductive functions in the ram per year per bull was 53.27 and correspondingly, and male goat with special reference to the the expected frozen doses produced per year per use of diagnostic ultrasound. Ph.D Thesis, bull could be 6,879.49. Zafar et al. (1988) reported Department of Large Animal Med. and yearly production to be 8,412 semen doses per bull Surgery, Royal Vet. College, University of

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London. London. Kumar, S., K.L. Sahni and G.S. Bisht. 1993. Bhakat, M., T.K. Mohanty, V.S. Raina, A.K. Correlation studies among various seminal Gupta and H.M. Khan. 2011. frozen semen characteristics of motile and static ejaculates production performance of murrah buffalo of Murrah Bulls. Indian J. Dairy Sci., 46: bulls. Buffalo Bull., 30: 157-162. 223-226. Bhat, V., T.G. Honnappa, B.M. Dubey and M. Nath, R. 1988. Cryopreservation of buffalo Devraj. 2002. Impact of age on frozen semen semen. M.V.Sc. Thesis, GB University of production potential in Murrah buffalo Agricultural and Technology, Pantnagar, bulls. In Proceedings of 9th International India. Congress on Biotechnology in Animal Nazir, M. 1988. Semen evaluation and Reproduction, Chennai, India. sperm morphology - monography on Brohi, N.A. 1993. Sexual behaviour and semen reproductive pattern of Riverine buffaloes characteristics of Kundhi buffalo bulls. and recommendations to improve their 1993. M.Sc. Thesis, Department of Anim. reproductive performance at small farmer Repro. Sindh Agriculture University level. PARC, Islamabad. Tandojam. Pant, H.C., R.K. Sharma, S.H. Patel, H.R. Shukla, Campbell, R.C., H.M. Dott and T.D. Glover. 1956. A.K. Mittal and R. Kasiraj. 2003. Testicular Nigrosin eosin as stain for differentiating development and its relationship to semen live and dead spermatozoa. J. Agr. Sci., 48: production in Murrah buffalo bulls. 1-8. Theriogenology, 60: 27-34. Christensen, P., P.B. Brockhoff and H. Lehn- Prajapati, K.B., P.K. Nagpaul, M.K. Chauhan, Jensen. 1999. The relationship between M.M. Kale and V.S. Raina. 2000. The effect semen quality and the non-return rate of of exercise on seminal attributes of Mehsana bulls. Reprod. Domest. Anim., 3: 503-507. buffalo bulls in different seasons. Indian J. Dhami, A.J. 1992. Comparative evaluation of Anim. Reprod., 21: 38-40. certain processing procedures in deep Pratap, N., V.N. Reddy, P.A. Sharma, T.G. freezing of cattle and buffalo semen under Honnappa, M. Devraj, A. Krishnaswamy tropical climate. Ph.D. Thesis, IVRI and V.K. Arora. 1999. Spermiogram and Deemed University, Izatnagar, India. biochemical studies in Murrah buffalo bulls. Javed, M.T., A. Khan and R. Kausar. 2000. Effect of Indian J. Anim. Reprod., 20: 156-158. age and season on some semen parameters Ram, S. 1988. Cryopreservation of semen of of Nili-Ravi buffalo (Bubalus bubalis) Murrah, exotic and crossbred bulls. M.V. Sc. bulls. Vet. Arhiv, 70: 83-94. Thesis, GB Pant University of Agricultural Koonjaenak, S., V. Chanatinart, S. Aiumlamai, and Technology, Pantnagar, India. T. Pinyopumimintr and H. Rodriguez- Rehman, Z.U., M.U. Samo, T.A. Qureshi, S. Khan, Martinez. 2007. Seasonal variation in semen M.S. Qureshi, F.A. Khan and S. Bahadadr. quality of swamp buffalo bulls (Bubalus 2012. Studies on the freezability of kundhi bubalis) in Thailand. Asian J. Andro., 9: 92- buffalo bull semen. J. Anim. Plant Sci., 22: 101. 18-23.

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Roy, B. 2006. Inluence of zinc on semen quality of seasonal variation on semen production and sexual behaviour of crossbred and of Nili-Ravi buffalo bulls. Buffalo J., 4: 61- murrah buffalo bulls. Ph. D. Thesis, NDRI 68. Deemed University, Karnal, India. Sahu, S.B. and R.K. Pandit. 1997. Grading of breeding Murrah bulls on sexual behavior and seminal characteristics including cryopreservation. Indian Vet. J., 74: 557- 569. Saxena, V.B. and S.S. Tripathi. 1978. Studies on the physico-chemical attributes and freezability of semen of crossbred bulls. Indian J. Anim. Sci., 48: 865-69. Shukla, M.S. and A.K. Mishra. 2005. Correlation between seminal characteristics in Murrah bulls. Indian J. Anim. Sci., 75: 263-266. Soderquist, L., L. Janson, M. Haard and S. Einarsson. 1992. Factors affecting the variation in sperm morphological abnormalities in Swedish dairy A.I. bulls. In Proceedings of 12th International Congress, The Hague, Netherlands. Tardif, S., J.P. Laforest, N. Cormier and J.L. Bailey. 1999. The importance of porcine sperm parameters on fertility in vivo. Theriogenology, 52: 447-459. Thomas, C.K. and N.S.R. Sastry. 2005. Livestock Production Management, 4th ed. Kalyani Publisher. 143p Tomar, N.S.,B.S. Mishra and C.B. Johari. 1966. Seasonal variations in reaction time and semen production, and prediction of some semen attributes on initial motility of spermatozoa in Hariana and Murrah bulls. Indian J. Dairy Sci., 19: 87-93. Tuli, R.K. 1984. Seasonal variation in seminal characteristics of Murrah buffalo bulls. Livestock Advisor, 9: 37-40. Zafar, A.H., N. Ahmed and S.K. Shah. 1988. Effect

72 Original Article Buffalo Bulletin (March 2016) Vol.35 No.1

SERUM CONCENTRATION OF CALCIUM, INORGANIC PHOSPHORUS AND MAGNESIUM IN CYCLIC, NON CYCLIC AND REPEAT BREEDER BUFFALOES

Qaisar Shahzad1,*, Muhammad Imran1, Hamayun Khan3, Armughan Ahmed Wadood2, Moazam Ali Khan1, Muhammad Binyameen1 and Amir Ahmad Niazi1

ABSTRACT INTRODUCTION

Nutritional deiciency including mineral Reproductive eficiency is the primary deiciency may decrease reproductive eficiency in factor affecting productivity of a dairy buffalo buffaloes. Therefore, aim of the present study was to and is greatly inluenced by late attainment of assess effect of minerals Calcium (Ca) Magnesium puberty, seasonal breeding, long calving intervals, (Mg) and inorganic phosphorus (Pi) on cyclicity increased number of services per conception, of Nili Ravi Buffaloes. The present experiment increased days open, uterine infections and was performed at Livestock Experiment Station, various obstetrical problems (Samad et al., 1987). Bhunikey. The female buffaloes (n=90) were Anoestrus and repeat breeding are the biggest divided into 3 groups; cyclic (n=30), non-cyclic factor decreasing reproductive eficiency. Among (n=30) and repeat breeders (n=30). Mineral proile the various factors that cause anoestrus and repeat was measured through serum of the buffaloes under breeding, major one is under-nutrition (Francos et study. Calcium and Magnesium were measured al., 1977; Bhaskaran and Patil, 1982). The minerals through calorimetric method while inorganic play vital role in development of reproductive phosphorus was measured through UV method. potential and maintenance of functional integrity The level of calcium was signiicantly (P<0.05) of the reproductive system in domestic animals higher in normal cyclic buffaloes than that of (Leathem, 1966). The calcium (Ca), Phosphorus, the non-cyclic and repeat breeders. The level of (P) and Magnesium (Mg) are important minerals magnesium was non-signiicant in normal cyclic in this respect. The exact mechanism by which and non-cyclic buffaloes, while it was signiicantly mineral deiciency reduces fertility is not clear (P<0.05) higher in repeat breeders as compared to (Luca et al., 1977). Calcium, inorganic phosphorus cyclic and non-cyclic buffaloes. Phosphorus was and magnesium may lead to reproductive failure not in balance in non-cyclic and repeat breeders. (Hidiroglo, 1979). The ovarian activities are most It is concluded from the present study that Ca: P prone to minerals imbalances and their deiciency should be 2:1. suppresses ovarian activity (Haq et al., 1999). Therefore, this study was conducted to assess Keywords: repeat breeder, non-cyclic, calcium, the effect of minerals (Calcium, Phosphorus, and inorganic phosphorus, magnesium Magnesium) on anoestrus and repeat breeding in

1Buffalo Research Institute, Pattoki, District Kasur, Pakistan, *E-mail: raoqaisarshahz [email protected] 2University of Veterinary and Animal Sciences, Lahore, Pakistan 3Department of Animal Health, University of Agriculture, Peshawar, Pakistan

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buffaloes. inorganic phosphorus will be computed as following.

MATERIALS AND METHODS Calcium Concentration (mg/dl) = Absorbance of sample x 9.82 Absorbance of standard Nili-Ravi buffaloes (n=90) of 3-10 years of age having similar BCS (Body Condition Score) were selected from Livestock Experiment Station, Magnesium Bhunikey. Concentration (mg/dl) = Absorbance of sample x 2.31 Absorbance of standard Buffaloes were grouped into three categories; Cyclic (n=30), Non Cyclic (n=30) and Repeat Breeder (n=30). Buffaloes having smooth Inorganic phosphorus and inactive ovaries in 10 days interval of rectal Concentration (mg/dl) = Absorbance of sample x 5.18 Absorbance of standard palpation were called as non cyclic. Buffaloes having no abnormality on palpation and not being pregnant after artiicial inseminations in three Statistical Analysis consecutive estrus period were called as repeat The data thus collected was analyzed by breeder. Analysis of Variance (ANOVA) by using SPSS Blood samples were collected from the version 13 (Steel and Torrie, 1982). jugular vein of the animal aseptically. The samples were kept at room temperature for 24 h for serum separation. The serum were collected and stored at RESULTS AND DISCUSSIONS -20ºC for further studies. Mean ± S.E values of calcium, magnesium, Analytical procedure and phosphorus among different Buffalo group at i) Diagnostic kits: Diagnostic kits bearing cat. LES Bhunikey, Pattoki shown in Table.1. No. Ca 590, Mag 570 and PH. 1016 (Randox Normal cyclic buffaloes had signiicantly International Lab. Ltd, UK) were used (P<0.05) higher calcium than non cyclic and repeat and standard procedures were applied for breeder buffaloes. The results of the present study estimating concentrations of serum calcium, are in line with Pasha et al. (2012) who reported magnesium and inorganic phosphorus. similar (9-11 mg/dl) calcium levels in the serum of ii) Analyses of samples: Colorimetric method buffaloes in Punjab. Results are not in coincidence was used for the estimation of serum calcium with Husnain et al. (1981), who reported the (Sarkar, 1967) and magnesium and UV calcium level in serum of milking buffaloes were method was applied for inorganic phosphorus slightly lower (6.70- 8.00 mg/dl). These results are concentration (Teiz, 1983). All these analyses not in line with the study of Hedaoo et al. (2008) were performed through spectronic-21. who reported that there is no difference of calcium iii) Computation of concentration: between normal cycling and anoestrus buffaloes. Concentration of calcium, magnesium and Normal cyclic buffaloes had similar levels 74 Buffalo Bulletin (March 2016) Vol.35 No.1

of magnesium to that of non cyclic, while repeat proile and soil mineral proile should be measured breeder buffaloes had higher levels of magnesium to sort out exact problem of the nutritional as compared to both cyclic and non cyclic buffaloes. deiciency. Results of the present study are in line with Hedaoo et al. (2008) who reported that magnesium has no effect on cyclicity of the buffaloes. Magnesium REFERENCES values in buffaloes blood were slightly higher than Pasha et al. (2012) who reported 2.68 mg/dl Haq, I.A., Z. Rehman and M.S. Butt. 1999. Role of in Punjab, Iqbal. (1990), who reported 2.57-2.58 minerals in animal’s reproduction, p. 51-54. mg/dl magnesium in blood serum of cattle and In PASF/PVMA Agro-Livestock Conference. Hussain (1991) who reported 1.75-280 mg/dl in Bhaskaran, R. and R.V. Patil. 1982. Effects of blood serum of cattle. Results are similar to Oba precalving feed level on birth weight, and Ramos (1988), who reported 3.84±1 mg/dl calving dificulty and subsequent fertility.J. magnesium in serum of cattle. Anim. Sci., 46: 1522-1528. Normal cyclic buffaloes had low levels of Francos, G., N. Davidson and E. Mayer. 1977. inorganic phosphorus than that of non cyclic and Inluence of some nutritional factors on repeat breeder buffaloes. Calcium and phosphorus incidence of repeat breeder syndrome in should be 2:1 in mammals. In the present in high producing dairy herds. Theriogenology, anoestrus and repeat breeder buffaloes, this ratio 7: 105-111. was high. Results in the present study were in line Hedaoo, K.M., K.P. Khllare, M.D. Meshram, with Hignett. (1959) who reported that phosphorus S.K. Sahatpure and M.G. Patil. 2008. higher than 2:1 may result in infertility. Comparative studies of certain Bio- It’s concluded from the above trial that chemical constitutents of normal cyclic and calcium and phosphorus imbalance may result in anoestrus surti buffaloes. Veterinary World, infertility while magnesium may have no effect on 1(4): 105-106. fertility. It’s further suggested that further studies Hidiroglou, M. 1979. Trace element deiciencies comprising blood mineral proile, fodder mineral and fertility in ruminants, A Review. J.

Table 1. Mean ± S.E values of calcium, magnesium, and inorganic phosphorus among different buffalo groups. Calcium (mg/dl) Magnesium (mg/dl) Inorganic Phosphorus Groups Mean ± SE Mean ± SE (mg/dl) Mean ± SE Non cyclic 10.55±0.43a 4.76±0.22a 5.39±0.28a Repeat breeder 10.01±0.42a 5.36 ±0.38b 5.55±0.32a Cyclic 11.77±0.37b 4.98±0.38a 4.64±0.33b

Mean values with in the same column bearing different superscripts differ signiicantly (P<0.05) among groups.

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Dairy Sci., 62: 1195-1206. Biochem., 20: 155-166. Hignett, S.L. 1959. Some nutritional and other Steel, R.D.G. and J.H. Torrie. 1982. Principals interacting factors which may inluence the and procedures of Statistics. Mc-Graw, Hil fertility of cattle. Vet. Rec., 71: 247. Koga Kusha Ltd. Book Co. Inc. New York, Husnain, Z.U., C.S. Ali, K.M. Ahmad and H.A. USA. Samad. 1981. Studies on the relationship Teitz, N.W. 1983. Clinical Guide to Laboratory between blood mineral level and fertility of Tests. W.B.Saunder Co., USA. buffaloes. Pak. Vet. J., 1: 141-144. Hussain, G. 1991. Mineral imbalances in dairy cattle of Pakistan. M.Sc. Thesis, Department of Animal Nutrition, College of Veterinary Sciences, Lahore, Pakistan. Iqbal, T. 1990. Mineral imbalances in buffaloes of Punjab. M.Sc. Thesis, Department of Animal Nutrition, College of Veterinary Sciences, Lahore, Pakistan. Leathem, J.H. 1966. Nutritional effect on hormone production. J. Anim. Sci., 25: 68-82. Luca, L.J. De., R.H. Silve, R.J. Grimoldi and E.G. Capaul. 1977. Fertility in cattle and the practical application of some bolld values, p. 972-974. In Proceedings of 20th World Veterinary Congress. Thessaloniki, Greece. Oba, E. and A.A Ramos. 1988. Qualitative and quantitative aspects of the crossbred Murrah buffalo blood in feedlot during the service period, p. 62. In Proceedings of 2nd World Buffalo Congress, Delhi, India Pasha, N.T., Z.M. Khan, U. Farooq, A.Y. Ditta, M. Ilyas and H. Ahmad. 2012. Macro-minerals status of buffaloes in rice zone of Punjab province. JAPS., 22(3): 319-323. Samad, H.A., C.S. Ali, N.U. Rehman, A. Ahmad and N. Ahmad. 1987. Clinical incidence of reproductive disorders in buffalo. Pak. Vet J., 7(1): 16-19. Sarkar, B.C.R. and U.S.P. Chauhan. 1967. A new method for determining micro quantities of calcium in biological materials. Anal.

76 Original Article Buffalo Bulletin (March 2016) Vol.35 No.1

DIAGNOSIS OF DERMATOPHILUS DERMATITIS AMONG BUFFALOES IN KERALA

P.V. Tresamol, M.R. Saseendranath and K. Vinodkumar

ABSTRACT INTRODUCTION

The present study reports prevalence of Dermatophilosis is an exudative, pustular dermatophilosis due to Dermatophilus congolensis dermatitis that affects domestic, aquatic and among buffaloes in Kerala. Five buffaloes presented wild animals and man, caused by Dermatophilus with skin lesions primarily on the lower limbs, udder congolensis. It is an economically important disease and tail were subjected to detailed investigations which causes considerable loss in terms of skin to identify the etiological factors. Skin swabs damage, reduced meat and milk production, culling and scabs were collected from the lesions under or death of affected animals and costs of control sterile conditions and were subjected to direct and treatment (Zaria, 1993). It has been reported microscopical and cultural examinations. Direct by the Food and Agricultural organization (FAO) microscopical examination of Giemsa and Gram’s to be one of the four major bacterial diseases which stained smears of scabs revealed typical tram track affect cattle and other animals in the tropical and appearance of Dermatophilus congolensis. Culture subtropical regions (Hashemi Tabar et al., 2004). of skin scabs in sheep blood agar yielded typical Diagnosis of the condition is by demonstration of greyish beta haemolytic adherent colonies. The typical tram track appearance of the organism in isolates were further conirmed by morphological stained skin scabs and conirmation by isolation appearance and biochemical reactions. Direct and identiication of organisms. This disease is microscopical examination of skin scrapings of worldwide occurrence but more prevalent in yielded negative results for fungal elements tropical and subtropical countries. The disease is and mites. This forms the preliminary report of a chronic dermatitis and could occur in any part dermatophilosis among buffaloes in Kerala. of the body and occasionally become generalised. Accurate diagnosis and early treatment are found Keywords: Dermatophilus congolensis, to be useful for better clinical recovery from the prevalence, buffaloes, Kerala condition. There are few reports of Dermatophilosis among buffaloes from India (Pal, 1995; Sharma et al., 1992). The present study forms the irst report of dermatophilosis from buffaloes in Kerala.

Department of Veterinary Epidemiology and Preventive Medicine, College of Veterinary and Animal Sciences, Mannuthy Kerala Veterinary and Animal Sciences University, Kerala, India

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MATERIALS AND METHODS method and the preliminary tests were done based on it. The morphological, cultural, biochemical Five Murrah buffaloes presented with and sugar fermentation tests of the isolates were dermatological problems during 2011 were included determined as per the methods described by Cowan in the study. Detailed clinical examination of these (1974). animals was carried out and type of lesions was recorded. Skin scabs and scrapings and impression smears from lesions were collected under sterile RESULTS AND DISCUSSIONS conditions for laboratory examination. Small pieces of skin were taken from the underside of the Detailed clinical examination of animals scabs and softened in few drops of distilled water revealed characteristic exudative dermatitis on a clean microscopic slide; a smear was made lesions with formation of scabs, crusts and issures and stained with Giemsa and Gram’s stains (Quinn with matted hair at their bases, suggestive of et al., 1994). The impression smears taken from the dermatophilosis (Figure 1). Similar types of lesions lesions were also stained with Giemsa’s stain and were described by most of the workers irrespective Gram’s stain and examined under the oil immersion of the species of the animals affected (Koney, objective of microscope. The skin scrapings were 1996; Gitao et al., 1998; Wabacha et al., 2007). All also subjected to direct microscopical examination the animals had lesions on the lower limbs, three using 10 percent potassium hydroxide to rule out had lesions on the udder (Figure 2) and two had fungal elements and mites. lesions on the tail (Figure 3). One of the animals Isolation of D. congolensis was carried had severe generalised lesions involving all these out using Haalstra’s technique (Haalstra, 1965). areas (Figure 4). Skin scabs were minced with a sterile scalpel Microscopical examination of Giemsa or blade and placed in glass bottles. One millilitre Gram’s stained smears of the scab material from the of sterile water was added to each specimen. The lesions revealed characteristic Gram positive septate bottles were allowed to stand open for three and a branching ilaments which were longitudinally as half hours at room temperature. Then the opened well as transversely divided to form spherical or bottle was transferred to candle jar, with a candle ovoid cocci in multiple rows, with typical ‘tram- was burned within the jar to obtain 10 to 20 percent track appearance’ suggestive of D. congolensis in carbon dioxide tension. Under CO2 tension the all samples (Figure 5). This distinctive morphology motile zoospores if present, were chemotactically of the organism was demonstrated by most of the attracted to the surface of the distilled water. After workers as the most practical diagnostic method for 15 minutes, the bottle was carefully removed and dermatophilosis (Abu-Samra, 1978; Quinn et al., a loopful taken from the water surface was seeded 1994). The organisms were observed in different on blood agar plates and incubated at 37oC in 20 forms depending on the stage of development percent carbon dioxide for 24 to 48 h. The plates varying from long branching ilaments, ilaments were examined for colonies of D. congolensis packed with zoospores and mature free zoospores (Quinn et al., 1994). released from the ilaments. Kaminski and Suter The isolates were stained by Gram’s (1976) and Hyslop (1980) described pleomorphic

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Figure 1. Lesions with thick scabs and issures on Figure 2. Lesions on the udder. limb.

Figure 3. Lesions on the tail. Figure 4. Severe generalised dermatophilus dermatitis.

Figure 5. Branching ilaments of D. congolensis in scabs(Gram’s stainx1000).

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Figure 6. Greyish white haemolytic colonies of Figure 7. Filaments and zoospores of D. congolensis D. congolensis in sheep blood agar. from culture (Gram’s stainx1000). nature of D. congolensis in stained smears of scabs reported for D. congolensis by several workers and stated that the organism might be seen in any (Pal, 1995; Mannan et al., 2009). form of the various stages of its lifecycle. All the ive isolates produced acid from Culture of the scab materials from all ive glucose, fructose and sucrose within 24 h of animals yielded typical beta haemolytic colonies of incubation. But variable results were obtained D. congolensis in sheep blood agar in presence of with maltose, mannitol and lactose. The isolates 10 percent carbon dioxide (Figure 6). There were were unable to produce acid from sorbitol and variations in the shape, colour and texture of the xylose. This is in agreement with previous indings colonies. Similar observations were also made by (Mannan et al., 2009; Shaibu et al., 2011). None Gordon (1964) and EL-Nageh (1971). of the isolates produced gas from the sugars. No Microscopical appearance of organisms fungal elements or mites could be detected on in Gram stained smears from colonies were also microscopical examination of skin scrapings using highly variable with Gram positive branching 10 percent potassium hydroxide. ilaments in different stages of segmentation, The results of the present study conirmed packets of coccoid forms, germinating spores or occurrence of Dermatophilosis among buffaloes combinations of the above forms depending on the in Kerala. The presence of predisposing factors age of the culture and strain of the isolate (Figure such as prolonged wetting, high humidity, high 7). The wet mount preparation of all the isolates temperature and various ectoparasites might have revealed motile zoospores. All the isolates were predisposed to the occurrence of the condition. haemolytic producing clear zones of haemolysis in An early and prompt diagnosis and treatment of seven percent sheep blood agar within a period of the condition has to be undertaken to reduce the 24 to 72 h of incubation. The isolates were positive economic loss to farmers. for catalase, oxidase and urease tests and were able to digest gelatin and Loeflers coagulated serum, indicating proteolytic activity. The isolates showed REFERENCES hydrolysis of starch and casein. All the isolates showed negative results with nitrate test and indole Abu-Samra, M.T. 1978. Morphological, cultural test. Similar biochemical characteristics were also and biochemical characteristics of

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Dermatophilus congolensis. Zbl. Vet. Med. Med., 7: 342-347 B., 25: 668-688. Pal, M. 1995. Prevalence in India of Dermatophilus Cowan, S.T. 1974. Cowan and Steel’s Manual for congolensis infection in clinical specimens Identiication of Medical Bacteria, 2nd ed. from animals and humans. Rev. Sci. Tech. Cambridge University Press, New York, OIE, 14: 857-863 USA. 416p. Quinn, P.J., M.E. Carter, B.K. Markey and El-Nageh, M.M. 1971. Comparison of strains of G.R. Carter. 1994. Clinical Veterinary Dermatophilus congolensis Van Saceghem Microbiology. Wolfe Publishing. USA. 1915 isolated from different species of 648p. animals. Ann. Soc. Bige. Med. Trop., 51: Shaibu, S.J., H.M. Kazeem, U.S. Abdullahi 239-246. and M.Y. Fatihu. 2011. Phenotypic and Gitao, C.G., H. Agab and A.J. Khalifalla. 1998. genotypic characterisation of isolates of Outbreaks of Dermatophilus congolensis Dermatophilus congolensis from cattle, infection in camels (Camelus dromedarius) sheep and goats in Jos, Nigeria. Afr. J. from the Butana region in Eastern Sudan. Microbiol. Res., 5: 467-474. Rev. Sci. Tech. OIE, 17: 743-748. Sharma, D.R., M.S. Kwatra and S.S. Dhillon. 1992. Gordon, M.A. 1964. The genus Dermatophilus. J. Dermatophilosis outbreak in buffaloes in Bacteriol., 88: 509-522. Punjab. Buffalo J., 3: 293-296 Haalstra, R.T. 1965. Isolation of Dermatophilus Wabacha, J.K., C.M. Mulei, N.P. Gitonga, M.J. congolensis from skin lesions in the Njenga, A.G. Thaiyah and J. Nduhiu. 2007. diagnosis of streptothricosis. Vet. Rec., 77: Atypical dermatophilosis of sheep in Kenya. 824-834. J. S. Afr. Vet. Assoc., 78: 178-181. Hashemi Tabar, G.R., M. Rad and M. Chavoshi. Zaria, L.T. 1993. Dermatophilus congolensis 2004. A survey on dermatophilosis in sheep infection (dermatophilosis) in animals and in the north of Iran. Iranian J. Vet. Res., 5: man. An update. Comp. Immunol. Microb., 97-101. 16: 179-222. Hyslop, N. 1980. Dermatophilosis (Streptothricosis) in animal and man. Comp. Immunol. Microb., 2: 389-404. Kaminski, G.W. and I.I. Suter. 1976. Human infection with Dermatophilus congolensis. Med. J. Australia, 1: 443-447. Koney, E.B.M. 1996. Dermatophilosis in Ghana: effect on livestock industry. Trop. Anim. Health Pro. 28: 3-8 Mannan, M.A., M.S.R. Khan, M.M. Rahman, F. Begum and M.Z. Uddin. 2009. Isolation and identiication of dermatophilus bacteria from the skin lesions of cattle. Bangl. J. Vet.

81 Buffalo Bulletin (March 2016) Vol.35 No.1 Original Article Buffalo Bulletin (March 2016) Vol.35 No.1

MANAGEMENT OF REPEAT BREEDING IN BUFFALOES UNDER FIELD CONDITIONS USING HORMONAL AND ANTIBACTERIAL THERAPIES

M.G. Butani1, A.J. Dhami2,*, R.G. Shah2, N.P. Sarvaiya3 and Ankita Killedar3

ABSTRACT intrauterine (i/ut) at 12-24 h post-AI, and Povidone plus Metranidazole (Ranvidone 20-40 ml) i/ut This study was carried out during for 2-4 days (AI in next cycle), the irst service breeding season on 125 repeat breeding buffaloes conception rates were 23.08, 34.78 and 33.33%, to evaluate the therapeutic eficacy of various respectively, and overall CRs 53.85, 65.22 and hormonal and non-hormonal drugs in improving 83.33% (P<0.01), with the fertile oestrus intervals their reproductive eficiency. Repeat breeding of 20.86±5.53, 12.20±4.49 and 17.20±8.17 days, buffaloes (112) were treated parenterally with 5 respectively. The results of cefriaxone were better different drugs, keeping 13 animals as untreated as compared to enroloxacin. The overall CRs for control, and results were compared with 22 normal the GnRH, progesterone and antibacterial therapies cyclic buffaloes. The conception rates in treatment were 76.00, 62.85 and 53.85 to 83.33% (P<0.01), cycle and overall of 3 cycles post-treatment were respectively (normal cyclic group 81.82%, repeat compared between different groups. For 25 and breeding control 38.46%), with signiicantly 32 repeat breeding buffaloes treated with 0.02 (P<0.05) shorter fertile oestrus interval in GnRH mg Gonadotropin Releasing Hormone (GnRH; and progesterone treated groups as compared to Receptal 5 ml i/m), just after artiicial inseminationantibiotics treated one. Thus, all these regimes, (AI) and 500 mg of hydroxy-progesterone caproate and GnRH in particular, are recommended to the (Duraprogen 2 ml i/m) on day 4th or 5th post- practitioners for their use in the ield to ameliorate AI, the conception rates (CRs) in the treatment the problem of repeat breeding in buffaloes. cycle were 60.00 and 43.75% and overall CRs within 3 cycles were 76.00 and 62.50% (P<0.05), Keywords: repeat breeding, buffaloes, hormonal/ respectively, with a mean treatment to fertile non-hormonal therapy, conception rate oestrus interval of 6.58±3.27 and 8.25±3.28 days. For 26, 23 and 6 repeating buffaloes treated with Enroloxacin (Inj. Bayrocin single shot 30 ml) i/m at the time of AI, Ceftriaxone (Inj. Vetacef 2 g)

Department of Animal Reproduction Gynaecology and Obstetrics, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat, India 1Amul Research and Development Association (ARDA), Amul, Anand, India 2Department of Animal Reproduction Gynaecology and Obstetrics, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat, India, *E-mail: [email protected] 3Reproductive Biology Research Unit, Anand Agricultural University, Anand, Gujarat, India

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INTRODUCTION under ield conditions.

The term repeat breeder or cyclic non- breeder describes the animal that has failed to MATERIALS AND METHODS conceive after 3 or 4 services of a fertile bull/ artiicial inseminations (AI). Repeat breeding is a This study was conducted under ield major constraint in dairy farming. It is an important conditions in villages of Anand district in Gujarat cause of low reproductive eficiency in buffaloes. (India). The buffaloes managed by the farmers The incidence of repeat breeding varies from individually at their door-step and brought to the 15-32% and seems to be lower in animals kept AI Centers of the concerned village co-operative individually on small-holdings than in large herds. societies for AI, pregnancy diagnosis and sexual Endocrine imbalance, nutrition, faulty breeding health control camps were initially screened management, early embryonic mortality and through gynaeco-clinical examinations. In all 125 infectious agents leading to clinical and sub-clinical repeat breeding buffaloes that had taken more endometritis are amongst the major causes of than 3 infertile services, even with good quality repeat breeding in dairy animals (Zemjanis, 1980). frozen-thawed semen, beyond 6 months to 1 year Fertilization failure is rare in females but zygote postpartum and conirmed by rectal palpation does not survive and therefore subsequent oestrus twice 10 days apart, were selected and subjected follows normally. Luteal dysfunction leading to to different therapeutic regimes (112), keeping inadequate progesterone production post-breeding 13 as untreated control. Moreover, 22 buffaloes could be a cause of embryonic death. Gonadotropin exhibiting spontaneous estrus within 90 days Releasing Hormone (GnRH) / Human Chorionic postpartum and inseminated without any treatment Gonadotropin (hCG) and/or progesterone served as normal cyclic controls. Buffaloes in estrus analogues have been successful to sustain early were inseminated by the concerned lay inseminator pregnancy and improve conception rate in repeat of the society. breeding bovines (Sreenan and Diskin, 1983; All the animals identiied were dewormed Dhami et al., 2009; Patel et al., 2014). Similarly using Albandazole 3000 mg (Helmiguard 3000, post-insemination antibiotics therapy intrauterine Vetcare India Ltd.) and were also treated for is beneicial in enhancing conception rate in repeat ectoparasites, if any, by using Flumethrin (Flupor, breeders of unknown etiology and particularly Vetnex-RFCL India Ltd). Owners of the ear-marked with low grade genital infection (Mahto et al., animals were supplied with mineral mixtures 2006; Dhami et al., 2009). However, in most of (Amul brand) for supplementing to their animals the reports only one protocol has been tested at a 50-55 g per day for 15 days. The following were time, and the literature on comparative eficacy of the treatment protocols used (Table 1). hormonal and antibacterial approach in the same Animals of all seven groups once condition is scarce. Hence the present study was inseminated were followed for 1 to 3 cycles planned to evaluate the comparative therapeutic post-treatment, and overall as well as cycle-wise eficacy of GnRH, Progesterone and Antibiotics/ conception rates and fertile oestrus intervals were Antiseptics at a time in repeat breeding buffaloes compared between groups by Chi-square test,

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and completely randomized design, respectively just after AI. The conception rates obtained were (Snedecor and Cochran, 1986). The results are 60 and 76% in the treatment cycle and overall of presented in Table 2 and also illustrated by Figure 3 cycles post-treatment, respectively, with a mean 1. treatment to fertile oestrus interval of 6.58±3.27 days. Maximum buffaloes conceived in the treatment cycle itself indicating beneicial effect of RESULTS AND DISCUSSION GnRH in inducing fertile ovulation and CL growth (Table 2, Figure 1). The present indings of 76 and Effect of gonadotropin releasing hormone 60% conception rate using GnRH as against 38.46 (GnRH) and 14.29% in untreated control group coincided Twenty ive repeat breeding buffaloes werewell with the reports of Ghulam et al. (2002); treated with 20 µg GnRH intramuscularly (i/m), Vijayarajan et al. (2007); Sharma and Dhami

Table 1. Different approaches used in the treatment of repeat breeding 6)(Gr. in 1buffaloes to under ield conditions. Sr. Treatment No. of Status of Repeat Breeding / Treatment Approaches No. Groups Buffaloes Normal Cyclic Animals Buserelin acetate-GnRH 20 Long oestrus, free from visible 1 GnRH 25 µg i/m simultaneous to AI genital infection (Receptal, 5 ml) Hydroxyprogesterone 500 Normal oestrus, apparently free 2 Progesterone 32 mg i/m 4th or 5th day post-AI from visible genital infection (Duraprogen, 2 ml) Enroloxacin 3 g i/m Normal oestrus, discharge free (Bayrocin 1 shot, 30 ml), AI 3 Enrofolxacin 26 from visible genital infection simultaneously or in next cycle Ceftriaxone 2 g i/uterine in Normal oestrus, discharge free 4 Ceftriaxone 23 20 ml DW (Vetaceph 2 g) 12- from visible genital infection 24 h post-AI Povidone + Metronidazole Repeat breeding with clear 5 Ranvidone 06 20-30 ml i/uterine for 2-3 unhealthy discharge days, AI in next cycle Untreated Normal oestrus, discharge free No any treatment, only AI 6 13 Control from visible genital infection and follow up Normal irst oestrus within 90 Normal Cyclic No any treatment, only AI 7 22 days postpartum, free from Control and follow up visible genital infection

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(2008); Dhami et al. (2009); Savalia et al. (2013) rise in conception rate over control group with in repeat breeding buffaloes and Patel et al. (2014) 250 and 500 mg progesterone supplementation, in crossbred cows. Stevenson et al. (1990) found respectively, on 4th day post-AI in repeat breeding overall conception rate of 32.1 vs 41.6% (P<0.01) buffaloes suggesting beneicial role of higher dose following AI alone and AI+100 µg GnRH i/m of therapy. According to Das et al. (1992), the most among hundreds of repeat breeding cows. Morgan critical period for the embryo survival was the late and Lean (1993) recorded 12.5% increase in overall blastocyst. The failure of blastocyst to implant conception rate with the use of GnRH (250 µg) in might be due to the progestational changes in the normal cows and up to 22.5% in repeat breeding endometrium at the appropriate time. Kastelic cows, while in other studies the conception rates of (1994) stated that most embryonic losses occur 60 vs 40% were found for GnRH treated (0.02 mg during early pregnancy, and the cause is usually i/m) vs untreated repeat breeding cows (Ata and unknown. Embryonic loss before 125 days is Tekin, 2001; Shelar et al., 2002). Further, Mandal usually preceded by, and may be caused by, luteal et al. (2004) found irst service conception rates of regression. Hence, hormonal treatment to increase 50.0 vs 37.5% and overall conception rates 87.5 plasma progesterone concentrations may improve vs 75.0% in GnRH (2.5 ml Receptal) treated vs pregnancy rates, particularly in repeat breeding untreated repeat breeders. The beneicial results cows. with GnRH injection at AI could be due to induction of timely ovulation with improved CL function. Eficacy of antibiotics/Antimicrobials Twenty six repeating buffaloes were treated Effect of progesterone supplementation with long acting enroloxacin intramuscularly at For 32 repeat breeding buffaloes treated the time of insemination. The conception rates in with 500 mg of hydroxy-progesterone caproate on treatment cycle, and overall of 3 cycles were 23.08 day 4th or 5th post-AI, the conception rate obtained and 53.85%, with the fertile oestrus interval of was 43.75% in the treatment cycle itself, and 20.86±5.53 days. Among 23 repeating buffaloes 62.50% overall, with a mean treatment to fertile treated with intrauterine infusion of 2 g Ceftriaxone oestrus interval of 8.25±3.28 days (Table 2, Figure 12 to 24 h post-AI, 34.78% buffaloes conceived in 1). These indings of irst service and overall treatment cycle with an overall conception rate of conception rates obtained with progesterone 65.22% after a mean treatment to fertile oestrus supplementation, as against 14.29 and 38.46% in interval of 12.20±4.49 days. Among 6 repeat untreated control group, are closely comparable breeding buffaloes with clear endometritis treated with the earlier reports of Kavani and Kodagali with intrauterine infusion of Ranvidone 20-30 ml, (1984); Awasthi et al. (2002); Kumar et al. (2003); 33.33% buffaloes conceived in post-treatment I Dhami et al. (2009) in cows and buffaloes. Sharma cycle, with an overall conception rate of 83.33% et al. (2004) and Patel et al. (2005) found conception after a mean treatment to fertile oestrus interval of rates of 66.7 vs 50.0% and 50.0 vs 33.3% for 4th day 17.20±8.17 days. The results of ceftriaxone were post-AI progesterone treated vs untreated repeat better as compared to enroloxacin treated group in breeding buffaloes and HF cows, respectively. terms of irst service (34.78 vs 23.08%) and overall Sharma and Dhami (2008) recorded 20 and 40% conception rates (65.22 vs 53.85%) and even for

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fertile oestrus interval (12.20±4.49 vs 20.86±5.53 treated with cephalaxin 4 g and ceftriaxone days). Further, the overall conception rate (83.33%) 2 g intrauterine 24 h post-AI as compared to with Ranvidone was signiicantly higher than untreated control group, while Kumar et al. (2004) the antibiotics treated groups without signiicant obtained 80% conception in repeat breeding cows difference in the time interval, but the number of treated with enroloxacin intramuscularly as animal included in this group was comparatively against only 20% in untreated control group and less (Table 2, Figure 1). 45% in normal breeding group. Rane et al. (2003) Present indings of varying conception obtained 71.67% conception rate within 33 days rate in treatment cycle and overall with different among 60 repeat breeding buffaloes treated with i/ modes of antibiotics therapy corroborated well ut enroloxacin at 1500 mg (15 ml) for 2 days at or partly with the previous report of Sharma and previous oestrus as per the sensitivity of isolates Dhami (2008); Dhami et al. (2009), who obtained of cervical mucus. Mahto et al. (2006) achieved signiicantly higher overall conception rates 50% CRs each with pre- and post-AI ceftriaxone within 3 cycle among repeat breeding buffaloes treatment in repeat breeding cows as compared to

Table 2. Fertility response following various hormonal and antibacterial treatments in repeat breeding buffaloes under ield condition.

Conception Rate No. of Treatment Cycle-wise Pregnancy Rate Treatment within 3 Cycles Animals to Fertile OI Group II III Treated No. %(Days) I Cycle Cycle Cycle GnRH 25 19 76.00b 6.58±3.27b 15 (60.00) 3 1 (Receptal) Progesterone 32 20 62.50c 8.25±3.28b 14 (43.75) 5 1 (Duraprogen) Antibiotic 26 14 53.85c 20.86±5.53a 6 (23.08) 5 3 (Bayrocin) Antibiotic 23 15 65.22c 12.20±4.49a 8 (34.78) 6 1 (Vetacef) Antiseptic* 6 5 83.33a 17.20±8.17a 2 (33.33) 2 1 (Ranvidone) Overall 112 73 65.18c 11.66±2.00ab 45 (40.18) 21 7 Treated Untreated 13 5 38.46d 13.40±5.47a 2 (15.38) 3 0 Control Normal 22 18 81.82a 7.00±2.40b 12 (54.55) 6 0 Cyclic Control

OI = oestrus induction. $ AIs were done in the next cycle following 2-4 days treatment with Ranvidone intrauterine. Values bearing common superscript within the column do not differ signiicantly (P>0.05).

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of different drug at AI in repeat breeding buffaloes 90 80 70

60 CR TRT CY CLE 50 40 30 CR OVERALL

Per cent Conception 20 10 0 Receptal DuraprogenBayrocin Vetacef Ranvidon RB ControlNormal Cyclic

Treatment Groups

Figure 1. Conception rate overall and in treatment cycle following use of different drug at AI in repeat breeding buffaloes.

only 16% in untreated group. The usage of many and another 18.75% and 6.25% conceived in other antibiotics and antiseptics have been reported post-treatment I and II cycles, respectively, with with variable beneicial effects among repeat an overall conception rate of 65.18% after a breeding and endometritic buffaloes and cows by mean interval from treatment to fertile oestrus as earlier workers, viz. Dhabale et al. (1997) as 50.0 11.60±2.00 days. Among 22 normal cyclic and vs 16.7% conception with gentamicin and Singh 13 repeat breeding control buffaloes that were et al. (2001) as 69.23% with cephalexin and 55.56 inseminated without any treatment on spontaneous to 64.28% with other antibiotics. Present results oestrus, 54.55% and 15.38% buffaloes, respectively, and many of the above researchers, thus, suggest conceived in I cycle. The overall conception rates that mild genital infection prevails in repeat for the two groups were 81.82% and 38.46%, breeders and it can be cleared with use of effective with mean fertile oestrus intervals of 7.00±2.40 antibiotics, either locally in uterus or parenterally and 13.40±5.47 days, respectively, from the day thereby improving conception rate, at par with the of irst AI. The differences between groups were normal fertile animals, and reducing the calving signiicant for all the traits (Table 2). interval to a desired goal. As regards relative eficacy of different treatment protocols of repeat breeding problem, the Comparison of treatment response in repeat conception rate in the irst (treatment) cycle itself breeders Vs. Normal cyclic group was the highest for GnRH treated group (60%), at Among 112 repeat breeding buffaloes par with normal cyclic group (54.55%), followed treated with hormonal and antibacterial drugs, by progesterone treated (43.75%) and antibiotics/ 40.18% buffaloes conceived in treatment cycle, antiseptic treated groups (23.08 to 34.78%). The 88 Buffalo Bulletin (March 2016) Vol.35 No.1

overall conception rates for the respective three though in a limited number of animals. protocols were 76.00, 62.85 and 53.85 to 83.33% (normal cyclic group 81.82%). The results with hormone therapy, particularly GnRH, were better CONCLUSIONS and comparable with the normal cyclic group, followed by antibiotics treated group, and all Our indings clearly support that the these results were signiicantly better or superior use of GnRH at the time of insemination and of than those of untreated control group. Moreover, progesterone therapy 4th or 5th day post-insemination cost-wise and looking to the period of response, deinitely improves conception rate by 10-20% use of GnRH was the most economic in repeat depending upon the cause in repeat breeding breeding buffaloes as compared to progesterone or buffaloes, and hence can be advocated to deal with antibiotics in the present study. this problem under ield conditions. Present results Studies on the comparative eficacies of with antimicrobials suggest that mild genital different treatment protocols of repeat breeding infection prevails in repeat breeders and it can cows or buffaloes on farm (Patel et al., 2005) or be cleared with use of effective antibiotics, either even ield conditions (Sharma and Dhami, 2008; infused locally in uterus or parenterally, thereby Dhami et al., 2009; Patel et al., 2014) are meagre. improving conception rates in repeat breeders and Patel et al. (2005) recorded conception rates achieving the optimum calving interval. of 66.66, 83.33 and 50.00% within two cycles following GnRH (0.02 mg), hCG (1500 IU) and Progesterone (500 mg) i/m treatments post-AI in ACKNOWLEDGEMENTS repeat breeding HF cows as against 33.33% in untreated control. Sharma and Dhami (2008) also We thank Dean of the Faculty for obtained signiicantly higher overall conception the facilities provided and the secretaries and rates with i/m use of GnRH (90%), at par with inseminators of Village Co-operative Societies, normal cyclic group (88.2%) and hydroxy- including animal owners for the co-operation progesterone caproate (80%), and i/ut cephalexin extended for this work. The inancial support (80%) and ceftriaxone (70%) in repeat breeding received through DBT project of RBRU, AAU, buffaloes than in untreated control group (40.00%). Anand is also gratefully acknowledged. The present relatively better indings with GnRH and progesterone suggested that ovulatory problem, endocrine imbalance and luteal insuficiency REFERENCES leading to fertilization failure and/or embryonic mortality may be major causes of repeat breeding Ata, A. and N. Tekin. 2001. Fertility and application in buffaloes under the study area. Antibiotics of GnRH in repeat breeder cows. Lalahan therapy was also to some extent beneicial, but the Hayvancilik Arastrima Enstitusu Dergisi, relatively low results could be due to resistance 41(1): 13-24. developed by the genital microlora against them, Awasthi, M.K., R.P. Tiwari and O.P. Mishra. 2002. since Ranvidone-an antiseptic gave the best results Effect of progesterone supplementation

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during mid luteal phase on conception Sinha. 2006. Eficacy of pre- and post-AI in repeat breeding cows. Indian J. Anim. administration of sterile ceftrioxone sodium Reprod., 23(1): 67-68. on conception rate in normal and repeat Das, R.K., U.K. Mishra and D.N. Mohanty. 1992. breeding crossbred cattle. Indian J. Anim. Physiological mechanism and implantation Reprod., 27(1): 18-20. failure. The Veterinarian, 16(9): 1. Mandal, D.D., S.K. Srivastava and P. Kumar. 2004. Dhabale, R.B., N.C. Sharma and A.K. Kumar. Effect of day of GnRH administration on 1997. Antibiotic sensitivity pattern of conception rate in buffaloes. Indian J. Anim. bacteria isolated from cervical mucus of Sci., 74(12): 1189-1191. repeat breeder buffaloes and therapeutic Morgan, W.F. and I.J. Lean. 1993. Gonadotropin measures. Buffalo J., 13(2): 215-222. releasing hormone treatment in cattle: a Dhami, A.J., M.G. Butani and S.K. Sharma. 2009. meta-analysis of the effects on conception Therapeutic management of repeat breeding at the time of insemination. Aust. Vet. J., 70 buffaloes with hormones and antibiotics (6): 205-209. under ield conditions.Intas Polivet, 10(1): Patel, J.A., A.J. Dhami, F.S. Kavani and N.P. 39-44. Sarvaiya. 2005. Effect of hormonal therapies Ghulam-Ahmad, M.A. Saeed and I.N. Bashir. at breeding on plasma progesterone proile 2002. Use of GnRH to improve conception and fertility in repeat breeding Holstein rate in repeat breeder buffaloes during the Friesian cows. Int. J. Cow Sci., 1(1): 52-59. low breeding season. Pak. Vet. J., 22(1): 42- Patel, K.R., A.J. Dhami, K.K. Hadiya, K.K. 44. Savalia, A. Killedar and S.B. Patel. 2014. Kastelic, J.P. 1994. Non-infectious embryonic loss Effect of mid-cycle PGF2α and GnRH at AI in cattle. Vet. Med., 89(6): 584-589. on conception rates, plasma progesterone Kavani, F.S. and S.B. Kodagali. 1984. Clinical and biochemical proile in repeat breeding investigation of repeat breeder buffaloes crossbred cows. Indian Journal of Field for ovulatory disturbances. Indian J. Anim. Veterinarians, 9(3): 5-11. Reprod., 5(2): 38-42. Rane, R.S., R.H. Jadhav, R.C. Mazkori and S.S. Kumar, R., J.B. Phogat, Inderjeet Singh, B. Kumar, Swami. 2003. Eficacy of intrauterine B., Umed Singh and R.K. Sethi. 2003. Enrogil in the treatment of repeat breeding Eficacy of post-insemination progesterone in buffaloes. Indian Vet. J., 80(2): 169-171. supplementation for enhancement of Savalia, K.K., A.J. Dhami, K.R. Patel and K.K. fertility in buffaloes. Bubalus Bubalis, 9(3): Hadiya. 2013. Inluence of controlled 76-82. breeding technologies on fertility and Kumar, S., M.H. Akhtar, G.P. Roy, Nitesh Kumarh plasma macro-minerals proile in conceiving and R.K. Asthana. 2004. Eficacy of and non-conceiving anoestrus and repeat enroloxacin (Quinintas) on the treatment breeding buffaloes. Indian Journal of Field of repeat breeding cows. Intas Polivet, 5(2): Veterinarians, 9(2): 28-35. 204-206. Sharma, A., R. Jindal and R.V. Singh. 2004. Mahto, D., M.P. Singh, B. Singh and M.P. Progesterone supplementation on

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conception and biochemical proile in repeat breeding buffaloes. Indian Vet. J., 81(9): 1036-1039. Sharma, S. and A.J. Dhami. 2008. Effect of post- insemination antibiotics and hormone therapy on fertility in relation to macro- minerals proile in repeat breeding animals. Indian J. Field Vets., 3(3): 1-6. Shelar, R.R., V.L. Deopurkar, S.A. Bakshi and S.R. Chinchkar. 2002. Eficacy of pre- insemination GnRH treatment on the conception rate in repeat breeder cows. Blue Cross Book, 19: 26-27. Singh, A.K., H. Akhtar, R.B. Singh, A.P. Singh, G.P. Rey and S.B. Verma. 2001. Eficacy of treatment of endometritis with chemotherapeutic agents in cows and buffaloes. Indian J. Anim. Reprod., 22: 57- 59. Snedecor, G.W. and W.G. Cochran. 1986. Statistical Methods, 8th ed. Iowa State University Press, Ames, Iowa, USA. Sreenan, A.M. and M.G. Diskin. 1983. Early embryonic mortality in the cow: its relationship with progesterone concentration. Vet. Rec., 112: 517-521. Stevenson, J.S., E.P. Call, R.K. Scoby and A.P. Phatak. 1990. Double insemination and gonadotropin releasing hormone treatment of repeat breeding dairy cattle. J. Dairy Sci., 73(7): 1766-1772. Vijayarajan, A., C. Chandrahasan and E. Napolian. 2007. Effect of pre- and post-AI substitution of GnRH in repeat breeding buffaloes. Indian Vet. J., 84: 940-943. Zemjanis, R. 1980. Repeat breeding or conception failure in cattle. In Morrow, D.A. (ed.) Current Therapy in Theriogenology, 1st ed. W.B. Sounders Co., Philadelphia, London.

91 Buffalo Bulletin (March 2016) Vol.35 No.1 Original Article Buffalo Bulletin (March 2016) Vol.35 No.1

EFFECT OF GONADOTROPIN RELEASING HORMONE (GnRH) PREPARATIONS ON INDUCTION OF ESTRUS AND FERTILITY IN BUFFALOES UNDER FIELD CONDITIONS IN HARYANA

Vikash, Meenakshi Virmani*, R.K. Malik and Pardeep Singh

ABSTRACT improves the estrus induction and pregnancy rates in buffaloes under ield conditions. The study was carried out to test the eficacy of Gonadotrophin Releasing Hormone Keywords: anestrus, buffalo, conception rate, (GnRH) protocols for induction of estrus and estrus, gonadotrophin releasing hormone, GnRH fertility in buffaloes. Impact of the GnRH preparations used by the ield veterinarians to treat a total of 499 buffaloes with history of INTRODUCTION anestrus and infertility belonging to different agro-climatic zones of Haryana was analyzed Buffalo is very sensitive to environmental during the period of study. The data of treated temperature and radiation due to black thick skin buffaloes so obtained was divided into two major and very few sweat glands. Although buffalo groups depending upon the treatment protocol is polyestrous animal, however there is distinct used, viz., GnRH group (n=300) and Controlled seasonal variation in display of estrus, conception Internal Drug Release (CIDR) + GnRH group rate and calving interval (Singh et al., 2000). (n=399). GnRH group animals were subdivided During summer, there is reduction in feed intake into GnRH alone, GnRH-PG (Prostaglandin F2α) along with alteration of the proile of reproductive and GnRH-PG-GnRH (Ovsynch) protocols. CIDR hormones. Lower circulating concentrations of + GnRH treated animals were categorized into FSH (Razdan et al., 1982), LH (Rao and Pandey, CIDR + GnRH, CIDR + GnRH-PG and CIDR 1982) and progesterone have been detected + GnRH-PG-GnRH (Ovsynch) protocols. The during summer along with higher prolactin levels overall estrus induction rate was recorded to be (Kaker et al., 1982). This results in weak estrus 100% in animals treated with Ovsynch, followed symptoms during summer months. Also longer by CIDR + GnRH (98.20±1.80%), then CIDR + inter-calving interval in buffalo due to prolonged GnRH-PG (96.87±3.12%) and CIDR + GnRH- postpartum anestrus (Barile, 2005), is mainly PG-GnRH protocols. However, the overall attributed to lower circulating concentration of conception rate was observed to be signiicantlyhypophyseal and gonadal hormones (Madan higher in animals of CIDR + GnRH group. It can et al., 1983) and suboptimal functioning of be concluded that GnRH in combination with hypothalamo-hypophyseal and gonadal axis (Rao Progesterone based CIDR protocol subsequently and Shreemannarayan, 1982).

Department of Veterinary Physiology and Biochemistry, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), Hisar, India, *E-mail: [email protected]

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Early re-establishment of cyclic ovarian (Livestock Census, 2007) to obtain the information activity after calving is essential because more related to different hormonal protocols used for the estrus cycles a female has before 30 days induction of estrus and fertility in buffaloes. At least postpartum, the fewer services per conception are a ten per cent of total hormonal preparations used required (Metwelly, 2001). The productive life of a by ield veterinarians were selected randomly and buffalo can be maximized if it is bred within 100- impact analysis of different hormonal preparations 150 days after parturition to produce a calf and start was done. a new lactation every year (Abdalla, 2003). The The hormones were supplied in the ield treatments given in the irst month postpartum in by the Department of Animal Husbandry and order to initiate normal estrus cycles, also improve Dairying, Haryana. In order to quantify the effect reproductive performance (Zain et al., 2001). Thus of different hormonal protocols used, data collected many managemental strategies and hormonal was broadly classiied into Group I (n=300) and regimens have been administered to stimulate Group II (n=199) with three subgroups in each, ovulation and resumption of normal cyclicity based on combination of hormones used. of anestrus in buffalo during peak breeding and low breeding periods (Singh and Singh, 1986; Group I: GnRH Group Aminudeen, 1991; Malik, 2005). These hormones 1. GnRH Alone (n=100) act directly on the reproductive organs or indirectly Injection Receptal® 2.5 ml was on the pituitary gland to stimulate the release of administered intramuscularly (I/m) and animals naturally occurring hormones, which in turn act on were then observed for heat. This was used in the reproductive organs. animals with follicular cyst on the ovaries. In view of above, the present study was 2. GnRH- PG Protocol (n=153) planned in order to test the eficacy of gonadotropin The irst injection of Receptal® 2.5ml was hormone (GnRH) in combination with other administered I/m on day 1 of treatment. Injection hormones for induction of estrus and fertility in Clostenol® 2 ml was administered I/m 7 days after anestrus buffaloes. the Receptal® injection. Animals were observed for estrus and inseminated 12 h after onset of estrus. 3. GnRH- PG- GnRH / Ovsynch Fixed Time MATERIALS AND METHODS Artiicial Insemination Protocol (Ovsynch FT AI) (n=47) The basic purpose of the study was to The irst injection of Receptal® 2.5 ml evaluate the eficacy of the hormonal preparations I/m was administered on the irst day of treatment being used for treatment of anestrus, induction followed by injection Clostenol® 2 ml I/m after 7 of estrus and fertility in buffaloes under ield days. A second injection of Receptal® 2.5 ml was conditions. The study constituted the survey of administered I/m 48 h after Clostenol® injection. different villages of the selected ten districts of All the animals were inseminated either at the time Haryana state (namely Hisar, Sirsa, Fatehabad, of second injection of Receptal® (0 h) or 12 h later. Kaithal, Karnal, Panipat, Sonipat, Jind, Rohtak and Bhiwani) known for higher buffalo population

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Group II: CIDR + GnRH Group RESULTS AND DISCUSSION 1. CIDR- GnRH protocol (n=58) CIDR device was inserted on the irst day Postpartum anestrus in buffaloes is of treatment and was removed on day 7. Heat was responsible for long calving intervals (Borghese et observed for 2-6 days and insemination was done al., 1994). A variety of hormones are being used 12 hours after observing heat. Injection Receptal® to treat reproductive disorders and to regulate the 2.5 ml was injected I/m at the time of AI. estrus cycle for timed breeding in buffaloes. These 2. CIDR- GnRH – PG protocol (n=111) hormones act directly on the reproductive organs On the irst day of treatment, CIDR was or indirectly on the pituitary gland to stimulate the inserted and injection Receptal® 2.5 ml was release of naturally occurring hormones, which injected I/m. On day 7, the CIDR was removed in turn act on the reproductive organs. Different and injection Clostenol® 2 ml was administered hormonal protocols give satisfactory pregnancy I/m. The animals were inseminated 12 h after heat rates, which are comparable to those achieved in observation. animals inseminated at natural estrus (De-Rensis 3. CIDR- GnRH - PG- GnRH / CIDR Ovsynch FT and Lopez, 2007). AI protocol (n=130) A total of 499 buffalo with history of On the irst day of treatment, the CIDR anestrus and infertility belonging to different was inserted intravaginally and injection Receptal® agro-climatic zones of Haryana were subjected to 2.5 ml was administered I/m. On day 7, CIDR was different hormonal treatment protocols. The treated removed and injection Clostenol® 2 ml was given buffaloes were divided into two groups: GnRH I/m. Injection Receptal® 2.5 ml was administered group and CIDR + GnRH group with three sub I/m 48 h after Clostenol injection and insemination groups in each. In the present investigation with the by clock was followed between 0 and 12 h after use of GnRH alone protocol, estrus induction and GnRH injection. conception rate were observed to be 90.53±3.61% Estrus induction rate, conception rate/ and 61.06±7.12% respectively (Table 1). Previously, pregnancy rate at 1st service (induced heat), Ramoun et al. (2012) observed 60% and 30%, conception rate/ pregnancy rate at 2nd service estrus induction and conception rate, respectively (spontaneous heat) and overall pregnancy rate with the administration of this protocol. They also were recorded in order to evaluate the response suggested that in order to get better results with of different hormonal treatment protocols used. GnRH, the nutritional requirement of animal should Comparative study of the treatment regimes was be fulilled for proper development of follicles for carried out for comparing their effectiveness action of these hormones. and eficacy for estrus induction and successful With the use of GnRH-PG protocol, conception. The data obtained in the study was estrus induction and overall pregnancy rates statistically analyzed using Duncan’s multiple were observed 81.03±7.92% and 62.68±7.78%, range test for comparing means in an analysis of respectively (Table 1). GnRH treatment would enhance ovulation and subsequent PGF treatment variance (p=0.05) to draw the scientiic inferences. 2α would induce luteolysis of CL and later on, ovulation. Hafez and Hafez (2000) suggested that

95 Buffalo Bulletin (March 2016) Vol.35 No.1 a a b b ab ab Overall Pregnancy

nd Estrus Conception at 2 Conception Conception at Conception Induced Estrus Induced 29 66.23±8.68 2 1.79±1.79 31 68.02±8.59 86 58.95±7.27 5 3.73±2.31 91 62.68±7.78 87 71.65±7.95 1 2.78±2.78 88 74.43±9.16 58 61.06±7.12 0 0.00±0.00 58 61.06±7.12 42 81.26±8.46 0 0.00±0.00 42 81.26±8.46 78 74.19±9.13 2 7.14±7.14 80 81.33±11.01 a a a a a ab Estrus Animals Induced to Induced Animals 100 96.87±3.12 N n (%Mean± SE) n (%Mean± SE) n (%Mean± SE) n (%Mean± SE) 47 47 100.00±0.00 111 100 91 90.53±3.61 No. of No. treated animals Name of Name Hormonal Protocol Used Protocol GnRH Alone GnRH CIDR-Ovsynch 130 124 95.74±2.87 Ovsynch Ovsynch AI FT GnRH-PG 153 120 81.03±7.92 CIDR-GnRH-PG CIDR-GnRH 58 54 98.20±1.80 Group GnRH GnRH Group CIDR+GnRH Group Means with different superscripts are signiicantly different (P<0.05). superscripts are signiicantly different with Means different Table 1. Comparison of different hormonal treatment protocols for induction and of estrus fertility in buffaloes. 1. Comparison of different Table

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the administration of PGF in early postpartum with subsequent increase in the intensity of estrus 2α period would reduce the incidence of subclinical (Azawi et al., 2012). The conception rate in present uterine infection and hasten the return to a suitable study is higher than 20% and 66.5% reported by uterine environment for fertilization and pregnancy. Azawi et al. (2012) and Hammam et al. (2009) Administration of PGF at 7-10 days postpartum in summer season respectively. The differences 2α was effective in facilitating the uterine involution in conception rate between the present study as and resumption of ovarian cyclicity and improving compared to others, could be due to the reason reproductive performance (Noakes et al., 2001). that the animals were treated during their normal The results of present study are in harmony with breeding season (autumn-winter). Hammam et al. (2009) and Yendraliza et al. (2011) In the animals treated with CIDR-GnRH- who observed 60% and 100% estrus induction rates PG combination, estrus induction of 96.87±3.12% and 67% and 100% pregnancy rates respectively. and pregnancy rate of 81.33±11.01% was observed A 100% induction of estrus in treated (Table 1). The results indicated that administration animal was observed by the use of Ovsynch FT AI of GnRH after removal of CIDR showed tighter protocol (Table 1). Such a higher percentage of estrus synchrony in estrus response and tended to increase induction might be due to the reason that GnRH the pregnancy rate in anestrus buffaloes (Naseer et – PGF -GnRH (Ovsynch) protocol synchronizes al., 2011). The results of this study are in agreement 2α follicular development, luteal regression and with the results obtained in beef cattle (Martinez et time of ovulation, thus permitting ixed time alAI., 2011), dairy cows (Thatcher et al., 2006) and after the second GnRH administration (Azawi et buffaloes (Azawi et al., 2012). al., 2012). Conception rate obtained in our study The estrus induction rates using CIDR- (68.02±8.59 %) with Ovsynch protocol treatment Ovsynch FT AI protocol were observed to be in buffalo is considered high when compared to 95.74±2.87% in the present study (Table 1). These other similar studies conducted by several workers results are in agreement with Baruselli et al. (2007) such as Irikura et al. (2003); Mialot et al. (1999) who also observed that 100% estrus induction rates and Baruselli et al. (1999) who found lower could be achieved in breeding season by combining conception rates in buffalo were 27.2, 36.1 and CIDR with Ovsynch protocol in anestrus buffaloes. 42.4%, respectively. The differences in conception The main action of GnRH used at the start of rates in the present study as compared to the others progesterone treatment in order to synchronize could be because buffaloes in our study were not emergence of a new cohort of follicles (Rhodes et speciically selected as true anestrus and also the al., 2003). Additionally, second injection of GnRH breeding season favors the higher estrus induction has the additional effect of inducing ovulation and and conception rates in buffaloes. the formation of a corpus luteum in a majority Using CIDR-GnRH protocol for of animals, resulting in elevated concentrations treatment, estrus induction rate was observed of progesterone in anestrus buffaloes. GnRH to be 98.20±1.80% with a conception rate of synchronizes the development and occurrence 81.26±8.46% (Table 1). CIDR causes an increased of follicles and resulted in more homogenous circulatory concentration of progesterone resulting follicular development. It also induces ovulation in increased sensitivity of hypothalamus to estrogen or luteinization of dominant follicle in non cyclic

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animals. However, the induced ovulation in non Azawi, O.I., M.D Ali, S.A. Oday, A. Salih, cyclic animals stimulated luteal tissue development A.S. Al-Hadad, S.J. Mouayad and A.S. and function resulting in the occurrence of cyclic Hussien. 2012. Comparative eficacy of activity (Bao et al., 2003). Inserting CIDR at the different CIDR protocols for the treatment initial GnRH injection of the Ovsynch program of postpartum anestrus in Iraqi buffaloes (Ovsynch plus CIDR) and then removing the Ibrahim. Vet. World., 5: 201-205. CIDR and injecting PGF has been demonstrated Bao, G.A., P.S. Baruselli, M.F. Marques. 2003. 2α to improve pregnancy rates in lactating buffaloes Pattern and manipulation of follicular (Ravikumar et al., 2011). The conception rate development in bos indicus cattle. Anim. in our study was observed to be 74.43±9.16% Reprod. Sci., 15: 307-326. when Ovsynch protocol was supplemented with Barile, V.L. 2005. Improving reproductive progesterone. Baruselli et al. (2007) and Azawi eficiency in female buffaloes.Lives. Prod. et al. (2012) observed 57.5 and 32% overall Sci., 92: 183-194. pregnancy rates, respectively. Baruselli, P.S., N.A.T. Carvalho, L.U. Gimenes and Thus in the present study, it was observed G.A. Crepaldi. 2007. Fixed-time artiicial that Ovsynch FT AI protocol produced maximum insemination in buffalo. Ital. J. Anim. Sci., estrus induction rate in anestrus buffaloes under 6: 107-118. ield conditions. However, inclusion of a CIDR Baruselli, P.S., E.H. Madureira, V.H. Barnabe, R.C. device in combination with GnRH resulted in Barnabe, J.A. Visintin, C.A. Oliveira and R. comparable estrus induction rate along with higher Amaral. 1999. Estudo da dinamica follicular conception rate in postpartum anestrus buffaloes. em bufalas submetidas a sincronizacao da ovulacao para inseminacao artiicial em tempo ixo. Arquivos da Faculdade de REFERENCES Veterinaria. UFRGS, 27: 210. Borghese, A., G.M. Terzano, V.L. Barile, G. Abdala, E.B. 2003. Improving the reproductive Annicchiarico, S. Allegrini, L. Esposito, performance of Egyptian buffalo cows by R. Di Palo, R. Boni, E. Seren and changing the managemental system. Anim. A. Parmeggiani. 1994. Puberty and Reprod. Sci., 75: 1-8. maintenance of ovarian cyclic activity in Aboul, E.M.B, F.E. Keraby and R. Khattab. 1985. buffalo. Agric. Ric., 153: 5-16. Effect of GnRH treatment on postpartum De-Rensis, F. and F. Lopez-Gatius. 2007. Protocol resumption of oestrus and ovulation in for synchronization estrus and ovulation buffaloes. Buffalo J., 1: 61-69. in buffalo (Bubalus bubalis). A review. Aminudeen, 1991. Effect of gonadotrophin Theriogenology, 67: 209-216. releasing hormone on postpartum Gordon, I. 1996. Controlled Reproduction in Cattle reproductive eficiency of cross-bred and Buffaloes. Wallingford, UK: CAB cows: Clinical and endocrine aspects. International. Ph.D. Thesis, CCS Haryana Agricultural Hafez, E.S.E. and B. Hafez. 2000. Physiology University, Hisar India. of reproduction in farm animals. Lea &

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Febiger, Philadelphia., 8: 59-93. heifers. Reprod. Domest. Anim., 47(2): 230- Hammam, A.M., A.O. Hegab, W. Scott and K.M. 237. Ibrahim. 2009. Improvement of fertility Metwelly, K.K. 2001. Postpartum anestrus in in Egyptian buffaloes during summer buffalo cows; cause and treatment, p. 259- season using different protocols for estrus 267. In Proceedings of 6th Sci. Cong. Egypt. synchronization. Mansoura, Vet. Med. J., Soe Cattle Disease. Assiut University, 11: 1-12. Egypt. Irikura, C.R., J.C.P. Ferreira, I. Martin, L.U. Mialot, J.P., G. Laumonnier, C. Ponsert, H. Cimenes, E. Oba and A.M. Jorge. 2003. Fauxpoint, N.E. Barassi, A.A. Ponter and F. Follicular dynamics in buffalo heifers Deletang. 1999. Postpartum suboestrus in (Bubalus bubalis) using the GnRH-PGF2α- dairy cows; comparison of treatment with GnRH protocol. Buffalo J., 20: 323-327. prostaglandin F2α or GnRH + prostaglandin Kaker, M.L., M.N. Razdan and M.M. Galhotra. F2α + GnRH. Theriogenology, 52: 901-911. 1982. Serum prolactin levels of non-cycling Naseer, Z., E. Ahmad, J. Singh and N. Ahmad. Murrah buffaloes (Bubalus bubalis). 2011. Fertility Following CIDR Based Sync Theriogenology, 17: 469. hronization Regimens in Anoestrous Nili- Livestock Census. 2007. 18th Livestock Census. Ravi Buffalo. Reprod. Domest. Anim., 4: Department of Animal Husbandry & 225-229. Dairying. State Goverment of Haryana, Noakes, D.E., T.J. Parkinson and G.C.W. England. India. 2001. Arthur’s Veterinary Reproduction and Madan, M.L., S.M.K. Naqvi, C.V. Triu, A.K. Suri Obstetrics, 8th ed. Baillier Tindall, London. and B.S. Prakash. 1983. Plasma estradiol- Ramoun, A.A., B.H. Serura, El-S.M. Fattouha, 17-β, progesterone and cortisol among S.A. Darweishb and H.A. Abou El-Ghait. anestrus rural animals. In Symposium on 2012. Enhancing follicular growth as a Animal reproduction in India. Society prerequisite for GnRH treatment of true for Study of Animal Reproduction, HAU, anestrum in buffaloes. Anim. Reprod. Sci., Hissar, India. 132(1-2): 29-35. Malik, R.K. 2005. Studies on ovarian follicular Rao, A.V. and O. Sreemannayanan. 1982. Clinical dynamics during early postpartum period, analysis of reproductive failure among anestrus condition and hormonal therapies female buffaloes (Bubalus bubalis) under for induction of estrus in Murrah buffaloes village management in Andhra pradesh. (Bubalus bubalis). Ph.D. Thesis, CCS Theriogenology, 18: 403-411. Haryana Agricultural University, Hisar Rao, L.N. and R.S. Pandey. 1982. Seasonal changes India. in plasma progesterone concentrations in Martinez, M.F., G. de. Nava, K.J. Demmers, D. buffalo cows (Bubalus bubalis). J. Reprod. Tutt, M. Rodriguez Sabarros, B. Smaill, Fertil., 66: 57-61. M. Corti and J. Juengel. 2011. Intravaginal Ravikumar, K., S.A. Asokan, C. Veerapandian and progesterone devices in synchronization A. Palanisamy. 2011. Ovarian status serum

protocols for artiicial insemination in beef progesterone (P4) level and conception rate

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in Ovsynch plus CIDR treated postpartum in buffaloes. Tamilnadu J. Vet. Anim. Sci., 7: 1-5. Razdan, M.N., M.L. Kaker and M.M. Galhotra. 1982. Serum FSH levels during estrus and a 4-week period following mating in Murrah buffaloes (Bubalus bubalis). Theriogenology, 17: 175-181. Rhodes, F.M., S. McDougall, C.R. Burke, G.A. Verkark and K.L. Macmillan. 2003. Treatment of cows with an extended postpartum anestrus interval. J. Dairy Sci., 86: 1876-1894. Singh, J., A.S. Nanda, G.P. Adams. 2000. The reproductive pattern and eficiency of female buffaloes. Anim. Reprod. Sci., 2: 593-604. Singh, L.P. and B.K. Singh. 1986. Studies on incidence and treatment of anestrus desi cow. Indian Vet. J., 63: 961-962. Thatcher, W.W., T.R. Bilby, J.A. Bartolome, F. Silvestre, C. R. Staples and J.E.P. Santos. 2006. Strategies for improving fertility in the modern dairy cow. Theriogenology, 65: 30-44. Yendraliza, B.P. Zesin, Z. Udin, Jaswandi and C. Arman. 2011. Effect of combination of GnRH and PGF2α for estrus synchronization on onset of estrus and pregnancy rate in different postpartum in swamp buffalo in Kampar regency. J. Indonesian Trop. Anim. Agric., 36: 9-13. Zain, A.E., A.K.H. Abdel-Razek and M.M. Anwar. 2001. Effect of combined using of GnRH and PGF2α on ooestrus synchronization and pregnancy rate in buffalo-cow. Assiut. Vet. Med. J., 45: 89.

100 Original Article Buffalo Bulletin (March 2016) Vol.35 No.1

FARMERS’ KNOWLEDGE ON PRODUCTIVE AND REPRODUCTIVE PERFORMANCES OF BUFFALO UNDER SMALLHOLDER FARMING SYSTEM

B.S. Meena*, H.C. Verma and Amit Singh

ABSTRACT having high level of knowledge about productive and reproductive practices. Whilst about 30.00 Dairying farming can be cost effective percent of respondent were having medium level when the animals rearing were directly inluenced of knowledge. Knowledge index on productive by the productive and reproductive parameters of and reproductive parameters of dairy animal was buffalo. Productive and reproductive performances calculated and observed that respondent were of dairy animals should be determined on the possessing 73.47 and 70.21 percent knowledge basis of average daily milk yield, lactation length, in study area. Though the respondent are facing lactation milk yield, peak yield, dry period, serious constraint in rearing, they were frequently service period, conception rate, pregnancy rate, updating their knowledge on productive and calving interval etc. These parameters should be reproductive practices as compared to Buffalo and maintained by the farmers so that the productivity indigenous cow based dairy farmers. could be increased of their herd. Hence, the study was conducted to measure the productive and Keywords: buffalo, dairying, farmers’ knowledge, reproductive performances of buffalo and dairy india, productive and reproductive performance animal and Farmers’ knowledge on these aspect in Faizabad district of Uttar Pradesh. For this purpose 150 farmers engaged in dairying were interviewed. INTRODUCTION The farmer was selected on the basis of at least completion of one lactation length of buffalo at Dairying sector is focussing to plays the time of investigation and each farmer must be a unique multi-faceted role in socio-economic rearing a buffalo in combination with crossbred cow development of rural households and contributes and indigenous cow at the time of investigation. about 3.88 percent to the Gross Domestic Product The overall Average daily milk yield and 21.58 percent to the Agricultural Gross (ADMY) and Lactation length (LL) was estimated Domestic Product in India (Annual Report, 2014- to be 5.75±0.65 litre/day/animal and 276±14 days/ 15). The 51% share of total milk production of animal. The average peak yield of buffalo in the 127.8 million tonnes (2011-12) was dominated ield condition was estimated to be 8.56±0.85 litre by 105.3 million buffaloes accounting for about /animals while Age at irst calving (AFC) was 51 percent of Asia’s and about 19 percent of 1288±122 days/ animals respectively. The study world’s bovine population. The current market reveals that the 41.00 percent of respondent were trend in livestock sector growth suggests that in

National Dairy Research Institute, Karnal, Haryana, India, *E-mail: [email protected]

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order to meet the emerging demand for livestock managed by farmers and to assess the knowledge based products basically milk, both in domestic of farmers on these parameters. and global markets, there is a need to reorient the production system by enhancing the eficiency and creating quality consciousness. India ranks MATERIAL AND METHOD irst in milk productionwhich is produced by its huge bovine population (304.5 million) and little The present study was purposively contribution from other species (Annual Report, undertaken in the state of Uttar Pradesh, which 2014-15). But the major concerns that is troubling is one of the largest state occupying irst position farmers is the low productivity per animal. in milk production with 23.00 percent of total Basically milk production (productivity) depends buffaloes (26.44 million), producing 20.10 million on four dimensions of animal husbandry practices tonnes of milk. Faizabad district from Uttar Pradesh i.e. breeding, feeding, health-care and management was selected purposively as the buffalo population practices. was fairly distributed in the region. Productive and Reproductive eficiency The region is not highly productive, small are important parameters, which inluences the farmers are heavily dependent on different species economics of milk production considerably. There of livestock for their livelihood. From the present are a large number of productive and reproductive study area six villages were selected randomly problem in the ield condition which render the from three randomly selected blocks. The animal with losses of reproductive function. Any information for this study was collected from 150 impairment in normal reproductive function results farmers, 25 from each selected village. The farmer into infertility or sterility of animal, leading to was selected on the basis of at least completion economic losses due to widening of dry period of one lactation length of buffalo at the time of and inter calving interval, reducing calving and investigation. The criteria for the selection of lactation during lifetime of animal (Agarwal et respondents was that each farmer must be rearing al., 2005). About 18-40 percent of cattle and a buffalo in combination with crossbred cow buffalo are culled and reach to abattoir mostly and indigenous cow at the time of investigation. due to infertility (Sharma et al., 1993).Total Finally, the respondents were grouped in three losses due to reproductive problems in buffaloes different dairy farming system i.e. Buffalo based were 39 percent of all the losses incurred by farmers, Crossbreed farmers and Indigenous based various disease conditions (Khan et al., 1995). farmer based on the criteria that maximum number Production and Reproduction are one of the of farmers are rearing the particular livestock. The most important considerations to determine the primary data was collected by personal interview proitability of cattle and buffalo. Productive and method using a structured interview schedule. The reproductive performances of dairy animals should respondents were interviewed, individually and the be determined on the basis of these parameters. data about animal performance and knowledge of Keeping in view these problem the following the respondents was collected. The collected data study was undertaken to ind out the Productive were tabulated, scored and analyzed in the light of and reproductive performance of buffaloes animals the set objectives.

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Knowledge of farmers on productive and daily milk yield of upgraded buffalo in ield which reproductive parameters of buffalo were reared in combination with other animals. It English and English (1961) deinedrefers to average milk yield of buffalo during the knowledge as a body of understood information lactation period of 305 days measured in liters/ possessed by an individual. Knowledge in the day. The result in table-1 shows that the ADMY present study refers to which information and of buffalo based farmers, cross breed based and understanding, the respondent has about the indigenous based farmers was 6.01±0.5, 5.79±0.56, improved dairy breeding and management practices and 5.45±0.61 litre/day/animal respectively. The (Productive and reproductive traits of dairy overall ADMY was estimated to be 5.75±0.65 animals). A knowledge a test was developed and litre/day/animal. The high ADMY of buffalo was standardized by following the procedure described due to the fact that buffalo are actually treated as by Linquist (1951). The farmers were exposed milk animal and due care was taken in feeding and to the test and knowledge score was obtained management of these animals. depending on Farmers’ recall memory. All possible care was taken to cover maximum aspect pertaining Lactation Length (LL) Productive and reproductive performances of dairy The optimum lactation length of dairy of animal. The respondents were classiied in terms animals is one of the best productive indicator of having low, medium and high knowledge level of dairy animals. LL is number of days a buffalo on the basis of cumulative square root frequency remain in milk from the date of calving to the date method. of dry. The data pertaining to present investigation the lactation length of Buffalo in different system Knowledge index was 281±19, 277±16, and 276±14 days/animal The knowledge index was measured by respectively. The overall LL of the buffalo in the using following formula area was 276±14 days/ animal. Murrah buffalo lactation length in the west Godavari was reported to be 299.91±5.01 which is quite similar to RESULTS AND DISCUSSION the above data (Suresh, 2013). LL was mainly inluenced by the parity of lactation. Singhet Productive and reproductive performances of al. (2011) has shown the positively correlation buffaloes in dairying farming system between the parity of lactation and lactation length The current status of the production of in Nilli-Ravi buffaloes. buffalo was ascertained with respect to various production and reproduction traits considered Lactation Milk Yield (LMY) under study were analysed and presented in the The lactation milk yield of the dairy animals following sub-heads: has positive relation with the overall performances of an animal. It is conceptualized as the average Average Daily Milk Yield (ADMY) total quantity of milk produced by an animal in Average milk yield of buffalo is an its lactation period of 200 days. A cursory look on important productive indicator showing average Table 1 reveal that the LMY of Buffalo reared in

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the buffalo based farming was highest. It shows the performances of dairy animals lower the age that the LMY was 1733.03±189, 1694.44±124, of irst calving better the performances of dairy and 1487.33±117 litre/animal respectively. Hitesh animals. AFC is the actual age of animals in et al., (2012) reported that 305 day milk yield in days at the time of its irst calving for buffaloes. Murrah Buffalo was 2147.6 ± 87.06 kg The author Buffalo reared under different system the age observed these results because buffaloes been the at irst calving was observed to be highest in the major milk producer and due care was taken in indigenous pattern (1308±108) followed by cross feeding and management of the animals. breed animals (1294±123) and least was observed in buffalo rearing system (1260±112) days/ animal Peak Yield (PY) respectively. Peak yield is measured as the highest milk The overall AFC of buffalo in the area was produced by the milch animal in it lactation length. stated as 1288±122 days/ animals respectively. The data present in Table 1 reveals that the average The study conducted by Bohra et al. (2007) in peak milk yield of Buffalo was highest in buffalo Uttarakhand state found that the AFC in buffalo based farming system (8.87±0.98) followed by was about 4.6 years. In general, AFC in Indian indigenous based (8.12±0.79) and cross breed dairy animal is much higher compared to their (8.64±0.85) based farming. The average peak yield exotic or crossbred counterparts which is largely of buffalo in the ield condition was estimated to be attributed to lack of selection for their traits from 8.56±0.85 litre/animals respectively. Suresh (2013) generation to generation. The Reduction in the stated that the overall peak milk yield of Buffalo age at irst calving leads to an increase in lactation was recorded as 13.97±1.13. yield and helps in improving the economy of dairy farmers. Shashidhara et al (1998) stated that age at Dry Period (DP) irst calving of 1301-1390 days was optimum for It refers to the number of days a cow getting maximum lactation milk yield and lifetime remained dry i.e. the interval between the dates milk yield in buffaloes which shows that the farmers of dry to the date of next calving. It was observed were having ideal management of buffaloes. from the table-1 that the dry period of Buffalo was 211±12, 230±14 and 237±16 days/animal. The Service Period average DP calculated was observed to be 226±13 The data pertaining to service period days/animal respectively. Similar inding were also was presented in the Table 1 and found that the observed by Thiruvenkadan et al. (2010) and stated service period of Buffalo was 189±15, 198±17 that the dry period of 250.5 ± 15.9 days in buffaloes. and 199±18 days/animal respectively. The average However Yadav et al. (2007) stated the lower dry service period of 139.91±2.96 days was reported period in the Murrah buffaloes maintained in its in Murrah buffalo at NDRI farm (Jamuna et al, home tract. 2013). The important cause of long service period as many heat period are lost due to unavailability Age At First Calving (AFC) of breeding bulls or artiicial insemination in the Late maturity and age at irst calving area. Abayawansa et al. (2011) also stated that are one of the most important reasons to losses poor detection of oestrus caused by low attention

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on buffalo with irst parity as they produce low conception rate in ield condition which might milk could be one of reasons to have comparatively relect their sound knowledge in reproductive longer intervals. aspect of buffalo or due to good management provided by them. Khan et al. (2009) observed Service Per Conception service per conception of about 2 in Murrah buffalo It is deined as an average number of at organized farm. Higher rate of service per insemination or natural service required by buffalo conception may be due to un-identiication of heat, to become pregnant. It was observed that the service post partum complication in the buffalo and may per conception of buffalo was highest in buffalo also indicative of poor postpartum management. based farming (1.84±0.52). Service per conception of buffalo in other system was 1.78±0.32 and Calving Interval 1.76±0.42 times/animal respectively. It was Calving interval (CI) is another important observed the farmers were attaining appropriate parameter for performances of the dairy animals

Table 1. Productive and reproductive performances of the Buffalo.

Buffalo Based Cross breed Indigenous cow Overall S. No. Parameters farmers based farmers based farmers performance (Buffalo = I09) (Buffalo = 45) (Buffalo = 33) (Buffalo = 187) Productive parameters Average daily 1 6.01±0.5 5.79±0.56 5.45±0.61 5.75±0.65 milk yield (l) Lactation Length 2 281±19 277±16 276±14 276±14 (Days) Lactation milk 3 1733.03±189 1694.44±124 1487.33±117 1587.60±113 yield (l)

4 Peak yield (l) 8.87±0.98 8.12±0.79 8.64±0.85 8.56±0.85

5 Dry period (Days) 211±12 230±14 237±16 226±13

Reproductive parameters Age at irst 6 1260±112 1294±123 1308±108 1288±122 calving (Days) Service period 7 189±15 198±17 199±18 189±16 (Days) Service per 8 1.84±0.52 1.78±0.32 1.76±0.42 1.76±0.72 Conception (no.) Calving interval 9 495±45 508±47 513±38 505±39 (Days)

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Table 2. Distribution of farmers based on knowledge on Productive and reproductive practice. n=150 S. No. Respondents Categories Frequency Percentage Knowledge on productive Low ( <17.8 ) 43 28.67 parameters

1 Range (13-25), Medium (17.8-21.78) 45 30.00

Mean (20.35) High ( >21.78) 62 41.33

Knowledge on Reproductive Low ( <27.96) 41 27.33 parameters

2 Range (22-39), Medium (27.96-33.60) 60 40.00

Mean (31.66) High ( >33.45 ) 49 32.67

Knowledge on productive and Low ( <45.4 ) 37 24.67 Reproductive parameters

3 Range (37-62), Medium (45.4-55.4) 58 38.62

Mean (52.07) High ( >55.4) 55 36.55

Figure 1. Knowledge index of farmers on productive and reproductive parameters.

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keeping in the mind the data was properly analyzed reveals similar inding that the farmers were having and state that 495±45, 508±47 and 513±38 days/ highest knowledge about reproductive disorders animals respectively. It was concluded that calving like abortion followed by Repeat breeding, interval of buffalo was 505±39 days/animal anoestrus, late maturity and retention of placenta. respectively. The lower calving interval of the He also concluded that nearly 65.41 percent of buffalo was due to the care taken by the farmers in respondent was considered as good knowledge on their feeding and maintaining the dry period. While reproductive disorders of dairy animals. the study of Thiruvenkadan et al. (2010) reports Knowledge Index of Farmers on the higher calving interval of 559.6±17.3 days in Productive and Reproductive Practices of Buffalo buffaloes. was calculated. The collected data was further analysed and the resulted presented in Figure 1 Knowledge of Dairy Farmers on Productive and reveals that the overall knowledge possessed by Reproductive parameters of Buffaloes farmers was 66.79 percent in the study area. It Knowledge is a body of understood was further observed that those respondents were information possessed by an individuals. Therefore having good knowledge (68.91%) on productive the knowledge possessed by individual regarding practices as compared to reproductive practices the productive and reproductive practices of (66.10%). It was also noticed that buffalo based buffalo had impact on the proitability of farmers.farmers were having knowledge up to the extent Farmer’s rate of adoption was greatly inluencedof 73.47 and 70.21 percent on productive and by the gain in knowledge. Hence an attempt has reproductive practices of dairy animals. been made to study the knowledge of farmers in The study inely concluded that majority relation to Productive and reproductive practices of of the farmers were having healthy knowledge buffalo. about performance on productive and reproductive The inding presented in Table 2 reveals that practices of buffalo. The buffalo based farmers about 41.00 percent of respondent were having high were having more knowledge on productive level of knowledge on productive practices of dairy (73.47%) and reproductive (70.21%) parameters animals. Whilst about 30.00 percent of respondent than their counterparts. So it can be concluded were having medium level of knowledge and almost from the above discussion that the performance of 28.67 percent respondent were possessed low level productive practices is comparatively better than the of knowledge. Thus it could be concluded that reproductive practices due to the more knowledge respondent in the study area were having high to on productive practice and low knowledge on medium level of knowledge in productive practices reproductive practice of farmer. of dairy animals. It is also reveals that the 40.00 percent of respondent were having medium level of knowledge on reproductive parameters of dairy REFERENCES animals. Whilst about 32.67 and 27.00 percent of respondent were having high and low level of Abayawansa, W.D., S. Prabhakar, A.K., Singh knowledge on the reproductive practices of dairy and P.S. Brar. 2011. Effect of climatic animals. The study of Subhash et al. (2013) also changes on reproductive performance of

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Murrah buffaloes in Punjab: A retrospective Lahore, Pakistan. analysis. Indian J. Anim. Sci., 81(4): 334- Linquist, E.F. 1951. Educational Measurements 339. Part II. American Council of Educational, Agarwal, S.K., S.K. Singh and R. Rajkumar. Washington, USA. 2005. Reproductive disorders and their Sharma, V.K., R.C. Gupta, S.K. Mishra, N.K. management in cattle and buffaloes: a Khurana and S.K. Khar. 1993. An abattoir review. Indian J. Anim. Sci., 75(7): 858-873. study of lesions in buffalo genetelia. Indian Annual Report. 2014-2015. Annual Report of Vet. J., 70: 1165. Department of Animal Husbandry, Dairying Shashidhara, T., S.S. Bhatln and M.M. Appanna. and Fisheries. Ministry of Agriculture, 1998. Breeding eficiency and optimum Government of India. levels of various reproductive traits in Surti Bohra, B., R.J. Sharma, S.K. Singh, R.S. aiswal and buffaloes. Indian J. Anim Sci., 68(1): 73-75. V. Singh. 2007. Productive and reproductive Singh, T.P., R. Singh, G. Singh, K.S. Das and S.M. performance of dairy animals in Almora Deb. 2011. Performance of production traits district of Uttarakhand. Indian J. Anim. Sci., in Nili-Ravi buffaloes. Indian J. Anim Sci., 77(7): 633-634. 81(12): 1231-1238. English, H.B. and A.C. English. 1961. Subhash, C., B.S. Meena, K.S. Kadian and S. Comprehensive Dictionary of Psychological Anoop. 2013. Knowledge of dairy farmers Analytical Terms. Green & Company, on reproductive disorders of dairy animals. Longman’s New York, USA. Vet. Pract., 14(1): 23-25. Pawar, H.N., G.R. Kumar and R. Narang. 2012. Babu, D.S. 2013. Production performance of Effect of year, season and parity on milk Murrah buffaloes under organized dairy production traits in Murrah buffaloes. farm production system in west Godavari Journal of Buffalo Science, 1(1): 122-125. district of Andhra Pradesh. Indian Journal Jamuna, V., A.K., Chakravarty, C.S. Patil, A.C. of Applied Research, 3(8): 674-675. Mahajan, S. Dash and V. Vohra. 2013. Thiruvenkadan, A.K., S. Panneerselvam, R. Decline in reproductive performance in Rajendran and N. Murali. 2010. Analysis high producing Murrah buffalo. J. Anim. on the productive and reproductive traits Res., 3(2): 203-208. of Murrah buffalo cows maintained in the Khan, H.M., T.K. Mohanty, V.S. Raina, A.K. Gupta coastal region of India. Applied Animal and M. Bhakat. 2009. Effect of peripartum Husbandry and Rural Development, 3(1): disorders on reproduction performance 1-5. traits in murrah buffaloes at an organized Yadav, B.S., M.C. Yadav, A. Singh and F.H. Khan. farm. Buffalo Bull., 28(4): 176-211. 2007. Study of economic traits in Murrah Khan, M.A., M.S. Khan., H.R. Chaudhry, J. Khan buffaloes. Buffalo Bull., 26: 10-14. and S. Saleem. 1995. Data analysis of epidemiological and economical losses and ranking order of buffalo and cattle disease in Punjab. Collage of Veterinary Science,

108 Original Article Buffalo Bulletin (March 2016) Vol.35 No.1

STANDARDIZING PREGNANCY RATE OF INDIAN MURRAH BUFFALOES FOR HIGHER MILK YIELD

V. Jamuna*, A.K. Chakravarty, Vijay Kumar, M.A. Mir and Vikas Vohra

ABSTRACT INTRODUCTION

Study revealed that increase in milk India is among few countries in the world, production has lead to decline in fertility which has contributed richly to the international performances of Murrah buffaloes. Data pertaining livestock biodiversity and improvement of livestock to 1224 lactation records of Murrah buffaloes genepool. India contributes about 63 percent of spread over a period 19 years were analyzed in total world buffalo milk and 95 percent of buffalo the study. It was observed that pregnancy rate milk in Asia comes from India. Among the various (fertility) depicted negative phenotypic association buffalo breeds in India, Murrah is the important with 305 days or less milk yield (-0.08±0.04), milch breed with superior genetic potential for milk wet average (-0.12±0.02) and test day ive milk production and constitutes around 19.5% of total yield (-0.09±0.03). It was observed that to achieve buffalo population in the country (FAO, 2012). The around 2000 kg MY, 7.5 kg WA and 7.7 kg in TD breed is versatile and has shown wide adoption for 5 MY, the level of pregnancy rate varied between milk production across the length and breadth of 30-50%. Under the present study an attempt was the country. made to quantify the decline of fertility with the A multitude of studies in dairy cattle have increase of milk production in Murrah buffaloes. shown that selection for higher milk yield alone is The per unit change in fertility with respect to associated with reduced health and fertility (Pryce milk yield in Murrah buffaloes, were studied et al., 1999). Fertility is economically important as using multiple regression analysis. Increasing it brings buffalo into lactation, reduces reproductive one kilogram milk yield in test day iveth (125disorders and maximizes the proitability by in time day) pregnancy rate reduced by about 1.3%, and calf crop. Most of the developed countries have increase in one kilogram milk yield per day in 305 already developed a national genetic evaluation for days or hundred kilogram in 305 days of Murrah female fertility along with the production traits and buffaloes pregnancy rate reduced by 0.9%. they have used fertility traits like Pregnancy Rate (PR) and Service Period (SP) for genetic evaluation Keywords: Life time, pregnancy rate, production, of dairy cattle (VanRaden, 2004; De Vries, 2010 and productivity, standardization Cabrera, 2011). Pregnancy rate (PR) measures the

Animal Genetics and Breeding Division, ICAR- National Dairy Research Institute, Karnal, Haryana, India, *E-mail: [email protected]

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percentage of non-pregnant animals that become production traits were recorded. The reproduction pregnant during each estrous cycle, because each traits included pregnancy rate (PR) and life time estrous cycle represents only one chance for an pregnancy rate (LTPR). The production traits animal to become pregnant. A comparison of included 305 days or less milk yield (MY), wet pregnancy rate with other fertility traits from 14 average (WA), test day ive milk yield (TD 5 MY), countries has been conducted by Interbull, 2007 life time 305 days or less milk yield (LTMY), life and the result indicated that pregnancy rate is time wet average (LTWA) and life time test day highly correlated with other fertility traits. These ive milk yield (LT TD 5 MY). correlations indicate that pregnancy rate can be In the present study, the normal lactation expected to improve fertility in animals (Jorjani, was considered as the period of milk production 2007). by a buffalo for at least 100 days or a minimum Conventional selection for milk has made of 500 kg milk produced and the animal calved, buffaloes more proitable producers in the country dried under normal physiological conditions. and continuous selection for increased milk The information of 1224 lactation records of 522 production has led to long service period in buffalo Murrah buffaloes were subjected to standardization result in reduced pregnancy rate and decrease in and normalization of traits, and subsequently 853 calf crop and milk yield on lifetime basis. Until lactation records were obtained (distributed as recently under Buffalo Improvement programme 404, 230, 138 and 81 completed irst, second, third in India, no efforts have been made in using genetic and fourth lactations) for production traits and selection of fertility to improve performances of 748 records (distributed as 340, 204, 126 and 78 buffaloes. The increase in milk production causes completed irst, second, third and fourth parity) for how much apparent decline in fertility in each fertility traits. lactation as well as in life time production has not been explored in Murrah buffaloes. The objective Statistical analysis of the study was standardization of level of fertility The genetic parameters for fertility and with respect to milk production and quantiies production traits were estimated by REML using the decline of fertility with the increase of milk Wombat software (Meyer, 2010) applying a production in Murrah buffaloes. repeatability animal model, Yijklm= µ + Ai + Sj + Pk th + Pal + AGm + eijklm where, Yijklm, ijklm observation of fertility and production traits; m, population

MATERIALS AND METHODS mean; Ai, Animal random effect; Sj, Fixed effect of jth season of calving (winter, summer, rainy and th Data Size: The breeding information autumn);Pk, Fixed effect of k period of calving (1 to th of 522 Murrah buffaloes scattered over a period 8); Pal, Fixed effect of l parity(1 to 4), AGm, Fixed of 19 years from January 1993 to October 2011, effect of mth age group of calving(<37 months, 37-

maintained at Dairy Cattle Breeding Division 51 months, >51 months); and eijklm, Random error is and Livestock Research Station, National Dairy normally and independently distributed with mean 2 Research Institute, Karnal were collected. The basic zero and variance (σ e). information of Murrah buffaloes, reproduction and

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Estimation of Pregnancy Rate of Murrah Murrah buffaloes, regression analysis was done in buffaloes the study using the General Linear Model (GLM) The Voluntary Waiting Period (VWP) is the procedure of SAS (SAS Institute 2009), presented period after calving during which no inseminations in Table 3. Following model was used: Yi = a + b1X1 occur, voluntarily left by the management for better + ei where Yi is pregnancy rate (PR), a is intercept, pregnancy rate. VWP in Indian Murrah buffaloes b1 is regression coeficient estimated, X1 is MY/ has been standardized as 63 days (Patil et al., 2013). WA/TD 5 MY and eij is Random error ~NID (0, 2 Pregnancy rate in each lactation was estimated as σ e). The relation of change in life time pregnancy PR = 21 / (Service Period – Voluntary Waiting rate with respect to per month milk yield in life

Period + 11). The constant factors 11 centralize time was analyzed using the regression model: Yi the measure of possible conception within each 21 = a + b1X1 + ei where Yi is life time pregnancy rate days time period. (LTPR), a is intercept, b1 is regression coeficient

estimated, X1 is per month milk yield and eij is 2 Estimation of life time traits Random error ~NID (0, σ e). An empirical estimation suggests that in buffaloes about 90.27 % was produced up to fourth lactation and thereafter milk production started to RESULTS AND DISCUSSION decline. Therefore, each life time performance trait was estimated as average of four parity /lactations. Fertility traits of Murrah buffaloes were Life time pregnancy rate (LTPR) was estimated as found inluenced by period and season of calving, average pregnancy rate of buffaloes completed four parity and age group of calving, while production parity. Life time production or productivity was traits were mainly inluenced by period of calving estimated as average production or productivity of and parity. Least-squares means of SP, LL and MY buffaloes completed four lactations. were estimated as 28±5.58 days, 286.06±1.72 days and 2078.20±31.21 kg, respectively. By keeping Standardization of level of fertility for optimum days to irst service or VWP as 63 days, the average milk production / productivity pregnancy rate (PR) and life time pregnancy rate Standardization of fertility and production/ (LTPR) of Murrah buffaloes were estimated as productivity were done parity wise, overall and 0.36±0.013 and 0.38±0.03, respectively. Wet life time (Table 1). For standardizing the level of average (WA) of Murrah buffaloes was estimated fertility with milk production and productivity, the as 7.29±0.06 kg. On analysis of test day milk, it pregnancy rate was classiied into seven classes was observed that TD 5 (125th day) MY had the with the increment of 10% in all parities, overall highest phenotypic association (0.79±0.31) with and life time. The number of buffaloes scattered in MY out of eleven test day milk yields in Murrah different classes of pregnancy rate were identiied buffaloes. The average LTMY, LTWA and LT and their corresponding average pregnancy rate in TD 5 MY were estimated as 2188.00±41.50 kg, relation to MY, WA and TD5 MY were estimated. 7.54±0.12 kg and 8.52±0.15 kg respectively. The To explore the per unit change in fertility fertility traits were more inluenced by environment with respect to milk production/ productivity in and management of the herd, as pregnancy rate

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encompasses buffalo’s ability to return to normal 2118.66 kg to 1994.96 kg, however the decline of reproductive status after calving, to display visible productivity was found less i.e. from 7.11 kg to signs of estrus, to conceive when inseminated and 7.04 kg and 8.52 kg to 8.45 kg in WA and TD 5 to maintain the pregnancy. MY. The heritability of PR, MY, WA and TD 5 MY were estimated as 0.02±0.005, 0.17±0.04, Level of fertility with milk production and 0.15±0.03 and 0.12±0.04. The pregnancy rate had productivity in overall and life time low but negative phenotypic associations with In overall lactations, with the increase of MY (-0.08±0.04), WA (-0.12±0.02) and TD 5 MY pregnancy rate from 34% to 45%, the corresponding (-0.09±0.03).The association of pregnancy rate production declined from 2053.98 kg to1946.64 with life time was 0.15±0.03. The buffalo fertility kg, productivity declined from 7.50 kg to 7.33 kg had negative correlation with milk yield but is a in WA, 7.82 kg to 7.63 kg in TD 5 MY depicted in major component of longevity, as pregnancy rate Figure 1, 2 and 3. However, with the increase of had positive association with life time. same pregnancy rate in life time, the productivity of life time reduced from 7.12 kg to 7.00 kg and 7.60 Level of fertility (PR) with milk production kg to 7.33 kg in WA and TD 5 MY, respectively. (MY) and productivity (WA/TD 5 MY) in The life time milk yield reduced from 2249.56 kg different parities to 2089.65 kg, as depicted in Figure 4, 5 and 6. The pregnancy rate of Murrah buffaloes From the perusal of data from different varied from less than 10% to more than 90% in each parities, overall and life time, pregnancy rate of of the four parity (Table 1). The average pregnancy Murrah buffaloes had been classiied into three rate ranged from 30% to 50%. In the irst parity, classes, i.e. the buffaloes with less than 30% it was observed that with the increase of average pregnancy rate, 31-50% pregnancy rate and pregnancy rate of Murrah buffaloes from 34% to more than 50% pregnancy rate. Average milk 45%, the level of average production was found productivity and production corresponding to three decreased from 2040.46 kg to 1993.32 kg, 7.15 kg ranges of pregnancy rate in different parities as well to 6.58 kg in WA and 8.52 kg to 6.59 kg when TD as in over all parities has been presented in Table 5 MY was considered. Almost similar trend were 2. Therefore, under Murrah Buffalo Improvement observed in other parities also. In the second parity, breeding programme in India, once the target is it was observed that with the increase of 35% to to obtain 2000 kg MY, 7.4 kg WA and 7.7 kg TD 46% pregnancy rate, MY reduced from 2113.40 5 MY, the level of pregnancy rate should be in kg to 2034.48 kg, where WA declined from 7.45 between 30-50%. kg to 7.32 kg and TD 5 MY declined from 7.95 kg to 7.81 kg. In the third parity, with the increase Linear evaluation of fertility and productivity / of 36% to 47% pregnancy rate, MY, WA and TD production in Murrah buffaloes 5 MY from 2008.02 kg to 1906.31 kg,7.29 kg to The percent change in of fertility with the 7.19 kg and 7.85 kg to 7.52 kg, respectively. In the corresponding unit increase of milk productivity fourth parity, though the increase of pregnancy rate and production was studied in different lactations, was from 33% to 46%, milk yield reduced from overall and life time in Murrah buffaloes (Table 3).

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The change in per unit (percent) of fertility with the genetic evaluation of Indian Murrah buffaloes the corresponding increase of per month milk and a sustainable level of fertility should be production in life time of Murrah buffaloes was also maintained for further improvement in milk yield analyzed using simple regression model. In Murrah of buffaloes. buffaloes, average life time was estimated as 4.10 years, average life time milk yield and average per month milk yield in life time were estimated as CONCLUSION 2188.00 kg and 151.44 kg, respectively. The results revealed that by increasing hundred kilograms of In the present study, fertility had found per month milk yield in life time, the pregnancy negatively associated with production traits of rate will reduce by about 13% in life time. Murrah buffaloes. The increase in milk production Scanty reports were available about the or productivity causes how much apparent decline literature reviewing the standardization of fertility in fertility in each lactation as well as in life time with milk yield. According to De Vries (2010) one production has been revealed in the study. It was month life time is worth 112 lbs (approx. 50.80 kg) found that to achieve around 2000 kg milk yield in milk in a lactation and 1.5% daughter pregnancy 305 days, 7.5 kg wet average and 7.7 kg in test day 5 rate in Holstein dairy cattle. He also reported that (125th day) milk yield, level of fertility (pregnancy one percent daughter pregnancy rate is worth 73 rate) should be in between 30-50%. Increasing one lbs (approx. 33.11kg) milk in 305-day lactation. kilogram milk yield in test day ive (125th day) Cabrera (2011) reported that improving pregnancy pregnancy rate reduced by about 1.3%, and increase rate by 5% would increase economic gain of milk in one kilogram milk yield per day in 305 days or production by 21.41 kg per year, while De Vries hundred kilogram in 305 days of Murrah buffaloes (2010) reported that improving pregnancy rate pregnancy rate reduced by 0.9%. By increasing by 1% will increase the milk production 7.13 kg hundred kilograms of per month milk yield in life per year, by 2.85 kg per year (Rogers and Cooper, time, the pregnancy rate reduced by about 13% in 2011), while Hansen (2007) reported an increase life time. In the present study, fertility had positive of 4.26 kg per year in cattle. In India, economic correlation with life time or productive life, which estimates of percent increase of pregnancy rate indicated that selection of buffaloes for fertility with milk yield have not been explored. In present along with milk production increases their life study, fertility had low negative association with time and proitability of herd. The study quantiies milk yield. The present study quantiies the decline the decline of fertility (pregnancy rate) with the of pregnancy rate with increase of lactation milk increase of milk production / productivity in Indian yield in Indian Murrah buffaloes. However, in Murrah buffaloes. The indings of the study clearly the absence of any direct selection pressure on depicts that importance should be given to fertility pregnancy rate of Murrah buffaloes due to low traits like pregnancy rate along with production heritability (h2 0.02), there has been downward traits for improving milk yield of buffaloes, thereby trend in fertility associated with the selection for increasing the economic eficiency of herd. milk yield. The study emphasis the importance of fertility (pregnancy rate) along with milk yield in

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Table 1. Parity wise, Overall and life time level of fertility (PR) with milk productivity (TD 5 MY / WA) and production (MY) in Murrah buffaloes. Range First parity Second parity of PR MY WA TD 5 MY PR MY WA TD5 MY N N PR (%) (%) (kg) (kg) (kg) (%) (kg) (kg) (kg) <10 33 9 2137.95 7.39 8.52 32 9 2255.46 8.00 8.37 11-20 88 14 2239.26 7.12 8.28 43 15 2036.66 7.63 8.12 21-30 58 25 2095.98 7.33 8.23 22 25 2234.58 7.70 8.08 31-40 26 34 2040.46 7.15 7.97 38 35 2113.40 7.45 7.95 41-50 13 45 1993.32 6.58 7.42 14 46 2034.08 7.32 7.81 51-60 12 55 1961.78 6.55 7.40 9 57 1986.27 7.25 7.84 >60 36 98 1821.37 6.44 6.59 33 100 1915.96 6.97 7.63 Third parity Fourth parity <10 15 9 2201.45 7.79 8.53 11 9 2594.79 8.03 10.25 11-20 34 15 2025.76 7.68 8.03 20 15 2199.22 7.56 8.52 21-30 15 25 2082.18 7.68 8.23 11 27 2150.68 7.34 8.61 31-40 19 36 2008.02 7.29 7.85 4 33 2118.66 7.11 8.52 41-50 6 47 1906.31 7.19 7.52 5 46 1994.96 7.04 8.45 51-60 6 54 1877.91 6.77 7.48 3 54 1989.39 6.97 7.52 >60 13 95 1772.31 6.38 7.30 10 97 1847.84 6.70 7.25 Overall Lifetime <10 82 9 2239.7 8.60 7.62 - - - - - 11-20 19 14 2144.4 7.95 7.65 13 15 2393.5 8.00 8.00 21-30 11 25 2104.2 8.10 7.58 15 26 2280.6 7.72 7.43 31-40 74 34 2053.9 7.82 7.50 10 34 2249.5 7.60 7.12 41-50 45 45 1946.6 7.63 7.33 12 45 2089.6 7.33 7.00 51-60 30 55 1923.2 7.26 7.16 14 71 2014.2 7.05 6.98 >60 12 95 1914.3 6.90 7.02 - - - - -

PR, Pregnancy Rate; TD 5 MY, Test day ive milk yield; WA, Wet Average MY, 305 Days or less Milk Yield.

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Table 2. Average milk production and productivity corresponding to pregnancy rate. Range of 305 Days or less Milk Yield (kg) PR (%) Overall First parity Second parity Third parity Fourth parity <30 2162.78 (119) 2124.95(39) 2431.05 (49) 2189.91(25) 2314.90(9) 30-50 2000.31 (388) 2024.75(179) 2165.12 (81) 1983.61(64) 2056.81 (42) >50 1918.81 (122) 1856.47(48) 1931.03 (57) 1805.54(19) 1981.62(13) Wet Average (kg) Overall First parity Second parity Third parity Fourth parity <30 7.63 (119) 7.24 (39) 7.90(49) 7.71(25) 7.27 (9) 30-50 7.44 (388) 6.98 (179) 7.41 (81) 7.25 (64) 7.04 (42) >50 7.05 (122) 6.46 (48) 7.03 (157) 6.50 (19) 6.77 (13) Test Day 5 Milk yield (kg) Overall First parity Second parity Third parity Fourth parity <30 8.13 (119) 8.03(39) 8.19 (49) 8.35 (25) 8.99 (9) 30-50 7.75 (388) 7.69 (179) 7.76 (81) 7.95(64) 8.32 (42) >50 7.37 (122) 6.76 (48) 7.34(157) 7.67 (19) 7.75 (13)

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Table 3. Parity wise regression of productivity and production on fertility (Pregnancy Rate) in Murrah buffaloes. MY WA TD 5MY Average Parity Models Intercept R2(%) b b b error I 0.2646 - - -0.008 0.30 0.13 First II 0.3061 - -0.017 - 2.47 0.15 III 0.4112 -0.00003 -- 0.70 0.08 I 0.4606 - - -0.011 0.70 0.02 Second II 0.4634 - -0.024 - 1.41 0.11 III 0.5520 -0.00007 -- 1.90 -0.04 I 0.4151 - - -0.011 0.70 0.02 Third II 0.4956 - -0.042 - 8.89 0.16 III 0.5180 -0.00009 -- 9.47 -0.01 I 0.3959 - - -0.017 2.47 0.13 Fourth II 0.5802 - -0.044 - 6.41 0.07 III 0.6721 -0.00014 0.14 0.07 I 0.3647 - - -0.013 0.7 0.09 Overall II 0.4192 - -0.009 - 0.2 0.0002 III 0.5047 -0.00009 - - 1.47 0.14 I 0.5829 -0.0002 1.88 -0.04 Lifetime II 0.6358 - -0.034 - 4.88 -0.02 III 0.8718 -0.023 - - 15.0 -0.01

MY, 305 Days or less Milk Yield; WA, Wet Average; TD 5 MY, Test day ive milk yield; b, regression co-eficient estimated; R2, coeficient of determination.

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Figure 1. Level of fertility (pregnancy rate) and milk yield upto four lactations of Murrah buffaloes.

Figure 2. Level of fertility (pregnancy rate) and wet average upto four lactations of Murrah buffaloes.

Figure 3. Level of fertility (pregnancy rate) and test day ive milk yield upto four lactations of Murrah buffaloes.

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Figure 4. Level of life time fertility (pregnancy rate) and life time milk yield in Murrah buffaloes.

Figure 5. Level of life time fertility (pregnancy rate) and life time wet average in Murrah buffaloes.

Figure 6. Level of life time fertility (pregnancy rate) and life time test day 5 milk yield in Murrah buffaloes.

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ACKNOWLEDGEMENT V. Jamuna and V. Vohra. 2013. Development of predictive model for daughter pregnancy rate and standardization of voluntary The authors express their sincere gratitude waiting period in Murrah buffalo. Trop. to Director and Vice Chancellor, National Dairy Anim. Health Pro., 46: 279-284. Research Institute, Karnal for providing all research Pryce, J.E., B.L. Nielsen, R.F. Veerkamp and G. facilities for successful completion of the study. Simm. 1999. Genotype and feeding system effects and interactions for health and fertility traits in dairy cattle. Lives. Prod. REFERENCES Sci., 57: 193-201. Rogers, W.J. and J.B. Cooper. 2011. Genetic Cabrera, V.E. 2011. The economic value of changes differences in fertility among U.S. dairy in 21-day pregnancy rate and what controls cattle breeds. Progressive Dairyman. 26: this value. Department of Dairy Science 112-117. University of Wisconsin-Madison. SAS Institute Inc. 2009. User’s guide, Release 9.2. De Vreis, A. 2010. Economic improvements of SAS Institute Inc., Cary, NC, USA. genetic improvements in milk production, Raden, V. 2004 Selection on net merit to improve reproduction and productive life. life time proit. J. Dairy Sci., 87: 3125-3131. Department of Animal Sciences, University of Florida (http://edis.ifas.ul.edu). Food and Agriculture Organization. 2012. Buffalo Production and Research. (www.fao.org. com) Hansen, J. 2007. Improving Dairy Cow Fertility through Genetics. Department of Animal Sciences, University of Florida, Gainesville. In Proceedings of 44th Florida Dairy Production Conference, Gainesville, USA. Interbull. 2007. Interbull routine genetic evaluation for female fertility traits. (www.interbull. org). Jorjani, H. 2007. International genetic evaluations for female fertility traits. Interbull Bulletin, 34: 57-64. Meyer, K. 2010. Wombat. A program for mixed model analyses by Restricted Maximum Likelihood. University of England, Armidale, Australia. Patil, C.S., A.K. Chakravarty, A. Singh,V. Kumar,

119 Buffalo Bulletin (March 2016) Vol.35 No.1 Original Article Buffalo Bulletin (March 2016) Vol.35 No.1

SERO-PREVALENCE OF BRUCELLOSIS IN BUFFALOES IN GUJARAT: AN ON-FARM CASE STUDY

B.J. Trangadia1 and R.M. Patel2

ABSTRACT INTRODUCTION

The present study was conducted in the Bovine brucellosis is caused by Brucella month of August, 2011 to determine the sero- abortus, which is responsible for heavy economic prevalence of brucellosis in organized herd of losses to the developing countries by causing buffaloes with a history of abortions located in late term abortions, infertility and reduced milk North Gujarat. For the purpose, a total of 117 serum production. In India, the high sero-prevalence of the samples were collected from buffaloes and were disease in buffaloes has been reported as 14.61% screened for the presence of antibodies against to 65.31% by various workers (Sharma and Sani, Brucella by Rose Bengal plate test. The overall 1995; Trangadia et al., 2010; Jagapur et al., 2013). sero-prevalence recorded as 25.64%. Age-wise Accurate diagnosis, quarantine and proper culling prevalence indicated that 30.53% animals reacted of the infected animals from the herd are required positive to the test was of above 60 months age. for control and prevention of the disease. Therefore, However, in less than 60 months of age groups, in the present study, an attempt was made to study only 4.55% animals were positive. Prevalence the prevalence of brucellosis in organized herd of of brucellosis in aborted animals was 72.00% as buffaloes with a history of abortion. compared to 13.04% in animals without history of abortion. The study indicated high-seropositivity of brucellosis in the farm and to prevent economic MATERIALS AND METHODS losses to the farm, it is necessary to develop and Sample details adopt various control measures. During the month of August 2011, a total of 117 whole blood samples were collected Keywords: brucellosis, sero-prevalence, buffaloes, from Mehsani buffaloes reared in a commercial Rose Bengal Test farm located in North Gujarat and the herd had a history of abortion and repeat breeding. Serum was

1Department of Veterinary Pathology, Vanbandhu College of Veterinary Science and Animal Husbandry, Navsari Agricultural University, Navsari, Gujarat, India, E-mail: [email protected], [email protected] 2Department of Veterinary Medicine, Vanbandhu College of Veterinary Science and Animal Husbandry, Navsari Agricultural University, Navsari, Gujarat, India

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separated and stored at -20oC until used. of 17.64% in buffaloes by RBPT. Contrary to these indings, Sutariyaet al. (2005) reported a Rose Bengal Plate Test (RBPT) comparatively low prevalence 7.76% in buffaloes RBPT antigen was procured from the Indian from samples screened from the State of Gujarat. Veterinary Research Institute (IVRI), Izatnagar, However, a higher sero-prevalence of 52.94% in Uttar Pradesh (UP), India. The test was performed buffaloes by RBPT was reported from animals according to procedure described by World reared in organized farms located in various Organization for Animal Health (OIE, 2008). The parts of India (Trangadia et al., 2010). Similarly, result was recorded after the mixture was rocked Chauhan et al. (2000) also reported a higher gently for 4 minutes at room temperature. Any sign incidence of brucellosis as 44% among buffaloes of agglutination was considered as positive. in Gujarat. Selective sampling of animals with the history of abortion, repeat breeding and retention of placenta may be responsible for the report of RESULTS AND DISCUSSION higher prevalence in the study (Mahajan et al., 2011). In the present study, the seroprevalence Age-wise sero-prevalence of brucellosis of brucellosis was recorded as 25.64 percent in was shown in Table 1. Out of 95 animals above 60 buffaloes by RBPT. In agreement to the present months age group, 30.53% animals (29) had shown study, Mahajan et al. (2011) reported a prevalence

Table 1. Age-wise sero-prevalence of brucellosis in buffaloes based on RBPT.

Numbers of Age of animals (months) Numbers of sera tested positive reactors (%) 13-60 22 1 (4.55) > 60 95 29 (30.53) Overall 117 30 (25.64)

Table 2. Prevalence of brucellosis in buffaloes with a previous history of abortion based on RBPT.

Numbers of Previous abortion history Numbers of sera tested positive reactors (%) No 92 12 (13.04) Yes <3 months gestation 4 2 (11.11) 3-6 months gestation 5 2 (11.11) >6 months gestation 16 14 (77.78) 25 18 (72.00) Overall 117 30 (25.64)

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positive reaction against brucellosis by RBPT, during the middle and late stages of pregnancy where as in the below 60 months age groups, only (Samartino and Enright, 1992). 4.55% (One out of 22 animals) animals reacted The results of the study indicated high positively. Rahman et al. (2011) reported a high sero-positivity of brucellosis among bovines prevalence (4.92%) in >48 months age group as housed in an organized farm. Such situation in the compared to 13-48 months age group in buffaloes farms warrants immediate attention and preventive by RBT (2.63%). It is possible that the higher measures like restricted movement of animals, prevalence of brucellosis among older cows may proper screening of animals before procurement, be related to their advanced age, as the organism quarantine the animals before entry to the main may remain latent or chronic for an unspeciied herd etc. should be adopted immediately. period before manifesting as clinical disease. Alternatively, aged animal have more chances of exposure to the bacteria and contracting disease. ACKNOWLEDGEMENT (Nicoletti, 1980). The authors are thankful to the owner of the The sero-prevalence of brucellosis with farm for their co-operation and help in collecting regards to the history of abortion is depicted the blood samples from the buffaloes. in Table 2. The prevalence of brucellosis was recorded 72% (18/25) in aborted animals as compared to 13.04% (12/92) in animals without history of abortion. The prevalence among animals REFERENCES carrying pregnancy of >6 months was 77.78% (14/18) and in each of 3-6 months and <3 months Chauhan, H.C., B.S. Chandel and N.M. Shah. 2000. of gestation was 11.11 percent (2/18). However, Seroprevalence of brucellosis in buffaloes both the animals aborted before completing the in Gujrat. Indian. Vet. J., 77: 1105-1106. 3 months of gestation had a history of the torsion. Ibrahim, A.E. and N. Habiballa. 1975. A survey Similarly, Ibrahim and Habiballa (1975) reported a of brucellosis in Messeriya cows of Sudan. prevalence of brucellosis as 14.2 percent in aborted Trop. Anim. Health Pro., 7: 245-246. cows. Rahman et al. (2011) reported a higher Jagapur, R.V., R. Rathore, K. Karthik and R. prevalence of brucellosis in animals with the Somavanshi. 2013. Seroprevalence studies history of abortion as 60% than in animals with no of bovine brucellosis using indirect-enzyme- abortion record (1.16%). Contrary to our indings, linked immunosorbent assay (i-ELISA) at a high seropositivity was recorded (19%) in organized and unorganized farms in three animals aborted in the 6th month of their gestations different states of India. Vet. World, 6(8): as compared to only 6 percent in the 9th month of 550-553. their pregnancy (Khan and Soomro, 2013). The Khan, S.A. and A.H. Soomro. 2013. Seroprevalence host mechanisms responsible for the increased of brucellosis in cattle and buffaloes in susceptibility to infection in advanced pregnancy district hyderabad. IJAVMS, Published are not known, but they may be related to the Online First: 14 Jan, 2013. Web Accessed differential susceptibility of placental trophoblasts on November 15, 2013.

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Mahajan S., R. Agrawal and N. Pande. 2011. A comparative evaluation of different diagnostic tests for brucellosis in buffaloes. Buffalo Bull., 30(1): 75-78. Nicoletti, P. 1980. The Epidemiology of bovine brucellosis. Adv. Vet. Sci. Comp. Med., 24: 75. OIE. 2008. Manual of Standards for Diagnostic Tests and Vaccines, 6th ed. Paris, France. Rahman, M.S., M.O. Faruk, M. Her, J.Y. Kim, S.I. Kang and S.C. Jung. 2011. Prevalence of brucellosis in ruminants in Bangladesh. Vet. Med-Czech., 56(8): 379-385. Samartino, L.E. and F.M. Enright. 1992. Interaction of bovine chorioallantoic membrane explants with three strains of Brucella abortus. Am. J. Vet. Res., 53: 359-363. Sharma, J.K. and S.S. Sani. 1995. Sero prevalence of brucellosis among farm animals of Punjab. Indian Vet. J., 72: 881-882. Sutariya, P.H., A.N. Kanani, H.J. Patel, M.J. Dave, G.S. Parmar, N.D. Parmar and R.B. Shukla. 2005. Estimation of prevalence rate of brucellosis in cattle and buffaloes by ELISA testing in Gujarat, 49p. Abstract presented at National Seminar of Assistant to States for Control of Animal Diseases (ASCAD). Trangadia, B.J., S.K. Rana, F. Mukherjee and V.A. Srinivasan. 2010. Prevalence of brucellosis and infectious bovine rhinotracheitis in organized dairy farms in India. Trop. Anim. Health Pro., 42: 203-207.

124 Original Article Buffalo Bulletin (March 2016) Vol.35 No.1

EFFECT OF ALFALFA FODDER SUPPLEMENTATION ON ENTERIC METHANE EMISSION MEASURED BY SULFUR HEXAFLUORIDE TECHNIQUE IN MURRAH BUFFALOES

P.K. Malik1,* and K.K. Singhal2

ABSTRACT pH, ammonia nitrogen and total volatile fatty acid production did not differ (p>0.05) among the Phyto-sources possessing different groups. A signiicant (p<0.05) decrease in acetate secondary metabolites are under investigation for production was also with concomitant increase the mitigation of enteric methane emission from in propionate production. Results revealed a non- livestock. Alfalfa (Medicago sativa) contains signiicant change in archaeal population, while considerable saponin which is known for methane protozoal population were adversely affected and reduction through anti-protozoal action. Therefore, about 20% less numbers were observed in test this study was undertaken to ascertain the effect of group. From the study it may be concluded that saponin containing alfalfa fodder (Medicago sativa; saponin from natural feed resources like alfalfa second cut) on enteric methane emission in Murrah fodder at a level of 6.0 g/kg DM can be used for buffaloes quantiied using sulfur hexaluoride the signiicant enteric methane reduction.

(SF6) technique. Twelve male Murrah buffalo calves were randomly divided into two groups of Keywords: enteric methane, leguminous fodder, six animals each. Buffalo calves in control group Murrah buffaloes, saponin, sulfur hexluoride were fed on wheat straw and concentrate based diet (R: C, 60:40), while animals in test group were supplemented with saponin containing alfalfa INTRODUCTION fodder (second cut, 30%) replacing wheat straw on w/w basis. Effect of alfalfa fodder supplementation Current atmospheric methane on rumen fermentation characteristics, archaeal concentration is 155% higher than the pre- and protozoal population were also studied. industrial concentration (IPCC 2007). Livestock Enteric methane emission in control and test group are the major contributor to anthropogenic methane buffaloes was reported as 78.09 and 61.39 g/d, wherein Indian livestock contributes substantially respectively. In this study, about 21% reduction to global enteric methane emission. Due to the huge in enteric methane emission was achieved on the dispension of methane, United Nation’s Food and feeding of saponin containing alfalfa fodder at Agriculture Organization recently stated livestock 30% level of the diet. However, dry matter intake, are the major threat for environment. As per one

1Animal Nutrition, ICAR-National Institute of Animal Nutrition and Physiology, Bangalore, India, *E-mail: [email protected] 2National Dairy Research Institute, Karnal, India

125 Buffalo Bulletin (March 2016) Vol.35 No.1 estimate, approximately 37% of anthropogenic MATERIALS AND METHODS methane and 12-13% of the total atmospheric methane is emitted from livestock. Asia is the Animals and feeding harbor for 179.5 million buffaloes and India alone Twelve male Murrah buffalo calves is possessing approximately 56.7 percent of the (BW 172.62±0.36 Kg) were selected from the total (FAO, 2008). During last 10 years, the world herd of institute and divided into two groups of buffalo population increased at a rate of 1.49% per six animals each. Necessary permission from year, wherein, in India and Asia, the increase was the Animal Ethical Committee was obtained for 1.53% and 1.45% per annum, respectively. Various conducting the experiment. Buffalo calves were agencies have promulgated the enteric methane kept in a well ventilated shed having a provision emission from Indian livestock in the tune of 7.2- for individual feeding. To keep the animals free 12.9 Tg/y (Malik et al., 2012). Buffaloes are the from external and internal parasites, butox 0.5% noteworthy emitters to this and contribute about (v/v) and albandazole (0.5 mg/kg BW) were given, 2.8 Tg/y (Singh, 1998). The need for curtailing the respectively. Buffalo calves were fed as per the methane emission from ruminants is mandatory Kearl (1982) to meet the nutritional requirement. from global warming and dietary energy loss point Animals in group I (control) was fed on wheat straw of view (Malik et al., 2013). and concentrate based ration (60:40). Concentrate Worldwide attempts have been made mixture comprising maize grain (33%), groundnut for the mitigation of livestock methane using cake (20.2%), mustard cake (12%), wheat bran halogenated methane analogues, antibiotics, fat (20%), deoiled rice bran (11%), urea (0.8%), and oils, organic acids, but the response from mineral mixture (2%) and common salt (1%) was majority of these was highly variable and adoption offered to the animals of control group. Animals rate due to high cost, reduced feed intake, toxicity under group II received the total mixed ration to inhabiting rumen microbes/host animal and consisting wheat straw, concentrate and second transitory effect was very low (Malik et al., 2012). cut alfalfa fodder (Medicago sativa) in the ratio Plant secondary metabolites such as saponin may of 30:40:40. Concentrate mixture for the group II be a potent agent in achieving the signiicant animals was prepared by mixing of maize grain reduction in methane emission from livestock. So (24%), groundnut cake (15%), mustard cake (8%), far in vitro studies have been conducted to evaluate wheat bran (42%), deoiled rice bran (8%), mineral the effect of saponin on methane production (Malik mixture (2%) and common salt (1%). Second cut and Singhal, 2008; Wang et al., 2011), and reports alfalfa (Medicago sativa) fodder was selected for for methane reduction in buffaloes on using saponin the supplementation to buffalo calves on the basis from natural sources is very limited. Keeping these of comparatively higher saponin content among facts in view, the study was under taken to assess irst three cuts. The saponin content of alfalfa fodder the effect of saponin containing alfalfa fodder was determined as per the method of Yosioka et al. supplementation on enteric methane emission in (1974). The CP was maintained about 12.0% of the buffaloes. diet for both control and test group.

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x Measuring methane emission using Sulfur QCH4 = QSF6 [{CH4}Y-{CH4}b] Hexaluoride (SF) tracer technique [SF ] 6 6 In vivo methane emission from Murrah

buffalo calves fed on control and test diet was QCH4 is methane emission rate; QSF6 is

measured using sulfur hexaluoride (SF6) trace the release rate of SF6; CH4b is the concentration technique of Johnson et al. (1994). Four successful of methane in background sample and CH4 Y is the methane gas collections from each individual methane concentration in PVC canister. buffalo were made to quantify the daily emission under control and test group. Before actual gas Rumen fermentation, archaeal and protozoal collection from the calves, PVC canisters were population ixed around the neck of each animal for 2 days to At the end of gas collection, rumen liquor acclimatize them. The gas was collected into PVC samples were collected from buffalo calves for canister through halters tied around the head and in three consecutive days through stomach tube front of nostrils. The initial and inal pressure of the (Figure 2). Rumen liquor was stained through PVC canisters weres measured at the time of tying four layers of muslin cloth and stored into an and removal. Brass Permeation tubes (brass, 1.25” insulated anaerobic container. Rumen liquor pH length, 3x 16” ID) with central window were illed was recorded using digital pH meter just after the with SF gas itted with Telon cap and 2µ brass frit 6 collection of liquor. However, ammonical nitrogen, using standard protocol. Frit was ixed to facilitate total volatile fatty acids (TVFA) and volatile fatty the release of SF from brass permeation tube kept 6 acids (VFA) fractionation were determined using at room temperature. Daily weight of permeation standard protocol of Conway (1950), Barnett and tube was recorded until the release rate of SF 6 Reid (1957) and Erwin et al. (1961), respectively. became constant, thereafter; the permeation tubes Methanogenic archaea as well as protozoal were placed into rumen. The SF and CH emitted 6 4 population were also enumerated in rumen liquor from each animal were collected into an evacuated samples collected from buffalo calves fed on yoke like PVC canister through a capillary tube control and test diet. Anaerobic media described ending just above the nostrils of the animal (Figure by Ranade and Gadre (1988) for methanogens was 1). A similar canister was hanged in animal shed used for enumerating the numbers while protozoal to record background concentration of CH . 4 numbers was enumerated through haemocytometry Concentration of CH and SF gases in collected gas 4 6 microscopic counting method. Data samples was determined using gas chromatograph were analyzed in SPSS 16 using one way Anova itted with Flame Ionization Detector (FID) and and means were compared for statistical difference. Electron Capture Detector (ECD) for methane and

SF6, respectively. The methane emission rate was determined from CH4 to SF6 ratio using release rate RESULTS AND DISCUSSION of SF6 as given in formula. Background methane was subtracted from methane concentration in the In vivo methane emission PVC canister. Saponin content (DM basis) of alfalfa

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Figure 1. in vivo enteric methane emission measurement in Murrah buffalo calves using SF6 technique.

Figure 2. Rumen liquor collection from buffaloes using stomach tube.

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fodder at three different cuts varied between emission in control and test group was 78.09 and 0.92±0.05 to 2.0±0.05 per cent with highest level at 61.39 g/d, respectively. Enteric methane emission second cut (2.0%). Due to higher saponin level and (g/d) in control group was signiicantly higher results from in vitro studies (data not presented), (P<0.05) than the test group. About 21% reduction second cut alfalfa fodder was selected for the in methane emission was reported in test group on supplementation at 30% level in wheat straw and the inclusion of saponin containing alfalfa fodder concentrate based diet to ascertain the effect on at 30% level replacing wheat straw in the diet enteric methane emission in Murrah buffalo calves. (Figure 3). Similarly, in vivo methane emission on Similar to the indings of this study, Cheeke and g/kg DMI was also signiicantly (P<0.05) lower in Shull (1985) also reported highest saponin level in test group than control (18.1 vs. 12.4 g). Enteric alfalfa fodder at second cut. methane emission on g/kg digestible dry matter Dry matter intake (DMI) and in intake basis was also signiicantly (p<0.05) lower vivo methane emission as affected by the in test group. Lower enteric methane emission supplementation of saponin containing alfalfa from the test group buffaloes may be attributed fodder in Murrah buffaloes is depicted in Table 1 to the saponin of alfalfa fodder. Average saponin and Figure 3, respectively. Data did not reveal any intake in test group was 47 g/d or 6.03 g/kg of signiicant change in dry matter intake between dry matter. Srivastava and Garg (2002) reported control and test group (Table 1). In vivo enteric 19.26 g methane emission per kg of DMI in methane emission in buffaloes in control and test crossbred calves fed on paddy straw and fodder groups is presented in Figure 3. Enteric methane based diet. Mohini and Singh (2001) recorded the

Table 1. Effect of saponin containing alfalfa fodder supplementation on dry matter intake (DMI) and fermentation pattern in buffaloes.

Parameter Control diet Test diet DMI Kg/d 7.40±0.28 7.79±0.19 Kg/100kg BW 2.77±0.16 2.94±0.08 g/kgW0.75 BW 111.8±0.46 118.6±0.40 Fermentation pH 6.40±0.18 6.30±0.18

NH3-N (mg/100ml) 30.92±1.37 28.62±1.33 TVFA (mM/l) 108.94±0.68 108.87±0.97 Acetate 74.13a±0.34 67.07b±0.35 Propionate 15.99b±0.74 17.70a±0.37 Butyrate 9.86±0.42 10.22±0.64 A:P 4.6:1 3.7:1

Values bearing a, b superscripts in a row differ signiicantly (p<0.05).

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methane emission in the range of 15.97 - 18.35 g/ fraction at the cost of acetate signiicantly (P<0.05) kg DMI from buffalo calves fed on maize fodder increased, while butyrate remain unaffected by the and straw based diet. Mao et al. (2010) also found supplementation. Data showed a shift in individual a reduction of 27.2% in methane emission from fatty acid production from acetate to propionate on lamb on the inclusion of saponin at 4.1 g/kg level the inclusion of alfalfa fodder. These results are in from Camellia scinensis. However, administration consonance of the indings of Diaz et al. (1993); of 5 g/kg of S. saponaria fruits to sheep for 21 days Hristov et al. (1999) who also reported similar trend

reduced CH4 emission by 7.8% only in a study of on the dietary incorporation of Sapindus saponaria Hess et al. (2004). and Yucca schdigera , respectively. Hu et al. (2006) Data from the study revealed that enteric also recorded same trend without affecting TVFA methane emission in buffaloes is inluenced by concentration on the incorporation of Yucca and the saponin from alfalfa fodder. Lower methane Quillaja saponin in corn meal and grass based diet. emission in test group was attributed to the anti- From these results, it may be concluded that the protozoal action of saponin of alfalfa, which saponin from alfalfa fodder to a level of 47 g/d or in turn puts a restriction on hydrogen transfer 6 g/kg DM did not affect the feed fermentability or to methanogens (Krumholz et al., 1983). dry matter intake. Mechanistically, saponin forms an irreversible complex with cholesterol which is an integral Effect on methanogen archaea and protozoa component of protozoal cell membrane and Results of the study revealed a non- therefore leads to cell lysis and death. signiicant effect of alfalfa fodder as such or its saponin (47g/d or 6.03g/kg DM) on rumen Effect on feed fermentability methanogen archaea. On the feeding of control Dry matter intake (DMI) and fermentation as well as test diet, their population remained characteristics as affected by the supplementation constant ~ 1.73 log CFU/ml (Figure 4), which of saponin containing alfalfa fodder in Murrah shows that the saponin did not have any direct buffaloes are presented in Table 1. Dry matter action on methanogen archaea and directly not intake (DMI) did not show any signiicant change accountable for methane reduction from buffalo due to the supplementation (Table 1). Similarly, calves. Results is in congruence of the indings of pH, ammonia nitrogen and total volatile fatty Wina et al. (2005) who did not ind any decrease acid also did not affect with saponin containing in methanogens number on the addition of saponin alfalfa fodder supplementation. Data from the from Sapindus rarak. On the contrary the protozoal study envisaged that saponin from alfalfa fodder population was adversely affected due to alfalfa at 6.03 g/kg DM (total intake 47g) did not have fodder supplementation (Figure 4). Protozoal any adverse effect on dry matter intake, and total population badly affected (P<0.05) in group II on volatile fatty acid production. the addition of saponin containing alfalfa fodder and Acetate production (P<0.05) in alfalfa the decrease was almost 20 per cent as compared to fodder supplemented buffaloes decreased control group (Figure 4). Entodinimorphs are the signiicantly (p<0.05) by 7 units (9.5%) asmost comparedvulnerable to dietary changes in cattle and to control (Table 1). On the other hand, propionate buffaloes (Bhatia et al., 1998) and most susceptible

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Figure 3. Effect of lucerne fodder supplementation on methane emission in Murrah buffaloes.

Figure 4. Effect of supplementation on rumen methanogens and protozoa.

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to the dietary saponin (Lu and Jorgenson, 1987). REFERENCES However, in present study no attempt was made for Barnett, A. and R.L. Reid. 1957. Studies on the the characterization of various protozoal population production of volatile fatty acids from grass affected by alfalfa saponin. These results are in by rumen liquor in an artiicial rumen. 1. agreement with indings of Navas-Camachoet The volatile acid production from fresh al. (1993); Diaz et al. (1993); Klita et al. (1996), grass. J. Agri. Sci., 48: 315-21. noted a signiicant reduction in ruminal protozoa Bhatia, S.K., K. Pradhan, S. Singh and S. Singh. following dietary incorporation of E. ciclocarpum, 1998. Microbes and their activities in rumen Sapindus saponaria and alfalfa root saponin, of cattle and buffaloes (Technical bulletin). respectively. Thus, the signiicant reduction in Published by CCS Haryana Agricultural methane emission in group II on the inclusion University, Hisar, India. of alfalfa fodder is attributed to comparatively Cheeke, P.R. and L.R. Shull. 1985. Natural lesser number of protozoa badly hit by saponin. Toxicants in Feeds and Poisonous Plants. Anti-protozoal effect of saponin is depend on the AVI Publishing Company, Inc., Westport, presence of cholesterol in protozoal cell membrane, Conn., USA. 492p. which possible made a selective susceptibility Conway, E.J. 1950. Microdiffusion Analysis and of ruminal protozoa to saponin and due to this Volumetric Error, 4th ed. London: Crosby reason methanogens (lacking in cell membrane Lockwood. 391p. cholesterol) did not directly hit by saponin. Diaz, A., O.M. Avendon and A. Escobar. 1993. Evaluation of Sapindus saponaria as a CONCLUSION defaunating agent and its effect on different ruminal digestion parameters. Lives. Res. and Rural Dev., 5: 1-10. It may be inferred from the study that Erwin, E.S, G.J. Marco and I.N. Emery. 1961. secondary metabolite saponins from natural feed Volatile fatty acid analyses of blood and sources like alfalfa may signiicantly reduce enteric J. methane emission in buffaloes and quite safe to rumen luid by gas chromatography. Dairy Sci., 44: 1768-1771. feed up to a level of 6.03 g/Kg of DM or 47 g/day FAO. 2008. The state of food and agriculture. Food without affecting dry matter intake. Methanogenic and Agriculture Organisation, Rome. http:// archaea is not affected by the alfalfa fodder apps.fao.org.page/collection. Fao.org/stat supplementation, while it has an adverse action on 2008. rumen protozoa which lead to less enteric methane Hess, H.D., M. Kreuzer, T.E. Diaz, T.E. Lascano, emission. There is a need to conduct instant research J.E. Carulla, C.R. Soliva and A. Machmuller. towards exploring the saponin containing phyto- 2003. Saponin rich tropical fruit affect sources and to optimize their safe level of inclusion fermentation and methanogenesis in in diet for the substantial methane reduction. faunated and defaunated rumenAnim. luid. Feed Sci. and Tech., 109: 79-94. Hess, H.D., R.A. Beuret, M. Lotscher, I.K.

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Hindrichsen, A. Machmüller, J.E. Carulla, saponins affect site and extent of nutrient C.E. Lascano and M. Kreuzer. 2004. digestion in ruminants. J. Nutr., 117: 919- Ruminal fermentation, methanogenesis 927. and nitrogen utilization of sheep receiving Malik, P.K, K.K. Singhal and R.A. Siddique. 2012. tropical grass hay-concentrate diets offered Mitigation strategies for enteric methane with Sapindus saponaria fruits and Cratylia emission with special reference to biological argentea foliage. Anim Sci., 79: 177-189. approaches: a review. Indian J. Anim. Sci., Hristov, A.N., T.A. McAllister, F.H. Van Herk, 82(8): 794-804. K.J. Cheng, C.J. Newbold and P.R. Cheeke. Malik, P.K. and K.K. Singhal. 2008. Inluence of 1999. Effect of Yucca schidigera on ruminal supplementation of wheat straw based total fermentation and nutrient digestion in mixed ration with saponins on total gas and heifers. J. Anim. Sci., 77: 2554-2563. methane production in vitro. Indian J. Anim. Hu, W.L, J.X. Liu, Yan-Qiu Guo, Yue-Min Sci., 78(9): 987-990. Wu. 2006. Effect of saponins on rumen Malik, P.K., K.K. Singhal, N.M. Soren, A. Thulasi, fennentation and methanogenesis in vitro, p. L. Jose and S.S. Santosh. 2013. Effect of 12l. In Proceedings of 12th AAAP Congress. graded level Egyptian clover (Trifolium Busan, South Korea (Abstract). alexandrium) supplementation on in vitro Inter Governmental Panel on Climate Change methane production and fermentation (IPCC). 2007. Summary for Policymakers. pattern. Indian J. Anim. Sci., 83(8): 802- Approved in Plenary XXVII, working 805. group contributions to the IV assessment Mao, H.L., J.K. Wang, Y.Y. Zhou and J.X. Liu. Report, Valentia, Spain. 2010. Effects of addition of tea saponins Johnson, K.A., M.T. Huyler, H.H. Westberg, and soybean oil on methane production, B.K. Lamb and B. Zimmerman. 1994. fermentation and microbial population in Measurement of methane emission from the rumen of Growing lambs. Livestock ruminant livestock using SF6 tracer Sci., 129: 56-62. technique. Environ. Sci. Tech., 28: 359. Mohini, M. and G.P. Singh. 2001. Methane Kearl, L. 1982. Nutrient requirements of ruminant production on feeding Jowar fodder based in developing countries. Utah State Univ. ration in buffalo calves. Indian J. Anim. Logam, USA. Nutr., 18: 204-209. Kilta, P.T., G.W. Mathison and T.W. Fenton 1996. Navas-Camacho, A., M.A. Laredo, A. Cuesta, Effect of alfalfa root saponins on digestive H. Anzola and J.C. Leon. 1993. Effect of function in sheep. J. Anim. Sci., 74: 1144- supplementation with a tree legume forage 1156. on rumen function. Liv. Res. Rural Dev., Krumholz, L.R., C.W. Forsberg and D.M. Veira. 5(2). 1983. Association of methanogenic bacteria Ranade, C.D.R. and R.V. Gadre. 1988. Media, p. with rumen protozoa. Can. J. Microbiology, 67-77. In Microbial Aspects of Anaerobic 29: 676-680. Digestion: Laboratory Manual, Pune: Lu, C.D. and N.A. Jorgensen. 1987. Alfalfa MACS Research Institute.

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Singh, G.P. 1998. Methanogenesis and production of greenhouse gases under animal husbandry system. Report of AP Cess fund project, National Dairy Research Institute, Karnal, India. Srivastava, A.K and M.R. Garg. 2002. Use of sulfur hexaluroide tracer technique for measurement of methane emission from ruminants. Indian J. Dairy Sci., 55(1): 36- 39. Wang, J.K., Y. Jun-An and J.X. Liu. 2011. Effects of tea saponins on rumen microbiota, rumen fermentation, methane production and growth performance-a review. Trop. Anim. Health Pro., DOI 10.1007/s11250-011- 9960-9968. Wina, E., S. Muetzel and K. Becker. 2005. The impact of saponins or saponin-containing plant materials on ruminant production-A review. J. Agric. Food Chem., 53: 8093- 8105. Yosioka, I., A. Inada and I. Kitagawa. 1974. Soil bacterial hydrolysis leading to genuine aglycone—VIII : Structures of a genuine sapogenol protobassic acid and a prosapogenol of seed kernels of Madhuca longifolia L. Tetrahedron, 30(6): 707-714.

134 Original Article Buffalo Bulletin (March 2016) Vol.35 No.1

HISTOLOGICAL EXPLORATION OF GRAAFIAN AND ATRETIC FOLLICLES OF BUFFALO OVARY: A SEASONAL STUDY

Jasvinder Singh Sasan1, Varinder Uppal2, Neelam Bansal2 and Anuradha2

ABSTRACT INTRODUCTION

The present research work was conducted Buffaloes are known for the poor to study distribution of Graafian and atretic follicles reproductive performance such as silent heat, low in buffalo ovary during different seasons of year. conception rate and long calving interval which For this purpose, 100 ovaries of adult Murrah causes heavy economic losses to farmers (Madan, buffaloes during different seasons (20 in each 1988). Further, the embryo transfer technique season) of year viz; winter (November - January), in buffalo has not proved to be much successful, spring (February - March), summer (April - June), mainly due to very poor superovulatory response rainy (July - August) and autumn (September - and poor recovery of viable embryo (Taneja et October) were collected from slaughter house. The al., 1991). The success of in vitro production of paraffin sections of 10 µm were cut and every 20th buffalo embryos has been hampered by factors section was stained with hematoxylin and eosin. In such as low quality of follicles on ovaries and poor present study, two types of follicles were observed oocyte recovery rate. In past it has been shown viz. young and mature Graafian follicles. Highest that environmental temperature plays an important number of normal follicles was during autumn role and buffaloes exhibit a distinct reproductive season whereas lowest number of normal follicles performance in different seasons (Shah, 1988). The was during summer season. The most common number of graafian follicles produced on the ovary type of atresia was antral. The ratio of normal to may be one of the important factor to determine the atretic follicle was maximum in summer (1: 3.10) reproductive efficiency of the animal as it is affected and minimum in autumn season (1: 2.19). by the environmental temperature. Although, the number of mature follicles have been counted Keywords: buffalo, follicular atresia, during follicular and luteal phases of estrous cycle histomorphometry, ovarian follicles, seasons (Danell, 1987 and Bansal, 2002) but seasonal study on such a count is lacking. So the present research was planned to count and to correlate the number of mature follicles in each season of year.

1Sher-e-Kashmir University of Agricultural Sciences and Technology (SKUAST), Jammu, India, E-mail: [email protected] 2Department of Veterinary Anatomy, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana, Punjab, India

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MATERIALS AND METHODS perpendicular to the plane of section (D3). Third

diameter D3 was calculated as Collection of ovaries

Left and right ovaries of 100 adult D3 = N x 10 x 20 Murrah buffaloes were collected during different seasons (20 in each season) of year viz; winter where (November - January), spring (February – March), “N” represents number of sections in summer (April - June), rainy (July - August) and which the particular follicle appeared autumn (September - October). Immediately after “10” represent the thickness of section in collection, the ovaries were fixed in 10% neutral µm buffered formalin (NBF). “20” represents every 20th serial section of ovary at which diameter was measured. Processing of ovaries Actual estimated diameter was calculated Ovaries were processed by acetone by dividing the mean follicle diameter by five. benzene schedule (Luna, 1968). The whole ovary was serially sectioned with a rotary microtome at a thickness of 10 µm. The serial sections were placed RESULTS AND DISCUSSION in a sequence on clean glass slides keeping track of section numbers and slide numbers. Every 20th Depending upon the size of antrum, two section of ovaries was stained with hematoxylin types of follicles were observed viz. young and and eosin (Luna, 1968). Subsequent sections were mature Graafian follicles. Mostly the follicles stained with Masson’s trichrome for collagen fibres, contained single oocyte but occasionally 2 or 3 Gridley‘s stain for reticular fibres and Verhoeff oocytes were also observed (Figure 1). The oocyte stain for elastic fibres. of the graafian follicle was round to oval with mean diameter of 71.47±8.18 µm. The nuclei were Number of follicles centrally or eccentrically placed. The antrum was The follicles which were ≥ 1mm in filled with eosinophillic colloidal fluid, the liquor diameter were observed and counted by a method folliculi. The membrana granulosa was covered reported by Danell (1987). Every 20th section of by basement membrane. The theca layer was ovary was placed in an enlarging apparatus and completely differentiated into theca interna and projected 5 times larger onto normal photographic theca externa (Figure 2) as reported earlier in cattle paper. After development, the photographs were (Rajakoski, 1960) and buffalo (Bansal, 2002). arranged in a series and fixed on a white sheet. Theca interna composed of mainly epithelial For describing the size of follicles, each particular cells with vesicular nuclei and small amount of follicle was given a similar number on all the fibroblasts and connective tissue fibres whereas photographs where it appeared. The mean follicle theca externa consisted of fibroblasts, connective diameter was determined as the average of three tissue and muscle fibres. Theca interna was measurements: the largest diameter (D1), the highly vascular as reported earlier (Danell, 1987; diameter perpendicular to D1 (D2) and diameter Bhardwaj and Roy, 1998).

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The mean size of Graafian follicle was cent atresia in Surti buffalo and Bansal (2002) who 443.18±72.1 µm. The average thickness of zona reported 71.77 per cent of atresia in buffalo ovary. pellucida was 5.34±0.48 µm. The mean size of The ratio of normal and atretic follicles was antrum was 395.83±39.26 µm. The membrana calculated during different seasons. It was 1:2.31 in granulosa, which enclosed the antrum was winter, 1:2.68 in spring, 1:3.10 in summer, 1:2.48 having mean thickness of 38.22±2.52 µm. The in rainy season and 1:2.19 in autumn, respectively. mean thickness of theca interna and externa was The data revealed that the ratio was highest in 63.03±3.4 µm and 59.25±5.45 µm, respectively. summer season as compared to other seasons. Graafian follicles (> 1 mm) were classified into five categories on basis of size viz. 1 –<2 Follicular atresia mm, 2 – < 3 mm, 3 – < 4 mm, 4 – < 5 mm and As the primordial follicles progress through ≥ 5 mm and has been depicted in Figure 13. The different stages of development, most of them never present study revealed that the follicles falling in 1 reach maturity and thus got degenerated at certain – < 2 mm group were present in maximum number point along the way. This process of follicular and these constituted 65.27%, 64.79%, 61.16%, degeneration is called follicular atresia and 53.33% and 64.89% of the total follicles during depends upon the variable degree of susceptibility winter, spring, summer, rainy and autumn season, of different cells to death (Rodgers and Irving- respectively. These follicles represented more than Rodgers, 2010). In the present study, atresia was half of the total number of follicles (≥ 1 mm in seen in follicles at all stages of development. The diameter). The findings corroborates well with the highest atresia was also observed in the follicles of earlier findings of Danell (1987) and Bansal (2002) range 1 - < 2 mm (Figure 14) as reported earlier by during different seasons. Danell (1987) and Bansal (2002) with maximum Percentage distribution of total number of value of 76.35% during summer season. Graafian follicles (normal and atretic) in the ovaries The follicular atresia of large follicles during different seasons has been summarized in was categorized as obliterative and cystic. The Table 1 which revealed highest number of normal obliterative atresia was further subdivided into follicles was during autumn season whereas lowest first degree and second degree as described by number of normal follicles was during summer Danell (1987) and Bansal (2002). In first degree of season. Similar types of findings were observed atresia, a number of pyknotic nuclei were observed by Sadeghinezhad and Hasanzadeh (2010) in river either in the layers of membrana granulosa close buffalo. Also, it was observed that although the to antrum or in the antrum itself but in close number of normal and atretic follicles were more proximity to the membrana granulosa whereas on the right sided ovary but the difference between the cells closest to the basal lamina were tightly distribution of follicles in left and right ovary was packed and appeared healthy (Figure 3). In some non-significant. The maximum per cent of atresia of the follicles, the upper layer of granulosa cells (75.62) was observed during summer followed was detached from the underlying granulosa cells by spring (72.89), rainy (71.25), winter (69.79) (Figures 4, 5). The pyknotic nuclei were rarely and autumn (68.73). These findings are almost seen in the granulosa cells close to the basement similar to Danell (1987) who reported 70.60 per membrane. The fibroblast present in the theca layer

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were spindle shaped and were orientated parallel to fibrosed or hyalinized. There was decrease in the membrana granulosa. This type of atresia has follicular size and antrum. In some of the follicles been described as antral atresia by Irving-Rodgers only 1-2 layers of granulosa cells was observed et al. (2001) and is comparable to first degree of so giving the follicle a classic string of pearl obliterative atresia described by Danell (1987) and orientation as described by Marion et al. (1968) Bansal (2002). With further advancement, there who assumed that these follicles had begun to was degeneration of granulosa layers with few expand on atresia. Rodgers et al. (2001) reported pyknotic nuclei in the antrum, granulosa cells and that number of layers of granulosa cells decreases cumulus. Later on, cumulus disappeared leaving a as follicles enlarge to reach a plateau. The number naked oocyte followed by in growth of connective of granulosa cell layer is both a function of net rate tissue in the antrum (Figures 6, 7) as described of granulosa cell replication and the rate of antrum earlier by Danell (1987) and Bansal (2002) in expansion. Thus, the string of pearls description buffalo ovary. When the antrum was completely used by Marion et al. (1968) arised by reduction in filled with connective tissue, the degenerated the number of layers of granulosa cells as the antrum follicle was termed as corpus atreticum (Figure 8). expands during follicular growth and not during In other type of atresia, there was atresia. Luteinized cystic type of atresia occurred destruction of the most basal layer of the granulosa infrequently in which there was degenerated cells whereas the most antral granulosa cells granulosa cells followed by luteinization of whole remained healthy and closely apposed to each of the theca interna cells (Figure 11). Since atretic other. There were very few pyknotic nuclei in the follicles produces high amount of progesterone as antrum or in membrana granulosa closest to antrum. compared to non-atretic follicles (Westhof et al., The cells nearer to antrum were often flattened so 1991) and also dibutyryl cyclic AMP significantly that overall antral surface of membrana granulosa stimulates progesterone production by cells of appeared smooth and regular. The cells in the basal atretic follicles. The increased concentration of layer of membrana granulosa were separated from progesterone leads to luteinization of follicles each other and from basal lamina by intercellular which is accompanied by cellular hypertrophy, spaces. With further advancement of atresia, there formation of diffusely distributed lipo-proteins was degeneration of granulosa cells (Figure 9) and and increased number of lipid droplets (Guraya, large apoptotic bodies (Figure 10) were observed 1997). Another type of cystic atresia observed in in the antrum. The spindle shaped fibroblasts of the present study was fibrosed type in which there theca interna were arranged randomly. This type of was degeneration of granulosa layer along with atresia observed during present study fits well as increased number of fibroblasts in theca interna basal atresia described earlier by Irving- Rodgers as reported earlier by Bansal (2002) in buffalo et al (2001) and is comparable to the second degree ovary. This type of atresia was maximum among of obliterative atresia as observed by Danell (1987) all the types of cystic atresia. In third type of cystic and Bansal (2002). atresia, theca cells were hyalinized along with In cystic type of atresia, both granulosa degeneration of granulosa cells (Figure 12). The and theca layers atrophied or only granulosa atresia occurs in a particular sequence as reported layer atrophied and theca layer may be luteinized, earlier by Guraya (1979) and Danell (1987) i e. first

138 Buffalo Bulletin (March 2016) Vol.35 No.1

A MG TI TE

1 2

A

A

3 4

MG

5 6

Figure 1. Growing follicle with three oocytes (arrow). H & E stain X10. Figure 2. Section of buffalo ovary showing layers of normal Graafian follicle; antrum (A), membrana granulosa (MG), theca interna (TI) and externa (TE). Masson’s trichrome stain X20. Figure 3. Photomicrograph of paraffin section of ovary showing sloughing of granulosa cells (arrow) close to antrum (A). H & E stain X40. Figure 4. Section of ovary showing loosening and sloughing of granulosa cells (arrow) close to antrum (A). Masson’s trichrome stain X40. Figure 5. Section of ovary showing complete sloughing of granulosa cells (arrow). Masson’s trichrome stain X20. Figure 6. Section of ovary showing degeneration of membrana granulosa (MG) cells. Masson’s trichrome stain X10.

139 Buffalo Bulletin (March 2016) Vol.35 No.1

Ct CA

7 8

AF

NF

9 10

11 12

Figure 7. Section of ovary showing connective tissue (Ct) in antrum. H & E stain X20. Figure 8. Section of ovary showing connective tissue scar, corpus atreticum (CA). H & E stain X10. Figure 9. Section of ovary showing wall of normal (NF) and atretic follicle (AF). Masson’s trichrome stain X20. Figure 10. Figure38 Photomicrograph of paraffin section of ovary showing apoptotic bodies (arrow) in basal type of atresia. H & E stain X 20. Figure 11. Photomicrograph of paraffin section of ovary showing luteinization of follicle. Masson’s trichrome stain X10. Figure 12. Photomicrograph of paraffin section of ovary showing hyalinization of follicular wall. Numerous pyknotic nuclei (arrow) can be seen. H & E stain X20.

140 Buffalo Bulletin (March 2016) Vol.35 No.1 Graph.1 Percentage of different categories of follicles (≥ 1 mm) during different seasons

Winter Spring 1 - < 2mm 2 - < 3mm 3 - < 4mm 1 - < 2mm 2 - < 3mm 3 - < 4mm 4 - < 5mm ≥ 5mm 4 - < 5mm ≥ 5mm 2% 1% 2% 0% 9% 8% 23% 25% 65% 65%

Summer 1 - < 2mm 2 - < 3mm 3 - < 4mm 4 - < 5mm ≥ 5mm 6% 3% 0%

30% 61%

Rainy Autumn 1 - < 2mm 2 - < 3mm 3 - < 4mm 1 - < 2mm 2 - < 3mm 3 - < 4mm 4 - < 5mm ≥ 5mm 4 - < 5mm ≥ 5mm

1% 3% 3% 6% 11% 9% 20% 53% 29% 65%

Figure 13. Percentage of different categories of follicles (≥ 1 mm) during different seasons.

141 Buffalo Bulletin (March 2016) Vol.35 No.1 Graph.2 Number of normal and atretic follicles (≥ 1 mm) in different categories during different seasons

Winter season Spring season

140 140 120 120 100 100 80 80 60 60 40 40 20 20 0 0 1 - < 2 - < 3 - < 4 - < ≥ 1 - < 2 - < 3 - < 4 - < ≥ 5mm 2mm 3mm 4mm 5mm 5mm 2mm 3mm 4mm 5mm

normal atretic normal atretic Summer season

120 100 80 60 40 20 0 1 - < 2 - < 3 - < 4 - < ≥ 2mm 3mm 4mm 5mm 5mm

normal atretic Rainy season Autumn season

160 100 140 80 120 100 60 80 40 60 20 40 20 0 0 1 - < 2 - < 3 - < 4 - < ≥ 1 - < 2 - < 3 - < 4 - < ≥ 2mm 3mm 4mm 5mm 5mm 2mm 3mm 4mm 5mm 5mm

normal atretic normal atretic

Figure 14. Number of normal and atretic follicles (≥ 1 mm) in different categories during different seasons.

142 Buffalo Bulletin (March 2016) Vol.35 No.1 1 : 2.5 Atretic 1 : 2.48 1 : 2.19 1 : 3.10 1 : 2.68 1 : 2.31 Normal : 240 339 242 284 288 total 1393 Overall 171 995 233 183 207 201 (71.25) (71.43) (68.73) (75.62) (72.89) (69.79) Total 69 59 77 87 398 106 (27.11) (28.75) (28.57) (31.27) (24.38) (30.21) Normal Atretic 137 724 170 122 148 147 Total 97 93 117 516 107 102 (70.81) (71.27) (68.82) (76.23) (72.30) (69.39) Right ovary 53 40 29 41 45 208 (31.18) (29.19) (28.73) (23.77) (27.70) (30.61) Normal Atretic 169 103 669 120 136 141 Total 74 90 99 116 479 100 (68.64) (71.84) (71.60) (75.00) (73.53) (70.22) Left ovary 53 29 30 36 42 190 (31.36) (28.40) (28.16) (25.00) (26.47) (29.78) Normal Atretic Total Rainy Spring Season Winter Autumn Summer

Values in parentheses indicate percentage calculated from total a row. Values Table 1. Percentage distribution of normal and atretic Graafian follicles (≥ mm) in left and right ovaries during different seasons. 1. Percentage distribution of normal and atretic Graafian follicles (≥ mm) in left right ovaries during different Table

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there was dissolution and pyknosis of granulosa So, the most important consequences cells followed by differentiation of theca interna of follicular atresia is to limit the number of into fibrous cells. Later on there was hyalinization ovulations or select the dominant follicle (Guraya, and dissolution of theca interna layer. 1997), thus reducing the number of offsprings. Although atresia may occur due to Secondly, follicular atresia contributes thecal excessive secretion of gonadotropins (Harman et type interstitial cells to the ovary which constitute al., 1975) or insufficient gonadotropins (Hirshfield, the steroidogenic tissue. Atretic follicles may 1991), excessive androgens (Harman et al., 1975), contribute significantly to intra-ovarian levels of excess or insufficient estradiol (McNatty, 1978) androgens and progesterone which can be utilized and endogenenous GnRH like substance within by non-atretic follicles to enhance estradiol the ovary (Birnbaumer et al., 1985), yet there are synthesis (Westhof et al., 1991). also certain other factors which regulate the cyclic appearance and atresia of dominant follicles and other follicles. These factors may include age, REFERENCES stage of reproductive cycle, pregnancy, lactation, hormones of extra-ovarian and intra-ovarian Adashi, E.V. 1994. Endocrinology of ovary. sources, a genetic programmed nutrition, ischemia Human Reproduction, 9: 815-27. and season (Danell, 1987). Sluss et al (1983) has Bansal, N. 2002. Gross and histomorphological suggested the possibility of FSH-binding inhibitors studies on follicular population in ovaries in the induction or propagation of follicular atresia of buffalo (Bubalus bubalis). Ph.D. by suppressing the responsiveness of granulosa dissertation, Punjab Agricultural University cells to FSH. Different studies have clearly Ludhiana, India. suggested that the follicles which are destined to Bhardwaj, R.L. and K.S. Roy. 1998. Age related undergo atresia in normal estrous cycle of cattle demographical study on the distribution of can be rescued by administration of exogeneous follicle type in the ovary of Indian buffalo. gonadotrophins (Drianocourt, 1987). The follicles Buffalo. J., 3: 395-399. undergo atresia due to lack of gonadotrophins at a Birnbaumer, L, N. Shabi, J. Rivier and W. Vale. key stage (at which the amount of FSH receptors 1985. Evidence for a physiological role of on granulosa cells and LH receptors on theca layer gonadotropin-releasing hormone (GnRH) becomes maximum and also aromatase activity or GnRH like material in the ovary. is increased) in folliculogenesis (Guraya, 1997). Endocrinology, 116: 1367-70. Actually the balance between estrogens and Danell, B. 1987. Oestrus behavior, ovarian androgens plays especially an important role in morphology and cyclical variation in determining whether a follicle becomes atretic or follicular system and endocrine pattern in not (Adashi, 1994). It has also been reported that water buffalo heifers. Swedish University metabolism of granulosa cells can be affected by of Agricultural Science, Uppsala, Sweden. any interference in the development of their LH Driancourt, M.A. 1987. Follicular dynamics receptors which possibly can induce follicular and intraovarian control of follicular atresia (Guraya, 1997). development in the ewe. In Roche, J. and D.

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O’Callanghan (eds.) Follicular Growth and reference to seasonal, cyclical and left-right Ovulation Rate in Farm Animals. Martinus variations. Copenhagen. Acta Endo., 34: Nijhoff, The Hague. 1-68. Guraya, S.S. 1979. Morphological and Rodgers, R.J. and H.F. Irving-Rodgers. 2010. histochemical observations on the buffalo Morphological classification of bovine ovaries during anoestrus. Indian. J. Anim. ovarian follicles. Reproduction, 139: 309- Sci., 49: 423-432. 318. Guraya, S.S. 1997. Ovarian Biology in Buffaloes Rodgers, R.J., H.F. Irving-Rodgers, I.L. Van and Cattle. Indian Council of Agricultural Wezel, M. Krupa and T.C. Lavranos. 2001. Research, New Delhi, India. Dynamics of the membrana granulosa Harman, S.M., J.P. Louvet and G.T. Ross. 1975. during expansion of the ovarian follicular Interaction of estrogen and gonadotropins antrum. Mol. Cell. Endocr., 171: 41-48. on follicular atresia. Endocrinology, 96: Sadeghinezhad, J. and S. Hasanzadeh. 2010. Study 1145-52. of ovarian follicular development during Hirshfield, A.N. 1991. Development of follicles in summer and winter seasons in river buffalo. the mammalian ovary. International Review Global Vet., 4: 582-86. of Cytology, 124: 43-101. Shah, S.N.H. 1988. Comparative studies of Irving-Rodgers, H.F., I.L. Van Wezel, M.L. seasonal influence on breeding behavior Mussard, J.E. Kinder and R.J. Rodgers. and conception rate of dairy buffalo and 2001. Atresia revisited: two basic patterns zebu cattle. In Proceedings of 11th Congress of atresia of bovine antral follicles. on Animal Reproduction and Artificial Reproduction, 122: 761-775. Insemination, 3: 538. Luna, L.G. 1968. Manual of Histological Staining Sluss, P.M, P.W. Fletcher and L. Richrt Jr. Methods of Armed Forces Institute of 1983. Inhibition of I125 –human follicle- Pathology, 3rd ed. McGraw Hill Book stimulating hormone binding to receptor by Company, New York, USA. a low molecular weight fraction of bovine Madan, M.L. 1988. Status of reproduction in follicular fluid: inhibitor concentration female buffalo. In Buffalo Production and is related to biochemical parameters of Health: A Compendium of Latest Research follicular development. Bio. Rep. 29: 1105- Information Based on Indian Studies. ICAR 1113. publication, New Delhi, India. Taneja, M., S.M. Toey, C.H. Pawshe, G. Singh, A. Marion, G.B., H.T. Gier and J.B. Choudary. 1968. Alli and G. Talwar. 1991. Ultrasonographic Micro-morphology of the bovine ovarine monitoring of ovarian follicular dynamics follicular system. J. Anim. Sci., 27: 451-465. in supraovulatory buffalo, p. 581-585. McNatty, K.P. 1978. Follicular fluid, p. 215-259. In Proceedings of Third World Buffalo In Jones, R. (ed.) The Vertebrate Ovary. Congress, Vol. 3. Varna, Bulgaria. Plenum Press, New York, USA. Westhof, G., K.F. Westhof, W.L. Braendle and Rajakoski, E. 1960. The ovarian follicular system G.S. diZerega. 1991. Differential steroid in sexually mature heifers with special secretion and gonadotropin response by

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individual tertiary procine follicles in vitro. Possible physiologic role of atretic follicles. Bio. Rep., 44: 461-468.

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

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