International Buffalo Information Center (IBIC)

Buffalo Bulletin ISSN: 0125-6726 (Print), 2539-5696 (Online)

Aims IBIC is a specialized information center on water buffalo. Established in 1981 by Kasetsart University (Thailand) with an initial financial support from the International Development Research Center (IDRC) of Canada. IBIC aims at being the buffalo information center of buffalo research community throughout 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 January-March, April-June, July-September and October- 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, Office of the University Library, Kasetsart University

Online available 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, Office of the University Library, Kasetsart University, Thailand Miss Supanee Hongthong International Buffalo Information Center, Office of the University Library, Kasetsart University, Thailand

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

Editor Dr. Sunpetch Sophon Thailand

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

Assistant Journal Manager Miss Kanchana Anuphan International Buffalo Information Center, Office of the University Library, Kasetsart University, Thailand Miss Jirawadee Wiratto International Buffalo Information Center, Office of the University Library, Kasetsart University, Thailand

Buffalo Bulletin 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 (July-September 2016) Vol.35 No.3

CONTENTS Page Case Report

An unusual case of ideopathic fibrinous pericarditis in Nili-ravi buffalo-A case report Muhammad Saqib, Ghazanfar Abbas and Mudassar Niaz Mughal...... 299

Original Article

Effect of lugol’s iodine on estrus induction and fertility response in true anestrus Jaffrabadi buffaloes A.R. Ahlawat, P.U. Gajbhiye, M.D. Odedra, V.B. Dongre and S.N. Ghodasara...... 303

Haemato-biochemical alterations during different stages of lactation in Mehshani buffaloes Hemen Das, A. Lateef, H.H. Panchasara and M. Ayub Ali...... 307

effect of early post-partum GnRH and PGF2 alpha administration on follicular activities in Murrah buffaloes M.V. Ingawale and S.A. Bakshi...... 317

Clinical, hemato-biochemical and therapeutic studies on rumen impaction in buffaloes A.K. Tripathi, J.S. Soodan and R.B. Kushwaha...... 325

Diagnosis of rabies in buffaloes: comparison of clinico-pathological, immunohistochemical and immunofluorescent techniques A.B. Beigh, B.S. Sandhu, C.K. Singh, K. Gupta and N.K. Sood...... 331

Epidemiology of ixodid ticks in buffaloes (Bubalus bubalis) of Punjab, India N.K. Singh and S.S. Rath...... 347

Epidemiological studies on gastrointestinal parasites of buffaloes in seven agro-climatic zones of Madhya Pradesh, India S. Nath, G. Das, A.K. Dixit, V. Agrawal, S. Kumar, A.K. Singh and R.N. Katuri...... 355

Studies on effect of non-genetic parameters on mortality pattern in Murrah buffaloes Nitin Mohan Gupta, M.L. Mehra and Puneet Malhotra...... 365

Buffalo Bulletin (July-September 2016) Vol.35 No.3

CONTENTS Page Original Article

Evaluation of breeding values Murrah buffalo bulls under organized farms Vijay Kumar and A.K. Chakravarty...... 371

Incidence and fertility status of hydatid cysts in buffaloes A. Sheeba, A. Sangaran, B.R. Latha and A. Raja...... 379

Seroepidemiological study of leptospirosis in buffaloes of south Gujarat, India J.M. Patel, P.D. Vihol, V. S. Dabas, M.C. Prasad, J.H. Patel, C.F. Chaudhari, N.B. Patel and K.M. Patel...... 383

Age related changes in the histomorphology of mandibular gland in prenatal buffalo (Bubalus bubalis) K. Raja, M.S. Lakshmi, G. Purushotham, K.B.P. Raghavende and T.S. Chandrasekhara Rao...... 389

β(1,4)-Galactosyltransferase-I gene polymorphisms in Pakistani Nili Ravi buffalo Aamir Sohail, Asif Nadeem, Masroor Ellahi Babar, Tanveer Hussain, Akhtar Ali, Wasim Shehzad and Maryam Javed...... 399

Investigation of response to selection for milk traits in dairy buffalo of Iran based on three sale situations Bahareh Taheri Dezfuli and Leonardo De Seno...... 405

Nutritional status and hemato-biochemical profile of anoestrus buffaloes of Malwa region of Madhya Pradesh Nagendra Patil, R.K. Jain and Dharmesh Tewari...... 417 Buffalo Bulletin (July-September 2016) Vol.35 No.3

CONTENTS Page Original Article

Developmental competence of buffalo (Bubalus bubalis) oocytes: effect of oocytes quality, protein additives, hormonal supplement and type of capacitating agents M.M. Waheed, K.H. El-Shahat and A.M. Hammam...... 427

Seminal plasma and sperm membrane proteins of buffalo and cattle bulls: A comparative study Shilpi Dixit, Vijay Pandey, Dilip Kumar Swain, Rajesh Nigam, Ambika Sharma, Deepak Sharma, Atul Saxena and Pawanjit Singh...... 437

Incidence of repeat breeding in varying breeds of buffaloes and cattle in different climatic conditions in Khyber Pakhtunkhwa (Pakistan) Amjad Khan, Muhammad Hassan Mushtaq, Mansur ud Din Ahmad, Abid Hussain, Asghar Khan, Ajab Khan and Habibun Nabi...... 445

Incidence, pain assessment and management of horn affections in buffaloes K. Rama Rao, Makkena Sreenu, K.B.P. Raghavender and P.V.S. Kishore...... 455

Evaluation of feeding practices and certain minerals status of lactating buffaloes in coastal zone of western India P.L. Sherasia, P.R. Pandya, S. Parnerkar, B.R. Devalia and B.M. Bhanderi...... 467

Gross morphological studies on major salivary glands of prenatal buffalo K. Raja, M. Santhi Lakshmi, G. Purushotham, K.B.P. Raghavender, T.S. Chandrasekhara Rao and D. Pramod Kumar...... 479

Case Report Buffalo Bulletin (July-September 2016) Vol.35 No.3

AN UNUSUAL CASE OF IDEOPATHIC FIBRINOUS PERICARDITIS IN NILI-RAVI BUFFALO-A CASE REPORT

Muhammad Saqib, Ghazanfar Abbas* and Mudassar Niaz Mughal

ABSTRACT seen in old, pregnant and recently parturated animals. Ingestion and penetration of any sharp An unusual case of idiopathic fibrinous object from gastrointestinal tract to heart usually pericarditis in Nili-Ravi buffalo was presented leads to development of this disease. Incidence at Veterinary Teaching Hospital, Department of of foreign body associated pericarditis higher in Clinical Medicine and Surgery (CMS), University buffalo than in cattle (Misk et al., 2001). Among of Agriculture Faisalabad. The clinical signs and animals, dogs and horses are usually affected by postmortem findings were suggestive of idiopathic idiopathic fibrinous pericarditis (Jestyet al., 2005). fibrinous pericarditis which are discussed in detail. This is infrequently seen condition in buffalo especially Nili-Ravi buffalo (Summet et al., 2012). Keywords: Nili-Ravi buffalo, clinical signs, postmortem findings, idiopathic, fibrinous pericarditis CASE PRESENTATION

A 7 year old, Nili-Ravi buffalo was INTRODUCTION presented in comatosed position at Veterinary Teaching Hospital, Department of Clinical Medicine Nili-Ravi buffalo is also known as Black and Surgery (CMS), University of Agriculture Gold of Pakistan, because of its adaptability in Faisalabad Pakistan. The buffalo was from a dairy hot climates and major share in milk and meat farm with 20 other milking animals. She had calved production. Inflammation of pericardium along 25 days earlier without any problem. Anamnesis with accumulation of fibrinous material is called did not reveal any significant abnormality but as pericarditis (Grunder, 2002). Clinical signs history of complete anorexia, scanty feces and associated with pericarditis are tachycardia, muffled sudden drop in milk production for the last 5 days. heart sounds, distention of jugular vein, edema She was treated by referring veterinarian with of submandibular, brisket and ventral abdominal fluid therapy, antibiotics, analgesics, laxatives region. This is usually a disease of developing and ruminotonics but buffalo did not respond; the countries because of poor managemental and condition of the animal had deteriorated despite feeding practices, affecting ruminants through loss therapy. On clinical examination, buffalo had of production and death. This condition is mostly severe tachycardia, dyspnoea and mild brisket

Department of Clinical Medicine and Surgery, Faculty of Veterinary Sciences, University of Agriculture Faisalabad, Punjab, Pakistan, *E-mail: [email protected]

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edema. Temperature, respiration and pulse rate Reticular wall was intact. No other lesions were were 39.2 oC, 45 breaths/minute, 120 beats/minute, detected in rest of the body. respectively. Before a diagnosis could be arrived, the animal died. Postmortem was conducted with aim of investigation of the cause of death. DISCUSSION

The clinical signs and postmortem NECROPSY FINDINGS findings were suggestive of fibrinous pericarditis as described by Radostits et al., (2007). Grossly, Thoracic and abdominal findings were heart and reticulum were normal, as there was insignificant except generalized edema most not a single spot or hole on the outer surface. prominent in thoracic region, along with foul The underlying cause of pericarditis was not of smelling straw colored fluid in pericardium having traumatic origin because of absence of fibrous flakes of fibrinous material giving gelatinous tract from reticulum to pericardium. Generally and hirsute like appearance to heart especially pericarditis has three forms, effusive, fibrinous and epicardium and pericardium. Grossly there was no constrictive pericarditis. Accumulation of protein detectable lesion or spots on heart except markedly rich fluid in the pericardial sac is called as effusive increased heart size and thickened pericardial wall. pericarditis. If there is fibrin deposition along with Reticulum was full of undigested fodder having protein rich fluid then it is termed as fibrinous four, 5 to 6 centimeter non penetrated metal wires pericarditis, while maturation of fibrin along with along with six, 3 to 4 centimeter blunt ended nails. the fibrosis of pericardium is known as constrictive

Figure 1. Accumulation of straw colored fibrinous material in pericardium of affected buffalo.

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pericarditis (Pekins et al., 2004). In ruminants, elsewhere (Radostits et al., 2007; Sumeet et al., incidence of fibrinous and septic pericarditis is 2012). However, this report lacks microbiological more in contrast to idiopathic and non septic and histopathological investigations that need to be pericarditis. In ruminants mostly pericarditis is investigated to understand the etiopathogensis of traumatic in origin, where a sharp object such as this condition. wire and nail penetrates into pericardium through perforation in reticulum. Postmortem examination of affected animal usually reveals foreign body REFERENCES penetrating into pericardium but sometime the penetrating object may fall back into reticulum after Gründer, H.D. 2002. Krankheiten des Herzens und contacting pericardium, so traumatic pericarditis des Herzbeutels, p. 159-181. In Dirksen, G., cannot be ignored in this case.Nonetheless, the H.D. Gründer and M. stöber (eds.) Innere heart was bereft of any healing or healed trauma. Medizinund Chirurgie des Rindes, 4th ed. Haematogenous route associated pericarditis has Parey Buchverlag. Berlin, Germany. been reported in colibacillosis, salmonellosis and Jesty, S.A., R.W. Sweeney, B.A. Dolente and V.B. some anaerobic infections. It is less common and Reef. 2005. Idiopathic pericarditis and is masked by signs of septicemia. Radostits et cardiac tamponade in two cows. J. Am. Vet. al. (2007) reported that physical penetration of Med. Assoc., 226: 1555-1558. pericardial sac is not essential for development of LeWinter, M.M and S. Kabbani. 2005. Pericardial pericarditis as in some cases traumatic mediastinitis diseases, p. 1757-1780. In Zipes, D.P., P. acts as a vehicle for the development of infection. Libby, R.O. Bonow and E. Braunwald (eds.) In humans, presence of large quantity of fibrinous Braunwald’s Heart diseases. A Textbook of material in the pericardial sac is considered to be a Cardiovascular Medicine, 7th ed. Saunders diagnostic parameter for pericarditis (Lewinter and Elsevier, New York, USA. Kabanni, 2005). Misk, N.A and M.A. Semieka. 2001. The Clinical signs, hematological analysis, radiographic appearance of reticular radiography, pericardiocentesis, ultrasonography diaphragmatic herniation and traumatic and postmortem findings are diagnostic tools for pericarditis in buffalo and cattle. Vet. Radiol. confirmation of pericarditis. Different control Ultrasound., 42: 426-430. measures such as routine administration of Pekins, S.L., K.G. Maqdesia, W.P. Thomas and S.J. magnets to pregnant animals, keeping ruminants Spier. 2004. Pericarditis and pleuritis caused away from construction areas, routine analysis by corynebacterium pseudotuberculosis in a of crops for metallic objects, effective feeding horse. J. Am. Vet. Med. Assoc., 224: 1133- and managemental strategies such as screening 1138. of fodder and processed feed before serving to Radostits, O.M., C.C. Gay, K.W. Hinchcliff and animals. This unusual case of idiopathic fibrinous P.D. Constable. 2007. Veterinary Medicine. pericarditis has not been reported in Nili-Ravi A Textbook of the Diseases of Cattle, Horse, buffalo in Pakistan. Our case study is based on Sheep, Pigs and Goats, 10th ed. Saunders clinical signs and postmortem findings as described Elsevier, PA. p. 189-391.

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Sumeet, S., G.S. Navjot and Varun. 2012. Ideopathic fibrinous pericarditis in a Nili-Ravi buffalo. Buffalo Bull., 31: 173-175.

302 Original Article Buffalo Bulletin (July-September 2016) Vol.35 No.3

EFFECT OF LUGOL’S IODINE ON ESTRUS INDUCTION AND FERTILITY RESPONSE IN TRUE ANESTRUS JAFFRABADI BUFFALOES

A.R. Ahlawat1, P.U. Gajbhiye1, M.D. Odedra1, V.B. Dongre2 and S.N. Ghodasara1

ABSTRACT INTRODUCTION

Anestrus is a functional disorder of Anoestrus is the major infertility problem reproductive cycle in cattle and buffalo which is in farm animals. It is important to note that anoestrus characterized by absence of overt signs of estrus is abroad term, which indicates the lack of oestrus and also affecting the livestock enterprise to a great expression at an expected time. The meaning extent. Incidence of anestrus is more in buffalo than depends on age, weight, breed and history. Delay in the cattle, and problem is severe during summer. The expression of oestrus is beyond accepted average present study was carried out to see the efficacy of in anoestrus. It must be understood that a period of lugol’s iodine for the initiation of ovarian cyclicity sexual quietness in animals is shown by complete in postpartum true anestrus buffaloes. Confirmation absence of oestrus cycles. The incidence and of true anestrus in 20 buffaloes was done by finding management of the anoestrus have been recognised smooth ovaries at rectal examination, out of 20, 10 as age old problems in cattle breeding and there buffaloes was treated with Lugol’s iodine (1:50) is wealth of documentation on various therapies ratio 30 ml I/U once only whereas, the remaining to induce estrus in cow and buffaloes. The main 10 buffaloes were serve as control for treated group native tract of Jaffarabadi buffaloes is Saurashtra and no treatment was given to such animals. The region of Gujarat. These animals though very good result for induction of estrus was 70% (7/10) and milkers have a very high age at first calving and a the conception rate was 85.71% (6/7). Lugol’s long inter- calving period. Delayed resumption of iodine treatment is cheaper and effective means postpartum estrous activity is a most vital factor of management of anestrus but response has been responsible for poor reproductive efficiency of variable. these animals. Various hormonal and non hormonal therapies have been been used for induction of Keywords: anestrus, conception rate, infertility, estrus and fertility in anestrous bovines by various ovarian-cyclicity workers. Ovarian massage is known to stimulate the cyclicity in some anoestrous cases. It is proved that the cervical stimulation with lugol’s iodine

1Cattle Breeding Farm, Junagadh, Agricultural University Junagadh (Gujarat), India, *E-mail: [email protected] 2Department of Animal Genetics and Breeding, College of Veterinary Science and Animal Husbandry, Junagadh Agricultural University, Junagadh (Gujarat), India

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at very low concentration as paint or intrauterine from the buffalo bulls stationed at farm. Artificial infusion gives better response in inducing oestrus. insemination was conducted 18 h after sign of estrus was clearly visible. The parameters measured were onset of estrus, duration of estrus, percentage of MATERIALS AND METHODS estrus and conception rate. Buffaloes were rectally palpated to confirm pregnancy 50 days after last The experiment was conducted at cattle AI. All the experimental animals were maintained breeding farm, Junagadh, Gujarat a total of 20 as a group and were housed in semi open system. postpartum pluriparous Jaffrabadi buffalo were Each animal was fed with 30-35 kg of green fodder, selected by rectal palpation. The age and the parity 3-5 kg of concentrate (Amul dhan and Cotton seed of the buffaloes ranged from 5 to 12 years. The cake). Management of these animals was nearly animals in experiment had parities ranging from similar and they were released extensively during 2 to 6 The buffaloes which did not any signs of the day for free grazing. estrus 90 days postpartum were included in the study. The animals were divided in two groups of ten animals each, Group I (treatment group) were RESULTS AND DISCUSSION given Lugol’s iodine (1:50) 30 ml I/U once only. Whereas the animals of Group II control) animals A total of seven animals (70%) out of served as control and no treatment was given. ten animals injected in group I, responded to Beginning from the day of injection all the animal the treatment. While out of the control group were observed for estrus twice a day at 7.00 am three animals exhibited heat signs. Out of the and 3.00 pm with a teaser bull. The buffaloes were seven animals bred six animals conceived with observed for oestrus activity next day onwards a conception rate of 85.71%. Out of the three a buffalo was said to be in standing estrus if it animals bred (control group) one animal conceived allowed the bull to mount. Any buffalo in standing (conception rate of 33.33%). Various workers have estrus was inseminated with good quality semen reported variable response ranging from (45%

Table 1. Efficacy of Lugol’s Iodine for induction of estrus in postpartum true anoestrus Jaffrabadi buffaloes.

Group I Group II Sl. No Attribute (Treatment group) (Control) 1 Total No of animal treated 10 10 2 Response (animal induced) 7 3 3 Percentage response (%) 70 30 4 Animal bred 7/7 3/3 5 Animal conceived 6/7 1/3 6 Conception rate 85.71% 33.3% 7 Average No of AI / conception 3 2.75

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to 91%) among cattle and buffaloes (Singh and anestrus.The animals given Lugol’s iodine had a Thakur 1999; Tomar 2004; Gupta et al., 2011). The better response percentage, faster heat induction period of postpartum anoestrus is usually longer in and better conception rate. buffalo than the cattle under similar management conditions (Jainudeen and Hafez, 1993), probably due to low LH secretion during early postpartum REFERENCES period (Perera, 2011) It is presumed that painting of Lugol’s iodine on posterior part of the cervix causes El-Shahat, K.H. and A. Badr. 2011. Comparative local irritation and brings about reflux stimulation Study on Efficacy of Different Medicaments at anterior pituitary for secretion of gonadotrophins on Postpartum Anestrus Dairy Cows. and consequently cyclicity. Lugol’s iodine is an Journal of Applied Biological Sciences, irritating solution and intrauterine infusion of 5(3): 59-63. Lugol’s solution (0.5 to 1.0%) causes hyperemia Gupta, R., M.S. Thakur and A. Sharma. 2011. (enhanced circulation) of uterine mucosa resulting Estrus induction and Fertility response in into degree of iodine absorption from uterus. The true anestrus buffaloes using lugol’s iodine. absorbed iodine probably increases the metabolic Veterinary World, 4(2): 77-78. rate of body through stimulating the thyroid Jainudeen, M.R. and E.S.E. Hafez. 1966. Control hormone secretion (Sanchez, 1995). Increased of estrus and ovulation in cattle with orally metabolic rate trigger the ovarian functions by active progestin and gonadotrophin. Int. J. enhancing the energy utilization (El–Shahat and Fertil., 11(1): 47. Badr, 2011). Injectable Lugol’s iodine has also Perera, B.M. 2011. Reproductive cycles of buffalo. been used with the same assumption (Sarkar, Anim. Reprod. Sci., 124: 194-199. 2005). Anestrus is a multifactorial problem but Sanchez, J.M. 1995. Iodine in bovine nutrition. its occurrence signals the inadequate nutrition, Nutr. Anim. Tropic., 2: 95-120. environmental stress, uterine pathology and Sarkar, A.K. 2005. Treatment of anestrus cow improper managemental practices. The return of with diluted logul’s iodine and massage estrus in the three animals of control group may on reproductive oragans-uncontrolled case be explained due to correction in any one of the study. J. Anim. Vet. Adv., 4(8): 734-736. factors. Singh, S.L. and M.S. Thakur. 1999. Estrus synchronization and fertility with the uterine infusion of prostaglandin F2 alpha CONCLUSION and lugol’s iodine in crossbred cows. Indian Journal of Animal Reproduction, 20(1): 12- Anestrus is a multi-causative factors 14. associated problem affecting livestock enterprise to Tomar, D.S. 2004. Studies on etiology and treatment a great extent. Diagnosis of the condition needs to be of subestrus in Murrah buffaloes. M.V.Sc prompt and at the earliest to prevent its occurrence and A.H., Thesis, JNKVV. Jabalpur, India. for effective treatment. Lugol’s iodine treatment is cheaper and effective means of management of

305 Buffalo Bulletin (July-September 2016) Vol.35 No.3 Original Article Buffalo Bulletin (July-September 2016) Vol.35 No.3

HAEMATO-BIOCHEMICAL ALTERATIONS DURING DIFFERENT STAGES OF LACTATION IN MEHSHANI BUFFALOES

Hemen Das1,*, A. Lateef2, H.H. Panchasara2 and M. Ayub Ali1

ABSTRACT was recorded to be the lowest in the early stage of lactation; whereas, the protein and creatinine The present study was carried out to concentrations were slightly higher in this stage. investigate the haemato-biochemical profile of No significant alteration in the concentration Mehshani buffalo, a native breed of Gujarat, India of Aspartate aminotransferase (AST), Alanine with the purpose of analyzing the physiological aminotransferase (ALT) and Alkaline phosphatase variations under the influence of different lactation (ALP) was noticed amongst the three groups of stages in terms of determining possible biomarkers buffaloes under the current study. Data generated to monitor the energetic balance and the metabolic during the current study may be useful as reference adequacy during lactation. Eighteen clinically values for the scientific community as this is the healthy lactating buffaloes were categorized into first study of its kind in case of Mehshani buffalo. three groups based on the length of their lactation: group I (early stage), group II (mid stage) and Keywords: haemato-biochemical, lactation stage, group III (late stage). Non significant variations mehshani buffalo were observed in case of the hematological parameters amongst the three groups of animals. The packed cell volume (PCV), RBC count and INTRODUCTION haemoglobin (Hb) concentration was lowest in the buffaloes of the early stage of lactation. Blood biochemical parameters vary during Other haematological parameters viz. different physiological stages of animals (Ahmad total leucocyte count (TLC), differential leucocyte et al., 2003). Pregnancy and lactation are two most count (DLC), mean corpuscular volume (MCV), important stages in the life of dairy animals, which mean corpuscular haemoglobin (MCH) and affect metabolism resulting in the alteration of the platelet count were recorded to be within the haemato-biochemical profile (Krajnicakova et al., normal limits. Similarly, the values of the blood 2003; Iriadam, 2007). There are numerous reports biochemical analytes varied apparently, but the on the effects of different phases of the reproductive differences were statistically non significant cycle and pregnancy on haemato-biochemical amongst the groups studied. The glucose level indices in domestic animal species including

1Department of Physiology and Biochemistry, College of Veterinary Science and Animal Husbandry, Central Agricultural University, Selesih, Mizoram, India, *E-mail: [email protected] 2College of Veterinary Science and Animal Husbandry, Sardarkrushinagar Dantiwada Agricultural University Sardarkrushinagar, Dantiwada, Gujarat, India

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buffalo (Jain et al., 2009). However, no such study Sardarkrushinagar Dantiwada Agricultural could be traced investigating the blood picture University, Sadarkrushinagar, Gujarat, India. The during different stages of lactation in Mehshani buffaloes were in various stages of lactation and buffalo, a unique milch breed of Gujarat, India. It based on the length of their lactation, the animals is well established that milk and milk components were identified as in early (7 to 105 days), mid (106 are directly and indirectly synthesized from blood. to 210 days) and late (211 to 315 days) lactational The rate at which blood flows to the mammary stage. Accordingly, they were categorized into gland is one of the key-factors in determining milk three different groups of six animals each viz. synthesis. group I (early lactation), group II (mid lactation) Approximately, 400 to 500 liters of blood and group III (late lactation). circulate through mammary gland to produce one liter of milk (Fernandez and Hoeffler, 1998). Collection of blood samples There is a 2 to 6 folds increase in blood flow in Blood samples were collected aseptically the mammary gland starting 2 to 3 days prepartum. from each animal of all the three groups by jugular During lactation, the mammary gland secretory vein puncture into collection tubes containing ­ cells utilize 80% of the blood metabolites for anticoagulants viz. K3EDTA and Lithium heparin milk synthesis depending on the infiltration of for hematological and biochemical analysis, precursors of milk components like amino acids, respectively. glucose and fatty acids (Piccione et al., 2009). Hence, blood biochemical parameters including Haematological analysis total protein, triglycerides, free fatty acids and Collected blood samples were analyzed urea are important indicators of the metabolic for different hematological parameters including activity in lactating animals (Karapehlivan et al., packed cell volume (PCV), haemeoglobin 2007). Since the milk yield and composition varies (Hb), mean corpuscular volume (MCV), mean across the length of lactation stage, it is, therefore, corpuscular hemoglobin (MCH), total erythrocyte imperative, to study haematolo-biochemical count (TEC), total leucocyte count (TLC), constituents during different stages i.e. early, mid differential leucocyte count (DLC) and platelet and late stage of lactation. Accordingly, the present count (PLT) using Automated Haematology study was undertaken to investigate the variations Analyzer (Cell-Dyn 3700, Abbott Diagnostics, in haemato-biochemical profile during different USA). stages of lactation in Mehshani buffalo. Biochemical analysis Blood samples were analyzed for different MATERIALS AND METHODS biochemical analytes viz. Glucose, Total protein, Blood Urea Nitrogen (BUN), Calcium (Ca), Experimental animals Creatinine, Total Bilirubin, Alkaline phosphatase Eighteen (18) clinically healthy lactating (ALP), Alanine aminotransferase (ALT), Mehshani buffaloes were selected from the Asparate aminotransferas (AST) by employing herd maintained at Livestock Research Station, Dry Chemistry discs/cartridges in Piccolo Xpress

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Chemistry Analyzer (Abaxis, USA). that lactose synthesis and milk yield show a linear positive correlation with glucose uptake and thus Data analysis the lactose synthesis potential is accompanied The results were statistically analyzed using by greater glucose uptake by lactating mammary one-way ANOVA as per the method of Snedecor gland (Afshar and Fathi, 2012). The total protein and Cochran (1994). P<0.05 was considered to be level (8.45 gdl-1) was found to be slightly higher statistically significant. in group I as compared to group II and group III animals. This observation is on the contrary to the finding of Yaylaket al. (2009), who recorded lower RESULTS AND DISCUSSION protein values in dry and early stages of lactation in case of Holstein cows. Krajnicakova et al. (2003) The results (mean±SE) of the biochemical also observed an increasing trend of total protein and haematological analysis have been presented level of serum with the progress of lactation in in Table 1 and Table 2, respectively. lactating goats and concluded that this is due to Table 1 reveals no significant difference the catabolism of protein for milk synthesis. The (P>0.05) in the concentrations of various variation may be attributed to the differences in biochemical constituents amongst the three species, nutrition, husbandry, environment and groups of lactating Mehshani buffaloes. This methods of assay (Beaunoyer, 1992; Osman and Al- finding corroborates the report of Hagawane et Busadah, 2003). However, Hagawane et al. (2012) al. (2012). Present study further indicated that reported highest protein value in the early stage of the mean concentration of blood glucose was lactation, which is comparable to current findings. lowest (40.67±2.04 mgdl-1) in early stage and The possible explanation for this phenomenon may increased subsequently as the lactation advances. be the haemoconcntration and water losses due The observed values of blood glucose for mid and to parturition. Further, earlier investigations have late stage of lactation were 42.5±4.57 mgdl-1 and clearly shown that the expression of major milk 46.37±4.31 mgdl-1, respectively. Current trend proteins increases dramatically and in a concerted of variations are consistent with earlier report way during the onset of lactation (Bionaz and Loor, in lactating ewes (Roubies et al., 2006) and in 2011). lactating mares (Heidler et al., 2002). In contrast, Similarly, the mean value of blood urea glucose levels were reported to be the same nitrogen (BUN) was also recorded to be higher throughout the three stages of lactation by Peterson in initial stage of lactation and decreased as the and Waldern (1981); whereas, Doornenbal et lactation progresses. This may hold good in al. (1988) reported somewhat higher (P<0.05) relation to observed apparently increased level glucose concentration at parturition that declined of total protein. The BUN values observed in the during lactation period. The lower level of blood present study at different stages of lactation were glucose recorded during early stage of lactation higher than those reported in earlier investigation may be ascribed to the utilization of large amount (Hagwane et al., 2012). Reinartz and Hofmann of blood glucose by mammary gland for the (1989) also found that serum urea concentration synthesis of lactose (Schultz, 1968). It is reported was significantly influenced by the lactation stage.

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Table 1. Mean±S.E. values of biochemical analytes at different stages of lactation. Parameters Early lactation Mid lactation Late lactation Glucose (mg/dl) 40.67±2.04 42.5±4.57 46.37±4.31 Total protein (g/dl) 8.45±0.13 8.12±0.22 8.01±0.29 Blood Urea Nitrogen (mg/dl) 22.67±1.3 20.5±1.58 18.89±2.25 Calcium (mg/dl) 7.0±0.93 8.1±0.54 8.19±0.23 Creatinine (mg/dl) 1.33±0.08 1.2±0.07 1.15±0.16 Total Bilirubin (mg/dl) 0.23±0.03 0.22±0.04 0.24±0.04 Alanine aminotransferase (ALT) (U/L) 66.67 ±5.35 72.67±6.37 67.5±5.07 Aspartate aminotransferase (AST) (U/L) 154.0±4.46 148.5±17.97 159.5±7.87 Alkaline phosphatase (ALP) (U/L) 178.67±81.50 168.67±47.03 165.56±45.50

(P<0.05; statistically non significant)

Table 2. Mean±S.E. values of hematological indices at different stages of lactation. Parameters Early lactation Mid lactation Late lactation WBC (K/µl) 9.63±0.65 8.24±0.79 9.55± 0.98 NEU (%) 30.83±2.19 28.28±1.58 29.12±2.01 LYM (%) 50.17±2.02 48.35±1.69 50.15±2.29 MONO (%) 7.17±0.40 7.18±1.03 8.95±1.96 EOS (%) 5.83±1.49 6.44±0.97 5.36±0.57 BASO (%) 0.841±0.15 0.746±0.16 0.644±0.17 RBC (M/µl) 5.67±0.98 6.38±0.35 7.49±0.41 HGB (g/dl) 12.35±4.48 14.1±4.17 13.73±2.84 PCV (%) 30.87±0.97 32.72±1.87 31.33±1.35 MCV (fL) 48.07±2.021 45.22±2.54 41.93±0.87 MCH (pg) 16.22±0.69 15.48±0.80 15.17±0.58 PLT (K/µl) 326.5±33.63 617.17±104.86 507.17±51.62 (P< 0.05; statistically non significant)

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It is recorded that the efficiency for utilization of may be hypothesized that the buffaloes gradually metabolisable protein for milk production (0.68) recover from the stress of parturition and excess is less than that of maintenance (1.00) (McDonald demand of calcium for initiation of lactation. On et al., 1995). So, as the milk production increases, the contrary, Ramakrishna (1991) recorded higher the overall protein utilization efficiency decreases, values (9.77±0.33 mgdl-1) of calcium in lactating which consequently leads to more drainage of buffaloes. Further, Yokus et al. (2004) also nitrogen in terms of urea through urine and milk concluded that the levels of Ca decreased slightly (Roy et al., 2003). An increase in urea value was in early pregnancy to late pregnancy and then further observed in the first 8 weeks of lactation increased during lactation period in sheep. (Ndibualonji and Godeau, 1993) and found to be Present study also indicated that the blood peak at 12 weeks postpartum, which decreased creatinine level was higher in group I as compared slowly thereafter (Rajcevic et al., 1993). However, to group II and group III buffaloes. The apparent other researchers found a different trend of variation increase in creatinine level at the early stage of in case of BUN. During the first month of lactation lactation may be ascribed to uterine involution lower milk urea (MU) concentration was recorded and myometrial protein degradation (Bell et al., by Carlsson et al. (1995). Likewise, Whitaker et 2000). Nonetheless, Peterson and Waldern (1981) al. (1995) also reported that cows in early lactation found no differences in creatinine concentrations often have much lower MU level. In contrast, no amongst the various stages of lactation, but relation was reported between urea concentration observed that creatinine levels rose in dry cows in milk and stage of lactation by Erbersdobler et al. with increasing days of pregnancy. Kronfeld (1990) and values were relatively constant between (1982), working with 21 Holstein herds, reported 200 to 300 mg/l. Similarly, Coustumier (1996) also the highest serum creatinine levels during the found no correlation between lactation stage and peak of lactation. Total bilirubin concentration urea levels except just after calving. Similar to our recorded in this study was found to be consistent study, Schepers and Meijer (1998) also observed throughout the lactation period indicating that its that stage of lactation had no significant influence concentration remains unaffected with the stage on BUN and thus on MU concentration. Hence, of lactation. Similarly, no significant alteration in in the light of varying observations of different the concentration of Aspartate aminotransferase researchers, a systemic and critical investigation (AST), Alanine aminotransferase (ALT) and may be established in this aspect. Alkaline phosphatase (ALP) was recorded amongst In this study, the drop in calcium (Ca) the three groups of buffaloes under the current level (7.0±0.93 mgdl-1) was more pronounced study. The concentration of AST was found to during early stage of lactation as compared to be highest (72.67±6.37 U/L) in the mid lactation mid and late stage. This may be due to excessive stage. Ling et al. (2003) observed that the blood drainage of blood calcium pool through colostrum concentration of AST increases between day 117 and milk during this stage. As the stage of and 151 of lactation (mid stage) in Holstein mares, lactation progresses, the blood calcium level is which is in accordance with the present findings. increased, which is in agreement with the findings Conversely, Yaylak et al. (2009) reported that the of Rowlands et al, (1975) and Nale (2003). It stage of lactation affects AST and ALT activities

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significantly. An increase in ALT, AST and GGT of lactation. (γ-glutamyl transferase) activity in the blood of It may be concluded that stage of lactation ewes during lactation is indicative of increase in does not play significant role in alteration of hepatic metabolism (Antunovic et al., 2004, 2011). haemato-biochemical profile in lactating Mehshani Further, AST and ALP are considered to be effective buffaloes. The limited sensitivity of these blood biomarkers to detect the energetic and mineral parameters to stage of lactation in clinically normal imbalance in Saanen dairy goats (Mundim et al., dairy animals is not surprising because, most of 2007). Changes in activities of these enzymes may these parameters are under the homeostatic control also be related to reduced dry matter intake around systems (Cozzi et al., 2011). Nonetheless, data parturition, which lead to hepatic lipidosis and alter generated during the current study may be useful the normal function of the liver (Greenfield et al., as reference values for the scientific community as 2000). However, no indications were found in the this is the first study of its kind in case of Mehshani literature to explain the relationship of the recorded buffalo. Further, blood profile has traditionally trends of variations in the concentrations of these been used to assess the metabolic health status of enzymes with different stages of lactation. the animals; hence the present investigation may Table 2 indicated that although the also be helpful in this regard. In addition, this observed values of the haematological parameters study may also assist the nutritionists to formulate varied apparently, the differences were statistically ration for optimum productivity of the Mehshani non significant. Similar types of observations buffaloes since blood-biochemical analytes are were also recorded by Hagawane et al. (2012). being widely considered to identify dietary causes The packed cell volume (PCV), RBC count and of diseases leading to low productivity. haemoglobin (Hb) concentration was found to be lowest in Mehshani buffaloes in early stage of lactation corroborating with those of Esievo REFERENCES and Moore (1979), who concluded that the concentrations of PCV RBC, Hb along with serum Afshar, M. and H. Fathi. 2012. Lactose in ruminants iron (SI), iron binding capacity (IBC) and serum feeding: A review. Annals of Biological albumin decreased in early lactation and rose to Research, 3(1): 645-650. pre-lactation levels by mid-lactation. Decline in Ahmad, I., A. Gohar, N. Ahmad and M. Ahmad. the number of RBC in the blood of ewes in the 2003. Haematological profile in cyclic, early lactation was also reported by Antunovic et non Cyclic and Endometritic Cross-Bred al. (2011). Other haematological indices such as cattle. International Journal of Agriculture the TLC, DLC, mean corpuscular volume (MCV), Biological, 5(3): 332-334. mean corpuscular haemoglobin (MCH) and Antunovic, Z., J. Novoselect, H. Sauerwein, M. platelet count were found to be within the limits Speranda1, M. Vegeraand and V. Pavic. of normal values laid for the buffaloes. Similar to 2011. Blood metabolic profile and some current investigation, non significant differences in of hormone concentration in ewes during various haematological indices were also reported different physiological status. Bulg. J. by Flores et al. (1990) during early and late stage Agric. Sci., 17(5): 687-695.

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Antunovic, Z., M. Speranda and Z. Steiner. 2004. stage of lactation and breed of the cows. J. The influence of age and the reproductive Anim. Physiol. An. N., 63: 1-7. status to the blood indicators of the ewes. Esievo, K.A. and W.E. Moore. 1979. Effects of Arch. Tierz. Dummerstorf., 47(3): 265-273. dietary protein and stage of lactation on Beaunoyer, D.E. 1992. Changes in Serum Enzyme the haematology and erythrocyte enzymes Activities after maximal exercise, p. 331- activities of high-producing dairy cattle. 333. In Proceedings of First International Res. Vet. Sci., 26(1): 53-58. Camel Conference, 2nd-6th ed. Publications. Fernandez, J.M. and J.P. Hoeffler. 1998. Transgenic New market Ltd. Expression, p.410. In Gene Expression Bell, A.W., W.S. Burhans and T.R. Overton. 2000. Systems: Using Nature for the Art of Protein nutrition in late pregnancy, maternal Expression, Academic Press, U.S.A. protein reserves and lactation performance Flores, A., R. Althaus, J.C. Toibero, O.J. Garnero in dairy cows. The Proceedings of the and L. Perren. 1990. Metabolic profile Nutrition Society, 59: 119-126. of dairy cow before and after parturition. Bionaz, M. and J.J. Loor. 2011. Gene networks Revista Argentia de Production Animal, driving bovine mammary protein synthesis 10(4): 289-293. during the lactation cycle. Bioinform. Biol. Greenfield, R.B., M.J. Cecava, T.R. Johnson Insights, 5: 83-98. and S.S. Donkin. 2000. Impact of dietary Carlsson, J., J. Bergstrom and B. Pehrson. 1995. protein amount and rumen undegradability Variations and breed, age, season, yield, stage on intake, peripartum liver triglyceride, of lactation and herd in the concentration of plsama metabolites and milk production urea in bulk milk and individual cow’s milk. in transition dairy cattle. J. Dairy Sci., 83: Acta Vet. Scand., 36: 245-254. 703-710. Coustumier, J.L.E. 1996. The balance of ration is Hagawane, S.D., S.B. Shinde and D.N. Rajguru. not the whole story. Production Laitiere 2012. Haematological and blood Moderne, 261: 96-97. biochemical profile in lactating Buffaloes in Cozzi, G., L.F. Gottardo, A.L. Stefani, B. Contiero, and around Parbhani city. Veterinary World, L. Moro, M. Brscic and P. Dalvit. 2011. 2(12): 467-469. Reference values for blood parameters in Heidler, B., H. Sauerwein, U. Heintges, J. Aurich, Holsteindairy cows. Effects of parity, stage W. Pohl and C. Aurich. 2002. Metabolic of lactation, and season of production. J. profiles and plasma leptin concentrations Dairy Sci., 94: 3895-3901. in lactating and non-lactating mares. Doornenbal, H., A.K.W. Tong and N.L. Murray. Theriogenology, 58: 557-561. 1988. Reference values of blood parameters Iriadam, M. 2007. Variation in certain in beef cattle of different ages and stages of haematological and biochemical parameters lactation. Can. J. Vet. Res., 52: 99-105. during the pre-partum period in Kilis does. Erbersdobler, H.F., S. Braasch and E.A. Trautwein. Small Ruminant Res., 73: 54-57. 1990. Concentrations of taurine, urea and Jain, R.K., D.D. Nimkar, C.M. Saksule and R.K. free amino acids in milk as influenced by Dhakad. 2009. Haemato-biochemical

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profile of haemoglobinuric buffaloes. before and after parturition. M.V. Sc. thesis Buffalo Bull., 28(4): 184-187. submitted to MAFSU, Nagpur, India. p. 29- Karapehlivan, M., E. Atakisi, M. Citil, O. Kankavi 34. and O. Atakisi. 2007. Serum sialic acid Ndibualonji, B.B. and J.M. Godeau. 1993. Changes levels in calves with pneumonia. Vet. Res. in plasma amino acids, urea and glucose Commun., 31: 37-41. in relation to the end of gestation and the Krajnicakova, M.E., V. Hendrichovscky and I. onset of lactation in low yielding dairy Maracek. 1993. Concentrations of total cows. Mededelingen Vande Faculteit Land lipid, cholesterol and progesterone during bouwwetenschappen Universitiet Gent, 58: oestrus synchronization and pregnancy in 1713-1717. sheep. Vet. Med-us., 38: 349-357. Osman, T.E.A. and K.A. Al-Busadah. 2003. Krajnicakova, M.E., E. Bekeova, G. Kovac, Normal Concentration of Twenty Serum M. kostecky, I. valocky, I. Maracek, I. Biochemical Parameters of She-Camels, Sutiakova and L. Lenhardt. 2003. Selected Cows and Ewes in Saudi Arabia. Pakistan clinical-biochemical parameters in the Journal of Biological Sciences, 6(14): puerperal period of goats. B. Vet. I. Pulawy, 1253-1256. 47: 177-182. Peterson, R.G. and D.E. Waldern. 1981. Kronfeld, D.S., S. Donoghue, R.L. Copp, F.M. Repeatabilities of serum constituents in Stearns and R.H. Engle. 1982. Nutritional Holstein-Friesians affected by feeding, age, status of dairy cows indicated by analysis of lactation and pregnancy. J. Dairy Sci., 64: blood. J. Dairy Sci., 65: 1925-1933. 822-831. Ling, K., H. Jaakson, J. Samarutel and A. Leesmae. Piccione G, G. Caola, C. Giannetto, F. Grasso, 2003. Metabolic status and body condition S.C. Runzo, A. Zumbo and P. Pennisi. 2009 score of Estonian Holstein cows and their Selected biochemical serum parameters in relation to some fertility parameters. ewes during pregnancy, post-parturition, Veterinariaja Ir. Zootechnika. T., 24: 94- lactation and dry period. Anim. Sci. Pap. 100. Rep., 27: 321-330. McDonald, P., R.A. Edwards, J.F.D. Greenhalgh Rajcevic, M., I. Jazbec and M. Ponikvas. 1993. and C.A. Morgan. 1995. Animal Nutrition, Urea in milk and blood as indicator of 5th ed. Addison Wesley Lonman, Efinburgh nutritive matter supply in high-yielding Gate, Harlow, Essex CM202JE, United dairy cows. Zbornik Biotehniske Fakultete Kingdom. Univerze Edvasda Kardelja V Ljubljani, Mundim, A.V., A.S. Costa, S.A.P. Mundim, E.C. Kmetijstvo, 62:179-184. Guimaraes and F.S. Espindola. 2007. Ramakrishna, K.V. 1991. Comparative studies on Influence of parity and stage of lactation certain biochemical constituents of lactating on the blood biochemical profile of Saanen and dry Murrha buffaloes. Livestock goats. Arq. Bras. Med. Vet. Zoo., 59(2): 306- Advisor, 14: 16-19. 312. Reinartz, M. and W. Hofmann. 1989. Serum urea Nale, R.A. 2003. Metabolic profiling in buffaloes 314 Buffalo Bulletin (July-September 2016) Vol.35 No.3

estimation in dairy herds. Prakt. Tierarzt, of seasonal and physiological variations on 70: 22-28. the serum major and trace element levels in Roubies, N., N. Panousis, A. Fytianou, P.D. sheep. Biol. Trace. Elem. Res., 101: 241- Katsoulos, N. Giadinis and H. Karatzias. 255. 2006. Effects of age and reproductive stage on certain serum biochemical parameters of chios sheep under greek rearing conditions. J. Vet. Med. A., 53: 277-281. Rowlands, G.J., R. Manston, M. Rita, Pocock and M.D. Sally. 1975. Relationships between stage of lactation and pregnancy and blood composition in a herd of dairy cows and the influences of seasonal changes in management on these relationships. J. Dairy Res., 42: 349-362. Roy, B.R., K. Mehla and S.K. Sirohi. 2003. Influence of Milk Yield, Parity, Stage of Lactation and Body Weight on Urea and Protein Concentration in Milk of Murrah Buffaloes. Asian Austral. J. Anim., 16: 1285-1290. Schepers, A.J. and R.G.M. Meijer. 1998. Evaluation of the utilization of dietary nitrogen by dairy cows based on urea concentration in milk. J. Dairy Sci., 81: 579-584. Schultz, L.H. 1968. Ketosis in dairy cows. J. Dairy Sci., 51: 1133-1140. Snedecor, G.W. and W.G. Cochran. 1989. Statistical Method, 8th ed. Oxford and IBH Publishing Co., New Delhi, India. Whitaker, D.A., J.M. Kelly and H.F. Eayros. 1995. Assessing dairy cow diets through milk urea tests. Vet. Rec., 136: 179-180. Yaylak, E., C. Yenisey and K. Seyrek. 2009. Effects of Lameness, Stage of lactation and body conditions score on some blood parameters in Holstein cows. Asian J. Anim. Vet. Adv., 4(5): 245-251. Yokus, B., D.U. Cakmr and D. Kurt. 2004. Effects

315 Buffalo Bulletin (July-September 2016) Vol.35 No.3 Original Article Buffalo Bulletin (July-September 2016) Vol.35 No.3

effect of Early Post-Partum GnRH and PGF2 alpha Administration on follicular activities in Murrah buffaloes

M.V. Ingawale* and S.A. Bakshi

Abstract weeks in Group I, II and III, respectively. From

the present study it can be concluded that PGF2a The present study was carried out administration during early PP hastens the initiation to examine the effect of GnRH and PGF2α of post-partum estrus in buffaloes and transrectal administration during early post partum period ultrasonography is good tool for monitoring the (PP) on ovarian activities and initiation of PP estrus slight follicular developments. in Murrah buffaloes. A total of thirty six Murrah buffaloes were selected and divided equally into Keywords: GnRH, PGF2α, ovarian activity, post- three groups comprising twelve buffaloes each. partum estrus Buffaloes in Group I were i.m. injected with GnRH as busreline acetate ( 10 µg) while those in group

II were i.m. injected with PGF2α as tiaprost- Introduction trometamol (0.750 mg) on day 14 PP. The buffaloes in Group III were kept as The buffalo has important role in livestock control. The experimental buffaloes were examined economy of Asia including India. Buffaloes are with 7.5 transrectal probe on day 14, 21 and 28 valued for milk, meat and draught power. Hence PP. On day 14 PP, 14 (38.88%) ovaries showed the importance of buffaloes to the economy small follicular development and 4 (11.11%) of this country is considerable and cannot be ones showed multiple follicular activity when underestimated (Madan, 2010). Low reproductive observed ultrasonographically. On day 21 post- efficiency in general and buffaloes in particular partum, 4 (11.11%) ovaries showed small follicular remains a major economic problem globally, and development, 11 (30.50%) ovaries showed multiple its incidence is higher (4.66 to 12.66%) in our follicular activity and 18 (50.00%) ones indicated country (Tomar and Ram, 1993). Failure to resume good follicular development when observed ovarian activity after calving is the main reason ultrsonographically. On day 28 PP, 3 (9.37%) for delay in conception in buffaloes (Parmar et al., ovaries showed small follicular development and 3 2012). Early post-partum (PP) breeding to reduce (9.37%) ones showed multiple follicular activities the calving interval in buffaloes would increase whereas 26 (81.25%) ones indicated good follicular their reproductive efficiency (Shah et al., 2002). development when observed ultrasonographically. Thus PP period is regarded as an important in the The post partum ovarian activity was initiated reproductive life of bovines (Fonesca et al., 1983). with average 3.5±0.150, 3.25±0.130 and 4±0.467 The low reproductive efficiency of buffaloes, as

Department of Animal Reproduction, PGIVAS, Akola (MS) India, *E-mail: [email protected]

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evident from delayed first PP estrus and conception, and remained high for 8 to 16 days PP. So delay prolonged service period resulting in extended in involution of uterus was due to short period of calving interval (Khasatiya et al., 2006). high prostaglandin F2 alpha metabolite release

Ovarian follicular growth resumes early whereas, long duration of PGF2α release resulted after calving with the formation of first dominant in short period for completion of uterine involution follicle, detected by ultrasonographcally within (Lindell, 1981). It has also positive effect on the 10.12±72 in Bulgarian Murrah buffaloes (Yotov uterine musculature tone (Lindell and Kindahl, and Atanasov, 2013). Although uterine involution 1983). So PGF2α injection in early PP period begins and ovarian follicular waves resumes soon (day 14) enhances the uterine involution and after parturition due to rise in FSH concentration reproductive efficiency in normal calved buffaloes (Schallenberger, 1985). However, dominant follicle (Nazir et al., 1994). This is promoted us to study of these waves fails to ovulate due to failure to the follicular dynamics and initiation of PP ovarian undergo final terminal maturation. Failure of PP activity ultrasonographically and per-rectally dominant follicles to undergo final maturation is following 14 PP injection with GnRH and PGF2α due to inadequate LH pulse frequency, which result in normally calved buffaloes. in low foolicular androgen production (Fortune, 1986) and inadequate oestrodiol positive feedback to induce LH surge (Peters et al., 1985), which is Materials and Methods perquisite for follicular terminal maturation prior to ovulation. Absence of LH pulses in early post- Experimental animals partum is primarily due to depletion of anterior The present research work was carried out pituitary LH stores. Following replenishment of using 36 post-partum (PP) Murrah buffaloes at M/S LH stores between days 15 and 30 PP absence B.G. Chitale Dairy, Research and Development of LH pulses is due to continued sensitivity Farm, Bhilawadi in Sangli district, over a period of the hypothalamic GnRH pulse generator to of ten months. The buffaloes were housed in a the negative feedback effect of estradiol-17b loose housing barn with four groups of twenty-four which results in absence of GnRH pulses. The buffaloes. The buffaloes were kept indoors and administration of GnRH will therefore overcome there was no open paddock in the barn. Each lot the inadequate secretion of pituitary LH in early had twenty-six resting places (1.2x2m) on one side post-partum period (Shah et al., 1990) and restore and a manure alley with Delta MasterTM manure ovarian function earlier within PP period. scraper (Delaval AB, Sweden) on the other hand Prostaglandins plays major role in side positioned towards the feed rack. Each lot had regulation of reproductive cyclicity (Singh and one automatic concentrate feeding station (AFS) Madan, 1985). The reproductive cyclicity and its and nine valve-controlled automatic water bowls. rhythm in terms of its reawakening during early The ordinary routine in the barn was adlib feeding PP period has been linked to temporal changes of of roughages three times a day. The roughages prostaglandins in particular (Perera et al., 1981). fed during the experiment consisted of fresh, cut Lindell et al. (1980) reported that prostaglandin and chopped sugercane, alfalfa, napier grass, metabolites increased at the time of parturition green maize and jowar straw which were chopped

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and transported to the barn in tractor trolley and as control group. dispensed manually into the feed troughs. A pre- All the experimental were observed for calculated quantity of concentrate mixture was ovarian activity on days 14, 21 and 28 PP per- fed to each buffalo based on milk yield, body rectally and ultrasonographically. The ovarian weight and pregnancy status. Concentrate was fed follicular development, corpus luteum development through the automatic concentrate feeding station and regression were monitored using real time, (AFS) in the barn. If the pre calculated amount B-mode ultrasonograhy machine (Aloka-900) with was not consumed, the residual was transferred 7.5 MHz linear array rectal transducer. The follicles to the next feeding. Residual amounts at the end appear as black anechoic, roughly circumscribed of a 24 h period were transferred to the next 24 h areas surrounded by hyperechoic ovarian stroma period. During milking, an in-parlor feeding (IPF) on the ultrasound image. The follicles observed system supplied a fixed amount of concentrates. ultrasonographically in the present study were The buffaloes were provided mineral mixture classified according to according to milk production and body weight of SFD (slight follicular development): the buffaloes. F<0.5cm or follicles as small as 3-4 mm, but few All the buffaloes were appropriately (<5) in number. vaccinated against foot and mouth disease and MSF (Multiple small follicles): F<0.5 cm haemorrhagic septicemia. They were also tested or follicles as small as 3-4 mm but many (>5) in annually to detect possibilities of Brucellosis, number. Johne’s disease and Tuberculosis and the positive GFD (Good follicular development): reactors were suitably disposed off. The fecal Bigger follicles >0.8cm and or oestrual follicles. samples and blood smears were also screened NFD (No follicular development): Absence periodically for detection of parasitic infestations of the above picture within ovarian stroma. and protozoan parasites, respectively. As a routine, The data was analyzed by employing all buffaloes were dewormed biannually. statistical design as recommended by Snedecor and Cochran (1994). Experimental design Total 36 Murrah buffaloes with second to seventh lactation were selected. These buffaloes RESULTS AND DISCUSSION were divided into three groups comprising twelve bufaloes and following treatments were given. The ultrasound imaging of ovaries in 36 PP buffaloes revealed ovarian activity with Group I Buffaloes were intramuscularly respect to follicular growth right from the first injected with 10 µg Busereline acetate (Intervet, day of examination (day 14 PP). The ovaries India) on day 14 PP. were characterized by growth and regression of Group II Buffaloes were intramuscularly several small (up to 5 mm) and medium sized injected with 0.750 Tiaprost trometamol (Intervet, (>5 and <10 mm in diameter) follicles until the India) on day 14 PP. detection of first PP dominant (≥10 mm) and/ Group III Buffaloes were kept untreated or ovulatory follicle during the study period.

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Follicular activities ovaries showed multiple follicular activity when A total 424 observations on ovaries were observed ultrasonographically. Thirteen (36.11%) carried out by per-rectally and ultrasonographically. ovaries indicated good follicular development The manual observations were compared with when observed per-rectally while 18 (50.00%) the ultrasonographical observations to study the ovaries indicated good follicular development comparative detectability of ovarian follicles by when observed ultrsonographically. On day 28 palpation per-rectum and by ultrasonography. The PP, 6 (16.16%) ovaries indicated small follicular overall observations during different periods for development observed per-rectally while 3 (9.37%) both the methods are presented in Table (1). ovaries showed small follicular development and According to Table (1), 6 (16.16%) 3 (9.37%) ovaries showed multiple follicular ovaries indicated small follicular development activity when noticed ultrasonographically. when observed per-rectally while 14 (38.88%) Furthermore, 26 (81.25%) ovaries indicated good ovaries showed small follicular development and follicular development observed per-rectally while 4 (11.11%) ovaries showed multiple follicular 26 (81.25%) ovaries indicated good follicular activity when observed ultrasonographically development when observed ultrasonographically. on day 14 PP. Moreover, two (5.55%) The above observations indicate that ovaries showed good follicular activity when small follicular development and good follicular observed ultrasonographically on day 14 PP. development is felt per-rectally, in all the ovaries. On day 21 PP, 15 (41.66%) ovaries indicated Follicular growth (small as well as good) is also small follicular development when observed noted on ultrasonographic examination in all per-rectally while 4 (11.11%) ovaries showed ovaries. Besides that the follicular growth which small follicular development and 11 (30.50%) was not palpated per-rectally was detected by

Table 1. Ovarian activity monitored by per-rectally and ultrasonographically of Murrah buffaloes following

14 days PP GnRH and PGF2α administration.

Day 14 PP Day 21 PP Day 28 PP Follicular Groups Per- Ultraono- Per- Ultraono- Per- Utrasono- Development rectally graphically rectally graphically rectally graphically SFD 3 5 6 1 0 0 Group-I MSF - 2 - 5 - 0 GFD 0 1 5 6 11 11 SFD 2 5 6 2 0 0 Group-II MSF - 2 - 4 - 0 GFD 0 1 4 6 9 9 SFD 1 4 3 1 6 3 Group-III MSF - 0 - 2 - 3 GFD 0 0 4 6 6 6

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ultrasonographic examination. This suggested that activity or prominent follicular growth, but it rectal examination is a reasonably fair indicator is possible that slight follicular development or to judge the good follicular development. But deeply situated follicles could be missed. Thus, ultrasound is better tool for classification of rectal palpation is a fairly good indicator of ovarian follicles depending upon its diameter. ovarian activity only for routine observations. With the help of ultrasonographical examination Ultrasonography should be considered as a tool it is possible to diagnose fairly good amount when daily/frequent monitoring of follicular of follicular activity which otherwise would activity is required. Thus ultrasonographical have missed on rectal palpation of the ovaries. ovarian scanning should be considered especially It is seen in the present study that higher in the research techniques and therapeutic purposes. percentage of follicles have missed or wrongly diagnosed by rectal palpation. The preference of Initiation of post-partum ovarian activity ultrasonography method to compare with rectal The week for initiation of ovarian activity palpation in current study is agreed with Pieterse was noted in the buffaloes from all groups (Table (1990) who compared between trans-vaginal 2). ultrasound and rectal palpation methods for It was that PP ovarian activity was initiated a diagnosis the ovaries in bovines. It is observed earliest (P<0.01) in the PGF2 treated buffaloes that ultrasonography permits a better estimation (Group II) with the average of 3.25 weeks, of number and size of the follicles, being similarly followed by GnRH treated buffaloes (Group I) and with observations in cows Hanzen et al. (2000). control group (Group III) with averages of 3.50 and The current observations that by manual 4 weeks, respectively. diagnosis of smaller follicles was not detected Observations regarding the initiation and diagnose of follicles (<5mm) size was more of ovarian activity in the present study was accurate. The ultrasonographic appearance of corroborated with those reported by Iqbal et ovarian structures in the present study was in line al. (2003) who observed initiation follicular with those described by Honparkhe et al. (2003) and development at days 21.20±5.71 after PGF2a Lohan et al. (2004) in buffaloes. Ovarian activity is treatment as compared with days 28.20±8.75 in noted on day 14 post-partum by ultrasonography control Nili-Ravi buffaloes. Lohan et al. (2004) more effectively than by rectal examination. observed large follicle>8.5 mm in 75% buffaloes It can be concluded that rectal palpation is and increases to more than 8.5 mm between day fairly good technique to diagnose good follicular 14-33 PP. Chaudhary et al. (1989) noticed that

Table 2. Average week of initiation of post-partum ovarian activity for all groups in Murrah buffaloes. Group I Group II Group III Particulars a (GnRH) (PGF2 ) (Control) Average 3.5±0.150 3.25±0.130 4±0.467** ** Significant at P<0.01

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interval from calving to detection of first palpable to elicit behavioral signs of estrus (Noakes et follicle was averaged 27.43±1.24 days. Sheldon et al., 2001). Behavioural signs of estrus did not al. (2000) showed >8 mm between days 14-28 day accompany the first PP ovulation in majority of PP. Concomitant with present study, Bekana et al. buffaloes. This finding agreed with the observation (1994) observed the resumption of cyclical ovarian reported by Savio et al. (1990) who showed that activity within a month in seven animals by rectal first PP ovulation occurred without overt estrus palpation as well as on ultrasound. More days behavior in 17 out of 18 dairy cows. Thus, the sign required for initiation of follicular activity than of first estrus is not a true reflection of onset of present findings were reported by Baruselli (1991), ovarian activity. Usmani (1992) and Shah (1999) in buffaloes. The early return to active follicular development in PP buffaloes, and the fact that conclusion some buffaloes ovulated between days 15 and 20 PP, demonstrated the ability of the ovary to It can be concluded that the slight resume early PP activity. It suggested that ovarian follicular developments is easily detectable responsiveness may not be the major reason for through transrectal ultrasonography being a good the variable duration of the PP anestrus period tool for monitoring of follicular activity. The commonly observed in buffaloes. Usmani et PGF2a administration during early PP hastens the al. (1985) recorded formation of first CL on day initiation of estrus in Murrah buffaloes. 23.8±1.7 after calving as indicated by plasma progesterone level in buffaloes. Arya and Madan, (2001) observed 19.67±3.23 and 19.17±4.53 days references for first ovulation in suckled and non-suckled Murrah buffaloes. The small (8.10±5.67) and large Aboul-Ela, M.B. and A.H. Barkawai. 1988. number (1.00±0.00) of follicles was detected day Pulsatile secretion of LH in cycling buffalo six post-partum in buffaloes (Lohan et al., 2004). heifers affected by season and stage of the The present finding of initiation of PP oestrus cycle. In The 11th International ovarian activity at comparatively earlier days Congress on Animal Reproduction and might be due to careful monitoring of ovarian Artificial Insemination in Dublin, Ireland. activity through ultrasound scanning of ovaries, Arya, J.S. and M.L. Madan. 2001. Post-partum good feeding, health and management practices of reproductive cyclicity based on ovarian the farm as well as due to the PGF2 alpha treatment steroids in suckled and weaned buffaloes. in early PP period. Buffalo J., 3: 361-369. Determination of ovulation by rectal Baruselli, P.S. 1991. Postpartum ovarian activity palpation during earlier days after calving is tedious and reproductive performance in buffaloes. and it may be easily missed. Moreover, most of Anais 9th Congresso Brasileiro de these studies were based on palpation of corpora Reproduces Animal Belo Horizonge Brazil, lutea formed following ovulation. It is accepted Brazil. that CNS requires prior exposure to progesterone Bekana, M.T. and H. Kindhal. 1994. Ultasonography

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of the bovine post-partum uterus with parturition and uterine involution in bovine. retained fetal membranes. Zbl. Vet. Med. A., Veterinary Bull., 051: 064414. 41: 653-662. Lindell, J.O. and H. Kindahl. 1983. Exogenous Chaudhary, M.A., M. Ahmed and R.A. Chaudhary. prostaglandin F2 alpha promotes uterine 1989. Postpartum ovarian functions and involution in the cows. Acta Veternaia oestrus interval in Nili-Ravi buffaloes. Pak. Scandinavica, 24(3): 269-274. Vet. J., 9(4): 155-158. Lindell, J.O., M. Kindahl and L.E. Edquist. 1980. Fonesca, F.A., J.H. Britt, B.T. Mcdainel, A.H. Uterine involution in relation to post partum Rakes and J.C. Wilk. 1983. Reproduction release of PGF2 alpha in cows. Proceeding traits in Holland and Jersey. Effect of age, of 9th International Congress of Animal milk yield and clinical abnormalities on Reproduction and AI . III symposia, Medrid, involution of cervix and uterus, ovulation, Spain. estrus cycle, detection of estrus, CR and Lohan, I.S., R.K. Malik and M.L. Kaker. 2004. days open. J. Dairy Sci., 66: 1128-1147. Uterine involution and ovarian follicular Fortune, J.E. 1986. Bovine theca and granulose cells growth during early postpartum period of interact to promote androgen production. Murrah buffaloes (Bubalus bubalis). Asian Biol. Reprod., 35: 292-299. Austral. J. Anim., 17(3): 313-316. Hanzen, C., M. Pieterse, O. Scenzi and M. Drost. Madan, M.L. 2010. Concern and conflicts in buffalo 2000. Relative accuracy of the identification production. In Proceedings of International of ovarian structures in cows by Buffalo Conference. New Delhi, India. ultrasonography and palpation per-rectum. Nazir, F., R.A. Chaudhry, T. Rahil and K.R. Chohan. Vet. J., 159: 161-170. 1994. Effect of prostaglandin F2 alpha Honparkhe, M., V.K. Gandotra, A.S. Nanda and at early postpartum on uterine involution S. Prabhkar. 2003. A comparison of rectal and subsequent reproductive performance palpation and ultrasonography for detection in suckled Nili-Ravi buffaloes. Buffalo J., of follicles and corpus luteum in pluriparous 10(3): 269-271. buffaloes. Ind. J. Anim. Repro., 24: 149-151. Noakes, D.E., T.J. Parkinsons, G.C.W. England Iqbal, S., M. Aleem and M.A. Sayeed. 2003. Role and G.H. Arthur. 2001. Arthur’s Veterinary of single injection of prostaglandin F2 Reproduction and Obstetrics, 1st ed. W.B. alpha on breeding efficiency of post-partum Saunders Company, Pennsylvania. buffaloes. Pak. Vet. J., 23(4): 197-201. Parmar, K.H., R.G. Shah, P.H. Tank and A.J. Dhami. Khasatiya, C.T., F.S. Kavani, A.J. Dhami, H.J. 2012. Strategies for improving reproductive Derashri, M.T. Panchal and P.M. Desai. efficiency of postpartum anestrus Surti 2006. Studies on puerperal events and buffaloes. Ind. J. Anim. Repro., 33(1): 47- reproductive efficiency following hormonal 50. therapy at day 42 post-partum in Surti Perera, B.M.A.O., H. Abeygumawardona, A. buffaloes. International Journal Agriculture Thamotharm, H. Kindahl and L.E. Edquist. and Biology, 8(1): 34-41. 1981. Peripheral changes of oestrogen, Lindell, J.O. 1981. Studies on induced abortion, progesterone and prostaglandin in water

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buffaloes. Theriogenology, 15: 463-467. on subsequent reproductive performances Peters, A.R., M.G. Pimental and G.E. Lamming. of dairy cows. Theriogenology, 54: 409- 1985. Hormone response to exogenous 419. GnRH pulses in post-partum dairy cows. J. Singh, M. and M.L. Madan. 1985. Circulating Reprod. Fertil., 75: 557-565. PGF2a during oestrus cycle among Pieterse, M.C., O. Szenci, A.H. Willemse, C.S.A. buffaloes. Proceddings of the 1st World Bajcsy, S.J. Dieleman and M.A.M. Taverne. Buffalo Congress, Cario Egypt. 8: 569-571. 1990. Early pregnancy diagnosis in cattle by Snedecor, G.W. and U.G. Cochran. 1994. Statistical means of linear array real-time ultrasound Methods, 8th ed. Affiliated East West Press. scanning of uterus and a qualitative p. 491. and quantitative milk progesterone test. Tomar, S.S. and R.C. Ram. 1993. Factors affecting Theriogenology, 33: 697-707. replacement rule and its comparision in a Savio, J.D., M.P. Boland, N. Hynest and J.F. Roche. herd of Murrah buffaloes. Indian Journal of 1990. Resumption of follicular activity in Diary Science, 48: 340-342. the early postpartum period of dairy cows. Usmani, R.H. 1992. Effect of past gravid uterine J. Repro. Fertil., 88: 569-579. horn on the pattern of resumption of ovarian Schallenberger, E. 1985. Gonadotropin and ovarian functions in postpartum Nili-Ravi buffaloes. steroids in cattle. III Pulsatile changes of Buffalo J., 8(3): 265-270. gonadotropin concentration in jugular vein Usmani, R.H., M. Ahmed, E.K. Inskeep, R.A. post-partum. Acta Endocrinologica, 109: Dailey, P.E. Lewis and G.S. Lewis. 1985. 37-43. The uterine involution and postpartum Shah, R.G. 1999. Hormonal and biochemical ovarian activity in Nili-Ravi buffaloes. profile in fertile and infertile postpartum Theriogenology, 24: 435-448. Surti buffaloes. Ph.D. Thesis, Gujarat Yotov, S.A. and A.S. Atanasov. 2013. Agricultural University, Anand, India. Ultrasonographic determination of follicle Shah, S.N.H, A.H. Willemase and D.F.M. Van-de- development and resumption of ovarian wiel. 1990. Reproductive performance of activity in post-partum Bulgerian Murrah Nili-Ravi buffaloes after single injection buffaloes during breeding season. Animal of GnRH early post-partum. Trop. Anim. and Veterinary Science, 1(5): 36-41. Health Pro., 22(4): 239-246. Shah, R.G., V.B. Kharadi, A.J. Dhami, P.M. Desai and F.S. Kavani. 2002. Effect of gonoadotrophin releasing hormone on reproducive performance and steriod profile of postpartum suckled Surti buffaloes. Indian J. Anim. Sci., 72(12): 1076-1082. Sheldon, I.M., D.E. Noakes and T.M. Dobson.2000. The influence of ovarian activity and uterine involution determined by ultrasonography

324 Original Article Buffalo Bulletin (July-September 2016) Vol.35 No.3

CLINICAL, HEMATO-BIOCHEMICAL AND THERAPEUTIC STUDIES ON RUMEN IMPACTION IN BUFFALOES

A.K. Tripathi1, J.S. Soodan2 and R.B. Kushwaha2

ABSTRACT INTRODUCTION

Ruminal impaction was studied in Rumen impaction mainly occurs buffaloes in and around Jammu region the majority due to feeding of poor quality hay, straw or of cases were of animals having ingested fibrous roughages deficient in protein and readily feed material, coarse grain, polythene and jute bags digestible carbohydrate, overeating of young and ropes and nonpenetrating metallic objects. grasses, ingestion of mouldy or decomposed Prominent clinical signs noticed were complete feed, polythene bags, ropes and other plastic cessation of rumination, impaction and atony of materials, and exposure to hot and dry weather rumen, hardening and pelleted mucous coated conditions (Radostits et al., 2010). The clinical dung, and inappetance to anorexia. Haematological signs include decreased rumen motility or rumen alterations revealed reversal of neutrophil atony, abdominal distension, anorexia, constipated to lymphocyte ratio. The diseased buffaloes feces, occasional diarrhoea, normal to increased had significantly higher bilirubin, aspartate temperature, increased pulse rate, hard consistency aminotransferase, glucose, blood urea nitrogen and of rumen and solid mass on left side on per rectal creatinine, levels and significantly lower calcium, examination (Nwity and Chaudhary, 1995). Present than the control values. The levels of alkaline study reports the clinical, hematobiochemical phosphatase, total protein, albumin, globulin and alterations and therapeutic studies in various cases phosphorus did not differ significantly from the of ruminal impaction in buffaloes. respective control values.

Keywords: buffalo, haematology, biochemical, MATERIALS AND METHODS rumen impaction

The study was conducted on twenty buffaloes suffering from ruminal impaction, presented at Division of Veterinary Clinic and

1Department of Clinical Medicine, College of Veterinary and Animal Sciences, UP Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan (DUVASU), Mathura, Uttar Pradesh, India, E-mail: [email protected] 2 Division of Veterinary Clinic and Teaching Hospital, Faculty of Veterinary Sciences and Animal Husbandry, Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu (SKUAST-J), R.S. Pura- Jammu, India

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Teaching Hospital Faculty of Veterinary Sciences The data were subjected to student’s t-test and and Animal Husbandry SKUAST-J, R S Pura- means and standard errors were calculated for Jammu during the study period from January 2008 to comparison between control and animals with December 2010. The diagnosis of rumen impaction rumen impaction (Snedecor and Cochran, 1994). was done on the basis of hard consistency of rumen Therapeutic management includes on per rectal examination (Grymer and Ames, 1981) exploratiory ruminotomy to evacuate the rumen and exploratory laparorumenotomy. Six apparently followed by fluid and electrolyte therapy, broad healthy buffaloes brought to the clinic for artificial spectrum antibiotics, mineral oils, antihistaminics, insemination used for studying normal parameters. NSAIDS and ruminotorics, ruminal cud transfer Each animal was thoroughly evaluated was done in some cases as and when required for for its general condition and hydration status. restoration of normal ruminal flora. The physical parameters (rectal temperature, heart rate, and respiration rate, colour of mucous membrane, muzzle status, rumen consistency RESULTS AND DISCUSSION and rumen motility) were recorded at the time of presentation. Quantity and consistency of faeces All the animals were in depressed condition in rectum, faecal colour, rumen consistency, and and rumination was suspended, muzzle was dry. rumen size were recorded on rectal examination. On visual examination of abdominal contour from Physical and microscopic examination of ruminal rear side, all the animals had unilaterally distended fluid was done for color, odour, concistency (left side) abdomen, and moderate degree (8-10%) pH and protozoal motility (Garry, 2002). of dehydration. Ruminal motility (1.40±0.25 per Blood samples (2 ml) were collected 5 minutes) was reduced significantly compared aseptically from jugular vein in EDTA coated vials. to healthy control (8.50±0.30 per 5 minutes). No Haemoglobin (Hb, g/dL), packed cell volume significant variation in rectal temperature and (PCV, %), total leukocyte count (TLC, per/µl) and respiration rate were noticed in comparison to differential leukocyte count (DLC, % and per/µl) healthy control. However, heart rate (72.60±4.26 were estimated by standard methods (Benjamin, per min) was significantly higher than healthy 1985). For glucose estimation blood samples were control (57.80±3.08). No abnormal sounds were collected in vials containing sodium fluoride. herd on auscultation of lung and heart. On per- Blood samples were also collected in rectal examination, the consistency of rumen acid free vials without any anticoagulant; serum was doughy to moderately hard in all cases, no was separated and transferred to a dry clean distension or ballooning of intestinal loops and in vial for storage at-20oC till further evaluation. majority of animals rectum was found empty and Following biochemical parameters were estimated few having constipated dry feces (pellets). These using diagnostic kit with help of autoanalyzer signs are similar to those earlier reported by Nwity viz Total bilirubin, aspartate aminotrasferase and Chaudhary (1995). (AST), alkaline phosphatase (ALP), glucose, Physical and microscopic examination total protein, albumin, blood urea nitrogen of rumen fluid in impaction cases revealed (BUN), creatinine, calcium and phosphorus. greenish brown to yellowish brown color, watery

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consistency, and pungent odor, normal pH, poor animals were earlier reported by the Nagarajan and (+) to nil protozoal motility in all cases, similar Rajmani, 1973. In the present study leukocytosis findings were reported by Garry, 2002. Exploratory and neutrophilia were observed which might have ruminotomy was done in all the cases and it was resulted from chronic irritation of the forestomach revealed that impacted materials in majority of wall by impacted feed materials, leaving the cases having ingested fibrous feed material, coarse wall exposed to secondary infection, which grain, polythene and jute bags and ropes and resulted in inflammation (Hailat et al., 1996). nonpenetrating metallic objects. Decreased lymphocytes could be due to release of Hematological studies revealed (Table corticosteroid as a result of stress (Feldman et al., 1) that mean TLC, and percent neutrophils were 2000). significantly higher whereas, the mean lymphocyte Biochemical studies revealed (Table was significantly lower. However, no significant 1) significantly higher mean Plasma values of variation was observed in the Mean Hb, Mean total bilirubin, AST, glucose, BUN, creatinine, PCV, in ruminal impaction cases than the healthy total protein, globulin and significantly lower control. No variation in the Hb and PCV values in mean plasma values of calcium in the ruminal the ruminal impaction cases than healthy control impaction cases than healthy control. However, no

Table 1. hemato-biochemical alterations in buffaloes with rumen impaction. Rumen Impaction (Mean±SE), Parameters Control (Mean±SE), n=6 n=20 Hb (g/dl) 9.80±0.35 8.74±0.76 PCV (%) 28.70±1.20 278.40±4.84 WBC (x 103/µl ) 5.45±0.56 8.65±0.38* Neutrophil (%) 32.60±3.46 61.60±4.19* Lymphocyte (%) 67.10±1.90 36.40±4.35* N/L ratio 0.49±1.82 1.69±0.96* AST (IU/L) 120.50±4.58 218.80±26.58* ALP (IU/L) 168.40±8.24 178.40±25.20 Total bilirubin (mg/dl) 0.42±0.10 3.12±0.68 Glucose (mg/dl) 60.32±8.50 120.20±18.80* BUN (mg/dl) 26.40±1.52 81.24±20.20* Creatinine (mg/dl) 0.50±0.05 2.30±0.60* Total protein (g/dl) 7.20±0.20 8.35±0.45 Globulin (g/dl) 3.48±0.14 4.40±0.24* Albumin (g/dl) 3.72±0.15 3.94±0.20 Calcium (mg/dl) 11.32±0.24 7.65±0.82* Phosphorus (mg/dl) 6.20±0.30 5.95±0.18

Means bearing *differ significantly at P≤0.05

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significant variation in the mean plasma values of All the cases affected with ruminal ALP, albumin and phosphorus were observed in impaction were treated successfully in a similar the ruminal impaction cases than healthy control. principle as suggested by Khose et al., 2010, The increased bilirubin level in present study may with slight modification. Initially the left para be attributed to absorption of toxic substances lumbar fossa was prepared for aseptic surgery. from rumen. Impaired uptake and excretion of The rumenotomy was performed as per standard bilirubin due to deranged liver function, as evident technique to evacuate the impacted ruminal content by increased liver enzymes, may have resulted and fresh rumen cud, along with 4 Rumentas in increased serum bilirubin concentration. The (Rumenotoric bolus) were introduced into the increased bilirubin may also be due to constipation rumen before closing it. The Laparotomy incision and starvation (Kaneko et al., 2008). The possible was closed as per standard technique. Post- cause of higher AST could be necrosis of liver operative management included Dextrose Normal due to toxemia from the damaged rumen mucosa Saline 10 liters I/V, Strepto-penicillin 5 gm I/M (7 (Garry, 2002). In rumen impaction the putrefied days), Melonex 15 ml I/M, Anistamina 10 ml I/M ingesta liberates toxic amines like histamine in and Tribivet 10 ml I/M for 3 days and Floratone rumen which after absorption into circulation 4 boli, Rumentas 2 boli orally bid for 7 days in increases BUN concentration (Dain et al., 1995). each cases. Skin sutures were removed on the 10th The increased BUN level could also be attributed post-operative day. All The buffaloes affected with to decrease in renal blood flow as a part of ruminal impaction recovered uneventfully. compensatory mechanism to maintain circulation in hypovolemia associated with dehydration (Kaneko et al., 2008) and same phenomenon may be held REFERENCES responsible for increased creatinine concentration. Moreover, during ruminal disorders there is failure Benjamin, M.M. 1985. Outline of Veterinary of urea cycling process and urea is not utilized by Clinical Pathology. Kalyani Publisher, New rumen microbes (Singh et al., 2001). The increase Delhi, India. p. 60-63, 71-75. in glucose level may be due to stress of impaction Dain, T.A, A.L. Neal and R.W. Dougherty. 1995. leading to adrenocorticosteroid release, which has The occurrence of histamine and tyramine glycogenolytic effect, causing hyperglycemia. in ruminal ingesta of experimentally urea Hypocalcaemia may be due to less fed sheep. J. Anim. Sci., 14: 930-955. assimilation of feed materials as a result of long Daniel, R.C.W. 1983. Motility of rumen and standing anorexia (Sethuraman and Rathore, 1979; abomasum during hypocalcemia. Can. J. Radostits et al., 2010). Daniel (1983) reported that Compart. Med., 47: 276-280. both rumen and abomasal motilities were similarly Feldman, B.F., J.G. Zinkl and N.C. Jain. 2000. reduced in hypocalcemia due to general effect of Indigestion in ruminants. In Garry, F.B. depression of levels of ionised calcium on smooth (ed.) Schalm’s Veterinary Haematology, 5th muscle contractibility. Similarly, reduced calcium ed. Lee and Febiger, Philadelphia. level in present study may have contributed to Garry, F.B. 2002. Indigestion in ruminants, p. decreased ruminal motility. 722-747. In Smith, B.P. (ed.) Large Animal

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Internal Medicine, 2nd ed. Mosby, St. Louis on secondary indigestion in bovines due and Baltimore. to traumatic reticulitis and diaphragmatic Grymer, J. and N.K. Ames. 1981. Bovine abdominal hernia. Indian J. Anim. Sci., 49: 703-706. pings. Clinical examination and differential Singh, N., R. Kumari and M.A. Akbar. 2001. diagnosis. Compendium on Continuing Ruminal pH as a regulator of blood Education of Practicing Veterinarians, 3: metabolites in lactating Murrah buffaloes. 311-318. Indian Vet. Med. J., 25: 253-256. Hailat, N., S. Nouth, A. Al-Darraji, S. Lafi, F. Al- Snedecor, G.W. and W.G. Cochran. 1994. Statistical Ani and A. Al-Manjali. 1996. Prevalence Methods, 8th ed. Iowa State University and pathology of foreign bodies (plastic) Press. Ames. Iowa, USA. in Awassi sheep in Jordan. Small Ruminant Res., 24: 43-48. Kaneko, J.J., J.W. Harvey and M.L. Bruss. 2008. Clinical Biochemistry of Domestic Animals. 6th ed. Academic Press, London. Khose, K.A., P.A. Jadhav and V.E. Mahajan. 2010. Ruminal Impaction in a Cow with Indigestible Foreign Bodies and its Surgical Management. Intas Polivet., 11(2): 189- 190. Nagarajan, V. and S. Rajamani. 1973. Alkaline indigestion and rumen putrefaction in cow. Indian Vet. J., 50: 1147-1151. Nwity, T.N.E. and S.V.R. Chaudhary. 1995. Ruminal impaction due to indigestible materials in the arid zone of Borno state of Nigeria. Pak. Vet. J., 15: 29-33. Pienkowski, M. 1969. Estimation of the functional state of liver in cows with acid indigestion. Annals universitatis, Marial CurieSkalodowska, Lublin Pulonia, Section, 18: 209-222. Radostits, O.M., C.C. Gay, K. Hinchcliff and P.D. Constable. 2010. Veterinary Medicine. A Textbook of the Diseases of Cattle, Horses, Sheep, Pigs and Goats, 10th ed. Saunders Elsevier. Sethuraman, V. and S.S. Rathore. 1979. Clinical, haematological and biochemical studies

329 Buffalo Bulletin (July-September 2016) Vol.35 No.3 Original Article Buffalo Bulletin (July-September 2016) Vol.35 No.3

DIAGNOSIS OF RABIES IN BUFFALOES: COMPARISON OF CLINICO-PATHOLOGICAL, IMMUNOHISTOCHEMICAL AND IMMUNOFLUORESCENT TECHNIQUES

A.B. Beigh1, B.S. Sandhu2, C.K. Singh2, K. Gupta2 and N.K. Sood2

ABSTRACT Keywords: buffaloes, FAT, IHC, formalin fixed, rabies The present study was envisaged to compare the sensitivity of detection of rabies virus antigen by application of Fluorescent Antibody INTRODUCTION Technique on fresh impression smear (Direct-FAT) and that on formalin fixed nervous tissue (Indirect- Rabies is a fatal zoonotic disease of FAT), histopathology and immunohistochemistry worldwide concern caused by a neurotropic (IHC) in the species which is highly significant for negative sense single stranded RNA (ssRNA) virus the economics of the dairy farmer i.e. buffalo. A of the genus Lyssavirus, Order Mononegavirales total of 28 cases of buffaloes suspected for rabies and of family Rhabdoviridae. Diagnosis of clinical were presented. Out of 28 cases, 18 (64.28%) cases rabies is difficult and is often not made until after were positive by direct-FAT, indirect-FAT, IHC death of the animal, so early diagnosis of rabies in and 60.71% (17/28) by demonstration of negri animals is necessary for timely administration of bodies and thus, histopathology revealed 94.4% post-exposure prophylaxis. At necropsy, rabies is sensitivity in comparison to direct- FAT. While as, usually diagnosed by subjecting fresh or formalin indirect-FAT, and IHC revealed 100% sensitivity in fixed nervous tissue samples to pathological comparison to direct-FAT. Percentage of neurons examination and the routine diagnostic methods positive for Negri bodies by H and E and IHC were used are fluorescent antibody test on brain 59.35% and 78.88% and average number of Negri impression smears and histopathological bodies detected per neuron by H & E and IHC examination of the brain for Negri bodies. These were 1.8 and 3.01, respectively. Important clinical inclusions are not present in all cases and the use signs in rabid animals were anorexia, circling/Head of fresh tissue samples for laboratory examination pressing, behavioural change and bellowing. Thus, is hazardous due to possible risk of contamination it is concluded that rabies detection in animals can of the environment with rabies virus. However, be accomplished from diagnosis of rabies from in many situations, only formalin-fixed tissue is fixed brain tissues which offers same sensitivity as available for post-mortem diagnosis due to lack detection of rabies in impression smears. of laboratory facilities or presentation of fixed rather than fresh tissues to the laboratory Warner

1Department of Veterinary Pathology, Sher-e-Kashmir University of Agricultural Sciences & Technology of Kashmir (SKUAST-K), Srinagar, India, E-mail: [email protected] 2Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India

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et al. (1997) and Abreu et al. (2012). Hence, Detection of rabies virus antigen in fresh tissue there is a need for a better method of diagnosis impression smear using fluorescein-labeled of rabies using formalin-fixed paraffin-embedded antibody (Direct-FAT) tissues. Immunohistochemistry, indirect FAT and The direct FAT is employed as diagnostic histopathology can be performed on such samples. technique because of its sensitivity, accuracy Immunohistochemistry and indirect FAT technique and speed as recommended by World Health improves diagnostic accuracy by promoting Organization (Meslin et al., 1996). A pair of thin visualization of the distribution of the infectious impression smears from each of brain tissue were disease agent in histological sections Deborah et al. prepared in grease-free labelled glass slides 1 cm in (1991). They provide sufficient amplification of the diameter, about 1.5 cm from each end, from either antibody-antigen interaction to enable detection of fresh tissue or preserved tissues kept at 20oC in antigens immunogenically altered by fixation. So, deep freeze. the present study was envisaged to establish the Control positive slides from known rabies comparison of sensitivity of routine detection with case and control negative from normal uninfected application of FAT on nervous tissue impression and unvaccinated animal were prepared along with smear with other techniques on formalin fixed the test smear. Impression smears were air dried nervous tissue. for 30 minutes at room temperature. Smears were fixed by immersing in coupling jars containing cold acetone in a deep freeze at 20oC for overnight. MATERIALS AND METHODS Acetone was drained off and impression smear slides were air dried at room temperature for 20 minutes. A total of 28 cases of buffaloes suspected Lyophilized, anti-rabies nucleocapsid Fluorescein for rabies were presented at Rabies Research-cum- isothiocyanate (FITC) conjugate acquired from Bio- Diagnostic laboratory, Department of Veterinary radMarnes-La-Coquette, France was reconstituted Pathology, Guru Angad Dev Veterinary and Animal with 3 ml of distilled water as recommended by Sciences University (GADVASU), Ludhiana from the manufacturer and centrifuged at 1500 rpm for various parts of Punjab. The data regarding age, 5 minutes for clarification. The clarified conjugate sex, history of bite, date of bite, source of bite and (0.1 ml) was added on the duplicate impression clinical signs was acquired as per the questionnaire smears on every slide for each tissue samples prepared for the purpose from owners of the and on positive and negative control slides. Then, animals. smears were covered with cover slips and slides incubated at 37oC for 30 minutes by placing in a Clinical samples for detection of rabies humidified chamber. Slides were washed twice in Brain samples were collected from dogs 0.01 M phosphate buffered saline (PBS) pH 7.5 suspected for rabies. Three pieces of each tissue for 5 minutes each. Thereafter, slides were air- sample were stored at 20oC, in 50% glycerol saline dried and mounted in 90% buffered glycerol (pH solution, and in 10% neutral buffered formalin 8.5). Slides were examined using an AHBT3 - RFC solution respectively. reflected light fluorescence attachment (Olympus, Japan).

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Detection of rabies virus antigen in formalin- used for immunohistochemical staining of brain fixed tissues using fluorescein-labeled antibody tissues sections. Maximum dilution of antibody at (Indirect-FAT) which these samples revealed positive reaction was The FAT of formalin-fixed tissues was 1:1000. performed as described by Warner et al., 1997 with Paraffin embedded tissues were sectioned the following modifications: The dewaxing and at 4 to 5µm thickness and mounted on Superfrost/ rehydration of tissue sections was carried out by Plus, positively charged microscopic slides (Fisher EZ-AR Common solution at 70oC for 10 minutes Scientific, USA). The slides were then placed in in microwave oven; Antigen retrieval was done in hot air oven to melt the paraffin at o 60 C for 30 citrate buffer (0.01 M, pH 6.0-6.2) EZ-RetrieverR minutes and stored till further use. Advanced SSTM System V.2.1 (BioGenex Laboratories Inc., San two step polymer Horseradish Peroxidase (HRPO) Ramon, California, USA) at different time and Immunohistochemical detection system (BioGenex temperature combinations-2 cycles-95oC for 10 Laboratories Inc., San Ramon, California, USA) minutes and at 98oC for 5 minutes, respectively; was used for staining of paraffin embedded tissue Thereafter, slides were washed with PBS washing sections as per recommendation of the manufacturer buffer (pH 7.2 to 7.6) for 3 times, 5 minutes each; with some modifications Pedrosoet al. (2008). The and allowed to dry at room temperature. dewaxing and rehydration of tissues sections were Then Fluorescein isothiocyanate (FITC) carried out by EZ-AR Common solution at 70oC conjugate (0.1 ml) was added on every paraffin for 10 minutes in microwave oven. embedded tissue sections. After this slides were incubated at 37oC for 60 minutes by placing in a Antigen retrieval humidified chamber. Then slides were washed with Antigen retrieval was done in EZ-ARTM 3 in PBS washing buffer (pH 7.2 to 7.6) for 2 times 5 EZ-RetrieverR System V.2.1 (BioGenex Laboratories minutes each, then twice more in deionized water at Inc., San Ramon, California, USA) at different time room temperature. The sections were air dried and and temperature combinations-2 cycles-95oC for 10 cover glasses were applied using aqueous mounting minutes and at 98oC for 5 minutes, respectively. The media FluoromountTM (SIGMA-ALDRICH, Saint slides were cooled and brought to room temperature, Louis, Missouri, USA). The slides were examined washed with PBS buffer (pH 7.2 to 7.6) for 3 times using a fluorescent microscope (Nikon, 800i, for 3 minutes each. The endogenous peroxidase Japan). activity was blocked by incubating slides with a

solution of 3% H2O2 in methanol for 25 minutes at Immunohistochemistry (IHC) room temperature in humidified chamber. Slides Antirabies polyclonal antisera (Rabbit) were washed with PBS buffer (pH 7.2 to 7.6) for 3 available in the Rabies Research-cum-Diagnostic times 3 minutes each and sections were encircled Laboratory of the department of GADVASU; with hydrophobic pen (Pap pen). Non-specific Ludhiana was used as primary antibody for protein binding was blocked using power block immunohistochemical studies. Different dilutions solution (BioGenex Laboratories Inc., San Ramon, of 1:50, 1:100, 1:500, 1:1000, and 1:2000 of California, USA) for 15 minutes in moist chamber. polyclonal antisera in PBS (pH 7.2 to 7.6) were Slides were incubated with primary

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polyclonal rabbit anti-rabies antibody (1:500 and were routinely processed through ascending grades 1:1000 dilution in PBS 1% BSA) for one and half of alcohol, cleared in benzene and embedded in hour in humidified chamber at room temperature. paraffin wax. The paraffin sections were cut at 4 For each staining a negative control was run on to 5 μ thickness and stained by haematoxylin and sister section in which primary antibody was eosin (H and E) method (Luna, 1968). Slides were replaced by PBS. Slides were washed with PBS examined by by BX61 Research Photomicrograph washing buffer (pH 7.2 to 7.6) for 3 times, 3 minutes Microscope System of Olympus Corporation, each subsequently. USA. The tissue sections were incubated with secondary antibody ImmPRESSTM UNIVERSAL Sensitivity comparison with direct-FAT REAGENT Anti-mouse/Rabbit Ig (Vector Sensitivity of various techniques was Laboratories Inc., Burlingame, U.S.A.) for 30 calculated in comparison with dFAT as per Perrin minutes at room temperature in humidified and Sureau (1987). chamber. Slides were washed with PBS washing buffer (pH 7.2 to 7.6) for 3 times, 3 minutes each. True positive Substrate 3, 3 ‘-diaminobenzidine (DAB) solution, Sensitivity = (True positive + false ×100 freshly prepared by mixing a drop of ImmPACTTM negative) DAB chromogen with 1 ml of ImmPACTTM DAB buffer (Vector Laboratories Inc., Burlingame, U.S.A) and 5 μl hydrogen peroxide. The antigen- RESULTS AND DISCUSSION antibody-peroxidase reaction was visualized by adding substrate 3, 3 ‘-diaminobenzidine (DAB) Clinical signs in rabid buffaloes solution on sections for 1 to 2 minutes. Sections In case of rabid buffaloes, anorexia was were washed in tap water for 5 minutes to stop the found in 94.44% (17/18) cases, followed by antigen-antibody-peroxidase reaction. Slides were behavioral change and pressing of head against counterstained with Gill’s haematoxylin (Merck, hard objects in 55.55% (10/18) cases, difficulty Germany) for 30 seconds and washed with running in feed intake and hyper-salivation and bellowing tap water for 5 minutes. Finally the sections were in 50% (9/18) cases (Figure1 and 2). Whereas, dehydrated in ascending grades of alcohol (70%, paralysis and fever in 38.88% (7/18). However, 80%, 90%, and absolute alcohol) and cleared in frequent micturition in 27.77% (5/18) cases. xylene for 2 minutes and mounted with DPX. Slides Non-recognizing owner 22.22% (4/18) and pica were examined under microscope (BX 61, Olympus were found in 11.11% (2/18) cases, respectively Corporation, Japan). (Table1). Similar symptoms have been reported by (Salem et al., 1995; Rissi et al., 2008; Pedroso et Histopathology al., 2009). All nervous tissues samples, viz. cerebellum, cerebrum, hippocampus, pons, medulla Direct FAT oblongata from dead animals were collected in 10% Out of 28 cases, 17 cases (60.71%) were neutral buffered formalin solution. These tissues

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diagnosed positive for the presence of rabies formalin for short and long period of time. Johnson viral antigen (Table2). Characteristic apple et al. (1980) suggested FAT on formalin fixed green immunofluorescence was observed intra- tissue as complementary to standard diagnostic cytoplasmic in neurons as well as in form of techniques. diffused fluorescence in the brain tissue smears (Figure 3). FAT is sensitive, specific, and easy to Immunohistochemistry perform, serves as standard diagnostic procedure Brain tissues were positive in 18 out of and is the preferred test for rabies diagnosis (Smith, 28 cases (64.28%) (Table 2), using polyclonal 1999; Whitfieldet al., 2001). antiserum by immunohistochemistry and it revealed 100% sensitivity in comparison to direct-FAT Indirect FAT (Table 4). No positive reaction was observed with Out of 28 cases, 18 (64.28%) were monoclonal antibody. The controls were negative found positive for rabies virus antigen (Table 2) and free of endogenous peroxidase (Figure 4). and revealed 100% sensitivity in comparison to A large amount of distinct, granular rabies viral direct-FAT on fresh tissue smears (Table 3). The antigen deposits stained as sharply demarcated viral antigen in formalin fixed tissue was visible brown precipitates of variable sizes were found as distinct apple green coloured intracytoplasmic within the Purkinje cells and in the neurons of the inclusion bodies and finely granular particles hippocampus, in the axons, in the processes of along dendritic arborization, axonal tracts and in neurons and in the stroma (Figure 5and 6). These the stroma (Figure 5 and 6). Similar finding have findings were similar as reported by (Gunawardena been reported by Swoveland and Johnson (1979), and Blakemore, 2007; Pedroso et al., 2009). Johnson et al.(1980), Reid et al.(1983), Umoh et al.(1984), Bourhy and Sureau (1990). Detection of Histopathology viral antigen was almost same in tissues stored in Out of 28 cases, 17 cases (60.71%) were

Table 1. Clinical signs in rabid buffaloes (Total positive cases=18). Symptom No. of animals Percentage Off feed 17 94.44 Hyper-salivation 9 50 Fever 7 38.88 Not recognizing owner 4 22.22 Circling/Head pressing 10 55.55 Difficulty in standing/paralysis 7 38.88 Difficult intake of food 9 50 Frequent micturition 5 27.77 Bellowing 9 50 Pica 2 11.11 Behavioral change 10 55.55

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Table 2. Comparison of Direct-FAT with other diagnostic techniques for detection of rabies virus antigen.

S. no. Case no. Direct-FAT Indirect-FAT IHC Histopathology 1 RL 25/10 + + + + 2 RL 26/10 - - - - 3 RL 33/10 + + + + 4 RL 36/10 + + + + 5 RL 38/10 - - - - 6 RL 41/10 + + + + 7 RL 02/11 + + + + 8 RL 04/11 + + + + 9 RL 10/11 - - - - 10 RL 17/11 + + + + 11 RL 25/11 - - - - 12 RL 28/11 + + + + 13 RL 31/11 + + + + 14 RL 33/11 + + + + 15 RL 36/11 - - - - 16 RL 37/11 + + + + 17 RL 03/12 - - - - 18 RL 04/12 - - - - 19 RL 10/12 + + + + 20 RL 11/12 - - - - 21 RL 14/12 + + + + 22 RL 16/12 + + + + 23 RL 17/12 + + + + 24 RL 18/12 - - - - 25 RL 19/12 + + + + 26 RL 24/12 + + + - 27 RL 25/12 + + + + 28 RL 27/12 - - - - % Test 64.28% 64.28% 64.28% (18/28) 60.71% (17/28) positive (18/28) (18/28)

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Table 3. Sensitivity of Indirect-FAT in comparison to Direct-FAT on brain tissue smears.

Direct-FAT Test Direct-FAT (Negative) Total (Positive) Indirect-FAT (Positive) 18 0 18 Indirect-FAT (Negative) 0 10 10 Total 18 10 28

True positive Sensitivity of IHC for brain sample = x 100 (True positive + False negative)

= 18/18+ 0 ×100 = 1800/18 = 100%

Table 4. Sensitivity of Immunohistochemistry (IHC) on Brain samples in comparison to FAT on fresh brain tissue smears.

Direct-FAT Test Direct-FAT (Negative) Total (Positive) IHC on brain (Positive) 18 0 18 IHC on brain (Negative) 0 10 10 Total 18 10 28

True positive Sensitivity of IHC for brain sample = x 100 (True positive + False negative)

= 18/18+ 0 ×100 = 1800/18 = 100%

Table 5. Sensitivity of Histopathology on Brain samples in comparison to FAT on fresh brain tissue smears.

Direct-FAT Test Direct-FAT (Negative) Total (Positive) Histopathology (positive) 17 0 17 Histopathology (negative) 1 10 11 Total 18 10 28

True positive Sensitivity of IHC for brain sample = x 100 (True positive + False negative)

= 17/17+ 1 ×100 = 1700/18 = 94.4%

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Table 6. Histopathological and Immunohistochemical evaluation of brain tissues for number of Negri bodies.

No. of neurons positive for No. of Negri bodies Sr. No. Case No. Negri bodies/100 neurons detected/100 neurons H &E IHC H & E IHC 1 RL 25/10 66 87 172 408 2 RL 33/10 46 95 125 381 3 RL 36/10 43 88 69 325 4 RL 41/10 77 80 114 230 5 RL 2/11 59 78 103 164 6 RL 4/11 69 83 119 233 7 RL 17/11 6 33 7 52 8 RL 28/11 14 90 18 217 9 RL 31/11 76 98 282 580 10 RL 33/11 87 100 152 495 11 RL 37/11 39 94 50 190 12 RL 10/12 80 96 146 426 13 RL 14/12 69 89 174 210 14 RL 16/12 41 72 45 141 15 RL 17/12 67 91 87 211 16 RL 19/12 75 86 150 219 17 RL 25/12 95 79 65 178 Total 1009 1341 1878 4044

Table 7. Comparison of histopathology and immunohistochemistry.

Parameter Histopathology (H& E) IHC Neurons having Negri bodies (n=1700) 1009 1341 %age of Neurons positive (Negri bodies) 59.35 78.88 Total number of Negri bodies detected 1878 4044 Average number of Negri bodies per neuron 1.8 3.01

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Figure 1. Rabies suspected buffalo exhibiting head pressing and paralysis.

Figure 2. Rabies suspected buffalo exhibiting hypersalivation.

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Figure 3. Impression smear drawn from hippocampus of a rabid buffalo showing apple green fluorescence in neurons. Direct FAT X 165.

Figure 4. Negative control of IHC-Section of cerebellum showing absence of reaction. IHC-One step polymer HRPO Technique - Original magnification x 400X.

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Figure 5. Section of hippocampus of rabid buffalo showing few Negri bodies with H&E stain- Original magnification x 1000X (A), corresponding IHC stained section - Original magnification x 1000X (B) showing brown coloured Negri bodies (arrow) and corresponding FAT stained section- Original magnification x 400X (C) showing more clearly green coloured Negri bodies (arrow).

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Figure 6. Section of hippocampus of rabid buffalo showing few Negri bodies with H&E stain- Original magnification x 1000X (A), corresponding IHC stained section - Original magnification x 1000X (B) showing brown coloured Negri bodies (arrow) and corresponding FAT stained section- Original magnification x 400X (C) showing more clearly green coloured Negri bodies (arrow).

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found positive for rabies by demonstration of a comparison of polyclonal and monoclonal negri bodies (Table 2) and thus, histopathology antibodies employed in detection of rabies antigen revealed 94.4% sensitivity in comparison to direct- in formalin fixed paraffin embedded tissue sections FAT (Table 5). Negri bodies appeared as single or using IHC revealed polyclonal antibody to be multiple, eosinophilic intracytoplasmic inclusions highly efficacious. within the Purkinje neurons, in the axons and in As compared to IHC specificity of FAT the neurons of the hippocampus (Figure 5 and 6). on formalin fixed tissue was more, this is because Which has been also reported by (Gonzalez and of nonspecific binding of polyclonal antibody Stephano, 1984; Salem et al., 1995; Lima et al., with nonspecific antigens in case of IHC. There 2005; Srinivasan et al., 2005; Pedroso et al., 2008 is also enhanced detection of viral antigen due to and Rissi et al., 2008). fluorescence of antigen-antibody complex. Thus, it can be concluded that IHC was more sensitive Comparison of immunohistochemistry and than histopathology but as sensitive as either of histopathology FAT procedures and proved to be a valid method FAT on formalin fixed tissue can be used for rabies diagnosis and can replace FAT where as an alternative to FAT on fresh tissue with the fluorescent microscopy is not available or when same sensitivity, when only formalin-fixed tissue fresh samples are not available for FAT. is available for post-mortem diagnosis. Hundred neurons per case were observed for negri bodies and number of negri bodies in positive neurons (Table ACKNOWLEDGEMENTS 6) and a comparison of IHC and histopathology were done (Table 7). With IHC 78.88% neurons Authors thank the Director of Research, were positive for negri bodies and 59.35% with Guru Angad Dev Veterinary and Animal Sciences H and E. It can be concluded that IHC established University, Ludhiana for providing financial many more virus infected cells than H and E stained support to conduct this study. We also thank Mr. sections which is same as reported by Feiden et al. Dan Singh, Mr. Kewal Singh, Mr. Dilchain Singh (1985). and staff of rabies diagnostic and histopathology Average number of negri bodies detected laboratory for their help, cooperation and support per neuron by IHC was 3.01 which were greater extended during the period of study. than H and E stained brain sections (1.8). The amount of antigen detected with IHC was much more abundant than histopathological findings REFERENCES (Figure 5 and 6) which are reported by several workers (Hamir et al., 1992; Martinez-Burnes, Abreu, C.C., P.A. Nakayama, C.I. Nogueira, L.P. 1997; Jogai et al., 2001 and Suja et al., 2004). Mesquita, P.F. Lopes, M.S. Varaschin, J.N. Palmer et al. (1985) reported that rabies antigen Seixas. E. Ferreira and P.S.J. Bezerra. 2012. with IHC was apparent in 62% of the brain area Domestic microwave processing for rapid in which inclusion bodies were not found in the immunohistochemical diagnosis of bovine corresponding H and E sections. In present study rabies. Histol. Histopathol., 27(9): 1227-

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1230. 2005. The heminested RT-PCR for the Bourhy, H. and P. Sureau. 1990. Laboratory study of rabies virus pathogenesis. J. Virol. methods for rabies diagnosis. Collection Methods, 124(1-2): 79-85. of Institute Louis Pasteur, Paris, France, p. Martinez-Burnes, J., A. Lopez, J. Medellin, D. 154-197. Haines, E. Loza and M. Martinez. 1997. An Deborah, M., E.G. Haines and Clark. 1991. Enzyme outbreak of vampire bat-transmitted rabies immunohistochemical staining of formalin- in cattle in northeastern Mexico. Canadian fixed tissues for diagnosis in Veterinary Vet. J., 38: 175-177. Pathology. Canadian Vet. J., 32(5): 295- Palmer, D.G., P. Ossent, M.M. Suter and E. Ferrari. 302. 1985. Demonstration of rabies viral antigen Feiden, W., U. Feiden, L. Gerhard, V. Reinhardt in paraffin tissue sections: Comparison of and A. Wandeler. 1985. Rabies encephalitis: the immunofluorescence technique with the Immunohistochemical investigations. Clin. unlabeled antibody enzyme method. Am. J. Neuropathol., 4(4): 156-164. Vet. Res., 46(1): 283-286. Gonzalez, C. and H.A. Stephano. 1984. Pedroso, P.M.O., E.M. Colodel, C.A. Pescador, L.P. Histopathology of the central nervous Arrudo and D. Driemeier. 2009. Clinical system in rabid dogs. Vet. Mexico, 15(1): and pathological aspects in cattle affected 39-52. by rabies with special reference to the rabies Gunawardena, G.S.P. de. S. and W.F. Blakemore. antigen mapping by immunohistochemistry. 2007. Immunohistochemical Detection of Pesquisa Vet. Brasil., 29(11): 899-904. Rabies Virus Antigen in the Brainstem and Pedroso, P.M.O., C.A. Pescador, P.M. Bandarra, Spinal Cord of Rabid Dogs in Sri Lanka. D.L. Raymundo, M.R. Borba, F. Wouters, Proceedings of the Peradeniya University P.S. Bezerra-Junior and D. Driemeier. 2008. Research Sessions, Sri Lanka, 12(1): 168. Standardization of immunohistochemistry Hamir, A.N., G. Moser and C.E. Rupprecht. 1992. technique for detection of rabies virus in Morphologic and immunoperoxidase study formalin-fixed and paraffin-embedded of neurologic lesions in naturally acquired tissue samples from central nervous system rabies of raccoons. Journal of Veterinary of cattle. Pesquisa Vet. Brasil., 28(12): 627- Diagnostic and Investigation, 4(1): 369- 632. 373. Perrin, P. and P. Sureau. 1987. A collaborative Jogai, S., B.D. Radotra and A.K. Banerjee. 2001. study of an experimental kit for rapid Rabies Immunohistochemical study of human enzyme immunodiagnosis. B. World Health rabies. Neuropathology, 20(3): 197-203. Ogan., 65(4): 489-493. Johnson, K.P., P.T. Swoveland and R.W. Reid, F.L., N.H. Hall, J.S. Smith and G.M. Baer. Emmons. 1980. Diagnosis of rabies by 1983. Increased immunofluorescent staining immunofluorescencein trypsin-treated of rabies-infected, formalin-fixed brain tissue histologic sections. Journal of American after pepsin and trypsin digestion. J. Clin Medical Association, 244: 41-43. Microbiol., 18: 968-971. Lima, K.C., J. Megid, A.V. Silva and A. Cortez. Rissi, D.R., R.A. Fighera, L.F. Irigoyen, G.D.

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Kommers and S.L.B. Claudio. 2008. Infectious Diseases, 140: 758-764. Occurrence of rabies in sheep in Rio Grande Umoh, J.U., C.D. Ezeokoli and A.E. Okoh. 1984. doSul, Brazil. Pesquisa Vet. Brasil., 28(10): Immunofluorescent staining of trypsinized 495-500. formalin-fixed brain smears for rabies Salem, S.A.H., E.I. Mashed and F.K. Hamoda. antigen: results compared with those 1995. Pathological and epidemiological obtained by standard methods for 221 studies on bovines in Kauobia Governorate. suspect animal cases in Nigeria. J. Hyg.- Proceeding 3th Science Congress Egypt Camb., 94: 129-134. Science Cattle Diseases, 2: 303-311. Warner, C.K., S.G. Whitfield, M. Fekadu and H. Smith, J. 1999. Rabies virus, p. 1099-1106. In Ho. 1997. Procedures for reproducible Murray, P.R., E.J. Baron, M.A. Pfaller, F.C. detection of rabies virus antigen mRNA and Tenover and R.H. Yolken (eds.) Manual genome in situ in formalin-fixed tissues. J. of Medical Microbiology, 7th ed. American Virol. Methods, 67: 5-12. Society for Microbiology, Washington, DC, Whitfield, C.G., M. Fekadu, J.H. Shaddock, M. USA. Niezgoda, C.K. Warner and S.L. Messenger. Srinivasan, A., E.C. Burton, M.J. Kuehnert, C. 2001. The rabies working group. A Rupprecht, W.L. Sutker, T.G. Ksiazek, comparative study of the fluorescent C.D. Paddock, J. Guarner, W.J. Shieh, antibody test for rabies diagnosis in fresh C. Goldsmith, C.A. Hanlon, J. Zoretic, and formalin-fixed brain tissue specimens. B. Fischbach, M. Niezgoda, W.H. El, L. J. Virol. Methods, 95: 145-151. Orciari, E.Q. Sanchez, A. Likos, G.B. Klintmalm, D. Cardo, J. LeDuc, M.E. Chamberland, D.B. Jernigan and S.R. Zaki. 2005. Transmission of rabies virus from an organ donor to four transplant recipients. The New England Journal of Medicine, 352: 1103-1111. Suja, M.S., A. Mahadevan, C. Sundaram, J. Mani, B.C. Sagar, T. Hemachudha, S. Wacharapluesadee, S.N. Madhusudana and S.K. Shankar. 2004. Rabies encephalitis following fox bite, histological and immuno-histochemical evaluation of lesions caused by virus. Clin. Neuropathol., 23(6): 271-276. Swoveland, P.T. and K.P. Johnson. 1979. Enhancement of fluorescent antibody staining of viral antigens in formalin fixed tissues by trypsin digestion. The Journal of

345 Buffalo Bulletin (July-September 2016) Vol.35 No.3 Original Article Buffalo Bulletin (July-September 2016) Vol.35 No.3

EPIDEMIOLOGY OF IXODID TICKS IN BUFFALOES (BUBALUS BUBALIS) OF PUNJAB, INDIA

N.K. Singh* and S.S. Rath

ABSTRACT microplus

A total of 3590 buffaloes of all age groups and sex were examined from various districts of INTRODUCTION Punjab state, India. The association between age, sex of host, season and the prevalence of ixodid ticks Ticks and the diseases they transmit on animal level were investigated by multivariate are widely distributed throughout the world, logistic regression. The overall prevalence of particularly in tropical and subtropical regions. ixodid ticks, Rhipicephalus (Boophilus) microplus, Losses attributable to ticks are caused either directly Hyalomma anatolicum anatolicum and mixed through tick worry, blood loss, damage to hides infestations were 57.68, 31.83, 19.08 and 6.76%, and udders and the injection of toxins, or indirectly respectively. The results of multivariate analysis through mortality or debility caused by the diseases showed that the prevalence was associated with transmitted by or associated with the ticks. The season (P<0.001; OR: 3.153; CI 95%: 2.69-3.69), global economic loss due to tick infestation has host age (P<0.001; OR: 2.13; CI 95%: 1.67-2.73) been estimated as US$ 14000 to 18000 million and sex (P<0.001; OR: 0.416; CI 95%: 0.24-0.71). annually and the cost of management of tick and The prevalence of ixodid ticks were highest tick borne diseases (TTBDs) in livestock of India is in monsoon season (74.48%) whereas, maximum as high as US$ 498.7 million per annum (Minjauw prevalence of R. (B.) microplus and H. a. anatolicum and Mc Leod, 2003). The most common combined were recorded in monsoon (44.73%) and summer effect of TTBDs in Indian dairy system is reduction (23.32%), respectively and the seasonal variation in milk yield i.e. loss of 14% of the lactation was significant (P<0.001). Further, higher tick (McLeod and Kristjanson, 1999) and quality of infestation was recorded in calves <6 months of hides for leather industry (Biswas, 2003). age and also in males. The findings of the current Punjab is having a significant percentage study would provide a basis for evolving effective of India’s buffalo heads and contributes nearly 9% control strategy for the management of ticks in to the Indian milk production. The state is situated buffalo population of the region. at the North West frontier of India and the climatic condition of the state is highly conducive for Keywords: buffalo, epidemiology, Hyalomma growth and development of ticks. In past, several anatolicum anatolicum, Rhipicephalus (Boophilus) sporadic reports on tick infestation patterns of dairy

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

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animals from Punjab had been published (Gill and tick samples were then transferred to plastic tubes Gill, 1977; Singh and Singh, 1999; Ghai et al., and were brought to the laboratory and separately 2008; Haque et al., 2011) but a comprehensive stored in 70% ethanol. Adult ticks were identified epidemiological study covering whole of the under a stereomicroscope, according to general state has not been explored so far. Therefore, the identification keys (Estrada-Penaet al., 2004). present study was undertaken to determine the epidemiological patterns of the ixodid tick in Statistical analysis buffaloes of Punjab state, India. All data analyses were performed by using statistical software program (SPSS for Windows, Version 19.0, USA). Association between the MATERIALS AND METHODS prevalence of ixodid tick infestation and various factors was carried out by Chi square (χ2-test). Location, geography and climate of study area Variables with significant association at P<0.05 The state of Punjab extends from the (two-sided) were subjected to the multivariate latitudes 29.30oN to 32.32oN and longitudes logistic regression model. The results were each 73.55oE to 76.50oE in the northwest region of India. expressed as P value and odds ratio (OR) with a It covers a geographical area of 50,362 km2 and lie 95% confidence interval (CI 95%). between altitudes 180 meters and 300 meters above sea level. The three major seasons in Punjab are summer (April to June; average rainfall 51.6 mm), RESULTS AND DISCUSSION monsoon (July to September; average rainfall 395.2 mm) and winter (October to March; average Ticks collected from the buffalo population rainfall 119.1 mm). of Punjab state were identified as Rhipicephalus The climate of the plains is excessively hot (Boophilus) microplus and Hyalomma anatolicum and dry in summers and winters are cool with some anatolicum. The prevalence of ixodid ticks, R. (B.) frosts. Average rainfall in Punjab is 565.9 mm and microplus, and H. a. anatolicum were 57.68%, ranges from about 915 mm in north to 102 mm in 31.83% and 19.08%, respectively. Most infestations south. (http://punjabonline.in/Profile/Geography/ were pure with single species as mixed infestations climate.asp). of both genera occurred less frequently (6.76%). These ticks were present on all over the body but Collection of ticks R. (B.) microplus showed preference for the areas Ticks were collected during February, 2010 with softer skin viz. groin, insides of shoulder and to August, 2011 from 3590 buffaloes of different thighs, around the anus and external genetalia, base villages covered under the eighteen districts. Animals of tail etc. Whereas, H. a. anatolicum were found of both sexes and all age groups were examined to be evenly distributed through out the body and and each animal examined was considered as one in some animals an atypical circular infestation sample. Ticks were searched by passing hands pattern by the nymphal stages was documented through the animal’s coat and collected manually (Figure 1). without damaging their mouthparts. The collected Results of the current study reveal that R.

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(B.) microplus and H. a. anatolicum are the ixodid of ticks and R. (B.) microplus was recorded as ticks infesting buffaloes of Punjab state, India. the predominant tick in all seasons of Punjab In contrast, much earlier reports from the region state. Significant association between prevalence document a large number of ixodid tick species of infestation with ixodid ticks in buffaloes and parasitizing buffaloes from the same geographical the season was observed (P<0.001; OR: 3.153; area (Gill and Gill, 1977; Miranpuri, 1988). The CI 95%: 2.69-3.69). The β value of-0.725 was change in the tick population in form of reduced recorded between the prevalence of ticks and the number of species may be related to the adaptation various seasons (summer followed by monsoon of better animal husbandry practices in the last two and winter) thus indicating a decrease in tick to three decades. Newer tick control measures and prevalence with decrease in ambient temperature availability of effective acaricides proved to be (Table 1). Several studies are on record with regard detrimental particularly for the survival of ticks to seasonal dynamics of ticks in India (Rajagopalan other than the two species reported in the study and Sreenivasan, 1981; Das, 1994; Vatsya et al., mainly due to their initial low frequencies in the 2008). population (Gill and Gill, 1977). Similar to the The highest infestation rate was recorded findings of the current study, recent studies on the in monsoon season (74.48%), followed by summer tick population of dairy animals from the region (66.64%) and least in winter (38.78%) with a also report R. (B.) microplus and H. a. anatolicum significant variation (P<0.001) (Table 2) in seasonal as the only tick species infesting dairy animals distribution as the hot and humid environmental (Singh and Singh, 1999; Sangwan et al., 2000; conditions in the monsoon is most conducive for Ghai et al., 2008; Haque et al., 2011). the development of various developmental stages of ticks. Whereas, the cold and dry conditions Seasonal dynamics of ixodid ticks of the winters are unfavourable for its survival The current study indicates that season and tick passes the winter as engorged females, plays a very important role in population dynamics nymphs, larvae and unfed adults by hiding into

Figure 1. Atypical circular infestation pattern of Hyalomma a. anatolicum in buffalo.

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the cracks and crevices (Chaudhuri, 1969) thus with limited use of acaricides leading to higher leading to low infestation levels. The trend was tick infestations. Also, low tick infestation on similar for the distribution of R. (B.) microplus adult cattle is probably due to resistance acquired with significant statistical variation (P<0.01) following repeated exposure from early life (Das, among seasons whereas; seasonal prevalence of 1994). Further, similar infestation pattern of ticks H. a. anatolicum showed entirely different trend had been reported in past (Nagar et al., 1978; with maximum prevalence in drier months with Manan et al., 2007). significant variation (P<0.001) as hot and dry weather is conducive for its development and Sex wise prevalence of ixodid ticks similar trend has been reported earlier (Bouattour The effect of sex of host on the prevalence et al., 1996; Estrada Pena, 2008). An earlier study of ixodid tick infestations was significant (P<0.01; also revealed the gradual overpowering of R. OR: 0.416; CI 95%: 0.24-0.71) with positive (B.) microplus on H. a. anatolicum in the winter correlation (β=4.42) and higher prevalence in season and is because of its wide distribution and males. The prevalence of R. (B.) microplus, H. a. prevalence (Haque et al., 2011). anatolicum and mixed infestation in both sexes were significantly variable (P<0.001) (Table 2). Prevalence of ixodid ticks in various age groups Although, Sutherst et al. (1983) reported that the of buffaloes milch animals because of the hormonal stress Animal age significantly affected the carry more ticks but in the current study a higher prevalence of infestation by ixodid ticks (P<0.001; tick infestation was encountered in males and can OR: 2.13; CI 95%: 1.67-2.73) but had a negative be attributed to the fact that male animals in this correlation (β=-2.61). Thus, among the different part of the country are neglected and least care is age group of buffaloes screened maximum tick provided with occasional use of acaricides. This is infestation was recorded in calves<6 months of due to the reason that males are now considered age (72.73%), followed by 6 months to 1 year useless by the farmers after the popularization of age group (61.30%) and least in >1 year age artificial insemination and use to motorized power group (55.53%). Similarly, significant difference for farm usage as informed by the owners through (P<0.001) was recorded in the prevalence of the questionnaire. R. (B.) microplus among different age groups It can hence be concluded that R. (B.) with highest prevalence in calves<6 months of microplus and H. a. anatolicum are the ixodid ticks age, whereas, prevalence of mixed infestations of buffaloes of Punjab state and the former being was higher in older animals (Table 2). The age the predominant one. Further, as the tick population of the host animal plays a significant role on the peaks at the monsoon followed by summer season infestation pattern of tick species (Manan et al., the control measures should be adopted accordingly 2007). Younger animals are more prone to tick to minimize the losses attributed to ticks and thus infestations and can be correlated with the fact increase the productivity of the animals. that the adult or the productive animals are given utmost care with better animal husbandry practices whereas the younger animals are least attended

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Table 1. Final logistic regression model for factors associated with ixodid ticks infestation in buffaloes on animal levels. Regression Standard Error Confidence Interval Variables P value Odds coefficient (β) (SE) (CI 95%) Season -0.725 0.06 0.000 3.153 2.69-3.69 Age -2.61 0.106 0.000 2.135 1.67-2.73 Sex 4.42 0.237 0.000 0.416 0.24-0.71

Table 2. Epidemiology of ixodid ticks in buffaloes of Punjab state, India. +ve for Mixed +ve for R. (B.) +ve for H. a. Groups Examined Positive infestation microplus anatolicum Summer 1256 837 (66.64) 143 (11.38) 401 (31.92) 293 (23.32) Monsoon 921 686 (74.48) 85 (9.22) 412 (44.73) 189 (20.52) Season Winter 1413 548 (38.78) 15 (1.06) 330 (23.35) 203 (14.36) χ2 value - 358.9* 101.4* 10.40** 27.9* <6 mon 352 256 (72.73) 16 (4.54) 167 (47.44) 73 (20.73) 6 mon – 1 yr 292 179 (61.30) 20 (6.84) 93 (31.84) 66 (22.60) Age > 1 yr 2946 1636 (55.53) 207 (7.02) 883 (29.97) 546 (18.53) χ2 value - 41.49* 294.1* 999.3* 663.0* Female 3514 2013 (57.29) 238 (6.77) 1115 (31.73) 660 (18.78) Sex Male 76 58 (76.32) 5 (6.57) 28 (36.84) 25 (32.89) χ2 value - 11.80** 223.4* 1034* 588.6* Total 3590 2071 (57.68) 243 (6.76) 1143 (31.83) 685 (19.08)

Figures in parenthesis are the % prevalence of infested ticks; *P<0.001, **P<0.01

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ACKNOWLEDGEMENTS Punjab State. Ann. Biol., 24: 95-100. Gill, H.S. and B.S. Gill. 1977. Qualitative district- The authors are thankful to The Director of wise distribution of adult ixodid ticks in Research, Guru Angad Dev Veterinary and Animal the Punjab state. Ixodid Ticks of Domestic Sciences University, Ludhiana for providing Animals in the Punjab State. PAU, Ludhiana. facilities to carry out the research work. Haque, M., Jyoti, N.K. Singh, S.S. Rath and S. Ghosh. 2011. Epidemiology and seasonal dynamics of ixodid ticks of dairy animals REFERENCES of Punjab state, India. Indian J. Anim. Sci., 81: 661-664. Biswas, S. 2003. Effects of ticks on animal Manan, A., Z. Khan, B. Ahmad and Abdullah. 2007. production system. In Proceedings the Prevalence and identification of Ixodid tick Natural Seminar Leather Industry in genera in frontier region Peshawar. J. Agric. Today’s Perspective. Kolkata, India. Biol. Sci., 2: 21-25. Bouattour, A., M.A. Darghoulh and L. BenMiled. McLeod, R. and P. Kristjanson. 1999. Tick Cost: 1996. Cattle infestation by Hyalomma ticks Economic Impact of Ticks and TBD to and prevalence of Theileria in H. detritum Livestock in Africa, Asia and Australia. species in Tunisia. Vet. Parasitol., 65: 233- International Livestock Research Institute 245. (ILRI), Nairobi, Kenya. Chaudhuri, R.P. 1969. Description of the Minjauw, B. and A. McLeod. 2003. Tick-borne immature stages of Hyalomma kumari and diseases and poverty. The impact of ticks redescription of the adults with notes on its and tick-borne diseases on the livelihood of hosts and distribution. Parasitology, 60: 43- small scale and marginal livestock owners 53. in India and eastern and southern Africa. Das, S.S. 1994. Prevalence of ixodid tick infestation Research report, DFID Animal Health on farm animals in Pantnagar, tarai of Uttar Programme, Centre for Tropical Veterinary Pradesh. J. Parasitol. Appl. Anim. Biol., 3: Medicine, University of Edinburgh, UK. 71-73. p. 59-60. Estrada-Pena, A. 2008. Climate, niche, ticks, and Miranpuri, G.S. 1988. Ticks parasitising the Indian models: what they are and how we should buffalo (Bubalus bubalis) and their possible interpret them. Parasitol. Res., 103: 87-95. role in disease transmission. Vet. Parasitol., Estrada-Pena, A., A. Bouattour, J.L. Camicas 27: 357-362. and A.R. Walker. 2004. Ticks of Domestic Nagar, S.K., R.N. Raizada and V.K. Saxena. Animals in Mediterranean Region. A Guide 1978. Studies on the rate of infestation of to Identification of Species. Bioscience Boophilus microplus on Indian cattle: Its Reports, London, UK. p. 43-131. activity and infestation differential. Indian Ghai, J.K., M. Singh and A. Singh. 2008. Population J. Anim. Sci., 48: 173-176. dynamics of ixodid ticks infesting cattle in Rajagopalan, P.K. and M.A. Sreenivasan. 1981. Bathinda and Hoshiarpur districts in the Ixodid ticks on cattle and buffaloes in the

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Kyasanur forest disease area of Karnataka state. Indian Vet. J., 65: 18-22. Sangwan, A.K., N. Sangwan and M.C. Goel. 2000. Progressive displacement of Hyalomma ticks by Boophilus microplus in Haryana. Journal of Parasitic Diseases, 24: 95-96. Singh, A.P. and A. Singh. 1999. Seasonal dynamics of ixodid ticks infesting the crossbred cattle of Ludhiana district. Indian Vet. J., 76: 167- 168. Sutherst, R.W., J.D. Kerr and G.F. Maywald. 1983. Effect of season and nutrition on the resistance of cattle to the tick Boophilus microplus. Aust. J. Agric. Res., 34: 329-339. Vatsya, S., C.L. Yadav, R.R. Kumar and R. Garg. 2008. Prevalence of ixodid ticks on bovines in foothills of Uttarkhand state: a preliminary report. Indian J. Anim. Sci., 78: 40-42.

353 Buffalo Bulletin (July-September 2016) Vol.35 No.3 Original Article Buffalo Bulletin (July-September 2016) Vol.35 No.3

EPIDEMIOLOGICAL STUDIES ON GASTROINTESTINAL PARASITES OF BUFFALOES IN SEVEN AGRO-CLIMATIC ZONES OF MADHYA PRADESH, INDIA

S. Nath1, G. Das1,*, A.K. Dixit1, V. Agrawal1, S. Kumar1, A.K. Singh2 and R.N. Katuri3

ABSTRACT August (79.68%) and lowest in the month of April (28.25%). Mean EPG of strongylewas 321.8 In the present study 3779 feacal samples and highest intensity of strongyle infection was of buffaloes were collected from the seven agro- recorded in the month of July (513.1). Coproculture climatic zones of Madhya Pradesh state, India. examination revealed that Haemonchus being the The study was conducted for a period of one year predominant (72.08%) nematode genus, followed from April 2011 to March 2012. The prevalence by Trichostrongylus (11.42%), Oesophagostomum of gastrointestinal (GI) parasitic infection was (10.08%), Bunostomum (3.75%) and Strongyloides 55.65% (2143). Amphistomes (28.10%) being the (2.67%). The current investigation provide basis most prevalent GI parasite followed by Strongyle to formulate strategic control measures against GI (25.59%), Schistosoma sp. (5.19%), Strongyloides parasitism. sp. (3.15%), Trichuris sp. (2.59%), Fasciola sp. (2.30%), Toxocara (0.66%) and Monezia sp. Keywords: buffalo, gastrointestinal parasite, (0.42%). Among non helmithic infection coccidian prevalence, strongyle showed prevalence of 19.00%. Out of the seven zones, zone V (Central Narmada valley) had the highest prevalence (61.46%) and the Hills of Jhabua INTRODUCTION zone XII had the lowest prevalence (50.42%). Prevalence in calves was more (59.78%) as World population of buffaloes (Bubalus compared to adult (54.36%). Season wise highest bubalis) is approximately 177.247 million of prevalence was observed in monsoon (73.41%) which 97% (171 million) are found in Asia. followed by winter (60.47%) and then summer India constitutes about 55.7% (98.7 million) of (36.22%). Prevalence of coccidiosis (25.00%) the total world buffalo population (FAO, 2008). was highest in winter. Monthly prevalence data Buffaloes are important livestock because of showed highest prevalence in the month of their multifunctional purposes, providing milk,

1Department of Veterinary Parasitology, College of Veterinary Science and Animal Husbandary, Jabalpur, Madhya Pradesh, India, *E-mail: [email protected] 2Department of Veterinary Parasitology, College of Veterinary Science and Animal Husbandary, Mathura, Uttar Pradesh, India 3Department of Veterinary Parasitology, College of Veterinary Science and Animal Husbandary, Tirupati, Andhra Pradesh, India

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meat and good quality hides. They are considered Madhya Pradesh which extend between latitude as “tractors” in Southeast Asia in agriculture 21.20oN-26.87oN and longitude 74.02oE-82.49oE farms, means of transportation and their dung act and considered as “Heart of India or Central India” as a good fertilizer and fuel (Liu et al., 2009). and stretches over an area of 3,08,252 sq km. The Gastrointestinal parasitism is one of the major average rainfall is about 1,370mm and having constraints of livestock industry, severely affecting subtropical climate. The M.P state has been divided the animal productivity, retarded growth and in 11 major agro-climatic zones and 50 districts increased susceptibility to other diseases. The (Figure 1). Present work has been carried out in 7 economic losses may run into millions of rupees zones out of the eleven agro-climatic zones. (Shah and Chaudhry, 1995). The study period was April 2011 to March But, the problem is neglected due to 2012. A total of 3779 faecal samples were collected its chronic and insidious nature (Sanyal, 1998). per rectally or immediately after defication The prevalence of gastrointestinal parasites and from the selected villages throughout the year at its severity depends on many factors such as monthly interval. These samples were collected in local environmental conditions and management sterile labeled polythene bags and brought to the practices (Regassa et al., 2006). Studies of Department of Veterinary Parasitology, College of epidemiological pattern of the parasitic diseases Veterinary Science& Animal Husbandry, Jabalpur. in different agro-climatic zones of the country These samples were subjected to flotation and empower us to develop measures for strategic and sedimentation technique (Soulsby, 1982). The tactical control of these diseases. Epidemiology positive samples were further checked for their and factors associated with prevalence of intensity by Mc Master technique as described gastrointestinal parasites in domesticated animals of by Thienpont et al. (1979). The samples positive the Indian subcontinent is described by Chowdhury for nematodes were examined for the generic and Tada (1994). The incidence of GI parasites in composition by glass tumbler method. The buffaloes has been reported from different states infective larvae were collected by Bearmann of India (Haque et al., 2011; Wadhwa et al., 2011; technique (Anon, 1977), cleaned and segregated Reddy et al., 2012; Singh et al., 2012 and Mir by repeated centrifugation and decantation. These et al., 2013). A few reports are available on the were identified as per the keys by Van Wyk et prevalence of GI parasites in buffalo of Madhya al. (2004). Data analysis was done according to Pradesh and adjoining area (Agarwal et al., 2002 Snedecor and Cochron (1980). and Pal et al., 2001). The present communication records such information pertaining to buffaloes of Madhya Pradesh to formulate control strategies RESULTS AND DISCUSSION against GI parasites. Overall prevalence Out of 3779 buffaloes coprologically MATERIALS AND METHODS examined during the period April 2011 to March 2012, 2103 (55.65%) were positive for different The study was carried out in the state of gastrointestinal parasitic infections (Table 1).

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The prevalent parasites constituted strongyles, (chhattisgarh Plains) showed the lowest prevalence Strongyloides sp. and Trichuris sp. amongst of amphistome (18.96%) and coccidia (13.36%) nematodes and amphistomes, Schistosoma sp., respectively (Table 2). Fasciola sp., Moniezia sp. and coccidian as non nematode GI parasites. Strongyles were the Age wise prevalence predominant (25.59%) nematodes, followed Faecal samples of 2879 adult buffalo and by Strongyloides sp. (3.15%) and Trichuris sp. 900 buffalo calf were examined coprologically. (2.59%). Likewise, Kashyap et al. (1997) reported Prevalence of GI parasitism in buffalo calves 40.3% prevalence of gastrointestinal helminthiosis (59.78%) was non-significantly higher compared to in cattle and buffaloes from Madhya Pradesh where the adult (54.36%) (Table 3). The result of present strongyle showed highest prevalence. Prevalence investigation were in accordance with Haque et al. of amphistomosis was highest (28.10%) amongst (2011) and Bilal et al. (2009) who reported higher the non-nematode parasites. The prevalence of prevalence of GI parasites in calves. In our country, coccidia, Schistosoma sp. and Fasciola sp. was calves are more prone to the parasitic infection 19.00%, 5.19% and 2.30% repectively (Table 1). due to inadequate attention towards management, Our study is in accordance with many workers treatment and disease control measures (Pfukenyi reporting higher prevalence of strongyle in dairy et al., 2007). Conversly Biswas et al. (2014) animals (Haque et al., 2011; Singh et al., 2012 reported higher infection in adult. The cause of and Singh et al., 2008). The higher range of contradiction may be due to exhausted immune prevalence of amphistomes in the present study system, different grazing pattern and managemental is in agreement with Yadav et al., (2004) and practices. Adult buffaloes show very low level of Kuchay et al., (2011). It may be due to factors such prevalence of Toxocara vitulorum (0.07%) there is as wallowing habit, easy dispersion of faeces in an inverse relationship between prevalence and age water and bulk ingestion of grasses near the water of animal (Halmandge et al., 2005). Coccidiosis source, increases the risk of amphistomosis due to was significantly higher in calves (P<0.01) than availability of intermediate host (Radostitis et al., adult animals as the later exhibited cellular 1994). immunity against coccidiosis as a result of the previous exposure to the oocysts (Soulsby, 1982). Agro-climatic zone-wise prevalence Moreover the practice of using coccidiostat or Out of the seven zones, Central Narmada coccidiocidal drug was very less in this region. Our Valley, zone IV had the highest prevalence (61.46%) finding was in accordance with Haqueet al. (2011) and the Hills of Jhabua zone XI had the lowest and Singh et al. (2012). Moneziosis was not seen in prevalence (50.42%) (Table 2, Figure 2) however adult buffaloes. Prevalence of amphistomosis was the difference is non-significant. Prevalence of insignificantly higher in adult buffaloes. strongyle was highest in zone I (31.11%) minimum in Zone III (19.67%). Zone II (Northen hills zones Season wise prevalence of Chhattisgarh) showed the highest prevalence The prevalence of G.I. parasitic infections of amphistome (36.46) and coccidian infection in summer, monsoon and winter season was (23.54%). Zone VIII (Satpura Plateau) and zone I 36.22%, 73.41% and 60.48%, respectively (Table

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4). The prevalence of gastrointestinal parasites was EPG was observed in the rainy season followed significantly higher in Monsoon season (P<0.01). by winter and summer. The present finding was The onset and advancement of monsoon rains have supported by the results of Mathur et al. (1996) and profound effect on incidence and seasonality of Waruiru et al. (2000) reporting the monsoon season gastrointestinal infection (Wadhwa et al., 2011). to be the highest intensity showing season. The Significant higher infection rate of strongyle and mean intensity of strongyle infection was highest amphistome infection, was seen in rainy season (362.1) in zone II and lowest in zone III (262.7). (P<0.01). Same results were seen in the study of Baseshankar and Maske (2000) but results of many Generic composition of nematode larvae scientists vary with the present findings and they Coprocultural examination of faeces have found different seasons for highest prevalence revealed that Haemonchus was the predominant at their working area. Biswas et al. (2013) in (72.08%) nematode genus, followed by Bangladesh, Samanta and Santra (2009) in West Trichostrongylus (11.42%), Oesophagostomum Bengal and Mir et al. (2013) in Jammu reported (10.08%), Bunostomum (3.75%) and Strongyloides summer to be the prevalent season for GI parasitic (2.67%). (Figure 3) This finding is consistent incidence. The environmental condition of these with the findings of earlier workers (Yadav et regions was hot and humid with normal rainfall al. 2008) who reported Haemonchus to be the during summer providing favourable conditions most common and pathogenic genus among for development and survival of pre parasitic stage various gastrointestinal nematodes causing high of the parasites where as during rains there was mortality and morbidity in India. Jamra et al. excessive rainfall declining the pastural growth of (2014) reported Haemonchus to be the most pre parasitic larvae. In Madhya Pradesh rainfall prevalent genera followed by Trichostrongylus occur mainly in monsoon providing suitable and Oesophagostomum. Climatic and geographic environment for the survival and propagation of condition of this region supports the propagation preparasitic stages of parasite. Coccidiosis was and growth of Haemonchus larvae. significantly higher in winter season (P<0.01) In order to formulate effective control because of the suitable temperature and humidity strategies against parasitic infection in any particular required for the oocystic sporulation at this time. region we have to know the status of infection which can be only possible by the surveys as conducted in Intensity of strongyle infection the present study. The results give a brief scenario The overall mean EPG (Egg Per Gram) about the prevalent parasites in Madhya Pradesh counts in the seven agro-climatic zones studied emphasizing amphistomes and strongyle to be were and found to be moderate (321.8) (Figure most prevalent GI parasites. Among non-helminth 2). Wadhawa et al. (2011) reported high overall parasites coccidian had the highest prevalence. It is mean EPG (684.61). It may be due to the climatic imperative that integrated strategies and measures and geographical variation. Highest intensity of be taken to control helminth infections in buffaloes strongyle infection was recorded in the month of in Madhya Pradesh and elsewhere. July (513.1). The month wise intensity of strongyle infection is presented in Figure 4. Highest mean

358 Buffalo Bulletin (July-September 2016) Vol.35 No.3 19.00 23.33 16.88 13.75 21.88 19.67 23.54 13.36 Coccidia Coccidia 0.00 0.00 0.83 0.63 0.00 0.83 1.04 0.42 Moniezia Moniezia 3.96 4.38 5.21 6.25 4.78 6.04 6.05 5.19 Schistosoma Schistosoma Positive for other GI parasites other Positive for 25.63 18.96 22.08 33.75 33.56 36.46 21.50 Positive for other GI parasites other Positive for 28.10 Amphistome Amphistome 2.71 1.04 2.08 3.96 2.22 2.71 1.46 Fasciola 2.30 Fasciola 0.00 1.25 0.63 1.46 0.00 1.04 0.84 Toxocara Toxocara 0.66 Toxocara 3.54 2.50 2.71 2.71 0.89 3.13 4.18 Trichuris Trichuris 2.59 Trichuris 5.21 2.71 3.33 3.33 1.56 3.96 3.34 Strongyloides Positive for GI Nematodes Positive for 3.15 Strongyloides Positive for GI Nematodes Positive for 31.11 25.42 26.88 27.29 26.46 19.67 27.50 Strongyle 25.59 Strongyle (%) 50.42 52.08 54.38 61.46 54.00 58.54 60.13 Positive (%) 55.65 Positive No. 480 480 480 480 900 480 479 Examined No. 3779 I V II IV XI III VIII Zone Examined Table 2. Prevalence (%) of GI parasitism in buffalo in different Agro climatic zones of M.P. in different 2. Prevalence (%) of GI parasitism in buffalo Table Table 1. Overall prevalence (%) of different GI parasitic infections in buffalo of M.P. GI parasitic infections in buffalo 1. Overall prevalence (%) of different Table

359 Buffalo Bulletin (July-September 2016) Vol.35 No.3 5.3* 13.82 22.06 25.00 36* 17.33 24.33 Coccidia Coccidia 0 0.24 0.79 0.48 1.78 Moniezia Moniezia 2.86 7.62 3.89 5.59 3.89 Schistosoma Schistosoma Positive for other GI parasites other Positive for Positive for other GI parasites other Positive for 1.16 17* 28.52 26.78 18.98 37.46 30.48 Amphistome Amphistome 2.11 2.36 1.43 3.02 1.75 Fasciola Fasciola 0.07 2.56 0.00 1.19 1.27 Toxocara Toxocara 2.36 3.33 1.91 4.21 2.14 Trichuris Trichuris 2.95 3.78 1.03 4.84 3.33 Strongyloides Positive for GI nematodes Positive for Positive for GI Nematodes Positive for Strongyloides 0.45 23* 25.32 26.44 11.44 40.16 25.00 Strongyle Strongyle (%) 54.36 59.78 8.16* 35* (%) 36.22 73.41 60.48 Positive Positive No 900 2879 No. 1259 1260 1260 Examined Examined Value Zone 2 Value 2 X Adult Young Season Summer Monsoon Winter X (*) P<0.01 Table 3. Age wise prevalence (%) of different GI parasites in buffalo of M.P. GI parasites in buffalo Age wise prevalence (%) of different 3. Table in M.P. 4. Seasonal prevalence (%) of GI parasitism in buffalo Table (*)P<0.01

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Figure 1. Different Agro climatic zones of M.P.

Incidence (%) All Zone mean FEC (epg) 50 600 45 40 500 35 400 30 25 300 20 200

Incidence (%) 15 10 (epg) FEC Mean 100 5 0 0 Apr- May- Jun- Jul- Aug- Sep- Oct- Nov- Dec- Jan- Feb- Mar- 11 11 11 11 11 11 11 11 11 12 12 12

Figure 2. Intensity of strongyle infection in buffalo of M.P.

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Figure 3. Mean generic composition of nematode larvae in buffalo.

ACKNOWLEDGEMENTS Basesahanker, S.B. and D.K. Maske. 2000. Epidemiology of helminthic infestation in cattle of eastern zone of Maharashtra State. The authors are highly thankful to the In The 11th National Congress of Veterinary Dean, College of Veterinary Science and Animal Parasitology, Bhubaneswar, India. Husbandry, NDVSU, Jabalpur for providing the Bilal, M.Q., A. Hameed and T. Ahmad. 2009. facilities and “All India Network Programme Prevalence of gastrointestinal parasites in on Gastrointestinal Parasitism” (ICAR) for fund buffalo and cow calves in rural areas of required for conducting the research work. Toba Tek singh, Pakistan. J. Anim. Plant Sci., 19(2): 67-70. Biswas, H., A.R. Dey, N. Begum and P.M. Das. REFERENCES 2013. Epidemiological aspects of gastro- intestinal parasites in buffalo in Bhola, Agrawal, R., R. Singh, M. Kumar and A.K. Bangladesh. Indian J. Anim. Sci., 84(3): Upadhyay. 2002. Epidemiological features 245-250. of parasitic diseases in some parts of Chowdhury, N. and I. Tada. 1994. Helminths Chhattisgarh state. Indian J. Vet. Med., 22: of domesticated animals in Indian 47-49. subcontinent, p. 73-120. In Helminthology. Anon. 1977. Manual of Veterinary Parasitological Springer-Verlag, Narosa Publishing House. Laboratory Techniques. Bulletin No. 18, FAO. 2008. Rome, Italy. STAT database.; www. Ministry of Agriculture, Fisheries and Food. fao.org. Her Majesty’s Stationary Office, London, p. Halmandge, S., M.D. Suranagi, A. Murugeppa, 5-50. Sudhindra and S.P. Kumar. 2005. Prevalence

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of ascariosis in buffalo calves in and around Pfukenyi, D.M., S. Mukaratirwa, A.L. Willingham Bidar. J. Vet. Parasitol., 19: 149-151. and J. Monard. 2007. Epidemiological Haque, M., Jyoti, N.K. Singh, P.D. Juyal, H. Singh, studies on parasitic gastrointestinal R. Singh and S.S. Rath. 2011. Incedence of nematode, cestode and coccidian infections gastrointestinal parasites in dairy animals of in cattle in the Highveld and lowveld western plains of Punjab. J. Vet. Parasitol., communal grazing areas of Zimbabwe. 25: 168-170. Onderstepoort J. Vet., 74: 129-142. Jamra, N., G. Das, M. Haque and P. Singh. 2014. Radostitis, O.M., D.C. Blood and C.C. Gay. 1994. Prevalence and intensity of strongyles in Veterinary Medicine, 8th ed. The English buffaloes at Nimar region of M.P. Int. J. Language Book Society, Bailliere Tindall, Agric. Sc Vet. Med., 2(1): 54-57. London. 625p. Kashyap, Z., R.S. Sisodia and P.C. Shukla. 1997. Reddy, M.V.B., P. Sasikala and A. Karthik. 2012. Incidence of gastrointestinal parasites in Prevalence of parasitic infections in cattle cattle and buffaloes in Malwa region of and buffaloes of Piler, Chittoor district of Madhya Pradesh. Haryana Vet., 36: 34-36. Andhra Pradesh. IJPHC., 6: 131-135. Kuchai, J.A., M.Z. Chishti, M.Z. Manal, S.A. Dar, Regassa, F., T. Sori, R. Dhuguma and Y. Kiros. R., Muzaffar, J. Ahmad and T. Hidayatullah. 2006. Epidemiology of Gastrointestinal 2011. Some epidemiological aspects of Parasites of Ruminants in Western Oromia, Fascioliasis among cattle of Ladakh. Global Ethiopia. Intern. J. Appl. Res. Vet. Med., Veterinaria, 7(4): 342-346. 4(1): 52-58. Liu, Y., F. Li, W. Liu, R.S. Dai, Y.M. Tan, D.S. He, Sanyal, P.K. 1998. Integrated gastrointestinal R.Q. Lin and X.Q. Zhu. 2009. Prevalence parasite management in dairy animals in of helminthes in water buffaloes in Hunan Gujarat by self medication. J. Vet. Parasitol., Province, China. Trop. Anim. Health Pro., 12: 17-20. 41: 543-546. Shah, H.L. and R.K. Chaudhry. 1995. Parasitism Mathur, A.C., S. Sarkar and P.K. Sanyal. in dairy animals in India: Present status and 1996. Epedemiology of gastrointestinal its impact, p. 1-5. In Proceeding Against nematodosis in buffalo compared to cattle Gastrointestinal Parasites in Dairy Animals in gangetic plains of eastern India. Buffalo in India Using Medicated Urea Molasses J., 12: 127-138. Blocks. NDDB, Anand, India. Mir, M.R., M.Z. Chishti1, M. Rashid, S.A. Dar, Singh, A., A.K. Gangwar, N.K. Shinde and R. Katoch, J.A. Kuchay and J.A. Dar. S. Srisvastava. 2008. Gastrointestinal 2013. Point Prevalence of Gastrointestinal parasitism in bovines of Faizabad. J. Vet. helminthiasis in large ruminant of Jammu, Parasitol., 22: 31-33. India. Int. J. Sci. Res. Pub., 3: 1-3. Singh, N.K., H. Singh, Jyoti, M. Haque and Pal, S., S. Roy and A.K. Pathak. 2001. Prevalence S.S. Rath. 2012. Prevalence of parasitic of gastrointestinal parasites in cattle and infections in buffaloes in and around buffalo from Chhattisgarh region. J. Vet. Ludhiana district, Punjab, India: A Parasitol., 15: 155-156. preliminary study. J. Buffalo. Sci., 1: 1-3.

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Samanta, A. and P.K. Santra. 2009. Prevalence of gastrointestinal helminthes in hot and humid zone of West Bengal. J. Vet. Parasitol., 23: 73-76. Snedecor, G.W. and W.G. Cochram. 1980. Statistical Methods, 7th ed. The Iowa State University Press, Ames, Iowa, USA. 593p. Soulsby, E.J.L. 1982. Helminths, Arthropods and Protozoa of Domesticated Animals, 7th ed. ELBS and Bailliere Tindal. 788p. Thienpont, D., F. Rochette and O.F.J. van Parijs. 1979. Diagnosing Helminthiasis by Coprological Examination. Beerse, Belgium, Janssen Research Foundation. 187p. Van Wyk, J.A., J. Cabaret and L.M. Michael. 2004. Morphological identification of nematode larvae of small ruminants and cattle simplified.Vet. Parasitol., 119: 277-306. Wadhwa, A., R.K. Tanwar, L.D. Singla, S. Eda, N. Kumar and Y. Kumar. 2011. Prevalence of gastrointestinal helminthes in cattle and buffaloes in Bikaner, Rajasthan, India. Vet. World, 4(9): 417-419. Waruiru, R.M., N.C. Kyvsgaard and S.M. Thamsborg. 2000. The prevalence and intensity of helminth and coccidial infections in dairy cattle in central Kenya. Vet. Res. Commun., 24: 39-53. Yadav, C.L., R.R. Kumar, S. Vatsya, R. Garg and P.S. Baneerjee. 2008. Epidemiological studies on gastrointestinal nematodosis in cattle and buffaloes. J. Vet. Parasitol., 22: 49-52. Yadav, A., J.K. Khajuria and A.K. Raina. 2004. Gastrointestinal parasitic infestation profile of bovines at R.S. Pura, Jammu. J. Vet. Parasitol., 18(2): 167-169.

364 Original Article Buffalo Bulletin (July-September 2016) Vol.35 No.3

STUDIES ON EFFECT OF NON-GENETIC PARAMETERS ON MORTALITY PATTERN IN MURRAH BUFFALOES

Nitin Mohan Gupta1,*, M.L. Mehra1 and Puneet Malhotra2

ABSTRACT is to boost the genetic improvement in important economic traits of animals. Disposal of large The theme of investigation was the herd number of animals from any herd due to various of 1230 Murrah breed buffaloes at the dairy reasons greatly affects its economy. While watching farm of Guru Angad Dev Veterinary and Animal at future genetic gain of the herd the intensive Sciences University, Ludhiana. The study was selection to decide an element of disposing off conducted for the episode of forty years 1971 to animals remains to be the tough job. Intensive 2010, to make out the mortality pattern at this selection remains directly proportional to the organized herd. The reasons for mortality were the quantum of selection differential in large sized herd. affections of Digestive and Respiratory systems, The number of born female calves during a year and also circulatory disturbances and the Unclassified when they reach at puberty it becomes replacement reasons. The share of mortality of buffaloes with stock. It is immense necessity to rear the progeny circulatory disturbances was principal i.e. 44.55%, of proven parents to encompass the healthy and followed by affections of digestive system 26.61%, high yielding animals and it is the fundamental of unclassified condition were on the tune of 17.95% any dairy animal improvement programme. Every and the least was accounted at the affections of farm has compulsorily replacements due to the respiratory system with only 10.89%. The seasonal death and culling on performance ground. The calf effect, period and parity have not any concern on disposal plays the important role in maintaining the mortality pattern of animal and also it was non- the herd strength and farm standards (Reddy and significant too. Nagarcenkar, 1989). The present investigation was undertaken in Murrah buffaloes with the objective Keywords: buffalo, mortality, parity, period, to know the major cause of mortality and effect of season season, period and parity on it.

INTRODUCTION MATERIALS AND METHODS

The main endeavour of the animal breeder The statistics pertaining to ancestry,

1Department of Animal Genetics and Breeding, Khalsa College of Veterinary and Animal Sciences, Amritsar, Punjab, India, *E-mail: [email protected] 2Department of Animal Genetics and Breeding, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India

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production and reproduction for the present to ith parity (i=1,2,3,4,5,6,7,8,9 and 10 & exploration were collected from the history- above parities). th cum-pedigree sheets, growth, production and Sj is the effect common to all animals died in j reproduction records respectively maintained at the season (j=1, 2, 3, 4, 5) and summer = 1, rainy dairy farm of GADVASU, Ludhiana. The data with = 2, autumn = 3, winter = 4 and spring= 5. th respect to these vital traits were composed over Pk is the effect common to all animals died in k an episode of 40 years, i.e. from 1971 onwards to period (k=1, 2, …………,8). 2 2010 and pertained to 1230 animals that were born eijkl is the random error, assumed to be NID (0, σ e). throughout the period of 1965 to 2010.

Statistical analysis RESULTS AND DISCUSSION The statistics on mortality for the present revision pertained to animals which died during Causes of mortality different parity, season and period. The effect The foremost causes for mortality were of various factors viz. parity, season and period acknowledged as disturbances in circulatory were studied on mortality to appraise relative system, which accounted for 44.55 percent of involvement of each factor. For estimating the entire mortality followed by affections of digestive effect of these factors, data on mortality of only system (26.93%) and affections of respiratory females were taken, so as to keep away the biasness system (10.89%) while unclassified condition in results, as males were transferred/ auctioned at accounted for 17.63% of total mortality. Similar early age. percentage (25.81%) of casualty due to affection of Since the frame of collected data follows digestive system was reported by Malhotra (2003) discrete allotment, for that reason it was required to in the herd of crossbred cattle. get transformed by means of Arcsine transformation Unclassified conditions accounted for and transformed data including sum of squares 17.63 percent of whole mortality which includes for various factors incorporated in the statistical NAD (Nothing Abnormality Detected) cases, model were analyzed by Least-squares analysis as be deficient in of proper diagnosis and putrefied explained by Harvey (1968). carcasses ensuing from overdue post mortem of The following statistical model was used animals. to study the variation in mortality (data from 1971 to 2010) due to parity, season and period. Season wise incidence of mortality Each year was alienated into five season’s

Yijkl = µ + Ri + Sj + Pk + eijkl viz. summer, rainy, autumn, winter and spring Where, (Table 2). Uppermost mortality was observed in Yijkl is the lth observation on mortality of animals winter (25%) followed by summer (24.03%) and belonging to ith parity, died in jth season and rainy season (23.4%) whereas, highest occurrence kth period. of mortality of 38.29% was observed by Patil et µ is the overall average of mortality percentage. al. (1992) during winter in Surti buffaloes. Parallel

Ri is the effect common to all animals belonging result was reported by Mourad and Rashwan

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(2001) and Rana et al. (2010) in winter in Murrah were 312 lactating animals (females) died out of buffaloes. Pooled percentage of these three seasons 1230 dead animals which constitutes about 25.36 constitutes round about three forth (72.43%) of total percent of whole mortality (Table 4). Greatest season wise mortality. Towering mortality during mortality was observed in first parity or lactation summer season may be owing to the less efficient (22.44%), which accounted for one fourth of total thermo-regulatory mechanism in buffaloes. The mortality, followed by third (16.35%), fourth black colour of buffalo absorbs supplementary (14.42) and second parity (13.14%). Least mortality heat during sunny days, as a result it directs to the was observed in 10th parity and above, this may be state of heat stress, which may be compensated due to the reason that a very few animals can reach by providing better managemental conditions. On up to this parity as most of the animals died due the other hand, higher frequency of fatality during to different diseases prior to reaching up to 10th rainy season may be owing to increased dampness and above parities. Out of all the diseases, bulk of and underprivileged sanitary environment due to the animals died owing to affections of circulatory heavy rainfall over tiny period of time. Smallest trouble (44.55%) followed next to affection of amount of mortality was evidenced in autumn digestive system (26.9%), unclassified condition (11.22%) followed by spring season (16.35%), constitutes (17.6%) and least mortality was due to which is possibly due to temperature which is affections of respiratory system which contributes neither too high nor too low in these seasons. The (10.89%) of entire mortality. The effect of parity effect of season on mortality was found to be non on mortality was established to be non significant significant (P>0.05); this is in agreement with the (P>0.05). findings of Khan et al. (2007).

Period wise incidence of mortality CONCLUSION The mortality accounts were taken from post-mortem records between 1971 to 2010. These The results of in hand study sketches the years were alienated into 8 periods of five year attention headed for circulatory disturbances and each (Table 3). The leader percentage of mortality affections of the digestive system which accounts of 26.28 percent was stuck between the period for more or less three fourth of full amount of 1975 to 1980, which alone constitutes round of mortality. Roughly one fourth of the whole about one fourth of total period wise occurrence mortality had been occurred all through in initial of mortality and the slightest of 6.74 percent of parity. All these factors designate the call for the mortality accounted between the periods of 1981 to improved management and anticipatory measures 1985. The effect of period on mortality was found to be followed for dropping the mortality, so as to be insignificant (P>0.05). additional number of animals will be available for future replacements, which will greatly enhance Parity/Lactation wise incidence of mortality selection intensity, selection differential and Parity wise mortality was calculated in moreover genetic gain at the farm. ten categories viz. first parity to tenth and above parities underneath four disease situation. There

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Table 1. Reasons for mortality in buffaloes.

Total number of Average number Disease Percentage of Causes of mortality animals died in of animal died code animals died 40 years per year A Affections of Digestive system 331 8.27 26.93a Affections of Respiratory B 134 3.35 10.89bc system C Circulatory disturbances 548 13.7 44.55a D Unclassified 217 5.42 17.63ac Total 1230 30.75 100.00

Table 2. Season wise incidence of mortality in buffaloes. Total number of Average number of Season Mortality (%) animal died animal died per year Summer 75 1.875 24.03a Rainy 73 1.825 23.40a Autumn 35 0.875 11.22bc Winter 78 1.95 25.00a Spring 51 1.275 16.35bc Total 312 7.8 100.00

Table 3. Period wise mortality in buffaloes.

Total number of Average number of Period Mortality (%) animal died animal died per year

1971-1975 29 0.725 9.29ab 1976-1980 82 2.05 26.28a 1981-1985 21 0.525 6.74ac 1986-1990 28 0.70 8.98ac 1991-1995 48 1.20 15.38bc 1996-2000 37 0.925 11.86a 2001-2005 38 0.95 12.18a 2006-2010 29 0.725 9.29ab Total 312 7.8 100.00

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Table 4. Parity wise incidence of mortality in Murrah buffaloes.

No. of Parity/ Mortality Code % of total animals Lactation died A B C D died 1 15 8 35 12 70 22.44a 2 12 3 15 11 41 13.14bc 3 16 3 24 8 51 16.35bc 4 14 5 21 5 45 14.42bc 5 11 7 13 5 36 11.54bc 6 9 3 11 1 24 7.70ac 7 4 3 9 3 19 6.10ac 8 1 0 4 6 11 3.53acd 9 2 2 3 1 8 2.54cd 10 & above 0 0 4 3 7 2.24cd Total died 84 34 139 55 312 100.00

A-Affections of Digestive system, B- Affections of Respiratory system, C-Circulatory disturbances, D- Unclassified condition Note: The means with at least one common alphabet as superscript do not differ significantly from each other.

Table 5. Analysis of Variance for factors affecting mortality in Murrah buffalo. Degree of Source Mean squares F value freedom Sire 99 57.51 15.55* Period 7 4.78 1.29NS Season 4 8.29 2.24NS Parity 9 6.35 1.71NS Residual 594 3.69 Total 713

* P < 0.05, NS = Non-significant

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REFERENCES

Harvey, W.R. 1968. Least Squares Analysis of Data with Unequal Subclasses Numbers. United States Department of Agriculture. ARS, USA. Khan, Z.U., S. Khan, N. Ahmad and A. Raziq. 2007. Investigation of mortality incidence and managemental practices in buffalo claves at commercial dairy farms in Peshawar city. Journal of Agricultural and Biological Science, 2(3): 16-21. Malhotra, P. and O.S. Parmar. 2005. Studies on Culling reasons vis-s-vis Milk production in Cows. Indian Journal of Dairy Science, 58(6): 433-435. Mourad, M. and S. Rashwan. 2001. Milk production of buffaloes and causes of calf mortality under a semi-intensive production system in Egypt. Revue d’Élevage et de Médecine Vétérinaire des Pays Tropicaux, 54(2): 139- 145. Patil, N.A., S.P. Kumar, S. Mallikarjunappa, K.R. Lakshmaiah and A.R.S. Bhat. 1992. Calf mortality in Surti buffaloes. Indian Vet. J., 69: 1018-1022. Rana, N., S. Khanna, A.A. Raut, S.R. Bhardwaj, A. Manuja, B. Manuja, A. Saini, S. Kakkar, K.L. Khurana and R.K. Sethi. 2010. Retrospective epidemiological analysis of mortality trends in neonatal and growing Murrah buffalo calves at an organized herd. Indian J. Anim. Sci., 80(10): 976-979. Reddy, K.M. and R. Nagarcenkar. 1989. Studies on disposal pattern in Sahiwal calves. Indian Journal of Dairy Science, 42: 280-288.

370 Original Article Buffalo Bulletin (July-September 2016) Vol.35 No.3

EVALUATION OF BREEDING VALUES MURRAH BUFFALO BULLS UNDER ORGANIZED FARMS

Vijay Kumar* and A.K. Chakravarty

ABSTRACT INTRODUCTION

In the present study, breeding information Selection of the superior sires with spread over a period of 14 years from 1995 to maximum accuracy is of utmost importance for any 2008, was collected from the history-cum-pedigree breed improvement programme, as sires are easily sheets and milk yield registers of Murrah buffaloes and rapidly disseminated in various herds under maintained in four centres of Network Project on progeny testing programme. Robertson and Randle Murrah Buffalo Improvement (National Dairy (1954) opined that as much as 61% of genetic Research Institute, Karnal; Central Institute for gain in dairy cattle resulted from selection of sires Research on Buffalo, Hisar and Guru Angad through two paths, i.e. bulls to breed cows and bulls Dev Veterinary and Animal Sciences University, to breed bulls. Hence, accurate selection of bulls Ludhiana and Choudhary Charan Singh Haryana used in artificial insemination (AI) programme is Agricultural University, Hisar). Data on first of prime importance for long-term genetic progress lactation traits of 832 Murrah buffaloes sired in the population. by 95 bulls were used for the study. Farm had The prediction of breeding values significant effect on FL305MY, while season and constitutes an integral part of most breeding year of calving did not affect significantly in the programmes for genetic improvement of the sire present study. Breeding value for first lactation 305 for different economic traits. The accuracy of days milk yield was estimated using best linear estimating the breeding value of an animal is the unbiased prediction (BLUP) method. The breeding major factor that affects the genetic progress due value of different bulls varied from 1630.40 kg in to selection. The sire evaluation based on milk fifth set to 2022.61 kg in seventh set. yield was most widely used criteria. To make rapid genetic progress in performances through selection Keywords: BLUP, breeding value, FL305MY, for traits of economic importance, the animals must murrah buffalo be chosen accurately for their superior breeding values. Over the times various methods have been used for sire evaluation, Henderson’s (1973) mixed model or best linear unbiased prediction (BLUP)

Department of Animal Genetics and Breeding, Mathura Veterinary University, UP Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan (DUVASU), Mathura, Uttar Pradesh, India, *E-mail: [email protected]

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procedure has become the method of choice for of observations (FL305MY), fixed effects (farm, evaluating the genetic worth of the bulls. BLUP season and period effect), random effect (sire is one of the accurate sire evaluation methods effect) and random error and X and Z are incidence to obtain unbiased estimates of breeding values matrices pertaining to fixed effects and random of sires (Mukherjee et al., 2007). The model of effects. analysis under BLUP takes into account, the fixed effect and relationship among animals. Therefore, the breeding values of animals are estimated with RESULTS AND DISCUSSION higher accuracy. The overall least squares mean of total first lactation milk yield (Table 1) was, however, MATERIALS AND METHODS lower than that reported by Patil (2011) and Geetha (2005) in Murrah buffalo. Higher than this was The Murrah bulls in 7 sets (11, 12, 15, 14, reported by Katneni (2007). Farm had significant 15, 16 and 12 bulls) were inducted for progeny effect on FL305MY in the present study. Centre- testing at Central Institute for Research on Buffalo wise least-squares means for 305MY for NDRI, (CIRB), Hisar, National Dairy Research Institute CIRB, GADVASU and CCSHAU were found to (NDRI), Karnal Guru Angad Dev Veterinary be 1792.45±31.94, 1684.71±37.88, 1941.02±42.90 and Animal Sciences University (GADVASU), and 1969.26±104.80 kg, respectively. Season Ludhiana, Choudhary Charan Singh Haryana and year of calving did not affect significantly Agricultural University (CCSHAU), Hisar. The the FL305MY of Murrah buffaloes in the present daughters of first 7 sets have completed their first study. lactation records. The first lactation 305 days milk The information on bulls along with their yield (FL305MY) records of 832 daughters of 95 breeding values is given in Table 2 to Table7. The bulls calved during 14 years from 1995 to 2008, breeding value of different bulls varied 1683.44 were used for this study. The period of 14 years to 1976.89 kg in first set, 1746.03 to 1952.91 kg was divided into 14 years. Each year of calving was in second set, 1720.94 to 1929.56 kg in third set, further classified into 2 seasons, viz. most calving 1730.12 to 1907.14 kg in fourth set, 1630.40 to season (January to June) and least calving season 2011.21 kg in fifth set, 1710.75 to 1981.79 kg in (July to December) based on calving pattern. All sixth set and 1709.76 to 2022.61 kg in seventh set. information was classified in four farms viz. NDRI, The highest breeding value was observed for sire CIRB, GADVASU and CCSHAU. The breeding 88 (set 7) followed by 66 (set 5) and 69 (set 6). value of sires was estimated by best linear unbiased Singh and Singh (1999) observed breeding value of prediction (BLUP) method as given by Henderson Murrah bulls between 1137.30 to 1329.01 kg using (1973). BLUP method. Pandey and Singh (1999) computed The model of BLUP estimation was breeding value of 52 Murrah bulls for first lactation considered as follows: milk yield by corrected contemporary daughter Y= Xb +Za + e average index and reported it ranges from 1349.62 where, Y, b, a and e denotes the vector to 1934.39 kg.

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Table 1. Least-squares means of first lactation milk yield in Murrah buffaloes. Factor No. of Observation FL305MY Overall (µ) 832 1846.86±35.94* Farm NDRI 305 1792.45±31.94b CIRB 314 1684.71±37.88a GADVASU 188 1941.02±42.90c CCSHAU 25 1969.26±104.80c Season of calving Least calving season 291 1881.96±43.57 Most calving season 541 1811.75±36.94 Year of calving 1995 5 2070.52±220.35 1996 19 1956.38±117.84 1997 33 1754.71±92.10 1998 9 1665.17±165.24 1999 77 1884.81±62.23 2000 66 1860.91±65.15 2001 67 1736.42±65.23 2002 72 1804.10±63.26 2003 108 1788.41±53.71 2004 91 1871.62±57.91 2005 81 1903.16±56.12 2006 87 1855.29±57.40 2007 89 1889.94±53.04 2008 28 1814.57±93.51

*Significant P <0.05; Values with different superscript differ significantly: Milk yields are in kg.

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Table 2. Breeding values of Murrah buffalo bulls in set 1. Sire No. of Daughters Breeding Value Rank 1 5 1946.02 11 2 6 1962.25 6 3 8 1976.89 4 4 18 1948.54 10 5 19 1856.7 44 6 18 1683.44 94 7 10 1694.78 93 8 4 1884.15 32 9 5 1840.46 53 10 2 1804.92 62 11 11 1819.11 58

Table 3. Breeding values of Murrah buffalo in set 2. Sire No. of Daughters Breeding Value Rank 12 9 1835.51 55 13 8 1889.71 30 14 11 1906.64 23 15 7 1952.91 7 16 2 1863.89 40 17 8 1882.1 33 18 11 1754.49 77 19 15 1927.16 15 20 10 1841.04 52 21 9 1749.77 79 22 9 1746.03 80 23 13 1771.69 74

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Table 4. Breeding values of Murrah buffalo bulls in set 3. Sire No. of Daughters Breeding Value Rank 24 9 1829.47 56 25 4 1929.56 14 26 8 1905.36 25 27 11 1775.59 73 28 3 1916.97 19 29 6 1920.06 18 30 4 1809.36 60 31 3 1767.56 75 32 2 1892.23 29 33 3 1915.45 20 34 5 1757.44 76 35 21 1781.17 71 36 11 1808.65 61 37 7 1720.94 88 38 9 1875.56 36

Table 5. Breeding values of Murrah buffalo bulls in set 4.

Sire No. of Daughters Breeding Value Rank 39 18 1860.85 43 40 9 1781.36 70 41 5 1845.41 49 42 9 1793.57 66 43 6 1796.92 65 44 5 1856.17 45 45 6 1730.12 85 46 11 1899.66 27 47 7 1878.6 34 48 9 1743.18 81 49 11 1875.07 37 50 5 1845.71 48 51 11 1907.14 22 52 8 1841.94 51

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Table 6. Breeding values of Murrah buffalo bulls in set 5.

Sire No. of Daughters Breeding Value Rank 53 16 1728.66 87 54 3 1848.07 47 55 12 1630.4 95 56 7 1789.89 68 57 6 1800.28 64 58 6 1790.09 67 59 6 1862.63 41 60 8 1842.65 50 61 3 1698.94 92 62 6 1838.57 54 63 11 1888.87 31 64 9 1803.98 63 65 22 1963.15 5 66 12 2011.21 2 67 12 1933.16 13

Table 7. Breeding values of Murrah buffalo bulls in set 6. Sire No. of Daughters Breeding Value Rank 68 10 1735.45 83 69 20 1981.79 3 70 7 1788.69 69 71 2 1710.75 90 72 3 1819.02 59 73 4 1905.32 26 74 8 1875.61 35 75 15 1867.7 38 76 14 1925.69 16 77 5 1728.86 86 78 7 1825.39 57 79 10 1949.13 9 80 10 1906.24 24 81 5 1951.98 8 82 4 1716.71 89 83 7 1739.52 82

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Table 8. Breeding values of Murrah buffalo bulls in set 7.

Sire No. of Daughters Breeding Value Rank 84 18 1861.04 42 85 8 1749.87 78 86 13 1780.19 72 87 8 1921.59 17 88 6 2022.61 1 89 4 1735.16 84 90 3 1709.76 91 91 13 1899.06 28 92 6 1909.69 21 93 13 1864.92 39 94 9 1848.93 46 95 17 1934.11 12

REFERENCES Murrah buffaloes. Indian J. Anim. Sci., 69: 1067-1069. Geetha, E. 2005. Studies on breeding values for Patil, C.S. 2011. Genetic evaluation of fertility persistency in Murrah buffaloes. M.Sc. in Murrah buffalo. M.V.Sc. Thesis, Thesis, National Dairy Research Institute, National Dairy Research Institute, Deemed Deemed University, Karnal Haryana, India. University, Karnal, India. Henderson, C.R. 1973. Proceedings of the Animal Robertson, A. and J.M. Randel. 1954. The Breeding and Genetics Symposium in performance of heifers got by artificial honor of Dr. Jay L. Lush. American society insemination. J. Agri. Sci., 44: 184-192. of Animal Science and American Dairy Singh, P.K. and B.P. Singh. 1999. Efficacy of Science Association, Chanpaign, Illinois, different methods in genetic evaluation of USA. Murrah sires. Indian J. Anim. Sci., 69: 1044- Katneni, V.K. 2007. Studies on breeding values 1047. for persistency in Murrah buffaloes. Ph.D. Thesis, National Dairy Research Institute, Deemed University, Karnal, India. Mukherjee, S., B.K. Joshi and G.K. Gaur. 2007. Comparison of sire evaluation methods In Friewal cattle. Indian J. Anim. Sci., 77: 773- 776. Pandey, A.K. and H. Singh. 1999. Comparison of different methods of sire evaluation in

377 Buffalo Bulletin (July-September 2016) Vol.35 No.3 Original Article Buffalo Bulletin (July-September 2016) Vol.35 No.3

INCIDENCE AND FERTILITY STATUS OF HYDATID CYSTS IN BUFFALOES

A. Sheeba, A. Sangaran*, B.R. Latha and A. Raja

ABSTRACT of animals slaughtered in Chennai. Hydatidosis in animals results in significant economic loss to the Study on the incidence of hydatidosis in meat industry through condemnation of infected food animals meant for human consumption such organs such as liver, lungs and other organs apart as buffaloes was done at the time of slaughter by from reduced quality of milk, meat and wool. inspecting the carcasses and viscera for the presence Hence, a study was done to know the incidence of hydatid cysts with particular reference to lungs, of the hydatid disease in slaughtered buffaloes in liver, spleen etc., Based on the observation, the Chennai as well as the organ wise involvement incidence of hydatid cysts in buffaloes examined and the fertility status of the hydatid cysts. was found to be 11.11 percent. With regard to the organ wise involvement, the presence of hydatid cysts was more in lungs, followed by liver and the MATERIALS AND METHODS fertility rate of hydatid cysts was high in lungs. Buffaloes were observed for the presence Keywords: hydatidosis, incidence, fertility, of hydatid cysts in lungs, liver and other organs at buffaloes the time of slaughter in the Corporation slaughter house, Chennai by inspecting the carcasses and viscera of the slaughtered animals. The visceral INTRODUCTION organs harbouring the hydatid cysts were collected and brought to the laboratory so as to ascertain the Hydatidosis, a zoonotic parasitic disease fertility or sterile nature of the hydatid cysts based of animals and man is caused by the larval stage on the presence or absence of protoscolices in the (metacestode) of the dog tapeworm Echinococcus hydatid cyst fluid. granulosus, the life cycle involving two mammalian hosts. Definitive hosts are carnivores such as dogs and the intermediate hosts are herbivores and RESULTS AND DISCUSSION omnivores wherein the development of the cysts occurs in liver, lungs and other organs. Incidence A total of 810 buffalo were screened of hydatidosis has been reported earlier by and observed for the presence of hydatid cysts at Sundaram and Natarajan (1960) by examination slaughter. Out of the 810 buffaloes, 90 buffaloes

Department of Veterinary Parasitology, Madras Veterinary College, Tamilnadu Veterinary and Animal Sciences University, Chennai, India, *E-mail: [email protected]

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showed the presence of hydatid cysts giving an and buffaloes, maximum number of fertile cysts overall incidence of 11.11% in buffaloes. Out of were recorded in liver (59%) followed by lungs 90 hydatid cysts observed in various organs, 57 and spleen (Sangaran and Lalitha, 2013). Variation hydatid cysts were recovered from lungs, 17 from in the cyst fertility might be due to the difference liver, 15 from lungs and liver and one hydatid cyst in tissue resistance among the visceral organs. from muscle. Forty two cysts were found fertile out The reason for higher prevalence of fertile cyst in of the 90 hydatid cysts observed. With regard to lungs in the present study might be due to softer fertility status of the cysts, 27 hydatid cysts from consistency of lungs and also large capillary fields lungs (64.30%), 7 hydatid cysts from liver (16.6%), encountered by the oncospheres (Urquhart et al., 7 hydatid cysts from lungs and liver (16.66%) and 1996). The result of the present study correlates one hydatid cyst from muscle (2.38%) were found well with the earlier observations of Kosalaraman to be fertile. and Ranganathan (1980) and Koshy (1984) on The prevalence of hydatidosis in cattle fertility nature of hydatid cysts. has been reported to vary from 7.6% (Deka et al., 1983) to as high as 56.6% (Himonas et al., 1994 and Daryani et al., 2009). The findings on the REFERENCES incidence of hydatid cysts as 11.11% in buffaloes in the present study correlates with the earlier reports. Beyhan, Y.E. and S. Umur. 2011. Molecular Variations in the prevalence could be due characterization and prevalence of cystic to the changes in the temperature, environmental echinococcosis in slaughtered water conditions, and the management practices buffaloes. Turkish Vet. Parasitol., 181: 174- adopted in rearing the animals. The present study 179. showed lower prevalence in comparison with the Daryani, A., M. Sharif, A. Amouei and M. earlier reports. Contrary to past decades, various Nasrolahei. 2009. Fertility and viability precautions and changing in behavior pattern such rates of hydatid cysts in slaughtered animals as awareness about the disease, routine deworming in the Mazandaram province, northern Iran. of dogs against tapeworms as well as decrease Trop. Anim. Health Pro., 20(2): 44-48. in the number of stray dogs could be the major Deka, D.K., G.C. Srivastava and R.C. Chhabra. reasons for the decrease in the incidence of hydatid 1983. Incidence of hydatidosis in ruminants. cysts (Beyhan and Umur, 2011). Indian J. Anim. Sci., 53: 200-202. With regard to fertility status of hydatid Himonas, C., A. Sotiriadou and E. Papadopoulos. cysts recorded from different viscera, it was 1994. Hydatidosis of food animals in observed that 64.30% of hydatid cysts from lungs Greece: prevalence of cysts containing were found to be fertile, followed by 16.66% from viable protoscolices. J. Helminthol., 68: liver. Kosalaraman and Ranganathan (1980) in 311-313. Madras had reported 35 percent of fertile cysts Kosalaraman,V.R. and M. Ranganathan. 1980. from lungs, 28% from liver. Koshy (1984) reported A survey of disease conditions of lungs of 20% of fertile cysts in liver, 28% in lungs and 36% buffaloes. Cheiron, 9: 281-284. in spleen. In case of food animals like sheep, goats Koshy, T.J. 1984. Taenid infections in dogs. Ph.D.

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Thesis, Tamil Nadu Agricultural University, Coimbatore, India. Sangaran, A. and L. John. 2013. Incidence and organ wise involvement of hydatidosis in buffaloes. Buffalo Bull., 32(Special Issue 2): 1009-1010. Sundaram, R.H. and R. Natarajan. 1960. A study on the incidence of hydatid disease in cattle in the city of Madras. Indian Vet. J., 37: 19- 24. Urquhart, G.M., J. Armour, J.L. Duncan and A.M. Jennings. 1996. Vet. Parasitol., 2nd ed. Blackwell Science Ltd, United Kingdom, p. 307.

381 Buffalo Bulletin (July-September 2016) Vol.35 No.3 Original Article Buffalo Bulletin (July-September 2016) Vol.35 No.3

SEROEPIDEMIOLOGICAL STUDY OF LEPTOSPIROSIS IN BUFFALOES OF SOUTH GUJARAT, INDIA

J.M. Patel1,*, P.D. Vihol1, V. S. Dabas2, M.C. Prasad1, J.H. Patel3, C.F. Chaudhari4, N.B. Patel5 and K.M. Patel6

ABSTRACT (00%) without significant difference (P≤0.05). In buffaloes out of 102 sera screened, 16 were positive A total of 102 serum samples were with one or more serovars. The highest number of collected randomly from buffaloes exposed with seropositivity was recorded against serovar Kaup different clinical conditions (abortion, repeat (17.39%). breeding, fever, mastitis, anorexia) suspected for leptospirosis and apparently healthy. These serum Keywords: buffaloes, Leptospirosis, samples were subjected to seroepidemiogical seroepidemiology, zoonosis, MAT study using microscopic agglurination test (MAT) having different serovars of Leptospira spp. The seroprevalence of leptospirosis among buffaloes INTRODUCTION was noted to be 15.69% (16/102). All the three districts of South Gujarat showed the presence Leptospirosis is an economically important of leptospiral antibodies without any significant widespread zoonotic disease caused by pathogenic difference (P≤0.05) with the highest rate in Tapi species of Leptospira interrogans occurs in man (50.00%) followed by Navsari (14.89%) and and different species of animals like cattle, buffalo, Surat (13.72%). Jafrabadi breed showed 50.00% sheep, goat, deer, pig, rodents, camel, horse, sealion, seropositivity followed by Surati (16.67%), shank, raccoon (Cutler et al., 2005; Cheema et al., Mehsana (15.00%) and Non-Descript (5.55%). In 2007). The causative agent is frequently excreted female buffaloes the seroprevalence positivity was though urine of infected individual and contaminate noted in 16.49% cases. However, none of male the environment, there by exposing human being exhibited seropositivity. In respect of age groups especially farmers, agriculture labour and animal the highest rate of seropositivity (19.23%) was holders. observed in age group of 1 to 4 years followed The state of Gujarat, Maharastra, Tamil by above 4 years (15.71%) and below 1 year Nadu, Kerala and Andaman and Nicobar Islands

1Department of Veterinary Pathology, *E-mail: [email protected], [email protected], 2Departmant of Veterinary Surgery and Radiology, 3Department of Veterinary Pharmacology and Toxicology, 4Department of Veterinary Gynaecology and Obstetrics, 5Department of Livestock Production and Management, 6Department of Animal Husbandry, Vanbandhu College of Veterinary Sciences and Animal Husbandry, Navasari Agricultural University, Eru cross road, Navsari, Gujarat, India

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are endemic in India and incidences in humans Microscopic agglutination test at Leptospirosis are perennially reported from these states during Reference Laboratory, Government Medical monsoon season (Maskey et al., 2006). Hence the College, Surat (Vijayachari et al., 2001) and present seroepidemiological study was conduct Project Directorate on Animal Disease Monitoring to determine the magnitude of occurrence pattern and Surveillance (PD-ADMAS), Bangalore using of leptospirosis in buffaloes, reared in rural areas standard procedure (WHO-OIE, 2013; Faine, of various district i.e. Navsari, Surat and Tapi of 1982). South Gujarat.

STATISTICAL ANALYSIS MATERIALS AND METHODS Chi-square test was used according to Animal sera WEB AGRI STAT PACKAGE software developed A total of 102 serum samples were by Jangam and Wadekar, ICAR research complex, collected randomly from different age groups and Goa for statistical analysis of data (Jangam and breeds of buffaloes (n=102) of either sex reared in Wadekar, 2012). villages of various districts (Navsari, Surat, Tapi) of south Gujarat (Table 1). Whole blood samples were collected from jugular vein directly or during RESULTS AND DISCUSSION slaughter of buffaloes in sterile 9.0 ml plain vacutainers. To obtain serum, whole blood was In the present study a total 102 sera were kept in slanting position in 9.0 ml plain vacutainers screened from three different districts of South until serum extracted out of the whole blood. Then Gujarat (Navsari, Tapi, Surat) for leptospiral these 9.0 ml plain vacutainers were centrifuged antibodies. The details of district, breed, sex and at 7000 rpm for 10 minutes. The straw coloured age wise seroprevalence results are depicted in serum was collected into 1.5 ml sterile cryo vials Table 1. and aliquoted and stored at -20oC for carriying out The seroprevalence of leptospirosis among MAT. buffalo was noted to be 15.69% (16/102) and was comparable to reported prevalence of 14.55% in Gujarat (Savalia, 2001), 14.7% in Uttaranchal MICROSCOPIC AGGUTINATION TEST (Agrawal et al., 2005) and 15% in Uttaranchal, (MAT) Tamil Nadu and Uttar Pradesh (Mariya et al., 2007). In contrast to above findings higher seroprevalence All the sera were tested for antibodies was 54.14% in Gujarat (Balakrishnan et al., 2011), against live antigens of Leptospira sp. serovars 26.66% in Andaman and Nicobar Islands (Varma Pyrogenes, Australis, Bankinang, Grippotyphosa, et al., 2001) and 88.8% (125/111) in Chennai Patoc, Pomona, Icterohaemorrhagiae, Hebdomadis, buffaloes (Selvaraj et al., 2010) have been reported. Canicola, Hardjo, Bellum, Bataviae, Tarassovi, All the three districts of South Gujarat Shermani, Kaup, Hurstbridge and Javanica by showed the presence of leptospiral antibodies

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without any significant difference (P≤0.05) with the seroprevalence in cattle, buffaloes and goats the highest rate in Tapi (50.00%) followed by and reported higher seropositivity in buffalo above Navsari (14.89%) and Surat (13.72%). Comparable 9.0 years of age and concluded that seropositivity findings of leptospirosis seroprevalence were increases with advancing age irrespective of animal reported by Savalia (2001) from Valsad (17.24%), species involved. Navsari (19.35%) and Surat (22.50%) districts. A total of 102 sera screened, 16 were No significant difference in seroprevalence positive with one or more serovars. In the present was observed among samples tested from the study highest number of seropositivity was different breeds of buffalo. Jafrabadi breed recorded against serovar Kaup (17.39%) followed showed 50.00% seropositivity followed by by Grippotyphosa (13.04%), Pomona (13.04%), Surati (16.67%), Mehsana (15.00%) and Non- Javanica (13.04%), Patoc (8.70%), Canicola Descript (5.55%). Contrary to this Balakrishnan (8.70%), Hardjo (8.70%), Bataviae (8.70%), et al. (2011) reported Murrah breed to be most Autumnalis/Bankinang (4.35%), Hurstbridge susceptible (58.25%) followed by Pandharpuri (4.35%). As against the present observations the (40.91%) and Jaffrabadi (37.50%). In the present most common serovars in Indian buffaloes reported study prevalence rate of leptospirosis in Jafrabadi by earlier workers from different states included breed of buffalo was higher (50.00%) than reported Hardjo (Agrawal et al., 2005; Balakrishnan et earlier by Balakrishnan et al. (2011). There is every al., 2011), Grippotyphosa (Varma et al., 2001) likelihood that the breed susceptibility reported by and Pomona (Selvaraj et al., 2005; Selvaraj et various workers is influenced by the sample size al., 2010). However serovars distribution seen in in particular area where specific breed might be South Gujarat region in present investigation was prevalent but need further elucidation. comparable with the findings of Savalia (2001) and In female buffaloes the seroprevalence Balakrishnan et al. (2011) who reported serovars positivity was noted in 16.49% cases. However, Hardjo, Grippotyphosa, Australis, Hebdomadis, none of male exhibited seropositivity. Possibly Ballum and Pomona. Other serovars reported from because a wide gap occurred in the number of different states of India enlisted Grippotyphosa, samples processed (Male-5 and Female-97). Pomona and Australis from Andaman and In respect of age groups (above 4 years, Nicobar (Varma et al., 2001), Hardjo, Javanica 1 to 4 years and below 1 year) the highest rate of and Australis from Uttaranchal (Agrawal et al., seropositivity (19.23%) was observed in age group 2005), Pomona, Hebdomadis, Tarassovi, Sejroe, of 1 to 4 years followed by above 4 years (15.71%) Australis, Pyrogenes, Autumnalis, Grippotyphosa, and below 1 year (00%) without significant Ballum, Javanica, Icterohaemorrhagiae, Canicola difference (P≤0.05). It was not in agreement with from Chennai (Selvaraj et al., 2005; Selvaraj et al., findings ofBalakrishnan et al. (2011) who observed 2010). maximum seropositivity (77.05%) among buffaloes Major areas of South Gujarat districts between 4 to 7 years (adult group) followed by are used for paddy and sugarcane cultivation above 7 years (75.00%, older age group) and below and are rich in natural vegetation with plenty of 4 year (26.67%) with significantly different among marshy lands and small ponds/water logging areas age group (P≤0.01). Agrawal et al. (2005) studied with almost neutral pH, suitable humidity and

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Table 1. Seroprevalence of leptospirosis in buffaloes. Percent Attributes No. of Tested No. of Positive Positive Region South Gujarat 102 16 15.69

Districts Navsari 47 07 14.89 Tapi 04 02 50.00 Surat 51 07 13.72 Total 102 16 15.69 χ2 = 5.99 NS(P<0.05) Breed wise Surati 60 10 16.67 Mehsani 20 03 15.00 Jafrabadi 04 02 50.00 Nondiscript 18 01 5.55 Total 102 16 15.69 χ2 = 7.82 NS(P<0.05) Sex wise Male 05 00 00 Female 97 16 16.49 Total 102 16 15.69 χ2 = 3.84 NS(P<0.05) Age wise <1 year 06 00 00 1-4 years 26 05 19.23 >4 years 70 11 15.71 Total 102 16 15.69 χ2 = 5.99 NS(P<0.05) Note: NS-Non significant at P<0.05

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temperature which are optimum for growth/survival pathogenic leptospires in animals by PCR of leptospires and perpetuation in the environment. based on lipL21 and lipL 32 genes. Indian Cultivated fields infested with rats, a carrier for J. Exp. Biol., 45: 568-573. leptospires (Collares-Pereira et al., 2000) also Collares-Pereira, M., M.L. Mathias, M. Santos-Reis, play an important role in spreading the infection. M.G. Ramalhinho and P. Duarte-Rodrigues. Prevalence in buffalo could be attributed to their 2000. Eur. J. Epidemiol., 16: 1151. habit of wallowing in water bodies contaminated Cutler, S.J., A.M. Whatmore and N.J. Commander. with infected urine (Treml et al., 2002) as one of 2005. Brucellosis-new aspects of an old the main sources of transmission of leptospira. disease. J. Appl. Microbiol., 98: 1270-1281. The present study concludes that Faine, S. 1982. Guidelines for the control of seroprevalence of leptosprosis among buffaloes leptospirosis. WHO. Offset Publication 67, in South Gujarat region was ranged from 14.55% Geneva, Italy. (Savalia, 2001) to 54.14% (Balakrishnan et al., Jangam, A.K. and P. Wadekar. 2012. Web Agri State 2011) with presently reported seroprevalence was Package. ICAR research complex for Goa. 15.69% without significant difference between age, Mariya, R., S.K. Srivastava and E. Thangapandian. breed, sex and different district. The continuous 2007. Seroprevalence of leptospiral presence of Leptospires in this area lead to potential antibodies in Bovine. Indian Vet. J., 84: zoonotic risk to slaughter house workers, meat 547-548. inspectors, animal holder, agriculture labour and Maskey, M., J.S. Shastri, K. Saraswathi, R. farmers. This study also determines the need for Surpam and N. Vaidya 2006. Leptospirosis continuous monitoring of leptospirosis in animal in Mumbai: Post-deluge outbreak 2005. and humans to combat this zoonotic infection. Indian J. Med. Microbi., 24: 337-338. Savalia, C.V. 2001. Seroprevalence of Leptospirosis in bovines of Gujarat. The Veterianarian, REFERENCES 25(2): 4-5. Selvaraj, J., B.M. Manohar, R. Govindarajan, Agrawal, R., M. Kumar, M. Kumar and S.K. V. Jayakumar, T.V. Meenambigai, C. Srivastava. 2005. Epidemiological pattern Balachandran and A. Koteeswaran. 2005. of leptospirosis in livestock of Uttaranchal Prevalence of leptospiral antibodies in state. Indian Journal of Comparative buffaloes (Bos bubalis) at slaughter. Indian Microbiology, Immunology and Infectious J. Comp. Microbiol. Immunol. Infect. Dis., Diseases, 26(2): 109-113. 26(2): 125-127. Balakrishnan, G., G.P. Roy, R. Govindarajan, Selvaraj, J., B.M. Manohar, R. Govindarajan, V. Ramaswamy and B.M. Manohar. V. Jayakumar, T.V. Meenambigai and C. 2011. Seroepidemiological studies on Balachandran. 2010. Seroprevalence of leptospirosis among bovines in organized leptospirosis in she-buffaloes (Bos bubalis) farm. IJAVMS., 5(6): 511-519. at slaughter in chennai, india. Short Cheema, P.S., S.K. Srivastava, R. Amutha, S. Singh, Communication. Buffalo Bull., 29(2): 95- H. Singh and M. Sandey. 2007. Detection of 98.

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Treml, F., M. Pejcoch and Z. Holesovska. 2002. Vet. Med. Czech., 47: 309. Varma, A., R.B. Rai, P. Balakrishnan, A. Gupta and K.A. Naveen. 2001. Seroprevalence of leptospirosis in animas of Andaman and nicobar islands. Indian Vet. J., 78: 936-937. Vijayachari, P., A.P. Suganan and S.C. Sehgal. 2001. Role of microscopic agglutination test (MAT) as a diagnostic tool during acute stage of leptospirosis in low and high endemic areas. Indian J. Med. Res., 114: 99- 106. World Organization for Animal Health (Office International des Épizooties-OIE). 2013. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. OIE, Paris; Chapter 2.1.9: 251-264.

388 Original Article Buffalo Bulletin (July-September 2016) Vol.35 No.3

AGE RELATED CHANGES IN THE HISTOMORPHOLOGY OF MANDIBULAR GLAND IN PRENATAL BUFFALO (BUBALUS BUBALIS)

K. Raja1,*, M.S. Lakshmi2, G. Purushotham2, K.B.P. Raghavende3 and T.S. Chandrasekhara Rao4

ABSTRACT Keywords: histomorphology, mandibular gland, prenatal buffalo The present work was undertaken on 42 buffalo embryos and fetuses ranging from 40 to 253 days to study the histomorphological INTRODUCTION features of mandibular salivary glands in the buffalo (Bubalus bubalis) during the prenatal Major salivary glands of various domestic period. The specimens were fixed and processed animals are paired structures, which includes for serial paraffin sectioning and the sections parotid, mandibular and sublingual glands. were subjected to different staining methods. The Salivary glands fulfill important role in the oral primordium of mandibular gland appeared first as biology by producing saliva to provide water for a solid epithelial bud from the oral epithelium at lubrication, as well as supplying electrolytes, the base of the tongue at 40 days. Ductal Lumen mucus, antibacterial compounds and various formation was observed first in the terminal buds enzymes to the oral cavity. Loss of salivary glands and primary cords of mandibular gland at 84 function can result in the wide spread deterioration days. The differentiation of terminal buds into of oral health (Hsu et al., 2010). Study of normal terminal tubules was completed at 91 days. The development of salivary glands will be helpful for typical compound tubulo - alveolar architecture both developmental anatomists and clinicians as of the gland was attained first at 125 days. The they are having important role in several dreadful gland was predominant in mucous type of acini diseases like rabies, foot and mouth disease and from 140 day onwards. The formation of capsule Herpes viral diseases. around the gland was evident at 125 days and it was well developed at 197 days. Differentiation of intercalated, intralobular and interlobular ducts was possible at 125 days. 1Department of Veterinary Anatomy, College of Veterinary Science, Korutla, India, *E-mail: [email protected] 2Department of Veterinary Anatomy, College of Veterinary Science, Rajendranagar, Hyderabad, Andhra Pradesh, India 3Department of Veterinary Surgery and Radiology, College of Veterinary Science, Rajendranagar, Hyderabad, Andhra Pradesh, India 4Faculty of Veterinary Science, Sri Venkateswara Veterinary University, Tirupati, India

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MATERIALS AND METHODS (Venkatakrishnan, 1994) in buffalo and during 6th week (Arey, 1965) in human. The migration of Total 42 buffalo embryos and fetuses epithelial bud into the surrounding mesenchyme ranging from 40 to 253 day of gestation (2.5 to as club shaped structure was noticed at 41 days 79.5 cm) “Curve Crown-Rump Length” (CVRL) (Figure 2) with surrounding mesenchyme in were collected from pregnant uteri immediately condensed form at 43 days. The appearance of 1hr after the slaughter from slaughter house of the anlagen as a solid club shaped structure from Hyderabad irrespective of the the age and sex. the oral cavity in relation with the developing The age of fetuses was determined on the basis of tongue was also reported by Venkatakrishnan their CVRL by using Soliman’s formula (1975). (1994), Mc Geady et al., (2006) and Santhi (2006) The whole embryos of earliest possible age (from in buffalo. The glandular mass was composed of 40 days i.e. 2.5cm CVRL) and fresh tissue pieces undifferentiating basophilic epithelial cells. The from the mandibular salivary gland of fetuses epithelial bud was attached to oral epithelium by from 84 day (12.4 cm CVRL) to 253 day (79.5cm a single epithelial stalk. The glandular mass began CVRL) were collected and fixed in 10% Neutral to branch into several terminal epithelial buds at Buffered Formalin(pH 7). Bouin’s fluids (Singh 45 days in dichomotous pattern surrounded with and Sulochana, 1997) also used for the fixation of large amount of dense mesenchyme. The terminal the gland because bouins fluid allows crisper and buds were formed by multilayered polyhedral cells better nuclear staining than 10% neutral-buffered with basophilic cytoplasm and spherical nuclei. formalin. The tissues were processed in routine The gland reached the space between the tympanic for paraffin sectioning of 5 to 8µm thickness and bulla and the angle of the mandible at 54 days. subjected to Mayer’s Haemotoxylin and Eosin At 84 days the gland was formed by groups method for routine histological developmental of luminized terminal buds (terminal tubules) and study, Van Gieson’s technique for collagen fibres, primary cords (Intercalated ducts) (Figure 3) with Masson’s Trichrome for connective tissue fibres dense mesenchyme and rich vasculature. However (Singh and Sulochana, 1997) staining techniques Venkatakrishnan (1994) reported the similar to study the histomorphological changes in the finding at 105 days in buffalo. The emergence of mandibular salivary gland will be observed by duct system was reported to be observed at 21 day using the Olympus microscope. in utero in the submandibular gland of rat (Ogawa et al., 2000). Most of the terminal tubules were lined with 2 to 3 layers of cells with central lumen RESULTS AND DISCUSSION from 91 days. The terminal tubules attained the structure of acini at 115 days in which the lining The primordium of mandibular salivary epithelium was changed to single layer (Figure 4), gland developed as a solid epithelial bud from the which was reported to be established at 5 weeks oral epithelium at the base of the tongue in linguo- postnatally in rat (Ogawa, 2000). gingival space at 40 days (Figure 1). Contrary to The cytoplasm of the acinar cells was this the primordium of the gland was reported to lightly basophilic with spherical basal nucleus. be observed at 45 days (Santhi, 2006) and 69 days Typical compound tubulo - alveolar nature of the

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gland was attained first at 125 days of foetal life of interlobular ducts between 140 and 188 days of (Figure 5). From 140 day onwards the gland was foetal life. predominantly mucous type along with serous The developmental changes in the demilunes. The cells of the mucous acini were parenchyma and stroma of the mandibular salivary pyramidal in shape with distinct cell borders gland of fetuses were gradual in buffalo, which is and basement membrane. The cytoplasm was in agreement with the findings of Venkatakrishnan lightly basophilic with flattened basal nuclei. The (1994) in buffalo, Arey (1965) and El- Mohandes cells lining the serous acini were pyramidal with et al. (1987) in man. The stromal content was spherical and darkly stained nuclei. Myoepithelial reduced and replaced by the parenchyma as the age cells were observed first around the acini and of the foetus advanced. The parenchyma and the intercalated ducts at 140 days between the basement duct system of the foetal mandibular gland nearing membrane and acinar cells. The developing the full term of pregnancy had achieved its identity mandibular gland was compound tubulo-alveolar to that of glands in adult buffalo.Differentiation of type with predominantly mucous alveoli during the intercalated and intralobular ducts was observed late foetal stage, which was reported to occur in all at 84 days of foetal age. The intercalated ducts foetal age groups of animals by Venkatakrishnan and striated ducts were lined by a double layered (1994) in buffalo. epithelium from 91 to 130 days with inner low The differentiation of embryonic columnar or cuboidal cells and outer flattened mesenchymal tissue into stroma was observed first cells. All types of ducts i.e., intercalated (acinar in foetal mandibular gland at 84 days (Figure 6). ducts), intralobular (striated ducts) and interlobular The gland showed primitive lobules separated by ducts were observed at 125 days (Figure 6) in stroma with fine collagen fibers at 91 days. The the developing mandibular gland. However the lobulation of the gland was distinct at 101 days differentiation of striated ducts, intercalated ducts (Figure 7). The lobules of the gland were reported and terminal buds was reported to occur at the to be formed during 9th to 10th week (Merida- time of the birth (Cutler and Chaudhry, 1973) and Velasco et al., 1993) in human beings and 14 to 26 at 16 weeks (El-Mohandes et al., 1987) in human days of prenatal development (Knopse and Bohme, being. The intercalated ducts were lined with 1995) in cat. The gland was highly vascular single layered cuboidal epithelium and were closer between 84 and 101 days. Dense lobulation of the nearest to the secretory end pieces from 140 days. gland with steep increase in the number of lobules The number of intercalated ducts increased as the and capsule formation was evident at 125 days foetal age increased. (Figure 5). The connective tissue septa were The intralobular ducts appeared first at formed at 131 days and were traversed by several 84 days in the form of solid epithelial cell cords. blood vessels, nerves and ducts. Capsule showed Double layered intralobular ducts composed of large amount of collagen fibres and few elastic inner and outer cuboidal cells were evident at 140 fibres at 197 days. The parenchyma of the gland days of foetal life (Figure 8). The mature striated was predominant in mucous acini from 125 days ducts were lined by a layer of tall inner columnar and 253 days (Figure 9). A large quantity of cells with flattened myoepithelial cells in the connective tissue was observed around the groups basal area. The inner cells lining the striated ducts

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Figure 1. Photomicrograph of cross section of foetal head showing the primordium of mandibular (M) salivary gland at 40 days. SL- Sublingual salivary gland , LN-Lingual nerve, T-Tongue H and E X 20.

Figure 2. Photomicrograph of cross section of 41 day buffalo foetal head showing the migration of epithelial bud of mandibular (M) gland into the mesenchyme. SL-Primordium of sublingual gland, O-Oral cavity, MG- Mandibular ganglion H and E X 20.

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Figure 3. Photomicrograph of cross section of 84 day foetal mandibular gland showing groups of terminal buds (TB), terminal tubules (TT) with dichomotus intercalated (ICD) and intralobular (LD) ducts. ILD-Interlobular ducts, EM-Embryonal mesenchyme, BV- Blood vessels Van Gieson’s method X 10.

Figure 4. Photomicrograph of cross section of 115 day foetal mandibular gland showing the earliest appearance of the mucous acini (A) lined with single layer epithelium (E). ICD-Intercalated duct, ILD-Interlobular duct, BV-Blood vessel H and E X 20.

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Figure 5. Photomicrograph of cross section of 125 day foetal mandibular gland showing compound tubulo- alveolar architecture predominant in mucous acini (A) and developing capsule (C) around the gland. LD-Intralobular duct, ILD-Interlobular duct, E- Excretory duct, L-Lobule Van-Gieson’s method X 10.

Figure 6. Photomicrograph of cross section of 84 day foetal mandibular gland showing the differentiation of stroma (ST), capsule (C) and septa (SE) from embryonic mesenchyme. TT-Terminal tubule Van-Gieson’s method X 10

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Figure 7. Photomicrograph of cross section foetal mandibular gland showing distinct lobulation (L) at 101 days. TT- Terminal tubule, ICD-Intercalated duct, LD-Intralobular duct, M-Mesenchyme H and E X 10.

Figure 8. Photomicrograph of cross section of 140 day foetal mandibular (M) gland showing Two layered intralobular ductal structures composed of inner and outer cuboidal cells. MA-Mucous acini, SA-Serous acini, SD-Serous demilune ---Goblet cells H and E X 20.

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Figure 9. Photomicrograph of cross section of 253 day foetal mandibular gland showing parenchyma predominant in mucous acini (MA). ICD-Intercalated duct, SD-Serous demilune, SA- Serous acini H and E X 10. showed intensely eosinophilic cytoplasm and with collagen fibres, elastic fibres, blood vessels darkly stained nuclei. These ducts showed a well and nerves. Findings of this work gave a valuable developed adventitia with abundant vasculature. informations and tremendous scope for the Two layered immature interlobular ducts clinicians and Developmental anatomists. were evident at 125 days. The tunica adventitia was prominent around the interlobular ducts with well developed collagen fibres, elastic fibres, REFERENCES blood vessels and nerves at 131 days. Groups of mature interlobular ducts were also prominent in Arey, L.B. 1965. Developmental Anatomy, 7th ed. the interlobular space from 140 days and lined with W.B. Saunders Company, Philadelphia. multilayered cuboidal or columnar epithelium with p. 411-419. abundant goblet cells (Figure 8). Cutler, L.S. and A.P. Chaudhry, 1973. The excretory ducts were lined by a double Differentiation of myoepithelial cells in layered columnar epithelium at 54 days. The the rat submandibular gland in vivo and in caruncula sublingualis was distinct at 56 days as vitro-An ultrastructural study. J. Morphol., reported by Santhi (2006) in prenatal buffalo. The 140: 343-354. excretory ducts were larger than the other ducts in El-Mohandes, E.A., K.G. Botros and A.A. Bondok. all age groups studied and were lined by stratified 1987. Prenatal development of the human cuboidal or columnar epithelium in advanced age submandibular gland. Acta Anatomica, groups. Vacuolated cells were seen occasionally. 130(3): 213-218. The ducts had a well developed adventitial layer Hsu, J.C.F., M. Kenneth and Yamada. 2010. 396 Buffalo Bulletin (July-September 2016) Vol.35 No.3

Salivary gland branching morphogenesis, India. recent progress and future opportunities. International Journal of Oral Science, 2(3): 117-126. Knospe, C. and G. Bohme. 1995. Zur Prsanatalen Entwicklung Derglandula Manibularis und Glandula parotis der katze. Anatomia, Histologia and Embryologia, 24: 1-6. Mc Geady, T.A., P.J. Quinn, E.S. Fitzpatrick and M.T. Ryan. 2006. Veterinary Embryology. Wiley-Blackwell Publishers, UK. p. 184- 204. Merida-Velasco, J.A., I. Sanchez-Montesinos, J.F. Espin, J.D. Garcia-Garcia, S. Garcia- Gomez and V. Roldan-Schilling. 1993. Development of the human submandibular salivary gland. Journal of Dental Research, 72(8): 1227-1232. Ogawa, Y., S. Toyosawa, T. Ishida and N. Ijuhin. 2000. Keratin 14 Immunoreactive cells in Pleomorphic Adenomas and Adenoid Cystic carcinomas of Salivary glands. Virchows Arch., 437(1): 58-68. Santhi, L., 2006. Prenatal development of the skull in the buffalo (Bubalus bubalis). Ph.D. Thesis, Sri Venkateswara Veterinary University. Singh, U.B. and S. Sulochana. 1997. A Practical Manual of Histopathological and Histochemical Techniques. Kothari Publication, Bombay. p. 1-41. Soliman, M.K. 1975. Studies on the physiological chemistry of the allantoic and amniotic fluids of buffaloes at the various periods of pregnancy. Indian Vet. J., 52: 106-112. Venkatakrishnan, 1994. Morphogenesis, histomorphology and histochemistry of the mandibular gland of the buffalo (Bubalus bubalis). Thesis, TANUVAS, Chennai,

397 Buffalo Bulletin (July-September 2016) Vol.35 No.3 Original Article Buffalo Bulletin (July-September 2016) Vol.35 No.3

β(1,4)-GALACTOSYLTRANSFERASE-I GENE POLYMORPHISMS IN PAKISTANI NILI RAVI BUFFALO

Aamir Sohail1, Asif Nadeem1,*, Masroor Ellahi Babar2, Tanveer Hussain2, Akhtar Ali2, Wasim Shehzad1 and Maryam Javed1

ABSTRACT INTRODUCTION

β4GalT-I interacts with α-lactalbumin The β 1, 4-galactosyltransferase-I to form the lactose synthase in mammary gland. (β4GalT-I) belongs to a family of enzymes Considering the biological function of the called galactosyl transferases. In the mammalian lactose synthase complex, β4GalT-I gene can be mammary gland, lactose is synthesized from considered as a candidate gene for milk production blood glucose and galactose by a lactose synthase in dairy animals. The bovine β4GalT-I gene, 3,283 enzyme (Strucken et al., 2015). The βeta 1, base pairs in length, is mapped on chromosome 8. 4-galactosyltransferase-I gene (β4GalT-I) interacts The present research work was planned to identify with α-lactalbumin, the calcium binding non- the polymorphism in β4GalT-I gene in Nili Ravi catalytic protein, in the golgi complex of mammary buffalo. Blood Samples were collected, DNA was secretory cells to form the lactose synthase extracted and primers were designed for PCR. complex (Ramakrishnan et al., 2002; Shahbazkia After amplification, PCR products were sequenced et al., 2010; Strucken et al., 2015). This enzyme is for the identification of allelic variation. Thirteen a membrane-bound glycoprotein which is widely polymorphic, six in coding and seven in non- distributed among in mammals, non-mammalian coding region of gene, were identified. This is a vertebrates and also in some plants (Powell and first report toward genetic screening of β4GalT-I Brew, 1974). Considering the biological function gene at molecular level in Nili Ravi buffalo. The of the lactose synthase complex, β4GalT-I gene can present study will provide a better selection to be a potential candidate gene for milk production develop association of identified polymorphisms in dairy animals. It is considered a housekeeping with production traits in buffalo population. gene in the biosynthesis of glycans which occurs in essentially all cell types (Shaper et al., 1998). Keywords: polymorphisms, β4GalT-I Gene, Nili β4GalT-I act as a cell surface component, adhesion Ravi, buffalo, Pakistan and recognition molecule, signal transducer, tumor marker (Berger and Rohrer, 2003) and also involve in angiogenesis, wound healing and collagen deposition in the skin (Shen et al., 2008). However, in spite of its involvement in various physiological

1Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences Lahore, Pakistan, *E-mail: [email protected] 2Virtual University of Pakistan, Lahore, Pakistan

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and biochemical reactions, little is known regarding All primers were amplified by touchdown PCR the presence of polymorphisms in dairy animals and protocol with annealing temperature (62-52°C) any consequent effects on production traits. It is a on Bio-Rad and peQ Lab thermocycler. After membrane-bound glycoprotein widely distributed the precipitation with 70% ethanol in dark, PCR in the mammals, non-mammalian vertebrates and products were sequenced through ABI prism 3100 also in some plants. Two distinct isoforms of the genetic analyzer (Applied Biosystems Inc., Foster β4GalT-I gene product have been reported in the City, CA). Sequencing results were analyzed human, mouse and in cattle. with BioEdit software (http://www.mbio.ncsu. The bovine β4GalT-I gene is 53,283 base edu/bioedit/bioedit.html). Pair wise alignment pairs in length, comprises of six exons and five of sequence was done with the help of blast2 introns and is located on chromosome 8. The sequence. Allelic and genotypic frequencies for the bovine β4GalT-I protein consists of four domains- identified polymorphisms were calculated using the cytoplasmic domain (residues 8-24), the bioinformatics software POPGENE (Yeh et al., transmembrane domain (residues 25-44), the stem 1999). region (residues 45-145) and the catalytic domain (residues 146-402) (Qasba et al., 2008). Thus, it was accepted that the polymorphisms which RESULT are located in the catalytic domain of the protein may affect some of the catalytic properties of the The overall sequence variation across enzyme. This study was aimed to identify the the bovine β4GalT-I locus is high. Thirteen probable polymorphism in the β4GalT-I gene of polymorphic sites were identified by using BLAST Nili Ravi buffalo breed of Pakistan. in local Nili Ravi buffalo breed. Aligned sequence represent that six identified substitutions were in coding region of the gene while seven were MATERIALS AND METHODS positioned in intronic (near to exonic) region of the gene. All these identified polymorphism Blood samples (n=50) of Nili Ravi can be used as strong association markers with buffaloes were collected from Livestock Production economic traits in Nili Ravi buffalo population. Research Institute (LPRI) Bahadarnagar, Okara The reported exonic polymorphisms do not bring and preserved in EDTA (0.5 M) coated falcon amino acid change and appeared as synonymous tubes. Genomic DNA was extracted from blood substitution. However, these sites may be related samples using inorganic method (Sambrook and to detect causative mutation or adjacent QTL. Russel 2001). DNA quantification was carried out The distribution pattern of alleles and genomic using 0.8% Agarose gel. 50 ng/uL of genomic DNA frequencies against each identified polymorphisms was used for the amplification of coding regions are presented in Table 1. However the identified of the β4GalT-I gene. PCR primers were designed polymorphic sites were considered breed specific from GenBank accession no. AC_000165.1 by and might be correlated to milk production and web based software “Primer3” (http://bioinfo. other economic traits. ut.ee/primer3-0.4.0/) (Untergasser et al., 2012).

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DISCUSSION fifty buffaloes, using PCR technique followed by Sanger sequencing. Altogethersixteen polymorphic The β4GalT-I encodes the catalytic part sites were identified across the whole bovine of lactose synthase enzyme which is responsible β4GalT-I locus. for lactose synthesis in the mammary gland. The Two distinct isoforms of the β4GalT-I gene whole coding region of the gene was screened for product have been reported in the mouse (Shaper the presence of allelic variation among a sample of et al., 1998) and similar variants have been

Table 1. Change in nucleotide, genotypic and allelic frequency of all identified polymorphisms in bovine β4GalT-I gene.

Chromosomal Change in SNP ID Genotype Frequency Allele Frequency Position Nucleotide GG AG AA G A GALT1 76203115 G>A 0.5806 0.0646 0.3548 0.5968 0.4032 CC CT TT C T GALT2 76183169 C>T 0.5484 0.3187 0.1329 0.6613 0.3387 CC CT TT C T GALT3 76183187 C>T 0.4516 0.2113 0.3371 0.4861 0.5139 TT TG GG T G GALT4 76183243 T>G 0.5881 0.2791 0.1328 0.8226 0.1774 TT TC CC T C GALT5 76183252 T>C 0.5137 0.3319 0.1544 0.4677 0.5323 TT TA AA T A GALT6 76183296 T>A 0.5741 0.2897 0.1362 0.3548 0.6452 TT TC CC T C GALT7 76183328 T>C 0.6134 0.1844 0.2022 0.4918 0.5082 AA AG GG A G GALT8 76183346 A>G 0.3147 0.2741 0.4112 0.3953 0.6047 TT CT CC T C GALT9 76183447 T>C 0.4918 0.3147 0.1935 0.5173 0.4827 CC CG GG C G GALT10 76180158 C>G 0.5178 0.1928 0.2894 0.5712 0.4288 GG AG AA G A GALT11 76180262 G>A 0.4617 0.2349 0.3034 0.4911 0.5089 CC CT TT C T GALT12 76179615 C>T 0.4143 0.2316 0.3541 0.6197 0.3803 CC CT TT C T GALT13 76179669 C>T 0.5349 0.2199 0.2452 0.5763 0.437

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identified in the bovine (Russo et al., 1990) and β4GalT-I gene single nucleotide polymorphisms human (Mengle-Gaw et al., 1991) homologues. in Nili Ravi buffalo breed of Pakistan. The Both of these isoforms, ‘‘short’’ and ‘‘long’’ are above discussion just explains the potential and differ only by an N-terminal sequence of 13 molecular basis of identified polymorphisms. residues (Shahbazkia et al., 2012). The amount of These SNPs may serve as a potent genetic reserve β4GalT-I enzyme in the lactating mammary gland for the development of molecular markers to assist increases during the lactation period to meet the selection in dairy breeding. demand for lactose synthesis. The most important mechanism by which this increase is ensured is the switch from the REFERENCES long to the short variant, which has the effect of raising β4GalT-I transcript level (Shaper et al., Berger, E.G. and J. Rohrer. 2003. 1998). Thus, allelic variation which affects the Galactosyltransferase-still up and running. transcription start codon probablydisturbs this Biochimie, 85: 261-274. mechanismand prevents the synthesis of higher Mengle-Gaw, L., M.F. McCoy-Haman and levels of lactose. D.C. Tiemeier. 1991. Genomic structure Shahbazkia et al., (2012) reported the exon and expression of human beta-1, 1 T→A (14Lys) transversion alters the second 4-galactosyltransferase. Biochem. Bioph. transcription start site of the codon and may have Res. Co., 176: 1269. impact on the gene expression. However, no such Powell, J.T. and K. Brew. 1974. Glycosyltransferases type of transversion was seen in this study. Other in the Golgi membranes of onion stem. polymorphisms (174 Thr and 220 His) in exon 2 Biochem. J., 142: 203. were identified in the catalytic domain that may Qasba, P.K., B. Ramakrishnan and E. Boeggeman. alter some of the catalytic properties of the enzyme 2008. Structure and function of -1, (Shahbazkia et al., 2012). In another study, Qasba et 4-galactosyltransferase. Curr. Drug al., 2008 reported that the Phe280 residue, together Targest., 9: 292. with Tyr286, Gln288, Tyr289, Phe360 and Ile363, Ramakrishnan, B., E. Boeggeman and P.K. Qasba. were concerned in the relations between β4GalT-I 2002. Beta-1, 4-galactosyltransferase and and α-lactalbumin and may alter this interaction lactose synthase: molecular mechanical and the properties of the lactose synthase complex. devices. Biochem. Bioph. Res. Co., 291(5): In our study all the identified substitutions 1113-1118. GTG> GTA (236Val), AAT> AAC (296Asp), Russo, R.N., N.L. Shaper and J.H. Shaper. 1990. GGA> GGG (302Gly), ATC> ATT (326 Ile), Bovine beta1-4-galactosyltransferase: two GTG> GTA (369Val), and ACG> ACA (387Thr) sets of mRNA transcripts encode two forms were synonymous in nature and located in the of the protein with different amino-terminal catalytic domain of enzyme. However, this Phe280 domains. In vitro translation experiments Tyr substitution was not identified in Nili Ravi demonstrate that both the short and the long buffaloes. forms of the enzyme are type II membrane- To conclude, it was the first screening of bound glycoproteins. J. Biolog. Chem., 265:

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3324. of Alberta, Edmonton. http://www.mbio. Sambrook, J. and D.W. Russell. 2001. Molecular ncsu.edu/bioedit/bioedit.html. Biological Cloning: A Laboratory Manual, 3rd ed. sequence alignment editor. Cold spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA. Shahbazkia, H.R., M. Aminlari and A. Cravador. 2012.Association of polymorphism of the β (1, 4)-galactosyltransferase-Igene with milk production traits in Holsteins. Mol. Biol. Rep., 39: 6715-6721. Shahbazkia, H.R., M. Aminlari, A. Tavasoli, A.R. Mohamadnia and A. Cravador. 2010. Polymorphisms of the β-1, 4 galactosyltransferase-I gene in Holsteins. Livest. Sci., 131: 297-300. Shaper, N.L., M. Charron, N.W. Lo and J.H. Shaper. 1998. 1, 4-Galactosyltransferase and lactose biosynthesis: recruitment of a housekeeping gene from the nonmammalian vertebrate gene pool for a mammary gland specific function. J. Mammary Gland Biol., 3: 315- 324. Shen, A., J. Qian, L. Liu, H. Liu, J. Chen, S. Niu, M. Yan, X. Chen, C. Shen and J. Gu. 2008. The role of [beta]-1, 4-galactosyltransferase-I in the skin wound healing process. Am. J. Dermatopath., 30: 10. Strucken, E.M., Y.C.S.M. Laurenson and G.A. Brockmann. 2015. Go with the flow-biology and genetics of the lactation cycle. Front. Genet., 6: 118. Untergasser, A., I. Cutcutache, T. Koressaar, J. Ye, B.C. Faircloth, M. Remm and S.G. Rozen. 2012. Primer3 - new capabilities and interfaces. Nucleic Acids Res., 40(15): 115. Yeh, F., C. Boyle, T. Rongcai, Z.Y. Ye and J.M. Xian. 1999. POPGENE, Version 1.31. A Microsoft window based free ware for population genetic analysis. University

403 Buffalo Bulletin (July-September 2016) Vol.35 No.3 Original Article Buffalo Bulletin (July-September 2016) Vol.35 No.3

INVESTIGATION OF RESPONSE TO SELECTION FOR MILK TRAITS IN DAIRY BUFFALO OF IRAN BASED ON THREE SALE SITUATIONS

Bahareh Taheri Dezfuli1 and Leonardo De Seno2

ABSTRACT when milk is produced for sale only milk. However, when Sarshir cream or Mozzarella making is added The aim of the present study was to estimate to the production, the selection for components and the index and individual responses to selection for milk volume is economically beneficial. milk, fat percentage and protein percentage for different breeding goals in three buffalo milk sale Keywords: buffalo, Iran, selection index, economic situations in Khuzestan State of Iran characterized weight, Sarshir cream by: 1) selling just milk, 2) selling milk and Sarshir cream and 3) selling milk and Mozzarella. The current payment policy is based exclusively on INTRODUCTION milk volume. Index responses to selection were calculated for three different breeding goals (BG): According to statistical data of Food and 1) milk yield (MY) exclusively; 2) milk yield and Agriculture Organization (FAO) in 2010, there milk fat percentage (MY+FP), 3) milk yield and is 650 thousand head of buffaloes in Iran that is milk fat and protein percentage (MY+FP+PP), 4) fat ranked as 16th country among the 43 countries percentage (FP) exclusively and 5) fat and protein which breed buffaloes (FAO, 2010). There are percentage (FP+PP). Index responses for the milk buffalo in three different regions of Iran: 1) cold sale situation were US$102.85 (BG1), US$103.30 highlands, 2) Mediterranean humid temperate and

(BG2), US$103.52 (BG3), US$5.26 (BG4) and 3) warm lowland. Khuzestan province in warm

US$5.94(BG5). For the Sarshir (local cream of lowland region of Iran is one of the provinces the region) sale situation, index responses were that have buffalo. The suitable climatic conditions

US$143.99 (BG1), US$143.97 (BG2), US$144.12 of Khuzestan for rearing buffalo (existence of

(BG3), US$2.78 (BG4) and US$2.80(BG5) and for important large rivers and ponds and also having a Mozzarella sale situation, index responses were special weather conditions) causes to employment

US$185.13 (BG1), US$185.08 (BG2), US$ 184.59 of more than 5 thousand rural families to buffalo

(BG3) US$4.54 (BG4) and US$9.22(BG5). The rearing (Anonymous, 2008). Relative importance results suggest that for the present circumstances, of buffalo products was the same in different selection for milk components is not advantageous areas of the province and in all regions buffalo are

1Animal Science Research Department, Khuzestan Agricultural and Natural Recourses Research and Education Center, Agriculture Research, Education and Extension Organization (AREEO), Ahwaz, Iran, *E-mail: [email protected] 2Agricultural Sciences Faculty, Grande Dourados Federal University, Dourados, MS, Brazil

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bred primarily for milk production, so that about The aim of this study was to estimate 40 percent of Khuzestan dairy products produced the total and component selection response for from buffalo (Anonymous, 2008). There is not milk production traits (milk yield, fat and protein government payment policy for buffalo milk and percentage) with different selection purposes in all of its milk is sold to the private sector with free current milk sale situations in Khuzestan province pricing system based on its volume. However, in of Iran. some cities, most buffalo owners separate fat from milk with completely traditional method and sell it as Sarshir (a local cream) along with remaining milk MATERIALS AND METHODS (skim) to market. Buffalo milk has special features because of high fat percentage (6 to 7%), and its In this study, the current status of the sale products are of high economic value. Therefore, of milk and fat (Sarshir) was used to calculate increasing production capacity of buffalo and the economic values of milk yield (MY) and fat full use of its genetic potential can be effective percentage (FP). Also, the proposed sale status of in improving buffalo production efficiency and Mozzarella cheese was used to calculate economic economic buffalo breeding, in addition to providing value of protein percentage (PP) beside milk yield a significant portion of needed protein. and fat percentage. Production, demographic, Generally, to achieve maximum genetic economic and management data of 30 buffalo progress, it is necessary to define the target traits herds with average of 30 head of buffalo cows for selecting animals (Groen, 1988; Ponzoni, 1988; were used to estimate the parameters required Bekman and Van Arendonk, 1993; Charfeddine, for the calculation of cost and revenue equations. 2000) and selecting these traits should be such Herds were selected from the main areas of that maximize economic profit of the production buffalo breeding, so that covered different weather system. In dairy animals, milk production and its conditions and the production and sale of milk and components have higher economic importance dairy products, and also different managements. than other traits that with changes in these traits, Management system of the herds in the study was the greatest changes in profitability is obtained. traditional and in all herds, milking is performed Komlosi et al. (2010) in a study of 15 traits of milk twice a day by hand and in the presence of calves production, growth and carcass characteristics of in order to expedite the removal of milk. Also, Hungarian Holstein-Friesian cows, reported milk cows after the morning milking and feeding are production as the most economically important taken to wetlands to bath for 2 to 3 hours.The trait. Seno et al. (2006) also to provide a good aim of the present study was to estimate the index selection program for buffaloes of Sao Paulo state and individual responses to selection for milk, fat in Brazil, studied the response of single trait and percentage and protein percentage for different multi trait breeding purposes determined based on breeding goals for three buffalo milk sale situations milk yield, fat and protein yield. The authors found in Khuzestan state of Iran characterized by: 1) that the high fat and protein percentages in buffalo selling just milk, 2) selling milk and Sarshir and 3) milk provide high economic returns to Brazilian selling milk and Mozzarella. The current payment farmers that produce their own Mozzarella cheese. policy is based exclusively on milk volume. Index

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responses to selection were calculated for three additional fat, the feed cost to produce one percent different breeding goals (BG): 1) milk yield (MY) of additional protein, variable costs include of exclusively; 2) milk yield and milk fat percentage the average cost of transporting, construction and (MY+FP), 3) milk yield and milk fat and protein maintenance, electricity, water, labor, health, and percentage (MY+FP+PP), 4) fat percentage (FP) the average cost of feed for one buffalo herd, and exclusively and 5) fat and protein percentage also, the fixed cost of herd with an average size of (FP+PP). 30 buffalo cows. During collecting data from the Revenues and costs for both present sales herds, measure the amount of feed used by each situations were considered for all herds: 1) sale of buffalo was not possible, feed intake and daily food only milk and 2) separation of milk fat to produce requirements of maintenance, growth, reproduction Sarshir, and sale it and remaining milk along and milk production of buffaloes were extracted with selling buffalo milk and 3) production and using Borghese (2005). sale of Mozzarella cheese substitute of milk and Economic weights of milk traits Sarshir. Sarshir or oily part of milk is produced were calculated in maximized profit breeding from milk fat particles with different sizes which perspective, using following equation: are spread on the surface of the milk by heating TP = TR-TC the milk. We produced Sarshir by 1 kilogram milk with different amount of fat percent. Based on this Where, TP is total annual herd profit per experiment the correlation between fat percentage head of buffalo cow ($), TR is total annual herd of milk and the amount of Sarshir was obtained revenues per head of buffalo cow ($) and TC total 0.72 and the amount of Sarshir increased 11 gram annual herd costs per head of buffalo cow ($). with increasing one percent of milk fat percentage. The current payment policy is based on To calculate the amount of Mozzarella cheese exclusively on milk volume. Milk and Sarshir prices produced (PKM) from one kg milk in the proposed were 0.73 US$/kg and 8.16 US$/kg, respectively. sale of the study, the equation or Mozzarella index Mozzarella cheeses price were considered equal proposed by Altiero et al. (1989) was used: to Sarshir because it was proposed to substitute Sarshir. Exchange rate at the time of calculations PKM - MY x ( 3.5 (%P)+1.23 (%F)-0.88 ), for this paper: 1 US$ = 12260 Rials (March 2013). 100 Economic values (Table 1) were calculated Where, MY is the milk yield and %F for milk yield, fat percentage and protein percentage, and %P are the milk fat and protein percentage, taking into accounts the differences of the three sale respectively. situations. For the calculation of economic values, The significant sources of income for the prices of production components and product considered buffalo herds were sale of milk, were obtained from questionary forms with going Sarshir and remaining low-fat milk (skim), sale of to each buffalo herd (Taheri Dezfuli et al., 2011). animals (calves, heifers, culled cows and sires) and Expected responses were calculated for fertilizer. The annual costs for herds were: the cost five different breeding goals (BG) in each sale of producing one kilogram of milk (food and non- situation: food costs), the feed cost to produce one percent of 1) Milk Yield (MY) exclusively

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2) Milk Yield (MY)+Fat Percentage (FP) breeding goal. After calculating the b values, the 2 2 3) Milk Yield (MY)+Fat Percentage (FP)+Protein index (σ I ) and breeding goal (σ H ) the standard

Percentage (PP) deviation were obtained from: σI = sqrt(b’Pb) and

4) Fat Percentage (FP) σH = sqrt(v’Cv), where C = matrix of (co)variances 5) Fat Percentage (FP)+Protein Percentage (PP) for traits in the breeding goal. The correlation

between the index and the breeding goal (rIH) was

given by σI/σH. Index responses to selection were calculated based on the selection index theory Index responses were then obtained from

(Hazel, 1943). Solutions for weighting factors (b) R = i × σI = i × rIH × σH, where i is the selection -1 were calculated as b = var (X) cov (X,A). X is intensity, σI is the standard deviation of the index, a vector with the information sources, var (X) is rIH is the correlation between the index and the a matrix with (co)variances between these sources breeding goal and σH is the standard deviation of and cov (X,A) is a vector of covariances between the breeding goal. each information source and the true genotype. The Individual responses to selection were also index equations are then Pb = Gv or b = P-1Gv. calculated for the studied traits. The average of P is used to describe the matrix with variances genetic and phenotypic parameters of traits used and covariances between the information sources in response calculations are presented in Table 2 in X. It is a variance-covariance matrix between (Taheri et al., 2012; Da-you et al., 2008; Rosati and the means of phenotypic observations. G is the Van Vleck, 2002; Thevamanoharan et al., 2000; matrix with covariances between X and A. Finally, Seno et al., 2010; Castillo et al., 2001; Tonhati et v is a vector with economic values of traits in the al., 2000; Tonhati et al., 2004; Aspilcueta-Borquis

Table 1. Economic values (EV), expressed in US$ for milk (MY), fat (FP) and protein percentage (PP) for Milk, Milk and Sarshir, and Milk and Mozzarella sale situations.

Sale Situation EVMY EVFP EVPP Milk 0.40 -15.92 -14.91 Milk and Sarshir 0.56 8.43 -12.60 Milk and Mozzarella 0.72 13.73 61.01

Table 2. Standard deviations (σp), heritabilities (bold), genetic (above diagonal), and phenotypic correlation coefficients (below diagonal) for milk yield (MY), fat (FP) and protein (PP) percentages.

Sale Situation σp MY FP PP MY 635.4 0.22 -0.08 -0.12 FP 0.93 -0.19 0.18 0.31 PP 0.32 -0.20 0.48 0.18

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et al., 2010). Progeny test was considered to select the production of Parmesan cheese, where protein best buffalo bulls with an average progeny size is of high value and payment system suggested by of 50 daughters per sire. Selection intensity was Badino dealing for the 1985 price and production assumed equal to one. circumstances for an optimal cheese production Relative selection efficiencies (RSE) were (Rozzi, 1989). The results showed that Parmesan expressed as a percentage of the expected responses payment system resulted in the highest marginal to selection obtained for BG1 with each selection value of milk with average composition in index, as follows: situations without herd output limitation. The selection indexes, calculated for

RSE2= (BG2/BG1) × 100, and the three studied sale situations and different

RSE3= (BG3/BG1) × 100. breeding goals are presented in Table 4. XM, XF

RSE4= (BG4/BG1) × 100. and XP refer to the average performance of sires’

RSE5= (BG5/BG1) × 100. daughters for milk yield and milk fat and protein percentage, expressed as deviations from the average performance of all females. XF and XP RESULTS AND DISCUSSION were standardized for the b value obtained for milk yield. The weighting factors for milk yield, The index and individual responses to fat percentage and protein percentage, standard selection, calculated for each breeding goal in the deviations of indexes and of breeding goals, three sale situations (Milk, Milk and Sarshir, Milk correlations between selection indexes and and Mozzarella), are presented in Table 5. breeding goals, according to the breeding goals, are In the case of milk sale situation, when presented in Table 3, for the three sale situations. breeding goal was only milk, the individual In general, the weighting factors of response for milk yield was 257.12 kg. For breeding traits showed magnitude and signals that were in goal 2 and 3, when we select with two and three- agreement with the breeding goals and economic trait indexes, the individual response for milk yield values of traits in the breeding goals. In the approximately decreased (256.96 and 256.91 kg, research of Seno et al., (2005), taking into account respectively), but decreasing amount is negligible. the differences of the two production systems About breeding goal BG2 and BG3, individual (milk and Mozzarella systems), economic values responses for fat and protein percentage were were calculated for MY, FY and PY as 0.26, -0.27 negative. In the fourth and fifth breeding goals, and -0.30 for milk production system and 1.12, as the results show due to the negative economic 6.90 and 20.14 in Mozzarella production system, weights for both fat and protein percent, selection respectively. Also, Pieters et al., (1997) calculated based on the attributes of the characters and for economic values of milk traits for three different these traits, reduced fat and protein percentage Italian payment systems. The first system is based (-0.3 and -0.06 %). on a payment system for regions, where milk is Regarding the milk sale situation, the produced for direct consumption or fresh dairy greatest economic response to the index was products, second system is based on using milk for observed for BG3 (US$103.52), when three studied

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traits were in the breeding goal, while the lowest advantage between two and three-trait indexes for index response was observed for BG1 (US$102.85) fat percentage response (-0.01 and -0.01%). when selection was only on the milk yield. This The expected index responses, in the milk was expected, taking into account the current and Sarshir sale situation, were US$143.99 (BG1), milk payment policy with no differential payment US$143.97 (BG2) and US$144.12 (BG3). Despite for milk components in this region and negative the positive economic weigh for fat percentage, correlations between fat and protein percentage and the individual responses to selection for FP were milk yield. However, the expected index response negative and similar for BG2 and BG3. It seems for breeding goal BG3 (US$103.52) was the same that negative correlation between fat percentage as BG2 (US$103.30) and both of them were close and milk yield and also greater response for milk to BG1 (US$102.85) and the differences were low. yield cause to decrease fat percent. In the fourth The relative efficiencies of selection were and fifth breeding goals, as the results show, 100.43 and 100.63% for BG2 and BG3, respectively, selection based on and for fat percent, increased fat indicating that the selection exclusively on each percentage as 0.3%, but the response for protein selection index (BG1, BG2, and BG3) would be percentage is negative. It was expected because in efficient. But this parameter was calculated as this case the protein percentage is not important. 5.11 and 5.77% for BG4 and BG5, respectively, The relative efficiency of selection for BG2 and indicating that selection exclusively on fat percent BG3 was 100% (99.98 and 100.09%, respectively). of fat and protein percent would not be efficient in This parameter was calculated as 1.93 and 1.94% this case. Totally, these results suggest that under for BG4 and BG5, respectively, indicating that the current payment policy, it is not desirable to selection exclusively on fat percent of fat and protein select on fat and protein percentage, given that the percent would not be efficient. So, comparing these revenues are based exclusively on milk sale. So, results suggest that with selling of Sarshir besides selection for milk is the easiest index and more the milk, selection on milk and fat percentage can suitable for milk sale situation. be desirable as easiest selection index. In the case of milk and Sarshir sale In the case of milk and Mozzarella sale situation, when breeding goal was only milk, the situation the individual response for milk was individual response for milk was approximately the also approximately the same for all three breeding same for three first breeding goals (257.10, 257.20, goals with increasing the number of traits in index 257.30 kg). The economic responses were also the (257.12, 257.30, 257.00 kg) with insignificant same for these three breeding goals. For BG2, with differences. The economic responses were also increasing the number of traits in the index, the the same for indexes of MY and MY & FP, in this individual result for milk did not change basically, sale situation. For BG2 with two-trait index, with but with adding fat and protein percentage in this adding fat and protein percentage in the index, index, the individual response for fat percentage the individual response for fat percentage was was improved (from -0.03 to -0.01%). In breeding improved (from -0.03 to -0.01%). The economic goal 3, the three-trait indexes (257.30 kg) were response also had slight increasing for this breeding superior to selection on milk and fat percentage goal. In breeding goal 3, selection on milk and fat for milk response. But there were not observed any percentage or milk, fat and protein percentage

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(-0.004 and -0.005%) were superior to selection Sarshir) show the importance of implementing index on only milk. For protein percentage also appropriate selection indexes for buffalo dairy there were not observed any advantage between herds taking into account the local production two and three traits (-0.01 and -0.01%). In this and market circumstances, with special reference case, the economic response increased for BG3 to regions where the Sarshir represents the main with adding traits in selection index. product of the buffalo dairy activity. Also, the In the milk and Mozzarella sale situations, incorporation of the Mozzarella-making process the expected index responses were US$185.13 on the farm level (as proposed sale situation) (BG1), US$185.08 (BG2) and US$184.59 (BG3). resulted in positive economic values for FP and PP, The individual responses to selection for FP and and consequently improved individual responses PP were negative but less than those in milk and for these traits. Also, at the individual responses

Sarshir sale situation. In this case, the relative obtained for BG3, it is observed that the individual efficiency of selection for BG2 and BG3 was responses for FP and PP were around 40 and 50%,

100%, too (99.97 and 99.70%, respectively). This respectively, superior to those of BG2. The results parameter was calculated as 2.45 and 4.98% for also suggest that the additional payment for fat BG4 and BG5, respectively. The greatest selection and protein percentage could benefit not only milk response in these two breeding goals was obtained producers, but also the industry. in milk and Mozzarella sale situation. Seno et al., (2006) in study of the index and In spite of the economic importance of individual responses to selection for milk (MY), fat milk components (fat and protein percentage) (FY) and protein (PY) yields for different breeding in the production of Mozzarella cheese, in this goals for two commercial buffalo milk production situation the results showed that the greatest systems in São Paulo State (1. all milk produced is response was observed when the breeding goal sold to the industry and 2. all milk produced is used included just the milk yield. But, in the production in the Mozzarella cheese-making process at the of Sarshir, the results showed that the responses farm), suggest that when milk is produced for sale were approximately the same for three breeding to the industry, selection for milk components is goals. Comparing the individual responses to not advantageous and when Mozzarella making is selection for FP and PP obtained for the milk and added to the system, the selection for components Mozzarella and milk and Sarshir sale situations, in and milk volume is the most economically milk sale situation if selection were based on these beneficial. two traits (BG2 and BG3), negative responses were obtained because of negative economic values. In two other situations, these individual responses also CONCLUSION were obtained negative because of their negative correlation with milk yield but its magnitude has The results obtained in the present study decreased from milk sale situation. suggest that for the milk sale situation, selection for The differences in both index and milk components is not advantageous. However, individual responses for milk production traits when the sale situation shifted to milk and Sarshir between the two sale situation (milk and, milk and or the manufacturing of Mozzarella cheese is

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Table 3. Weighting factors (b) for milk Yield (MY), fat (FP) and protein (PP) percentage, standard deviations

of indexes (σI) and of breeding goals (σH), correlations between selection indexes and breeding

goals (rIH), according to the breeding goal (BG), for the three sale situations. B Sale Situation BG σ σ r MY FP PP I H IH 1 0.5954 - - 102.85 119.22 0.86270 2 0.5968 -7.306 - 103.30 119.89 0.86164 Milk 3 0.5979 -9.939 3.905 103.52 120.18 0.86138 4 - -22.35 - 5.26 6.28 0.83787 5 - -22.41 -17.43 5.94 7.17 0.82773 1 0.8336 - - 143.99 166.91 0.86270 2 0.8373 33.05 - 143.97 166.67 0.86377 Milk & Sarshir 3 0.8385 30 10.06 144.12 166.88 0.86366 4 - 11.84 - 2.78 3.33 0.83787 5 - 12.47 -20.19 2.80 3.23 0.86399 1 1.072 - - 185.13 214.59 0.86270 2 1.077 46.56 - 185.08 214.23 0.86392 Milk & 3 1.079 40.5 123.3 184.59 213.46 0.86475 Mozzarella 4 - 19.28 - 4.54 5.42 0.83787 5 - 17.9 84.14 9.22 11.21 0.82258

Table 4. Selection indexes standardized for the b value obtained for milk yield (MY), for the three sale situations (Milk, Milk and Sarshir cream and Milk & Mozzarella) and different breeding goals.

Sale Situation BG Index b value(MY)

1 1 (Xmy) 0.5944

2 1 (Xmy) - 12.24 (Xfp) 0.5968

Milk 3 1 (Xmy) - 16.62 (Xfp) + 6.53 (Xpp) 0.5979

4 - 22.35 (Xfp) -

5 - 22.41 (Xfp) - 17.43 (Xpp) -

1 1 (Xmy) 0.8330

2 1 (Xmy) + 39.47 (Xfp) 0.8373

Milk and Sarshir 3 1 (Xmy) + 35.78 (Xfp) + 12 (Xpp) 0.8385

4 11.48 (Xfp) -

5 12.47 (Xfp) – 20.19 (Xpp) -

1 1 (Xmy) 1.072 2 1 (X ) + 43.23 (X ) 1.077 Milk and my fp 3 1 (X ) + 37.53 (X ) + 114.27 (X ) 1.079 Mozzarella my fp pp 4 19.28 (Xfp) -

5 17.9 (Xfp) + 84.14 (Xpp) -

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Table 5. Individual* and expected index responses to selection for Milk, Milk and Sarshir, and Milk and Mozzarella sale situations. Individual Response Index Sale Situation BG MY (kg) FP (%) PP (%) Response ($) 1 257.10 - - 102.85 2 256.90 -0.03 - 103.30 Milk 3 256.90 -0.03 -0.01 103.52 4 - -0.3 - 5.26 5 - -0.3 -0.06 5.94 1 257.10 - - 143.99 2 257.20 -0.01 - 143.97 Milk and Sarshir 3 257.30 -0.01 -0.01 144.12 4 - 0.3 - 2.78 5 - 0.3 -0.03 2.80 1 257.12 - - 185.13 2 257.30 -0.01 - 185.08 Milk and 3 257.00 -0.005 -0.01 184.59 Mozzarella 4 - 0.3 - 4.54 5 - 0.23 0.10 9.22

*For milk yield (MY), fat percentage (FP) and protein percentage (PP).

adopted, selection for components and milk Anynoumus. 2008. Buffalo breeding in Khuzestan, volume is the most beneficial from decreasing fat p. 23-20. Publications committee of the and protein percentage. The differences in index agriculture promotion and exploitation of responses for milk production traits between the the Khuzestan province. three sale situations (Milk, Milk and Sarshir, Milk Aspilcueta-Borquis, R.R., R.C. Sesana, M.H.M. and Mozzarella) suggest that it is necessary to Berrocal, L.D.O. Seno, A.B. Bignardi, take into account the local production and market L.El Faro, L.G. de Albuquerque, G.M.F. circumstances, when designing breeding programs de Camargo and H. Tonhati. 2010. Genetic for buffalo milk production situations in Iran. parameters for milk, fat and protein yields in Murrah buffaloes (Bubalus bubalis Artiodactyla, Bovidae). Genet. Mol. Biol., REFERENCES 33(1): 71-77. Bekman, H. and J.A.M. Van Arendonk. 1993. Altiero, V., L. Moio and F. Addeo. 1989. Previsione Derivation of economic values for veal, della resa in Mozzarella sulla base del beef and milk production traits using profit contenuto in grasso e proteine del latte di equations. Livest. Prod. Sci., 34: 35-56. bufala. Sci. Tecn. Latt. Cas., 40: 425-433. Borghese, A. 2005. Buffalo Production and

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Research. REU Technical Series 67. FAO J.A.M. Van Arendonk. 1997. Consequences Regional Office for Europe: 316p. of differences in pricing systems between Catillo, G., B. Moioli and F. Napolitano. 2001. regions on economic values and revenues Estimation of Genetic parameters of some of a national dairy cattle breeding scheme productive and reproductive traits in Italian in Italy. Livest. Prod. Sci., 49: 23-32. buffalo. Genetic evaluation with BLUP- Ponzoni, R.W. 1988. The derivation of economic Animal model. Asian Austural. J. Anim., values combining income and expense in 14(6): 747-753. different ways: an example with Australian Charfeddine, N. 2000. Economic aspects of defining Merino sheep. J. Anim. Breed. Genet., 105: breeding objectives in selection programmes. 143-153. CIHEAM, Options Méditerranéennes, Rosati, A. and L.D. Van Vleck. 2002. Estimation Seminaires Mediterraneens: Série A, 43: of genetic parameters for milk, fat, protein 9-17. and Mozzarella cheese production in Italian Da-you, F.A.N., X.U. Shang-zhong, L.I. Jun- river buffalo population. Livest. Prod. Sci., ya, R.E.N. Hong-yan and Y.A.N.G. Xue- 74: 185-190. li. 2008. Genetic and statistical analysis Rozzi, P. 1989. Indici economici adottati dall’Anafi between production traits and secondary nella se lezione; da1 IX Congresso ANAFI. traits in Chinese Semintal. Acta Vet. et Bianco Nero giugno 1989, 6: 23-27. Zoote. Sin., 39(8): 1025-1032. Seno, L.O. 2005. Valores econômicos para as Dezfuli, B.T., A.N. Javaremi, M.A. Abbasi, J. características de produção de leite de Fayazi and M. Chamani. 2011. Economic búfalos (Bubalus bubalis) no Estado de São Weights of Milk Production Traits for Paulo. Dissertação (Mestrado em Genética Buffalo Herds in the Southwest of Iran e Melhoramento Animal), Faculdade Using Profit Equation. World Applied Estadual Paulista, Jaboticabal. Sciences Journal, 15(11): 1604-1613. Seno, L.O., V.L. Cardoso and H. Tonhati. 2006. FAO STAT. 2010. Statistical Division, Food and Responses to selection for milk traits in Agriculture Organization f the United dairy buffaloes. Genet. Mol. Res., 5(4): 790- Nations. Available on: http://faostat.fao.org. 796. Groen, A.F. 1988. Derivation of economic values Seno, L.O., V.L. Cardoso, L. El Faro, R.C. in cattle breeding. A model at farm level. Sesana, R.R. Aspilcueta-Borquis, G.M.F. Agr. Syst., 27: 195-213. de Camargo and H. Tonhati. 2010. Genetic Hazel, L.N. 1943. The genetic basis for constructing parameters for milk yield, age at first calving selection indexes. Genetics, 28: 476-490. and interval between first and second Komlósi, M., J. Wolfová and B. Wolf. 2010. calving in milk Murrah buffaloes. Livestock Economic weights of production and Research for Rural Development, 22(2). functional traits for Holstein-Friesian cattle Taheri, D.B., A.N. Javaremi, M.A. Abbasi, J. in Hungary, J. Anim. Breed. Genet., 127(2): Fayazi and M. Chamani. 2012. Performance 143-153. study and estimating genetic parameters Pieters, T., F. Canavesi, M. Cassandro, E. Dadati and of production and reproduction traits of

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Khuzestani buffaloes. Iran J. Vet. Res., 8(3): 45-53. Thevamanoharan, K., W. Vandepitte, G. Mohiuddin and M. Shafique. 2000. Genetic, phenotypic and residual correlation between various performance traits of Nili-Ravi buffaloes. Buffalo Bull., 19: 80-86. Tonhati, H., M.F.C. Muñoz, J.Á. Oliveira, J.M.C. Duarte, T.P. Furtado and S.P. Tseimazides. 2000. Parâmetros genéticos para a produção de leite. Gordura e proteína em bubalinos. R. Bras. Zootec, 29: 2051-2056. Tonhati, H., M.F.C. Muñoz, J.M.C. Duarte, R.H. Reichert, J.Á. Oliveira and A.L.F. Lima. 2004. Estimates of correction factors for lactation length and genetic parameters for milk yield in buffaloes. Arq. Brasileiro de Med. Vet. e Zootec., 56(2): 251-257.

415 Buffalo Bulletin (July-September 2016) Vol.35 No.3 Original Article Buffalo Bulletin (July-September 2016) Vol.35 No.3

NUTRITIONAL STATUS AND HEMATO-BIOCHEMICAL PROFILE OF ANOESTRUS BUFFALOES OF MALWA REGION OF MADHYA PRADESH

Nagendra Patil, R.K. Jain and Dharmesh Tewari*

ABSTRACT 8.13±0.38 and 9.59±0.25 mg/dl; Inorganic-P (iP) 5.06±0.14 and 5.07±0.17 mg/dl; Total-P 24±2.21 In Malwa region of Madhya Pradesh and 17.50±1.42 mg/dl, respectively. Trace mineral reproductive failure (anoestrus) is a major problem levels in heifers and lactating buffaloes were Fe in buffaloes under field conditions due to under 22.05±0.58 and 17.68±0.84ppm; Zn 1.04±0.05 and feeding and non availability of balanced ration. To 0.87±0.05 ppm; Cu 0.90±0.05 and 0.77±0.04 ppm; find out the nutritional causes behind anoestrus, Mn 0.84±0.05 and 1.14±0.10 ppm; Co 1.92±0.41 thirty anoestrus buffaloes {10 heifers (average and 1.13±0.09 ppm, respectively. body wt. 262.80±22.51 Kg)+20 buffaloes (average Results indicated that values of Hb, body wt. 461±10.83 Kg, milk yield 7.62±0.48 litre/ blood glucose, total protein, Ca, iP, Zn were h/d)} were selected randomly that have normal marginally low but Cu was deficient in heifers, genitalia from ten villages of Indore district. while levels of iP, Cu and Zn were marginally low Average daily feed intake of each animal was in lactating buffaloes. Fe levels were found high recorded and proximate principles, major elements in both heifers and lactating buffaloes. It may be like Calcium (Ca) and Phosphorus (P) and trace concluded that dietary deficiency reflected the elements like iron (Fe), zinc (Zn), manganese (Mn), hemato-biochemical profile of anoestrus buffaloes copper (Cu) and cobalt (Co) in available feedstuffs and a strategic supplementation is needed to these were determined to find out nutrient availability. animals for exploitation of their genetic potential Deficiency of various nutrients was calculated by for optimum production and reproduction. comparing with the standard requirements of the animals. Blood samples were also collected from Keywords: nutritional status, blood profile, the animals and analyzed for different haemato- anoestrus, buffalo, Malwa, Madhya Pradesh biochemical constituents. The average values for different blood parameters in heifers and lactating buffaloes were hemoglobin (Hb) 10.32±0.19 and INTRODUCTION 11.15±0.32 g/dl; blood glucose, 50.62±1.80 and 59.29±1.05 mg/dl; plasma protein 5.06±0.19 Reproductive failure of dairy animals is and 6.33±0.18 g/dl, respectively. Major mineral the major area of concern now days in all over the profile in heifers and lactating buffaloes were Ca, country, which causes a huge economical loss to the

Department of Animal Nutrition, College of Veterinary Science and Animal Husbandry, Narendra Deva University of Agriculture Technology, Kumarganj, Faizabad, Uttar Pradesh, India, *E-mail: [email protected]

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dairy owners. Among the various factors affecting MATERIALS AND METHODS it, nutrition is one of the most important factors which receive less attention than what actually it Thirty buffaloes {10 heifers (average body should be. For normal development and activity of wt. 262.80±22.51 Kg)+20 buffaloes (average body reproductive organs, feeding of balanced ration is wt. 461±10.83 Kg, milk yield 7.62±0.48 litre/h/d)} of utmost importance, because most field cases of with normal genitalia (by per rectal examination) reduced fertility or sterility are of nutritional origin. and without any clinical infection, showing The interaction between nutrition and reproduction anoestrus were selected randomly from 10 villages needs particular attention in our country, to (Borkhedi, Harsola, Kevti, Piplihamalhar, Umaria, overcome nutritional inadequacies either in terms Panda, Rau, Rangwasa, Sonvay and Bhaslai) around of quantitative or qualitative nutrient deficiencies/ Veterinary College, Mhow of Indore District (from imbalances. Malwa region of Madhya Pradesh). Body weights The nutritional deficiency causes several (Kg) of the animals were determined by recording infertility conditions in buffaloes and the highest the length (inch) and girth (inch) of each animal (50.26%) prevalence was observed for anoestrus, and then putting them in Shaeffer’s formula (Sastry while cases of repeat breeder, metritis, pyometra et al., 1982). Feed offered and residues left of and prolapse were only about (25.69%) among each animal were weighed with the help of spring the common field cases Malwa region of Madhya balance at both times (morning and evening) for Pradesh (Shukla et al., 2007). Thus anoestrus three consecutive days. Then average feed intake remains a major condition which constitutes about of each animal was calculated. The representative half of the reproductive problems occurring in samples of each feed were subjected to proximate buffaloes. analysis (AOAC, 1995), Ca and P (Talpatra et al., In Malwa region of Madhya Pradesh 1940) content and trace mineral estimations by most of the farmers rear crossbred cattle and Atomic Absorption Spectrophotometer. buffaloes as the dairy animal and anoestrus is Milk yield (litre) of each lactating animal the major reproductive problem among dairy was measured during milking (morning and evening) animals. Farmers are following traditional feeding for three consecutive days. After that average milk practices, usually cotton seed cake is the only yield was calculated. Availability of DM, DCP, concentrate source fed to lactating cattle along TDN, major (Ca and P) and trace elements (Fe, Cu, with straw (wheat/ masoor/ gram/ soybean) and Mn, Zn and Co) for each animal was calculated mineral supplementation is a rare inhabitant among on the basis of chemical composition of feedstuffs the farmers (Mudgal et al., 2003). Other factors and their intake. Selenium, carotene and vitamin associated with anestrous are energy deficiency E intake were worked out using values given in (Ling et al., 2007) and minerals also play important the literature. Finally, the nutrient availability of role in the regulation of hormones and enzymes individual animal was compared with the standard for initiation of estrus (Dhoble and Gupta, 1986). nutrient requirements calculated out for specific This nutritional deficiency also reflects the blood body weight and productivity of individual animal profile. So that blood profile of anoestrus buffaloes with the help of feeding standards (Kearl, 1982) of Malwa region was evaluated in this experiment. and thus the deficiencies/ excess to specific nutrient

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was worked out. been reported by Mudgal et al. (2003) and Tiwary et Blood samples of around 15 to 20 ml were al., (2007). Between the groups deficiency of major collected from all 30 anoestrus buffaloes (heifer nutrients was observed in heifers for digestible and lactating) from jugular vein in a sterilized crude protein which was only about 48% to that of plastic tubes containing anti-coagulant heparin the requirements, while limited deficiency (11%) solution (0.2 mg/ml of blood). was seen in lactating buffaloes and which may be The tubes containing blood samples associated with the supply of concentrate to the were kept in ice and brought to the laboratory. In lactating buffaloes. Similar observations were also laboratory blood was analyzed for hemoglobin recorded by earlier workers (Sinha, 1982; Sohal et (Oser, 1979), glucose (Folin and Wu, 1920) and al., 1982; Mudgal et al., 2003 and Tiwary et al., total phosphorus (AOAC, 1995) and remaining 2007). The lower levels of energy and /or protein blood was centrifuged for separating the plasma. may be associated with the ovarian inactivity and The plasma sample then stored in glass vials for anoestrus (Wiltbank et al., 1965) as negative energy analysis of protein (Teitz, 1986), Ca (Ichaylova and balance depresses the ovarian activity by inhibiting Iikova, 1971), inorganic P (Fiske and Subbarow, pulsatile LH release (Butler and Smith, 1989). 1925) and trace minerals (Fe, Zn, Cu, Mn and Co) When the major minerals were compared using atomic absorption spectrophotometer [Perkin it showed that Calcium was the element supplied Elmer Aanalyst 100, USA] after wet digestion. in excess (33 to 283%) to the requirement and which may be due to supply of higher amounts of leguminous straws in their ration. This reduces the RESULTS AND DISCUSSION availability of phosphorus on one hand and over supply the calcium on other hand as leguminous The chemical composition of specific straws having a wider Ca: P ratio and having a fair feed ingredients being consumed by the animals deficiency of element phosphorus. Phosphorus is has been presented in Table 1. The feeds being necessary for normal energy and phospholipids offered to the heifers were mainly the agricultural metabolism as well as normal skeletal development by products including wheat straw, gram straw, and its severe deficiency may delay the onset of masoor straw and soybean straw, while wheat bran, puberty and postpartum anoestrus and increased cotton seed cake or at some places concentrate incidence of cystic follicles because of inactive mixtures were also being used additionally in the ovaries, whereas moderate and low conception ration of lactating buffaloes. rates (Pugh et al., 1985). Many other workers also In Table 2, the availability of different have found lower levels of inorganic phosphorus nutrients to the animals was worked out and in serum of anoestrus heifers /buffaloes than cyclic compared with the standard requirement and animals (Naidu and Rao, 1982; Kumar et al., 1992 hence the excess or deficiency of specific nutrient and Dutta et al., 2001). has been presented. As compared to the standard Among trace elements the supply of iron requirements (Kearl, 1982), availability of DM was and cobalt remained on plus side, while of zinc and about 4-6% less, which may be associated with the copper remained deficient, while Mn was deficient deficiency of TDN too. Similar findings was also only in buffaloes but not in heifers. The presence

419 Buffalo Bulletin (July-September 2016) Vol.35 No.3 mixture 2.66±0.32 0.86±0.05 3.50±0.18 6.08±1.48 0.32±0.05 0.17±0.02 16.16±2.70 15.19±1.36 49.29±2.51 13.98±1.79 28.83±2.49 18.82±1.37 Concentrate 258.45±5.94 Cotton 0.55±0.05 4.25±0.22 0.18±0.02 0.22±0.01 0.51±0.02 Seed cake 22.60±0.42 10.12±0.80 10.22±0.64 27.15±1.72 35.75±1.15 41.64±3.03 15.80±0.56 275.24±16.96 3.45±0.11 0.69±0.17 9.99±0.92 4.51±0.74 0.21±0.01 0.48±0.10 0.61±0.05 11.68±1.16 13.99±0.52 68.71±1.63 71.37±3.22 63.40±8.19 Wheat bran 298.51±15.23 Straw Soybean 0.18±0.02 6.14±0.34 6.59±0.38 0.80±0.06 0.94±0.04 0.69±0.06 41.77±1.48 45.52±1.74 67.63±2.38 10.01±0.70 26.43±1.37 0.24±0.015 461.99±62.70 straw Masoor Masoor 1.50±0.11 0.71±0.06 6.52±0.32 8.84±0.30 4.86±0.41 1.46±0.07 4.17±0.23 0.05±0.00 36.91±0.82 46.21±0.87 87.07±7.35 23.24±1.92 605.28±8.05 straw Gram 0.72±0.06 6.24±0.24 0.63±0.50 7.98±0.34 4.36±0.29 8.41±0.28 1.54±0.08 2.44±0.15 0.04±0.00 39.16±0.74 45.95±1.20 15.68±0.85 364.14±12.8 0.16±0.03 3.95±0.22 0.99±0.06 7.91±0.36 0.23±0.02 0.06±0.01 5.84±0.16 11.64±0.64 33.08±0.69 50.31±0.66 39.81±1.18 13.48±1.12 414.76±6.47 Wheat straw P (%) P Ca (%) CP (%) CP TA (%) TA EE (%) CF (%) Zn(ppm) NFE (%) Fe (ppm) Co (ppm) Cu (ppm) Mn (ppm) Feedstuffs AIA (%) AIA Table 1. Macro and micro nutrient contents of feedstuffs (DM basis). 1. Macro and micro nutrient contents of feedstuffs Table

420 Buffalo Bulletin (July-September 2016) Vol.35 No.3 (%) 6 (-) 11 (-) 11 31 (-) 39 (-) 17 (-) 17 (-) 24 (-) 46 (-) 33 (+) 286 (+) 534 (+) 290 (+) /Excess Adequate Deficiency 2.4 6.06±0.26 4.68±0.38 11.33±0.47 57.96±4.98 24.41±2.15 91.40±4.09 61.93±6.39 Body weight 294.24±21.01 396.24±24.83 684.69±39.97 461±10.83 kg 11418.95±0.20 3801.73±239.87 Availability For For Availability Buffaloes 15 12 600 480 120 480 772 35.8 7.28 46.4 1.20 21000 1.2-3.6 production (7% fat) production Body Wt and 8 lits /day Body Requirements for 500 kg for Requirements (%) 4 (-) 20(-) 38 (-) 32 (-) 34 (-) 26 (-) 52 (-) 52 (-) 48 (+) 828 (+) 283 (+) 315 (+) / Excess Adequate Deficiency Body wt. 9.49±1.54 8.16±1.28 2.83±0.21 2.45±0.51 0.78±0.06 5.75±0.43 57.38±8.92 44.61±3.22 Heifers 5717.81±45 118.24±13.74 353.96±38.74 179.32±21.80 262.80± 22.51 Availability for for Availability 2779.56±252.64 15 12 15 179 374 3.55 59.9 0.59 5.99 299.5 239.6 12000 0.6-1.8 Body wt. for 300 kg for Requirements Requirements P (g) P Ca (g) Fe (mg) Se (mg) Zn (mg) DCP (g) DCP Cu (mg) Co (mg) Mn (mg) TDN Kg) Vit E (IU) Vit Vit A (IU) A Vit DMI (Kg) Parameters Table 2. Daily requirements and availability of nutrients in anoestrus buffaloes. Table

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of zinc is highly essential for certain enzymatic animals. activities related to reproduction and indirectly it The average values of blood Hemoglobin, may act through the pituitary to influence the release glucose and total phosphorus and plasma levels of of gonadotropic hormones or directly through Protein and different major and micro elements complexing with specific legend in gonads (Miller, of the anoestrus buffalo heifers and lactating 1979). Deficiency of copper may also be reflected buffaloes are shown in Table 3. It was observed on reproductive behavior as well as performance that Hb contents in anoestrus heifers (10.32±0.19 of animals. Inactive ovaries, delayed oestrus and g/dl) and buffaloes (11.15±0.32 g/dl) were lower early embryonic death have been reported to occur than the normal value (13.14±0.06 g/dl) as reported due to deficiency of copper (Hidiroglou, 1979 and by Das et al. (2003). It may be due to deficient Singh and Vadnere, 1987). levels of copper. Similar lower values (8.79-10.24 Due to practice of least greens supply in g/dl) were also observed by other workers (Sharma farmers vitamin A remained the most deficient et al., 1983 and Perumal et al., 2007). The average among the animals and vitamin A is very important values of blood glucose and total protein were for maintaining the health status of epithelial also found lower in anoestrus lactating buffaloes tissue of the reproductive tract. The deficiency of and in heifers compared to the values of healthy vitamin E was only observed in heifers and which animals (Mandal et al., 2002 and Nayyar et al., indicates the lacking of concentrate in their ration, 2003). Similar findings were reported by different but not in lactating buffaloes. The negative impact workers (Sharma et al., 1983; Tandle et al., 1998; of insufficient vitamin E was also been observed Jani et al., 2001; Sharma et al., 2004; Singh and on ovulation rates (Harrison et al., 1984) and Singh, 2005; Singh and Singh, 2006, Perumal et postpartum activities (Arechiga et al., 1994) of the al., 2007). In contrast to above findings Giri and

Table 3. Hemato-biochemical profile* of anoestrus buffaloes. Parameters Heifers Buffaloes Hemoglobin (g/dl) 10.32±0.19 11.15±0.32 Glucose (mg/dl) 50.62±1.80 59.29±1.05 Total Phosphorus (mg/dl) 24±2.21 17.50±1.42 Total Protein (g/dl) 5.06±0.19 6.33±0.18 Calcium (mg/dl) 8.13±0.38 9.59±0.25 Inorganic Phosphorus (mg/dl) 5.06±0.14 5.07±0.17 Iron (ppm) 22.05±0.58 17.68±0.84 Zinc (ppm) 1.04±0.05 0.87±0.05 Copper (ppm) 0.90±0.05 0.77±0.04 Manganese (ppm) 0.84±0.05 1.14±0.10 Cobalt (ppm) 1.92±0.41 1.13±0.09

* The values of hemoglobin, Glucose and Total Phosphorus were reported in blood, while others in plasma.

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Yadav (2001) and Jagathesan et al. (2006) reported Nutritional approaches to prevent that Hemoglobin, glucose and total protein values reproductive disorders in dairy animals. were in normal range in anoestrus animals and In Sharma, K. 2007. Lead paper in which may be associated with change in feeding 23rd Annual convention of ISSAR and practices of those animals. National Symposium on Challenges in The plasma concentration of total P, Fe, Improving Reproductive Efficiency of Mn and co were within range in heifers (McDowell Farm and Pet Animals, Orissa University of et al., 1984) but Ca (8.13±0.38 mg/dl), inorganic Agriculture and Technology-Bhubaneswar. phosphorus (5.06±0.14 mg/dl) and Zn (1.04±0.05 Theriogenology, 41: 1251. ppm) and Cu (0.90±0.05 ppm) were below the Butler, W.B. and R.D. Smith. 1989. Interrelationship normal values (Underwood, 1977; Prasad and between energy balance and poetpartum Rao, 1997; Das et al., 2002; Mandal et al., 2002; reproductive function in dairy cattle. J. Das et al., 2003 and Sharma et al., 2004). In Dairy Sci., 72: 767. lactating buffaloes the values of Ca (9.59±0.25 Das, A., T.K. Ghosh and S. Haldar. 2003. Mineral mg/dl), total P (17.5±1.42 mg/dl), Fe, Mn and Co distribution in soil, feeds and grazing cattle were in range but iP, Zn, and Cu were marginally of different physiological stages in the red low (Underwood, 1977; McDowell et al., 1984; laterite and new alluvial agro climatic zones Mandal et al., 1996; Paul et al., 2000 and Yadav of West Bengal. Indian J. Anim. Sci., 73(4): et al., 2002). The concentration of Fe in plasma of 448-454. heifers (22.05±0.58 ppm) and lactating buffaloes Das, S., S. Basu, A.K. Sahoo, A.K. Sarkar and (17.68±0.84 ppm) observed many times higher R.N. Pal. 2002. Comparative study of than the reported normal values (Underwood 1977; certain serum macro and micro minerals Prasad and Rao, 1997; Das et al., 2002; Das et al., in anoestrus and normal cyclic rural cross 2003 and Sharma et al., 2004) indicates the higher bred cows. Indian J. Anim. Health, 41(2): levels of it in the feed ingredients used. 99-102. It may be concluded that dietary deficiency Dhoble, D. and S.K. Gupta. 1986. Serum calcium reflected the hemato-biochemical profile of and inorganic phosphorus levels during anoestrus animals and a strategic supplementation postpartum anoestrus in buffaloes. Indian J. is needed to these animals for exploitation of their Anim. Health, 25: 123-126. genetic potential for optimum production and Dutta, A., B.C. Sarmah and K.K. Baruah. 2001. reproduction. Concentration of serum trace elements in cyclic and anoestrus heifers in lower Brahmaputra valley of Assam. Indian Vet. REFERENCES J., 78: 300-302. Fiske, C.H. and Y.S. Subbarao. 1925. Determination AOAC. 1995. Official Methods of Analysis, 15th ed. of inorganic phosphorus in blood. J. Biol. Association of Official Analytical Chemists, Chem., 66: 375. Washington DC, USA. Folin, O. and H. Wu. 1920. A system of blood Arechiga, C., F.O. Ortiz and P.J. Hansen. 1994. analysis. Supplement IA simplified and

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improved method for determination of course on role of minerals and vitamins in sugar. J. Biol. Chem., 41: 367. livestock health and production. In Kumar, Giri, S.C. and B.P.S. Yadav. 2001. Role of H. 2004. Reproductive disorders due to phosphorus in improving the conception deficiency of minerals and vitamins in rate in cows and heifers. Indian J. Dairy Sci., livestock. IVRI, Izatnagar ,U.P., Indian J. 54(2): 106. Dairy Sci., 45(5): 278-280. Harrison, J.H., D.D. Hancock and H.R. Conard. Kutty, C.I. and K. Ramachandran. 2003. Bovine 1984. Nutritional approaches to prevent infertility a field oriented categorization reproductive disorders in dairy animals. based on investigation among crossbred In Sharma, K. 2007. Lead paper in 23rd cattle in a district of Kerala. Indian J. Anim. Annual convention of ISSAR and National Sci., 73(2): 35-37. Symposium on Challenges in Improving Ling, K., A. Waldmann, J. Samarutel, H. Jaakson, Reproductive Efficiency of Farm and Pet T. Kart and A. Leesmae. 2007. Field trial on Animals, Orissa University of Agriculture the relation ship of blood metabolites and and Technology-Bhubaneswar. J. Dairy body condition score with recurrence of Sci., 67: 123. luteal activity in Holstein cows. J. Vet. Med. Hidiroglou, M. 1979. Trace element deficiency and A., 54(7): 337-341. fertility in ruminants: A review. J. Dairy Miller, W.J. 1979. Dairy Cattle Feeding and Sci., 62: 1195-1206. Nutrition. Academic Press, New York, Ichaylova, V. and P. Iikova. 1971. Photometric USA. determination of micro amounts of calcium Mudgal, V., M.K. Mehta, A.S. Rane and S. with arsenazo III. Anal. Chem. Acta., 53: Nananavati. 2003. A survey on feeding 194. practices and nutritional status of dairy Jagatheesan, P.N.R., N. Selvaraju, V.R.S. Kumar animals in Madhya Pradesh. Indian J. Anim. and C. Chandrahasan. 2006. Blood Nutr., 20(2): 217-220. biochemical profile and estrus induction Naidu, K.V. and A.R. Rao. 1982. Short course on to arguments fertility in anoestrus Murrah role of minerals and vitamins in livestock buffaloes. Tamil Nadu Journal of Veterinary health and production. In Kumar, H. 2004. and Animal Science, 2(1): 10-12. Reproductive disorders due to deficiency of Jani, R.G., B.R. Prajapati, P.R. Patel and M.R. minerals and vitamins in livestock. IVRI, Dave. 2001. Certain haematological and Izatnagar, U.P., Indian Vet. J., 59: 781. biochemical changes in normal fertile and Oser, B.L. 1979. Blood analysis. Hawk’s infertile cross bred cows. Indian Journal of Physiological Chemistry, 14th ed. Tata Dairy and Biosciences, 12: 22-25. Mcgraw Hill Publishing Co. Ltd., New Kearl, L.C. 1982. Nutrient Requirements of Delhi. p. 1054-1093. Ruminants in Developing Countries. Perumal, P., S. Das, D.N. Mohanty and S.K. Senapati. International Feedstuffs Institute. Utah state 2007. A study on certain haematological university, Logan, Utah, USA. values of repeat and regular breeder cows. Kumar, H., G.D. Singh and H.C. Pant. 1992. Short (Abst.) In 23rd Annual convention of ISSAR

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and National Symposium on Challenges Izatnagar, U.P. Indian J. Anim. Reprod., 8: in Improving Reproductive Efficiency of 46-49. Farm and Pet Animals, Orissa University of Sinha, M.N. 1982. Gap analysis in relation to Agriculture and Technology-Bhubaneswar. feeding recommendation. Annual Report. Prasad, K.S.N. and S.V.N. Rao. 1997. Blood National dairy Research Institute, Karnal, mineral profile of anestrous and repeat India. p. 168-169. breeder cross bred cows-A field study. Sohal, T.S., J.N. Singh and J.P. Singh. 1982. Indian J. Anim. Nutr., 14(2): 135-137. Impact of intrusive extension education, Pugh, D.G., R.G. Almore and T.R. Hembri. 1985. the adoption of scientific diary farming Recent Advances in Animal Reproduction by landless rural families around Karnal. and Gynecology, p. 103-121. In Nanda, Indian J. Diary Sci., 35: 447-453. A.S. (ed.) USG Publishers and Distributors, Talapatra, S.K., S.C. Ray and K.C. Sen. 1940. Ludhiana. Studies on dual purpose fodder crop. In Sastry, N.S.R., C.K. Thomas and R.A. Singh. 1982. Bhaid, M. V. 1960. M.V.Sc. Thesis, Mathura Farm Animal Management and Poultry Veterinary College (U.P.) Indian Journal of Production, 2nd ed. Vikas Publishing House Veterinary Science and Animal Husbandry, Pvt Ltd. New Delhi, India. 10: 243. Sharma, M.C., M.P. Yadav and C. Joshi. Tandle, M.K., U.S. Briadar, M. Amanullah, 2004. Minerals - Deficiency Disorders, S.S. Honnappagol, S.M. Kartikesh, S.D. Therapeutic and Prophylactic Management Sonwale and Jagjiwanram. 1998. Blood in Animals. Publisher IVRI, Izatnagar (UP). biochemical profiles in cyclic and anoestrus Sharma, M.C., U. Shanker, O.P. Gupta and R.P. Deoni cow. Indian J. Dairy Sci., 51(1): 66- Verma. 1983. Haematological studies 68. in normal cyclic, anoestrus and repeat Teitz, N.W. 1986. Textbook of Clinical Chemistry. breeding cross breed cows. Indian Vet. Med. Publisher. W.B. Saunder, Philadelphia. p. J., 7: 153-155. 582-584. Singh, A.S. and O.N. Singh. 2005. Assessment of Tiwary, M.K., D.P. Tiwari, A. Kumar and B.C. haemetological and biochemical parameters Mondal. 2007. Existing feeding practices, in cross-bred heifers with anoestrus or nutrient availability and reproductive status estrus. Indian J. Anim. Sci., 75(12): 1372- of dairy cattle and buffaloes in Haridwar 1376. district of Uttarakhand. Indian J. Anim. Singh, A.S. and O.N. Singh. 2006. Haemetological Nutr., 7: 177-185. studies on crossbred heifers with anoestrus. Underwood, E.J. 1977. Trace Elements in Human Indian J. Anim. Res., 40(2): 170-172. and Animal Nutrition, 4th ed. Academic press, Singh, S. and S.V. Vadnere. 1987. Short course on New York, USA. role of minerals and vitamins in livestock Wiltbank, J.N., J. Bond, E.J. Warwick, R.E. Devis, health and production. In Kumar, H. 2004. A.C. Cook, W.L. Reynolds and W.H. Hazen. Reproductive Disorders Due to Deficiency 1965. Influence of total feed and protein of Minerals and Vitamins in Livestock. IVRI, intake on reproductive performance in the

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beef female through 2nd calving. Agriculture Research. Service, U.S.D.A., Washington, D.C., USA. Tech. Bull., 1314.

426 Original Article Buffalo Bulletin (July-September 2016) Vol.35 No.3

DEVELOPMENTAL COMPETENCE OF BUFFALO (BUBALUS BUBALIS) OOCYTES: EFFECT OF OOCYTES QUALITY, PROTEIN ADDITIVES, HORMONAL SUPPLEMENT AND TYPE OF CAPACITATING AGENTS

M.M. Waheed1, K.H. El-Shahat2 and A.M. Hammam3

ABSTRACT progressively enhanced developmental competence of buffalo oocytes. Four experiments were conducted to study the effect of media supplements on in-vitro Keywords: buffalo, oocytes, eCG, heparin, maturation, cleavage and embryo development of caffeine buffalo oocytes. In experiment 1, oocytes were cultured in TCM-199+10% fetal calf serum (FCS) o and kept at 39 C under 5% CO2 for in-vitro embryo INTRODUCTION development. In experiment 2, excellent quality oocytes were subjected to TCM-199 enriched with The application of superovulation and either 10% FCS or estrous buffalo serum (EBS; embryo transfer in buffaloes has been slow 20 to 40 pg/ml) and then fertilized using frozen (Mehmood et al., 2011; Kandil et al., 2012). In- semen in TALP medium containing heparin (0.02 vitro production of buffalo embryos has been mg/ml) and caffeine (3.89 mg/ml). In experiment gaining attention for its research and commercial 3, oocytes were cultured in-vitro maturation (IVM) applications (Mehmood et al., 2011). In-vitro medium supplemented or not with 20 IU/ml equine embryo production (IVEP) would be an effective chorionic gonadotropins (eCG). Experiment 4 was technique to improve the efficacy of transferable carried out to examine the suitable capacitating embryos (Di Francesco, 2010). Aspiration and agent, either heparin or caffeine or both. Excellent slicing methods were mostly used for the recovery and good quality oocytes produced higher (P<0.05) of buffalo oocytes from abattoir ovaries (Di maturation and morula development rates. In-vitro Francesco, 2010). The efficacy of this method maturation and cleavage rates were significantly was compared on the basis of cumulus oocyte higher (P<0.05) in IVM medium plus EBS or complexes per ovary (COCs/ovary; Cremonesi et eCG. Heparin and caffeine produced significantly al., 2010). However, these techniques are severely (P<0.05) higher embryo developmental rates. In hampered by poor recovery of total oocytes and in- conclusion, excellent quality oocytes cultured vitro maturation and fertilization (IVMF; Masudul with either EBS or eCG and fertilized with buffalo Hoque et al., 2011). Conditions during in- vitro spermatozoa capacitated with heparin and caffeine maturation, fertilization and embryo culture

¹Department of Clinical Studies, College of Veterinary Medicine and Animal Resources, King Faisal University, Kingdom of Saudi Arabia, E-mail: [email protected] 2Department of Theriogenology, Faculty of Veterinary Medicine, Cairo University, Egypt 3Department of Animal Reproduction and Artificial Insemination, National Research Center, Egypt

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(IVMFC) are believed to play a pivotal role in the Experimental design acquisition of development competence of embryos Experimental 1: Effect of oocytes quality (Alvarez et al., 2013). Therefore, the present study on IVMFC of buffalo oocytes. Oocytes (n=320) aimed to increase the developmental competence of were cultured in TCM-199 plus 10% FCS and 50 buffalo’s oocytes by studying the effect of oocytes ug/mL gentamicin and covered with mineral oil in quality, protein additives, hormonal supplement CO2 incubator containing 5% CO2 and 95% relative and type of capacitating agents. humidity at 39oC. Maturation rate was assessed either by the degree of cumulus mass expansion (Srinivasa Prasad et al., 2013) or by staining with MATERIALS AND METHODS 1% aceto-orcein stain (1% orcein in 45% glacial acetic acid) for observation of the 1st polar body All chemicals used in the study were (Prentice-Biensch et al., 2012). reagent grade (Sigma-Aldrich, American Samoa, USA) or as otherwise indicated. Sperm capacitation and IVF One 0.5 mL straw of frozen buffalo semen Collection and culture of oocytes was thawed in a water bath at 37oC for 30 seconds. Ovaries were collected at a local abattoir Spermatozoa were washed twice by centrifugation within 20 to 30 minutes after slaughter of buffaloes in TALP medium supplemented with 3.89 mg/mL and transported in a warm saline solution (0.9% sodium caffeine benzoate and 0.02 mg/mL heparin. NaCl) to a well-equipped laboratory within 2 h. Sperm pellet was suspended in 2mL TALP medium At the laboratory, ovaries were washed 3 times enriched with 20 mg/mL bovine serum albumin in normal saline containing 100 IU/mL penicillin (BSA) plus the above mentioned additives. The and 100 ug/mL streptomycin. Non atretic antral sperm cell concentration was adjusted to 2×106 /mL follicles (2 to 6 mm diameter) were aspirated sperm cells (Di Francesco, 2010). A 100 ul aliquot with an 18- gauge needle connected to a 10 of the sperm cell suspension was placed into a four mL disposable syringe. The aspiration medium well cultured dish and covered with warm mineral consisted of modified phosphate buffer (M-PBS) oil. After maturation, oocytes were washed in the enriched with sodium pyruvate (0.036 g/mL), 10% same sperm suspension medium and then 15 to 20 fetal calf serum (FCS) and the above mentioned oocytes were transferred into the sperm suspension antibiotics. Follicular oocytes were recovered and droplet and cultured under the previous conditions counted under stereomicroscope. The recovered in CO2 incubator for 5 h. oocytes were washed 3 times in IVM medium. After fertilization, oocytes were washed 3 According to the number of cumulus cell layers and times in IVM medium and then cultured for 6 to 7 ooplasm morphology, oocytes were divided into days in CO2 incubator. The cleavage rate and the three groups as adapted after Dadashpour Davachi frequency of morula and blastocyst were recorded. et al. (2012): (1) Excellent COCs; (2) Good POCs Experimental 2: Effect of protein additives (partial oocytes complexes); (3) Fair DO (denuded (EBS) on cleavage and embryo developmental rates oocytes). of buffalo oocytes. Only excellent quality oocytes (n=237) were washed 3 times in IVM medium then

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cultured for maturation in either TCM-199+10% was accomplished as previously mentioned in FCS that considered as a control group or in TCM- experiment 1. 199+10% estrous buffalo serum (EBS; Jamil et al., 2007). IVM medium was supplemented with 50 ug/mL gentamicin. In both groups, fertilization STATISTICAL ANALYSIS was performed using frozen thawed buffalo semen capacitated in TALP medium containing Experiments were repeated five times. heparin (0.02mg/ml) and sodium caffeine benzoate Data were pooled and analyzed by Chi-square test (3.89mg/ml). Maturation, cleavage and embryo using SPSS 22.0 statistical software (2013). developmental rates were carried out as in experiment1. Experimental 3: Effect of gonadotropin RESULTS (eCG) added to the maturation medium on cleavage and embryo developmental rates of buffalo oocytes. Experiment 1: The effect of oocytes oocytes (n=290) were classified into two groups; quality on maturation, cleavage and developmental Group 1 washed 3 times in TCM-199 enriched rates of IVF buffalo oocytes is shown in Table 1. with 10% FCS without gonadotropins and served Maturation rate and cleavage rates in COCs and as a control and Group 2 in which IVM medium POCs groups was significantly (P<0.05) higher was supplemented with 20 IU/ml equine chorionic than with Do type (Figures 1 and 2). In addition, the gonadotropins (eCG; Intergonan®, Intervet, proportion of embryo that developed to the morula Holland). Fertilization, maturation, cleavage and and blastocyst stage was significantly (P<0.05) embryo developmental rates were performed as in higher in COCs and POCs groups than in DO type. experiment 1. Experimental 4: Selection of suitable Experiment 2: Table 2 denotes that capacitating agent added to TALP medium. This addition of EBS to the culture medium (TCM-199) experiment (n=210 fertilized oocytes) was carried produces a significantly (P<0.05) higher maturation out to examine the suitable capacitating agent and cleavage rates than those cultured in the same added to TALP medium, either heparin (0.02 mg/ medium supplemented with FCS. The proportion of ml) or caffeine (3.89 mg/ml) or both. IVM/IVFC embryos in term of morula and blastocyst doesn’t

Table 1. Effect of oocytes quality on the maturation, cleavage and Embryo development rates (%). No. of Embryo development rates Oocytes Maturation Cleavage oocytes (%) quality rate (%) rate (%) cultured Morula Blastocyst COCs 100 70 (70.00)a 35 (50.00)a 15 (42.85)a 10 (28.57)a POCs 100 65 (65.00)a 30 (46.15)a 10 (33.34)a 5 (16.67)b DO 120 40 (33.34)b 10 (25.00)b 1 (10.00)b 0 (00.00)c

Percentages with dissimilar superscripts in the same column are significantly different at P<0.05

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Table 2. Effect of estrous buffalo serum (EBS) added to the culture medium (TCM-199) on the maturation, cleavage and embryo developmental rates (%). No. of Embryo development rates Culture Maturation Cleavage oocytes (%) condition rate (%) rate (%) cultured Morula Blastocyst TCM-199+ 100 45 (45.00)a 15 (33.34)a 4 (26.67)a 3 (20.00)a 10% FCS TCM- 137 100 (72.99)b 50 (50.00)b 15 (30.00)a 10 (20.00)a 199+10% EBS

Percentages with dissimilar superscripts in the same column are significantly different at P<0.05

Table 3. Influence of commercially available source of gonadotropins (eCG) on in vitro maturation, cleavage and embryo developmental rates (%). Embryo development rates Culture No. of oocytes Maturation Cleavage (%) condition cultured rate (%) rate (%) Morula Blastocyst TCM-199+ 2 1 143 84 (58.74)a 25 (29.76)a 10% FCS (8.00)a (4.00)a TCM-199+10% FCS+20 IU 147 110 (74.82)b 50 (45.46)b 10 (20.00)b 8 (16.00)b eCG

Percentages with dissimilar superscripts in the same column are significantly different at P<0.05

Table 4. Influence of heparin and/or sodium caffeine benzoate addition on the cleavage andembryo developmental rates (%) of buffalo oocytes.

No. of fertilized Cleavage rate Embryo development rates (%) Treatment oocytes (%) Morula Blastocyst Heparin (H) 60 20 (33.34)a 2 (10.00)a 2 (10.00)a Caffeine (C) 70 25 (35.72)a 3 (12.00)a 2 (8.00)a H+C 80 45 (56.25)b 15 (33.34)b 10 (22.23)b

Percentages with dissimilar superscripts in the same column are significantly different at P<0.05.

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Figure 1. Buffalo embryo developed in-vitro to 4-cell stage.

Figure 2. Buffalo embryo developed in-vitro to morula stage.

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significantly vary between EBS and FCS groups. The result of this experiment indicated that, addition of EBS to the IVM medium progressively Experiment 3: As shown in Table 3, enhanced the developmental competence of maturation, cleavage and embryo developmental buffalo oocytes as compared to FCS additives to rates up to blastocyst are significantly (P<0.05) the same medium. This is in agreement with the higher when eCG added to IVM medium compared result of Jamil et al. (2007) in buffalo. A possible with hormonal free medium. explanation for the beneficial role of ESB might be a result of its relatively high LH and estradiol levels Experiment 4: Table 4 shows that (Terzano et al., 2012). LH hormone may affect the the addition of heparin together with sodium cytoplasmic maturation of oocytes by increasing caffeine benzoate to TALP medium resulted in the calcium distribution within the ooplasm and significantly (P<0.05) higher cleavage and embryo promote glycolysis, combined with an increased developmental rates compared with the use of mitochondrial glucose oxidation metabolism heparin or caffeine alone. within the oocytes (Silverstre et al., 2007). The beneficial effects of EBS for oocyte maturation may also act via cumulus cells or directly on the DISCUSSION oocytes. EBS contains a number of known growth factors that have an important role in the regulation In the present study, data showed that good of oocyte maturation, and it also prevents the quality buffalo oocytes surrounded by multilayer of hardening of the zona pellucida (Jamil et al., 2007). compact investment with a homogenous ooplasm Moreover, Alm et al. (2002) stated that the higher had a significantly higher maturation cleavage, and maturation rates of equine oocytes could be due to developmental rates up to blastocyst compared with the increased concentration of insulin like growth oocytes of poor quality. This finding identifies the factor-1 in estrous mare serum (more than twice essential role of cumulus cells in promoting normal that found in FCS). cytoplasmic maturation of oocytes necessary for In the present study, IVM of buffalo fertilization and embryo development of buffalo oocytes in TCM-199 medium supplemented oocytes. Our results are similar to those previously with eCG increased maturation cleavage, and reported for buffalo oocytes (Gasparrini, 2013). The developmental rates up to blastocyst as compared presence of cumulus cells surrounding the oocyte to control medium. These findings run parallel to is essential to facilitate the transport of nutrients those previously reported for buffalo (Hegab et al., and signals into and out of oocytes (Kharche 2009). eCG as a source of gonadotropin (more FSH and Birade, 2013). The cumulus cells improve and less LH) stimulation lead to the generation of fertilization rate first by providing a capacitating- positive factors that acted on the oocytes to override inducing mechanism and secondly by facilitating the inhibitory influence and induced germinal the interaction between capacitated spermatozoa vesicle breakdown (Wang et al., 2009). Therefore, and the zona pellucida surface (de Souza et al., cAMP dependent protein kinase regulated by 2013). However, Zhang et al. (2012) found that cumulus cells following FSH- stimulation play cumulus cells had no influence on fertilization. a role in the complex mechanism of chromatin

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condensation leading to meiotic resumption in ACKNOWLEDGEMENT bovine oocytes (Tatemoto and Terada, 1998). Moreover, FSH enrichment to the culture medium The study was conducted in the Veterinary enhances early embryonic development (Anderiesz Research Division, National Research Centre, et al., 2000). Dokky, Egypt. In this study, the addition of heparin and caffeine to TALP medium resulted in a significantly higher cleavage and embryo developmental rates as REFERENCES compared to the addition of heparin or caffeine alone to the same medium. This is in agreement with the Alm, H., W. Mlodawska, H. Torner, S. Blottner, T. results reported in buffalo by Scholkamy (2002). Greising, F. Becker and A. Okolski. 2002. The role of caffeine may be through increasing the Influence of two different sera on dynamic concentration of cAMP which accelerate the rate of meiosis and extrusion of polar body of capacitation (Breininger et al., 2010); whereas, during in vitro maturation of horse oocytes. heparin appeared to be necessary for capacitation Theriogenology, 58: 735-738. and acrosome reaction (Cormier and Bailey, 2003). Álvarez, C., C. García-Garrido, R. Taronger Heparin stimulates the conversion of proacrosin to and G. González de Merlo. 2013. In acrosin and also it was reported to be responsible vitro maturation, fertilization, embryo for change of calmodulin and calmodulin binding development and clinical outcome of protein at capacitation (Leclerec et al., 1990). human metaphase-I oocytes retrieved from It seems therefore that, there is a synergistic stimulated intracytoplasmic sperm injection action for both heparin and caffeine in penetration cycles. Indian J. Med. Res., 137(2): 331- of oocytes in-vitro, which depends on their 338. compensatory action to induce capacitation and/or Anderiesz, C., A.P. Ferraretti, C. Magli, A. to increase penetration of oocytes (Tajik and Niwa, Fiorentino, D. Fortini, L. Gianaroli, G.M. 1998). Jones and A.O. Trounson. 2000. Effect of recombinant human gonadotrophins on human, bovine and murine oocyte meiosis, CONCLUSIONS fertilization and embryonic development in vitro. Hum. Reprod., 15(5): 1140-1148. Excellent quality oocytes cultured in Breininger, E., P.D. Cetica and M.T. Beconi. 2010. IVM medium supplemented with either protein Capacitation inducers act through diverse additives (EBS) or hormonal supplement (eCG) intracellular mechanisms in cryopreserved and fertilized with capacitated buffalo spermatozoa bovine sperm. Theriogenology, 74(6): in TALP medium enriched with heparin and 1036-1049. caffeine progressively enhanced developmental Cormier, N. and J.L. Bailey. 2003. A differential competence of buffalo oocyte. mechanism is involved during heparin-and cryopreservation-induced capacitation of bovine spermatozoa. Biol. Reprod., 69(1):

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177-185. Biotechnol, 4: 170-182. Cremonesi, F., K. Anderson and A. Lange- Leclerc, P., M.A. Sirard, J.G. Chafouleas and Consiglio. 2010. Efficacy of tuohy needle R.D. Lambert. 1990. Decreased binding of in oocytes collection from excised mare calmodulin to bull sperm proteins during ovaries. Vet. Med. Int., 2010. http://dx.doi. heparin induced capacitation. Biol Reprod., org/10.4061/2010/102591. 42: 483-489. Dadashpour, D.N., H. Kohram and S. Zainoaldini. Masudul Hoque, S.A., S.K. Kabiraj, M.A.M. 2012. Cumulus cell layers as a critical Yahia Khandoker, A. Mondal and K.M.A. factor in meiotic competence and cumulus Tareq. 2011. Effect of collection techniques expansion of ovine oocytes. Small Rumin. on cumulus oocyte complexes (COCs) Res., 102: 37-42. recovery, in vitro maturation and fertilization Di Francesco, S. 2010. Effect of season on of goat oocytes. Afr. J. Biotechnol., 10(45): reproductive performances in buffalo 9177-9181. species (Bubalus bubalis). Ph.D. Thesis, Mehmood, A., M. Anwar, S.M.H. Andrab and Napoli, Italy. p. 166. M. Afzal. 2011. In vitro maturation and Gasparrini, B., 2013. In vitro embryo production fertilization of buffalo oocytes: the effect of in buffalo: yesterday, today and tomorrow. recovery and maturation methods. Turkish Buffalo Bull., 32(1): 188-195. J. Vet. Anim. Sci., 35(6): 381-386. Hegab, A.O., A.E. Montasser, A.M. Hammam, Prentice-Biensch, J.R., J. Singh, B. Alfoteisy E.M.A. Abu El-Naga and S.M. Zaabel. and M. Anzar. 2012. A simple and high- 2009. Improving in vitro maturation and throughput method to assess maturation cleavage rates of buffalo oocytes. Anim. status of bovine oocytes: comparison of Reprod., 6(2): 416-421. anti-lamin A/C-DAPI with an aceto-orcein Jamil, H., H.A. Samad, N.U. Rehman, Z.I. Qureshi staining technique. Theriogenology, 78(7): and L.A. Lodhi. 2007. In vitro Maturation 1633-1638. and Fertilization of Riverine Buffalo Scholkamy, T.H. 2002. Biochemical aspects in Follicular Oocytes in Media Supplemented media and their supplements used for with Oestrus Buffalo Serum and Hormones. oocytes in vitro maturation, cleavage and Acta Vet. Brno., 76: 399-404. sperm capacitation in Egyptian buffalo. Kandil, O.M., A.S.S. Abdoon, D. Kacheva, C.H. Ph.D. Thesis, Cairo University, Egypt. Karaivanov, M.F. Fadel, N.A. Hemeida, Silvestre, M.A., I. Alfonso, E. García-Mengual, B. Georgiev, T.I. Maslev, W.M. Ahmed I. Salvador, C.C. Duque and I. Molina. and H.R. Badr. 2012. Successful embryo 2007. Effect of recombinant human follicle- transfer in Egyptian buffaloes. Glob Vet., stimulating hormone and luteinizing 8(4): 320-327. hormone on in vitro maturation of porcine Kharche, S.D. and H.S. Birade. 2013. oocytes evaluated by the subsequent in vitro Parthenogenesis and activation of development of embryos obtained by in mammalian oocytes for in vitro embryo vitro fertilization, intracytoplasmic sperm production: A review. Advances Biosci injection, or parthenogenetic activation. J.

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Anim. Sci., 85(5): 1156-1160. Zhang, A., B. Xu, Y. Sun, X. Lu, Z. Niu, Q. Chen, SPSS, 2013. Statistical Package for Social Y. Feng and C. Xu. 2012. The effect of Sciences, SPSS Inc., Chicago, IL, USA human cumulus cells on the maturation Copyright © for Windows, version 22.0. and developmental potential of immature Souza de, J.M.G., N. Duffard, M.J. Bertoldo, Y. oocytes in ICSI cycles. J. Assist. Reprod. Locatelli, E. Corbin, A. Fatet and V.J.F. Gen., 29: 313-319. Freitas. 2013. Influence of heparin or the presence of cumulus cells during fertilization on the in vitro production of goat embryos. Anim. Reprod. Sci., 138(1-2): 82-89. Srinivasa, P.C.H., A. Palanisamy, V.S. Gomathy, S. Satheshkumar, A. Thangavel and G. Dhinakar Raj. 2013. Effect of TCM- 199 and synthetic oviductal fluid (SOF) medium and cysteamine supplementation to in vitro maturation media on maturation, cleavage rate and subsequent embryonic development of buffalo oocytes. Buffalo Bull., 32(3): 182-188. Tajik, P. and K. Niwa. 1998. Effects of caffeine and/or heparin in a chemically defined medium with or without glucose on in vitro penetration of bovine oocytes and their subsequent development. Theriogenology, 49(4): 771-777. Tatemoto, H. and T. Terada. 1998. Involvement of cumulus cells stimulated by FSH in chromatin condensation and activation of maturation promoting factor in bovine oocytes. Theriogenology, 49: 1007-1020. Terzano, G.M., V.L. Barile and A. Borghese. 2012. Overview on reproductive endocrine aspects in buffalo. J. Buffalo Sci., 1: 126-138. Wang, C., B. Xu, B. Zhou, C. Zhang, J. Yang, H. Ouyang, G. Ning, M. Zhang, J. Shen and G. Xia, 2009. Reducing CYP51 inhibits follicle-stimulating hormone induced resumption of mouse oocyte meiosis in vitro. J Lipid Res., 50: 2164-2172.

435 Buffalo Bulletin (July-September 2016) Vol.35 No.3 Original Article Buffalo Bulletin (July-September 2016) Vol.35 No.3

SEMINAL PLASMA AND SPERM MEMBRANE PROTEINS OF BUFFALO AND CATTLE BULLS: A COMPARATIVE STUDY

Shilpi Dixit1, Vijay Pandey1,*, Dilip Kumar Swain1, Rajesh Nigam1, Ambika Sharma1, Deepak Sharma1, Atul Saxena2 and Pawanjit Singh2

ABSTRACT semen characteristic and seminal plasma proteins reported by Sharma et al. (2015). The seminal plasma proteins and sperm Besides proteins of seminal plasma, sperm membrane proteins of six each Bhadawari buffalo surface proteins reported to have important role in and Hariyana cattle were isolated by using the recognition of zona proteins for binding to zona protein isolation kits and protein profiling were and plasma lemma of ovum and also in acrosome carried out by 1-D SDS-PAGE. reaction for successful fertilization (Jagadish et al., 2005). It has been reported that, the loss of integrity Keywords: buffalo, cattle, SDS-PAGE, seminal of the sperm plasma membrane is frequently plasma protein, sperm membrane protein associated with infertility in male, despite normal semen parameters (Rajeev and Reddy, 2004). Looking to the importance of buffalo in INTRODUCTION India, it becomes necessary to understand the structural and functional attributes of the buffalo Seminal plasma is complex fluid spermatozoa and its comparison with that of containing a wide variety of both organic and cattle which will help in better understanding of inorganic components, among which proteins are molecular features that make the buffalo sperm an important part of the high-molecular-weight less fertile than cattle. In the scenario of climate substances. The protein composition of mammalian change, indigenous animals are of choice for better seminal plasma varies in species and has important dissemination of gemplasm and it urges the science effect on sperm functions such as sperm motility to have a picture of the protein profiles of the semen (Henricks et al., 1998), viability (Brandon et al., as a whole to make it more suitable for artificial 1999) and freezability (Asadpour et al., 2007), insemination. Keeping in this mind the present sperm capacitation and fertilization (Rodriguez study was designed for comparative evaluation et al.,1998) and also serve to protect sperm from of protein profiles of seminal plasma and sperm damage or to maintain their longevity which were membrane in buffalo and cattle bull semen. evident from the correlation observed among

1College of Biotechnology, *E-mail: [email protected], 2College of Veterinary Science and Animal Husbandry, Deen Dayal Upadhyaya Veterinary and Animal Science University (DUVASU), Mathura, India

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MATERIALS AND METHODS done after one day of sample processing.

Experimental animals Preparation of sperm membrane extract Semen samples were collected by an The sperm membrane proteins were artificial vagina from six each sexually mature extracted by method described by Cheema and Haryana cattle and Bhadawari buffalo bulls (2 to Babbar (2008) with some modification. The sperm 4 years old) from the District Dairy Demonstration pellets were washed three times in PBS (pH 7.4) farm of College of Veterinary Science and and resuspended in lysis buffer containing 1.5% Animal Husbandry, Mathura. The animals were (w/v) Tris [hydroxylmethyl] aminomethane buffer maintained in nearly identical nutritional and (pH 6.8), 20% (w/v) sucrose, 10% (w/v) SDS, management conditions throughout the period of 5% (v/v) β-mercaptoethanol and 0.05% (w/v) study. The animals utilized were clinically healthy bromophenol blue (3’3”5’5” tetrabromophenol- and regularly dewormed and vaccinated against sulfonephthalein). The mixture was kept in boiling common ailments. water bath for 5 minutes and centrifuged at 5000 rpm for 10 minutes and protein extraction was Semen evaluation carried out by using the Triprep extraction kit Ejaculates were collected twice a week (Fisher Scientific). Proteins were recovered by from each bull in morning hours (8:00 to 9:00 centrifugation at 10,000 rpm for 10 minutes, re- AM) and total six ejaculates were collected from suspended in phosphate buffered saline (PBS) each bull during the study period. Immediately and stored at -20oC until further analysis by SDS- after collection, semen volume was determined PAGE. with a graduated plastic tubes and concentration of spermatozoa (million/ml) was determined by SDS-polyacrylamide gel electrophoresis (SDS- automatic sperm counter. PAGE) SDS-PAGE was performed for separation Preparation of seminal plasma and determination of molecular weight of The seminal plasma was prepared by seminal plasma and sperm membrane proteins. centrifugation and protein extraction was carried Proteins samples were subjected to SDS-PAGE out by using the Triprep extraction kit (Fisher as per method of Laemmli (1970), using a 12% Scientific). Fresh semen was centrifuged at 5000 polyacrylamide gel. The relative molecular rpm for 10 minutes. The total protein was estimated weights were determined by using the (broad range by spectrophotometric method at 280 nm (protein’s molecular weight markers of Merck, Germany), absorbance at 280 nm). After the estimation of total Gel documentation and analysis system (Gel-doc. protein, the protein extraction was carried out as Model- Alpha imager TM1220, Alpha Innotech per the protocol of the kit. Proteins were recovered Corporation, USA). by centrifugation at 10,000 rpm for 10 minutes, re- suspended in phosphate buffered saline (PBS) and Image analysis and statistical analysis stored at -20oC until further analysis of seminal Gel images were analysed to determine plasma proteins. The SDS-PAGE analysis was molecular weight and relative protein content

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using the Gel doc system. Data were analyzed (2007) in Surti and Dhanju et al. (2001) in Murrah using SPSS software program (SPSS version 16.0 buffaloes. The concentration of sperm membrane for Windows). The results were presented as mean extract protein showed significant difference ± standard error of mean (S.E.M) and analysis of (P<0.05) in buffalo and cattle bull spermatozoa. significance was attributed at P<0.05. The concentration of membrane protein extracted from sperms of buffalo and cattle in present study RESULTS AND DISCUSSION was observed lower than the reports of Cheema and Babbar (2008) in crossbred cattle and Dhanju The results of semen characteristics and et al. (2001) in buffalo bulls. The variation in the protein concentration of seminal plasma and concentration of extracted proteins may be due to sperm membrane extract of buffalo and cattle genetic variability in the animals or may be due to bulls are depicted in Table 1 and 2, respectively. variations in extraction methods used by different The statistical analysis of the result did not show authors. significant difference in ejaculate volume and Table 2 shows the protein profile of seminal sperm concentration in buffalo and cattle bull plasma and sperm membrane protein of buffalo semen while protein concentration of seminal and cattle bulls. The electrophoretogram of buffalo plasma as well as sperm membrane extract showed semen revealed 24 protein bands ranging between significant difference (P<0.01) in buffalo and cattle 6.0 to 200 kDa in seminal plasma and 14 protein bull semen. The lower ejaculate volume and sperm bands ranging from 16.0 kDa to 205 kDa in sperm concentration was observed in buffalo compared to membrane extract proteins. Nine protein bands of cattle bull in present study corroborate the reports molecular weight 20, 26.5, 36.5, 38, 44, 66, 70, of Khalek et al. (2008) in Nilliravi buffalo and 72 and 84 kDa were observed common in seminal Holestein cattle bulls. In spite of the insignificance plasma and sperm membrane of buffalo which of differences between the two species in semen indicated that these proteins are structural as well characteristics, there was tendency of lower values as secretory in nature. Asadpour et al. (2007) and of ejaculate volume and sperm concentration in Sharma et al. (2014) revealed 25 protein bands on buffalo than cattle bulls (Table 1).Variation in semen SDS - PAGE analysis of seminal plasma in buffalo volume and sperm concentration might be due bulls, and most of the bands were observed to be to differences in frequency of collection, season, comparable with results of present study. Selvaraju nutrition, management, genetics, reproductive et al. (2010) and Dhanju et al. (2001) also reported health status and age of bulls (Soderquist et al., protein bands of comparable molecular weight in 1992). Variations can also be due to skill of semen seminal plasma and sperm membrane of buffalo collector/attendant and temperature of AV. spermatozoa, respectively. The reports of these The seminal plasma protein concentration authors simulate the findings of present study. in present investigation showed a significant SDS-PAGE of cattle seminal plasma difference (P<0.01) between buffalo and cattle revealed 13 protein bands ranging from 6.5 kDa bull semen. Comparable seminal plasma protein to 204 kDa. The proteins of comparable molecular concentration was observed by Arangasamy et al. weight were reported by Jobim et al. (2004) and (2005) and Singh et al. (1995) in cattle and Nendre Bellin et al. (2012) in seminal plasma of different

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Table 1. Mean semen characteristics of Haryana cattle and Bhadawari buffalo bulls. Semen attributes Cattle Buffalo Semen volume (ml) 4.038±0.22a 2.958±0.18a Sperm concentration (106/ml) 1736.944±60.46a 1678.05±86.68a Seminal plasma protein (gm/dl) 7.86±0.34a 4.63±0.16b Sperm membrane protein (mg/109sperms) 2.81±0.25a 4.42±0.63b

Means bearing at least one common superscript alphabet in one parameter did not differ significantly (P≥0.05), otherwise significant at 5% level (P<0.05).

Table 2. SDS-PAGE protein profile of seminal plasma and sperm membrane of cattle and buffalo bull semen (kDa). Cattle Buffalo S. No. Seminal Plasma Sperm Membrane Seminal Plasma Sperm Membrane 1 6.5 6.5 6.5 16 2 8.5 8.5 12.5 20 3 18.5 12 18.5 22.5 4 26.5 22.5 20 26.5 5 43 25 24 36.5 6 66 26.5 26.5 38 7 70 32.5 28 42 8 75 34.5 32 44 9 84 38 35 66 10 88 43 36.5 70 11 96 48 38 72 12 160 58 40.5 84 13 204 66 44 174 14 69 46 205 15 70 48 16 84 60 17 174 66 18 70 19 72 20 84 21 86 22 96 23 184 24 200

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breeds of cattle. Bellin et al. (1996) reported profile indicates species specific variations may proteins of 75, 84, 66 kDa molecular weight in account for their variable fertility and freezability seminal fluid of cattle bulls. Fernandezet al. (2009) of buffalo and cattle semen. Further studies may reported eight bands ranging from 15 to 63kDa. be carried out by employing the modern tools to Out of these eight bands, two similar proteins (22 characterize these proteins and their putative role in and 25kDa) reported by Fernandez et al. (2009) in the regulation of variable fertility and freezability Bos taurus taurus bulls simulates the findings of in buffalo and cattle bull semen. This will help present study. The cattle sperm membrane proteins in the formulation of extenders during semen revealed 17 protein bands ranging between 6.5 to processing so as to increase the post thaw quality 174 kDa. Five proteins of comparable molecular of the buffalo semen. weight (84, 66, 48, 24 and 12 kDa) were reported by Bellin et al. (1996) in sperm membrane of vasectomised bulls. Cheema et al. (2011) reported ACKNOWLEDGEMENTS proteins of comparable molecular weight (10, 25, 40, 65, and 70 kDa) in sperm membrane of buck. Authors are thankful to Vice Chancellor, The reports of these authors simulate the findings UP Pandit Deen Dayal Upadhyaya Pashu Chikitsa of present study. Proteins observed other than these Vigyan Vishvavidyalya evam Gau Anusandhan proteins in present study may be specific to cattle Sansthan, (DUVASU), Mathura, for providing bulls. Seven protein bands of molecular weight 6.5, necessary facilities to carry out this work. The 8.5, 26.5, 43, 66, 70, and 84 kDa were observed authors are also thankful to Dean, College of common in seminal plasma and sperm membrane Biotechnology and College of Veterinary Science, of cattle which indicated that these proteins are Mathura for his cooperation and guidance in structural as well as secretory in nature. carrying out the research work. The protein bands of molecular weight 6.5, 10 and 12 kDa in cattle and 26.5, 36.5 and 174 kDa in buffalo sperm membrane were observed to REFERENCES be most abundant proteins. Seven protein bands (6.5, 18.5, 26.5, 66, 70, 84 and 96 kDa) in seminal Arangasamy, A., L.P. Singh, N Ahmed, M.R. plasma and seven protein bands (22.5, 26.5, 38, 66, Ansari and C.G. Ram. 2005. Isolation and 70, 84, and 174) in sperm membrane were detected characterization of heparin and gelatin similar in both the species in present investigation. binding buffalo seminal plasma proteins Proteins detected other than these proteins in and their effect on cauda epididymal present study may be said to be species specific spermatozoa. Anim. Reprod. Sci., 90: 243- proteins. 254. In conclusion, not all the seminal plasma Asadpour, R., S. Shoushtari, S. Rezaii and M. and sperm membrane proteins are similar in buffalo Ansari. 2007. SDS-PAGE of buffalo bull and cattle only seven proteins are observed to be seminal plasma proteins and their relation similar in seminal plasma and sperm membrane of with semen freezability. Anim. Reprod. Sci., buffalo and cattle semen. The difference in protein 102: 308-313.

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Bellin, M.E., H.E. Hawkins, J.N. Oyarzo, R.J. spermatozoa: influence on sperm motility. Vanderboom and R.L. Ax. 1996. Monoclonal Biol. Reprod., 59: 330-337. antibody detection of heparinbinding Jagadish, N., R. Rana, R. Selvi, D. Mishra, M. proteins on sperm corresponds to increased Garg, S. Yadav, J.C. Herr, K. Okumura, fertility of bulls. J. Anim. Sci., 74: 173-182. A. Hasegawa, K. Koyama and A. Suri. Bellin, M.E., J.N. Oyarzo, H.E. Hawkins, H. Zhang, 2005. Characterization of a novel human R.G. Smith, D.W. Forrest, L.R. Sprott and sperm associated antigen 9 (SPAG9) R.L. Ax. 2012. Fertility-associated antigen having structural homology with c-Jun on bull sperm indicates fertility potential. J. NH2-terminal Kinase interacting protein. Anim. Sci., 76: 2032-2039. Biochem. J., 389(1): 1-39. Brandon, C.I., G.L. Heusner, A.B. Caudle and R.A. Jobim, M., E. Oberst, C. Salbago, D. Souza, V. Fayrer-Hosken. 1999. Two-dimensional Wald, F. Tramontina and R. Maltos. 2004. polyacrylamide gel electrophoresis of 2D SDS-PAGE of bovine seminal plasma equine seminal plasma proteins and their proteins and their relation with semen correlation with fertility. Theriogenology, freezability. Theriogenology, 61: 255-266. 52: 863-873. Khalek, A.E., M.B. Aboul-Ela, F.A. Soheir and Cheema, R.S., A.K. Bansal, G.S. Bilaspuri and V.K. E. Dandooush. 2008. Semen quality of Gandotra. 2011. Correlation between the holstein and buffalo bulls after filtration proteins and protein profile(s) of different using sephadex column. Saudi J. Biol. Sci., regions of epididymis and their contents in 15(1): 91-97. goat buck. Anim. Sci. Papers Report, 29(1): Laemelli, U.K. 1970. Cleavage of structural 75-84. proteins during the assembly of the head of Cheema, S.R. and B.K. Babbar. 2008. Sperm bacteriophage T4. Nature, 220: 680-685. membrane/seminal plasma proteins reflects Nandre, R.M. 2007. Effect of preservation of semen quality in cross-bred cattle bulls. Ind. spermatozoa at sub-zero temperature on J. Anim. Res., 42(4): 242-247. DNA integrity by comet Assay. M.V.Sc. Dhanju, C.K., R.S. Cheema and S.P. Kaur. 2001. Thesis, Anand Agricultural University, Effects of freezing on proteins and protein Gujrat, India. profiles of sperm membrane extracts and Rajeev, S.K. and K.V.R. Reddy. 2004. Sperm seminal plasma of buffalo bulls. Asian- membrane protein profiles of fertile Austr. J. Anim. Sci., 14(12): 1678-1682. and infertile men: identification and Fernandez, C.E., F.F. de Souza, J.A. Souza-Neto characterization of fertility-associated and P.E.M. Ribola. 2009. Heparin-Binding sperm antigen. Human Reprod., 19(2): 234- Proteins of Seminal Plasma in Nellore Bulls. 242. Ciência Rural, Santa Maria. 39: 275-278. Rodríguez, M.H., I. Iborra, P. Martínez and J.J. Henricks, D.M., A.J. Kouba, B.R. Lackey, W.R. Calvete. 1998. Immunoelectroscopic Boone and S.L. Gray. 1998. Identification imaging of spermadhesin AWN epitopes on of Insulin-like growth factor I in bovine boar spermatozoa bound in vivo to the zona seminal plasma and its receptor on pellucida. Rep. Fert. Dev., 10: 491-497.

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Sharma, L., V. Pandey, R. Nigam, A. Saxena and D. K. Swain. 2015. Association of semen attributes and seminal plasma proteins of buffalo bulls. J. Anim. Res., 5(1): 119-123. Sharma, L., V. Pandey, R. Nigam, P. Singh, A. Saxena and D.K. Swain. 2014. Seasonal variations in seminal plasma proteins of buffalo. Repro. Dom. Anim., 49: 387-391. Selvaraju, S., T.S. Subramani, B.S. Raghavendra and J.P. Ravindra. 2010. Effect of IGF-I on spermatozoa membrane protein profile and correlation between seminal plasma IGF-I and antioxidant enzymes in buffalo semen. Indian J. Anim. Sci., 80(12): 1171-1174. Singh, M.P., A.K. Sinha and B.K. Singh. 1995. Effect of cryoprotectants on certain seminal attributes and on the fertility of buck spermatozoa. Theriogenology, 43: 1047- 1053. 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 12th International Congress. The Hague, Netherlands.

443 Buffalo Bulletin (July-September 2016) Vol.35 No.3 Original Article Buffalo Bulletin (July-September 2016) Vol.35 No.3

INCIDENCE OF REPEAT BREEDING IN VARYING BREEDS OF BUFFALOES AND CATTLE IN DIFFERENT CLIMATIC CONDITIONS IN KHYBER PAKHTUNKHWA (PAKISTAN) Amjad Khan1,*, Muhammad Hassan Mushtaq1, Mansur ud Din Ahmad1, Abid Hussain2, Asghar Khan3, Ajab Khan4 and Habibun Nabi5

ABSTRACT (23.71%) cattle and buffaloes. Significant variations in incidence of RB with season were Change in climatic factors poses formidable observed. The results also elicit the significant challenge to the livestock sector development in impact of monthly mean temperature, humidity, Pakistan. Repeat breeding (RB), defined as adult average annual rainfall, altitude and breed on buffalo and cow’s failure to conceive from more the incidence of RB. The culling percentage was than 3 times regularly spaced AI or natural services significantly (P<0.05) higher in repeat breeder in the absence of any detectable reproductive buffaloes (77%) than in cattle (23%). In conclusion abnormalities, is a costly problem for the dairy the result shows that exotic and cross bred cattle sector. An active surveillance was conducted breeds were more efficient reproductively than aimed to address the impact of climate change buffaloes and non descriptive breeds of cattle in on incidence of RB in different cattle and buffalo varying environmental conditions. It was also breeds in Khyber Pakhtunkhwa (KPK), Pakistan. concluded that RB is a multi-factorial problem that Through multistage cluster sampling 3 different involves a number of intrinsic and extrinsic factors climatic and geographic clusters were selected. fixed to the animal. Total of 1167 animals were included in the study. Out of total 586 were cows and 581 were buffaloes. Keywords: repeat breeding, geographic, The sampled population was stratified on parity primiparous, multiparous, incidence basis into primiparous and multiparous cow’s and buffalo sub-groups. The overall incidence of RB was calculated 27.33%. INTRODUCTION RB incidence was significantly (P<0.05) higher in buffaloes (33.04%) than in cattle Repeat breeding (RB) has been considered (21.67%). from many decades one of the most important Whereas RB in multiparous (29.28%) were reproductive disorder in cattle and buffaloes. significantly (P<0.05) higher than primiparous Incidences of RB in lactating large dairy

1Department of Epidemiology and Public Health, University of Veterinary and Animal Sciences, Lahore Pakistan, *E-mail: [email protected] 2University of Poonch Rawlakot Azad, Jammu and Kashmir, India 3Department of Clinical Medicine and Surgery, 4Department of Pathology, University of Veterinary and Animal Sciences, Lahore, Pakistan 5Veterinary Research Institute (VRI), Peshawar, Khyber Pakhtunkhwa (KPK), Pakistan

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animals varied among regions, management and Acclimation is a phenotypic reaction by environments. Internationally, declined have been the animals developed within the environment to an reported in calving rates to 1st service from 60% individual source of stress (Fregley, 1996). To cope to 40% over the past twenty five years (Bulman et with the thermal challenges that leads to reduced al., 1978). Environment all over the world is the feed intake and many physiological functions major factor in these days affecting reproductive alterations i.e. productive and reproductive and productive efficacy respectively. Breeding efficiency (Beede and Collier, 1986; Wolfenson et efficiency in buffaloes is affected greatly by al., 2000) reported that more that 50% of bovine season. Previous studies have shown a certain population is being located in the tropical region tendency of having better performance in cool and estimated economic losses in about 60% of the months, July to February (70 to 80% conception dairy farms due to heat stress around the world. It rate) in buffaloes. It is also known that buffaloes compromises oocyte growth in dairy animals by are activated sexually by decrease in day length altering progesterone level, follicle-stimulating and temperature (Agrawal et al., 2003). As they hormone, secretion of luteinizing hormone (LH) have poor thermal regulation system. That’s why and dynamics throughout the estrus cycle (Ronchi it is important to protect them in summer from et al., 2001). extreme heat allowing them wallowing and also in According to the Intergovernmental Panel winter from extreme cold, that may give chance to on Climate Change (IPCC) evaluation reports, many other diseases to attack the animal (Ramesh about 0.6oC increase has occurred in average et al., 2002). global temperature since the industrialization and A lower number of services per conception in future increase of 2 to 4.5oC is almost expected are needed during the July to February. RB by the end of 21th century (IPCC, 2007). Although syndrome is a major cause of economic losses and climate changes at the global level, its positive deprived reproductive performance in the dairy impacts as well as negative, will be experienced at sector (Bartlett et al., 1986; Bage et al., 2002). It the local level. contributes to lower the dairy profit by insemination The average annual temperature in South costs and wasting semen, increasing culling, Asia by the end of 21th century could go up from increasing interval to conception and replacement 3.5 to 5.8oC because this region occurs in the arid costs and also reduces fertility (Gustafsson et al., and semi-arid zone (IPCC, 2007). Therefore the 2002). The incidence of RB ranges from 10.1 to South Asian region will be more affected by the 24% mostly in exotic cattle breeds (Bartlett et al., consequential climate change effects. In Pakistan 1986; Bage et al., 2002; Gustafsson et al., 2002). these impacts are already visible particularly since Though, specific causes of RB are not clear but it 1990 Pakistan meteorological department (PMD has found to be a multi-factorial problem involving records). For having great topographic contrasts a number of intrinsic as well as extrinsic one and as a result the climate of the Pakistan has (Gracia et al., 2007). Ever since numerous factors large temporal and spatial variations. In Pakistan affect incidence of RB in dairy animals, therefore no such research has conducted before addressing it is intricate to make generalizations about major the impact of changing climatic factors at different causes (Silvia, 1994). altitudes on incidence of RB in different breeds of

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cattle and buffaloes. This research will also provide clinical reproductive disorders. The animals were clue for future research to specify most significant excluded on the following conditions. risk factors responsible for incidence of RB. • Having any detectable reproductive disorder. • If not available for three MATERIALS AND METHODS consecutive AI or natural services. • If the owner is not willing. Study design and site selection • No farm animal was included in An active surveillance based study was the study. conducted in account to pile the incidence and impact of climate change on Repeat breeding Sampling technique and Sample size calculation (RB) in different breeds of cattle and buffaloes in Through multistage cluster sampling the Khyber Pakhtunkhwa (KPK) Pakistan. The target study frames were selected. Then the end sample population in the study area was about 808068 was drawn through simple random method for lactating, 184229 dry and 97664 not yet calved higher accuracy of results. The number of cow’s buffaloes and 6059041 lactating cows, 743852 and buffaloes to be sampled for estimate prevalence dry and 419547 cows that were not yet calved with a confidence interval of 95% was estimated (Livestock census, 2006). Administratively the using the formula by Thrusfield, (1995). northern province of Pakistan KPK is divided into three agricultural zones i.e. Semi-Arid, Sub- N = 1.962 ×Pexp (1− Pexp)/d2 Humid and Humid region (Fig 1). Eight districts were selected randomly, at least two from each Where n = required sample size, region and one from Federally Administered Tribal Pexp = expected prevalence, and Areas (FATA) (Figure 1). Geographically KPK d = desired absolute precision. could be separated into two zones: the northern and the southern one. The northern zone is winters Since the prevalence estimates of RB in the with heavy rainfall and moderate summers. While area were not available, therefore a 50% random southern zone is arid with hot summers and estimate was chosen. The minimum number of cold winters with scanty rainfall. The climate of cattle and buffaloes needed to be included from the KPK varies immensely as compared to its size, study site calculated was 384. More animals were encompassing the majority of many types of selected to take care of excluded animals during climates found in the country. study.

Repeat breeder’s definition and exclusion Data collection criteria Data regarding cattle and buffaloes from A cow or buffalo was considered as a smallholder dairy owners of the rural areas of KPK repeat breeder when it did not conceived after were obtained on a predesigned questionnaire. Only three inseminations or natural services (in case of house hold animals were observed purposely to see buffaloes mostly), apart from having no detectable the climatic impact under the local management

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practices in the study area. Various surveys were the multiparous cattle and buffaloes; 23.71% in encountered from December 2012 to mid December primiparous and 29.28% in multiparous. Average 2013 to observe the status of RB in the included rainfall, altitude and monthly average mean sampled animals at different seasons. Data about temperature showed a significant impact on the each cow and buffalo: breed, parity, season of heat incidence of RB especially in the cows breed. and services, any reproductive disorder (pyometra, Different breeds were included in the study both fetal membranes retention, endometritis, ovarian from the cattle and buffalo population prevailing cysts and urovagina) was recorded. in the study area. Most of the breeds are used to bring from the Punjab province of Pakistan into Statistical analyses this area for milk purpose. The results illustrated Analysis was performed by using the higher cumulative incidence rate of (35.87%) in statistical package (SPSS 16.0). Chi square test the non descriptive breed of cattle. While the most was used to calculate the association between efficiently reproductive and highly conceptive the categorical variations studied in the study breed were exotic breeds of cattle (Table 1). The i.e. repeat breeding with season, climatic region, local breed of cattle (Achai) was also found to parity, animal species, breed of the animal, average be highly conceptive as compared to the non mean temperature and culling status of the repeat descriptive ones. Same was the case with buffalo breeders. population; in which the local Aza kheli breed was found with the lesser incidence rate of RB than Nili ravi and non descriptive buffalo breeds. RESULTS Seasonal variation was observed; significantly higher (P<0.05) RB incidence rate A total of 1216 animals were included in of (34%) in the late summer (July to September). the present study, in which 49 animals were lost While a higher conception rate of almost 83% in follow up not falling in the inclusion criteria. from the month of January to April (Table 1). The Thus 586 cows and 581 buffalo’s were included in most important factor that was found in the present the study. The study population was also stratified study was the culling practice of the farmers after on parity basis to see the impact of climate change an animal gone to be a RB. Total of 200 (62.69) in relation to age of the animal. Total of 409 animals were culled out of 319 RB; 154 (77%) primiparous, 758 multiparous cows and buffaloes buffaloes and 46 (23%) cows. Amongst the culled were selected (Table 1). Statistical analysis showed animals 146 (73%) were multiparous RB while (27.33%) cumulative incidence of repeat breeding 54 (27%) were primiparous ones. A significant (RB); in cows (21.67%) and in buffalo (33.04%) (P<0.05) difference of culling for species and significantly (P<0.05) higher than in cattle. The parity was observed in the present study (Figure 2). result elicits the insignificant impact (P>0.05) of Seasonal variation with culling was also observed. climatic region on the incidence of RB (Table 1). Parity of the animal was evaluated for the incidence of RB in different climatic conditions. It was found significantly (P<0.05) higher in

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DISCUSSION 2009). Table 1 depicts cumulative incidence The present study was conducted to of RB in different climatic regions showing in understand the impact of extent of change in the significant (P>0.05) variation. This is because of climatic factors i.e. monthly mean temperature, the overall incidence calculated for both the species seasonal rainfall, humidity variation at different studied. There was significant variation observed altitudes and geographical conditions. In this in the incidence of RB in between the species and study, all the cows and buffaloes considered as also amongst the different breeds based on parity. RB had normal estrous cycles and returning to It was confirmed; RB incidence in primiparous sexual cycle within 18 to 24 days, but did not got (23.71%) lesser than multiparous (29.28%). This conceived, accordingly to the findings of (Allen substantial variation could be due to the stress et al., 1996).The reproductive performance of of high production and change in environmental various breeds of cattle and buffalo’s was also conditions on the aged animals as compared to evaluated in different environmental conditions. young ones (primiparous). These estimates were The overall cumulative incidence recorded in the in coincidence with that of (Rabbani et al., 2010) present study was 27.33%. This was much higher reporting (28.35%) of RB incidence in 2nd lactating than the previous cumulative incidence recorded cows. Though Yusuf et al. (2010) reported higher by Rabbani et al. (2010) in Faisalabad district conception rate in 1st parity cows and higher RB (Pakistan). The difference may be due the time incidence totally opposite to the results of the span or environmental conditions as well different present study. In this study we concluded higher management practices in the two study areas. Also conception rate in primiparous cows and buffaloes the later study was conducted in one of the hotter that multiparous ones (Robert et al., 2011) reported regions of the country where buffalo are considered (26.61%) incidence of RB in primiparous cows to be more adoptive to the hot environmental almost in acceptance with the results of our study. conditions as compared to the climate of KPK. To our knowledge, in Pakistan no accessible data The cumulative incidence in the buffalo (33.04%) exists on about the incidence of RB in different population was significantly (P<0.05) higher than cattle and buffalo breeds. Breed variation was cow’s (21.67%). This difference can be attributed recorded in the present study in both the species to the difference in nature of two different species that varied 15 to 35% in cattle and 26 to 34% in in terms of reproductive physiology. As previously buffaloes; that falls in the range reported by Robert discussed by Vale et al., (1988) and Danell (1987) et al., (2011) of 0.00% to 42.42% on basis of breed that higher RB incidence in buffalo population variations. might be due to low level of steroidal hormones Seasonal variation was significantly and high progesterone level. The silent heat in recorded among all the breeds of both the buffalo is also the most reasonable cause of higher species (Table 1). Such environmental stresses RB incidence. The incidence of RB in cows was on reproduction have also been explained earlier in agreement with the reported incidence by Yusuf by Dobson et al. (2000) and Gwazdauskas et al. et al. (2010). The results of our study were also (1981) that heat stress shortens the duration and similar to that of (Perez et al., 2007; Kendall et al., intensity of estrous expression leading to silent

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Table 1. Definitions and descriptive statistics of repeat breeding in different breeds of cattle and buffaloes, in KPK, Pakistan (2012-2013). *RB Variable code Description Values/level P-value Incidence[%(n/N)] Host specie Cattle/Buffaloes Cattle 21.67 (127/586) <0.05 Buffaloes 33.04 (192/586) Geography Climate based Semi-Arid 28.23(83/294) 0.071 Sub-Humid 22.15(76/343) Humid Region 29.94 (159/531) Breed Cattle breeds Zebu cattle (Achai) 20.68 (6/29) <0.05 Cross bred 18.06 (58/321) Exotic Breeds 15.23 (16/105) Non descriptive 35.87 (47/131) Buffalo breeds Nili Ravi 34.59 (137/396) <0.05 Aza-kheli 26.60 (29/109) Non descriptive 34.21 (26/76) Season Winter (Dec-March) 16.61(53/319) <0.05 Summer (Apr-June) 23.00(73/319) Monsoon (July-Sep) 34.00(107/319) Post Monsoon (Oct-Mid Dec) 26.29 (86/319) Age Cow’s Parity Primiparous 16.45 (38/231) <0.05 Multiparous 25.07 (89/355) Buffalo’s Parity Primiparous 33.14 (59/178) 0.500 Multiparous 33.00 (133/403) *n=number of repeat breeders, N= total animals observed.

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Figure 1. Geographic representation of the study Area KPK, Pakistan.

Figure 2. Showing the culling status of repeat breeders in the cattle and buffalo population in the studied animals.

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ovulation. These variations are also similar to the REFERENCES findings of (Agrawal et al., 2003). The astonishing major factor that is the Choudhary, P.C., B. Prasad and S.K. Misra. 1981. economic loss for the smallholder dairy farmers in Note on the use of rumen liquor in the this region of the study observed was the culling of treatment of chronic alkaline indigestion in RB, s. Where 62.70% repeat breeders were culled in cows. Indian J. Anim. Sci., 51: 356-360. one year period that in much higher than (39.17%) Agarwal, K.P. 2003. Augmentation of reproduction reported by Rabbani et al. (2010) from Pakistan in buffaloes, p. 121. In 4th Asian Buffalo five years back. As can be judged in Figure 2 higher Congress, New Delhi, India. culling percentage is for multiparous buffalo repeat Allen, J.F. 1996. Separate sexes and the breeders as compare to cows. It is because of the mitochondrial theory of aging. J. Theor. higher RB incidence rate for that group and low Biol., 180: 135-140. conception rate after calving. Also the RB season Bage, R., H. Gustafsson, B. Larsson, M. Forsberg, played an important role in mass culling because of Rodrıguez and H. Martinez. 2002. Repeat feed scarcity at that season. The longer the animal breeding in dairy heifers: follicular will take to conceive the greater chance it has to be dynamics and estrous cycle characteristics culled. These involuntary culling of RB reduces the in relation to sexual hormone patterns. rural farmer’s profitability significantly because it Theriogenology, 57: 2257-2269. is never correlated with dairy production. Beede, D.K. and R.J. Collier. 1986. Potential nutritional strategies for intensively managed cattle during thermal stress. J. CONCLUSION Anim. Sci., 62: 543-554. Bartlett, P.C., J.H. Kirk and E.C. Mather. 1986. To increase the reproductive performance Repeated insemination in Michigan Holstein of cattle and buffaloes in future in warming Friesian cattle: incidence, descriptive environmental challenges strategies shall be epidemiology and estimated economic adopted, making selection of breeds for adequate impact. Theriogenology, 26: 309-322. production purpose. Accordingly the genetics must Bulman, D.C and G.E. Lamming. 1978. Milk match the environment in future. progesterone levels in relation to conception, repeat breeding and factors influencing acyclicity in dairy cows. J. Reprod. Fertil., ACKNOWLEDGEMENT 54: 447-458. Danell, B. 1987. Long-term study of reproductive The authors are highly thankful to the performance in golden eagles in relation farmers of rural areas for their full cooperation to food supply in boreal Sweden. Ph.D. during study period. Thesis, Swedish University of Agricultural Sciences, Sweden. Dobson, H. and R.F. Smith. 2000. What is stress, and how does it affect reproduction. Anim.

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Reprod. Sci., 60-61: 743-752. Anim., 42: 449-456. Fregley, M.J. 1996. Adaptations: some general Rabbani, R.A., I. Ahmad, L.A. Lodhi, N. Ahmad characteristics, p. 3-15. In Fregley M.J. and G. Muhammad. 2010. Prevalence and C.M. Blatteis (eds.) Handbook of of various reproductive disorders and Physiology, Section 4: Environmental economic losses caused by genital prolapse Physiology, Vol. I. Oxford University Press. in buffaloes. Pak. Vet. J., 30(1): 44-48. Garcia-Ispierto, I., F. López-Gatius, P. Santolaria, Ramesh, V., T.T. Vanan and A. Varadhrajan. 2002. J.L. Yániz, C. Nogareda and M. López- Improvement of reproductive performances Béjar. 2007. Factors affecting the fertility of of buffaloes. Pashudhan, 17(01): 1-4. high producing dairy herds in northeastern Robert, Z., S. Tkalcic, V. Buic, I. Papal, D. Geres Spain. Theriogenology, 67(3): 632-638. and M. Samardzija. 2011. Repeat breeder Gustafsson, H. and U. Emanuelson. 2002. syndrome in dairy cows: influence of breed Characterization of the repeat breeding and age on its prevalence and the success of syndrome in swedish dairy cattle. Acta. Vet. a hormone therapy. Turk. J. Vet. Anim. Sci., Scand., 43: 115-125. 35(6): 405-411. Gwazdauskas, F.C., W.W. Thatcher, C.A. Kiddy, Ronchi, B., G. Stradaioli, S.A. Verini, U. M.J. Pape and C.J. Wilcox. 1981. Hormonal Bernabucci, N. Lacetera, P.A. Accorsi, A. pattern during heat stress following Nardone and E. Seren. 2001. Influence of PGF2alpha tham salt induced luteal heat stress and feed restriction on plasma regression in heifers. Theriogenology, 16: progesterone, estradiol-17β LH, FSH, 271-285. prolactin and cortisol in Holstein heifers. Intergovernmental Panel on Climate Change: AR4 Livest. Prod. Sci., 68: 231-241. (IPCC). 2007. The Intergovernmental Panel Silvia, W.J. 1994. Embryonic mortality and repeat on Climate Change 4th Assessment Report. breeder cows, p. 151-160. In Proceedings, Jackson Institute, University College, National Reprod Symposium, 27th Annual London. www.ipcc.ch. Conference of American Association of Kendall, N.R., A.P.F. Flint and G.E. Mann. 2009. Bovine Practitioners, Pittsburgh, PA. Incidence and treatment of inadequate Thrustfield, M. 2007. Veterinary Epidemiology, 3rd postovulatory progesterone concentrations ed. Wiley-Blackwell. 624p. in repeat breeder cows. Vet. J., 181: 158- Vale, G.W., O.M. Ohashi, J.S. Sousa and H.F.L. 162. Rebiero. 1988. Clinical reproductive Livestock Census. 2006. http://www.pbs.gov. problems of buffaloes in Latin America. pk/sites/default/files/aco/publications/ In Proceedings of the 2nd World Buffalo pakistan-livestock cencus2006/pak-11a. Congress, New Delhi, India. 2: 206-217. pdf. Wolfenson, D., Z. Roth and R. Meidan. 2000. Perez-Marın, C.C. and F. Espana. 2007. Oestrus Impaired reproduction in heat-stressed expression and ovarian function in repeat cattle: basic and applied aspects. Anim. breeder cows, monitored by ultrasonography Reprod. Sci., 60-61: 535-547. and progesterone assay. Reprod. Domest. Yusuf, M., T. Nakao, R.B. Ranasinghe, G. Gautam,

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S.T. Long, C. Yoshida, K. Koike and A. Hayashi. 2010. Reproductive performance of repeat breeders in dairy herds. Theriogenology, 73: 1220-1229.

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INCIDENCE, PAIN ASSESSMENT AND MANAGEMENT OF HORN AFFECTIONS IN BUFFALOES

K. Rama Rao1, Makkena Sreenu2, K.B.P. Raghavender3 and P.V.S. Kishore4

ABSTARCT INTRODUCTION

Pre and post operative pain in animals Horns of buffaloes are massive, angular suffering with horn affection should be attended and well developed with a wider base as compared to relieve stress on the animal as it may affect its to cattle. The thickness of the horn shell increases production. The incidence and, pain evaluation was towards apex until it becomes solid. The corium is studied. Out of 126 cases 76 (60.3%), 26 (20.16%), traversed by numerous blood vessels. The horn is 14 (11.11%), 9 (7.14) and 1 (0.79%) were fractures, prone to various affections like avulsion, fracture, avulsion, septic horn, overgrown horns and overgrowth, sepsis, fissures and cancer. Pre and horn cancer respectively. The incidence of horn post operative pain in animals suffering with horn affections were 62 (49.2%) 55 (43.7%) and 9 (7.1%) affection should be attended to relieve stress on the right, left and both horn affections. The common animal as it may affect its production. Most of these pain symptoms like restlessness, twitching of ears, affections do not respond to the routine medical shaking of head, bruxism, rubbing against fixed management and demand amputation of the horn objects, pawing at the affected site and evading (Sreenu and Kumar, 2006). This Paper reports about the affected site were observed in horn affections. the incidence, pain symptoms and management of Conservative and surgical Management of various various horn affections in buffaloes. affections of horn in buffaloes was recorded and discussed. MATERIALS AND METHODS Keywords: incidence, pain symptoms, horn affections, buffaloes The overall incidence of horn affections in buffaloes was recorded in terms of number of cases presented to the Department of Veterinary

1Veterinary Dispensory, Gollapudi Krishna (Dt), Andhra Pradesh, India 2Department of Veterinary Surgery and Radiology, NTR College of Veterinary Science, Sri Venkateswara Veterinary University Gannavaram, Krishna (Dt), Andhra Pradesh, India, E-mail: [email protected] 3Department of Veterinary Surgery and Radiology, College of Veterinary Science, Rajendra Nagar, Hyderabad, Andhra Pradesh, India 4Department of Veterinary Anatomy and Histology, NTR College of Veterinary Sciecne, Gannavaram, Krishna (Dt), Andhra Pradesh, India

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Surgery and Radiology, NTR College of Veterinary In the present study higher incidence of Science, Gannavaram, Andhra Pradesh, India fractures was recorded followed by avulsion, septic over a period of one and a half years i.e. January horn, overgrown horns and a case of cancer. The 2010 to August 2011 with available and relevant higher incidence of fractures was in accordance technical records. The various ailments, particular with the observations of Shivaprakash et al. (2007), to horn affections were tabulated and incidence of Salgar (2008) and Mistry (2009) while Sreenu and the each affection was noticed to arrive in terms Kumar (2006) and Mahida et al. (2009) reported of percentages. The clinical signs of the various higher incidence of Avulsion. The higher incidence horn affections, side of horn affected and parity of fractures among horn affections in the present were recorded. The various affections associated study might be due to vigorous and infighting with horn in buffaloes were recorded and the pain nature of buffaloes. The site of fracture was at tip, exhibited by the animals with a particular affection middle, lower third and base. The higher incidence was evaluated using pain score system modified of fractures at the base was more due to curly horns as per the procedure (appendix) of Holton et al. in buffaloes of this region which are mostly graded (2001). The horn affections recorded were treated murrah and its crosses, which might be locked during with appropriate method and discussed. fighting. The incidence of right side horns affection was more compared to the left in the present study. Similar observations were also made by Sreenu and RESULTS AND DISCUSSION Kumar (2006). On the contrary, Deshpande (1983) recorded involvement of left horn more than that Incidence of the right.Mahida et al. (2009) observed highest Out of 126 cases of horn affections 76 overgrown horns/ misshapen horns followed by (60.3%), 26 (20.16%), 14 (11.11%), 9 (7.14) and avulsion of the horn.The avulsion of the horn was 1 (0.79%) were fractures, avulsion, septic horn, noticed to be the major affection of the horn in overgrown horns and horn cancer respectively. The Surti buffaloes whereas the Mehsana buffaloes had site of fracture was at tip, middle, lower third and more incidence of the overgrown horns misshapen base in 8 (10.5%), 11 (14.5%), 22 (30%) and 35 horns. Naik et al., 1969; Kaul and Kalra, 1973 and (46%) respectively out of 76 cases of fracture. Somvanshi, 1991 stated that incidence of horn Out of 126 cases 62 (49.2%) 55 (43.7%) and cancer is a rare condition in buffaloes. Only one 9 (7.1%) were right, left and both horn affections. case was observed in the present study and it is in In avulsion cases 13 (50%) and 13 (50%) were accordance with Kumar and Tilagar (2000) who on right and left side. Among the fracture cases recorded a bilateral sqamous cell carcinoma of 40 (52.6%) and 36 (47.4%) were right and left the horn, while Salgar (2008) reported that four affections. Out of 14 cases of septic horn 6 (42.9%) Mehsani buffaloes suffered from horn cancer. and 8 (57.1%) were right and left horn affections. Damodaran and parthasarathy (1979) reported The over grown horns reported in the present study a case of neoplastic growth in a murrah buffalo were bilateral in 100 percent of the cases. In one bull. The observations suggestive of a very distinct buffalo with horn cancer it was observed to be a animal husbandry practices followed in the area left horn affection. where in the livestock owners are not interested in

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rearing the male as all the cases studied during the the horn at its lower third showed pain symptoms present study were only the female buffaloes. like animal trying to evade aside on palpation of affected area (100%), rubbing of the affected part Pain symptoms against fixed objects (71.42%), twitching of ears/ The pain symptoms exhibited by the shaking of head (71.42%) , restlessness (42.85%) buffaloes with horn affections were given in Table bruxism (28.57%) and decreased appetite and 1. water intake (28.57%). Avulsion of the horn: The prominent pain The symptoms like animal trying to evade symptom observed in the buffaloes suffering with the side on palpation of affected area (84.61%), Avulsion of the horn included restlessness (87.5%) rubbing of the affected part against fixed objects followed by twitching of ears (75%), decreased (76.92%), twitching of ears/shaking of head appetite and water intake (75%), rubbing the (76.92%) and restlessness (61.53%) decreased affected part against fixed objects (37.5%), and appetite and water intake (30.76%) and bruxism piloerection (37.5%) while all the animals tried (23.07%) were observed in buffaloes suffering with to evade aside on palpation of affected area. The the fracture of the horn at its base. The symptom symptoms like dilatation of pupil, abnormal posture like pilo erection was noticed in only one buffalo and bruxism were not observed with any animal. (7.69%) when the fracture was at the base of the Fracture of the horn: The symptom horn. observed in the buffaloes suffering with fracture of Septic horn: The symptoms like twitching the of the horn included pilo erection , dilatation of ears/shaking of head (100%) rubbing of the of pupil, restlessness, twitching of ears/shaking of affected part against fixed objects (100%), animal head, bruxism , rubbing of the affected part against tries to evade the side on palpation of affected fixed objects, tries to evade aside on palpation of area (75%), restlessness (50%) decreased appetite affected area and decreased appetite and water and water intake (50%) and bruxism (25%) were intake. observed in buffaloes suffering with septic horn. The pain reflexes varied with the site of the Over grown horn: The prominent pain fracture. The symptoms like animal trying to evade symptom observed with over grown horn in all the side on palpation of affected area in 100 percent buffaloes was animal trying to evade the side on and twitching of ears/shaking of head in 50 percent palpation of affected area. of the buffaloes were seen when the fracture was Horn Cancer: The symptoms like at the tip. twitching of ears/shaking of head (100%) rubbing The symptoms like animal trying to evade of the affected part against fixed objects (100%), the side on palpation of affected area (100%), animal tries to evade the side on palpation of rubbing of the affected part against fixed objects affected area (100%) and restlessness (100%) were (66.66%), twitching of ears/shaking of head observed in buffaloes suffering with horn cancer. (66.66%) and restlessness (33.33%) were noticed Pain score index and grading In avulsion in buffaloes suffering with the fracture of the horn cases the average pain score index was 12.00±0.46 at its middle third. and the pain was graded as severe. The average The buffaloes affected with fracture of pain score index was 3.50±0.50, 6.67±0.88,

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7.14±0.34 and 6.54±0.31 in the fracture at the conditions and is also supported through the tip, middle third, lower third and base of the horn changes in biochemical parameters. Sandford et respectively. The pain was graded as moderate in al. (1986) also stated that behavior was commonly all the fractures while the average pain score index used to recognize and assess the pain and distress in in septic horn was 9.40±1.03 and the pain was animals. Anil et al. (2002) mentioned that behavior graded as moderate. The average pain score index was a more sensitive indicator of pain than other in over grown horn cases was 2.50±0.50 and the physiological measures. According to Broom pain was graded as low. In the horn cancer case the (2000) pain is an aversive feeling or sensation average pain score index was 11.00±0.00 and the associated with actual or potential tissue damage pain was graded as severe. and resulting in physiologic, neuroendocrine, and In the present study the pain symptoms behavioral changes that are indicative of a stress observed in the buffaloes suffering from horn response. affections includes restlessness, rubbing the affected part against fixed objects, twitching of ears/shaking of head, decreased appetite and water MANAGEMENT OF HORN intake, trying to evade the side on palpation of AFFECTIONS affected area, dilatation of pupil, bruxism and pilo erection. The findings are in partial agreement with The animals brought for treatment were the report of George (2003). The pain symptoms evaluated to adopt treatment options. The animals are much in avulsion and cancer which might with avulsion showed loss of outer shell (Figure 1) be due to the exposed corium considered to be with severe excitement or resistance on touching, the sensitive part of the horn. The pain reflexes fracture at tip (Figure 2), middle (Figure 3), lower varied with the site of the fracture. The symptoms third (Figure 4) and base (Figure 5) and few cases observed with other conditions are specific to were fractures with discharging pus (Figure 6). A the problem. Assessment of individual animal case of horn cancer (Figure 7) and few cases were behavioral changes in response to pain is very overgrown horns causing pressure necrosis at the subjective and can be influenced by differences in back of the poll region (Figure 8) where the horn individual perception and interpretation. touches the skin was observed in the present study. According to the pain score index recorded, severe pain scores were noticed in avulsion Avulsion of the horn followed by horn cancer, septic horn and fractures All the buffaloes with avulsions were with moderate pain which might be due to sudden treated as outpatient cases by applying tincture exposure of the core in avulsions and induration of benzoin seal over the core of the horn after thorough the animal for slow growing cancer. The average irrigation with 1: 5,000 potassium permanganate pain score index in over grown horn cases was under surface analgesia with 4% Lignocaine graded as low as this is not a serious pathological Hydrochloride. problem. The behavioral changes observed due to pain were more in the cancer followed by avulsion Fracture of the horn which indicates more distress to animal in these The fracture cases at the tip were treated

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by applying Zinc oxide paste and bandage as tissues with antiseptic dressing using povidone conservative therapy. In cases which had complete iodine. fracture at middle or last third or the base of the horn exposing the communication of frontal Horn cancer sinus amputation was carried out. Two case of the A case of horn cancer was recorded in chronic fractures showed deep fissure and were a buffalo during the period of study which had effectively treated with packing off the cavitary a history of fracture at the base, one and half a area with Zinc oxide and wax (Figure 9). Due to month earlier. It was treated by horn amputation. anatomical peculiarity of the buffaloes the horns In this case there were discharges from the nostrils with incomplete fractures were also amputated to varying from frank blood in the initial days to avoid unnecessary complications (Figure 10).Two mucoid and purulent discharges in later stages.The cases with both fracture and avulsions were also management of horn affections vary depending on amputated (Figure 11 and Figure 12). the affection. The conditions reported under present study were managed with appropriate measures. Septic horn All cases of avulsion were successfully treated on Animals with septic horn (Figure 13) were outpatient basis, by spraying 4% lignocaine spray given prophylactic antibiotic therapy with 5g of as surface analgesic and applying tincture benzoin streptopenicillin for a period of 5 days along with seal over the stump of the horn after thorough antiseptic bandage using tincture benzoin so as to irrigation with mild antiseptics like (1: 5,000) maintain antibiotic levels at the time of surgery potassium permanganate as reported by Sreenu and and also to curtail the production of pus at the Kumar (2006). Verma and Kumar (1999) treated operative site. None of these four cases showed any avulsion of horn by covering it with an antiseptic postoperative sepsis or wound dehiscence which dressing with pine tar and carbolic acid in oil suggested that the adopted therapy yielded good soaked bandage and the authors opined that the results. avulsion of horn was not a serious condition except that there was profuse bleeding. The response was Over grown horn good in the present study as the tincture benzoin The overgrown horns were noted to cause has the properties of adhesiveness, antiseptic and varying degrees of damage ranging from simple styptic. In the present study treatment was given by discomfort to severe degree punctured wounds on spraying 4% lignocaine spray as surface analgesic the frontal, occipital or cervical regions based on effectively abolished the pain as evidenced by the the shape of the horn and angle of curvature and absence of pain symptoms. Verma and Kumar duration of contact (Figure 14). In some cases (1999) opined that cornual nerve block should be suppuration was also recorded as complications performed before treating avulsion of horn to avoid of soft tissue damage due to the overgrown horns. pain. Trimming of overgrown horns was done at a point The fracture cases at the tip were treated just above the junction of upper and middle third by packing off the cavity area with Zinc oxide using a hack saw without reaching the core. This as means of conservative therapy. In cases that was followed by treatment of the damaged soft deserved surgery, amputation by flap/modified flap

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Figure 1. Avulsion of horn in a buffalo. Note the Figure 2. Fracture at the tip of the horn in a buffalo. exposed core.

Figure 3. Fracture at the middle third of horn in a Figure 4. Facture at the lower third of horn in a buffalo. buffalo.

Figure 5. Fracture at the base of the horn in a Figure 6. Septic horn discharging thick pus in a buffalo. buffalo.

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Figure 7. Cancerous growth at the base of the horn Figure 8. Overgrown horns causing pressure in a buffalo. necrosis at the occipital region in a buffalo.

Figure 9. Deep fissure as a sequelae to fracture of Figure 10. incomplete fracture in a buffalo. the horn in a buffalo.

Figure 11. An old fracture of the horn at its lower Figure 12. A recent fracture of the horn at its third along with avulsed horn in a buffalo. lower third along with avulsed horn in a buffalo.

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Figure 13. Septic horn showing pus discharge from Figure 14. Punctured wounds at occipital region the base of the horn in a buffalo. due to over grown horns in a buffalo. method was carried out. Balappanavar (2005) used discomfort to severe degree punctured wounds on bamboo sticks as external splints for stabilization the frontal, occipital or cervical regions based on of the fractured horn at the base with intact skin. the shape of the horn and angle of curvature and The normal horn was used to stabilize the fractured duration of contact. In some cases suppuration horn in a criss cross pattern using splints. Sreenu was also recorded as complications of soft tissue and Kumar (2006) treated horn fractures effectively damage due to the overgrown horns. In the present by flap method of amputation of horn whereas study, trimming of overgrown horns was done at a Patil et al. (2007) treated a case of horn fracture point just above the junction of upper and middle using external splints using iron bars, rings, nuts third using a hack saw without exposing the core. and bolts. The treatment options followed in cattle This was followed by treatment of the damaged to treat fractures such as application of plaster soft tissues with routine antiseptic dressing using of paris bandage with or without splints, usage povidone iodine. Oheme and Prier (1974) opined of aluminium wire were not practcable due to that trimming of horn in bovine practice was anatomical variations of the horn in buffaloes as essential in case of its excess growth otherwise suggested by Sreenu and Kumar (2006). Animals it causes pressure sore of the head as well as with septic horn were given prophylactic antibiotic obstruction in vision. Sreenu and Kumar (2006) therapy with 5g of streptopenicillin for a period of suggested sawing the curved portion of the horn 5 to 7 days in addition to antibiotic bandage so as to without touching the corium. maintain antibiotic levels at the time of surgery and A case of horn cancer, recorded in the also to curtail suppuration at the operative site if present study was treated by horn amputation by they warrant amputation. This procedure was also flap method as reported by Angelo and Das (1970). followed by Sreenu and Kumar (2006) and Mahida Kumar and Thilagar (2000) who reported a case of et al. (2009). Mistry (2009) observed leucocytosis bilateral horn cancer in a buffalo mentioned that in septic horn of buffaloes which could be attributed the incidence of horn cancer was rare in buffaloes. to the trauma and inflammation. Various authors worked to treat horn cancer in The overgrown horns were noted to cause bovine with use of 5% liquor formaldehyde after varying degrees of damage ranging from simple amputation in cattle (Pandya,1932), flap method of

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APPENDIX Multidimensional pain scale for assessing pain in horn affections in buffaloes Details of animal: Case No: Species: Breed: Sex: Calvings: Case History: Clinical examination: Pain scale:

Score Objective A) Physiological Dilatation of pupil Yes:1 No:0 assessment Piloerection Yes:1 No:0 Respiratory rate Within reference range : 0 Above the reference range : 0 Heart rate Within reference range : 0 Above the reference range : 0 Temperature Within reference range : 0 Above the reference range : 0 B) Behavioral Posture Normal:0 Rigid:1 Arched back:2 Any abnormal:3 Behavior patterns Comfortable: 0 Restlessness:1 Twitching of ears/Shaking of head: 2 Bruxism: 3 Rubbing against fixed objects:4 Pawing at the site: 5 Vocalization Absent:0 Bellow/grunt:1 Mental state Change in mental state: yes:1 No:0 Evoked behavior No reaction on palpation of area:0 Trying to evade aside:1 Activity Appetite Normal: 0 Reduced: 1 Absent: 2 Water intake Normal: 0 Reduced: 1 Absent: 2 Total pain index 1-5 Low pain 6-10 Moderate pain >10 severe C) Biochemical Plasma cortisol C-reactive protein

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amputation (Angelo and Das, 1970), radiotherapy scale to measure acute pain in dogs. Vet. after horn amputation (Joslin, 1972), flap method Rec., 148(17): 525-531. of horn amputation following triphining of frontal Ithaca, NY. and C.A. Joslin. 1972. After loading sinus (Mohanty et al., 1972), administration of methods in radiotherapy. In Halnan, K.E. autogenous vaccine (Pachauri and Singh, 1978), (ed.) Recent Advances in Cancer and flap method of horn amputation using wire saw Radiotherapeutics. Churchill, Livingstone, (Kumar and Thilagar, 2000), flap method (Yadav London. p. 353. et al., 2002) amputation of horn by rising ventral Kaul, P.L. and D.S. Kalra. 1973. Incidence of horn flap after resection of horn (Sreenu and Kumar cancer in Haryana state. Haryana Agric. 2006) and modified flap method horn amputation Univ. J. Res., 3: 161-165. (Mahida et al., 2010). Kumar, R. and S. Thilagar. 2000. An unusual case of bilateral horn cancer in a buffalo. Indian Vet. J., 77: 48-49. REFERENCES Mahida, H.K., P. Tank, M.A. Dhami, D.O. Joshi, A.S. Karle and H.S. Vedapathak. 2009. Angelo, S.J. and S.C. Das. 1970. Amaurosis in Epidemiological status of surgical affections dog-a case report. Indian Vet. J. 47(3): 268- of the buffalo horn at hospital population 270. and ambulatory villages. Indian Journal of Anil, S.S., L. Anil and J. Deen. 2002. Challenges of Veterinary Surgery, 30(2): 192-193. pain assessment in domestic animals. J. Am. Mistry, J.N. 2009. Surgical affections of horn in Vet. Med. Assoc., 220: 313-319. Mehsani buffaloes and their management. Balappanavar, B.R. 2005. External splints for the Indian Journal of Veterinary Surgery, 30(2): treatment of horn fracture of cattle. The 112-113. Indian Cow: The Scientific and Economic Naik, S.N., C.R. Balakrishna and H.P. Randella. Journal, 2(6): 434-435. 1969. Epidemiology of horn cancer in Broom, D.M. 2000. The evolution of pain. Vlaama Indian Zebu cattle: Breed incidence. Brit. Diergeneeskundig Tijdschrift, 69: 385-411. Vet. J., 125: 222-230. Damodaran, S. and K.R. Parthasarathy. 1979. Oheme, F.W. and J.E. Prier. 1974. Text Book Indian Vet. J., 49: 649. of Large Animal Surgery, Williams and Deshpande. 1983. Studies on horn cancer in cattle. Wilkins Co., Philadelphia, p. 1208. M.Sc. Thesis, Marathwada Agricultural Pachauri, S.P. and N.P. Singh. 1978. University, Parbhani, India. Clinicopathological studies on horn cancer George, L.W. 2003. Pain control in food animals. in bovines. Phillipp. J. V. Med., 17: 181- In Steffey, E.P. (ed.) Recent Advances 184. in Anesthetic Management of Large Pandya, B.S. 1932. Liquor formaldehyde in cases Domestic Animals. International Veterinary of horn cancer. Indian Vet. J., 9: 147. Information Service. Patil, A.S., S.L. Patil, P. Suvarna and C.M. Holton, L.L., J. Reid, E.M. Scott and A.M. Nolan. Sajjanavar. 2007. Malignant melanoma 2001. Development of a behaviour-based over nasal ridge in a bullock and repair of

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horn fracture using external splints in two bullocks. Indian Journal of Veterinary Surgery, 28(1): 44. Salgar, B.S. 2008. Surgical affections of horn in Mehsani buffaloes and their management. MSc. Thesis abstract. Indian Journal of Veterinary Surgery, 30(2): 128. Sandford, J., R. Ewbuck, V. Motony, W.D. Tavernor and Uvarovo. 1986. Guidelines for the recognition and assessment of pain in animals. Vet. Rec., 118: 334-338. Shivaprakash, B.V., D. Kumar and S.M. Usturge. 2007. Dehornig with primary closure, a preferred technique over traditional amputation and open healing: Long term study on 500 cases. Indian Journal of Veterinary Surgery, 28(1): 24-25. Sreenu, M. and Kumar, N.R. 2006. Affections of horn in buffaloes Indian Vet. J., 83: 1206- 1207. Somavanshi, R. 1991. Horn cancer in Indian cattle. Veterinary Bulletin, 61: 901-911. Verma, R. and N. Kumar. 1999. Affections of horn and their management. Intas Polivet, 2(2): 134-139. Yadav, G.U., P.T. Jadhao, S.D. Moregaonker, A.V. Bhikane and R.R. Mugale. 2002. An unusual horn cancer in a cow. A case report. Indian Vet. J., 79: 515-516.

465 Buffalo Bulletin (July-September 2016) Vol.35 No.3 Original Article Buffalo Bulletin (July-September 2016) Vol.35 No.3

EVALUATION OF FEEDING PRACTICES AND CERTAIN MINERALS STATUS OF LACTATING BUFFALOES IN COASTAL ZONE OF WESTERN INDIA

P.L. Sherasia*, P.R. Pandya, S. Parnerkar, B.R. Devalia and B.M. Bhanderi

ABSTRACT required through area specific mineral mixture, for obviating deficiency in the ration of buffaloes were To evaluate the nutritional status of given to the farmers of Porbandar district. buffaloes (n=31), samples of feeds, fodder and blood serum were analyzed for proximate and Keywords: feedstuffs, nutritional status, buffalo, certain mineral contents collected from the selected porbandar, India villages of coastal zone (Porbandar district) of western (Gujarat state) India. Average intakes of protein and metabolizable energy were 101 and INTRODUCTION 111% of requirement, respectively. Calcium (Ca) content ranged from 0.22 to 1.74% in roughages, India possesses the world’s largest as compared to 0.02 to 0.19% in concentrates. livestock population having 57% of the world’s Average phosphorus (P) content in concentrates buffalo (Bubalus bubalis) population. Currently, (0.42%) was almost three times higher than that India is the largest milk producing country in the of roughages (0.14%). Copper (Cu) level was world and buffalo is the main milk producing recorded low in most of the feed resources. animal, contributing more than 50% of the total Straws of jowar (Sorghum bicolor) (8.11 milk production in India with an average lactation ppm), wheat (Triticum aestivum) (5.71 ppm) and yield of ~1300 kg. This low yield is mainly due to bajra (Pennisetum glaucum) (9.82 ppm) were found feeding of poor quality feed resources, particularly low in zinc (Zn). Manganese (Mn) content in feeds crop residues and agro industrial by-products fed and fodder ranged from 9.65 to 73.0 ppm. Average to animals in rural households. Moreover, buffalo blood serum levels of Cu, Zn and Mn in buffaloes milk has a much higher fat content at 6 to 7% in were 0.63, 0.79 and 0.05 ppm, respectively. As comparison to 3.1 to 4.5% in cow milk. compared to critical level of Cu (0.65 ppm) and Accurate assessment of nutritional status Zn (0.80 ppm) in blood serum, more than 60% of dairy buffaloes is invaluable in modern livestock of the animals screened showed low Cu and Zn production. For efficient production, reproduction status. Based on the calculated intakes of protein, and maintenance of normal health in dairy animals, energy, Ca, P, Cu, Zn and Mn from various feed it is essential to provide protein, energy and resources, suggestions for correcting supply of minerals according to their requirement. Limited protein and energy, and extent of supplementation information was available about the extent of

Animal Nutrition Research Department, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat, India, *Email: [email protected]

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nutrients availability from different feeds and forma developed for the purpose. While selecting fodder fed to lactating buffaloes in western zone farmers, due care was taken to ensure that selected of India. Therefore, present study was undertaken farmers were evenly distributed in the village and to evaluate the existing feeding practices to know truly represent animal management practices of the the status of protein, energy and certain minerals in village. The recorded parameters from each farmer lactating buffaloes of Porbandar district of Gujarat included number of livestock, land area, irrigated state and to recommend necessary modifications. facilities, fodder and other crops being grown etc. In addition, information regarding the amount and types of feeds and fodder offered to their milch MATERIALS AND METHODS animals, rate of actual daily feed intake, milk yield and fat percent, physiological status of animal etc Sampling procedure were collected with the fair degree of precision on a The survey was conducted in Porbandar questionnaire, using standard sampling procedure. and Ranavav tehsils of Porbandar district in Western India. Two villages were selected at random from Analytical methods each tehsil, for collection of samples of feeds, Composite samples of green and dry fodder and blood serum (Figure 1). In each village, fodder, individual concentrate ingredients and about 15 dairy farmers were selected at random. homemade concentrate mixtures were collected The selected farmers were interviewed and the from the surveyed area. Samples were dried in desired information was collected in the pro- oven, ground (1 mm) and stored in airtight bags

Figure 1. Map of Porbandar district.

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until analysis. The amount of dry matter (DM), animal owners/farmers reared their animals on crude protein (CP) and Metabolizable energy grazing and supplementation of wheat straw (ME) available to buffaloes were calculated from (Triticum aestivum), maize straw (Zea mays), the records of intake of feeds and fodder, using groundnut gotars (by-products of leguminous digestibility coefficients/nutritive values given by crops) or local grasses (a mixture of leguminous ICAR (1998). Average body weight of buffaloes and non-leguminous species in varying in the district was considered at 500 kg and proportions) collected from the wasteland, as the requirements for protein (Kearl, 1982), energy basal roughages. Tur gotar (by product of Cajanus (Mandal et al., 2003), calcium (Ca), phosphorus cajan) was also fed, but availability was seasonal. (P), copper (Cu), zinc (Zn) and manganese (Mn) Some of the farmers had employed the practice (NRC, 2001) were worked out. of feeding with cultivated fodder like lucerne Feed and fodder samples were analyzed (Medicago sativa) or jowar (Sorghum bicolar), for proximate constituents (AOAC, 2007). For bajra (Pennisetum typhoides) and maize (Zea mineral analysis, samples were prepared and mays). Green fodder availability was only for digested using 5 ml concentrated HNO3 plus 1 ml limited period due to water scarcity and frequent concentrated HCl, by microwave digestion method drought in the area. Those farmers, who did not and total volume of mineral extract was made to have irrigation facilities, were feeding local green 25 ml with de-ionized water. Blood from jugular grasses and cotton balls with leaves available at vein was collected from the individual buffalo and the time of cotton crop harvesting. Whole carrot centrifuged at 2000 rpm for 10 minutes, to harvest (Daucus carota) plants, as subsidiary green fodder the serum. Serum samples were preserved in deep was available for feeding to milch buffaloes. freeze till further analysis. All the samples were The practice of feeding compounded analyzed for Ca, P, Cu, Zn and Mn, by Inductively cattle feed was rare in the area. Concentrates were Coupled Plasma-Optical Emission Spectrometer; mostly offered twice a day, at the time of milking. Optima 3300 RL, Perkin Elmer, Waltham, MA, Interestingly, it was observed that none of the USA. farmers supplemented the ration of animals with Statistical analysis mineral mixture, except for therapeutic purpose or Data were statistically analyzed using on recommendation of the veterinary practitioners. SAS 9.3 software package (2012), SAS Institute, However, some of the farmers were supplementing USA as per Snedecor and Cochran (1994). Overall common salt to their animals. These observations differences between treatment means were are consistence with the reports from Gujarat state considered significant when P<0.05. The data have (Garg et al., 2002; Chavda, 2003; Ghogale, 2003; been presented as mean±S.E. Bhanderi, 2007). Some rich progressive farmers/ cattle owners were also feeding concentrates to pregnant (for steaming up) and growing RESULTS AND DISCUSSION animals. Crushed wheat (Triticum aestivum), bajra (Pennisetum typhoides), cottonseed cake Current feeding practices (Gossypium spp.) and edible oil (approximately Survey revealed that most of the dairy 100 g) were commonly used for steaming up.

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Proximate composition differed (P<0.05) between villages of Porbandar Chemical composition revealed that whole tehsil. Average energy intake as % of requirement cottonseed and cottonseed cake were good source in the district (111) was slightly higher than that of protein and energy (Table 1). Amongst green of in Porbandar tehsil, whereas, lower than that of fodders, lucerne (18.4%) had highest CP, followed in Ranavav tehsil. Lal et al. (1998) reported 43.23 by whole carrot plant (11.3%), bajra (10.1%) and Mcal/d ME intake against the requirement of 46.44 jowar (8.6%). Straws were low in CP content, Mcal/d by lactating buffaloes yielding 10 to 14 kg except gotars of groundnut and tur. Silica content milk/day. The average ME intake (37.40 Mcal/d) was highest in jowar straw (5.8%), followed by by buffaloes in the district was in agreement with maize straw (4.3%). Data are in agreement with these authors. To fulfill the nutrient requirements the reports of Anonymous (2006) and Bhanderi of buffaloes in villages Rajpar and Vadwala, about (2007). 250 g protein meal may be included in the ration. The buffaloes in the district were fed adequately Nutritional status in terms of protein, whereas energy intake was Average DM intake (DMI) of buffaloes in slightly higher than the requirements. the district was 15.1 kg/d. The DMI of buffaloes in different tehsils, as well as in different villages Minerals profile of feeds and fodder within tehsils did not differ significantly (Table 2). The average Ca content ranged from 0.22 Lal et al. (1998) reported similar values of DMI to 1.74% in roughages as compared to 0.02 to (15.7 kg/d) by lactating buffaloes yielding 10 to 14 0.19% in concentrates (Table 3). Average P level in kg milk/d. Similarly, Singh et al. (2001) reported concentrates (0.42%) was almost three times higher 15.3 kg DMI/d by Murrah buffaloes yielding 7 to than that of roughages (0.14%). Similar finding 18 kg milk/d in their native breeding tract. were also reported by Ramana et al. (2001), Udar et Average protein intake in villages of al. (2003) and Garg et al. (2008). About 100, 28.6 Porbandar and Ranavav tehsils was 0.88 and and 50.0% samples of concentrates, green fodder 0.90 kg/d, respectively, which did not differ and dry fodder, respectively, were found to be significantly. However, the same differed (P<0.05) below the critical level of Ca. All samples of green between villages within tehsils. Protein intake as and dry fodders were also found to be deficient in percent of requirement in the district was 101. P (Table 4). Lal et al. (1998) reported 1.28 kg protein intake Lucerne (26.7 ppm) and carrot leaves (24.2 as against requirement of 1.20 kg/d by lactating ppm) were good source of Cu as compared to buffaloes yielding 10 to 14 kg milk/d. Lal et al. bajra, maize and jowar green. Tur gotar had highest (1999) reported protein intake of 1.25 kg/d in Cu content (12.5 ppm) amongst the dry fodder, Murrah buffaloes yielding 16 to 18 kg milk/d. however, most of the concentrate feed ingredients Average protein intake in the district (0.89 kg/d) were low in Cu. Zinc content was found to be very was lower as recorded by these workers. low in wheat straw (5.71 ppm). These values are The ME intake in Porbandar and Ranavav in agreement with values reported by Yadav et al. tehsils was 36.21 and 38.53 Mcal/d, respectively, (2002) and Mandal et al. (2004). Wheat bran (51.6 which did not differ significantly. However, the same ppm) was a good source of Zn. Mn levels in the

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Table 1. Chemical composition of feeds and fodder (% on DM basis; mean±S.E.).

Feedstuffs CP EE CF NFE Ash Silica Grains/seeds Bajra (7) 10.3 ± 0.71 3.3 ± 0.25 1.5 ± 0.06 82.3 ± 0.45 1.5 ± 0.05 0.7 ± 0.04 Wheat (6) 8.9 ± 0.48 1.4 ± 0.02 1.4 ± 0.07 86.7 ± 0.51 1.5 ± 0.01 0.5 ± 0.04 Maize (6) 8.8 ± 0.40 2.8 ± 0.27 2.5 ± 1.13 83.9 ± 3.14 2.0 ± 0.74 0.5 ± 0.41 Whole cottonseed 22.3 ± 0.21 18.2 ± 0.68 18.6 ± 0.74 37.1 ± 0.33 3.7 ± 0.17 0.6 ± 0.15 (10) Brans and cakes Wheat bran (4) 16.3 ± 0.32 3.3 ± 0.06 5.0 ± 0.08 71.3 ± 0.25 4.1 ± 0.14 0.8 ± 0.04 Cotton seed cake 27.0 ± 1.08 5.7 ± 0.53 21.2 ± 0.27 42.1 ± 0.21 4.0 ± 0.05 0.5 ± 0.06 (16) Green fodder/grasses Jowar green (4) 8.6 ± 0.31 1.8 ± 0.05 23.4 ± 0.46 60.6 ± 0.28 5.5 ± 0.26 2.6 ± 0.32 Maize green (5) 6.5 ± 0.60 1.3 ± 0.03 27.5 ± 0.96 58.2 ± 0.36 6.5 ± 0.15 2.3 ± 0.77 Whole carrot plant 11.3 ± 0.35 3.5 ± 0.18 12.3 ± 0.32 60.4 ± 0.27 12.5 ± 0.22 2.7 ± 0.05 (4) Lucerne green (7) 18.4 ± 0.95 2.2 ± 0.05 28.1 ± 1.49 41.7 ± 3.74 9.6 ± 0.54 0.5 ± 0.07 Cotton ball with 5.5 ± 0.71 1.1 ± 0.06 26.0 ± 0.37 61.2 ± 0.40 6.3 ± 0.26 3.8 ± 0.20 leaves (3) Bajra green (4) 10.1 ± 0.84 2.5 ± 0.05 28.3 ± 0.70 45.8 ± 0.28 13.3 ± 0.36 1.4 ± 0.08 Local grass (3) 5.9 ± 0.80 3.1 ± 0.20 35.4 ± 3.45 40.1 ± 0.55 15.4 ± 2.84 6.8 ± 0.11 Dry fodder Groundnut gotar 9.0 ± 0.80 1.4 ± 0.08 24.8 ± 1.24 56.2 ± 0.43 8.6 ± 0.38 3.2 ± 0.35 (7) Jowar straw (8) 3.9 ± 0.28 1.2 ± 0.14 26.8 ± 0.36 60.9 ± 0.39 7.2 ± 0.57 5.8 ± 0.46 Tur gotar (3) 8.8 ± 0.22 3.0 ± 0.10 27.6 ± 0.28 51.5 ± 0.35 8.7 ± 0.36 0.2 ± 0.03 Maize straw (10) 4.9 ± 0.18 0.6 ± 0.04 31.6 ± 2.36 55.0 ± 0.46 7.9 ± 0.67 4.3 ± 0.36 Wheat straw (8) 3.2 ± 0.28 0.8 ± 0.06 39.4 ± 3.36 46.6 ± 0.44 9.9 ± 1.67 2.3 ± 0.26 Bajra straw (6) 3.1 ± 0.88 0.9 ± 0.14 26.6 ± 3.36 59.5 ± 0.31 9.9 ± 1.67 3.3 ± 0.30

Figures in the parentheses indicate number of samples analyzed.

471 Buffalo Bulletin (July-September 2016) Vol.35 No.3 ME ± 6.23 ± 4.34 ± 3.42 ± 3.24 a a b b 116 ± 3.98 116 115 ± 4.34 115 100 111 111 ± 3.95 111 106 116 ± 2.45 ± 4.25 a b Protein Intake as % of requirement 97.6 110 97.7 ± 5.35 100 ± 4.73 101 ± 2.10 98.9 ± 4.38 104 ± 3.50 ME (Mcal/d) 7.63 ± 0.62 7.50 ± 0.45 7.74 ± 0.64 7.79 ± 0.82 7.67 ± 0.64 7.77 ± 0.79 7.57 ± 0.51 Requirement (lg/d) Protein 0.85 ± 0.12 0.87 ± 0.09 0.87 ± 0.06 0.90 ± 0.10 0.87 ± 0.12 0.89 ± 0.07 0.86 ± 0.12 ± 0.75 ± 0.46 a b ME (Mcal/d) 34.19 38.95 ± 0.44 38.24 ± 0.75 38.11 37.37 ± 0.42 36.21 ± 0.67 38.53 ± 0.36 Intake ± 0.11 ± 0.06 ± 0.08 ± 0.12 (kg/d) a b a b Protein 0.91 0.83 0.85 0.89 ± 0.08 0.88 ± 0.10 0.96 0.90 ± 0.07 P <0.05) DMI (kg/d) 14.2 ± 0.66 14.6 ± 0.43 15.2 ± 0.84 15.1 ± 0.61 14.7 ± 0.73 16.3 ± 0.49 15.5 ± 0.46 MY (kg/d) 9.4 ± 1.22 8.6 ± 1.65 9.7 ± 0.32 9.3 ± 0.80 9.5 ± 0.71 9.6 ± 0.88 9.1 ± 0.94 Particular values differ significantly in a column ( values differ Degam Vadwala Rajpar District average Tehsil Tehsil average Bileshwar Tehsil Tehsil average Table 2. Nutritional status of lactating buffaloes in Porbandar district. 2. Nutritional status of lactating buffaloes Table a,b

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Table 3. Minerals profile of feeds and fodder (DM basis; mean±S.E.). Feedstuffs/ Ca (%) P (%) Cu (ppm) Zn (ppm) Mn (ppm) critical level < 0.30 < 0.25 < 8.0 < 30.0 < 40.0 Grains/seeds Bajra (7) 0.04 ± 0.01 0.29 ± 0.01 5.58 ± 0.09 32.4 ± 0.47 13.2 ± 2.07 Wheat (6) 0.04 ± 0.00 0.29 ± 0.01 4.68 ± 0.18 23.0 ± 0.72 32.5 ± 1.08 Maize (6) 0.02 ± 0.00 0.21 ± 0.01 3.07 ± 0.20 17.6 ± 0.54 9.93 ± 0.32 Whole cottonseed 0.15 ± 0.01 0.40 ± 0.01 7.77 ± 0.29 27.1 ± 0.49 9.65 ± 0.21 (10) Brans and cakes Wheat bran (4) 0.11 ± 0.00 0.84 ± 0.01 12.7 ± 0.43 51.6 ± 1.44 48.9 ± 1.69 Cotton seed cake 0.19 ± 0.02 0.48 ± 0.01 8.74 ± 0.43 31.6 ± 1.44 19.0 ± 1.69 (16) Green fodder/grasses Jowar green (4) 0.41 ± 0.01 0.15 ± 0.01 8.60 ± 0.25 23.8 ± 0.18 50.9 ± 2.27 Maize green (5) 0.22 ± 0.01 0.12 ± 0.00 12.9 ± 0.24 26.5 ± 0.89 44.7 ± 1.96 Whole carrot plant 1.03 ± 0.05 0.23 ± 0.02 24.2 ± 0.26 20.5 ± 0.57 40.8 ± 0.89 (4) Lucerne green (7) 1.22 ± 0.06 0.22 ± 0.01 26.7 ± 0.61 23.2 ± 1.36 40.2 ± 2.21 Cotton ball with 0.36 ± 0.03 0.17 ± 0.01 8.41 ± 0.57 28.8 ± 0.40 30.7 ± 0.84 leaves (3) Bajra green (4) 0.26 ± 0.02 0.15 ± 0.01 14.3 ± 0.57 32.4 ± 0.40 30.2 ± 0.84 Local grass (3) 1.58 ± 0.11 0.13 ± 0.01 15.1 ± 0.54 24.4 ± 1.48 73.0 ± 2.10 Dry fodder Groundnut gotar (7) 1.74 ± 0.09 0.15 ± 0.02 9.82 ± 0.45 16.6 ± 1.27 54.1 ± 5.38 Jowar straw (8) 0.52 ± 0.05 0.11 ± 0.02 6.04 ± 0.25 8.11 ± 0.76 15.9 ± 0.65 Tur gotar (3) 1.07 ± 0.14 0.18 ± 0.03 12.5 ± 0.72 16.1 ± 1.44 23.0 ± 2.80 Maize straw (10) 0.28 ± 0.03 0.10 ± 0.02 5.64 ± 0.25 13.4 ± 0.16 24.3 ± 0.65 Wheat straw (8) 0.28 ± 0.04 0.08 ± 0.02 6.81 ± 0.85 5.71 ± 1.28 28.9 ± 7.65 Bajra straw (6) 0.24 ± 0.00 0.09 ± 0.01 6.34 ± 0.12 9.82 ± 0.08 34.3 ± 0.83

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Table 4. Feedstuffs having below critical levels of minerals. Feed stuff/ Ca P Cu Zn Mn critical level (< 0.30%) (< 0.25%) (< 8.0 ppm) (< 30.0 ppm) (< 40.0 ppm) All, except Wheat, maize, All, except Maize cottonseed cake, Concentrate All (100%) cottonseed wheat bran (16.7%) wheat bran (50%) (83.3%) (66.7%) Jowar green, Maize green, All, except Cotton ball, Maize green, Green fodder Bajra green All (100%) bajra green bajra green Whole carrot (28.6%) (85.7%) (28.6%) plant (42.5%)

Maize straw, All, except All, except Wheat straw, groundnut gotar Dry fodder All (100%) All (100%) groundnut Bajra straw and tur gotar gotar (83.3%) (50%) (66.7%)

Table 5. Minerals content in blood serum of buffaloes. Ca (mg/dl) P (mg/dl) Cu (ppm) Zn (ppm) Mn (ppm) Particular < 8.0 < 4.50 < 0.65 < 0.80 < 0.02 8.75±0.23 5.31±0.23 0.63±0.8 0.79±0.03 0.05±0.00 Buffaloes (6.33-12.19) (4.65-6.89) (0.54-1.43) (0.53-0.97) (0.04-0.12) % of buffaloes 47.0 56.0 57.6 66.7 0.0 showing deficiency

Figure in the parenthesis indicate range.

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district ranged from 15.9 to 73.0 ppm in roughages ACKNOWLEDGEMENTS and from 9.65 to 49.0 ppm in concentrate feeds. The authors are grateful to the Department Mineral profiles of blood of Animal Nutrition, College of Veterinary Science The average blood serum Ca and P were 8.75 and Animal Husbandry, Anand Agricultural and 5.31 mg/dl, respectively (Table 5). Buffaloes University and the Management of National Dairy screened in the district showed 47 and 56% lower Development Board, for providing necessary serum Ca and P, respectively. These findings facilities and financial support to carry out this are similar to those of Ramana et al. (2001) and research work. Mandal et al. (2004). Average serum Cu and Zn content were 0.63 and 0.79 ppm, respectively. As compared to critical level of Cu (0.65 ppm) and Zn REFERENCES (0.80 ppm) in blood serum (Cuesta et al., 1993) more than 50% of the buffaloes screened showed Anonymous, 2006. Progress Report Presented in low Cu and Zn values. The Mn content of blood the Meeting of Research Sub-Committee on serum was within the normal range. The lower Animal Production. Published by Animal concentration of these minerals in feeds and fodder Nutrition Research Department, Faculty of might have resulted in lower level in blood serum Veterinary Science and Animal Husbandry, (Bhattacharya et al., 2004). However, blood serum Anand Agricultural University, Anand mineral levels are not always true indicators of Campus, Anand, India. mineral deficiency as minerals may be mobilized AOAC. 2007. Official Methods of Analysis, 18th ed. from the target tissue, during low dietary intake Association of Official Analytical Chemists, and complex interrelationships (McDowell et al., Washington, D.C., USA. 1993). Hence, regular supplementation of mineral Bhanderi, B.M. 2007. Assessment of feeding mixture in the ration of buffaloes is necessary. practices and status of certain minerals in dairy animals of Jamnagar district of Gujarat state. M.V.Sc. Thesis, Anand CONCLUSION Agricultural University, Anand, Gujarat, India. Study revealed that majority of lactating Bhattacharya, B.N., B.C. Sarmah, A. Baruah, K.K. buffaloes in Porbandar district was deficient in Baruah, K.C., Goswami, R N and D.J. essential minerals like Ca, P, Cu, Zn and Mn. It is Kalita. 2004. Mineral profile of lactating necessary to supplement these deficient minerals, cattle of two agro-climatic zones of Assam through supplementation of area specific mineral under field conditions. Indian J. Anim. Sci., mixture along with protein meal in the ration 74: 1206-1207. of buffaloes. Deficient trace minerals may be Chavda, M.R. 2003. Assessment of feeding supplemented in the form of chelates, for better practices and status of certain minerals minerals bio-availability. in dairy animals of Patan district of north Gujarat. M.V.Sc. Thesis, Gujarat

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Agricultural University, Sardarkrushinagar, Mandal, A.B., S.S. Paul and N.N. Pathak. 2003. Gujarat, India. Nutrient requirements and feeding of Cuesta, P.A., L.R. McDowell, W.E. Kunkle, F. buffaloes and cattle, 1st ed. International Bullock, A. Drew, N.S. Wilkinson and F.G. Book Distributing Co., Lucknow, U.P., Martin. 1993. Seasonal variation of soil India. and forage mineral concentrations in North Mandal, A.B., P.S. Yadav and V. Kapoor. 2004. Florida. Communication in Soil Science and Mineral status of buffaloes under farm Plant Analysis, 24(3-4): 335-347. feeding condition of Faridabad district of Garg, M.R., B.M. Bhanderi and S.K. Gupta. Haryana state. Indian J. Anim. Nutr., 21: 2008. Effect of supplementing certain 104-110. chelated minerals and vitamins to overcome McDowell, L.R., J.H. Conrad and F.G. Hembry. infertility in field animals. Indian J. Dairy 1993. Minerals for grazing ruminants Sci., 61(1): 181-184. in tropical regions. Animal Science Garg, M.R., B.M. Bhanderi and P.L. Sherasia. Department, Centre for Tropical Agriculture, 2002. Trace minerals status of feeds and University of Florida. The U.S. Agency for fodders in Junagadh district of Gujarat. International Development and Caribbean Indian J. Dairy Sci., 55: 154-158. Basin Advisory Group (CBAG). Ghogale, P.P. 2003. Assessment of feeding NRC. 2001. Nutrient Requirements of Dairy Cattle, practices and status of certain minerals 6th ed. National Research Council, National in dairy animals of Sabarkantha district Academy of Science, Washington, DC. of North Gujarat. M.V.Sc. Thesis, Gujarat Ramana, J., C.S. Prasad, N.K.S. Gowda and K.S. Agricultural University, Sardarkrushinagar, Ramachandra. 2001. Levels of micro- Gujarat, India. nutrients in soil, feed, fodder and animals ICAR. 1998. Nutrient Requirements of Livestock of North East transition and dry zones of and Poultry. Indian Council of Agricultural Karnataka. Indian J. Anim. Nutr., 18: 235- Research, New Delhi, India. 242. Kearl, L.C. 1982. Nutrient Requirements of SAS. 2012. Base SAS 9.3 Procedures Guide: Ruminants in Developing Countries. Statistical Procedures, 2nd ed. SAS Institute International Feedstuffs Institute, Utah Inc. Cary, NC, USA. State University, UMC 46, Logan, Utah Singh, J., K.K. Yadav and A.B. Mandal. 2001. 84322 USA. Feeding plane of milch Murrah buffaloes in Lal, D., V.B. Dixit, U. Arora and T.R. Chauhan. its native breeding tract. Buffalo J., 17(1): 1999. Nutritional status of high yielding 1-12. buffaloes – A case study. Indian J. Anim. Snedecor, G.W. and W.G. Cochran. 1994. Prod. Manage., 15(1): 39-41. Statistical Methods, 6th ed. Oxford and IBH Lal, D., V.B. Dixit, T.R. Chauhan and V.S. Solanki. Publication Company, New Delhi, India. 1998. A critical analysis of feeding system Udar, S.A., S. Chopde and R.N. Dhore. 2003. of lactating buffaloes in Hisar. Indian J. Mineral profile of soil, feeds and fodder and Anim. Prod. Manage., 14(3): 164-166. buffaloes in Western Agro-climatic Zone of

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Vidarbha. Anim. Nutr. Feed Techn., 3: 165- 172. Yadav, P.S. A.B. Mandal and D.V. Dahiya. 2002. Feeding pattern and mineral status of buffaloes in Panipat district of Haryana state. Anim. Nutr. Feed Techn., 2: 127-138.

477 Buffalo Bulletin (July-September 2016) Vol.35 No.3 Original Article Buffalo Bulletin (July-September 2016) Vol.35 No.3

GROSS MORPHOLOGICAL STUDIES ON MAJOR SALIVARY GLANDS OF PRENATAL BUFFALO

K. Raja, M. Santhi Lakshmi*, G. Purushotham, K.B.P. Raghavender, T.S. Chandrasekhara Rao and D. Pramod Kumar

ABSTRACT morphological, salivary glands, mandibular duct

Gross morphological study was conducted on the major salivary glands of 42 buffalo foetuses MATERIALS AND METHODS ranging from 73 to 253 days. The mandibular gland was the largest salivary gland during the prenatal The prenatal specimens of unknown age, life. Dense compact lobulation of the parotid and irrespective of the sex, nutritional status of the mandibular glands was observed first at 155 days of mother were collected from slaughter houses the foetal age. The mandibular gland was covered by the CVRL (Curved Crown Rump Length) of specimens confluence of linguo-facial, occipital and maxillary was measured to estimate the age of by Soliman’s veins laterally and developing thymus caudally (1975) formula. Gross morphological changes throughout the prenatal period. Differentiation of were studied in feasible foetal age from of 73 days dorsal and ventral parts of the sublingual gland was to 253 days regarding shape, size, location and observed at 108 days of foetal age. relation of the parotid, mandibular and sublingual Major salivary glands of various domestic salivary glands. animals are paired structures, which includes parotid, mandibular and sublingual glands. Salivary glands fulfill important role in the oral RESULTS AND DISCUSSION biology by producing saliva for lubrication, as well as supplying electrolytes, mucus, antibacterial The parotid gland was situated along the compounds and various enzymes to the oral cavity. caudal border of the masseter muscle extending Loss of salivary glands function can result in the from the region of the external auditory canal to wide spread deterioration of oral health (Hsu the level above the angle of mandible. It was in the et al., 2010). Study of normal development of form of a pyramid with loosely arranged multiple salivary glands will be helpful for both anatomists lobules at 73 day foetal age. However the shape and clinicians as they are having important role of the gland was found to be evident as triangular in several dreadful diseases like rabies, foot and with a broad base and apex during 84 days to 253 mouth disease and other viral diseases. days. Gradual change in the shape may be due to growth of the gland during the prenatal life. The Keywords: buffaloes, Bubalus bubalis, Gross base of the gland was superior with a notch placed

Department of Veterinary Anatomy, College of Veterinary Science, Rajendranagar, Andhra Pradesh, India, *E-mail: [email protected]

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around the external auditory canal, while the apex foetuses of 36 to 53 days (Guizetti and Radlanski, was situated little above the angle of the mandible 1996). The duct of the parotid gland was opened (Figure 1). Subsequently the gland reached the into the mouth cavity at the level of upper 2nd space between the base of the ear, vertical ramus erupting cheek tooth till 145 days, the same was of the mandible and sterno-mandibularis muscle in evident at the level of upper erupting 3rd cheek mid and late foetal stages (Figure 2). tooth from 155 days to 253 days of foetal life. The colour of the parotid gland varied The mandibular gland was the largest from light yellow to light brown during the prenatal among the three major salivary glands in prenatal period, variation may be attributed to increased buffalo, while the parotid gland was reported to be vasculature to the gland. The weight of the parotid the largest major salivary gland in human beings gland ranged from 2.0 to 7.1 g during 84 to 253 during prenatal life (Attie and Sciubba, 1981). days. The length and width of the parotid gland It was long, narrow and curved throughout the ranged between 0.8 to 2.1cm and 0.5 to 2.4 cm prenatal period (Figure 1). respectively from 84 to 253 days. Gradual increase The mandibular gland was light yellow in length, width and weight of the gland was due in colour during the prenatal period. It was long, to increased proliferation of ducts, increased narrow and curved and extended from the region lobulation and connective tissue formation during of tympanic bulla to the level above the angle of the foetal stage. Compact lobulation and adult the mandible behind the parotid salivary gland in characteristic features of the gland were attained at early age groups (Figure 1). However the position 155 days (Figure 3). of the gland gradually changed to that of adult at The lateral surface was covered by parotid 123 days, but it was located caudo-medial to the fascia, developing parotido - auricularis muscle parotid salivary gland as reported in day old kid by and a muscle extending from the zygomatic arch Rauf et al. (2004) which continued throughout the to cervical fascia. The gland was reported to be mid and late foetal age groups. The average length, attached superiorly to the zygomatic arch. The width and weight of the mandibular gland ranged middle part of the gland was penetrated by the from 1.23 to 2.84 cm, 0.45 cm to 1.6 cm and 2.1 to maxillary vein from lateral to medial surface 8.00 g respectively during the prenatal period from throughout the prenatal life (Figure 1). 84 to 253 days. The medial surface was uneven and The mandibular gland was covered related to great cornu of hyoid, digastricus, laterally by fascia and the confluence of the occipito-hyoideus and sterno-mastoideus muscles, external jugular vein with maxillary, linguo-facial external carotid artery, external jugular vein and and occipital veins (Figure 1), which agree partly its tributaries, facial nerve and its branches. The with the findings of Rauf et al. (2004) in one anterior border was in contact with the parotid lymph day old kid. The gland was related to the larynx, node above and masseter muscle below throughout division of the common carotid artery, external the prenatal period (Figure 1). The medial surface carotid artery, 9th ,10th and 11th cranial nerves, of the parotid gland showed impressions for the stylo-hyoideus muscle and great cornu of hyoid mandibular salivary gland, parotid lymph node and medially and developing thymus caudally. The masseter muscle as reported in human embryos and anterior extremity was narrow and placed at the

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level of the angle of the mandible above the sterno- close association with the sublingual duct opened mandibularis muscle and the posterior extremity at closely along the side of monostomatic sublingual the level of fossa atlantis as reported in domestic duct at the caruncula sublingualis. animals (Budras and Habel et al., 2003). However The sublingual gland was the smallest the anterior extremity was carried to the level of among the major salivary glands (Figure 3). It was root of the tongue in mid and late age groups. composed of two parts, the dorsal (polystomatic) The mandibular gland showed loosely and ventral (monostomatic) parts. The dorsal and arranged lobules at 73 days. Dense compact ventral parts of the gland were distinguishable lobulation of the gland was observed between 155 at 108 days of foetal age. The dorsal part of the to 253 days of prenatal life (Figure 3). Mandibular gland was arranged in a chain of lobules from lymph node was placed above the anterior the palato-glossal arch to the incisive part of the extremity of the gland, a little cranial to or at the mandible, while the ventral part was lying beneath angle of the mandible. At 253 days the mandibular the mucosa of the floor of the mouth above the gland attained the characteristic shape, colour and mylohyoid muscle between the mandible laterally position to that of adult. and the muscles of tongue medially. The dorsal The mandibular duct was well developed and ventral parts of the gland were distinguished at 84 days but not amenable for dissection. It was at 108 days. The colour of the dorsal part varied found to be leaving the gland at lower third of the from light yellow to light brown during prenatal inferior border between 84 to 123 days and middle development, while the ventral part appeared as of the concave border between 145 to 253 days. light yellow between 189 to 253 days of prenatal The duct running on the floor of the mouth cavity in life.

Figure 1. Photograph of 84 day buffalo foetal head showing parotid (P) and mandibular (M) salivary glands and their relation to adjacent structures. PL- Parotid lymph node, EJ- External jugular vein, SM- Sterno-mandibularis, D-Dorsal buccal nerve, MA-Masseter muscle,E-External ear, T- Thymus.

481 Buffalo Bulletin (July-September 2016) Vol.35 No.3

Figure 2. Photograph of 145 day buffalo foetus showing compact lobulation of parotid gland (P) and loosely arranged lobules of mandibular (M) salivary glands. E- External auditory canal, EJ- Ext jugular vein, T -Thymus,SM- Sterno mandibularis muscle, MA- Masseter muscle

Figure 3. Photograph showing dense compact lobulation in foetal parotid (P), mandibular (M), and sublingual (L) salivary glands at 155 days in buffalo.

482 Buffalo Bulletin (July-September 2016) Vol.35 No.3

The ventral part of the gland was elongated Recent Progress and Future Opportunities. and situated beneath the mucosa of the floor of the Int. J. Oral. Sci., 2(3): 117-126. mouth above the mylohyoideus muscle between Latshaw, W.K. 1987. The Veterinary Developmental the mandible laterally and the muscles of tongue Anatomy. B.C. Decker Co., INC, Toronto. medially. The ventral part of the sublingual gland p. 128-129. was drained by only one duct, which opened Rauf, S.M.A., M.R. Islam and M.K. Anan. 2004. along the side of mandibular duct at caruncula Macroscopic and Microscopic study of the sublingualis as reported by Latshaw (1987) in mandibular salivary gland of black bengal domestic animals. But the ducts of the dorsal part goats. Bang. J. Vet. Med., 2(2): 137-142. of sublingual gland were not visible to the naked Soliman, M.K. 1975. Studies on the physiological eye during the prenatal period. chemistry of the allantoic and amniotic The weight, length and width of the ventral fluids of buffaloes at the various periods of part of sublingual gland ranged from 1.45 g to 2.65 pregnancy. Indian Vet. J., 52: 106-112. g, from 1.7 to 4.2 cm and 0.25 to 1.1 cm from 84 days to 253 days of embryonic life. Increase in the size and weight of the gland was due to increased proliferation of ducts, increased lobulation and connective tissue formation.

REFERENCES

Attie, J.N. and J.J. Sciubba. 1981. Tumors of Major and Minor Salivary Glands, Clinical and Pathologic Features in Current Problems in Surgery. Medical Book Publishers Inc, Chicago, p. 80-81. Budras and Habel. 2003. Colour Atlas of Bovine Anatomy, 1st ed. Schlutersche Verlagsgesellschaft mb H and Co. KG., Hans - Bochler - Alle 7, 30173 Hannover, p. 38-39. Guizetti, B. and R.J. Radlanski. 1996. Development of the submandibular gland and its closer neighbouring structures in Human embryos and fetuses of 19-67 mm CRL. Ann. Anat., 178: 509-514. Hsu, J.C.F., M. Kenneth and Yamada. 2010. Salivary gland branching morphogenesis,

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