STUDIES ON THE EFFICACY OF OVSYNCH AND MODIFIED OVSYNCH PROTOCOLS ON THE CONCEPTION RATE IN REPEAT BREEDER COWS KANTHARAJ, S.

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1 STUDIES ON THE EFFICACY OF OVSYNCH AND MODIFIED OVSYNCH PROTOCOLS ON THE CONCEPTION RATE IN REPEAT BREEDER COWS KANTHARAJ, S. DEPARTMENT OF VETERINARY GYNAECOLOGY AND OBSTETRICS VETERINARY COLLEGE, HEBBAL, BANGALORE KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR MAY, 2015 i

2 STUDIES ON THE EFFICACY OF OVSYNCH AND MODIFIED OVSYNCH PROTOCOLS ON THE CONCEPTION RATE IN REPEAT BREEDER COWS Thesis submitted to the KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR in partial fulfillment of the requirements for the award of the degree of Doctor of Philosophy IN VETERINARY GYNAECOLOGY AND OBSTETRICS By KANTHARAJ, S. DEPARTMENT OF VETERINARY GYNAECOLOGY AND OBSTETRICS VETERINARY COLLEGE, HEBBAL, BANGALORE KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR MAY, 2015 ii

3 KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR DEPARTMENT OF VETERINARY, GYNAECOLOGY AND OBSTETRICS VETERINARY COLLEGE, HEBBAL, BANGALORE CERTIFICATE This is to certify that the thesis entitled Studies on the efficacy of Ovsynch and modified Ovsynch protocols on the conception rate in repeat breeder cows submitted by Dr. KANTHARAJ, S. I. D. No. DVHK-1210 in partial fulfilment of the requirements for the award of Doctor of Philosophy in VETERINARY GYNAECOLOGY AND OBSTETRICS of the Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar is a record of bonafide research work carried out by him during the period of his study in this University under my guidance and supervision. And the thesis has not previously formed the basis for the award of any degree, diploma, associateship, fellowship or other similar titles. Bangalore May, 2015 Dr. V. CHANDRASHEKARA MURTHY Major Advisor Associate Professor Department of Veterinary Gynaecology and Obstetrics Approved by: Chairman : (Dr. V. CHANDRASHEKRA MURTHY) Nominated External Examiner : (Dr. K. KULASEKAR) Members : 1. (Dr. SURESH S. HONNAPPAGOL) 2. (Dr. U. KRISHNAMOORTHY) 3. (Dr. V. GIRISH KUMAR) 4. (Dr. S. G. RAMACHANDRA) iii

4 Dedicated to My Beloved Parents and Wife iv

5 ACKNOWLEDGEMENT Gratitude is when memory is stored in the heart and not in the mind The success of any venture depends upon the people who helped in its formation. I take this opportunity to thank the number of people who helped me in this work. Primarily my guide Dr. V. Chandrashekara Murthy, Associate Professor, Department of Veterinary Gynaecology and Obstetrics, Veterinary College, Bangalore who guided me from the very planning of this research, timely advice, constant encouragement and inspiring moral support. It was a great pleasure and privilege for me to have him as my guide and I shall remain ever grateful to him for his benevolent outlook and generosity of mind. My sincere and heartfelt regards goes to Dr. A. Krishnaswamy, Professor and Head, Department of Veterinary Gynaecology and Obstetrics, Veterinary College, Bangalore for his moral support and valuable suggestions that helped me in completing the research work and thesis. I am indebted to him. I am equally grateful to my Advisory Committee Members, Dr. Suresh S. Honnappagol, Animal husbandry Commissioner, Government of India, Dr. U. Krishnamoorthy, former Professor and Head, Department of LPM, Veterinary College, Bangalore, Dr. S. G. Ramachandra, Chief Research Scientist, Central Animal Facility, Indian Institute of Science, Bangalore and Dr. V. Girish Kumar, Professor and Head, Department of Veterinary Biochemistry, Veterinary College, Bangalore for their valuable suggestions and encouragement throughout the period of study. I am equally grateful to Dr. T.G. Honnappa, Associate Professor, Department of Veterinary Gynaecology and Obstetrics, Veterinary College, Bangalore for his moral support, constant support to write the thesis in time and his valuable help in statistical analysis. I extend my sincere thanks to Dr. G. Sudha, Dr. Ravindranath and Dr. Narasimha Murthy for their kind cooperation and moral support during the period of study. I am extremely thankful to the Dean of Rajiv Gandhi Institute of Veterinary Education and Research, Pondicherry for deputing me to pursue my higher studies at Bangalore. I take this opportunity to thank my teachers and colleagues at Pondicherry v

6 Dr. M. S. Raju, Professor and Head, Dr. D. Antoine, Professor and Dr. K. Murugavel, Associate Professor of the Department of Veterinary Gynaecology and Obstetrics, RIVER who were constantly encouraging me both personally and officially throughout the period of my study. I thank my Ph.D Colleagues Dr. T.R. Lakshmikanth, Dr. C. Jayakumar, Dr. K. S. Shwetha and Dr. Mahesh Dodamani, Dr. G. Ananda Mannegar and Dr. G.S. Naveen for the fun time I had with them and the moral support provided by them is countless. I should thank them for being with me in all situations and would like to continue my relation with them forever. I thank Dr. Avinash Warundeo Lakkawar and Sridhar for making my presence in Bangalore a memorable one. I thank my colleagues Dr. Sathish Mannapur, Dr. Imam, Dr. Kanthesh, Dr. Abbas and Dr. Sunil for making my work and stay memorable during my entire period of study. A word of thanks should be mentioned to my friends Dr. Sahadev, Dr. Deepti, Dr. Sunita, Dr. Rudresh, Dr. Mahendra, Dr. Sandeep and Dr. Sarkar for the wonderful time I had with them. All the non-teaching staff members, Siddharam(CM), Rajanna, Satish, Vijayalakshmi, Manju and his family of the Department of Veterinary Gyanecology and Obstetrics for their cooperation and help when needed. Special thanks to Dr. Manjunath and Dr. Baramappa, Veterinary Assistant Surgeons of the Animal Husbandry Department, Government of Karnataka who toiled with me under hot sunlight to get me the research cases and for the constant support rendered by them to complete my research work. I have to thank my friends Dr. Sumanth, Dr. Vivek, Dr. Bharathiraja and Dr. Senthil of IVRI, Bangalore who have made my life in Bangalore most memorable. Special heartfelt thanks are there for my mother, wife, son, sisters and uncles without whom it wouldn t have been possible for me to reach my goal. I am grateful to them for the sacrifice they made for me, specially my mom, wife in particular and son. In this recounts, I may not have thanked many people. This doesn t mean that I am ungrateful to them, it just means that I have a lousy memory. Bangalore May 2015 (Dr. S. Kantharaj) vi

7 CONTENTS CHAPTER TITLE PAGE No. I INTRODUCTION 1-5 II REVIEW OF LITERATURE 6-46 III MATERIALS AND METHODS IV RESULTS V DISCUSSION VI SUMMARY VII BIBLIOGRAPHY VIII ABSTRACT 141 vii

8 LIST OF TABLES Table No Topic Haemo-biochemical attributes (Mean ± SE) in repeat breeding control and treatment groups Time (Mean ± SE) of onset of estrus following PGF 2 α administration in cows subjected to various treatments Serum progesterone concentrations (Mean ± SE) at first GnRH in all the treatment groups Serum progesterone concentrations (Mean ± SE) in repeat breeding control and treatment groups at the time of A.I Basal and suprabasal progesterone concentrations in all the repeat breeder cows Conception rate (percentage) for repeat breeder control and treatment cows Page No viii

9 LIST OF FIGURES Figure No Topic Blood haemoglobin levels of repeat breeder cows in control and treatment groups Blood glucose levels of repeat breeder cows in control and treatment groups Serum calcium concentrations of repeat breeder cows in control and treatment groups Serum phosphorus concentrations of repeat breeder cows in control and treatment groups Time of onset of estrus after PGF 2 α administration in repeat breeder cows with various treatments Serum progesterone concentrations at first GnRH in all the treatment groups Serum progesterone concentrations in repeat breeding control and treatment groups at the time of A.I Conception rate for repeat breeder cows in control and treatment groups Page No ix

10 LIST OF PLATES Plate No Topic Page No 1 Progesterone releasing intra vaginal device (Triu-B) for use in cattle 73 2 Insertion of progesterone releasing intra vaginal device (Triu-B) in a cow 73 3 Uterine cytology by lavage technique revealing the endometrial cells 74 4 Uterine cytology by lavage technique revealing PMNs with endometrial cells Pregnancy diagnosis at 30 days post AI by ultrasonography 75 x

11 LIST OF ABBREVIATIONS A.I AIDE ATP BCS CIDR CL DIM FTAI GnRH h HAI Hb LH ng/ml P/AI PGF 2 α PMN PRID RBH TAI TET Triu-B VH Artificial Insemination Artificial Insemination at Detected Estrus Adenosine triphosphate Body Condition Score Controlled Internal Drug Release Corpus Luteum Days in Milk Fixed Time Artificial Insemination Gonadotrophin Releasing Hormone hours Observed Heat Artificial Insemination Haemoglobin Luteinizing Hormone nanogram per ml Pregnancy per Artificial Insemination Prostaglandin F 2 alpha Polymorphonuclear cells Progesterone Releasing Intra vaginal Device Repeat Breeding Heifers Timed Artificial Insemination Timed Embryo Transfer Progesterone Impregnated Intra vaginal Device Virgin Heifers xi

12 SAI MAI OVS-FTAI OVS-HAI Single Artificial Insemination Multiple Artificial Inseminations Ovsynch with Fixed Time Artificial Insemination Ovsynch with Artificial Insemination at Observed estrus POVS-FTAI Progesterone Based Ovsynch with Fixed Artificial Insemination POVS-HAI Progesterone Based Ovsynch with Artificial Insemination at Observed estrus xii

13 Introduction

14 I. INTRODUCTION Repeat breeder cow comprises a heterogeneous group of sub fertile cows, without any anatomical abnormalities or infections of the reproductive tract, that exhibit a variety of reproductive disturbances in a consistent pattern during the course of 3 or more consecutive estrous cycles of normal duration (17-25 days). Repeat breeding is defined as a condition in which cattle and buffaloes which have regular estrous cycles but have failed to become pregnant following three or more breeding (Bartlett et al., 1986; Roberts, 1986) despite, they come normally in heat and show clear estrus signs with no clinically detectable reproductive disorders (Ahmed et al., 2010; Yusuf et al., 2010). Considering the great demands on dairy cow production (which ideally requires obtaining a calf per cow per year), repeat breeder cow has an important impact on dairy cattle economics. The etiology of repeat breeding in dairy cows is unclear and multifactorial. Potential causes of repeat breeding includes fertilization failure (29-41%), embryonic mortality (21-35%), defective luteal secretion of progesterone and other hormonal imbalances, errors in estrus detection, various defects in sperm or egg function and nutritional imbalances (Kim et al., 2007). Huge economic losses are encountered due to high incidence (20-39%) of repeat breeding (Nanda and Singh, 2008). Exploration of possible causes and measures for restoring fertility in repeat breeding animals has been the objective of reproductive biologists since the beginning. In

15 2 spite of good progress made, the causes of conception failure are largely not well understood and repeat breeding remains the biggest problems of the dairy industry. To reduce the negative effects of repeat breeding on the farm profitability, an effective therapy must be established after a proper diagnosis. A common recommendation for low fertility cows is a double Artificial Insemination (AI). Researchers have postulated that double AI might benefit lateovulating cows (Wilcox and Pfau, 1958) or repeat breeding due to gross errors in detection of estrus (O Farrell et al., 1983). Researches were carried out on double AI during the estrous period (Bostedt, 1976; Stevenson et al., 1990; Shukla and Misra, 2008) or multiple AI during spontaneous estrus (Singh et al., 2005) in repeat breeder cows and heifers with varying conception rates. Rodrigues et al. (2010) opined that problems associated with repeat breeders can be eliminated by the use of hormonal protocols designed to induce timely follicular development, ovulation and acceptable pregnancy rates. Estrus synchronization is the manipulation of the reproductive process so that cows can be bred with normal fertility during a short, predefined interval. This control facilitates breeding in two ways: it reduces and in some cases eliminates the labor of detecting estrus, and it allows the producer to schedule the breeding. Numerous hormonal treatments have been used for synchronization of estrus and ovulation to improve results in repeat breeding condition. Pursley et al. (1995) were the first to use a protocol for synchronization of the estrus and the ovulation in cows with fixed time artificial insemination (FTAI). Ovsynch

16 3 is a protocol based on the administration of GnRH, PGF 2α and GnRH (Pursley et al., 1995) to schedule the insemination time. Ovsynch protocol with FTAI has been developed, tested and intensively used in dairy cows and it yielded overall conception rates similar to those obtained after breeding to detected estrus (Pursley et al., 1997, 1998; Stevenson et al., 1999). Even though Ovsynch protocol has been studied both in lactating cows and repeat breeder cows, up to 30 % of the cows may not be synchronized to Ovsynch (Peters and Pursley, 2003). Variation among dairy cows in their synchronization rate to Ovsynch was attributed primarily to the stage of estrous cycle in which Ovsynch is initiated. Vasconcelos et al. (1999) and Moreira et al. (2000) reported that conception rates were greater with the Ovsynch protocol initiated during the early luteal phase (i.e. between day 5 and 12 of the estrous cycle) in cows and heifers respectively. Several studies have examined the effect of exogenous progestin supplementation on fertility in lactating dairy cows. The benefit of supplementing progesterone in lactating dairy cows through an Ovsynch protocol was demonstrated in a larger study (Melendez et al., 2006). Exogenous progesterone administration between the first GnRH and PGF 2 α treatment improved conception rate in dairy cattle (Stevenson et al., 2006; Galvao and Santos, 2010). Drost (2007) and Azawi et al. (2012) reported a higher pregnancy rate than Ovsynch protocol when progesterone (Controlled Internal Drug Release-CIDR) based Ovsynch protocol was used in repeat breeding buffaloes. Efficiency of Ovsynch for effectively synchronizing ovulation and fertility potential in dairy cows can be accomplished by its modifications. A modification of

17 4 Ovsynch protocol namely Ovsynch-56 allowed more time for follicular maturation and further improved conception rate by 10 per cent compared to the original Ovsynch. In this modification, the time from PGF 2 α administration to the second GnRH treatment was extended from 48 hours to 56 hours, maintaining 16 hour interval from the second GnRH to fixed time A.I. (Brusveen et al., 2008). Although Ovsynch treatment allows for acceptable pregnancy rates without heat detection, it does not necessarily eliminate the need for heat detection. Ovsynch-treated animals should be observed closely for return to estrus 18 to 24 days later. Additionally, up to 20 per cent of treated cows will display standing estrus between days six and nine of the Ovsynch protocol (Geary et al., 2000; De Jarnette et al., 2001). Conception rate of these animals will likely be compromised if bred strictly on a timed A.I. basis. Kasimanickam et al. (2006) reported 17 per cent of cows detected in estrus after the PGF 2α administration but before the second GnRH administration in CIDR-based Ovsynch and Co-Synch study. They reported that acceptable pregnancy rates can be achieved with or without inclusion of estrus detection in these protocols. There is paucity of literature on application of Ovsynch in repeat breeder cows with reference to the stage of the cycle at which it was initiated. Literatures are scanty on the information about the conception rate in repeat breeders inseminated at observed estrus or fixed time A.I in Ovsynch protocol and its modifications. Hence, the present study was attempted with the following objectives.

18 5 1. To study the efficacy of Ovsynch protocol to synchronize the estrus and ovulation in repeat breeder cows. 2. To study the efficacy of modified Ovsynch protocols to synchronize the estrus and ovulation in repeat breeder cows. 3. Evaluate the conception rate between Ovsynch and modified Ovsynch protocols in repeat breeder cows. 4. Compare the conception rate of Ovsynch and the modified Ovsynch protocols in repeat breeder cows. 5. To evaluate and compare the conception rate obtained from A.I. at observed estrus or FTAI in Ovsynch and modified Ovsynch protocols in repeat breeder cows.

19 Review of Literature

20 II. REVIEW OF LITERATURE Dairy cattle production requires great intensification, which has been demonstrated to affect negatively on the reproduction. One calf by cow and year is the reproductive objective in these animals. It means that cows must get pregnant after AI, maintain the pregnancy, have parturition after 270 days approximately, and wait for a period of days to be successfully inseminated again. Nevertheless, this is not always attained and cows must be re-inseminated during several consecutive cycles. In this context appears the repeat breeder cow, comprising a heterogeneous group of sub-fertile animals that exhibits a variety of reproductive disturbances in a consistent pattern during the course of three or more consecutive estrous cycles of normal duration (17-25 days). Any of these disturbances may affect the delicate interplay of estrous behavior, hormone patterns, and ovarian dynamics, which in synchrony with the uterine functions finally determine the outcome of mating or AI (Bage, 2002). A repeat breeder cow is one that has normal or nearly normal estrous cycles and estrus periods and has been bred thrice or more times to a fertile bull, yet failed to conceive (Roberts, 1971). Repeat-breeder cattle are defined as sub-fertile animals which fail to conceive with at least three services and have no anatomical or infectious irregularities. Repeat breeding syndrome is one of the frustrating gynaecological maladies of crossbred cows, leading to infertility (Rangnekar et al., 2002). It affects the reproductive efficiency and economy of milk production in dairy animals.

21 7 The incidence of repeat breeding as reported by Singh et al. (1981) was per cent. The incidence of repeat breeding, a major factor involved in reduced fertility, varies among various countries and ranging from 18 to 24 per cent (Stevenson et al., 1990). On the contrary, Sharma et al. (1991) reported a higher incidence (51.79 per cent) of repeat breeding in dairy cows. Huge economic losses are encountered due to high incidence (20-39 per cent) of repeat breeding (Nanda and Singh, 2008). Repeat breeding cow syndrome causes considerable economic losses in dairy cattle associated to reproductive failure; drop in milk yield and calves, and treatment cost. Numerous treatments have been studied to solve and prevent this syndrome at herd or individual level. To reduce the negative effects of repeat breeding on the farm profitability, an effective therapy must be established after a proper diagnosis. Estrus synchronization is the manipulation of the reproductive process so that females can be bred with normal fertility during a short, predefined interval. This control facilitates breeding in two important ways: it reduces and in some cases eliminates the labor of detecting estrus, and it allows the producer to schedule the breeding. Protocol that synchronize follicular dynamics, corpus luteum (CL) regression and ovulation, and allow for timed artificial insemination (TAI) results in improved reproductive performance because all animals are inseminated whether they show estrus or not (Colazo and Ambrose, 2013).

22 8 2.1 SINGLE AI DURING OBSERVED ESTRUS: Pathak et al. (1986) reported a conception rate of 37.7 per cent in repeat breeder cows inseminated once during the observed estrus. Similarly, Stevenson et al. (1990) obtained a conception rate of 32.1 per cent in repeat breeding dairy cattle. But, Rayos (1995) observed a relatively higher conception rate of 50 per cent in repeat breeder cows when single insemination was carried out. Awasthi et al. (2002) conducted single insemination using frozen semen in repeat breeder cows between 12 to 18 hours post estrus and obtained a conception rate of 20 per cent. Whereas, Rangnekar et al. (2002) reported 40 per cent conception rate in repeat breeders inseminated at estrus as per standard AM-PM rule. Similarly, Shelar et al. (2002) observed 40 per cent conception rate in repeat breeders inseminated after 10 to 12 h of commencement of estrus. Villarroel et al. (2004) observed an overall conception rate of per cent in repeat breeder Holstein Friesian cows that did not receive any treatment and inseminated once during the observed estrus. Amiridis et al. (2009) obtained per cent conception rate in repeat breeder cows when artificial insemination was carried out once after overt estrus. A conception rate of 37.5 per cent was reported for repeat breeder cows that received single AI during observed estrus (Iftikhar et al., 2009). Kendall et al. (2009) compared hcg treatment with control repeat breeders and reported that the pregnancy

23 9 rate in repeat breeder cows treated with hcg (45.2 %) was numerically higher than that seen in control repeat breeder cows (41.3 %). Patel et al. (2010) observed a conception rate of per cent for control repeat breeder cows inseminated once during estrus. Whereas, Rajesh Kumar et al. (2010) reported a very low first service conception rate of per cent in repeat breeder cows. More et al. (2012) reported a conception rate of 25 per cent for repeat breeding pluriparous Deoni cows inseminated during estrus without any treatment. Ergene (2013) compared the pregnancy rate of progesterone releasing intra vaginal device (PRID), Ovsynch treatment with control repeat breeder cows and concluded that the pregnancy rate was 56.5 %, 36.3 % and 33.3 %, respectively. Ravikumar (2013) reported per cent conception rate for repeat breeders inseminated during spontaneous estrus. Whereas, Vijayarajan and Meenakshisundaram (2013) observed a conception rate of only 25 per cent in repeat breeding Holstein Friesian crossbred cows. 2.2 MULTIPLE AI DURING OBSERVED ESTRUS: Anovulation and delayed ovulation are the major cause of repeat breeding in dairy cows and remain undiagnosed unless serious attention is given through repeated rectal examination. When delayed ovulation occurs, the poor tubal milieu and altered contractility compromises sperm survival and impairs proper gamete transport. The reproductive failure due to ovulatory disturbances is primarily due to the deficiency of Luteinizing Hormone (LH) secretion at appropriate time after estrus in

24 10 cows (Khanna and Sharma, 1992). Asynchrony in timing of insemination with respect to ovulation results in low pregnancy rate due to fertilization failure (Hunter, 1994). Therefore the importance of insemination timing with respect to ovulation has repeatedly been emphasized for ensuring fertilization (Martinez, 2001). Hence, the success of insemination depends not merely on the detection of estrus, but also on the timing of ovulation related to insemination. Therefore, single insemination following AM-PM rule may lead to poor conception due to shortage of motile/fertile spermatozoa. This problem might be alleviated if high numbers of fertilizable spermatozoa are available at ovulation. A common recommendation for repeat breeding cows is a double AI at the same estrus. Some researchers suggest that double AI might improve poor pregnancy rates resulting from late ovulation (Wilcox and Pfau, 1958) or gross errors in detection of estrus (O Farrell et al., 1983). Bostedt (1976) reported an increase in pregnancy rate from 9.5 % to 52.9 %, in cows that ovulated 24 h after first AI, when a single second AI was performed 24 h after first AI. However, Stevenson et al. (1990) found a marginal increase in pregnancy rate from 32.1 per cent to 33.5 per cent when a single second AI was performed 12 to 16 h after first AI and concluded that double AI failed to improve pregnancy rates more than did a single AI followed by GnRH treatment. Hernandez-Ceron et al. (1993) observed that conception rate for repeat breeder cows were 34.6 per cent with double insemination. Dodamani and Honnappagol (2004) reported a conception rate of per cent when HF x JR crossbred repeat breeder cows were inseminated with frozen thawed semen twice at an interval of 12 h.

25 11 Singh et al. (2005) compared the conception rate in Repeat Breeding Heifers (RBH) with Virgin Heifers (VH) when AI was carried out at every 6 h interval during their spontaneous estrus, starting from the onset of estrus. The total number of AI was significantly higher in RBH compared with VH (P<0.05) with a mean value in the pregnant RBH of 9.7 ± 0.88 and 6.5 ± 0.50 in the non-pregnant RBH. The conception rate was 60 per cent in both RBH and VH. They concluded that repeated inseminations, every 6 h from onset of behavioral estrus to spontaneous ovulation, yielded a conception rate in RBH comparable to that of controls (VH). Shukla and Misra (2008) reported a conception rate of per cent when repeat breeding crossbred cows were re-inseminated after 8 to 16 h of the first AI. Ashok (2013) conducted inseminations twice at 6 to 12 h interval after the onset of estrus in repeat breeders and obtained a conception rate of 30 per cent. Similar conception rate (30 %) was reported by Sowmya (2013) in infertile cows. Likewise, Kumar et al. (2014) observed a conception rate of 30 per cent when second AI was repeated at interval of 12 h after the first insemination in repeat breeding dairy cattle. 2.3 ESTRUS SYNCHRONIZATION PROTOCOLS TO AUGMENT FERTILITY Synchronization of estrus implies the manipulation of the estrous cycle or induction of estrus to bring a large percentage of a group of females into estrus at a short, predetermined time (Odde, 1990). It is one of the advanced management process through which the human errors and management costs could be minimized. It helps in fixing the breeding time within a short predefined period and thereby scheduling the parturition time at the most favourable season, when the newborns can be reared in a suitable

26 12 environment with ample of food for enhancing their survivability. Moreover, estrus synchronization provides more economic returns by improving the production efficiency in animals Ovsynch protocol: Soon after recognition of GnRH as a tool to regulate follicular growth and ovulation, protocols incorporating GnRH and PGF 2 α were developed for synchronization of estrus (Thatcher et al., 1989; Twagiramungu et al., 1992) and ovulation (Pursley et al., 1995; Twagiramungu et al., 1995; Schmitt et al., 1996). Ovsynch is a protocol based on the administration of GnRH, PGF 2 α and GnRH (Pursley et al., 1995) to schedule the insemination time. The first injection of GnRH causes ovulation/luteinization of any functional dominant follicle present on the ovary and induces the subsequent emergence of a new follicular wave approximately 1.5 to 2 days later (Pursley et al., 1995). The newly emerged follicular wave develops and undergoes selection and dominance during the following seven days. On day 7, PGF 2 α administered induces luteolysis, thus allowing further growth and maturation of the dominant follicle. Finally, a second GnRH injection administered 48 hours after PGF 2 α induces a preovulatory LH surge that triggered ovulation within an 8 h period, beginning approximately 24 h after the injection (Pursley et al., 1995). This synchronization protocol allows for a TAI (Pursley et al., 1997) because estrus detection efficiency is a major factor limiting reproductive performance of large dairy farms (Pursley et al., 1997; Lopez-Gatius, 2003). The real estrus detection rate is very low in large dairy herds in

27 13 which the number of cows managed per worker is high, resulting in decreased accuracy and effectiveness of the detection (Lopez-Gatius, 2003; Rabiee et al., 2005). The conception rate for Ovsynch protocol in lactating dairy cows was 29.0 per cent (Pursley et al., 1998), 35.6 per cent (Stevenson et al., 1999), 31.5 per cent (Bartolome et al., 2000), 20.6 per cent in Holstein cows (Ill-Hwa Kim et al., 2003), 23.5 per cent (Portaluppi and Stevenson, 2005), 27.7 per cent (Stevenson and Phatak, 2005) and 33 per cent (Stevenson et al., 2006). Similarly, the conception rate for Ovsynch protocol in postpartum dairy cows was 40 per cent (Gunaseelan, 1996), 22.4 per cent (Moreira et al., 2001), 20.9 per cent (Chebel et al., 2006), 31 per cent (Aali et al., 2008), 18 per cent in buffaloe heifers (Ghuman et al., 2009) and 22 per cent (Bahrami et al., 2012). There is paucity of literature on conception rate of Ovsynch in repeat breeder cows. Kasimanickam et al. (2005) studied the conception rate in observed heat artificial insemination (HAI) with FTAI in repeat breeder cows and reported a conception rate of 30.1 per cent and 22.3 per cent in HAI and FTAI, respectively. Similarly, Celik et al. (2009) reported that the pregnancy rate after AI in Ovsynch protocol was 27.2 per cent for repeat breeder Holstein cows. On the contrary, Vijayarajan et al. (2009) reported a higher conception rate of 50 per cent for repeat breeding cross bred cows subjected to Ovsynch. Ergene (2013) obtained a conception rate of 36.3 per cent in repeat breeder cows treated with Ovsynch protocol and inseminated at observed estrus. Whereas, Ravikumar

28 14 (2013) observed a higher conception rate of per cent when repeat breeding dairy cattle were subjected to Ovsynch protocol with FTAI. Biradar et al. (2014) reported 50 per cent conception rate for Ovsynch in nondescript repeat breeder buffaloes. They concluded that Ovsynch protocol is more efficacious in settling pregnancy than Co-synch in non-descript repeat breeder buffaloes (50.0 % vs. 37.5%) Progesterone based Ovsynch Protocol One of the causes of poor fertility in high producing dairy cows is inadequate progesterone, a key hormone of pregnancy. Lack of exposure to progesterone before spontaneous or GnRH induced ovulation results in greater risk for short luteal phases, which is characterized by luteolysis around day 10 of the estrous cycle (Rhodes et al., 2003). This is the time when the embryo does not produce sufficient IFN-τ to block the luteolytic cascade, resulting in reduced proportion of pregnant cows (Inskeep, 2004). Reproductive performance of cows receiving the Ovsynch protocol improves when progesterone is administered during the 7 days between the first GnRH and PGF 2 α injections. Progesterone should prevent premature estrus and ovulation during the period in which spontaneous luteolysis may occur in small percentages of cows whose dominant follicles are not responsive to the first GnRH injection (Twagiramungu et al., 1992; Pursley et al., 1995; Roy and Twagiramungu, 1999; Vasconcelos et al., 1999; Xu and Burton, 2000). The primary benefit of inclusion of progesterone in GnRH-based programs is that it guarantees that females will be exposed to progesterone during the period between day 0 and day 7. This progesterone exposure will result in normal (21

29 15 days) rather than short (10 days) cycles in previously anestrous cows. A second benefit to inclusion of progesterone is that it prevents premature estrus and ovulation that are inherent to these systems. The progesterone released will prevent estrus and ovulation between day 0 and 8 (Ambrose et al., 2010; Islam, 2011). El-Zarkouny et al. (2004) compared the pregnancy per artificial insemination (P/AI) in 182 lactating dairy cows treated with the Ovsynch protocol with or without the addition of a Controlled Internal Drug Release (CIDR) device. Pregnancy rate were 59.3 vs % at 29 d and 45.1 vs % at 57 d after TAI for the Ovsynch + CIDR vs. Ovsynch, respectively. They concluded that inclusion of CIDR inserts to TAI protocols improved P/AI. The pregnancy rate in normal breeding cattle was 65 per cent when progesterone was added to the Ovsynch protocol (Kim et al., 2005). The benefit of supplementing progesterone in lactating dairy cows subjected to an Ovsynch protocol was also demonstrated in a large study in Mexico. In lactating dairy cows presynchronized with two PGF 2α 14 days apart and subjected to Ovsynch and Ovsynch + CIDR protocol later, Melendez et al. (2006) concluded that P/AI were greater in cows subjected to Ovsynch + CIDR than Ovsynch alone (31 versus 23%). In yet another study, Stevenson et al. (2006) observed that the use of a CIDR in cows treated with Ovsynch led to a 10 per centile increase in P/AI (50 versus 40 %). They concluded that a CIDR insert during the Ovsynch protocol increased fertility in lactating cows having low serum progesterone before PGF 2 α injection. Mecitoglu et al. (2012) evaluated the conception rate in lactating dairy cows by using Ovsynch and modified Ovsynch protocol during summer. The modified Ovsynch group received the

30 16 Ovsynch protocol along with an ear implant containing progesterone (3mg norgestomet; Crestar) between the first GnRH and PGF 2 α injection. In addition, a third GnRH was injected into cows in the modified Ovsynch group. The P/AI at 31 d and 62 d were greater in the modified Ovsynch group than Ovsynch group (46.2 %; 41.5% vs %; 34.7 %). On the other hand, 3 of 6 experiments reported in 5 articles reported that a CIDR insert during the TAI protocols had no improved effect on P/AI in lactating dairy cows (El-Zarkouny et al., 2004; Galvao et al., 2004; Stevenson et al., 2006). Kim et al. (2007) used CIDR insert during the Ovsynch protocol and reported that the pregnancy rate following TAI was 32.1 per cent in repeat breeder dairy cows. They concluded that CIDR based Ovsynch protocol resulted in synchronous follicular wave emergence, follicular development, synchronous ovulation in repeat breeder cows, particularly in cows with a highly extended postpartum interval. Similarly, Keskin et al. (2010) observed a conception rate of 44 and 40.6 per cent at 31 and 62 days, respectively in repeat breeder cows subjected to progesterone (ear implant) based Ovsynch protocol along with a third GnRH administered 7 days after TAI. On the other hand, Ghuman et al. (2011) reported a higher conception rate of 61 per cent when Ovsynch plus CIDRbased FTAI protocol was initiated during the luteal phase in repeat-breeder crossbred dairy cattle.

31 Ovsynch-56 protocol: A common question among dairy producers is the optimal interval between PGF 2 α and the second GnRH treatment, and the optimal time to inseminate cows that have been synchronized with the Ovsynch protocol. In a preliminary study, Peters et al. (1999) examined the effect of delaying the second GnRH on synchrony of ovulation in dairy cows subjected to a 7-day Ovsynch. In this study, from a total of 11 cows treated with the second GnRH between 56 and 60 h after administration of PGF 2 α, ten cows ovulated within 24 h (between 72 and 96 h after administration of PGF 2 α). They concluded that GnRH administration between 56 and 60 h following PGF 2 α administration resulted in the tightest synchrony of ovulation. Although the early studies of Peters et al. (1999) showed that cows subjected to Ovsynch will have a more synchronous ovulation if the administration of second GnRH is between 56 to 60 h after PGF 2 α, the effect of this approach on P/AI in lactating dairy cows was not investigated until recently (Brusveen et al., 2008). Brusveen et al. (2008) evaluated altering the time of the second GnRH injection and AI during Ovsynch in lactating dairy cows and observed that P/AI was lower in cows receiving GnRH at 48 h (Cosynch-48, 29.2 %) or 72 h (Cosynch-72, 25.4 %) after PGF 2 α and inseminated concurrently that in those given GnRH at 56 h and inseminated 16 h later (Ovsynch-56, 38.6 %). Moreover, P/AI for first service was higher in Presynch/Ovsynch-56 (44.8 %) than Presynch/Cosynch-48 (36.2 %). Nebel et al. (2008) reported that cows receiving Cosynch-72 had lower fertility than cows receiving Ovsynch-56.

32 18 In an effort to compare the effectiveness of Cloprostenol sodium versus Dinoprost tromethamine for Ovsynch-56, Martins et al. (2011) compared the Presynch/Ovsynch-56 with two different types of PGF 2α (Dinoprost tromethamine and Cloprostenol sodium at each PGF 2α of Presynch/Ovsynch-56) and observed that P/AI of cows treated with cloprostenol sodium tended to be greater than dinoprost tromethamine (40 vs. 35 %, respectively). They also concluded that cows with greater serum concentrations of progesterone at the time of PGF 2α of Ovsynch-56 had a greater chance of luteolysis and pregnancy. Bahrami et al. (2012) compared the conception rate of Ovsynch-48 and Ovsynch- 56 in dairy cows and reported a higher conception rate for Ovsynch-56 (32 %) than Ovsynch-48 (22 %). Similarly, Mecitoglu et al. (2012) obtained a conception rate of 37.3 per cent for lactating dairy cows subjected to Ovsynch-56 during summer Timed Artificial Insemination (TAI) Versus Observed Heat Artificial Insemination (HAI) in Ovulation synchronization protocols: Considering that the expression of estrus is very poor in lactating dairy cows, and overall estrus detection efficiency is far from acceptable, TAI protocols can overcome deficiencies in estrus detection. Although acceptable conception rate are a prerequisite for the success of TAI protocols, only herds with poor estrus detection efficiency will substantially benefit from regular implementation of TAI. Generally conception rates on TAI are lower than for visual observation and insemination. Poorer fertility after TAI often was attributed to insufficient synchrony of estrus and ovulation to allow appropriate timing of AI relative to ovulation (Fogwell et al., 1986).

33 19 Fogwell et al. (1986) recorded a 22 per cent lower conception rate when a single, timed breeding at 80 h post PGF 2α was compared to breeding 12 h after detecting a synchronized estrus. Pursley et al. (1994) examined reproductive efficiency of lactating dairy cows that received periodic PGF 2 α and were bred at observed estrus compared with cows that received TAI and observed that first AI conception rates were similar for the two groups. Burke et al. (1996) compared the effectiveness of a TAI (Ovsynch) and an AI at detected estrus (AIDE) (Control; GnRH-PG) and reported that the conception rate was higher (41.5 %) for control cows than cows in the TAI group (26.5 %). Differences (control vs. TAI) were not detected for overall pregnancy rates by 120 days postpartum (58.8 ± 4.7 % vs ± 4.4 %). They concluded that timed AI was an effective alternative for reproductive management of lactating dairy cows. However, Schmitt et al. (1996) compared similar protocols in heifers and observed that conception rates were 23 per cent lower for heifers under TAI which was due to a higher frequency (15.5 %) of short inter estrous intervals. Stevenson et al. (1996) reported that conception rate for FTAI after the Ovsynch protocol (35.3 %) was lower compared with control cows inseminated at estrus after PGF 2 α (47.1 %). On the other hand, Pursley et al. (1997) summarized that one FTAI at a synchronized ovulation provided similar pregnancy rates per AI as did AI following the AM-PM rule after estrus had been induced by PGF 2 α in lactating cows, but the FTAI was not effective for heifers because of the lack of synchronization.

34 20 Stevenson et al. (1999) reported that actual conception rates tended to be lower (22.1 %) for cows treated with Ovsynch 33 and fixed-time AI than for cows treated with GnRH + PGF 2 α and inseminated at observed estrus (35.8 %). Kasimanickam et al. (2005) observed that the conception rate in repeat breeder cows for HAI and FTAI (30.1 % vs %) were not statistically significant (P>0.1). Kasimanickam et al. (2010) compared the pregnancy rates in cows bred at observed estrus with or without second GnRH administration in fixed-time progesteronesupplemented Ovsynch and Co-synch protocols and concluded that acceptable pregnancy rates can be achieved with or without inclusion of estrus detection in the Ovsynch-CIDR (48.3 %) and Co-synch-CIDR (51.8 %) protocols. Further, they reported that among cows detected in estrus, cows that received a second GnRH yielded similar pregnancy rates when compared with cows that did not receive the second GnRH. The cows in Ovsynch- CIDR protocol that were inseminated after detection of estrus had a 7 per cent increase in the pregnancy rate when they received the second GnRH of the Ovsynch. Ergene (2013) obtained a pregnancy rate of 36.3 per cent when repeat breeder cows were subjected to Ovsynch protocol and inseminated after detection of estrus. Bruno et al. (2014) evaluated the pregnancy per AI in cows inseminated on detected estrus and at fixed time in Ovsynch-56 and GGPG protocol and observed that the pregnancy per AI was 39.0 per cent (estrus detected) and 28.7 percent (TAI) in Ovsynch-56. The corresponding pregnancy per AI was 40.5 per cent (estrus detected) and 28.3 per cent (TAI) in GGPG group.

35 Stage of estrous cycle at initiation of Ovulation synchronization protocols: It has been demonstrated that the success of Ovsynch program is influenced by the number of follicular waves or length of the follicular wave (Pursley et al., 1997) and the stage of the estrous cycle when the first GnRH is administered (Vasconcelos et al., 1999; Moreira et al., 2000). The initiation of the Ovsynch treatment at mestestrus stage of the estrous cycle may lead to failure of synchronization of a new follicular wave by the first GnRH since the follicle present at the time of first GnRH will be small and fail to ovulate. If dominant follicle is too small at the time of first GnRH injection, it does not respond to GnRH induced LH surge because of the lack of LH receptors in the granulosa cells (Twagiramungu et al., 1995; Xu et al., 1995). Consequently, the dominant follicle will undergo early stage of atresia at the time of second GnRH. Thus the poor quality of the preovulatory follicle and the subsequent development of the aged oocyte may affect the pregnancy rate in dairy cattle (Wishart, 1977; Mihm et al., 1994). In addition, a markedly lower fertility was also found due to ovulation of a large persistent follicle in cows under treatments that provide only low progesterone concentration prior to ovulation (Savio et al., 1993; Stock and Fortune, 1993). Low fertility in these persistent follicles is attributed to the premature oocyte activation (Revah and Butler, 1996) and effect of low progesterone on subsequent uterine function (Shaham-Albalancy et al., 1997). When Ovsynch is initiated at late luteal phase of the estrous cycle, the animal may undergo premature CL regression and estrus is observed before the second injection of

36 22 GnRH. Since the normal CL regression starts Day 16 of the estrous cycle (Ginther et al., 1989), the CL will undergo spontaneous regression 3.2 d before the injection of PGF 2 α by the normal endogenous release of endometrial PGF 2 α. Vasconcelos et al. (1999) observed that ovulation in response to the first injection of GnRH is important for the success of the Ovsynch program, particularly for cows in the late estrous cycle. During the proestrus stage of the estrous cycle, initiation of Ovsynch leads to incomplete regression of induced accessory CL by PGF 2 α. The CL that developed under a low progesterone environment may be on borderline of being responsive to an injection of prostaglandin (Watts and Fuquay, 1985). Incomplete regression of CL following PGF 2 α in Ovsynch is associated with a lower pregnancy rate (Moreira et al., 2000). Vasconcelos et al. (1997) recorded higher pregnancy rates in cows initiated Ovsynch protocol during the early luteal phase when compared to cows initiated the treatment during first 3 days or after Day 13 of the estrous cycle. These findings are however inconsistent with those of Keister et al. (1999), who noted similar reproductive performance in dairy cattle when Ovsynch treatment was initiated at random or on Day 7 of the estrous cycle. However, Moreira et al. (2000) after a detailed investigation concluded that the early luteal stage of the estrous cycle (day 5 to 12) was the optimal period for initiating the Ovsynch program. Pregnancy rate were improved in dairy cows when Ovsynch was started on Day 12 (Cartmill et al., 2001) or Day 14 (Jordan et al., 2002) after prostaglandin administration, since most cows would be in early diestrus before the beginning of the Ovsynch protocol.

37 23 Based on the reports that the luteal phase was the optimal time of Ovsynch protocol onset in terms of conception rate, Moreira et al. (2001) presynchronized cows using two prostaglandin doses given 14 days apart to initiate the Ovsynch protocol at the targeted early luteal phase. Presynchronization was found to increase the pregnancy rate in cyclic lactating dairy cows, but not in anestrus cows, given their lack of prostaglandin responsive CL. Bartolome et al. (2002) obtained similar pregnancy rates between cows with and cows without CL following Ovsynch protocol, by presynchronizing cows with palpable CL using prostaglandin 14 days before initiation of Ovsynch and for cows without palpable CL, using GnRH 8 days before beginning of Ovsynch. Murugavel et al. (2003) observed that early postpartum dairy cows with high (>1ng/ml) progesterone concentration at onset of treatment (Presynch/Ovsynch) resulted in a significantly improved pregnancy rate (42.1 %) than cows with low (<1ng/ml) progesterone concentration (0 %). The conception rate recorded for repeat breeder cows treated with Ovsynch protocol at random stage of the cycle was 22.3 per cent (Kasimanickam et al., 2005) 27.2 per cent (Celik et al., 2009), 50 per cent (Vijayarajan et al. 2009), 36.3 per cent (Ergene, 2013) and per cent (Ravikumar et al., 2014). Kim et al. (2007) initiated progesterone (CIDR) based Ovsynch protocol at random stage of the estrous cycle and recorded a conception rate of 32.1 per cent in repeat breeder dairy cows. Similar conception rate (44 %) was reported by Keskin et al. (2010) for repeat breeder cows.

38 24 Ghuman et al. (2011) observed an appreciably high conception rate (61 %) when repeat breeder cows were subjected to CIDR based Ovsynch during the luteal phase of the estrous cycle. On the other hand, Bisinotto et al. (2015) observed that incorporating a single intra vaginal insert (CIDR) to the Ovsynch protocol increased progesterone concentrations in plasma by 1.3 ng/ml, but did not benefit fertility in lactating dairy cows that have CL at the initiation of the Ovsynch protocol Body Condition Score (BCS) Body condition score is the measure of the nutritional status of animals and is an important factor influencing the reproductive performance (Baruselli et al., 2001). It influences the length of postpartum anestrus and thus the proportion of cows cycling at the start of the breeding season. Cows need to be in a positive energy balance to resume normal estrous cycles. For optimum reproductive performance, BCS of 2.5 (on five point scale) was required at breeding in cattle and a BCS of 3.5 was required at calving (Edmonson et al., 1989). For every increase in BCS (1.7 to 4.3) at 65 days postpartum, conception and pregnancy rate increased 13 and 16 per cent, respectively, which suggested that signs of behavioral estrus were stronger and that fertility improved in cows as BCS increased. Similarly, plasma concentrations of progesterone increased by 2.2 ng/ml for every increase in BCS. The increase in plasma concentrations of progesterone also was associated with an increase in conception and pregnancy rates (Burke et al., 1996). Similarly, Stevenson et al. (1999) reported that cows with a greater BCS had 8 to 10 per cent greater conception rates for each unit increase in body condition and also had greater

39 25 concentration of progesterone. Wiltbank et al. (2005) showed that as BCS declined, the proportion of cows that were not ovulating increased; and for cows with BCS of 2.5 or less, the percentage of cows that were not ovulating was 80 per cent. Several studies have looked at the relationship between BCS and reproductive performance of cows on an Ovsynch program. Stevenson et al. (1996) observed that cows with BCS >2.5 had higher conception rates (33.3 %) than did those cows with BCS <2.5 (30 %) treated with Ovsynch protocol. Moreira et al. (2000) reported that the pregnancy rate at 45 d following TAI was 11.1 per cent for cows with BCS less than 2.5, while the pregnancy rate for cows with BCS equal to or greater than 2.5 was 25.6 per cent. Mattos et al. (2001) found positive influence of BCS on the conception rate following Ovsynch treatment. On the contrary, Ghuman et al. (2011) observed that no difference was observed in BCS of repeat breeder cattle that conceived or not. 2.4 ENDOMETRIAL CYTOLOGY DURING ESTRUS TO RULE OUT SUBCLINICAL ENDOMETRITIS: Subclinical endometritis is defined as the presence of >18 per cent polymorphonuclear (PMN) cells in uterine cytology samples collected days postpartum, or >10 per cent PMNs in samples collected at d Cows with subclinical endometritis do not have uterine discharge, however, the severity of the disease is still considered sufficient to impair reproductive performance (Sheldon et al., 2006). It is an inflammation of uterine mucosa in the absence of clinical signs of endometritis, which causes significant reduction of reproductive performance (Sheldon et al., 2009).

40 26 It was first described as cytological endometritis considering the presence of PMN in the endometrial lumen (Gilbert et al., 1998), and then standardized by Kasimanickam et al. (2004) based on its negative effects on reproductive performance. No gold standard exists for the diagnosis of subclinical endometritis, which turns the task into a challenging one. Nevertheless, uterine cytological evaluation is the most used tool for diagnosis of subclinical endometritis (Kasimanickam et al., 2004). Cytological examination of the reproductive tract is often used to evaluate possible reproductive lesions in humans and domestic animals. Endometrial cytological examination in mares (Wingfield-Digby, 1987) and cows (Gilber et al., 1998; Hammon et al., 2001) are accepted diagnostic techniques. Endometrial and inflammatory cells are collected by a guarded cotton swab, uterine biopsy, uterine lavage or cytobrush techniques to evaluate endometrial cytological examination (Kasimanickam et al., 2004; Ahmadi et al., 2005; Kasimanickam et al., 2005b; Ahmadi and Nazifi, 2006) especially as an aid in the diagnosis of acute endometritis. A technique that yields well-preserved cells representative of a large uterine surface area without causing harm to the reproductive tract is required for consistent and reliable cytological studies. Subclinical endometritis can be diagnosed accurately by either cytologically (uterine lavage and cytobrush) or histologically (biopsy) (Javed and Khan, 1991; Lewis, 1997; Kasimanickam et al., 2005b). Kasimanickam et al. (2005b) compared the cytobrush and lavage techniques for the assessment of endometrial cytology in clinically normal postpartum dairy cows and observed that the mean per cent of PMN cells significantly differed between the

41 27 techniques at 20 to 33 days in milk (DIM) but not at 34 to 47 DIM. They concluded that cytobrush technique can be used successfully and reliably to obtain endometrial samples than lavage technique. To differentiate between cows with endometritis and healthy cows, different thresholds of 5%, 10% and 18% PMN cells were used (Kasimanickam et al., 2004; Drillich et al., 2005; Gilbert et al., 2005; Plontzke et al., 2010; Baranski et al., 2012; Ghasemi et al., 2012). Barlund et al. (2008) compared various methods for diagnosing endometritis between 28 and 41 days postpartum in dairy cattle and reported that cytobrush technique was the most reliable method of diagnosing endometritis in cattle. The risk of nonpregnancy at 150 days postpartum was 1.9 times higher in cows with more than 8 per cent PMNs identified using cytobrush technique than in cows with less than 8 per cent PMNs. Cows with endometritis had a 17.9 per cent lower first service conception rate and a 24 day increase in days open. Oral et al. (2009) studied the diagnosis of postpartum endometritis using cytobrush, vaginoscopy and trans-rectal ultrasonography methods. Cytologically, 6 out of 31 cows were diagnosed non-endometritic. They suggested that combination of vaginal discharge score, transrectal ultrasonography methods together with cytobrush technique were useful for the diagnosis of endometritis in cows. There are few papers dealing with cytological diagnosis of subclinical endometritis around breeding. Kaufmann et al. (2009) demonstrated that cows with high

42 28 proportions of endometrial PMN (>15 % PMN) 4 h after AI have a decreased first service conception rate. Salasel et al. (2010) obtained endometrial samples from the uterus by using the lavage technique in the luteal phase of the estrus cycle and reported that conception rate in the subsequent AI was 5 per cent for cows with subclinical endometritis ( 3% neutrophils) and 47 per cent for cows without subclinical endometritis ( 3% neutrophils). Benbia et al. (2013) reported that the percentage of PMN cells in cows during follicular phase and luteal phase was 0.33 ± 0.21 and 2.33 ± 1.11, respectively by cytobrush technique. Janowski et al. (2013) performed cytobrush cytology in repeat breeder cows using the threshold of 10 % PMN and observed that 45 (40.2%) of 112 clinically healthy repeat breeding cows were diagnosed as having subclinical endometritis. Madoz et al. (2013) subjected postpartum cyclic dairy cows for Ovsynch protocol and collected endometrial cytology on day 0, 4, 11 and 18 of the estrous cycle (corresponding to estrus, metestrus, diestrus and proestrus, respectively). They observed that the percentage of PMN did not vary with the stage of the estrous cycle. Moreover, in their study, PMN counts were below any of the reported thresholds for most of the dairy cow (4%).

43 TIME OF ONSET OF ESTRUS AFTER PGF 2 α IN OVULATION SYNCHRONIZATION PROTOCOLS: Stevenson et al. (1996) reported that the mean interval from PGF 2 α to estrus in PGP protocol was 3.0 ± 0.2 days, whereas, it was 3.2 ± 0.2 days in the control group where only two PGF 2 α injections were given 14 days apart. The mean interval from the second PGF 2 α injection to estrus was 3.1 ± 0.2 days for both the treatments. Stevenson et al. (1999) observed that the mean onset of estrus after PGF 2 α injection in lactating dairy cows was 54 ± 13, 64 ± 3.7 and 55 ± 4.4 h in Ovsynch-33, GnRH + PGF 2 α and Ovsynch-48 protocols. Ill-Hwa Kim et al. (2003) compared the Ovsynch protocol (GPG) with progesterone based protocol (CPG) and observed that the proportion of cows with premature estrus prior to injection of PGF 2 α tended to be or were greater in the GPG group (12.9 %, 4/31) than in the CPG group (0 %, 0/31). Similarly, Galvao et al. (2004) reported that incorporation of CIDR into the synchronization protocol eliminated cows in estrus before the final PGF 2 α injection (0 vs %). Aral and Colak (2004) reported that the time taken for onset of estrus after PGF 2 α injection in Ovsynch protocol was 62.6 h. Cavalieri et al. (2004) found that the mean interval from cloprostenol injection to estrus for lactating dairy cows treated with CIDR for 7 or 8 days prior to cloprostenol was 2.82 days (1.5-3 d)

44 30 Larson et al. (2006) observed that the time from PGF 2 α injection to estrus for those cows exhibiting estrus was similar among control (52.8 ± 0.9 h), Select Synch +TAI (51.5 ± 0.9 h), and Select Synch + CIDR with TAI (53.4 ± 0.8 h). Regardless of the treatment, interval from 48 to 60 h after PGF 2 α injection yielded the greatest estrus response. Of cows detected in estrus, 34.8 per cent exhibited estrus during this interval. Honparkhe et al. (2011) reported that repeat breeder cows subjected to CIDR based estrus synchronization protocol had their estrus expressed between h after CIDR removal. Islam (2011) noted that some cows (8%) exhibit estrus up to 48 h before PGF 2 α (day 6) when a single dose of GnRH and PGF 2 α were injected on day 1 and 8, respectively. They opined that the early estrus was fertile and cows can be inseminated 12 h after estrus detection. The peak estrus response occurs 2-3 days after PGF 2 α with a range of 1-5 days. With this system, a minimum of 5 days of estrus detection after PGF 2 α and 2 days preceding PGF 2α is required to detect most estrus. Ammu et al. (2012) in a study conducted in Gir cows observed per cent estrus response with mean onset of estrus after PGF 2 α as ± 1.23 h using Ovsynch protocol in anestrus/subestrus cows. Ergene (2013) reported that the mean onset of estrus in repeat breeder cows subjected to progesterone based protocol (PRID) and Ovsynch protocol was 2.00 ± 0.41 and 1.12 ± 0.23 day from the end of the treatment, respectively.

45 31 Biradar et al. (2014) observed that all the non-descript repeat breeder buffaloes treated by Cosynch and Ovsynch protocols exhibited estrus signs (100 %) within h after PGF 2 α injection. Savalia et al. (2014) evaluated the CIDR and Ovsynch protocols on postpartum anestrus buffaloes and mid-cycle PGF 2 α injection in repeat breeder buffaloes and reported that the mean time of onset of behavioral estrus after PGF 2 α injection was ± 6.46 and ± 6.15 h, ± 4.50 h, respectively. 2.6 HAEMATOLOGICAL PARAMETERS IN NORMAL AND REPEAT BREEDER COWS Blood glucose profile Fertility requires adequate nutrition and energy reserves. Starvation, wasting and obesity are associated with reproductive system abnormalities and infertility. It is evident that the energy balance plays a significant role in reproductive function and fertility. Normal blood levels of various biochemical components are essential for normal function of various systems in the body including reproductive system. Deficiency or excess of certain biochemical constituents during estrus in blood serum may affect fertility status of cows (Dutta et al., 1991). Changes in several biochemical components have been blamed for reproductive failures. Blood glucose, cholesterol and protein appear to be some of the key nutrients affecting fertility and cyclicity in farm animals (Qureshi, 1998).

46 32 It has been reported that hypoglycaemia (<40 mg/dl) adversely affected the hypothalamo-hypophyseal function and reduces the fertility (McClure, 1968 and Lamond, 1970). Roberts (1971) suggested that hypoglycaemia at oestrus or shortly after service exerts a harmful effect on conception by lowering glucose and glycogen level resulting lack of energy of spermatozoa or fertilized ova in cattle. In repeat breeder cows the concentration of glucose in blood was reported to be 30 to 59 mg/dl which was found to be low in comparison to that recorded in fertile cows (McClure, 1968; Downie and Gelman, 1976). Dutta et al. (1991) reported that the mean blood glucose level in repeat breeder cows was ± 0.51 mg/dl with range of mg/dl. Manjunatha et al. (2001) reported that the mean serum glucose concentration at estrus, early luteal phase and late luteal phase as ± 3.67, ± 4.35, ± 3.91 and ± 2.22, ± 1.94, ± 4.43 mg/dl in normal cyclic and repeat breeder cows, respectively. They observed that significantly higher glucose concentration was observed during estrus (P<0.05) and early luteal phase (P<0.01) in cyclic animals compared to repeat breeders. Similarly, Chandrahar et al. (2003) observed that the average blood glucose was significantly lower in repeat breeder cows than in normal fertile cows (60.43 ± 0.73 vs ± 0.87 mg/dl). Nejad and Cheraghi (2003) noted that the mean serum glucose value in repeat breeder cows (60.29 ± 1.53 mg/dl) was significantly lower than the values recorded for normal cyclic cows (79.84 ± 1.20 mg/dl). Blood glucose levels of primiparous and pluriparous repeat breeders were ± 2.44 and ± 2.01 mg/dl, respectively. Likewise, Mondal and Paul (2012) reported that the mean serum glucose levels were low

47 33 (p<0.05) in repeat breeding cows (42.8 mg/dl) than the normal cyclic cows (49.2 mg/dl). Guzel and Tanriverdi (2014) reported that the mean blood glucose concentration was significantly higher in normal cows (65.00 ± 6.27 mg/dl) when compared to repeat breeder cows (44.71 ± 5.17 mg/dl). Kumar (2014) observed a significantly decreased levels of plasma glucose level (55.18 ± 3.10 mg/dl) when compared to normal cyclic animals (60.27 ± 2.28 mg/dl). Shiraz Khan et al. (2010) evaluated the blood glucose concentration in repeat breeder cows as ± 3.54, ± 3.54, ± 6.60, ± 3.19 and ± 4.39 mg/dl on day 0, 5, 10, 15 and 20 of the estrous cycle, respectively. The repeat breeding cows showed significantly higher (P < 0.01) glucose levels on day 5 of the cycle and day 10, whereas significantly lower (P <0.01) level was recorded on day 20 of the estrous cycle when compared to day 5 and 10 of the cycle. Ali et al. (2014) reported that pregnant cows exhibited relatively lower blood glucose concentration in contrast to non-pregnant cows on day 1 (pre-insemination) (40.88 ± 1.07 and ± 0.79 mg/dl). Similarly, the blood glucose content measured on day 21 (post-insemination) was higher in non-pregnant group (41.86 ± 0.96 mg/dl) as compared to pregnant group (40.15 ± 1.24 mg/dl). They observed that the correlation coefficient between blood glucose level and conception rate was statistically nonsignificant (p>0.01) on day 1 and 21. On the other hand, Sharma et al. (1983), Kumar et al. (1986) and Awasthi and Kharche (1987) did not note significant differences in blood glucose level between normal fertile cows and repeat breeders.

48 Blood Haemoglobin (Hb) profile: Tissue oxygenation of reproductive tract and in turn the normal cyclicity is dependent on the hemoglobin level in the circulation. During estrus, sufficient concentration of Hb in blood is required for the proper transportation of oxygen and nutrients to the vital organs, as the same is required for metabolic activities of the gonadal cells. Though the importance of Hb level has not been directly implicated in reproductive disorders, yet a decrease in Hb value is indicative of certain systemic disorders which could indirectly affect the functional activity of the reproductive organs. A low level of Hb influences tissue oxygenation of the reproductive tract, which in turn could affect the cyclicity (Ramakrishna, 1997). Blood et al. (1989) stated that the Hb values in normal healthy cows would range from 8-15 g/dl. According to Das (1993) the average Hb value of normal fertile cow was ± 0.61 g per cent and in animals with impaired fertility it was 6.86 ± 0.75 g per cent. Pariza et al. (2009) reported a significantly (p<0.01) lower value of Hb in repeat breeder cows (9.6 ± 1.1 g/dl) than cyclic cows (13.1 ± 2.6 g/dl). Similar observations were made by Sebasthin et al. (2012) in repeat breeder (9.7 ± 0.16 g/dl) and cyclic buffaloes (12.0 ± 0.13 g/dl). Mondal and Paul (2012) reported that the average values for Hb in repeat breeder cows was 9.29 g/dl whereas, it was 9.64 g/dl in cyclic cows. Reddy et al. (2012) observed that the mean haemoglobin concentrations in control repeat breeder cows was 9.80 ± 0.30 g/dl and further opined that the heamoglobin concentration did not vary between the

49 35 control and treatment groups. Perumal et al. (2013) observed that Hb values of normal cyclic cows (11.66 ± 2.88 g/dl) significantly differed (p<0.05) from repeat breeder cows (10.64 ± 1.51 g/dl) at estrus. Sahoo et al. (2014) studied the Hb profile in repeat breeder cows at various stages of the estrous cycle and reported Hb values of 9.3 ± 0.63, 9.38 ± 0.59, 9.43 ± 0.45 and 9.46 ± 0.44 g/dl on day 0, 7, 14 and 21 of the estrous cycle. Singh et al. (2014) reported that the mean heamoglobin concentrations in normal cows as 9.73 ± 0.16 g/dl, whereas in anemic cows it was 6.7 ± 0.11 g/dl. 2.7 BLOOD MACRO MINERAL PROFILE IN NORMAL AND REPEAT BREEDING COWS: Normal levels of vital haemato-biochemical constituents are of atmost importance for maintaining functional integrity of the reproductive system. Any change in haematological and biochemical parameters may adversely affect the reproductive efficiency of livestock. Minerals like calcium, phosphorus and magnesium play an intermediate role in the action of hormones and enzymes at sub cellular levels in an integrated fashion in initiation of oestrus in young growing heifers (Haq et al., 1999). Tessel (1967) stated that the animals sometimes do not respond to the treatment for hormonal imbalance or for disease of the reproductive failure might be due to deficiency or imbalance of single or combination elements causing enzymatic dysfunctions associated with fertility of animals. The low concentration of circulatory minerals resulted in impaired reproductive function leading to cessation of cyclic activity

50 36 (Martson et al., 1972). Normal levels of various minerals can influence the reproductive performance of ruminants. Reproductive failure may be induced by deficiencies or imbalances of macro and micro minerals (Hidiroglou, 1979). It is also reported that macro minerals such as calcium and phosphorus are essential for the growth and reproduction and are involved in a large number of physiological and biosynthetic processes in the body (Hurley et al., 1980). Sharma et al. (1984) stated that certain biochemical and mineral constituents in blood serum during estrus period had also been found to be associated with the fertility status of cows and their reproductive behavior. The deficiency or disturbed proportions of these minerals results in ovarian hypo function (Dhoble and Gupta, 1986), which might well, be reflected in lower blood levels of them. In yet another study, Kumar et al. (1986) stated that certain biochemical constituents in blood during estrus have been found to be associated with the fertility status of cows and their reproductive behavior. Mineral deficiencies might be responsible for lowering conception rate. The role of minerals in herd fertility is indisputable and play potentially significant role. Corah and Ives (1991) reported that the deficiencies of minerals induced or predisposed animals to repeat breeding and anestrus. The macro minerals that are of particular importance are calcium and phosphorus. Thus, the blood profile might have potential in characterizing the problem and diagnosing deficiency conditions (Eltohammy et al., 1989 and Jain et al., 2003). Repeat breeders had significantly lower blood concentrations of calcium and phosphorus than cows with normal fertility (Rupde et al., 1993). Burle et al. (1995)

51 37 reported that the concentrations of calcium and phosphorus were significantly higher in cycling cows than in repeat breeders and anestrus cows. Several studies have also demonstrated that the mineral deficiency and imbalances influenced the ovarian activity of ruminants and often incriminated as causes for poor reproduction (Corah and Ives, 1991, Ansotegui et al., 1999 and Boland, 2003) and mineral deficiency greatly affect reproduction (Hostetler et al., 2003, Ahmet et al., 2008) Serum calcium concentration: Enthia et al. (1982) reported a slightly higher (non-significant) serum calcium levels in repeat breeder than in normal cyclic cows, but Shanker et al. (1983) found the serum calcium level was higher in normal cyclic than in repeat breeder. While Awasthi and Kharache (1987) did not report any appreciable effect of calcium deficiency on reproductive performance of crossbred cows. In yet another study, Saba et al. (1987) reported higher serum calcium concentrations in cows with repeat breeding than the values in those without repeat breeding. Awasti and Kharche (1987) observed higher serum calcium level of 8.93 ± 0.63 mg % in repeat breeders as compared to concentration of 7.86 ± 0.42 mg % in normal cycling cows. Similarly, Bukhari and Ali (1987) reported that the mean concentration of calcium was significantly lower in repeat breeder group of animal (7.58 ± 1.46 mg/100 ml) than the normal cyclic group of animals (9.56 ± 1.68, 8.81 ± 1.73 mg/100ml) respectively. In another study Burley (1991), Shrikhande and Vhora (2000) reported that the regular breeder cows showed significantly high levels serum calcium (9.32 mg/dl) than those of repeat breeder cows (9.15 mg/dl)

52 38 McClure (1994) observed the mean calcium concentration of 9.3 and 9.9 mg/dl in normal and sterile animals, nevertheless, Burle et al. (1995) reported lowest serum calcium level in anoestrus as compared to levels in normal cyclic and repeat breeder Sahiwal cows. Kaneko et al. (1997) stated that the lower level of serum calcium in repeat breeder was attributed to less gut absorption and low serum calcium prevented release of matured ova during oestrus on account of inefficient muscular contraction. Further studies indicated that hypocalcaemia in cows was associated with poor ovarian activity and infertility as a result of impaired blood flow to ovaries (Kamgaprour et al., 1999). Numerous studies have reported significantly lower serum calcium concentrations in repeat breeder cows (Bukhari and Ali, 1987; Burley, 1991; Rupde et al., 1993; Balakrishnan and Balagopal, 1994; McClure, 1994; Kaneko et al., 1997; Shrikhande and Vhora, 2000; Seifi et al., 2005 and Ruginuso et al., 2011). Contrary to findings above, Enthia et al. (1982), Saba et al. (1987), Awasti and Kharche (1987) and Chandrakar (1999) have reported significantly higher serum calcium level in repeat breeder as compared to normal cyclic cows. In other studies, non-significantly lower serum calcium level in Sahiwal cows (Burle et al., 1995) and repeat breeding buffaloes (Chaurasia et al., 2010) were reported. While Das et al. (2002) reported that the serum calcium concentration in normal cyclic and repeat breeder cow were 10.5 ± 0.44 and 10.0 ± 0.32 mg/dl respectively and no significant difference in serum calcium level between repeat breeding and normal cyclic cows was observed. Similarly, Butani et al. (2011) have also reported no significant differences between repeat breeder and normal cyclic cows.

53 39 Kumar and Sharma (1993) stated that the low profile of plasma calcium in the repeat breeder cows could be due to some metabolic disturbance possibly described earlier causing poor absorption of calcium from gut. Since calcium is required for neuromuscular excitability, muscle contractions and transmission of nerve impulse at cellular level. Seifi et al. (2005) reported that the concentrations of calcium in fertile and infertile cows were 2.34 and 2.17 mmol/l, respectively and Pandey et al. (2009) reported that plasma calcium to phosphorus ratio was significantly disturbed in the repeat breeder cows. Kalita and Sarmah (2006) reported that the mean concentration of serum calcium ± 0.21 and 9.87 ± 0.29 mg/dl respectively in normal cyclic and repeat breeding cows. The mean serum calcium concentration was reported as ± 0.59 mg/dl, 9.40 ± 0.60 mg/dl in cows (Ceylan et al., 2008 and Pandey et al., 2009) and 9.40 ± 0.60 mg/dl in repeat breeding buffaloes (Chaurasia et al., 2010) and the concentrations of serum phosphorus in normal fertile counter parts reported respectively were 8.45 ± 0.65 mg/ dl, ± 0.30 mg/dl and 9.30 ± 0.29 and they concluded that the calcium levels were significantly lower in repeat breeder cows as compared to fertile cows Serum phosphorus concentration: Several studies have documented the role of phosphorous in regulating fertility in dairy cows (Simesen, 1970; Hewett, 1974; Bansal et al., 1978; Morrow, 1980; Hurley et al., 1980; Ahmet et al., 2008). Deficiency of phosphorous influences at the level of pituitary and ovary and might interfere with fertilization causing early embryonic death (Simesen, 1970), associated with fertility problems (Hewett, 1974 and Bansal et al.,

54 ), lowered conception rate, irregular oestrus, anoestrus, decreased ovarian activity, increased incidence of cystic follicles and increased, services per conception and depressed fertility (Morrow 1980), cause disturbance in pituitary-ovarian-axis (Bhaskaran and Abdullah Khan, 1981), ovulation (Bhaskaran and Patil, 1982) and blocking action on the pituitary gland (Herrick, 1977) and consequently on the ovarian function (Herrick and John, 1977), while increased phosphorus level was related to the improvement of ovarian activity ( Hurley et al., 1980 ) Number of studies have reported significantly lower serum phosphorus concentration in repeat breeder cows (Simesen, 1970; Bhaskaran and Abdllakhan, 1981; Bukhari and Ali, 1987; Eltohammy et al., 1989; Khan and Iyer 1993; Islam et al., 1994, Ramakrishna, 1996; Tandle et al., 1997; Shrikhande and Vhora, 2000; Seifi et al., 2005; Kalita and Sarmah, 2006; Das et al., 2009 and Chaurasia et al., 2010). Bhaskaran and Abdullakhan (1981) reported that the repeat breeders had a significantly lower (3.73 ± 0.29 mg %) levels of inorganic phosphorous than normal cyclic (5.06 ± 0.19 mg %) cows. While Bukhari and Ali (1987) reported that the mean plasma phosphorus concentration of 5.11 ± 0.93 mg/100 ml in repeat breeder cows and Eltohammy et al. (1989) concluded that low calcium, phosphorus and magnesium levels in serum were associated with infertility. Studies by Khan and Iyer (1993) suggested that lower serum phosphorus in repeat breeding was associated with lower estrogen secretion. McDowell (1992) reported that, a phosphorus shortage is more likely to occur than a calcium deficiency under conditions where forages were used more abundantly.

55 41 Khan and Iyer (1993) reported that the serum inorganic phosphorus was 6.07 ± 0.15 and 4.84 ± 0.10 mg/100 ml, in regular breeders and repeat breeder cows, respectively and similar findings were also reported by Islam et al. (1994). Significantly higher serum inorganic phosphorus values (4.65 mg/dl) were recorded in cyclic cows as compared to value of 4.50 mg/dl in repeat breeders (Shrikhande and Vhora, 2000). While, Seifi et al. (2005) observed a serum phosphorus concentrations of 1.84 and 1.42 mmol/l respectively in fertile and infertile cows. Kalita and Sarmah (2006) reported that the mean serum phosphorus concentration of 4.71 ± 0.19 and 3.65 ± 0.21 mg/dl respectively in normal cycle and in repeat breeding cows. The mean serum phosphorus concentration was reported as 5.19 ± 0.22 mg/dl, 2.80 ± 0.40 mg/dl in cows (Ceylan et al., 2008 and Pandey et al., 2009) and 4.37 ± 0.16 mg/dl in repeat breeding buffaloes (Chaurasia et al., 2010) and the concentrations of serum phosphorus in normal fertile counter parts reported respectively were 6.30 ± 0.23 mg/dl, 6.20 ± mg/dl and 5.29 ± 0.14 mg /100 ml and they concluded that the phosphorus levels were significantly lower in repeat breeder cows as compared to fertile cows. However, Joy-George and Nair (1995) observed no significant variations in the mean concentration of phosphorus between fertile and repeat breeder cows. Du Plessis et al. (1999) and Chaurasia et al. (2010) reported that the deficiency of phosphorus in particular, influences at the level of pituitary and ovary, and might interfere with fertilization causing early embryonic death, thereby produces aberrations in the normal reproductive rhythm since phosphorus is essential for transfer of biological energy especially through ATP and deficiency of it might arrest the phenomenon of

56 42 fertilization, this in turn, might cause early embryonic death resulted in the repeat breeding. 2.8 SERUM PROGESTERONE PROFILE IN NORMAL AND REPEAT BREEDING COWS: Deopurkar et al. (1985) estimated serum progesterone concentration of Gir cows during different phases of estrous cycle. The mean serum progesterone level was found to be 0.28 ± 0.32 ng/ml on the day of estrus. They observed that minimum level was on the day of estrus with a steady and gradual increase in the progesterone levels up to day 12, was on the higher side from days 12 to 16 of the estrous cycle with a peak value on day 14, and then sudden drop in the serum progesterone level from day 18 onwards. Wiltbank et al. (2012) reported that high circulating concentrations of progesterone near AI has been shown to be detrimental to fertility in dairy cattle, but the underlying physiological mechanisms that reduce fertility are not well understood. Near the time of AI, it is critical that progesterone concentrations are below a critical value, which appears to be about 0.4 ng/ml during Ovsynch-type protocol in which ovulation before FTAI is induced with GnRH. Further, they opined that even small increase in progesterone near the time of AI were associated with dramatic reductions in fertility, either in cows bred to natural estrus or after timed AI protocols. Birnie et al. (1997) opined that the conception rate in cows following induced estrus was positively correlated with plasma concentration of progesterone during the days preceding the luteolysis. Kumaresan et al. (2000) studied the influence of plasma progesterone level at estrus on conception rate in bovines. Conception rate was and

57 per cent in cattle and buffaloes, respectively that were inseminated when progesterone levels were low (<0.1 ng/ml) at estrus. Vasconcelos et al. (1999) initiated Ovsynch protocol in dairy cows at 1-4, 5-9, and days of the estrous cycle and reported that the mean progesterone concentration at the start of the treatment (first GnRH injection) was 0.2 ± 0.0, 2.0 ± 0.2, 3.0 ± 0.2 and 1.6 ± 0.6 ng/ml, respectively. They concluded that cows initiated Ovsynch protocol during the early luteal phase had better pregnancy rates when compared to cows initiated the treatment during first 3 days or after Day 13 of the estrous cycle. Similarly, Moreira et al. (2000) initiated Ovsynch protocol in heifers at d 2, 5, 10, 15, 18 of the estrous cycle and reported a mean progesterone concentration of 0.8 ± 1.3, 6.2 ± 1.3, 14.1 ± 1.4, 14.0 ± 1.3 and 1.6 ± 0.3 ng/ml, respectively at the initiation of Ovsynch protocol. They concluded that the early luteal phase (between day 5 and 12 of the estrous cycle) was the most appropriate time for initiating the Ovsynch protocol to obtain greater pregnancy rates. Martinez et al. (2000) observed that the plasma progesterone concentrations following CIDR insertion in cows were near to luteal level (5 to 7 ng/ml) by 24 h and then decreased to 4 ng/ml after 2 to 3 days, where they remained static until CIDR removal on day 7. Plasma progesterone concentration declined by 12 h after CIDR removal. Chander et al. (2002) reported that the plasma progesterone on day 10 post AI in normal cycling cows was 2.92 ± 0.71 ng/ml, whereas it was 2.12 ± 0.06 ng/ml in repeat breeding cows.

58 44 El-Zarkouny et al. (2004) noted that the average progesterone concentration in CIDR-based Ovsynch decreased from 2.6 ± 0.2 ng/ml upon its removal to 2.0 ± 0.2 ng/ml at least 1 to 2 h after its removal. Average progesterone concentrations were 1 ng/ml in both the treatments (Ovsynch and CIDR-based Ovsynch) at the time of PGF 2 α injection. Average progesterone concentration decreased (P< 0.01) to 0.5 ± 0.2 ng/ml on the day of second GnRH in response to the PGF 2 α injection. They concluded that addition of the CIDR into the Ovsynch protocol did not increase serum progesterone concentration on the day of removal of CIDR (day of PGF 2 α injection) beyond that of Ovsynch alone. `Likewise, Stevenson et al. (2006) observed that the supplementation of CIDR via the CIDR insert did not increase relative serum concentrations of progesterone in cows treated CIDR-based Ovsynch protocol. Concentrations of progesterone just before CIDR removal did not differ between Ovsynch (2.2 ± 0.3 ng/ml) and OVS + CIDR cows (2.4 ± 0.3 ng/ml). Concentration of progesterone at CIDR removal did not differ between cycling and non-cycling cows. Decline in progesterone concentration was only 0.5 ± 0.1 ng/ml when blood was collected between CIDR removal and 1 to 2 h later when the PGF 2 α was given suggesting that progesterone contribution of the CIDR insert to peripheral serum concentration of progesterone was small. Proportion of cows in which serum progesterone approached basal (proestrus) concentrations by 48 h after PGF 2 α was affected by treatment (83-99 % in OVS +CIDR vs % in Ovsynch). Kim et al. (2007) treated repeat breeder cows with CIDR-based Ovsynch protocol and reported that the mean serum progesterone concentration was 8.5 ± 1.1, 12.9 ± 1.8, 2.8 ± 0.4 and 2.6 ± 0.6 ng/ml on days 0, 7, 8 and 9, respectively, whereas, the mean

59 45 progesterone values for estradiol based CIDR+ Ovsynch protocol was 10.0 ± 2.5, 9.1 ± 2.2, 3.2 ± 0.8 and 2.6 ± 0.6 ng/ml on d 0, 7, 8 and 9, respectively. The mean serum progesterone concentration did not differ between the two groups in repeat breeder cows (P>0.05). Son et al. (2007) studied the effects of a CIDR-based timed embryo transfer (TET) in lactating repeat breeder cows. The repeat breeder cows received a CIDR device and 2 mg estradiol benzoate (Day 0), a 25 mg PGF 2 α injection at the time of CIDR removal on day 7 and a 1 mg estradiol benzoate injection on day 8. Cows then received TET on day 16 using frozen-thawed blastocysts or morula embryos. The mean serum progesterone on Day 0 (6.0 ± 0.8 ng/ml) was maintained at a level that was comparable to Day 7 of the luteal phase (9.2 ± 1.6 ng/ml). The progesterone concentration then decreased on Day 8 (1.4 ± 0.3 ng/ml) and subsequently increased on Day 16 (5.5 ± 0.5 ng/ml). Celik et al. (2009) treated repeat breeder cows with Ovsynch protocol and Ovsynch + β-carotene (β-carotene was administered along with the first GnRH injection) and evaluated the serum progesterone concentration on days 0, 7, 9 and at the time of AI. No significant differences were observed between the groups for the serum progesterone concentration on the day of the first GnRH injection. The progesterone concentration was significantly higher in the carotene group on the day of PGF 2 α. No significant differences could be seen between the groups on the serum progesterone concentration on the day of second GnRH and at the time of AI.

60 46 Similarly, Ravikumar et al. (2014) evaluated the progesterone concentrations in different estrus synchronization protocols in repeat breeder cows and reported the mean progesterone concentrations on days 0, 7, 9 and at the time of AI as 1.24 ± 0.21, 2.72 ± 0.37, 0.41 ± 0.24 and 0.67 ± 0.08 ng/ml, respectively in repeat breeder cows treated with Ovsynch protocol. Further, they concluded that the mean serum progesterone concentration at the time of AI did not differ significantly between control and various treatment groups.

61 Materials and Methods

62 III. MATERIALS AND METHODS The present study was designed to determine the efficacy of Ovsynch and modified Ovsynch protocols on the conception rate in repeat breeder cows when initiated during the early diestrus stage (day 5-12) of the estrous cycle. 3.1 Experimental animals: The study was carried out between January 2014 to October 2014 and 77 repeat breeder cows aged 3-8 years in their first to fourth lactation with BCS between 2.5 and 3.5 presented to Veterinary Dispensaries in and around Bangalore and also to the clinical service complex of the Department of Veterinary Gynaecology and Obstetrics, Veterinary College, Bangalore were randomly selected. The total number of services for the repeat breeder cows in the present study ranged from 3-7 with an average number of 4.5 services. The number of days these animals were not pregnant at the time of presentation for treatment ranged from 150 to 240 days. The criteria for selection of these repeat breeder cows was as follows: 1. These animals should have calved at least once. 2. They should have a clear estrus discharge and have a history of normal or nearly normal estrus periods. 3. Should have a BCS between 2.5 to 3.5 and 4. Should have no palpable abnormalities of the reproductive tract.

63 Study groups: The repeat breeder cows were randomly divided into 7 groups consisting of 11 cows in each group. All the cows were subjected to thorough clinico-gynaecological examination to ascertain the stage of estrous cycle based on trans-rectal palpation for the presence of CL and correlating with the history obtained from the owner regarding the day of last estrus. Cows in all the groups were subjected to estrus synchronization protocols anywhere between day 5-12 of the estrous cycle except Group-I and Group-II Group-I (Single Artificial Insemination) (SAI): Repeat breeder cows in this group were subjected to a single A.I. with good quality frozen thawed semen at observed estrus using the AM-PM rule. The animals in this group served as the control Group-II (Multiple Artificial Inseminations) (MAI): Cows in this group were done multiple A.I. (2-3) with good quality frozen thawed semen at 8-12 h interval during the observed estrus using the AM-PM rule, until the estrus signs subside Group-III (Ovsynch with Fixed Time Artificial Insemination) (OVS-FTAI): Repeat breeder cows in this group received 10 µg GnRH 1 intramuscular on day 0 (day of start of the treatment) and 500 µg of PGF 2 α 2 intramuscular on day 7. A second 1 GnRH analogue (Buserelin acetate) (Receptal ) Each ml solution contains mg Buserelin acetate equivalent to mg buserelin. Presentation-10 ml vial, MSD. 2 PGF 2 α Injection (Cloprostenol sodium) (Estrumate ) Each ml solution contains 263 µg of cloprostenol sodium equivalent to 250 µg of cloprostenol. Presentation-20 ml vial, MSD.

64 49 dose of 10 µg of GnRH was administered on day 9 and FTAI with good quality frozen thawed semen was carried out at h after the second GnRH injection. The time taken for the onset of estrus after PGF 2 α was recorded Group-IV (Ovsynch with Artificial Insemination at observed estrus) (OVS- HAI): Repeat breeder cows in this group received 10 µg GnRH intramuscular on day 0 (day of start of the treatment) and 500 µg of PGF 2 α intramuscular on day 7. A second dose of 10 µg of GnRH was administered on day 9 and A.I. was carried out with good quality frozen thawed semen during the observed estrus using the AM-PM rule. The time taken for the onset of estrus after PGF 2 α was recorded Group-V (Progesterone based Ovsynch with Fixed Time Artificial Insemination) (POVS-FTAI): Repeat breeder cows in this group received 10 µg GnRH intramuscular on day 0 (day of start of the treatment). Additionally, an intra vaginal progesterone insert (Triu- B ) 3 (Plate No: 1 and 2) was applied in situ in the vagina for seven days. 500 µg of PGF 2 α was administered intramuscularly on day 7 and Triu-B was removed from the vagina on the same day. A second dose of 10 µg of GnRH was administered on day 9 and FTAI with good quality frozen thawed semen was carried out at h after the second GnRH injection. The time taken for the onset of estrus after PGF 2 α was recorded. 3 Triu-B (Progesterone impregnated intra vaginal device) Each device has 3 medicated rings containing Progesterone IP 186 mg and one additional ring with Progesterone IP 400 mg. Presentation- 10 units/carton, VIRBAC.

65 Group-VI (Progesterone based Ovsynch with Artificial Insemination at observed estrus) (POVS-HAI): Repeat breeder cows received 10 µg GnRH intramuscular on day 0 (day of start of the treatment). Additionally, an intra-vaginal progesterone insert (Triu-B) was applied in situ in the vagina for seven days. 500 µg of PGF 2 α was administered intramuscular on day 7 and Triu-B was removed from the vagina on the same day. A second dose of 10 µg of GnRH was administered on day 9 and A.I with good quality frozen thawed semen was carried out at the observed estrus using the AM/PM rule. The time taken for the onset of estrus after PGF 2 α was recorded Group-VII (Ovsynch-56) (OVS-56): 10 µg of GnRH was administered to all the repeat breeder cows on day 0 (day of start of the treatment). 500 µg of PGF 2 α was administered intramuscular on day 7. A second dose of 10 µg of GnRH was administered at 56 h after the PGF 2 α injection, maintaining h interval from the second GnRH injection to FTAI (Brusveen et al., 2008). The time taken for the onset of estrus after PGF 2 α was recorded. 3.3 Blood Sampling: Blood samples about 1-2 ml with and without anticoagulant was collected in sterilized plastic tubes by jugular vein puncture from all the repeat breeding cows during the early diestrus stage for estimation of hemoglobin, blood glucose, serum calcium and phosphorus whereas, 2 ml blood was collected for estimation of progesterone in a separate vial during early diestrus in group of repeat breeder cows subjected to hormonal

66 51 treatments. The blood sample was also obtained on the day of A.I. from repeat breeder cows of all the groups for estimation of progesterone. Blood samples for estimation of serum calcium, phosphorus and progesterone were kept as a slant for 6-8 h for separation of serum. The expressed serum was centrifuged at 3000 rpm for 10 minutes and it was transferred into sterilized serum vials in two equal aliquots. All serum samples were labeled and stored at -20 C until analyzed Haemoglobin (Hb) estimation: The Hb concentrations in all the repeat breeder cows of the control and treatment groups were determined using an auto analyzer (MINDRAY BC-2800 VET) and expressed as g/dl Glucose estimation: The blood glucose concentration was determined spectrophotometrically according to the method described by Braham and Trinder (1972) and expressed as mg/dl Serum calcium and phosphorus estimation: The concentrations of calcium, phosphorus in serum were determined spectrophotometrically (TRIVITRON LABMATE 10 plus double beam spectrophotometer) using standard diagnostic kits (Accucare- calcium and phosphorus reagent, Lab-Care Diagnostic (India) Pvt. Ltd.) and expressed as mg/dl.

67 Serum progesterone estimation: The progesterone concentrations in serum samples of all the repeat breeder cows were determined by epro Check 2.0 (Minitube, Germany) instrument using progesterone test kit for bovine serum. The serum progesterone concentration was estimated based on Enzyme Linked Immunosorbent Assay (ELISA) as per the manufacturer s instructions and expressed as ng/ml. 3.4 Endometrial Cytology at the time of AI to rule out subclinical endometritis: Endometrial cytology was performed in all the repeat breeder cows before A.I. by uterine lavage technique as described by Salasel et al. (2010) to rule out subclinical endometritis. Twenty ml of normal saline was infused into the uterine lumen of each repeat breeder cow through a sterile A.I. sheath connected to a 20 ml syringe. Transrectal massage of the uterus was performed for 5-10 seconds before the fluid was withdrawn. The recovered fluid was centrifuged at 1000 rpm for 15 minutes. After discarding the supernatant, the sediment was used to prepare the smear. The smears were fixed in methanol and stained with Giemsa stain and was examined under microscope using 400x magnification, and a differential count of endometrial cells, PMNs and squamous cells based on their morphology (Plate No: 3) using a minimum of 100 cells were recorded. The diagnosis of subclinical endometritis was arrived when the endometrial cytology revealed the threshold of 10 % PMNs (Plate No: 4) in the uterine smears (Kasimanickam et al., 2004). Nine repeat breeder cows out of a total of that had more than 10 % PMNs in the uterine cytology were excluded from the study.

68 Pregnancy diagnosis: Repeat breeder cows in control group and in various treatment protocols were subjected to pregnancy diagnosis by trans-rectal ultrasound scanning using linear array probe with 7.5 MHz frequency on day 30 post A.I. (Plate No: 5) and confirmed by rectal examination at day 45 post A.I. to evaluate the conception rate. 3.5 Statistical Analysis: Mean values (± SE) for various haemo-biochemical parameters and progesterone levels for repeat breeder cows of seven groups were computed. In order to observe the magnitude of variation in these parameters among cows of the seven groups, the data were analyzed statistically using one way analysis of variance under completely randomized design (Steel and Torrie, 1980). Duncan s multiple range test (Duncan, 1955) was applied for multiple means comparison, wherever necessary. The conception rate between the control and different treatments groups were analyzed by Chi-square test. Results were considered to be statistically significant when P values are less than 0.05 (P < 0.05).

69 Results

70 IV. RESULTS A total of 86 randomly selected repeat breeder cows were utilized for the present study. Nine out of 86 repeat breeder cows were found to be positive for subclinical endometritis based on endometrial cytology using lavage technique. Hence, these nine cows were not utilized for the study. The results obtained from the 77 repeat breeder cows were utilized for the study and the various parameters studied were presented in tables. 4.1 Haemo-biochemical attributes in repeat breeding control and treatment groups: The data pertaining to the haemo-biochemical attributes in repeat breeding control and treatment groups are presented in Table Haemoglobin: The mean Hb concentration for repeat breeder cows subjected to single artificial insemination (SAI) was 9.28 ± 0.40 g/dl. The corresponding values were 9.66 ± 0.40 g/dl in multiple artificial insemination group (MAI), ± 0.68 g/dl in cows subjected to Ovsynch with fixed time artificial insemination (OVS-FTAI), 9.17 ± 0.27 g/dl in cows subjected to Ovsynch with artificial insemination at observed estrus (OVS-HAI), 8.84 ± 0.39 g/dl in cows subjected to progesterone based Ovsynch with fixed time artificial insemination (POVS-FTAI), 9.14 ± 0.38 g/dl in cows subjected to progesterone based Ovsynch with artificial insemination at observed estrus (POVS-HAI) and 9.20 ± 0.30 g/dl in cows subjected to Ovsynch-56 (OVS-56). The mean Hb concentrations did not differ significantly (P>0.05) between the different groups (Table 1, Fig: 1).

71 Blood glucose: The mean glucose concentrations of untreated control and treated repeat breeder cows in the present study are presented in Table 1, Fig: 2. As observed from the table, the mean blood glucose concentrations of repeat breeder cows were ± 2.76, 46.27± 1.99, ± 2.41, ± 2.11, ± 1.38, ± 1.49 and ± 1.23 mg/dl in SAI, MAI, OVS-FTAI, OVS-HAI, POVS-FTAI, POVS-HAI and OVS-56, respectively. Comparison of the mean blood glucose concentrations revealed non-significant variations between the groups (P>0.05) Serum calcium: In the present study, the mean serum calcium concentrations of repeat breeder cows were 8.52 ± 0.28, 9.14 ± 0.21, 9.59 ± 0.21, 9.17 ± 0.16, 9.13 ± 0.15, 9.31 ± 0.24 and 9.24 ± 0.08 mg/dl in SAI, MAI, OVS-FTAI, OVS-HAI, POVS-FTAI, POVS-HAI and OVS-56, respectively. The mean serum calcium concentration of 8.52 ± 0.28 mg/dl observed for cows in SAI differed significantly (P<0.05) as compared to the mean level of 9.59 ± 0.21 mg/dl in OVS-FTAI cows. However, comparison of the mean serum calcium concentrations between the other groups did not reveal any significant variations (P>0.05) (Table 1, Fig: 3) Serum phosphorus: The mean serum phosphorus concentrations of untreated control and treated repeat breeder cows are depicted in Table 1, Fig: 4. The mean serum phosphorus concentrations of 4.69 ± 0.21, 4.56 ± 0.18, 4.61 ± 0.19, 4.50 ± 0.19, 4.26 ± 0.14, 4.47 ± 0.20 and 4.36 ± 0.16 mg/dl were recorded for SAI, MAI, OVS-FTAI, OVS-HAI, POVS-

72 56 FTAI, POVS-HAI and OVS-56 treated repeat breeder cows, respectively. Further, statistical analysis revealed no significant (P>0.05) differences in the mean phosphorus concentrations among the different groups studied. 4.2 Time of onset of estrus following PGF 2 α administration: The mean time for onset of estrus after PGF 2α administration among repeat breeder cows belonging to OVS-FTAI, OVS-HAI, POVS-FTAI, POVS-HAI and OVS- 56 groups are portrayed in Table 2, Fig: 5. The mean time for onset of estrus of ± 2.10 h was recorded for repeat breeder cows in OVS-FTAI which was significantly higher as compared to ± 2.02, ± 0.99, ± 0.65 and ± 2.44 h for OVS-HAI, POVS-FTAI, POVS-HAI and OVS-56 repeat breeder cows, respectively. Further, the mean time of onset of estrus after PGF 2 α administration showed a progressive and a significant (P<0.05) decline in OVS-HAI cows as compared to POVS- FTAI and POVS-HAI cows. However, the comparison of the mean time of onset of estrus after PGF 2 α administration in the latter two groups was not significant. Similarly, no significant differences were observed in the time of onset of estrus after PGF 2 α administration among OVS-HAI and OVS-56 cows. 4.3 Serum progesterone concentrations at first GnRH in all the treatment groups: In the present study, the mean serum progesterone concentrations at the time of first GnRH injection in repeat breeder cows belonging to different treatment groups were 2.95 ± 0.60, 1.83 ± 0.21, 2.33 ± 0.33, 2.61 ± 0.41 and 2.36 ± 0.41 ng/ml in OVS-FTAI, OVS-HAI, POVS-FTAI, POVS-HAI and OVS-56 treated repeat breeder cows, respectively. Although, the mean serum progesterone concentrations ranged from 1.83 ±

73 to 2.95 ± 0.60 ng/ml in the different treatment groups, there was no significant (P>0.05) variations among the treatment groups (Table 3, Fig: 6). 4.4 Serum progesterone concentrations in repeat breeding control and treatment groups at the time of A.I: The mean serum progesterone concentrations at A.I. of repeat breeding control and treatment groups in the present study were 0.61 ± 0.06, 0.54 ± 0.07, 0.56 ± 0.08, 0.46 ± 0.07, 0.47 ± 0.06, 0.52 ± 0.09 and 0.39 ± 0.07 ng/ml in SAI, MAI, OVS-FTAI, OVS- HAI, POVS-FTAI, POVS-HAI and OVS-56 treated cows, respectively. Analysis of the data revealed that there was no significant difference (P>0.05) in the mean serum progesterone concentrations between the groups at the time of A.I (Table 4, Fig: 7). 4.5 Basal and suprabasal progesterone concentrations in repeat breeding control and treatment groups at the time of A.I: In the present study, the progesterone concentrations recorded in repeat breeding control and treatment groups at the time of A.I were categorized into basal and suprabasal concentrations. Among the 77 repeat breeder cows studied, 31 repeat breeder cows had basal progesterone concentrations of 0.4 ng/ml and 46 repeat breeder cows had suprabasal progesterone concentrations of 0.5 ng/ml (Table 5). 4.5 Conception rate: The conception rate of repeat breeder control and repeat breeder cows subjected to different synchronization protocols are presented in Table 6, Fig: 8. The results of the present study revealed that, out of 11 untreated repeat breeder cows in SAI group, two

74 58 cows were found pregnant with a conception rate of per cent. The conception rate of 27.27, 36.36, 36.36, 36.36, and per cent were recorded for the repeat breeder cows in MAI, OVS-FTAI, OVS-HAI, POVS-FTAI, POVS-HAI and OVS-56 groups, respectively. The highest conception rate of per cent was recorded for the repeat breeder cows in POVS-HAI and OVS-56 groups. The Chi-square analysis revealed significant (P<0.05) variations in the conception rate for the control and different treatment groups. Regardless of the control and the treatment groups, an overall conception rate of per cent (27 out of 77) was obtained in the repeat breeder cows in the present study.

75 59 Table 1: Haemo-biochemical attributes (Mean ± SE) in repeat breeding control and treatment groups Parameter Group-I (SAI) Group-II (MAI) Group-III (OVS-FTAI) Group-IV (OVS-HAI) Group-V (POVS-FTAI) Group-VI (POVS-HAI) Group-VII (OVS-56) Haemoglobin (g/dl) 9.28 ± ± ± ± ± ± ± 0.30 Glucose (mg/dl) ± ± ± ± ± ± ± 1.23 Serum calcium (mg/dl) Serum phosphorus (mg/dl) 8.52 ± 0.28 b 9.14 ± 0.21 ab 9.59 ± 0.21 a 9.17 ± 0.16 ab 9.13 ± 0.15 ab 9.31 ± 0.24 ab 9.24 ± 0.08 ab 4.69 ± ± ± ± ± ± ± 0.16 Note: Means bearing any one common superscript in row do not differ significantly with each other (P>0.05) Group-I : Single Artificial Insemination (SAI) Group-II : Multiple Artificial Inseminations (MAI) Group-III : Ovsynch with Fixed Time Artificial Insemination (OVS-FTAI) Group-IV : Ovsynch with Artificial Insemination at observed estrus (OVS-HAI) Group-V : Progesterone based Ovsynch with Fixed Time Artificial Insemination (POVS-FTAI) Group-VI : Progesterone based Ovsynch with Artificial Insemination at observed estrus (POVS-HAI) Group-VII : Ovsynch-56 (OVS-56)

76 60 Table 2: Time (Mean ± SE) of onset of estrus following PGF 2α administration in cows subjected to various treatments Parameter Group-III (OVS-FTAI) Group-IV (OVS-HAI) Group-V (POVS-FTAI) Group-VI (POVS-HAI) Group-VII (OVS-56) Onset of estrus after PGF 2α (h) ± 2.10 a ± 2.02 b ± 0.99 d ± 0.65 d ± 2.44 bc Note: Means bearing any one common superscript in row do not differ significantly with each other (P>0.05) Group-III : Ovsynch with Fixed Time Artificial Insemination (OVS-FTAI) Group-IV : Ovsynch with Artificial Insemination at observed estrus (OVS-HAI) Group-V : Progesterone based Ovsynch with Fixed Time Artificial Insemination (POVS-FTAI) Group-VI : Progesterone based Ovsynch with Artificial Insemination at observed estrus (POVS-HAI) Group-VII : Ovsynch-56 (OVS-56)

77 61 Table 3: Serum progesterone concentrations (Mean ± SE) at first GnRH in all the treatment groups Parameter Group-III (OVS-FTAI) Group-IV (OVS-HAI) Group-V (POVS-FTAI) Group-VI (POVS-HAI) Group-VII (OVS-56) Serum progesterone (ng/ml) 2.95 ± ± ± ± ± 0.41 Group-III Group-IV Group-V Group-VI Group-VII : Ovsynch with Fixed Time Artificial Insemination (OVS-FTAI) : Ovsynch with Artificial Insemination at observed estrus (OVS-HAI) : Progesterone based Ovsynch with Fixed Time Artificial Insemination (POVS-FTAI) : Progesterone based Ovsynch with Artificial Insemination at observed estrus (POVS-HAI) : Ovsynch-56 (OVS-56)

78 62 Table 4: Serum progesterone concentrations (Mean ± SE) in repeat breeding control and treatment groups at the time of A.I Parameter Group-I (SAI) Group-II (MAI) Group-III (OVS-FTAI) Group-IV (OVS-HAI) Group-V (POVS-FTAI) Group-VI (POVS-HAI) Group-VII (OVS-56) Serum progesterone (ng/ml) 0.61 ± ± ± ± ± ± ± 0.07 Group-I Group-II Group-III Group-IV Group-V Group-VI Group-VII : Single Artificial Insemination (SAI) : Multiple Artificial Inseminations (MAI) : Ovsynch with Fixed Time Artificial Insemination (OVS-FTAI) : Ovsynch with Artificial Insemination at observed estrus (OVS-HAI) : Progesterone based Ovsynch with Fixed Time Artificial Insemination (POVS-FTAI) : Progesterone based Ovsynch with Artificial Insemination at observed estrus (POVS-HAI) : Ovsynch-56 (OVS-56)

79 63 Table 5: Basal and suprabasal progesterone concentrations in all the repeat breeder cows Particulars Number of cows Total number of cows Conceived Total number of cows not conceived Basal progesterone ( 0.4 ng/ml) Suprabasal progesterone ( 0.5 ng/ml)

80 64 Table 6: Conception rate (percentage) for repeat breeder control and treatment cows Pregnancy Status Group-I (SAI) Group-II (MAI) Group-III (OVS-FTAI) Group-IV (OVS-HAI) Group-V (POVS-FTAI) Group-VI (POVS-HAI) Group-VII (OVS-56) Total No of animals Pregnant Total number of animals Conception rate (%) X 2 value at 6 d.f * Note: * Significant at 0.05 level Group-I : Single Artificial Insemination (SAI) Group-II : Multiple Artificial Inseminations (MAI) Group-III : Ovsynch with Fixed Time Artificial Insemination (OVS-FTAI) Group-IV : Ovsynch with Artificial Insemination at observed estrus (OVS-HAI) Group-V : Progesterone based Ovsynch with Fixed Time Artificial Insemination (POVS-FTAI) Group-VI : Progesterone based Ovsynch with Artificial Insemination at observed estrus (POVS-HAI) Group-VII : Ovsynch-56 (OVS-56)

81 Haemoglobin (g/dl) 65 Fig 1: Blood Hemoglobin levels of repeat breeder cows in control and treatment groups G-I (SAI) G-II (MAI) G-III (OVS-FTAI) G-IV (OVS-HAI) G-V (POVS-FTAI) G-VI (POVS-HAI) G-VII (OVS-56) Group-I : Single Artificial Insemination (SAI) Group-II : Multiple Artificial Inseminations (MAI) Group-III : Ovsynch with Fixed Time Artificial Insemination (OVS-FTAI) Group-IV : Ovsynch with Artificial Insemination at observed estrus (OVS-HAI) Group-V : Progesterone based Ovsynch with Fixed Time Artificial Insemination (POVS-FTAI) Group-VI : Progesterone based Ovsynch with Artificial Insemination at observed estrus (POVS-HAI) Group-VII : Ovsynch-56 (OVS-56)

82 Glucose (mg/dl) 66 Fig 2: Blood glucose levels of repeat breeder cows in coontrol and treatment groups G-I (SAI) G-II (MAI) G-III (OVS-FTAI) G-IV (OVS-HAI) G-V (POVS-FTAI) G-VI (POVS-HAI) G-VII (OVS-56) Group-I : Single Artificial Insemination (SAI) Group-II : Multiple Artificial Inseminations (MAI) Group-III : Ovsynch with Fixed Time Artificial Insemination (OVS-FTAI) Group-IV : Ovsynch with Artificial Insemination at observed estrus (OVS-HAI) Group-V : Progesterone based Ovsynch with Fixed Time Artificial Insemination (POVS-FTAI) Group-VI : Progesterone based Ovsynch with Artificial Insemination at observed estrus (POVS-HAI) Group-VII : Ovsynch-56 (OVS-56)

83 Calcium (mg/dl) 67 Fig 3: Serum calcium concentrations of repeat breeder cows in control and treatment groups G-I (SAI) G-II (MAI) G-III (OVS-FTAI) G-IV (OVS-HAI) G-V (POVS-FTAI) G-VI (POVS-HAI) G-VII (OVS-56) Group-I : Single Artificial Insemination (SAI) Group-II : Multiple Artificial Inseminations (MAI) Group-III : Ovsynch with Fixed Time Artificial Insemination (OVS-FTAI) Group-IV : Ovsynch with Artificial Insemination at observed estrus (OVS-HAI) Group-V : Progesterone based Ovsynch with Fixed Time Artificial Insemination (POVS-FTAI) Group-VI : Progesterone based Ovsynch with Artificial Insemination at observed estrus (POVS-HAI) Group-VII : Ovsynch-56 (OVS-56)

84 Phosphorus (mg/dl) 68 Fig 4: Serum phosphorus concentrations of repeat breeder cows in control and treatment groups G-I (SAI) G-II (MAI) G-III (OVS-FTAI) G-IV (OVS-HAI) G-V (POVS-FTAI) G-VI (POVS-HAI) G-VII (OVS-56) Group-I : Single Artificial Insemination (SAI) Group-II : Multiple Artificial Inseminations (MAI) Group-III : Ovsynch with Fixed Time Artificial Insemination (OVS-FTAI) Group-IV : Ovsynch with Artificial Insemination at observed estrus (OVS-HAI) Group-V : Progesterone based Ovsynch with Fixed Time Artificial Insemination (POVS-FTAI) Group-VI : Progesterone based Ovsynch with Artificial Insemination at observed estrus (POVS-HAI) Group-VII : Ovsynch-56 (OVS-56)

85 Onset of estrus (h) 69 Fig 5: Time of onset of estrus after PGF2 administration in repeat breeder cows with various treatments G-III (OVS-FTAI) G-IV (OVS-HAI) G-V (POVS-FTAI) G-VI (POVS-HAI) G-VII (OVS-56) Group-III : Ovsynch with Fixed Time Artificial Insemination (OVS-FTAI) Group-IV : Ovsynch with Artificial Insemination at observed estrus (OVS-HAI) Group-V : Progesterone based Ovsynch with Fixed Time Artificial Insemination (POVS-FTAI) Group-VI : Progesterone based Ovsynch with Artificial Insemination at observed estrus (POVS-HAI) Group-VII : Ovsynch-56 (OVS-56)

86 progesterone (ng/ml) 70 Fig 6: Serum progesterone concentrations at first GnRH in all treatment groups G-III (OVS-FTAI) G-IV (OVS-HAI) G-V (POVS-FTAI) G-VI (POVS-HAI) G-VII (OVS-56) Group-III : Ovsynch with Fixed Time Artificial Insemination (OVS-FTAI) Group-IV : Ovsynch with Artificial Insemination at observed estrus (OVS-HAI) Group-V : Progesterone based Ovsynch with Fixed Time Artificial Insemination (POVS-FTAI) Group-VI : Progesterone based Ovsynch with Artificial Insemination at observed estrus (POVS-HAI) Group-VII : Ovsynch-56 (OVS-56)

87 Progesterone (ng/ml) 71 Fig 7: Serum progesterone concentrations repeat breeding control and treament groups at the time of A.I G-I (SAI) G-II (MAI) G-III (OVS-FTAI) G-IV (OVS-HAI) G-V (POVS-FTAI) G-VI (POVS-HAI) G-VII (OVS-56) Group-I : Single Artificial Insemination (SAI) Group-II : Multiple Artificial Inseminations (MAI) Group-III : Ovsynch with Fixed Time Artificial Insemination (OVS-FTAI) Group-IV : Ovsynch with Artificial Insemination at observed estrus (OVS-HAI) Group-V : Progesterone based Ovsynch with Fixed Time Artificial Insemination (POVS-FTAI) Group-VI : Progesterone based Ovsynch with Artificial Insemination at observed estrus (POVS-HAI) Group-VII : Ovsynch-56 (OVS-56)

88 Conception rate (%) 72 Fig 8: Conception rate for repeat breeder cows in control and treatment groups G-I (SAI) G-II (MAI) G-III (OVS-FTAI) G-IV (OVS-HAI) G-V (POVS-FTAI) G-VI (POVS-HAI) G-VII (OVS-56) Group-I : Single Artificial Insemination (SAI) Group-II : Multiple Artificial Inseminations (MAI) Group-III : Group-IV : Ovsynch with Fixed Time Artificial Insemination (OVS-FTAI) Ovsynch with Artificial Insemination at observed estrus (OVS-HAI) Group-V : Progesterone based Ovsynch with Fixed Time Artificial Insemination (POVS-FTAI) Group-VI : Progesterone based Ovsynch with Artificial Insemination at observed estrus (POVS-HAI) Group-VII : Ovsynch-56 (OVS-56)

89 73 Plate 1: Progesterone releasing intra vaginal device (Triu-B) for use in cattle Plate 2: Insertion of progesterone releasing intra vaginal device (Triu-B) in a cow

90 74 Plate 3: Uterine cytology by lavage technique revealing the endometrial cells Plate 4: Uterine cytology by lavage technique revealing PMNs with endometrial cells

91 75 Plate 5: Pregnancy diagnosis at 30 days post AI by ultrasonography