By Tadesse Gugssa Kebede

Transcription

1 EFFECTS OF PROSTAGLANDIN ADMINISTRATION FREQUENCY, ARTIFICIAL INSEMINATION TIMING AND BREED ON FERTILITY OF COWS AND HEIFERS IN EASTERN ZONE OF TIGRAY REGION, ETHIOPIA By Tadesse Gugssa Kebede A Thesis Submitted to the College of Veterinary Medicine, Mekelle University, in Partial Fulfillment of the requirements for the Degree of Master of science in Veterinary Reproduction and Obstetrics June, 2015 Mekelle, Ethiopia 1

2 DECLARATION This is to certify that this thesis entitled Effects of prostaglandin administration frequency, artificial insemination timing and breed on fertility of cows and heifers in eastern zone of Tigray region, Ethiopia submitted in partial fulfillment of the requirements for the award of the degree of Masters of Science in Veterinary Reproduction and Obstetrics to the School of Graduate Studies, Mekelle University through the Department of Gynaecology and Obstetrics, done by Mr. Tadesse Gugssa Kebede (Id. No. CVM/PR84988/06) is an authentic work carried out by him under our guidance. The matter embodied in this project work has not been submitted earlier for award of any degree or diploma to the best of our knowledge and belief. Name of student: Mr. Tadesse Gugssa Signature Date Name of advisors: 1. Dr. Gebregiorgis Ashebir (DVM, MVSc) 2. Dr. Yayneshet Tesfay (PhD) Signature Date Signature Date Name of chairperson: Dr. Etsay Kebede Signature Date Name of Internal Examiner: Dr. Niraj Kumar (MVSc) Signature Name of External Examiner: Dr. Zelalem Tesfay Signature Date Date 2

3 DEDICATION I dedicate this thesis manuscript to my late father Gugssa Kebede for his ambition to get his talent through his children and to my mother Tarekesh Adhanom for nursing her children with affection and love. 3

4 EFFECTS OF PROSTAGLANDIN ADMINISTRATION FREQUENCY, ARTIFICIAL INSEMINATION TIMING AND BREED ON FERTILITY OF COWS AND HEIFERS IN EASTERN ZONE OF TIGRAY REGION, ETHIOPIA By Tadesse Gugssa Kebede BOARD OF EXTERNAL EXAMINERS Advisors: Signature: 1. Dr. Gebregiorgis Ashebir (DVM, MVSc) 2. Dr. Yayneshet Tesfay (PhD ) 4

5 BIOGRAPHICAL SKETCH The author was born in Tigray on May 08, He attended his elementary and secondary school education at Agazi primary and Secondary School in Adigrat. After completion of his high school education, he joined Addis Ababa University College of Veterinary Medicine, Debre-zeit, in September 1989 and graduated with Diploma in Animal Health in Soon after graduation he was employed by the Ministry of Agriculture and Rural Development and served for eleven years as Animal Health Assistant expert, Team Leader and Animal Health Coordinator at district level. The author joined Mekelle University College of veterinary medicine, in 2002 to study his undergraduate degree. In the year 2006 he graduated with BSc in Veterinary Science, after graduating, the author was employed by Bureau of Agricultural and Rural Development, Tigray region, where he served as regional animal health and artificial insemination higher expert for about twelve years. The author joined the School of Graduate Studies of Mekelle University, College of veterinary medicine in October 2013 to pursue MSc degree in Veterinary Reproduction and Obstetrics through financial funding from ILRI/LIVES project. I

6 AKNOWLEDGEMENTS Above all, I would like to render my utmost praise to the Almighty GOD, for giving me the opportunity for this postgraduate study and for giving me health, energy, and favorable situations to carry out the research work. I would like to extend due regards to the ILRI/LIVES project for sponsoring me for this post-graduate study and research work. Had it not been for the promising support of the LIVES Project, it would not have been possible to conduct the research in the three study areas of the Eastern zone, Tigray Region, Ethiopia. I have special honor and am very grateful to my advisors Dr. Yayneshet Tesfay (PhD) and Dr. Gebregiorgis Ashebir (MVSc) for their unreserved supports and guidance throughout the research work. This thesis would not have been possible without the enthusiasm, knowledge, guidance, tenacity, and, perhaps most importantly faith that I received from my academic advisors, their encouragement and all-rounded supports to perform this work from the very beginning to end. I also would like to express my honest gratitude to Dr. Berihu G/kidane ( MVSc, Dean of CVM), Proffesor Melaku Tefera, Dr.Alemayoh Lemma (PhD),Dr. Yohanes Hagos (MSc.), Dr. Niraj Kumar (MVSc), Dr. Yisehak Tsegaye (MVSc), Mr. Alemselam Brhanu (MSc, PhD candidate), Mr. Desalew Tadesse (MSc), Dr. Berihun Afera (MVSc), Mr. Nugus Abebe (MSc) and Mr. Mussie Girmay (MSc) for their invaluable support and constructive comments they have made on my research proposal and for the final thesis write-up. My genuine thanks goes to my organization, Tigray region Bureau of Agriculture and Rural Development for giving me a study leave and all round support. Many special thanks are due to Mr. Kiros Bitew (MA), Deputy Tigray Region Government State Administration and Head of Bureau of Agriculture and Rural Development, Dr. Gebrezigabher Gebreyohannes (PhD), State Minister Animal Resource Development Sector, Mr. Jemal Gidey (MSc.), Deputy Bureau Head and Livestock Development and Health process Owner, Mr. Fissiha Bezabih (MSc.), Deputy Bureau Head and Extension II

7 Process Owner and Mr. Getachew Ferede (BA degree), Deputy Bureau Head and Coordinator of Support Process Owners, in TBoARD for overall transport facilitation and help in my thesis work. I would like to appreciate the supports given to me by the Mekelle University College of Veterinary Medicine. I am very much indebted to all those who genuinely and affably devoted their times to contribute to the research by participating in animal selection, synchronizing, questionnaire filling, pregnancy diagnosis and related programs. These include relevant professionals from regional and wereda agricultural offices, and private sectors. Moreover, the cooperation of artificial insemination technicians and that of farmers in each study district was unforgettable. I would like to thank the University of Edinburgh for donating Rapid Progesterone Heat Detection Test (RPHDT) to the College of Veterinary Medicine which enabled me to conduct the study. Most sincere appreciation is also due to my colleague and friend, I would like to express my heartfelt thanks for their regular advises and comments to my thesis work. I also appreciate the valuable assistance of colleagues, Mr. H.Slassie G. Mariam (MSc), Mr. Haftay Abreha (MSc), Mr. Anteneh Zewdie (MA), Dr. Mekonnen H/slassie (MVSc), Mr. Tilahun G.her (BA), Mr. Amanual W.gerima (MA), Mr. Girmay Belay (BA), Mr. Jeilu Jemal (MSc), Mr. Moab Temesgen (BA) Mr. Tesfay G.mariam (BSc.), Goiteom Eyasu (BSc.), Medhanye Tekle (BSc), Bisrat Mesfin (MSc), Alemate Hagos (BSc), G/hiwet Hagos, Haftay Girmay, Alem G/Mariam, Temesgen Hagos, Kahase Birhane, Tesfu Gebru, and Meseret Hagos during the course of field work and analysis. My best friends and class mates, Dr. Bahlibi W.gebreal, Dr.Atakilty Hadush, Dr.Haftom Yemane, Dr.W/melak, Dr.Juhar Mohammed, Dr.Million would also deserve heartfelt gratitude for their support and for the good time we spent together. Last but not the least; I owe special thanks to my family W/ro Tarekech Adhanom, Mr. Solomon Gugssa with his family, Mr. Desta Gugssa with his family, Enquanayehush Gugssa and my sons and daughters Binyam, Solomon, Burtukaun and Selam Tadesse for their honest and continuous support during my study times. III

8 TABLE OF CONTENTS BIOGRAPHICAL SKETCH... I AKNOWLEDGEMENTS... II TABLE OF CONTENTS... IV LIST OF TABLES... VI LIST OF FIGURES... VII LIST OF APPENDIXES... VIII LIST OF ABBREVIATIONS... IX ABSTRACT... X 1. CHAPTER I: INTRODUCTION BACKGROUND AND JUSTIFICATION PROBLEM STATEMENTS RESEARCH OBJECTIVES General Objective Specific Objectives RESEARCH QUESTIONS CHAPTER II: LITERATURE REVIEW HISTORICAL BACKGROUND OF ESTROUS SYNCHRONIZATION IN CATTLE ESTROUS BEHAVIOR AND ITS IMPORTANCE IN REPRODUCTION ACTIVITY IN CATTLE PROSTAGLANDINS PROSTAGLANDIN BASED ESTROUS SYNCHRONIZATION PROTOCOL One shot prostaglandin Two shot prostaglandin USE OF PROSTAGLANDIN IN ESTRUS SYNCHRONIZATION OF DAIRY COWS FERTILITY FOLLOWING PROSTAGLANDIN INDUCED ESTRUS IN DAIRY CATTLE PROGESTERONE ANALYSIS USING MILK SAMPLES FACTORS INFLUENCING THE EFFECTS OF PROSTAGLANDIN TREATMENT Stage of Estrous Cycle at the Time of Prostaglandin Treatment Effect of Progesterone Level on Synchronized Estrus Effect of Different Prostaglandin Analogues on Estrus Response and Fertility Breed and Season FACTORS LIMITING THE USE OF PROSTAGLANDIN IN DAIRY COWS Effect of Accuracy in Rectal Palpation of CL Number of Cows in Synchronized Estrus Level of Progesterone during Prostaglandin Treatment Variation in Duration of Onset of Estrus CHAPTER III: MATERIALS AND METHODS LOCATION AND DESCRIPTION OF STUDY AREAS STUDY ANIMALS IV

9 3.3. STUDY DESIGN AND SAMPLING STRATEGIES Sample Size Determination Experimental Design Experimental Procedures Single and Double Prostaglandin Injection Time of Insemination RPHDT and AIT Corpus Luteum Detection through Rectal Palpation DATA COLLECTION Estrus Response of Cows and Heifers Conception Rate Efficiency of Artificial Insemination Technicians Independent Variables STATISTICAL ANALYSIS ETHICAL CONSIDERATION CHAPTER IV: RESULTS EFFECT OF BREED AND FREQUENCY OF PROSTAGLANDIN INJECTION ON HEAT EXPRESSION Estrus expression Rate Duration of estrus response after PGF 2α injection EFFECT OF BREED AND FREQUENCY OF PROSTAGLANDIN INJECTION ON CONCEPTION RATE COMPARISON OF FIXED TIME AI WITH AI AT DETECTED HEAT ON CONCEPTION RATE RAPID PROGESTERONE HEAT DETECTION TEST CHAPTER 5: DISCUSSION ESTRUS RESPONSE RATE CONCEPTION RATE EVALUATION OF THE EFFECTIVENESS OF FIXED TIME AI AND AI AT DETECTED ESTRUS ON CONCEPTION RATE COMPARISON OF THE EFFICIENCY OF AI TECHNICIANS ON CL DETECTION THROUGH RECTAL PALPATION WITH THAT OF RAPID PROGESTERONE HEAT DETECTION TEST (RPHDT) CONCLUSION AND RECOMMENDATIONS CONCLUSION RECOMMENDATIONS REFERENCES APPENDIXES V

10 LIST OF TABLES Table 1: Sources and functions of the major reproductive hormones and commercial products... 9 Table 2: Summarized lists of independent variables with their description and expected effects on the dependent variables Table 3: Estrus response rate of local and crossbred cows and heifers after single and double dose injection of PGF 2α Table 4: Estrus response rate of local and crossbred cattle after single dose injection of PGF 2α.. 36 Table 5: Conception rate result of Local and Crossbred, Cows and Heifers after single and double dose PGF 2α Injection Table 6: Conception rate result after single dose PGF 2α injection Table 7: Conception rate result after double dose PGF2α injection Table 8: Fixed AI Versus AI at Detected estrus Table 9: Rapid Progesterone Heat Detection Test (RPHDT) result Table 10: Results of a quadratic discriminant analysis VI

11 LIST OF FIGURES Figure 1: Location map of the study area Figure 2: Single dose PGF 2α and breeding according to estrus. (Upper), double dose PGF 2α and breeding according to estrus. (Middle), Double dose PGF 2α and timed AI (bottom) Figure 3: Estrus response rate of local and crossbred cattle after single and double dose injection of PGF 2α Figure 4: Time of Estrus Response after Single and double dose PGF 2α Injection Figure 5: Time of Estrus Response after Single PGF 2α Injection Figure 6: Time of Estrus Response after double PGF 2α Injection Figure 7: Estrus duration for single injection of PGF 2α before 48 and after 72 hours Figure 8: Estrus duration for double injection of PGF2α before 48 and after 72 hours Figure 9: Conception rate of local and crossbred cattle after single and double dose injection of PGF 2α Figure 10: Comparison of AI Technician efficiency with RPHDT VII

12 LIST OF APPENDIXES Appendix I: Investigation format for collecting data from selected individual heifers and cows for synchronization and hormonal analysis Appendix II: Elevation Map of the study area (Peasant association) Appendix III: Rapid progesterone heat detection test summary format Appendix IV: Different photos taken during the study Appendix V: Ethical Clearance Letter VIII

13 LIST OF ABBREVIATIONS AI AIED AIT BoFED CI CL EER FTAI FSH GDP GLM GnRH ILRI IPMS LH LIVES M.a.s.l. MoA OR P4 PGF2α RPHDT SNNPR SPSS TBoARD USAID WOARD WoFP Artificial Insemination Artificial Insemination at Estrus Detection Artificial Insemination Technician Bureau of Finance and Economic Development Confident Interval Corpus Luteum Estrus Expression Rate Fixed Time Artificial Insemination Follicular Stimulating Hormone Gross Domestic Product Generalized Linear Model Gonadotropin Releasing Hormone International Livestock Research Institute Improving the Productivity and Market Success Luteinizing Hormone Livestock and Irrigation Value Chains for Ethiopian Smallholders Meter above sea level Ministry of Agriculture Odds Ratio Progesterone Prostaglandin F Two Alpha Rapid Progesterone Heat Detection Test Southern Nations, Nationalities, and Peoples' Region Statistical Package for Social Science Tigray Bureau of Agricultural and Rural Development United States Agency for International Development Wereda Office of Agriculture and rural development Wereda Office of Finance and Planning IX

14 ABSTRACT The study was conducted to evaluate and compare the effect of single and double dose prostaglandin on estrus response and conception of crossbred (Holstein Friesian x Zebu) and local (Zebu) cattle in Eastern zone of Tigray, Ethiopia. The study also evaluated and compared efficiency of AI technicians to detect cuprous luteum through rectal palpation and reproductive status by progesterone level determination in milk using Rapid Progesterone Heat Detection Test (RPHDT). A total of 240 crossbred postpartum (>60 days) cows and heifers were assigned for single (group 1, n=120) and double (group 2, n=120) dose PGF2α protocols. Estrus response and conception rate was compared between protocols, breeds and parity. Reproductive stage of animals was confirmed prior to estrus synchronization and efficiency of AI technicians on detection of CL through rectal palpation assessed using RPHDT. Among cows/heifers synchronized using both PGF2α protocols, 87.2% (157/180) were detected in estrus on visual observation and rectal palpation. Overall conception rate among animals in heat was 62.7% (96/153). Out of the 120 cows/heifers synchronized using single dose PGF2α injection, 84.2% were detected in estrus with a conception rate of 59.6% (59/99). The overall estrus response rate of crossbred and local cattle was 84.2% and 93.3% in group 1 and group 2, respectively. No significant difference was found in estrus response between breeds in both groups. Conception rates in crossbred and local cattle in group 1 were 58.5% (31/53) and 60.9% (28/46), respectively. Whereas, the conception rates in local and crossbreds in group 2 were 63% (17/27) and 74% (20/27), respectively. No significant difference was found in conception rate between breeds in both treatment groups. The conception rates of cows and heifers in group 2 which were inseminated after heat detection and fixed time insemination were 68.5% (37/54) and 48.9% (23/47), respectively. The conception rate of cattle inseminated at detected heat was significantly higher (P<0.05) than those inseminated at fixed time. Among 90 animals milk progesterone level was determined using RPHDT prior to synchronization, 57% had high progesterone level, while 43% low progesterone. On average, one technician misclassified 4.6 cows out of 10 cows presented for corpus luteum detection through rectal palpation. In conclusion, the overall estrus response and conception rate in the study area was high. Single dose PGF2α protocol is recommended for estrus synchronization in cattle in the region as well as the country, as it is comparatively cost effective, has less number of visits to farms and less laborious. RPHDT can assist rectal palpation to evaluate reproductive stage of animals, hence provide proper breeding management. Refreshment trainings should be given to AI technicians on rectal palpation to improve detection of CL and subsequently improve reproductive performance of cattle in the region. Keywords: AI, breed, double dose, PGF2α, Single dose, Synchronization X

15 1. CHAPTER I: INTRODUCTION 1.1. Background and Justification Livestock production is an integral part of the agricultural activities in Ethiopia. The livestock sector contributes about 12-16% of the total national Gross Domestic Product (GDP), 30-35% of the agricultural GDP, 15% of export earnings, and 30% of agricultural employment in the country (Land O'Lakes, 2010). Moreover, livestock contributes about 60-70% of the livelihoods of the Ethiopian population (Tessema, et al., 2010). Although the country holds the largest livestock population in Africa, production is too low mainly due to poor genetic performance, nutrition, management, infertility, reproductive disorders and diseases accompanied with lack of veterinary and Artificial Insemination (AI) professionals hindering the growth of the dairy industry in the country (Mureda and Mekuriaw, 2007; Mekonnen, et al., 2010; Bitew and Prasad, 2011; Haileselassie, et al., 2011). Estrus (heat) detection has been cited as the most important factor affecting the reproductive success of artificial insemination programs. However, proper control of the time of estrus is difficult, since peak estrus activity often occurs at night, and determination of the actual onset of standing estrus may be difficult without 24 hour observation (Aulakh, 2008). The commonly used method of estrus detection for cow breeding is mainly visual inspection which makes estrus detection unsatisfactory in most of the dairy farms (Tsadik, et al., 2008).Visual detection is less efficient way as a result of which, in most cases, cattle remain unobserved when they came in to estrus. The case becomes severe when cows come into estrus in the evening when it is usual that people become less active and go for rest. Similarly, rectal palpation is the only feasible, available and routine methods of diagnosing pregnancy (Labago, 2007) as well as pathology of the reproductive organs or reproductive status (Tsadik, et al., 2008). Although rectal palpation is the cheapest method and results can be found quickly, inaccurate diagnosis of pregnancy at an early stage, in accurate reproductive status and loss of embryo /fetuses are the common disadvantages of this method (Franco, et al., 1

16 1987). Rectal palpation is usually carried out late, after 60 days post-services by artificial insemination technicians or other animal health professionals. Poor practice and lack of experience in the field of artificial insemination (AI) and pregnancy diagnosis has been one of the main problems aggravating the poor reproductive performance. This results in loss of milk production, low calf crop as well as unnecessary feeding and management costs. This is because of mis-diagnosing non pregnant cows and heifers as if they are pregnant and vice versa. All leads the owners to take inappropriate management decision and practices such as extended calving interval, abortion and selling of pregnant cows by mistake (Lobago, 2007). Anestrous and repeat breeding are among the major and common problems affecting the reproductive performance of dairy cattle in Tigray (Tsadik, et al., 2008). It has been reported that heifers never bred until they are three or more years old. Similarly, postpartum anestrous leads to very long inter-calving intervals in local and crossbred cattle (Mukasa-Mugerwa, et al., 1991; Tsadik, et al., 2008). Additionally, ovarian cyst (follicular or luteal cyst) is one of the common factors causing sub fertility/infertility in some of the anoestrus and repeat breeding dairy cows (Tsadik, et al., 2008). Cystic ovarian disease cause extended calving interval (Vanholder, et al., 2006) by disrupting the normal estrous cycle of the animal. Poor farm management, nutrition and health condition of cattle in smallholder farms have also reported to affecting the fertility of dairy cattle in Tigray region (Tsadik, et al., 2008). Fertility is an important factor for the production and profitability in dairy herds (Gokhan, et al., 2010). A calving interval of 12 to 13 months is generally considered to be economically optimal, but often difficult to achieve. To meet this goal cows must cycle and become pregnant within an average of 85 days postpartum. However, a long postpartum anoestrous period is a very common problem in cows reared in a tropical environment (Million, et al., 2011). In dairy cattle, detection of estrus can be difficult due to a number of factors including the incidence of silent estrus. Hormonal treatments designed to control both luteal and follicular function has permitting efficient synchronizations of time of ovulation. Thus, the AI can be performed in a large number of animals on a fixed schedule without the 2

17 need for detection of estrus. Using these management techniques, the fixed-time artificial insemination (FTAI) can overcome the problem of accurate estrus detection and help in reducing the incidence of repeat breeding. In addition, with FTAI in cattle operations, it is possible to facilitate management practices and commercialization, and to reduce the time and semen wasting with animals inseminated at incorrect times (Letícia, et al., 2011). The benefits of using technological options and approaches to improve supply of desirable animal genetic material that incorporates estrus synchronization and AI can be tremendous. These systems allow producers to reach certain production or economic goals quicker than natural service and can open the doors to value added markets as well by shortening and concentrating the calving and breeding season; inducing anestrous cows and pre-pubertal heifers to cycle; introducing new genetics into the herd; increasing calf performance and weaning weights with earlier birthdates; enabling more cows to be artificially inseminated to a genetically superior bull and decreasing the labor cost for heat detection (Bambal and Jais, 2011). Therefore, it is essential to introduce and implement appropriate technologies for improving the existing genetic makeup of dairy cattle generally in the region and particularly in the study area Problem Statements Infertility is the main influencing factor that adversely affects the production and productivity of local and crossbred cows and heifers in Ethiopia. Consequently, cow and calf production and productivity continues to be unsatisfactory. Calving interval is generally longer than 12 months in most cows, including crossbreds, kept by small holder farmers. Heifers are reported to have a higher age at first calving (Shiferaw, et al., 2003) and rarely calve every months after the first calving. In an attempt to reverse this situation, Tigray Bureau of Agriculture and Rural Development (TBoARD) in collaboration with other stakeholders conducted a mass synchronization scheme addressing 58,676 cows and heifers in three years (2011 to 2014), using single 3

18 prostaglandin F2α injection in 33 districts of the region. According to this report, the mass synchronization results showed low performance with a synchrony and conception rates of 85% (ranges from %) and 31.5% (ranges from %), respectively. This extremely low performance was mainly due to lack of skilled Artificial Insemination technicians, using of fixed time insemination in the synchronization protocol, unplanned strategic feed supplementation of synchronized cattle and animal selection problems (TBoARD, 2014). Technically, Synchronization programs for lactating cattle and the effectiveness of this treatment depends mainly on the presence of a functional Corpus Luteum (CL) on the ovary during the application and this can be easily verified by progesterone analysis. But, the skill, knowledge and practice exhibits that this process is not in place. Unfortunately, no research was done on the efficiency of AI technicians on corpus luteum detection through rectal palpation. Similarly, there is lack of information in the local breeding practice, effect of prostaglandin, on estrus synchronization of local and crossbred dairy cattle as well as the benefit of farmers from this technology. Therefore, this research is designed to fill these main gaps and come up with relevant possible recommendations useful for the dairy development program Research objectives General Objective The overall objective of this research is to investigate the effect of single and double dose prostaglandin on estrus response and conception rate of local and crossbred cows and heifers in the study area Specific Objectives To evaluate the effect of breed and frequency of prostaglandin injection on heat expression of cows and heifers. 4

19 To evaluate the effect of breed and frequency of prostaglandin injection on conception rate of cows and heifers. To evaluate the effect of frequency of prostaglandin injection on duration of heat expression of cows and heifers. To evaluate the effectiveness of fixed time AI and AI at detected estrus on the conception rate dairy cows and heifers. To compare the efficiency of AI technicians on CL detection through rectal palpation with that of Rapid Progesterone Heat Detection Test (RPHDT) in the study areas Research questions On the basis of the objectives formulated above the following research questions are raised to be investigated. What is the difference on estrus response and conception rates following single and double dose prostaglandin hormone administered to local and crossbred cows and heifers? Is there any difference on estrus duration time after single and double dose injection of PGF2α? What is the success rate of AI when given at fixed time and following the detection of reliable estrus in cows and heifers? Are AI technicians efficient enough in detecting CL in cows using rectal palpation? 5

20 2. CHAPTER II: LITERATURE REVIEW 2.1. Historical Background of Estrous Synchronization in Cattle Estrus synchronization is a farm management technique used to bring a group of animals in a same stage of estrus cycle so that they may come into estrus and ovulate at the same time. It is a good management tool for programmed breeding that can help beef and dairy producers to improve production and reproduction efficiency and economic returns. It can help shorten the breeding and calving seasons and produce calves more uniform in age and weight (Fike, et al., 1999). Hence leading to programmed feeding and easier management of the cows being at the same stage of gestation. Finally, the calving will also be easy and convenient due to expected date of calving spread over a shortest possible period (Sattar, 2002). The history of estrous cycle synchronization and the use of artificial insemination in cattle is a testimony to how discoveries in basic science can be applied to advance the techniques used for livestock breeding and management (Beal, 2002). The first successful synchronization of estrus in cattle was reported in 1948 (Christian and Casida, 1948). Since then more concentration was focused towards research on estrous synchronization and development of estrous synchronization products. Synchronizing estrous cycles of domestic cattle depends on control of the functional life span of the corpus luteum (Hansel and Convey, 1983). There are two ways to facilitate control of the corpus luteum that result subsequently in estrus and ovulation. The first method involves long term administration of a progestin with subsequent regression of the corpus luteum during the time the progestin is administered (Britt, 1987). Estrus and ovulation occur within 2 to 8 days after progestin withdrawal. The second method involves the administration of a luteolytic agent that shortened the normal life span of the corpus luteum. This is accompanied generally with estrus and ovulation within 48 to 120 hours after injection. 6

21 It is known that no enough selection and improvement for productivity has been performed on the indigenous cattle. Nevertheless, the indigenous cattle are known to have special merit of coping with the harsh environments of the country. On the other hand, the high performing exotic cattle cannot cope with the harsh environments of the country. Therefore, improvement on the indigenous cattle for productivity without losing traits, which are essential for survival, has been proposed (MoA, 1996). Artificial insemination (AI) and embryo transfer (ET) management programs are highly dependent upon accurate heat detection procedures to achieve successful results. Conducting two to three daily visual heat detection observations of the cattle herd during the AI breeding season could lead to economic benefits to beef and dairy producers. Efficient heat detection, however, is time-consuming, labor-intensive, and requires good management and recordkeeping. Undetected heats in an AI program play a significant role in lowering reproductive efficiency by increasing the number of open days, which in turn results in longer calving intervals and ultimately reduces the net return to the producer (DuPonte, 2007). The use of AI in Ethiopia is growing but estrus detection is difficult owing to poorly expressed estrus of Zebu breeds (Mukassa-Mugerwa, et al., 1989). Similarly, Tegegne, et al.,1989, Bekele, et al.,1991) have shown that the short duration and low intensity of estrus signs in Ethiopian Zebu cattle caused most estrus detection failures which indicates a need for the use of current advances in AI, such as estrus synchronization Estrous Behavior and its Importance in Reproduction Activity in Cattle A successful estrous synchronization program requires an understanding of the estrous cycle. Estrous behavioral events are physiological changes leading to ovulation and sexual receptivity. This periodic pattern of sexual receptivity is the result of an organized and complex series of changes that occur in the reproductive system of cattle. Bovine estrus has been described as a short period (approximately 15 to 18 h) of sexual receptivity that is manifested every 18 to 24 days, with ovulation occurring 10 to 14 h after the cessation of behavioral signs of estrus (Esslemont, et al., 1980; Allrich, 1993). 7

22 Estrogen production by the developing follicle results in a surge in the release of LH and FSH from the pituitary which stimulates maximum estrogen production by the follicle. The behavioral changes that occur at this time are used as the primary indicators of estrus. Estrous behavior expression in female cattle has a significant influence on the success of artificial breeding. The target of getting one calf annually per cow may only be achieved with proper detection of estrus and subsequent successful breeding. Standing behavior is one of several visual estrus symptoms (Diskin and Sreenan, 2000; Roberts, 1986). It shows that the female cattle are clearly in estrus (Yaniz, et al., 2006; DuPonte, 2007). Cows enter standing estrus gradually; secondary signs that an animal is getting close to standing estrus will progress until the animal stands to be mounted (Diskin and Sreenan, 2000) such as following, standing with, head resting, sniffing, nuzzling, licking, and grouping with other cows in or near estrus. These animals are active, nervous, restless, bawling, walking and searching (Allrich, 1993). However, none of the above behavior alone is a positive determination of standing estrus except standing to be mounted by a bull or another cow/heifer which is the only conclusive sign that an animal is in standing estrus and ready to be inseminated (Perry, 2004). Many factors affect estrus expression in cattle. Some of them related to management and to the cows (Colman, 1993). Reports from Dransfield, et al. (1998) and Stevenson, et al. (1998) showed that cow related factors (age or parity) contribute largely to the low detection rates. Difference between breeds in estrous behavior and duration of expression of standing heat and environmental stress (Gwazdauskas, et al., 1981) reported to affect expression of estrous behavior. The systems of estrous control that are used to synchronize or induce heat are designed to manipulate various components or functions of the estrous cycle. In order to manipulate various components or functions of the estrous cycle to synchronize or induce heat, it is necessary to understand the estrous cycle (Michael and Thomas, 2005; Roberts, 1986). The primary glands or tissues that control the estrous cycle are the hypothalamus, pituitary, ovary, and uterus. Each of these components of the reproductive system secretes chemical compounds called hormones, which regulate its own function, or the 8

23 function of other components. Many hormones are involved in control of the estrous cycle, and release into the bloodstream can be measured experimentally (Michael and Thomas, 2005; Soren, et al., 2012). The major hormones which are most commonly manipulated or administered to animals to synchronize estrus, are outlined in Table 1. Table 1: Sources and functions of the major reproductive hormones and commercial products Hormone Source Function Commercial Products GnRH Hypothalamus Releases FSH and LH Cystorelin, Factrel, Fertagyl, OvaCyst FSH Anterior pituitary Stimulates development of ovarian follicles Estrogen Ovarian follicle Stimulates behavioral estrus and the LH surge LH Anterior pituitary Stimulates rupture of a follicle (ovulation) Folltropin Not used in current systems Not used in current systems Progesterone Corpus luteum Maintains pregnancy Melengestrol acetate MGA ), Intravaginal Progesterone Releasing Insert (CIDR ) Prostaglandin Uterus Regress Corpus luteum Lutalyse, Estrumate, (Adapted from Michael and Thomas, 2005.) ProstaMate, In Sync 2.3. Prostaglandins PGF2α is an endogenous hormone produced by uterine endometrium. Its lipids consisting of 20-carbon unsaturated hydroxyl fatty acids derived from arachidonic acid and is responsible for luteolysis, or degradation of the CL in cattle (Lauderdale, et al., 1974). During the normal estrous cycle of a non-pregnant animal, PGF2α is produced and 9

24 released from the uterus 16 to 18 days after previous estrus. This release of PGF2α is responsible for regression of the CL. The CL is a structure in the ovary that produces progesterone and prevents occurrence of estrus. The release of PGF2α from the uterus is the triggering mechanism that eventually brings the animal to return to estrus every 21 days (Lauderdale, et al., 1974). An injection of a synthetic PGF2α will mimic natural PGF2α release to cause CL regression. Synchronized regression of the CL will synchronize a decline in progesterone and result in the final growth of the dominant follicle to produce estradiol and behavioral heat (Diskin, et al., 2002; Michael and Thomas, 2005). In cyclic females, estrus occurs within 2 to 6 days after intramuscular injections of prostaglandin F2α (Lutalyse ) or one of its analogues (ProstaMate, Estrumate, estroplan, In-Synch ) (Islam, 2011). Anestrous cows and prepubertal heifers will not respond to an injection of PGF2α since no CL exists. If represent a major portion of the herd, response rates could be quite low. Estrous-cyclic females can respond to injections between days 7 and 16 of cycles in the presence of a functional corpus luteum. The CL is a gland that develops in the ovary and secretes the hormone progesterone into the cow s blood. Estrous-cyclic females at days 0 to 6 and 17 to 21 of their cycles are without functional CLs and do not respond to injections. Research has shown that a higher percentage of cattle treated with PGF2α during the late luteal phase (Days 10 to 17) exhibited estrus than those treated during the early luteal phase (Days 5 to 9).It has also been shown that the closest synchrony of estrus occurs when cattle are at a similar stage of the estrous cycle when PGF2α is administered (Diskin, et al., 2002; Michael and Thomas, 2005). The effectiveness of PGF2α to induce estrus is dependent upon the presence of a responsive CL. This typically occurs from Days 5 to 17 (heifers) and 7 to 17 (cows) of the estrous cycle, however, responses are usually greatest, intermediate and least for cows in the late (Days 14 19; 95.7%, 202/211), middle (Days 10 13; 86.4%, 291/337) or early (Days 5 9; 76.9%, 173/225) stages of the estrous cycle, respectively (Xu, et al., 1997). Intervals to estrus following treatment with PGF2α are dependent on the stage of follicle development at the time of treatment; cows with mature follicles at the time of luteolysis 10

25 enter estrus sooner than cows with immature follicles (Roche, et al., 1999). Two-dose treatment protocols have been developed to ensure that most cows have a CL responsive to PGF2α at the time of treatment with the second dose of PGF2α Prostaglandin based Estrous Synchronization Protocol One shot prostaglandin Option 1: shows a single injection of PGF2α is given to cyclic females, and then these females are bred as express estrus. The disadvantage of this program is that 20-25% of the females will not respond to the injection, but the advantages are the lower cost of one injection and that females are only handled once other than for breeding (Islam, 2011). Option 2: Second one shot option requires detection of estrus before any PGF2α treatment is administered. The producer detects estrus for 5 days and breeds each cow as exhibits estrus. The cows that have not exhibited estrus by the fifth day are given an injection of prostaglandin, which should induce to come into estrus in about 3 to 5 days (Michael and Thomas, 2005). This option represents the greatest savings in cost and labor associated with treatments because only one injection is given and not all the cows will need it. In addition, detecting estrus for 5 days gives the producer some idea of the total number of cows that are cycling. During this 5-day period, approximately 20 to 25 percent of the cows should show estrus (4 to 5 percent per day). All cows that are cyclic should show estrus within five days after the PGF2α injection. This is the most popular protocol that uses only PGF2α to synchronize estrus and can result in more than 90% of cyclic cows being bred during the first 10 days of the season. If 4 to 5 percent of the cows are not exhibiting estrus each day, then the cows are probably not cycling. This will allow time to evaluate the effectiveness of the estrous synchronization program. The disadvantage of this program is that it requires 5 days of accurate detection of estrus before prostaglandin treatment is administered. This program is recommended because of the opportunity to determine the reproductive status of the herd before animals are treated for synchronization (Islam, 2011). 11

26 Two shot prostaglandin The two injection programs for synchronization with PGF2α are designed to increase the proportion of females with a CL that is responsive to regression with PGF2α. Option 1 uses two injections of prostaglandin spaced 14 days apart. Detection of estrus is not required before or between injections. All cycling cows should respond to the second injection regardless of what stage of the estrous cycle were in when the first injection was administered. Remember the non-cycling cows will not generally respond to prostaglandin products. The advantage of this option is that more cows should come into estrus at any given time than with the one shot options. The disadvantage is that it involves the cost and labor of administering two injections of prostaglandin to all cows (Michael and Thomas, 2005; Păcală, et al., 2009). Option 2 the second two-shot prostaglandin injection option is give the first injection, and breed all females exhibiting estrus and then give the second injection to only females that were not breed. This option lowers expense and handling, but results in two synchronized groups instead of one and a longer breeding period. Timed insemination instead of estrous detection may be used, but conception rates are generally lower than with estrous detection. Short-term calf removal may improve the response in cyclic postpartum cows (Michael and Thomas, 2005; Păcală, et al., 2009) Use of Prostaglandin in Estrus Synchronization of Dairy Cows In the early 1970s several workers pioneered the luteolytic effect of prostaglandin F2α (PGF2α) in cattle (Rowson, et al., 1972). Subsequent research efforts then attempted to improve the reproductive efficiency of dairy cattle by inducing estrus with PGF2α (Seguin, et al., 1978; Plunkett, et al., 1984). Several studies demonstrated the capacity of PGF2α and its synthetic analogues, to trigger the regression of a mature CL in the ovary, thus provoking and synchronizing estrus (Lauderdale et al., 1974; Stevenson and Pursley, 1994). When PGF2α was administered to cows with a functionally mature CL, 85 to 95% reached estrus within 7 days of treatment (Macmillan and Henderson, 1983; Armstrong, et al., 1989; Folman, et al., 1990; Rosenberg, et al., 1990); 70 to 90% showing signs of 12

27 estrus 3 to 5 days after treatment (Ferguson and Galligan, 1993). Further, an enhanced estrus response and normal fertility were reported when PGF2α was given at the late, rather than early to middle stage of the luteal phase (Tanabe and Hann, 1984; Watts and Fuquay, 1985; Xu, et al., 1997). Thus, the 14 days interval double prostaglandin regimen seems to show an improved response over the 11 days protocol, since two treatments given 14 days apart ensures that most animals are in the late luteal stage (cycle Day 11 to 14) when they receive the second PGF2α dose (Folman, et al., 1990; Rosenberg, et al., 1990; Young, 1989). Considerable research has been carried out in order to develop technologies to synchronize and efficiently detect estrus. In the past, reproductive management protocols have focused on the synchronization of estrus using PGF2α. These were very successful when cows were bred after a detected estrus. Detection of estrus increases and management of AI is more efficient when estrus is synchronized with PGF2α in contrast to daily detection of estrus (Stevenson and Pursley, 1994). Nevertheless, synchronization with PGF2α does not control the time of AI because estrus detection is still required. Lucy, et al., (1986) showed that cows receiving a fixed time AI at 72 to 80 hr after a second injection of PGF2α resulted in pregnancy rates considerably lower (P < 0.05) compared to cows receiving AI at a detected estrus alone. Low pregnancy rates related to timed AI following treatment with PGF2α may be explained by the variation in time of ovulation with respect to time of AI. This variation in time of ovulation is due to the deviation in stage of the pre-ovulatory follicle at the time of PGF2α injection (Pursley, et al., 1997). If a fully developed and functional dominant follicle is present at PGF2α injection, the time to and variation in time to ovulation, or estrus are significantly less than if the dominant follicle is early in development (Cavalieri, et al., 2008). Estrus synchronization protocols have been used to reduce labor and time associated with estrus detection and artificial insemination. Enhanced regulation of estrus depends on controlling the corpus luteum as well as follicular development. Prostaglandin F2α given in two doses, 14 days apart, has been used as a method of estrus synchronization which has limited time required for detecting estrus. Cows synchronized with two prostaglandin 13

28 injections, 14 days apart, have produced pregnancy rates of 84% (Folman, et al., 1990). Despite successful conception rates, intense estrus detection is necessary for 2 to 5 days following treatment. In order to increase the efficiency of AI in the dairy industry, producers must be presented with an AI program designed to coordinate follicular maturation and luteal regression closely such that ovulation can be predicted. This would eliminate the need for the detection of estrus, permitting insemination at a prescribed time. (Cavalieri, et al., 2008). Two PGF2α injections, separated by 14 days, would offer partial synchronization of follicular development before luteal regression. If a group of cycling cows is in random stages of the estrous cycle at the time of the first of two PGF2α administrations, luteolysis should be induced in those cows in day 5 to 15 of the estrous cycle, while the rest remain unaffected. At the second PGF2α administration, the initially responsive group should be in the early stages of a new cycle, and the remainder would be in a broader range of the estrous cycle, allowing for a leuteolytic response to PGF2α (Lauderdale, 2002; Wiltbank, et al., 2002). This practice would yield a greater number of cows that will have a maturing second wave dominant follicle capable of ovulating in response to a gonadotropin-releasing hormone (GnRH) induced lutenizing hormone (LH) surge than would a GnRH administration given at a random stage of the estrous cycle. A controlled release of GnRH capable of stimulating an LH surge would greatly reduce the time span of ovulations within a group of synchronized cows and greatly benefit reproductive management when breeding at a fixed time. Therefore, following the PGF2α dosage, a timed insemination protocol such as OvSynch could be incorporated. OvSynch consists of a GnRH injection, PGF2α administration 7 days later followed in 48 hr by administration of GnRH (Pursley, et al., 1997). The combination of the two protocols (two PGF2α injections 14 days apart followed by OvSynch) is known as PreSynch. 14

29 2.6. Fertility Following Prostaglandin Induced Estrus in Dairy Cattle Several researchers have noted normal or above normal fertility following synchronization of estrus with PGF2α in cows (Macmillan and Day, 1982; Lucy et al., 1986; Wenzel, 1991). Young and Henderson (1981) found no significant difference in conception rates after a double 11 days interval treatment regime using a prostaglandin analogue among cows inseminated only once at the fixed time of 75 to 80 hr (46%), cows inseminated twice at 72 and at 96 hours (47%) and control untreated cows (50%). Neither were differences found in cows timed AI following double 14 days PGF2α treatment compared to natural estrus (Macmillan, et al., 1977; Roche and Prendiville, 1979). However, reduced conception rates due to variations in the time of ovulation have been noted after timed AI, either following single (Fetrow and Blanchard, 1987; Archbald, et al., 1992) or double (Waters and Ball, 1978; Stevenson, et al., 1987) PGF2α administration, compared to AI at detected estrus. Reproductive performance in dairy cattle was also improved following double 14 days PGF2α treatment without assessing ovarian status when compared to a single dose based on detecting a CL by rectal palpation or by milk progesterone enzyme immunoassay (Heuwieser, et al., 1997). Tenhagen, et al., (2000) observed that timed insemination following double 14 days PGF2α treatment reduced the number of days open in lactating dairy cows when compared to AI performed at observed estrus. Fertility is high following PGF2α synchronization. Most studies indicate that conception rates are similar for beef cows or heifers synchronized with PGF2α and those bred after a naturally occurring heat. In one of the largest experiments (3,443 head) Moody and Lauderdale (1977) reported that cows or heifers bred 12 hr. After detection of a PGF2αsynchronized estrus had a conception rate of 59%. Untreated cows and heifers in the same herds achieved a 62% conception rate when bred 12 hr. After a natural heat. While some studies have demonstrated a tendency for animals treated with PGF2α late in the estrous cycle to have higher fertility, that trend has been inconsistent. 15

30 2.7. Progesterone Analysis Using Milk Samples Progesterone analysis of milk samples can be used to study the postpartum ovarian activity in the dairy cow. Firstly, a period of low progesterone levels after calving occurs when the cow exhibits a period of anoestrus (Lamming and Bulman, 1976). This period is followed by an increased progesterone level, which is indicative of the first postpartum ovulation. The cavity of the ovulated follicle is gradually filled with progesteronesecreting luteal cells, which forms the corpus luteum. The corpus luteum then dominates the estrus cycle during the luteal phase with high progesterone levels for about 14 days from about the fourth day after ovulation. After that, the corpus luteum is degenerated and a new ovulation can occur unless the cow becomes pregnant and the corpus luteum is maintained during the pregnancy (Peters and Ball, 1995). Since prostaglandins are being used more frequently in synchronization programs for lactating cattle and the effectiveness of this treatment depends on the presence of a functional corpus luteum on the ovary progesterone analysis is useful in verifying if a corpus luteum is present. In the mid-1980 s, kits for performing the progesterone enzyme immunoassay procedure became commercially available to dairy producers and veterinarians. This procedure is considered a cow side test since it can be performed on the farm or in a veterinary clinic (Friggens, et al., 2008). It is important, however that the farmer receives proper instruction on milk sampling assay procedures, and interpretation of the results. This enzyme immunoassay is designed to determine relative rather than absolute concentrations of progesterone, and results are classified as either low or high. In most kits, assays produce a color reaction that can be read visually or through an electronic scanner (Peters and Ball, 1995) Factors Influencing the Effects of Prostaglandin Treatment Stage of Estrous Cycle at the Time of Prostaglandin Treatment Since, induction of estrus was brought about by the luteolytic effect of PGF2α on the mature CL, the success of PGF2α primarily depends on the presence of a mature functional 16

31 CL in the ovary. (Kristula, et al., 1992). Therefore, the stage of estrous cycle at the time of administration of the drug influence the ability of prostaglandin to induce luteolysis in cows (Johnson, 1978; Jackson, et al., 1979; Hansen, et al., 1987). The stage of follicular wave development at the time of PGF2α treatment appears to be the factor determining the time of estrus onset (Ferguson and Galligan, 1993; Adams, 1994; Twagiramungu, et al., 1995). Thus, the time elapsed between PGF2α treatment and the onset of estrus depends on the stage of the estrous cycle at the time of PGF2α treatment (Macmillan and Henderson, 1983; Stevenson, et al., 1984; Tanabe and Hann, 1984). In the same way, Kastelic and Ginther (1991) reported that the time from the administration of PGF2α to ovulation is dependent on the maturity of the most recently emergent dominant follicle. The time of ovulation is therefore dependent on the size of this follicle at luteolysis, because a small dominant follicle takes longer to grow into an ovulatory follicle. Kastelic and Ginther (1991) also reported that when dominant follicle had reached the static phase, the time from treatment to ovulation was 3 days, and if a new dominant follicle emerged at the time of luteolysis, the time from treatment to ovulation was 4.5 days. Several studies have reported that the stage of estrous cycle at the time of prostaglandin administration greatly influences the conception rate in dairy cows. Armstrong (1988) reported that the conception rate among the cows treated on Day 13 (71 %) was significantly higher when compared to the cows treated on Day 8 (46 %) Effect of Progesterone Level on Synchronized Estrus The level of progesterone levels prior to ovulation following the administration of prostaglandin affect the fertility of cows in synchronized estrus. Folman, et al., (1990) found that cows conceiving to AI at induced estrus had higher progesterone levels during the preceding luteal phase than those not conceiving. However, Gyawu, et al., (1991) showed that excessively long periods of high progesterone prior to insemination can suppress fertility. It has also been reported that estrus was manifested in more percentage of cows (84 %) that had high progesterone concentrations, > 3.1 ng/ml. the day of the last PGF2α injection than did cows with low progesterone levels (56 %). 17

32 Effect of Different Prostaglandin Analogues on Estrus Response and Fertility The fertility of estrus, induced with different analogues of prostaglandin was reported to be similar to that of estrus induced with PGF2α (Martinez and Thibier, 1984; Seguin, et al., 1985). However, El-Menoufy and Abdou (1989) reported that the estrus synchronization rate was higher in cows treated with cloprostenol (90 %) when compared to cows treated with prostaglandin (82%). Schams and Karg (1982) compared the luteolytic action of alfaprostol, cloprostenol, prosolvin and tiaprost in heifers and reported that there was difference among the various analogues concerning their luteolytic action on the CL. Wenzel, (1991) reported that a greater proportion of cows with unobserved estrus show luteolysis and behavioral estrus when treated with PGF2α and fenprostalene than cows treated with cloprostenol (Colazo, et al., 2002) Breed and Season Use of PGF2α for synchronization of estrus had less success in Bos indicus when compared to Bos taurus (Hardin and Randel, 1982). Hansen, et al., (1987) reported that Brahman heifers required higher dose of alfaprostol than Brahman cows for synchronization of estrus. Interval to estrus after PGF2α is affected by age and breed (Burfening, et al., 1978) and season (Britt, 1979). (Britt, 1979) recorded the influence of season in response to PGF2α affects the estrous behavior and conception rate. They recorded a high conception rate when synchronization program was conducted during July (50 %) than in December (20 %). The effect of genotype (breed) on mounting activity and duration of expression of estrus were reported in many previous experiments. Plasse, et al., (1970) reported that duration of sexual receptivity in B. taurus females varied from 4 to 48 hr with means reported between and hr, while in B. indicus cows the mean duration of estrus was short (6.70 h) which also vary from 2 to 22 hr. Furthermore, Rae, et al. (1999) found difference in duration of sexual receptivity between B. taurus and B. indicus breeds which is being short for B. indicus compared to B. taurus. These, variations and short duration of estrus observed in B. indicus breed, although affected by climatic factors (Lamothe, et al., 1995), a good part due to genetics. 18

33 2.9. Factors Limiting the Use of Prostaglandin in Dairy Cows The luteolytic action of PGF2α is used considerably as a drug for estrus synchronization and controlled breeding schemes with the objective to improve the reproductive performance of dairy cows. However, a proportion of failures occur, mainly cows not exhibiting estrus within the expected time period following the injection of PGF2α (Wenzel, 1991). The reasons for the failure of luteolytic action of PGF2α are reviewed below Effect of Accuracy in Rectal Palpation of CL Gynaecological examination by way of rectal palpation of ovary is often done to detect a mature CL before PGF2α administration (Wenzel, 1991). One major reason for decrease in the success of estrus synchronization following administration of prostaglandin is due to the unreliability of CL palpation by rectal examination (Ott, et al., 1986). The accuracy of rectal palpation in determining the presence or absence of mature CL has been reported by various authors (Watson and Munro, 1980; Mortimer, et al., 1983). Even though the handling serum (Vahdat, et al., 1979; Fahmi, et al., 1985) and plasma (Vahdat, et al., 1984) samples have been shown to affect progesterone assay results, the concentration of progesterone in plasma (Boyd and Munro, 1979), Serum (Mortimer, et al., 1983) or milk (Watson and Munro, 1980) was used as the standard against which palpation for the presence or absence of mature CL was judged. Ott, et al., (1986) showed that there was only 77 % agreement between diagnosis of CL by experienced palpator and the progesterone concentration. Further, they reported that identification of a CL by rectal palpation was 85 % accurate and no CL was false as many times as it was true. Whereas, Seguin, et al., (1978) and Dailey, et al., (1986) reported palpation error up to 6 % during identification of a CL by rectal palpation. Similarly, Kelton, et al., (1991) reported that the success of estrus synchronization depends on the accurate identification of a mature CL by rectal palpation Number of Cows in Synchronized Estrus Another reason that affects the potential use of PGF2α in improving the pregnancy rate in 19

34 the herd is due to the presence of often a very high number of cows in estrus at a given time after the administration of PGF2α for synchronized estrus, which reduces the estrus detection efficiency in the herd (Seguin, et al., 1985) Level of Progesterone during Prostaglandin Treatment It has been shown that there is a positive correlation between the level of progesterone in plasma (Lucy, et al., 1986; Folman, et al., 1990; Stevens, et al., 1993) or in milk (Dailey, et al., 1986) and the conception rate in PGF2α induced estrus indicating that the conception rate in cows following PGF2α injection has been positively correlated with the plasma concentration of progesterone that is reached during the days preceding the luteolysis (Folman, et al., 1990). Stevens, et al., 1993, observed that the efficacy of prostaglandin as luteolytic agent is reduced when it is administered along with GnRH Variation in Duration of Onset of Estrus Another limiting factor in the use of PGF2α, is the variation in the duration of onset of estrus after the injection of the drug and estrus is not being precisely synchronized. This duration of onset of estrus following the injection of PGF2α ranges from 2 to 5 days in cattle (Watts and Fuquay, 1985; Dailey, et al., 1986). When PGF2α is administered to the cows having functionally mature CL, 85 to 95 % of the cows would be in estrus within Day 7 of injection (Macmillan and Henderson, 1983; Armstrong, et al., 1989; Folman, et al., 1990; Rosenberg, et al., 1990) and 70 to 90 % of these cows will exhibit the estrus on Day 3 to 5 after the injection of PGF2α (Ferguson and Galligan, 1993). This variation in the time of ovulation is the major obstacle, which causes substantially lower pregnancy rate per AI in timed insemination when compared to AI after a detected estrus induced by PGF2α in lactating dairy cows (Lucy, et al., 1986; Stevenson, et al., 1987; Archbald, et al., 1992). The synchronization protocols are measured by synchronization rate (the percentage of females detected in estrus compared with the total number treated), conception rate (the percentage of females becoming pregnant compared with those exhibiting estrus and inseminated during the synchronized period) and pregnancy rate (the percentage of 20

35 females becoming pregnant compared with the total number treated) (Lucy, et al., 2004; Lamb, et al., 2006). 21

36 3. CHAPTER III: MATERIALS AND METHODS 3.1. Location and Description of Study Areas The study was conducted in eastern zone of Tigray region Northern Ethiopia in the Livestock and Irrigated Value Chain for Ethiopian Smallholders (LIVES) project three districts (Kilte-awlaelo, Atsbi Wemberta and Ganta-afeshum) from October 15, 2014 to March, The districts where the study conducted are described as follows. Ganta Afeshum district is found in Eastern zone of Tigray region, in North Ethiopia about 117 km far away to the north from Mekelle. It is located geographically at 14 o 24' and 14 o 21'N Latitude and 39 o 13' and 39 o 37'E Longitude. It shares borders with Gulo- Mekeda, Hawzien, Saesi-Tsaedaemba, and Ahferom weredas in the North, South, East, and West, respectively. The altitude of the district ranges from 1800 to 3200 m.a.s.l. (WOARD, 2014). The study site or tabia (Peasant association) was Baati May Mesanu. Farmers practice mixed farming system comprising crop, livestock, and agro-forestry sub-systems. Livestock husbandry is the main integral part of the farming system of the district (WOARD, 2014). According to the national agro-ecological zonation, the study area falls under the Central Cereal Production Zone, classified as wheat and barley production area with uni-modal rainfall pattern (USAID, 2000). The annual rainfall of the wereda varies from 350 to 650 mm while the rainfall pattern is erratic and unpredictable. Out of the total annual rainfall greater than half falls between July and August. The mean minimum and maximum temperature ranges from 8 to 25 0 C (WOARD, 2014). Atsbi Wemberta district is located in Eastern zone of Tigray National Regional State at ' and ' north latitude and ' and ' east longitude. The altitudinal range of the district is from 1000 to 3200 m.a.s.l. (WOARD, 2014). The study site or tabia (Peasant association) is Golgol-naele. Livestock are integral component of the farming system. Livestock feed is a major limiting factor in the area. In areas where the altitude is below 2600 m.a.s.l, apiculture is an important marketable commodity and thus important sources of household income in the area. The average annual temperature of the district is between C. Most of the rain is mono-modal and concentrated in 22

37 summer (June to September). Nearly all the cereals and legumes are planted during this period. The area sometimes receives short rains within November to March (known as belg) as bimodal rainfall. Generally, the district is characterized by low rainfall which ranges mm (WOARD, 2014). Kilte Awlaelo district is situated in Eastern administrative zone of Tigray and one of the seventh rural districts of the eastern zone found in the south of the eastern administrative zone. It is found at distance of 45 km to north of Mekelle, capital of the region. Geographically, it is located between ' ' N latitude and ' ' E longitude (WOFP, 2014). The altitude ranges from meters above sea level. The study site or tabia (Peasant association) is Genfel. The livelihood of all inhabitants of the district fully depends on subsistent agriculture. The dominant types of agriculture in the district is mixed crop livestock farming system, but small scale rain fed crop farming is the main pillar of livelihood in the district. The area exhibits uni-modal type of erratic and unreliable rainfall distribution which occurs between June and August ranging from m.m. Besides to the major rain fed farming, irrigation is also practiced in some areas especially since 2005 due to the high attention given by the government to water harvesting and utilization (WOARD, 2014). Annual temperature varies from c maximum monthly average temperature is May/June, whereas the minimum temperature is in October and December (WOFP, 2014). 23

38 Figure 1: Location map of the study area (Source: BoFED, 2015) Study Animals The experimental animals includes a total of 240 local and crossbred cows (parity ranging between 1 and 5 and postpartum period > 60 days) and heifers having body weight above 230 kg were selected and used. Two breeds local (n= 120) and crossbred (n=120) were used. Their body condition score at the beginning of the experiment was 4-6 on a scale of 1 to 9 which is 1=emaciated and 9=obese (Roche, et al. 2009). This body condition score was subjectively give to females to describe overall body condition, fat cover and flesh over the ribs, loin and tail head. From each three district one potential station (tabia) was selected purposively. 120 local and 120 crossbred cows and heifers were selected from the three districts. The animals were selected purposely based on availability of feed, body condition, age, health status 24

39 and absence of pregnancy during synchronization. Pregnancy diagnosis was conducted using rectal palpation before starting the experiment to avoid the risk of abortion by PGF2α. Animals were diagnosed for the presence of any reproductive disorder clinically. Finally, 120 animals were assigned for single dose synchronization protocol, 60 for double dose synchronization protocol and the remaining 60 Animals for fixed time AI regime after double injection of the PGF2α hormone. Table 2: Existing dairy cattle population in the three study weredas S.No Wereda Cross-bred Local breeds Total cows heifers Sub total cows Heifers Sub total 1 Ganta-afeshum Atsbiwemberta Kilte-awlaelo Total Source: WOARD (Ganta-afeshum, Atsbiwemberta, and Kilte-awlaelo weredas, 2014) Study Design and Sampling Strategies Sample Size Determination Multi-stage sampling techniques were applied in sample selection processes. In the first stage, the study areas comprising of three districts of the LIVES project were selected purposively on the basis of availability of local and crossbred of cows and heifers, long time experience in artificial insemination services, availability of feed, and accessibility of the weredas. Finally, sample size was determined by using the following formula. The sample size determination formula provided by Yemane (1967) to determine the required. 25

40 n = N 1+N (e) 2 Where n = the sample size N = the population size e = the level of precision Based on the above formula the minimum sample size required for the study was 123 local and crossbred cows and heifers. But, for convenience the current study was carried out on a total of 240 local and crossbred cows and heifers which is more than double than the minimum sample size required for the study. In the second stage, from three district one potential tabia was selected purposively. In the third stage, 120 local and 120 crossbreds of cows and heifers were selected purposively from each district. Study subjects were purposively selected based on estrus synchronization through hormonal treatment is naturally and technically practical if and only if some pre-conditions or pre-requisite characters that must be fulfilled by the selected cows and heifers for estrus synchronization. Table 3: Distribution of experimental Animals in the study areas Districts G/afeshum Stations (Tabias) Baati may mesanu Single Injection Local Crossbred Total Double Injectio n at ED Double Fixed Time AI Single Injection Double Injection at ED Double Fixed Time AI K/awlaelo Genfel A/wenberta Golgolnaele Total

41 Experimental Design The study design was experimental with two treatment groups running in three steps. In the first step, the two treatment groups that are local breed cows/ heifers and crossbred cows/heifers were selected on the basis of their suitability for estrus synchronization through hormonal treatments. In the second step, each treatment group was assigned in to single and double shot frequency of hormonal injections. In the third step, the single and double dose PGF2α protocols were sub grouped on the basis of breeding techniques as breeding at fixed time and breeding according to estrus detection. Experimental animals were randomly assigned to one of the two treatment protocols (1=single PGF2α injection; and 2=double PGF2α injection). Prior to the start of the experiment, reproductive organs of animals were palpated per rectum to confirm the reproductive stage, presence of mature corpus luteum and whether they were free from any obvious reproductive tract abnormalities. Animals allocated to protocol 1 (n=120) were injected (2ml) PGF2α (Synchromate, Bremer Pharma GMBH, Germany, 1 ml solution of Synchromate contains cloprostenol 0.263mg equal to cloprostenol 0.250mg,) intramuscular (IM) on Day 0. Animals were observed for any external symptoms of estrus (for example, clear vaginal discharge, mounting other cattle, allowing other cattle to mount them, and other related signs) after 24 hrs following the treatment. Animals allocated to protocol 2 (n=120) were injected (2ml) PGF2α (Synchromate) on Day 0, which was followed by administration of same dose of PGF2α (Synchromate) IM on day 14. Animals were observed for estrus expression after 24 hours of the second injection of PGF2α. Animals were recorded whenever observed in estrus with assigned value. The following figures illustrate the single and double dose injection and time schedule of synchronization protocols. The first group was given single administration of PGF2α IM and bred according to estrus. Whereas, the second group was injected with two doses of 2ml PGF2α IM each at 14 days interval, estrus was detected after second administration of PGF2α. In both treatment groups, animals were inseminated either following visual observation of heat signs and rectal palpation, or inseminated at fixed time (without heat 27

42 detection) after the second injection of PGF2α. Figure 2: Single dose PGF2α and breeding according to estrus. (Upper), double dose PGF2α and breeding according to estrus. (Middle), Double dose PGF2α and timed AI (bottom). (Adapted from Richard and Richard, 2010.) Accessories Hormone (PGF2α) (BREMER PHARMA GMBH, GERMANY), RPHDT dipstick (Ridgeway Science Ltd, UK), Icebox, syringes and needles, semen (National Artificial Insemination Center, ETHIOPIA), AI equipment s, savlon, test tubes, ear tag applicator, ear tag marker and ear tags were used. 28

43 Experimental Procedures Single and Double Prostaglandin Injection Single shot of PGF2α was given for 120 cows/ heifers (group 1) and double shot for 120 cows/heifers (group 2) based on body condition, parity level and pedigree information. Females from the experimental groups were synchronized with synthetic analogues of PGF2α (Synchromate) using the following experimental protocols: The first group was injected with intramuscular administration of a single dose of 500μg PGF2α, then detection of females in heat in the next 5 days and AI. The second group was injected with intramuscular administration of two doses of 500μg PGF2α each at 14 days interval, heat detection after the second dose of PGF2α and fixed time AI were used. Estrus synchronization in these cows and heifers was done by the exogenous administration of PGF2α. It is synthetic analogue of prostaglandin structurally related to PGF2α containing Cloprostenol sodium mg equivalent to cloprostenol mg/ml, manufactured by BREMER PHARMA GMBH GERMANY. The production and expiry dates were noted and has dose of 500 μg, i.e., 2 ml intramuscularly in cows. Rectal examination was done prior to administration of PGF2α to exclude the chances of pregnancy and to detect the presence of corpus luteum Time of Insemination To know more precisely the estrus onset, animals were monitored starting 24 hours after PGF2α administration 3 times a day (in the morning, at noon and late in the evening). Animals were artificially inseminated when they were detected in heat after single dose injection. In animals treated with two doses of PGF2α, heat detection were performed only after the second administration of PGF2α (Figure 2, middle). For double dose administration of PGF2α the animals were inseminated at fixed time (at 48 and 72 hours) and after estrus detection. 29

44 RPHDT and AIT Corpus Luteum Detection through Rectal Palpation The test was conducted using 10 to 20 ml of milk from 90 lactating dairy cows were collected to detect the level of progesterone (Zdunczyk et al., 2002) after AI technicians checks the presence of CL by rectal palpation to screen the lactating cow for synchronization from same cow. Milk samples were collected from clinically healthy udder and teats by hand milking of the four quarters after discarding the first milk drops (after fourth milk drop). For RPHDT no additives were added to the raw whole milk as the test was conducted at the farms immediately after collection. First, raw whole milk samples were shacked very well. Milk progesterone level was determined by shaking the entire milk sample and then sub-sampling 0.5 ml of whole raw milk. Sampling was done using separate pipettes for each cow. Then, Dipstick (P4 Rapid, Ridgeway Science Ltd, Gloucestershire, UK) was placed in to the test tubes containing sample and left for a maximum of 10 minutes, according to manufacturer s instruction. Findings were interpreted as: When the dipstick showed only one strong line indicates high progesterone level - the cow was not in heat, it may be pregnant or in its diestrous stage (luteal phase) and there was functional corpus luteum. Whereas, when the dipstick showed two strong lines indicates very low P4 level - the cow was definitely not pregnant and there was no functional corpus luteum. When the dipstick showed one strong top line and one faint line it means that low P4 level - the cow may be in its metestrous or proestrous stage (going out or approaching to estrus) Data Collection Estrus Response of Cows and Heifers Cows and heifers were observed for estrus signs at 24, 48, 72, 96, and > 96 hours and the number of cows and heifers with no response after hormonal treatment were considered as anestrus in each treatment group. The time interval from the PGF2α administration to the onset of estrus, for the females treated with a single dose of PGF2α and two doses of PGF2α at 14 days was recorded for cows and heifers that manifested heats at 24,48,72,96 30

45 and greater than 96 hours. Cows that exhibit estrus were artificially inseminated by experienced technician using frozen semen after PGF2α injection Conception Rate The number of pregnant cows and heifers after artificial insemination was computed as a percentage of cows and heifers exhibiting estrus during the synchronized period in each treatment group. Pregnancy diagnosis was conducted by rectal palpation 60 days after AI. The percentage of females that became pregnant of those exhibiting estrus and inseminated during the synchronized period was evaluated after insemination at fixed time and after estrus detection in all experimental animals Efficiency of Artificial Insemination Technicians PGF2α is only effective if administered between days 6 to 17 of the oestrous cycle when functional corpus luteum is available in one of the ovaries. By using RPHDT in milk of lactating dairy cows it is possible to evaluate the CL detection efficiency of artificial insemination technician (Dobson and Fitzpatrick, 1976; Friggens, et al., 2008). First AI technicians checked the presence of CL through rectal palpation to screen the lactating cows for synchronization. Then to perform comparative evaluation of the efficiency of AI technicians milk was collected from 90 lactating cows for 9 AI technicians and RPHDT dipstick was placed in to the test tubes containing sample and left for 10 minutes and the presence of CL was evaluated based on high or low P4 level (Colour of the dipstick) Independent Variables The independent variables of importance in this study were those variables which were thought to have influence on the dependent variables of the study. These include breed, body condition, parity, frequency of injection, time of heat expression and time of insemination of the sampled cows and heifers (Table 4). 31

46 Table 2: Summarized lists of independent variables with their description and expected effects on the dependent variables Variable Description Variable type Value Breed Breed of the dairy cows and heifers categorical 1= if local breed, 2= if cross-bred PGF2α injection Frequency of injection of the dairy cows and heifers categorical 1= if Single dose injection, 2= if double dose injection Time estrus response AI method Time of heat expression of the dairy cows and heifers Time of insemination of the dairy cows and heifers categorical 1=if After 24, 2= 48, 3= 72, 4= 96 and 5= > 96 hours categorical 2= At detected oestrus 1= at fixed time insemination (at 48 and 72 hours) 3.5. Statistical Analysis Both descriptive and inferential statistics were used to analyze the data. The descriptive analysis includes percentage and frequency distribution. Reproductive performance analysis was also be conducted by using Generalized Linear Models (GLM) of SPSS version 20 to test for the significance of the reproductive effect of the independent variables between local and crossbred cows and heifers. To analyze the effect of breed and frequency of prostaglandin injection on heat expression and conception rate of cows and heifers, GLM called Binary Logistic Model was used as the response (dependent) variables expressed on binary basis and expressed as odds ratio and 95% confidence interval. To analyze data on duration of estrus expression was analyzed using a GLM called ordinary Logistic Model because the response variables were ordered according to the time taken to express heat. 32

47 To analyze the effectiveness of fixed time AI and AI at detected estrus on the conception rate of cows and heifers, GLM called binary Logistic Model and expressed as odds ratio and 95% confidence interval. To analyze the ability of AI technicians to correctly classify the presence/absence of active corpus luteum as verified by RPHDT kit was analyzed using quadratic discriminant function, and there was an assumption of no equal covariance matrices among AI technicians and descriptive Statistics such as frequencies and percentages was applied. The statistical model used in this analysis was logistic regression model and expressed as: Log [πij / (1- πij)] = β0 + β1 X1ij + β2x2ij + + βnxnij + eij Where πij is the probability of the presence of the event of interest (estrus response, duration of estrus response and conception rate) (1-πij) is the probability of the event not happening β0 is the log odd of the intercept β1, β2 βn are the regression coefficients to be estimated from the data X1ij, X2ij... Xnij are the independent variables (Breed, frequency of PGF2α, AI methods, Parity) The quantities eij is random error In all the comparisons, the level of significance was set at α< Modelling was continued until all the main effects or interaction terms were significant according to the Wald statistic at P < Ethical Consideration The Tigray national regional state science and technology agency health ethical review committee had been critically reviewed the proposal in the context of research ethics and conclude that, there is no ethical problem on the objectives and methodology of the proposal and authorized to implement the research project in the field work. The ethical clearance paper is attached in the appendix V. 33

48 4. CHAPTER IV: RESULTS 4.1. Effect of breed and frequency of Prostaglandin injection on heat expression Estrus expression Rate The study showed that, among 180 cows (n=119) and heifers (n=61) synchronized using both protocols of PGF2α injection, 87.2% (n=157) of them were detected to be in estrus on visual observation and rectal palpation while, 12.8% (n=23) did not manifest heat in both single and double dose PGF2α injection protocols. 83.3% (n=75) and 91.1% (n=82) of local and crossbred cows manifested estrus, respectively. The estrus response was higher in crossbreds (91.1%) than local breeds (83.3%). The odds of estrus response in crossbreds was 2.1 times more likely than local breeds (OR=2.1; 95%CI: 0.822, 5.11). Higher proportion (93.3%) of estrus response was measured among cows and heifers that received double injection. The odds of estrus response in cows and heifers that received double injection was 2.6 times more likely compared with females that received single injection (OR=2.6; 95%CI: 0.853, 8.125). The result of the study also showed that, from 61 heifers and 119 cows synchronized using both protocols of PGF2α injection, 77% (n=47) and 92.4% (n=110), respectively, showed estrus. The odds of estrus response in cows was 3.6 times more than heifers, and this difference was statistically significant (OR=3.6; 95%CI 1.473, 8.993). 34

49 Table 3: Estrus response rate of local and crossbred cows and heifers after single and double dose injection of PGF2α Characteristics No N (%) Estrus Response Yes N (%) Odds ratio (95% CI) P-value PGF2α Injection Single 19(15.8) 101(84.2) Single (reference) - Double 4(6.7) 56(93.3) 2.6 (0.853, 8.125) Total 23 (12.8) 157 (87.2) Breed Local 15(16.7) 75(83.3) Local (reference) - Cross 8(8.9) 82(91.1) 2.1 (0.822, 5.11) Total 23 (12.8) 157 (87.2) parity Heifer 14(22.9) 47(77.1) Heifer (reference) Cows 9(7.6) 110(92.4) 3.6(1.473,8.993) Total 23(12.8) 157(87.2) Values in parentheses are percentage. Among 120 cows and heifers synchronized using single shot injection of PGF2α, 84.2% (n=101) of them came to heat as visually observed and 15.8% (n=19) did not manifest heat. The estrus manifestation rate of the local and crossbreds treated with single injection of PGF2α were 78.3% (n=47) and 90% (n=54), respectively. Crossbred cattle was found higher proportion (90%) of estrus response than local breeds after single dose of PGF2α injection. The odds of estrus response in crossbreds were found to be 2.5 times more likely than local breeds (OR=2.5; 95%CI: 0.876, 7.066). 35

50 Table 4: Estrus response rate of local and crossbred cattle after single dose injection of PGF2α Characteristics Estrus Response Breed No, N (%) Yes, N (%) Odds Ratio (95% CI) P-value Local 13(21.8) 47(78.3) Local(reference) - Cross 6(10) 54(90) 2.5 (0.876, 7.066) Total 19 (15.8) 101 (84.2) Values in parentheses are percentage. The estrus expression rate of local and crossbreds treated with double dose PGF2α injection were 93.3% (n=28) for both breeds and 6.8 %( n=4) did not manifest heat. There was no significant difference between the local breeds detected in estrus compared to the crossbreds. Estrus expression rate of local and crossbred cows and heifers synchronized with single and double shot hormonal treatment programs are summarized in Fig.3. 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% ESTRUS RESPONSE100% Figure 3: Estrus response rate of local and crossbred cattle after single and double dose injection of PGF2α 36

51 Duration of estrus response after PGF2α injection From 180 local and crossbreeds treated with single and double doses of PGF2α, 12.8% (n=23) animals did not manifest heat, while 8.9% (n=16), 18.3% (n=33), 31.1% (n= 56) 17.2% (n= 31) and 11.7% (n=21) manifested heat at 24, 48, 72, 96, and >96 hours respectively (Fig. 4). Figure 4: Time of Estrus Response after Single and double dose PGF2α Injection About 15.8% (n=19) of them did not manifest heat, 10% (n= 12) of the cows and heifers manifested heat at 24 hours, and 27.5% (n=33) of them showed heat at 72 hours after single shot PGF2α injection (Fig. 5). 37

52 Figure 5: Time of Estrus Response after Single PGF2α Injection Among cows and heifers placed under double dose PGF2α injection about 6.7% (n=4) of them did not manifest heat, where as 6.7% (n= 4) and 38.3% (n=23) of cows and heifers manifested heat at 24 and 72 hrs. respectively (Fig.6). Figure 6: Time of Estrus Response after double PGF2α Injection The analysis for single dose PGF2α injection and estrus manifestation showed that among 120 local and crossbred cattle, 24.2% (29), and 60% (72) of them manifested heat before 24hrs and after 72 hrs of PGF2α administration, respectively. However, the rest 15.8% 38

53 (19) animals did not manifested heat after administration single dose PGF2α injection (Fig.7). Figure 7: Estrus duration for single injection of PGF2α before 48 and after 72 hours From 60 local and crossbreds treated with double doses of PGF2α, 6.7% (n=4) animals did not manifest heat, while 35% (n= 21) and 58.3% (n= 35) of them manifested heat before 48 hrs. and after 72 hrs, respectively (Fig. 8). 39

54 Figure 8: Estrus duration for double injection of PGF2α before 48 and after 72 hours 4.2. Effect of breed and frequency of Prostaglandin injection on Conception Rate Among 153 animals inseminated after treatment with single and double dose injection of PGF2α 62.7% (n=96) of them conceived, where as 37.2% (n=57) animals did not conceived. A total of five animals were sold from all experimental animals. The conception rate of the local and crossbreds animals inseminated after single and double dose injection of PGF2α were 59.6% (n=59) and 68.5 %( n=37) respectively. Higher proportion (68.5%) of conception rate was recorded among cows and heifers that received double injection. The odds of conception rate in cows and heifers that received double injection was 47% more likely to conceive compared with females that received single injection (OR=1.47; 95% CI: 0.732, 2.973). The conception rate of 73 local and 80 crossbreds treated with single and double dose injection of PGF2α were 61.6% (n=45) and 63.7% (n=51) respectively.the conception rate was higher in crossbreds (63.7%) than local breeds (61.6%). The odds of conception rate in crossbreds was 9% more likely to conceive than local breeds (OR=1.09; 95%CI: 0.567, 2.108). The result of the study also showed that, from 47 heifers and 106 cows synchronized using both protocols of PGF2α injection, 67% (n=31) and 61.3% (n=65), respectively, 40

55 conceived. The odds of conception rate in cows was 19% less likely to conceive than heifers, (OR=0.81; 95%CI: 0.398, 1.679). Table 5: Conception rate result of Local and Crossbred, Cows and Heifers after single and double dose PGF2α Injection Conception rate Characteristics Negative N (%) Positive N (%) Odds ratio (95% CI) P-value PGF 2α Injection Single 40(40.4) 59(59.6) Single(reference) - Double 17 (31.5) 37(68.5) 1.47 (0.732, 2.973) Total 57 (37.2) 96 (62.7) Breed Local 28(38.4) 45(61.6) Local(reference) - Cross 29(36.2) 51(63.7) 1.09 (0.567, 2.108) Total 57 (37.2) 96 (62.7) Parity Heifer 16(34.1) 31 (67) Heifer(reference) Cow 41(38.9) 65(61.3) 0.81 (0.398,1.679) Total 57(37.2) 96(62.7). Values in parentheses are percentage. The conception rate of 46 local breed and 53 crossbreds inseminated after single dose PGF2α injection, were 60.9% (n=28) and % (n=31) respectively. The conception rate was higher in local breeds (60.9%) than crossbreds (58.5%). The odds of conception rate in crossbreds was 10% less likely to conceive than local breeds. (OR=0.90; 95%CI 0.404, 2.027). Table 6: Conception rate result after single dose PGF2α injection Conception rate Characteristics Negative N (%) Positive N (%) Odds Ratio (95% CI) P-value Breed Local 18(39.1) 28(60.9) Local(reference) - Cross 22(41.5) 31(58.5) 0.90 (0.404, 2.027) Total 40(40.4) 59 (59.6) Values in parentheses are percentage. 41

56 The conception rate of 27 local and 27 crossbreds treated with double dose injection of PGF2α were 63% (n=17) and 74.1% (n=20) respectively. The conception rate was higher in crossbreds (74.1%) than local breeds (63%). The odds of conception rate in crossbreds was 68% more likely to conceive than local breeds. (OR=1.68; 95%CI 0.525, 5.373). Table 7: Conception rate result after double dose PGF2α injection Conception rate Characteristics Negative N (%) Positive N (%) Odds Ratio (95% CI) P-value Breed Local 10(37.1) 17 (63) Local(reference) - Cross 7(25.9) 20 (74.1) 1.68 (0.525, 5.373) Total 17(31.5) 37 (68.5) Values in parentheses are percentage Comparison of Fixed time AI with AI at Detected heat on Conception Rate Comparing the conception rate of 54 cows and heifers treated with double dose injection and inseminated after heat detection and 47 cows and heifer fixed time inseminated after double dose injection. 68.5% (n=37) and 48.9% (n=23) were conceived respectively. AI at detected estrus was found higher proportion (68.5 %) of conception rate than fixed time AI after double dose of PGF2α injection. The odds of conception rate AI at detected estrus was found to be 2.3 times more likely to conceive than Fixed time AI. Beside, this difference was statistically significant (OR=2.3; 95% CI 1.009, 5.107). The conception rate was higher in crossbreds (64.3%) than local breeds (53.3%) after double dose of PGF2α injection at FTAI and AIED. The odds of conception rate in crossbreds was 57% more likely to conceive than local breeds (OR=1.57; 95% CI 0.706, 3.509). 42

57 Table 8: Fixed AI Versus AI at Detected estrus Conception rate Characteristics Negative N (%) Positive N (%) Odds ratio (95% CI) P-value AI methods Fixed 24(51.1) 23(48.9) Fixed (reference) - At ED 17 (31.5) 37(68.5) 2.3 (1.009,5.107) Total 41 (40.6) 60 (59.4) Breed Local 21(46.7) 24(53.3) Local(reference) - Cross 20(35.7) 36(64.3) 1.57 (0.706, 3.509) Total 41 (40.6) 60 (59.4) Values in parentheses are percentages. Conception rate of local and crossbred cows and heifers synchronized with single and double dose hormonal treatment programs are summarized in Fig. 9. Figure 9: Conception rate of local and crossbred cattle after single and double dose injection of PGF2α 43

58 4.4. Rapid Progesterone Heat Detection Test Among animals tested for progesterone level prior to synchronization using RPHDT 57% (51/90) had high progesterone level, while 43% (39/90) showed low progesterone level. Table 9: Rapid Progesterone Heat Detection Test (RPHDT) result Progesterone Level Frequency Percent weak strong Total Values in parentheses are percentage. The efficiency of nine (9) AI technicians participated on detection of active corpus luteum through rectal palpation with the RPHDT was evaluated, and among 9 AI technicians, one technician scored 90% similarity with the RPHDT, while two technicians had the lowest score (40%). Out of ten cows, the minimum and maximum numbers of cows misclassified were 1 and 6, respectively, with 36.6% coefficient of variation. On average one technician misclassified 4.6 cows out of 10 cows presented for corpus luteum detection. 44

59 Figure 10: Comparison of AI Technician efficiency with RPHDT Results of a quadratic discriminant analysis in Table 12 shows, the proportion of correctly identified active corpus luteum by AI technicians was (57%) similarity with the RPHDT. Table 10: Results of a quadratic discriminant analysis Valid N (list wise) Corpus luteum detection by Artificial Inseminator Mean Std. Deviation Un weighted Weighted Yes RPHDT Result Total RPHDT Result

60 5. CHAPTER 5: DISCUSSION 5.1. Estrus Response Rate Largely abundant, clear and watery mucus discharge was observed following treatment with PGF2α. Higher proportion (93.3%) of estrus response was measured among cows and heifers that received double injection. Even though, there was no statically significant, cows and heifers that received double injection was 2.6 times more likely to give estrus response compared with females that received single injection. Regarding breed there was no statistical significance recorded, the likelihood of crossbred cows and heifers to manifest estrus was higher. Remember the non-cycling and unhealthy cows will not generally respond to prostaglandin products. The result with single dose PGF2α obtained from this experiment is higher than other previous works reported by (Păcală, et al., 2009) from the 70 females with known estrous cycle hormonal stimulated with a single dose of PGF2α, 67.1% (n= 47) manifested heats. It is also higher than what was reported in Hawassa-Dilla Milk shed, SNNPR (76.1%) conducted in mass synchronization campaign (IPMS, 2011). Though this result seems a little bit lower comparing it with previous work in the region, that reported 100% for Adigrat-Mekelle Milkshed in Tigray (Tegegne, et al., 2012), This result obtained from single dose PGF2α injection is agrees with other previous works reported by (Macmillan and Henderson, 1983; Armstrong, et al., 1989; Folman, et al., 1990; Rosenberg, et al., 1990); who indicated that, PGF2α administration to cows with a functionally mature CL, 85 to 95% reached estrus within 7 days of treatment and (Ferguson and Galligan, 1993) Also reported cyclic animals treated with single PGF2α dose showed 70 to 90% signs of estrus 3 to 5 days after treatment. The single dose of PGF2α injection also agrees with the results of Murugavel and his colleagues (2010) who confirmed 70 to 90% estrus rate within 2 to 5 days when PGF2α was administered to cows with a functional corpus luteum. In this study, higher number of crossbred manifested estrus than local breed this is in agreement with Bo, et al., (2003) who reported poor estrus expression of Bos indicus breed in tropical environment. Further, higher estrus response rate of crossbred may be 46

61 due to the higher care given for this group of animals such as giving good quality feed and close supervision by family members. Among animals treated with double dose PGF2α at 14 days interval 93% of them exhibited estrus, which is higher than the report of Păcală, et al., (2009), who reported 88.2% estrus expression rate. Enhanced estrus response was reported when PGF2α was given at the late, rather than early to middle stage of the luteal phase (Tanabe and Hann, 1984; Watts and Fuquay, 1985; Xu, et al., 1997). Since two treatments given 14 days apart ensures that most animals are in the late luteal stage (cycle Day 11 to 14) when they receive the second PGF2α dose (Folman, et al., 1990; Rosenberg, et al., 1990; Young, 1989). This practice resulted for a greater number of cows that have a maturing second wave dominant follicle capable of ovulating in response to a gonadotropin-releasing hormone (GnRH) induced lutenizing hormone (LH) surge than would a GnRH administration given at a random stage of the estrus cycle. It is noted that from total 120 cows received single dose of PGF2α majority (71.3%) showed estrus after 72 hrs. and 28.7% showed after 48 hrs. the present study was higher than Păcală, et al., (2009), 68% who reported cows exhibit heat after 72 hours with single dose PGF2α administration. Further, among 60 animals received two doses of PGF2α more than half (62.5%) animal s exhibit estrus after 72 hrs, where as 37.5% of them showed before 48 hrs. This is higher than the report of Păcală, et al., (2009), who record of 28.9% cows showed estrus before 48 hrs with double dose administration of PGF2α. In the present experiment, large number of animals from both experimental groups manifested heat after 72 hours of hormonal treatment. Estrus response to treatment was impressive, it might be due to strict follow up of the animals and technicians also participated in detecting estrus through palpation per rectum after day two of treatment, Selection of cows in good body condition and with functional corpus luteum was also crucial factor for such a good estrus response. 47

62 5.2. Conception Rate From a total of (153) cows and heifers received PGF2α from both protocols a considerable portion (62.7%) conceived. Higher proportion (68.52%) of conception rate was found among cows that received double injection. Cows subjected to double injection were more likely to conceive compared to cows with single injection. The conception rate of the total animal treated with single shot injection was 59.6% (59/99). Findings were similar with some previous work done in Awassa milkshed 57.7 % (n=94) and 61.7 % (n=119) Adigrat milk shed (Tegegne et al., 2012).The conception rate result obtained from this experiment is extremely higher than the three years work ( ) reported by Tigray Bureau of Agriculture and Rural Development (TBoARD, 2014) in collaboration with other stakeholders conducted a mass synchronization scheme addressing 58,676 cows and heifers, using single PGF2α injection in 33 weredas of the region. The mass synchronization results showed low performance with conception rates of 31.5% (TBoARD, 2014). Selection of cows with good body condition score, free from diseases and with functional ovaries, and close follow-up and proper heat detection might have contributed to the observed higher conception rates. The conception rate was higher in crossbreds (74.1%) than local breeds (63%) treated with double dose injection of PGF2α. The conception rate result obtained from this experiment were 68.5% (37/54) in agreement with 70.5% conception rate following second PGF2α administration reported by Xu, et al., (1997) in dairy cows Evaluation of the Effectiveness of Fixed Time AI and AI at Detected Estrus on Conception Rate AI at detected estrus resulted in higher conception rate (68.52 %) than fixed time AI (48.9%) after double dose of PGF2α injection. where findings were statistically significant (OR=2.3; 95%CI 1.009, 5.107). This result in agreement with Lucy et al., (1986) has shown that cows receiving fixed time AI at 72 to 80 hours after a second 48

63 injection of PGF2α resulted in pregnancy rates considerably lower (P < 0.05) compared to cows receiving AI at a detected estrus alone. From the findings of this study it can be inferred that fixed time AI has advantage of time saving for AI technician and the farmers, but the conception rate of fixed time AI is lower than insemination after detected estrus. This may be due to synchronization with PGF2α does not control the time of AI because estrus detection was still required. Low pregnancy rates related to timed AI following treatment with PGF2α may be explained by the variation in time of ovulation with respect to time of AI. This variation in time of ovulation may be due to the deviation in stage of the pre-ovulatory follicle at the time of PGF2α injection (Pursley, et al., 1997) Comparison of the efficiency of AI technicians on CL detection through rectal palpation with that of Rapid Progesterone Heat Detection Test (RPHDT) Comparing the efficiency of Nine (9) AI technicians who participated on detection of active corpus luteum through rectal palpation with the Rapid Progesterone Heat Detection Test, the minimum and maximum numbers of cows misclassified were 1 and 6, respectively, with 36.6% coefficient of variation. On average one technician misclassified 4.6 cows out of 10 cows presented for corpus luteum detection. Seguin, et al., (1978) and Dailey et al. (1986) reported palpation error up to 6% during identification of a CL by rectal palpation. The result obtained from this experiment is extremely lower than the 77% agreement between diagnosis of CL by experienced palpator and progesterone concentration reported by Ott, et al., (1986). Similarly, they reported that identification of a CL by rectal palpation was 85 % accurate. Kelton, et al., (1991) also reported that the success of estrus synchronization depends on the accurate identification of a mature CL by rectal palpation. Prostaglandin based synchronization requires the presence of active CL on the ovary. The simplest method to detect CL on the ovary is rectal palpation during synchronization. This result indicated that rectal palpation to detect ovarian status needs experienced 49

64 technician and vigorous training assisted with hormonal and visual aids (ultrasound imaging) before allowing less experienced AI technicians in ovary palpation. One major reason for the decrease in the success of estrus synchronization following administration of PGF2α could be due to the unreliability of CL palpation by rectal examination (Ott, et al., 1986). The accuracy of rectal palpation in determining the presence or absence of mature CL has been reported by various authors (Watson and Munro, 1980; Mortimer, et al., 1983). The concentration of progesterone in plasma (Boyd and Munro, 1979), Serum (Mortimer et al., 1983) or milk (Watson and Munro, 1980) was used as the standard against palpation to detect the presence or absence of mature CL was judged. 50

65 6. CONCLUSION AND RECOMMENDATIONS 6.1. Conclusion This study has shown that PGF2α based estrous synchronization could be implemented under smallholder farmer s condition. The overall estrus response and conception rate in the study area was high. Crossbred cattle showed higher estrus response than local breed cattle, though findings did not vary significantly. Crossbreed cattle have been given relative priority in feeding, housing, provision of clean water, medical care and other management practices. Farmers and technicians can use either of the two synchronization protocols, however double dose PGF2α injection requires comparatively longer time interval and costly as PGF2α is injected twice. The conception rate of AI applied following estrus detection is higher than timed AI. Fixed time AI, on the other hand, has an advantage of time saving as farmers do not require to observe their animals to show signs of estrus. Stage of estrous cycle at the time of PGF2α injection greatly influences the duration of estrus expression. Moreover, the efficiency of the AI technicians on detection of active corpus luteum through rectal palpation as compared with the RPHDT was low Recommendations Based on this study the recommendations were set as fellows, The single dose PGF2α synchronization protocol should be implemented with great care on appropriate animal selection and management to get high estrus response rate and highly requires setting proper heat detection mechanisms to maximize the conception rate of the animals. Fixed time insemination after synchronization with double dose injection protocol of PGF2α was showed low conception rate. So, to go for higher conception rate, it is better to implement heat detection after double dose synchronization protocol. 51

66 Strict and continuous supervision of heat detection should be done after 48 hours of hormonal injection, for animals synchronized using both single and double dose injection protocols. Hands on practice are needed using visual and hormonal aids for artificial insemination technicians to improve their expertise in corpus luteum detection. The use of RPHDT at farm level and at veterinary clinics should be implemented and encouraged to assist rectal palpation and avoid false diagnosis when rectal palpation alone is used. MY FUTURE PLANS I have evaluated estrus response and conception rate of the synchronized animals but I will intend to evaluate the calving rate of the local and crossbreds cows. Model farmers and AI technicians will be trained on the application of RPHDT to diagnose their animals and animals in the farm or veterinary clinics to diagnose their reproductive stage. Pushing the government to introduce RPHDT and ultra sound particularly to the region as well as to the country. Providing refreshment training on detection of CL through rectal palpation by using RPHDT to AI technician based on my findings. 52

67 REFERENCES Adams, G.P. (1994): Control of ovarian follicular wave dynamics in cattle: implications for synchronization and super stimulation. Theriogenology 41: Allrich, D. R. (1993): Estrous behavior and detection in cattle. Veterinary Clinics of North America 9: Archbald, L.T., Tran, T., Massey, R. and Klapstein, E. (1992): Conception rates in dairy cows after timed-insemination and simultaneous treatment with gonadotropinreleasing hormone and/or prostaglandin F2 α. Theriogenology 37: Armstrong, J. D. (1988): The effects of prostaglandin administration to dairy cows on day 8 and day 13 of the oestrous cycle. Proceedings of the 11 th International Congress on Animal Reproduction and Artificial Insemination (Dublin). pp Armstrong, J. D., O Gorman, J. and Roche, J. F. (1989): Effects of prostaglandin on the reproductive performance of dairy cows. Veterinary Records 125: Aulakh, B.S. (2008): In vivo Sex fixing in Dairy Animals to produce female progenies, Proceedings of the 15 th congress of FAVA, Bangkok, Thailand. Pp.243. Bambal, A. M. and Jais, P. (2011): To study oestrus synchronization in crossbred animals and buffaloes in Navsari district, MDTTC Vasudhara Dairy. Beal, W.E. (2002): Estrous synchronization of cyclic and anestrous cows with Synchro- Mate-B. In: Fields MJ, Sand RS, Yelich JV. (eds.), Factors Affecting Calf Crop: Biotechnology of Reproduction, CRC Press, and London. pp Bekele, T., Kasali, O.B. and Alemu, T. (1991): Reproductive problems in crossbred cattle in Central Ethiopia. Animal Production Science 26: Bitew, M. and Prasad, S. (2011): Study on major reproductive health problems in indigenous and crossbred cows in and around bedelle, south west Ethiopia. Animal 53

68 and veterinary Advances 10 (6): Bo, G.A., Baruselli, P.S. and Martinez, M. F. (2003): Pattern and manipulation of follicular development in B. indicus cattle. Animal Reproduction Science 78: BoFED (Bureau of Finance and Economic Development) (2015): Map of Tigray region, department of planning, Tigray, Ethiopia Boyd, H. and Munro, C. D. (1979): Progesterone assays and rectal palpation in pre-service management of a dairy herd. Veterinary Records 104: Britt, J. H. (1987): Induction and synchronization of ovulation. In: ESE Hafez (eds.), Reproduction in Farm Animals. 5 th Edition, Lea and Febiger, Philadelphia, PA. pp Britt, J. H. (1979): Prospects for controlling reproductive processes in cattle, sheep, and swine from recent findings in reproduction. Journal of Dairy Science 62: Burfening, P., Anderson, D. C., Kinkie, R. A., Williams, J. and Friedrich, R. L. (1978): Synchronization of estrus with PGF2α in beef cattle. Journal of Animal Science 47: Cavalieri, J., Smart, G., Hepworth, M., Ryan, K. and Macmillan, L. (2008): Ovarian follicular development and hormone concentrations in inseminated dairy cows with resynchronized estrus cycles. Theriogenology 70: Christian, R. E. and Casida, L. E. (1948): The effects of progesterone in altering the estrus cycle of the cow. Journal of Animal science 7: 540. Colazo, M. G., Martínez, M. F., Kastelic, J. P. and Mapletoft, R. J. (2002): Effects of dose and route of administration of cloprostenol on luteolysis, estrus, and ovulation in beef heifers. Animal Reproduction Science 72:

69 Colman, D. A. (1993): Detecting estrus in dairy cattle. USA National Dairy Database, Reproduction Collection. Available source: edu/edres/topiclagrenv/ndd l- reproduces. Dailey, R. A., Price, J. C., Simmons, K. R., Meisterling, E. M., Quinn, P. A. and Washburn, S. P. (1986): Synchronization of estrus in dairy cows with prostaglandin F2αand estradiol benzoate. Journal of Dairy Science 69: Diskin, M.G. and Sreenan, J. M. (2000): Expression and detection of estrus in cattle. Reproduction Nutrition Development 40: Diskin, M.G., Austin, E. J. and Roche, J. F. (2002): Exogenous hormonal manipulation of ovarian activity in cattle Domestic Animal Endocrinology Ireland; 23: Dobson, H. and Fitzpatrick, R. (1976): Clinical application of the progesterone in-milk test. British Veterinary Journal 132: Dransfield, M. B. G., Nebel, R. L., Pearson, R. E. and Warnick, L. D. (1998): Timing of insemination for dairy cows identified in estrus by a radio telemetric estrus detection system. Journal of Dairy Science 81: DuPonte, M. W. (2007): The Basics of Heat (Estrus) detection in cattle, cooperative extension service, Collage of Tropical Agriculture and Human resources, University of Hawai at manoa, livestock management LM-15. < hawaii.edu/freepubs>. September 4, El'Menoufy, A. A. and Abdou, M. S. S. (1989): Heat and conception rate in dairy cows after synchronization of oestrus with PGF2α or its synthetic analogue. Indian Journal of Animal Science 59 (5): Esslemont, R. J., Glencross, R. G., Bryant, M. J. and Pope, G. S. (1980): A quantitative study of pre-ovulatory behavior in cattle (British Friesian heifers). Applied Animal Ethology 6:

70 Fahmi, H. A., Williamson, N. B., Tibary, A. and Hegstad, R. L. (1985): The influence of some sample handling factors on progesterone and testosterone analysis in goats. Theriogenology24: Ferguson, J.D. and Galligan, D.T. (1993): Prostaglandin synchronization programs in dairy herds (part I). Compendium Continuing Education for the Practicing Veterinarian 15: Fetrow, J. and Blanchard, T. (1987): Economic impact of the use prostaglandin to induce estrus in dairy cows. Journal of the American Veterinary Medicine Association 190: Fike, K. E., Wehrman, M. E., Lindsey, B. R., Bergfeld, E. G., Melvin, E. J., Quintal, J. A., Zanella, E. L., Kojima, F. N. and Kinder, J. E. (1999): Estrus synchronization of beef cattle with a combination of melengestrol acetate and an injection of progesterone and 17β-estradiol. Journal of Animal Science, 77: Folman, Y., Kaim, M., Herz, Z. and Rosenberg, M. (1990): Comparison of methods for the synchronization of estrous cycles indairy cows. Journal of dairyscience73: Franco, J. O., Drost, M., Thatcher, M. J., Shile, V.M. and Thatcher, W.W. (1987): Fetal survival in the cow after pregnancy diagnosis by palpation per rectum. Theriogenology 27: Friggens, N., Bjerring, M., Ridder, C., Højsgaard, S. and Larsen, T. (2008). Improved detection of reproduction status in dairy cows using milk progesterone. Reproduction of Domestic Animals 43: Galina, C.S. and Arthur, G.H. (1990): Review on cattle reproduction inthe tropics. Part 4 Oestrous cycles. Animal Breeding 58: Gokhan, D., Sariban, M. K., Fikret, K. and Ergun, Y. (2010): The comparison of the pregnancy rates obtained after the ovysnch and double dose PGF2α +GnRH 56

71 applications in Lactating Dairy cows, Journal of Animal and Veterinary Advances 9 (40): Gwazdauskas, F. C., Thatcher, W. W., Kiddy, C. A., Paape, M. J. and Wilcox, C. J. (1981): Hormonal pattern during heat stress following tham salt induced luteal regression in heifers. Theriogenology 16: Gyawu, P., Ducker, M. J., Pope, G. S., Saunders, R.W., Wilson, G. D. A. (1991): The value of progesterone, oestradiol benzoate and cloprostenol in controlling the timing of oestrus and ovulation in dairy cows and allowing successful fixed time insemination. British Veterinary Journal 147: Haileselassie, M., Kalayou, S., Kyule, M., Asfaha, M. and Belihu, K. (2011): Effect of brucella infection on reproduction conditions of female breeding cattle and its public health significant in western Tigrai, Northern Ethiopia. Veterinary medicine.international 2: 1-7. Hansel, W. and Convey, E. M. (1983): Physiology of the estrous cycle. Journal of Animal science 57: 404. Hansen, T.R., Randel, R. D. and Peterson, L. A. (1987): Bovine corpus luteum regression and estrous response following treatment with alfaprostol. Journal of animal Science 64: Hardin, D. R. and Randel, R. D. (1982): The effect of cloprostenol and cloprostenol + hcg on corpus lutea and serum progesterone in Brahman cows. Theriogenology 17: Heuwieser, W., Oltenacu, P. A., Lednor, A. J. and Foote, R. H. (1997): Evaluation of different protocols for prostaglandin synchronization to improve reproductive performance in dairy herds with low estrus detection efficiency. Journal of Dairy Science 80: IPMS (Improving Productivity and Market success of Ethiopian Farmers) (2011): 14 th progress report. Addis Abeba, Ethiopia. Pp

72 Islam, R. (2011): Synchronization of estrus in cattle: A review. Veterinary World 4: Available at: (Accessed on Oct 28, 2014). Jackson, P. S., Johnson, C.T., Furr, B. J. and Beattie, J. F. (1979): Influence of stage of oestrous cycle on time of oestrus following cloprostenol treatment in the bovine. Theriogenology 12: Johnson, C.T. (1978): Time of onset of oestrus after the injection of heifers with cloprostenol. Veterinary Records 103: Kastelic, J. P. and Ginther, O.J. (1991): Factors affecting the origin of theovulatory follicle in heifers with induced luteolysis. Animal Reproduction Science 26: Kelton, D. F., Leslie, K. E., Etherington, W. G., Bonnett, B. N. and Walton, J. S. (1991): Accuracy of rectal palpation and of a rapid milk progesterone enzyme immunoassay for determining the presence of a functional corpus luteum in subestrous dairy cows. Canadian Veterinary Journal 32: Kristula, M. R., Bartholomew, R., Galligan, D. and Uhlinger, C. (1992): Effects of a prostaglandin F2 α synchronization program in lactating dairy cattle. Journal of dairy science 75: Labago, F. (2007): Reproductive and lactation performance of dairy cattle in the Oromia central highlands of Ethiopia with special emphasis on pregnancy period. Doctoral thesis Swedish university of agricultural sciences. Lamb, G. C., Larson, J. E., Geary, T. W., Stevenson, J. S., Johnson, S. K., Day, M. L., Ansotegui, R. P., Kesler, D. J., DeJarnette, J. M. and Landblom, D. (2006): Synchronization of estrus and artificial insemination in replacement beef heifers using GnRH, PGF2α and progesterone. Journal of animal science 84: Lamming, G. E. and Bulman, D. C. (1976): The use of milk progesterone radioimmunoassay in the diagnosis and treatment of subfertility in dairy cows. British Veterinary Journal 132:

73 Lamothe, C., Montiel, F., Fredriksson, G. and Galina, C. S. (1995): Reproductive performance of Zebu cattle in Mexico. Influence of season and social interaction on the timing of expressed estrus. Tropical Agriculture Trinidad. 72: Land O Lakes (2010): The Next Stage in Dairy Development for Ethiopia. Dairy Value Chains, End Markets and Food Security, Addis Ababa, Ethiopia. pp Lauderdale, J. W., Seguin, B. E., Stellflug, J. N., Chenault, J. R., Thatcher, W. W., Vincent, C. K. andloyancano, A. F. (1974): Fertility of cattle following PGF2α injection.journal of animal science38: Lauderdale, J. W. (2002): Use of prostaglandin F2α in cattle breeding. Factors Affecting Calf Crop: Biotechnology of Reproduction, CRC Press, and London. Pp Letícia, Z. O., Vera, F.M., Hossepian, d. L., Clara, S. O., Benner, G. A., Hugo, B.G. and Ricarda, M. d. S. (2011): Fertility Rates Following Fixed-Time Artificial Insemination in Dairy Heifers in a Practical Progesterone-Based Protocol. Acta Veterinary Science 39 (2): 964. Lucy, M. C., McDougall, S. and Nation, D. P. (2004): The use of hormonal treatments to improve the reproductive performance of lactating dairy cows in feedlot or pasture-based management systems. Animal Reproduction Science 82: Lucy, M. C., Stevenson, J. S. and Call, E. P. (1986): Controlling first service and calving interval by prostaglandin F2 alpha, gonadotropin-releasing hormone, and timed insemination. Journal of Dairy Science 69: Macmillan, K. L. and Day, A. M. (1982): Prostaglandin F2α. A fertility drug in dairy cattle? Theriogenology 18: Macmillan, K. L. and Henderson, N. V. (1983): Analysis of the variation in the interval from an injection of prostaglandin F2α to oestrus as a method of studying patterns of follicle development during diestrus in dairy cows. Animal Reproduction Science 6:

74 Macmillan, K. L., Curnow, R. J. and Morris, G. R. (1977): Oestrus synchronization with a prostaglandin analogue: I. Systems in lactating dairy cattle. New Zealand Veterinary Journal 25: Martinez, J. and Thibier, M. (1984): Fertility in anoestrous dairycows followingtreatment with prostaglandin F2α or the synthetic analogue fenprostalene. Veterinary Records 115: Mekonnen, T., Bekana, M. and Abayneh, T. (2010): Reproductive performance and efficiency of artificial insemination smallholder dairy cows/heifers in and around Arsi-Negelle, Ethiopia. Livestock research for rural development 22 (3):1-9 Michael, L. D. and Thomas, W. G. (2005): Hand book of estrous synchronization. The Ohio State University, Ohio Agricultural Research and Development Center, Ohio. Western Region Publication 14:1-41. Million, T., Theingthan, J., Pinyopummin, A., Prasanpanich, S. and Azage, T. (2011): Oestrus Performance of Boran and Boran X Holstein Fresian Crossbred Cattle Synchronized with a protocol based on Estradiol benzoate or Gonadotrophin- Releasing Hormone. Kasetsart Journal (Natural Science) 45: MoA (Ministry of Agriculture) (1996): Livestock breed development subprogram, First Draft. Addis Ababa, Ethiopia. Moody, E. L. and Lauderdale, J. W. (1977): Fertility of cattle following PGFα on controlled ovulation. Journal of animal science 45:189. Mortimer, R. G., Olson, J. D., Huffman, E. M., Farin, P. W., Ball, L. and Abbitt, B. (1983): Serum progesterone concentration in pyometritic and normal postpartum dairy cows. Theriogenology 19: Mukasa-Mugerwa, E., Tegegne, A., Mesfin, T. and Teklu, Y. (1991): Reproductive efficiency of Bosindicus (Zebu) cows under artificial insemination management in Ethiopia. Animal Reproduction Science 24: Mukassa-Mugerwa E., Tegegn A. Mattoin M., and Cecchini G. (1989): Effect of Estrus 60

75 Synchronization with Prostaglandin F2α in Ethiopian highland Zebu (Bos indicus) cows. Animal Production 48: Mureda, E. and Mekuriaw, Z. (2007): Reproductive performance of crossbred dairy cows in Eastern lowlands of Ethiopia. Livestock research for rural development 19 (161):3 Murugavel, K., Yániz, J. L., Santolaria, P., López-Béjar, M. and López-Gatius, F. (2010): Prostaglandin Based Estrus Synchronization in Postpartum Dairy Cows: An Update. Schering Plough Animal Health, Spain. Pp Ott, R.S., Bretzlaff, K. N. and Hixon, J. E. (1986): Comparison of palpable corporalutea with serum progesterone concentrations in cows. Journal of the American Veterinary Medicine Association 188: Păcală, N., Bencsik, I., Dronca, D., Acatincăi, S., Petroman, I., Cornelia, P., Ada, C., Carabă, I., Alexandra, B. and Papp, S. (2009): Researches regarding the effect of PGF2α administration interval on cow s estrus synchronization. Banat s University of Agricultural Science and Veterinary Medicine, Timisoara. 54: Perry, G. (2004): Detection of standing estrus in cattle. Available Source: agbiopubs.sdstate.edu/articles FS921B.pdf Peters, A.R. and Ball, P.J.H. (1995): Reproduction in cattle (2 nd edition). Blackwell Science Ltd. Oxford, United Kingdom. Pp.234. Plasse, P., Warnick, A. C. and Koger, M. (1970): Reproductive behavior of B. indicus females in a subtropical environment. Length of estrous cycle, duration of estrus, time of ovulation, fertilization and embryo survival in grade Brahman heifers. Journal of Animal Science 30: Plunkett, S. S., Stevenson, J. S. and Call, E. P. (1984): Prostaglandin F2αfor lactating dairy cows with a palpable corpus luteum but unobserved estrus. Journal of dairy Science 67:

76 Pursley, J. R., Wiltbank, M. C., Stevenson, J. S., Ottobre, J. S., Garverick, H. A. and Anderson, L.L. (1997): Pregnancy rates per artificial insemination for cows and heifers inseminated at a synchronized ovulation or synchronized estrus. Journal of dairy Science 80: Rae, D.O., Chenoweth, P. J., Giangreco, M. A., Dixon, P. W. and Bennett, F. L. (1999): Assessment of estrus detection by visual observation and electronic detection methods and characterization of factors associated with estrus and pregnancy in beef heifers. Theriogenology 51: Richard, J. and Richard, N. (2010): Synchronization of estrus in beef cattle. Institute of agriculture and natural resource; University of Nebraska-Lincoln extension; Pp1-12 Roberts, S. J. (1986): Veterinary Obstetrics and Genital Diseases. (3 rd edition), David and Charles Inc., North Pomfret. Roche, J. F. and Prendiville, D. J. (1979): Control of estrus in dairy cows with a synthetic analogue of prostaglandin F2 alpha. Theriogenology 11: Roche, J. F., Austin, E. J., Ryan, M., O Rourke, M., Mihm, M. and Diskin, M.G. (1999): Regulation of follicle waves to maximize fertility in cattle. Journal of Reproduction and Fertility 54: Roche, J. R., Friggens, J. K., Kay, M.W., Fisher, K., Stafford, J. and Berry, D. P. (2009): Body condition score and its association with dairy cow productivity, health and welfare. Journal of Dairy Science 92: Rosenberg, M., Kaim, M., Herz, Z. and Folman, Y. (1990): Comparison of methods for synchronization of estrous cycle in dairy cows. 1. Effects on plasma progesterone and manifestation of estrus. Journal of dairy Science 73: Rowson, L. E. A., Tervit, R. and Brand, A. (1972): The use of prostaglandins for synchronization of estrus in cattle. Journal of Reproduction and Fertility 29:

77 Sattar, A. (2002): Estrus synchronization in exotic herd at livestock experiment station, Bhunikey (Pattoki), District Kasur Pakistan, International Journal of Agriculture and Biology 4(4): Schams, D. and Karg, H. (1982): Hormonal responses following treatment with different prostaglandin analogues for estrous cycle regulation in cattle. Theriogenology 17: Seguin, B. E., Gustafsson, B. K., Hurtgen, J. P., Mather, E. C., Refsal, K. R., Westcott, R. A. and Whitmore, H. L. (1978): Use of the prostaglandin F2α analog cloprostenol (ICI 80,996) in dairy cattle with unobserved estrus. Theriogenology 10: Seguin, B. E., Momont, H. and Baumann, L. (1985): Cloprostenol and dinoprost tromethamine in experimental and field trails treating unobserved estrus in dairy cows. Bovine Practice. 20: Shiferaw, Y., Tenhagen, B. A., Bekana, M. and Kassa, T. (2003): Reproductive performance of crossbred cows in different production systems in the central highlands of Ethiopia. Tropical Animal Health and production 35: Soren, P. R., Michelle, F. E., Misty, A. E., Joshua, B. E., Julie, A. G. and Andrew, S. L. (2012): Estrus synchronization and artificial insemination programs for beef cattle. Alabama Cooperative Extension System. Available at: (Accessed on Oct 15, 2014). Stevens, R. D., Seguin, B. E. and Momont, H. W. (1993): Simultaneous injection of PGF2α and GnRH into diestrous dairy cows delays return to estrus. Theriogenology 39: Stevenson, J. S. and Pursley, J. R. (1994): Use of milk progesterone and prostaglandin F2 alpha in a scheduled artificial insemination program. Journal of dairy Science 77: Stevenson, J. S., Schmidt, M. K. and Call, E. P. (1984): Stage of estrous cycle, time ofinsemination, and seasonal effects on estrus and fertility of Holstein heifers after 63

78 prostaglandin F2α. Journal of dairy Science 67: Stevenson, J. S., Lamb, G. C., Kobayashi, Y. and Hoffman. D. P. (1998): Luteolysis during two stages of the estrous cycle: subsequent endocrine profiles associated with radio telemetrically detected estrus in heifers Journal of Dairy Science 81: Stevenson, J. S., Lucy, M. C. and Call, E.P. (1987): Failure of timed inseminations and associated luteal function in dairy cattle after two injections of prostaglandin F2α Theriogenology 28: Tanabe, T. Y. and Hann, R. C. (1984): Synchronized estrus and subsequent conception in dairy heifers treated with prostaglandin F2α. I. Influence of stage of cycle at treatment. Journal of Animal Science 58: TBoARD (Tigray Bureau of Agriculture and Rural Development). (2014): Reported on the international conference on enhancing economic growth and public health through livestock development and one health approach, Tigray, Ethiopia Tegegne, A., Estifanos, A., Tera, A. and Hoekstra, D. (2012): Technological options and approaches to improve smallholder access to desirable animal genetic material for dairy development: IPMS experience with hormonal oestrus synchronization and mass insemination in Ethiopia. Tropentag. Pp 1-4. Tegegne, A., Warnick, A. C., Mukassa-Mugerwa, E. and Ketema, H. (1989): Fertility of Bos indicus and Bos indicus x Bos taurus crossbred cattle after estrus synchronization. Theriogenology 31: Tenhagen, B.A., Drillich, M. and Heuwieser, W. (2000): Synchronization of lactating cows with prostaglandin F2alpha: insemination on observed oestrus versus timed artificial insemination. Journal of Veterinary Medicine 47: Tessema, Z. K., Mihret, J. and Solomon, M. (2010): Effect of defoliation frequency and cutting height on growth, dry matter yield and nutritive value of Napier grass (Pennisetumpurpureum (L.) Schumach). Grass and Forage Science 65 (4):

79 Tsadik, G., Eshetu, L., Tadesse, G., Birhanu A. and Khar, S.K. (2008): Efficacy of norgestomet estradiol valerate- PMSG combination in the reproductive performance of crossbred cattle in Ethiopia. Proceedings of the annual research review day of the college of veterinary medicine, Mekelle University, pp Twagiramungu, H. L., Guilbault, A. and Dufour, J. J. (1995): Synchronization of ovarian follicular waves with a gonadotropin-releasing hormone agonist to increase the precision of estrusin cattle: a review. Journal of Animal science 73: USAID (United State Agency for International Development) (2010): Ethiopian Livelihoods: the livelihood integration Unit. An Atlas of Vahdat, F., Hurtgen, J. P., Whitmore, H. L., Johnston, S. D. and Ketelsen, C. L. (1979): Effect of time and temperature on bovine serum and plasma progesterone concentration. Theriogenology 12: Vahdat, F., Seguin, B. E., Whitmore, H. L. and Johnston, S. D. (1984): Role of blood cells in degradation of progesterone in bovine blood. American Journal of Veterinary Research 45: Vanholder, T., Opsomer, G. and Kruif, A. (2006): Aetiology and pathogenesis of cystic ovarian follicles in dairy cattle: a review. Reproduction and Nutrition Development 46: Waters, R. J. and Ball, R. (1978): Commercial ovulation control and fixed timed artificial insemination in cattle. Veterinary Records 103: Watson, E. D. and Munro, C. D. (1980): A re-assessment of the technique of rectal palpation of corpora lutea in cows. British Veterinary Journal 136: Watts, T. L. and Fuquay, J. W. (1985): Response and fertility of dairy heifers following injection with prostaglandin F2α during early, middle or late diestrus. Theriogenology 23: Wenzel, J. G. W. (1991): A review of prostaglandin F products and their use in dairy reproductive herd health programs. Veterinary Bulletin 61:

80 Wiltbank, M. C., Gümen, A. and Sartori, R. (2002): Physiological classification of anovulatory conditions in cattle. Theriogenology 57: WOARD (Wereda Office of Agriculture and Rural Development) (2014): Annual report, department of planning, Ganta-afeshum wereda, Tigray, Ethiopia. WOARD (Wereda Office of Agriculture and Rural Development) (2014): Annual report, department of planning, Atsibi-wemberta wereda, Tigray, Ethiopia. WOARD (Wereda Office of Agriculture and Rural Development) (2014): Annual report, department of planning, Kilte-Awlaelo wereda, Tigray, Ethiopia WOFP (Wereda Office of Finance and Planning) (2014): Annual report, Kilte-Awlaelo wereda, Tigray, Ethiopia Xu, Z. Z., Burton L. J. and Macmillan, K. L. (1997): Reproductive performance of lactating dairy cows following estrus synchronization regimens with PGF2α and progesterone. Theriogenology 47: Yaniz, J. L., Santolaria, P., Giribet, A. and Lopez-Gatius, F. (2006): Factors affecting activity at estrus during postpartum period and subsequent fertility in dairy cows. Theriogenology 66: Yemane, T. (1967): Statistics an introductory (2 nd Ed.), New York. Young, I. M. (1989): Dinoprost 14 day oestrus synchronization schedule for dairy cows. Veterinary Records 124: Young, I. M. and Henderson, D. C. (1981): Evaluation of single and double artificial insemination regimes as methods of shortening calving intervals in dairy cows treated with dinoprost. Veterinary Records 109: Zdunczyk, S., Mwaanga, E. S., Malecki-Tepicht, J., Baranski, W. and Janowski, T. (2002): Plasma progesterone levels and clinical findings in dairy cows with postpartum anoestrus. 66

81 APPENDIXES Appendix I: Investigation format for collecting data from selected individual heifers and cows for synchronization and hormonal analysis Code number Part one: House hold head characteristics 1. Owner name sex male female Age in year s 2. Address: Wereda tabia Phone No. 3. Educational level years of schooling 4. Marital status: Married Single Divorced Widowed Part two: cow s or heifer s characteristics 5. Cow s /heifer s I.D. 6. Breed: Local cross 7. Age of the cow/heifer 8. Body condition Score (1-9) 9. Types of feed the cows or heifers are fed: (wheat bran) Hay straw concentrate industrial by product Green feed Mixed concentrate, hay, straw, green feed and Stover others 10. How many times do the animals get feed per day? 11. How do you feed your animals? indoor feeding free grazing others 12. Do the animals get water regularly? Yes No: If yes, how many times a day? 13. Number of births (Parity) 67

82 Part three: Reproductive performance 14. Age of 1 st service 15. Number of services per conception 16. Gestation period 17. Dry period (dry off period) 18. Age of 1 st calving 19. Date of last calving 20. Calving interval Part four: production performance 21. Milk yield per day ( liter ) 22. Milk yield per lactation ( liter) 23. Lactation length ( month ) Part five: estrous detection and insemination (past) 24. Date of the animal come to estrous after calving 25. When did the animal show signs of estrous? Morning Afternoon Evening 26. When the animal was mated or inseminated? Morning Afternoon Evening 27. Date of last mating or insemination? 28. Does the animal come back to estrous again? Yes No, if yes, how many times does it come back to estrous after insemination? 29. When does it come back to estrous for the last time? 30. What method of mating do you use to breed the animal before? Natural mating AI synchronization + AI 31. How many times the animal was mated or inseminated when it was came to estrous? Once twice three times more than three times Part six: synchronization, hormonal analysis and insemination 32. Do the animal checked for presence of pregnancy? Yes No 68

83 If yes, when what was the result? Pregnant Not pregnant 33. Examination of ovaries for CL detection through rectal palpation: A. Structures found on the ovaries i. Left ovary CL ii.right ovary CL B. Other events (Like pyometra., fetal maceration, mummification, etc ) 34. RPHDT (Rapid Progesterone Heat Detection Test) I. First test- Date Number of lines? Test line colour? Invisible Light blue Dark blue P4 level. High moderate Low 35. Reproductive status of the animal according to progesterone test? Corpus luteam present absent 36. Based on condition scoring, rectal palpation and hormonal test, does the animal fit for estrous Synchronization? Yes No 37. Synchronization of estrous using PGF2α: (a.) Date of PGF2α injection: Single injection Double injection (b.) Estrous response of the animal: Positive Negative (c.) Time of AI Fixed AI; at 48 hrs at 72 hrs After estrous detection at 24 hrs 48 hrs 72 hrs 96 hrs > 96 hrs Date of inseminations: Fixed AI After estrus detection Bull No (ID) d. Pregnancy diagnosis 69

84 i. Date of pregnancy diagnosis (60 days post insemination) ii. Result of PD Pregnant Not pregnant Part seven: Husbandry practices 38. Do your animals encountered any reproductive diseases or problems in the past? Yes No 39. If yes, what reproductive diseases or problems were observed? 40. Housing condition of the farm? No Poor Moderate Good 41. Hygienic condition (Shelter, feed and water) No Poor Moderate Good 42. Do you use veterinary services? No Yes 43. What kind of genetic potential improvement practices do you use? Selection with in local breeds Up grading through cross breeding with exotic breeds Others 44. Do you have any marketing problems in your farm? No Yes If yes, why? 45. Do you get any extension service relating to dairy production? No Yes If no, why? 46. W hat are the existing opportunities for the development of dairy production in your farm? 70

85 47. What are the most important constraints for the development of dairy production in your farm? Appendix II: Elevation Map of the study area (Peasant association) (Source: BoFED, 2015). 71

86 Appendix III: Rapid progesterone heat detection test summary format AIT name and code Wereda Service year of AIT Educational level AIT Training Course duration Date of examination S. Cows ID CL DETECTION BY RPHDT result Remark No AIT Left ovary Right ovary

87 Appendix IV: Different photos taken during the study Partial view of material preparation and mobilizing for cattle owners 73

88 Partial view of material preparation and ear tagging for selected animals 74

89 Partial view of supervision by Mekelle University and LIVES project advisors 75

90 Partial view of mobilizing and giving orientation for cattle owners 76

91 Partial view of rectal palpation and PGF2α administration in progress 77

92 Partial view of heat expression after PGF2α administration in progress 78

93 Partial view of artificial insemination in progress 79

94 Partial view of questionnaire filling and semen motility evaluation in progress 80

95 Partial view of farm visiting and monitoring in progress 81

96 Partial view of milk collection and RPHDT test in progress 82

97 Partial view of Pregnancy diagnosis in progress in the study area 83