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

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 ___________________

E-mail: tgtade32@gmail.com

Name of advisors:

1. Dr. Gebregiorgis Ashebir (DVM, MVSc) 2. Dr. Yayneshet Tesfay (PhD)

Signature __________________ Signature _________________

Date ______________________ Date ____________________

Name of chairperson:

Dr. Etsay Kebede

Signature ______________________

Date _________________________

Name of Internal Examiner: Name of External Examiner:

Dr. Niraj Kumar (MVSc) Dr. Zelalem Tesfay

Signature ____________________ Signature _____________________

Date _______________________ Date _________________________

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.

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

1. _____________________________________________________

2._____________________________________________________

Advisors: Signature:

1. Dr. Gebregiorgis Ashebir (DVM, MVSc) ____________________

2. Dr. Yayneshet Tesfay (PhD ) ____________________

I

BIOGRAPHICAL SKETCH

The author was born in Tigray on May 08, 1970. 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 1990. 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.

II

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

III

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.

IV

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 .................................................................................................. 1

1.1. BACKGROUND AND JUSTIFICATION ......................................................................................... 1

1.2. PROBLEM STATEMENTS .......................................................................................................... 3

1.3. RESEARCH OBJECTIVES ........................................................................................................... 4

1.3.1. General Objective ............................................................................................................ 4

1.3.2. Specific Objectives ........................................................................................................... 4

1.4. RESEARCH QUESTIONS ............................................................................................................ 5

2. CHAPTER II: LITERATURE REVIEW ..................................................................................... 6

2.1. HISTORICAL BACKGROUND OF ESTROUS SYNCHRONIZATION IN CATTLE ............................... 6

2.2. ESTROUS BEHAVIOR AND ITS IMPORTANCE IN REPRODUCTION ACTIVITY IN CATTLE ............ 7

2.3. PROSTAGLANDINS ................................................................................................................... 9

2.4. PROSTAGLANDIN BASED ESTROUS SYNCHRONIZATION PROTOCOL ...................................... 11

2.4.1. One shot prostaglandin .................................................................................................. 11

2.4.2. Two shot prostaglandin .................................................................................................. 12

2.5. USE OF PROSTAGLANDIN IN ESTRUS SYNCHRONIZATION OF DAIRY COWS ........................... 12

2.6. FERTILITY FOLLOWING PROSTAGLANDIN INDUCED ESTRUS IN DAIRY CATTLE ................... 15

2.7. PROGESTERONE ANALYSIS USING MILK SAMPLES ............................................................... 16

2.8. FACTORS INFLUENCING THE EFFECTS OF PROSTAGLANDIN TREATMENT ............................. 16

2.8.1. Stage of Estrous Cycle at the Time of Prostaglandin Treatment ...................................... 16

2.8.2. Effect of Progesterone Level on Synchronized Estrus ...................................................... 17

2.8.3. Effect of Different Prostaglandin Analogues on Estrus Response and Fertility ................ 18

2.8.4. Breed and Season .......................................................................................................... 18

2.9. FACTORS LIMITING THE USE OF PROSTAGLANDIN IN DAIRY COWS ...................................... 19

2.9.1. Effect of Accuracy in Rectal Palpation of CL .................................................................. 19

2.9.2. Number of Cows in Synchronized Estrus ........................................................................ 19

2.9.3. Level of Progesterone during Prostaglandin Treatment .................................................. 20

2.9.4. Variation in Duration of Onset of Estrus ........................................................................ 20

3. CHAPTER III: MATERIALS AND METHODS ........................................................................ 22

3.1. LOCATION AND DESCRIPTION OF STUDY AREAS ................................................................... 22

3.2. STUDY ANIMALS .................................................................................................................... 24

V

3.3. STUDY DESIGN AND SAMPLING STRATEGIES ......................................................................... 25

3.3.1. Sample Size Determination ............................................................................................ 25

3.3.2. Experimental Design...................................................................................................... 27

3.3.3. Experimental Procedures ............................................................................................... 29 3.3.3.1. Single and Double Prostaglandin Injection ................................................................................29 3.3.3.2. Time of Insemination ...............................................................................................................29 3.3.3.3. RPHDT and AIT Corpus Luteum Detection through Rectal Palpation........................................30

3.4. DATA COLLECTION ............................................................................................................... 30

3.4.1. Estrus Response of Cows and Heifers ............................................................................. 30

3.4.2. Conception Rate ............................................................................................................ 31

3.4.3. Efficiency of Artificial Insemination Technicians ............................................................ 31

3.4.4. Independent Variables ................................................................................................... 31

3.5. STATISTICAL ANALYSIS......................................................................................................... 32

3.6. ETHICAL CONSIDERATION .................................................................................................... 33

4. CHAPTER IV: RESULTS ........................................................................................................... 34

4.1. EFFECT OF BREED AND FREQUENCY OF PROSTAGLANDIN INJECTION ON HEAT EXPRESSION . 34

4.1.1. Estrus expression Rate ................................................................................................... 34

4.1.2. Duration of estrus response after PGF2α injection .......................................................... 37

4.2. EFFECT OF BREED AND FREQUENCY OF PROSTAGLANDIN INJECTION ON CONCEPTION RATE

40

4.3. COMPARISON OF FIXED TIME AI WITH AI AT DETECTED HEAT ON CONCEPTION RATE........ 42

4.4. RAPID PROGESTERONE HEAT DETECTION TEST ................................................................... 44

5. CHAPTER 5: DISCUSSION........................................................................................................ 46

5.1. ESTRUS RESPONSE RATE ....................................................................................................... 46

5.2. CONCEPTION RATE ............................................................................................................... 48

5.3. EVALUATION OF THE EFFECTIVENESS OF FIXED TIME AI AND AI AT DETECTED ESTRUS ON

CONCEPTION RATE ............................................................................................................................ 48

5.4. COMPARISON OF THE EFFICIENCY OF AI TECHNICIANS ON CL DETECTION THROUGH RECTAL

PALPATION WITH THAT OF RAPID PROGESTERONE HEAT DETECTION TEST (RPHDT) ..................... 49

6. CONCLUSION AND RECOMMENDATIONS .......................................................................... 51

6.1. CONCLUSION ......................................................................................................................... 51

6.2. RECOMMENDATIONS ............................................................................................................. 51

REFERENCES ..................................................................................................................................... 53

APPENDIXES ...................................................................................................................................... 67

VI

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 ........................................................................................................ 32

Table 3: Estrus response rate of local and crossbred cows and heifers after single and double dose

injection of PGF2α ..................................................................................................................... 35

Table 4: Estrus response rate of local and crossbred cattle after single dose injection of PGF2α .. 36

Table 5: Conception rate result of Local and Crossbred, Cows and Heifers after single and double

dose PGF2α Injection ................................................................................................................. 41

Table 6: Conception rate result after single dose PGF2α injection .............................................. 41

Table 7: Conception rate result after double dose PGF2α injection ............................................ 42

Table 8: Fixed AI Versus AI at Detected estrus ........................................................................ 43

Table 9: Rapid Progesterone Heat Detection Test (RPHDT) result ............................................ 44

Table 10: Results of a quadratic discriminant analysis .............................................................. 45

VII

LIST OF FIGURES

Figure 1: Location map of the study area .................................................................................. 24

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). .................. 28

Figure 3: Estrus response rate of local and crossbred cattle after single and double dose injection

of PGF2α ................................................................................................................................... 36

Figure 4: Time of Estrus Response after Single and double dose PGF2α Injection ...................... 37

Figure 5: Time of Estrus Response after Single PGF2α Injection ............................................... 38

Figure 6: Time of Estrus Response after double PGF2α Injection ............................................... 38

Figure 7: Estrus duration for single injection of PGF2α before 48 and after 72 hours .................. 39

Figure 8: Estrus duration for double injection of PGF2α before 48 and after 72 hours ............... 40

Figure 9: Conception rate of local and crossbred cattle after single and double dose injection of

PGF2α........................................................................................................................................ 43

Figure 10: Comparison of AI Technician efficiency with RPHDT ............................................ 45

VIII

LIST OF APPENDIXES

Appendix I: Investigation format for collecting data from selected individual heifers and

cows for synchronization and hormonal analysis ........................................................... 67

Appendix II: Elevation Map of the study area (Peasant association) ............................. 71

Appendix III: Rapid progesterone heat detection test summary format ......................... 72

Appendix IV: Different photos taken during the study .................................................. 73

Appendix V: Ethical Clearance Letter ........................................................................... 84

IX

LIST OF ABBREVIATIONS

AI

AIED

Artificial Insemination

Artificial Insemination at Estrus Detection

AIT Artificial Insemination Technician

BoFED Bureau of Finance and Economic Development

CI Confident Interval

CL Corpus Luteum

EER

FTAI

Estrus Expression Rate

Fixed Time Artificial Insemination

FSH Follicular Stimulating Hormone

GDP Gross Domestic Product

GLM Generalized Linear Model

GnRH Gonadotropin Releasing Hormone

ILRI International Livestock Research Institute

IPMS Improving the Productivity and Market Success

LH Luteinizing Hormone

LIVES Livestock and Irrigation Value Chains for Ethiopian

Smallholders

M.a.s.l. Meter above sea level

MoA Ministry of Agriculture

OR Odds Ratio

P4 Progesterone

PGF2α Prostaglandin F Two Alpha

RPHDT Rapid Progesterone Heat Detection Test

SNNPR Southern Nations, Nationalities, and Peoples' Region

SPSS Statistical Package for Social Science

TBoARD Tigray Bureau of Agricultural and Rural Development

USAID United States Agency for International Development

WOARD Wereda Office of Agriculture and rural development

WoFP Wereda Office of Finance and Planning

X

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

1

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.,

2

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

3

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.

1.2. 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 12-13 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

4

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 75-95 %) and 31.5% (ranges from 12.39-57.14 %),

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 cross-

bred 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.

1.3. Research objectives

1.3.1. 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.

1.3.2. Specific Objectives

To evaluate the effect of breed and frequency of prostaglandin injection on heat

expression of cows and heifers.

5

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.

1.4. 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?

6

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.

7

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.

2.2. 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).

8

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

9

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

GnRH

Source

Hypothalamus

Function

Releases FSH and LH

Commercial Products

Cystorelin®, Factrel®,

Fertagyl®, OvaCyst®

FSH Anterior pituitary Stimulates development

of ovarian follicles

Folltropin®

Estrogen Ovarian follicle Stimulates behavioral

estrus and the LH surge

Not used in current

systems

LH Anterior pituitary Stimulates rupture of a

follicle (ovulation)

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®,

ProstaMate®, In Sync®

(Adapted from Michael and Thomas, 2005.)

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

10

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

11

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.

2.4. Prostaglandin based Estrous Synchronization Protocol

2.4.1. 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).

12

2.4.2. 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).

2.5. 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

13

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

14

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.

15

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 PGF2α-

synchronized 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.

16

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 progesterone-

secreting 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).

2.8. Factors Influencing the Effects of Prostaglandin Treatment

2.8.1. 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

17

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 %).

2.8.2. 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 %).

18

2.8.3. 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)..

2.8.4. 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 13.60 and 19.30 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.

19

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.

2.9.1. 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.

2.9.2. Number of Cows in Synchronized Estrus

Another reason that affects the potential use of PGF2α in improving the pregnancy rate in

20

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).

2.9.3. 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.

2.9.4. 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

21

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

Lamb, et al., 2006).

22

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, 2015. 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 14o 24'

and 14o 21'N Latitude and 39o13' and 39o 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 0C (WOARD, 2014).

Atsbi Wemberta district is located in Eastern zone of Tigray National Regional State at

13036' and 14006' north latitude and 39039' and 39048' 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 between10-300C. Most of the rain is mono-modal and concentrated in

23

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 300-600 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 13046' - 13059' N latitude and 390 36'-390 42' E

longitude (WOFP, 2014). The altitude ranges from 1900 – 2460 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

350-450m.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 17 - 230c

maximum monthly average temperature is May/June, whereas the minimum temperature

is in October and December (WOFP, 2014).

24

Figure 1: Location map of the study area

(Source: BoFED, 2015).

3.2. 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

25

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 2190 753 2943 14140 5547 19687 22630

2 Atsbiwemberta

878 531 1409 21454 5609 27063 28472

3 Kilte-awlaelo

818 254 1072 26503 11131 37634 38706

Total 3886 1538 5424 62097 22287 84384 89808

Source: WOARD (Ganta-afeshum, Atsbiwemberta, and Kilte-awlaelo weredas, 2014).

3.3. Study Design and Sampling Strategies

3.3.1. 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.

26

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 Stations

(Tabias)

Local Crossbred Total

Single

Injection

Double

Injectio

n at ED

Double

Fixed

Time AI

Single

Injection

Double

Injection

at ED

Double

Fixed

Time AI

G/afeshum Baati may

mesanu

20 10 10 20 10 10 80

K/awlaelo Genfel 20 10 10 20 10 10 80

A/wenberta Golgol-

naele

20 10 10 20 10 10 80

Total 60 30 30 60 30 30 240

27

3.3.2. 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

28

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.

29

3.3.3. Experimental Procedures

3.3.3.1. 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 0.263 mg equivalent to cloprostenol 0.250 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.

3.3.3.2. 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.

30

3.3.3.3. 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).

3.4. Data Collection

3.4.1. 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

31

and greater than 96 hours. Cows that exhibit estrus were artificially inseminated by

experienced technician using frozen semen after PGF2α injection.

3.4.2. 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.

3.4.3. 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).

3.4.4. 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).

32

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

Time of heat expression of

the dairy cows and heifers

categorical 1=if After 24, 2= 48, 3= 72,

4= 96 and 5= > 96 hours

AI method Time of insemination of

the dairy cows and heifers

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.

33

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< 0.05. Modelling was

continued until all the main effects or interaction terms were significant according to the

Wald statistic at P < 0.05.

3.6. 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.

34

4. CHAPTER IV: RESULTS

4.1. Effect of breed and frequency of Prostaglandin injection on heat expression

4.1.1. 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).

35

Table 3: Estrus response rate of local and crossbred cows and heifers after single and

double dose injection of PGF2α

Characteristics

Estrus Response

Odds ratio (95% CI)

P-value

No

N (%) Yes

N (%)

PGF2α Injection

Single 19(15.8) 101(84.2) Single (reference) -

Double 4(6.7) 56(93.3) 2.6 (0.853, 8.125) 0.092

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) 0.123

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) 0.005

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).

36

Table 4: Estrus response rate of local and crossbred cattle after single dose injection of

PGF2α

Characteristics Estrus Response

No, N (%) Yes, N (%) Odds Ratio (95% CI) P-value

Breed

Local 13(21.8) 47(78.3) Local(reference) -

Cross 6(10) 54(90) 2.5 (0.876, 7.066) 0.087

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.

Figure 3: Estrus response rate of local and crossbred cattle after single and double dose

injection of PGF2α

0%10%20%30%40%50%60%70%80%90%

100%

ES

TR

US

RE

SP

ON

SE

37

4.1.2. 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).

38

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%

39

(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).

40

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,

41

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

Characteristics

Conception rate

Negative N (%) Positive N (%) Odds ratio (95% CI) P-value

PGF2α Injection

Single 40(40.4) 59(59.6) Single(reference) -

Double 17 (31.5) 37(68.5) 1.47 (0.732, 2.973) 0.276

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) 0.788

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) 0.584

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 58.49 % (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

Characteristics

Conception rate

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) 0.810

Total 40(40.4) 59 (59.6)

Values in parentheses are percentage.

42

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) 0.381

Total 17(31.5) 37 (68.5)

Values in parentheses are percentage.

4.3. 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).

43

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) 0.047

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) 0.266

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

44

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

39 43.3

51 56.7

90 100.0

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.

45

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

Corpus luteum detection by Artificial

Inseminator

Mean

Std.

Deviation

Valid N (list wise)

Un weighted Weighted

Yes RPHDT Result .57 .498 90 90.000

Total RPHDT Result .57 .498 90 90.000

46

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

47

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.

48

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 (

2011- 2014) 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.

5.3. 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

49

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).

5.4. 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

50

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.

51

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.

6.2. 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.

52

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.

53

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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:

Hay straw concentrate industrial by product

(wheat bran)

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) _________________

68

Part three: Reproductive performance

14. Age of 1st service _____________________

15. Number of services per conception ___________________

16. Gestation period _______________________

17. Dry period (dry off period) _____________________

18. Age of 1st 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

69

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

70

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?

_________________________________________________________

_________________________________________________________

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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).

72

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.

No

Cows ID CL DETECTION BY

AIT

RPHDT result Remark

Left ovary Right ovary

01

02

03

04

05

06

07

08

09

10

73

Appendix IV: Different photos taken during the study

Partial view of material preparation and mobilizing for cattle owners’

74

Partial view of material preparation and ear tagging for selected animals

75

Partial view of supervision by Mekelle University and LIVES project advisors

76

Partial view of mobilizing and giving orientation for cattle owners

77

Partial view of rectal palpation and PGF2α administration in progress

78

Partial view of heat expression after PGF2α administration in progress

79

Partial view of artificial insemination in progress

80

Partial view of questionnaire filling and semen motility evaluation in progress

81

Partial view of farm visiting and monitoring in progress

82

Partial view of milk collection and RPHDT test in progress

83

Partial view of Pregnancy diagnosis in progress in the study area

84

Appendix V: Ethical Clearance Letter

85

Assurance of principal investigator

The under signed agrees to accept responsibility for the scientific, ethical and technical

conduct of the research project and for the provision of required progress reports as per

terms and conditions of the University in effect at the time of grant, if grant is awarded as

the result of this application

Name: Tadesse Gugssa Kebede (BSc, MSc student MU-CVM)

Date: ________________________ Signature: _____________________________

Approval

Advisors:

Name Signature

Dr. Gebregiorgis Ashebir (Main advisor) _________________

Dr. Yayneshet Tesfay (Co-advisor) _________________

Head, Department of Veterinary Medicine

Name: ________________________________________

Signature: _____________________________________

Date:_________________________________________

Stamp:

College Research Council Coordinator:

Name: ________________________________________

Signature: _____________________________________

Date:_________________________________________

Stamp:

For Office Use

Amount of Approved Budget ___________________

Period of Allocation ___________________________