synchronization of estrus and ovulation …...viii acknowledgments i thank the god almighty for...

i

SYNCHRONIZATION OF ESTRUS AND OVULATION IN

POSTPARTUM ACYCLIC CROSSBRED COWS

By

N.RAMALAKSHMI B.V.Sc. & A.H.

TVM/13-49

Thesis Submitted to the

SRI VENKATESWARA VETERINARY UNIVERSITY

In partial fulfillment of the requirements

For the award of the degree of

MASTER OF VETERINARY SCIENCE

In the faculty of Veterinary Science

(VETERINARY GYNAECOLOGY AND OBSTETRICS)

DEPARTMENT OF VETERINARY GYNAECOLOGY AND OBSTETRICS

COLLEGE OF VETERINARY SCIENCE

SRI VENKATESWARA VETERINARY UNIVERSITY

TIRUPATI – 517 502 [A.P.] INDIA

NOVEMBER, 2015

ii

CERTIFICATE

Dr. N. RAMALAKSHMI, (TVM/13-49) has satisfactorily prosecuted

the course of research and that the thesis entitled “SYNCHRONIZATION

OF ESTRUS AND OVULATION IN POSTPARTUM ACYCLIC

CROSSBRED COWS” submitted is the result of original research work and

is of sufficiently high standard to warrant its presentation to the examination.

I also certify that the thesis or part thereof has not been previously submitted

by her for a degree of any university.

Date : Place : Tirupati

(Dr. M.SRINIVAS) Chairman of Advisory Committee

Associate Professor Department of Veterinary Gynaecology

and Obstetrics NTR College of Veterinary Science

Gannavaram 521101 [A.P.]

iii

CERTIFICATE

This is to certify that the thesis entitled “SYNCHRONIZATION OF

ESTRUS AND OVULATION IN POSTPARTUM ACYCLIC

CROSSBRED COWS” submitted in partial fulfillment of the requirements

for the degree of Master of Veterinary Science (Veterinary Gynaecology and

Obstetrics) of the Sri Venkateswara Veterinary University, Tirupati, is a

record of the bonafide research work carried out by N. RAMALAKSHMI

(TVM/13-49) under our guidance and supervision. The subject of the thesis

has been approved by the Student’s Advisory Committee.

No part of the thesis has been submitted by the student for any other

degree or diploma. The published part has been fully acknowledged. All

assistance and help received during the course of the investigations have been

duly acknowledged by the author of the thesis.

(Dr. M. SRINIVAS) Chairman of the Advisory Committee Thesis approved by the Student’s Advisory Committee CHAIRMAN : Dr. M. SRINIVAS ____________________

Associate Professor Department of Veterinary Gynaecology and Obstetrics NTR College of Veterinary Science

Gannavaram– 521101. MEMBER : Dr. K. MOULI KRISHNA ____________________ Professor and University Head

Department of Veterinary Gynaecology and Obstetrics College of Veterinary Science Tirupati – 517502.

MEMBER : Dr. B. SREEDEVI ____________________ Professor

Department of Veterinary Microbiology College of Veterinary Science Tirupati – 517502 .

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TABLE OF CONTENTS

Chapter No Title Page No

I INTRODUCTION 1-2

II REVIEW OF LITERATURE 3-35

2.1 Estrus synchronization 3

2.1.1 Ovsynch Protocol 6

2.1.2 Ovsynchwith PRID protocol 11

2.1.3 PRID with PG protocol 15

2.2 Estrus induction efficiency 21

2.2.1 Estrus Response 21

2.2.2 Duration of estrus 22

2.2.3 Intensity of estrus 23

2.3 Serum progesterone assay 23

2.4 Effect of estrus synchronization protocols on conception rate

27

2.4.1 Breeding at FTAI 27

2.4.2 Conception Rates 28

III MATERIALS AND METHODS 36-40

3.1 Experimental animals 36

3.1.1 Selection of Animals 36

3.2 Design of the study 36

3.3 Evaluation of estrus 37


3.5 Fertility 40

3.6 Statistical analysis 40

IV RESULTS 41-48


v

4.1.1 Estrus response 41

4.1.2 Duration of estrus 41

4.1.3 Intensity of estrus 41


4.3 Fertility 43

4.3.1 First service conception rate 43

4.3.2 Second service conception rate 43

4.3.3 Overall conception rate 43

V DISCUSSION 49-59


5.1.1 Estrus Response 49

5.1.2 Duration of Estrus 51

5.1.3 Intensity of Estrus 51


5.3 Fertility 54

VI SUMMARY 60-62

LITERATURE CITED 63-74

vi

LIST OF FIGURES

FIGURE NO PARTICULARS Page No.

1 Image showing TRIU-B device applicator 38

2 Image showing applicator and TRIU-B device 38

3 Insertion of TRIU-B device 39

4 Clipped tail of TRIU-B device 39

5 Withdrawal of TRIU-B device 39

6 Comparison of estrus response 45

7 Comparison of intensity of estrus 45

8 Comparison of duration of estrus 45

9 Comparision of mean serum progesterone

concentrations (ng/ml) in various synchronization

protocols on the day of initiation of treatment day ‘0’.

47

10 Comparision of mean serum progesterone

concentrations (ng/ml) in various synchronization

protocols on the day of AI.

47

11 Comparison of conception rates 48

vii

LIST OF TABLES

TABLE

NO

TITLE PAGE NO

1 Effect of synchronization on estrus induction 44

2 Analysis of variance for duration of estrus 44

3 Serum progesterone (Mean±SE) on day ‘0’ and on day

of AI

46

4 Analysis of variance for means (±SE) of serum

progesterone on day ‘0’ and day of AI

46

5 Fertility response in crossbred acyclic cows

synchronized with 3 different protocols

48

viii

Acknowledgments

I thank the God Almighty for showering his abundant blessings on me

which made me to accomplish the task.

I feel it a great privilege for having worked under the guidance of

Dr. M. Srinivas, Associate Professor, Department of Veterinary Gynaecology and

Obstetrics, NTR College of Veterinary Science, Gannavaram for his continuous

help, valuable guidance and endless encouragement during the course of

investigation. The untiring interest, critically going through the manuscript and

also spending time in correction, execution, compilation and preparation of the

thesis are invaluable.

I accord my sincere thanks to Dr. K. Mouli Krishna, Professor and

University Head, Department of Veterinary Gynaecology and Obstetrics, College

of Veterinary Science, Tirupati and member of Advisory Committee for his

guidance, deliberate counsel and help extended during presentation of thesis.

I am extremely thankful to Dr. B. Sreedevi, Professor, Department of

Veterinary Microbiology, College of Veterinary Science, Tirupati and member of

Advisory Committee for her suggestions and cooperation during presentation of

thesis.

I am greatly indebted and extend to my sincere thanks to

Dr. G. Suman kumar, Veterinary Assistant Surgeon, Masters in Veterinary

Gynaecology and Obstetrics, for his whole hearted co-operation and constant

encouragement at all stages of my work.

I wish to extend my gratitude to Dr. K. VeeraBrahmaiah, Professor and

Dr. Allu Teja, Assistant Professor, Department of Veterinary Gynaecology and

Obstetrics, College of Veterinary Science, Tirupati for their timely help, unstinted

ix

cooperation, constant encouragement and affection rendered during the period of

my study.

I wish to convey my sincere thanks to my seniors Dr. G. Padmanabham,

Dr. Y.Pavani, Teaching Assistant and Dr. Jaganath reddy, Teaching Assistant,

Dr. Vishal, Ph.D, Department of Veterinary Gynaecology and Obstretics, Dr.

Shruthi from Department of Veterinary Pathology for her wonderful love, support

and affection towards me, Dr. Jyothi sri from Department of Veterinary

Parasitology and Dr. Radhika, Dr. Lahari, Dr. Sudheer from Department of

Microbiology, Dr. Dhayana from Department of Veterinary Anatomy and Dr.

Roshan baig from Department of Veterinary Animal Husbandary and Extension,

College of Veterinary Science, Tirupati for their constant suggestions and

encouragement during the period of study.

It is the correct time to express my heartfelt thanks to my affectionate

colleagues Dr. A. Geetha reddy, Dr. P. Kalyan Venkatesh, Dr. Yedukondalu, Dr.

Hemkunar, Dr. Praveen kumar, Dr. Satish and Dr. Praveen Raj and seniors and

friends of veterinary Dr. Y.Prathiba, Dr. SivaNagaraju, Dr. Divya,

Dr.Sivajyothi, Dr. Indira, Dr. Mohan kishore, Dr. Gopikrishna, Dr.G. Srinivas,

Dr. Vamsikrishna, Dr. Chandrasekhar naik, Dr.Manoj, Dr. Sudharshan,

Dr.Sudhakar, Dr. Mahesh, Dr. Deepa, Dr. Asha Kumari, Dr. Sailu madhuri, Dr.

Sudheer, Dr. Yeswanth, Dr. Rama Chandra rao, Dr. Ramu, Dr.Prasanna and Dr.

Praveen(chano) for their affection, moral support, cheerful and generous help and

pleasant company.

I express warmest thanks to my juniors Dr.Anil, Dr.Ravikanth, Dr.

Preethi, Dr. Harini, Dr. Prasuna, Dr. Praveen Kumar, Dr.Hanuman, Dr. Harish,

Dr. Tejaswi, Dr. Prakash, Dr. Suvarna, Dr.Harshini, Dr. Sravani Pragna and

Shoba for their co-operation and affectionate help during my research work.

Lots of thanks to my dalzz lovely cute schoolmates Dr. Meena, Sowmya,

Dr.Sireesha, Gaddipati Anusha (gaddi) , Naga raju and Raghu vamsi for their pure

love and affection till today

x

I thank Mrs.Syamalamma, Mrs.Rajyalakshmi, Mr.Sekhar,

Mr.Chengalarayulu, Mr. Anand, Mr. Ramana and Mrs. Danamma for their

cooperation and affectionate help during my research work.

I thank Sri Venkateswara Veterinary University for providing monetary

support in the form of stipend to prosecute my post graduation study.

It is time to surface out my profound love and affectionate gratitude to my

beloved parents Sri. N.Anjana Rao and Smt.K. Rani and my sisters younger one

Dr. N. Alekhya and youngest Ms. Srilekha who constantly guided and moulded

me into present position and whose boundless love, unparalleled affection, moral

support is a constant source of motivation for me in shaping up my career. Where

emotions are involved, words cease to express affection, encouragement and

cooperation of my lovable family friends Dr.Anurupa, Dr. Srilatha, Dr.

Phaneendra, Dr. Vijay, Dr.Chaithanyamani, Dr. Sahitya, Dr.Kusuma, Dr.

Sailaja, Dr. Viswaja, Dr. Priyanka Roberts , Dr. Shiny, Dr. Priyanka, Dr. Dileep,

Dr. Mahendra, Dr. Asha, Dr. Poori, Dr. Swapna, Dr.Watsyavi and Dr. Bharath

without which, I would have never come to this stage of career building.

I place on record my apologies and sincere thankfulness to the unmentioned

personalities, who have played a role in this study and preparation of the

manuscript.

Ramalakshmi….

xi

DECLARATION

I, Dr. N. RAMALAKSHMI (TVM/13-49), hereby declare that the

thesis entitled “SYNCHRONIZATION OF ESTRUS AND OVULATION

IN POSTPARTUM ACYCLIC CROSSBRED COWS” submitted to Sri

Venkateswara Veterinary University, Tirupati for the degree of MASTER

OF VETERINARY SCIENCE is the result of original research work done

by me. It is further declared that the thesis or any part thereof has not been

published earlier in any manner.

Date : (N.RAMALAKSHMI)

Place : Tirupati TVM/13-49

xii

Name of the author : N. RAMALAKSHMI

Title of the thesis : “SYNCHRONIZATION OF ESTRUS AND OVULATION IN POSTPARTUM ACYCLICCROSSBRED COWS”

Degree to which it is Submitted : MASTER OF VETERINERY SCIENCE

Faculty : Veterinary Science

Department : Veterinary Gynaecology and Obstetrics College of Veterinary Science, Tirupati.

Major Advisor : Dr. M.SRINIVAS Chairman of Advisory Committee Associate Professor Dept. of Vety. Gynaec. & Obstetrics NTR College of Veterinary Science Gannavaram 521101 [A.P.]

University : SRI VENKATESWARA VETERINARYUNIVERSITY, TIRUPATI.

Year of submission : November, 2015

ABSTRACT

The present Research work was undertaken by utilizing 24 postpartum

acyclic crossbred cows maintained under diverse (village) managemental

conditions. The work was designed to study the efficacy of different

synchronization protocols viz., Ovsynch (Group I), Ovsynch+PRID (Group

II) and PRID+PG (Group III) based on estrus response, duration of estrus,

intensity of estrus, serum progesterone concentration and conception rate.

The estrus response and duration of estrus after synchronization of

postpartum acyclic crossbred cows inGroup I, Group II and Group III was

xiii

75.00, 100.00 and 87.50 percent; 50.62±8.39hrs, 68.00±2.61hrs and

58.63±9.36 hrs,respectively.

Intensity of estrus exhibited was high of (62.5%) when synchronized

with Ovsynch+PRID protocol followed by PRID+PG (50.0%) andOvsynch

(25.0%) protocols. In Ovsynch (12.5% weak, 62.5% intermediate and 25%

intense) Ovsynch+PRID, (0% weak, 37.5% intermediate and 62.5% intense)

and PRID+PG, (12.5% weak, 37.5% intermediate and 50% intense).

The mean serum progesterone concentrations in Group I, Group II and

Group III on day ‘0’ were 2.56±1.04ng/ml, 1.66±0.24 ng/ml and 1.43±0.32

ng/ml,respectively and at the time of AI they were 7.60±0.87 ng/ml,

1.86±0.28 ng/ml and 2.50±0.65 ng/ml, respectively. The difference in the

serum progesterone concentrations amongst all the three synchronization

protocols was non-significant (P>0.05) recorded on day ‘0’ and day of AI.

The first service conception rate was 50% and 50% in crossbred

acyclic cows synchronized with Ovsynchand PRID+PG protocols,

respectively while it was comparatively lower in Ovsynch+PRID protocol

(37.50%). The overall conception rate in Ovsynch, Ovsynch+PRID and

PRID+PG protocols was 50%, 50% and 62.50%.

It was concluded from the present study that Ovsynch+PRID protocol

obtained better estrus exhibition, while PRID+PG was found to be effective in

getting optimum conception rate with fixed time artificial inseminationin

postpartum acyclic crossbred cows.

xiv

LIST OF SYMBOLS AND ABBREVIATIONS

AI : Artificial Insemination

PRID : Progesterone ReleasingIntravaginal Device

CL : Corpus Luteum

d : Days

FTAI : Fixed Time Artificial Insemination

GnRH : Gonadotropin Releasing Hormone

h/hrs : Hours

LH : Luteinizing Hormone

mm : Millimeter

ml : Millilitre

ng : Nano gram

µg : Micro gram

PGF2α : Prostagladin2α

SE : Standard error

TAI : Timed Artificial Insemination

Vs : Versus

% : Percent

< : Less than

> : Greater than

± : Plus or Minus

≥ : Greater than or equal to

1

CHAPTER-I

1.INTRODUCTION

Crossbreeding programme was introduced in India during late 1970’s

as 50 percent Bostaurus cross-bred cowsas these cows had 1.8 times higher

lifetime milk yields and a 1.2 times higher number of total lactations.

However, it had registered a negative correlation on reproductive traits

(Galukandeet al., 2013).

Assisted reproductive techniques like Estrus synchronization has been

promoted historically as a labor saving tool for those producers who want to

capitalize on the superior genetics available through use of AI. However, the

labor saving aspect is meagerwhen compared to the economic returns

available when estrus synchronization was used as a “reproductive

managemental tool”. Thus, estrous cycle was manipulated to bring large

number of females into estrus at a predetermined time to enhance the

reproductive efficiency by shortening the calving interval. This was done by

adopting reproductive management techniques like synchronization of

estrus/ovulationwith fixed timed artificial insemination yielding optimum

conception rates (Islam, 2011). Results of many of the synchronization

protocols depend on body condition scoreas it was the measure of nutritional

status of animals and was an important factor(Baruselliet al., 2011).

Ovsynchwas a fixed-time AI synchronization protocol that has been

developed, tested and used extensively in dairy cattle as it allows acceptable

synchrony of follicular development, regression of corpus luteum and

1

2

synchronizes ovulation allowing effective FTAIwith no need for heat

detection with optimum pregnancy rates (Stevensonet al., 2006). During

natural estrous cycle prostaglandin F2αreleased from the uterus was a

triggering factor that resulted in luteolysis and helped the diestrus open cowto

return to estrus every 21 days, this mechanism was used artificially to shorten

the life span of the CL by administration of PGF2α in several synchronization

protocols for induction of estrus in postpartum acyclic cows (Mel DeJarnette,

2004).

Administration of GnRH helped in termination of ovarian follicle

development of one wave and emergence of new follicle of next wave leading

to the maturing as dominant follicle within 7 days after the injection, thus

helping in synchronization of estrus/ovulation (Twagiramunguet al., 1995).

Use of exogenous progestagen like PRID along with Ovsynchprotocol

increasedthe pregnancy rates and decreased days open which was helpful to

bring the animals to fertile estrus (JagirSingh et al., 2009).

Perusal of literature revealed that limited studies were conducted to

evaluate the efficacy of different estrus synchronization protocols to

synchronize estrus and ovulation in postpartum acyclic crossbred cows under

rural conditions. Hence, the present study was undertaken to study the

efficacy of different estrus synchronization protocols.

3

CHAPTER-II

2. REVIEW OF LITERATURE

2.1 ESTRUS SYNCHRONIZATION

Whittier et al. (1989) expressed that prostaglandins and its analogues

were approved and being used to improve the reproductive efficiency in dairy

herds as it increased the conception rates by effective observation of cyclicity

and detection of estrus.

GnRH along with progesterone and PGF2α resulted in an acute

treatment for programmed follicular development and ovulation (Thacther et

al., 1993). Interval of 6 or 7 days between Buserelin and PGF2α had

satisfactory synchronization with regards to fertility (Coleman et al., 1991 and

Guilbault et al., 1991) when compared with two PGF2α injections at 11 days

apart (Coleman et al., 1991). Gonadotrophin releasing hormone (GnRH)

occupies a central role in the reproductive function and has a potential for

fertility control in mammals (Diskin et al., 2002).

Estrus detection is one of the most important factors affecting the

reproductive success of artificial insemination programs, an increase in basic

understanding to manipulate ovarian follicular and corpus luteum dynamics

over the last decade have developed better results in synchronization protocols

and these protocols have a potential to enhance pregnancy rates and successful

artificial insemination programs (Rajamahendran et al., 2001).

Use of various hormonal combinations like estrogen and GnRH and

steroids like estradiol, progesterone with PGF2α were used for the regulation

3

4

of lifespan of the corpus luteum, controlling follicular wave emergence and

ovulation (Larson and Ball, 1992 and Thatcher, 2001).

Progesterone therapy was the treatment of choice for inducing cyclicity

in postpartum anoestrus cows, where hormones progesterone and

GnRH+PGF2α were used for the onset of estrus as in most of the anoestrus

cows there was no progesterone in the animal’s circulation upto 45 days of

calving due to which there was failure of ovulation and CL formation

(Mozaffari et al., 2014). Progestagens yielded satisfactory results in crossbred

cows under tropical conditions for induction of estrus and ovulation (Soto et

al., 1998 and 2002).

Progesterone releasing inserts have been used to synchronize estrus where

85% of the cattle exhibited estrus between 36 and 60 hrs after removal of the

device (Cavalieri et al., 2004).

Widespread acceptance of follicular wave emergence and luteal

regression using GnRH and PGF2α was seen in ovsynch protocol for dairy

herd management, while a second dose of GnRH after 48 h of administering

PGF2α induced ovulation and allowed fixed time insemination after 16 h

(Kasimanickam et al., 2004).

The ability to precisely control the time of ovulation after a

programmed follicular development and corpus luteum regression

sequentially permits a timed insemination which synchronizes with estrus and

helps in overcoming the errors in heat detection (Ozturk, 2009).

5

Synchronization of estrus could be accomplished with the injection of

prostaglandin alone, but this needed correct identification of the ovarian status

of cows as prostaglandin is sensitive to functional corpus luteum aged

between 8 to 17 days of the estrus cycle. Progesterone therapy may reduce

fertility upto 14%, but short time progesterone exposure (≤14 days) was

beneficial. GnRH addition in progesterone and prostaglandin synchronization

programme was more helpful to induce estrus as this protocol synchronized

estrus in delayed pubertal heifers and postpartum cows. Newer methods of

synchronizing estrus with GPG protocol preceded by progesterone treatment

were effective with high fertility (Islam, 2011).

Synchronization of estrus shortened the calving interval, increased

uniform calving dates, reduced the time devoted for estrus detection and

facilitated the use of AI (Waldmann et al., 2006) and thereby enhancing the

economic returns to farmers (Islam, 2011).

Estrus synchronization, ovulation synchronization and AI are

reproductive management tools that have been available to farmers for more

than 30 years. These tools remain the most important and widely applicable

reproductive biotechnologies available for beef cattle operations (Seidel,

1995). Ideal synchronization program should have similar or more conception

rate than the natural conception rate and the selection of the protocol should

be on the basis of its managemental capabilities and expectations of the farmer

(Vasconcelos et al., 1999).Synchronization of estrus is one of the advanced

6

managemental process through which the humane errors and managemental

costs could be minimized (Islam, 2011).

2.1.1 Ovsynch Protocol

Wolfenson et al.(1994) reported that use of Buserelin (8µg) on day 12

and seven days prior to synchronization of estrus with PGF2α altered follicular

development and produced preovulatory follicles which were more

homogenous, more estrogen-active and more dominant with a greater size

difference between the preovulatory and subordinate follicles prior to estrus

when compared to untreated cows.

A protocol using GnRH and PGF2α called Ovsynch was developed to

synchronize ovulation in dairy cattle (Pursley et al., 1995; Pursley et al., 1998

and Moreira et al., 2000a) and with a goal of synchronizing the ovulation

within 8 h period (from 24 to 32 h after the second GnRH treatment) enabling

TAI without the detection of estrus (Pursley et al., 1995) and with pregnancy

rates in the range of 30 to 40 % (Burke et al.,1996; Pursley et al., 1997a and

Stevenson et al., 1999). In Ovsynch protocol, GnRH injection was

administered on day 0followed by PGF2 after 7 days and a second injection

of GnRH after 48h with TAI at 72 h after PGF2.Synchronization with

Ovsynchprotocol was successful to detect estrus, efficient artificial

insemination and also helped the producers to enhance reproductive

efficiently as this protocol caused precised ovulations (Pursley et al., 1995).

The series of injections, synchronized ovulation for timed AI with

uncompromised pregnancy rates (Pursley et al., 1997) and this administration

7

of GnRH 6 days prior to PGF2 treatment improved the precision of estrus as

reported by Pursley et al., (1995). Thus, Ovsynch is an effective planned

breeding program developed for lactating dairy cows which allowed TAI

without estrus detection (Burke et al., 1996; Pursley et al., 1997a, 1996 and

1995 and Schmitt et al., 1996).

Geary et al., (1998) reported that Ovsynch protocol was capable of

inducing fertile ovulation in anoestrusand cycling beef cows. In addition,

Rabiee et al. (2005) and Kacar et al. (2008)also have recommended that

Ovsynch with TAI as one of the advanced reproductive tool for

improvementof reproduction in lactating dairy cows.

Ovarian follicles in cattle grow in distinct wave-like patterns with

generally one follicle becoming dominant (Fortune et al., 1988). Estrogens

and GnRH induce atresia or ovulation of dominant follicles and synchronize

the emergence of a new follicular wave (Macmillan and Thatcher, 1991; Bo et

al., 1994; Wolfenson et al., 1994 and Burke et al., 1999).

Controlling follicular waves with a single injection of GnRH at random

stages of the estrous cycle induces release of LH leading to ovulation or

luteinization of the dominant follicle(s) (≥10 mm) (Garverick et al., 1980; Bao

and Garverick, 1998 and Sartori et al., 2001). GnRH has synchronously

terminated most stages of ovarian follicle development to be followed by a

synchronized emergence of a new follicular wave leading to the presence of a

maturing dominant follicle 7 days after the injection (Twagiramungu et al.,

1992 and 1995). Further studies of Silcox et al.(1995) revealed that first

8

GnRH injection in Ovsynch induced a new follicular wave with a dominant

follicle that ovulated in response to the second GnRH injection given on

day 9.

Luteal tissue that formed after the GnRH administration was capable of

undergoing PGF2induced luteolysis 6 or 7 days later (Twagiramungu et al.,

1995). Follicular waves are controlled with an injection of GnRH followed 7 d

later by an injection of PGF2 to lyse the CL. After PGF2 a second injection

of GnRH was administered 48 h later to initiate ovulation of a dominant

follicle followed by TAI after 16 h (Lamb et al., 2010).

Vasconcelos et al. (1999) and Navanukraw et al. (2004) reported that

the ability of GnRH-PGF2based protocols to precisely synchronize estrus

and / or ovulation was dependent upon the stage of the follicle at the time of

the initial GnRH injection and is the major limitation in synchronization

programs.

Bello et al. (2006) observed that the ovulatory response after the first

GnRH administration was a critical factor for successful synchronization of

ovulation in Ovsynch protocol and also stated that high ovulation rate after the

first GnRH treatment increased the likelihood that cows will have a functional

dominant follicle capable of ovulation during the final GnRH treatment.

Previous studies have shown that response to first GnRH in Ovsynch protocol

was 45-90% and the synchronization rate was 90.1% (Pursley et al., 1995;

Vasconceles et al., 1999; Bello et al., 2006; Galvao and Santos 2010 and

Keskin et al., 2010). Atkins et al. (2008) had linked the size of the dominant

9

follicle at the time of the second GnRH with the ability of the first GnRH to

reset follicular development at appropriate stage of the estrous cycle.

Thatcher et al. (1993) reported that GnRH-induced turnover of follicles

or induction of a new follicular wave will be most efficient if ovulation was

induced in response to the first administration of GnRH and resetting

follicular development which could produce a new dominant follicle

containing an oocyte of greater fertility (Mihm et al., 1994). The cows

ovulating after the first GnRH treatment in Ovsynch have higher rates of

pregnancy than those of non responders (Bello et al., 2006 and Cirit et al.,

2007).

Perry et al. (2005) reported that follicles of smaller than normal

diameter ovulated and resulted in decreased fertility and correlated that these

females might have had decreased preovulatory concentrations of

estradiol.Lamb et al. (2010) also recorded similar findings when the interval

between GnRH and PGF2 administration was 7 days.

Vasconcelos et al. (1999) reported that the overall synchronization rate

in Ovsynch protocol was 85% and there were clear differences in response

according to the day of protocol initiation. They have also recorded that 64%

of cows ovulated to the first GnRH and 93% of the cows showed low

progesterone at second GnRH after PGF2 administration.

The stage of the estrous cycle when Ovsynch was initiated also

affected synchronization and conception rate. Initiation of Ovsynch protocol

in cows during the early cycle had higher synchronization rates but lower

10

pregnancy rates per AI possibly due to presence of large follicles than in cows

during the mid cycle which had the smallest ovulatory follicle and the greatest

pregnancy rate per AI(Vasconcelos et al., 1999 and Aali et al., 2008).

Moreira et al. (2000a) reported that initiating the Ovsynch protocol in

dairy heifers on days 15 of the estrous cycle exhibited estrus before the second

GnRH injection whereas no estrus behavior was observed when the protocol

was initiated on d 2, 5, 10 (or) 18 of the estrous cycle.

Pursley et al. (1995) recorded that ovulations occurred 24-32 h after the

second GnRH treatment in cattle. In subsequent studies,Pursley et al. (1998)

reported that conception rate was reduced, if AI was performed at 32 h after

the second GnRH treatment in the Ovsynch protocol or just after ovulation

compared with AI done at 8-24 h after detected estrus in dairy cows.

Momicilovic etal.(1998) observed that better reproductive performance was

observed in cows inseminated at synchronized ovulation rather than in cows

inseminated at synchronized estrus period.Geary et al. (2001) reported greater

conception rates with Ovsynch in cyclical cows when compared to anoestrus

cows. In another study Peters and Pursley (2003) reported that administration

of second GnRH in Ovsynch protocol at 0 h or within 24 h after PGF2

resulted in poor fertility compared to its injection at 36 or 48 h after PGF2.

Dejarnette (2004) reported that though Ovsynch allowed acceptable

pregnancy rates with no heat detection, it does not eliminate the need for heat

detection and also opined that natural heats can occur on any given day and as

many as 20% of cows will display standing estrus between days 6 and 9 of the

11

Ovsynch protocol and conception rates in these animals will be compromised

if bred strictly on a timed AI basis.The disadvantage of this protocol is that

approximately 5 to 15% of lactating cows exhibit estrus before or immediately

after the PGF2 injection (Kojima et al., 2000 and Lamb et al., 2001). These

cows might have been in the late stages of the estrous cycle when the

synchronization protocol was initiated (Geary et al., 2000).

Moreira et al. (2000b) observed incomplete luteal regression rate (21.0

%) 48 h after injection of PGF2which resulted in decreased pregnancy after

TAI (Burke et al., 1996, Peters et al., 1999 and Moreira et al., 2000).Kim et

al. (2003) also reported that 12.9% of the cows exhibited premature estrus

prior to injection of PGF2 in Ovsynch protocol andbased on serum

progesterone concentrations (48 h after the injection of PGF2)approximately

25.8 % of the cows in Ovsynch group had incomplete luteal regression.

2.1.2 Ovsynch with PRID protocol

The basic Ovsynch protocol, with numerous variations have been

tested and developed where they have become standard components in current

breeding management has TAI to avoid practical difficulties associated with

detection of estrus.

Failure of response to the initial GnRH injection in the Ovsynch

protocol has resulted in lowered TAI pregnancy rates due to asynchronous

ovarian responses (Vasconcelos et al., 1999; Dejarnette et al., 2001 and Bello

et al., 2006) and inability of the GnRH to turn over dominant follicles late in

the estrous cycle leading to premature estrus in 8-10 % of treated cows (Geary

12

et al., 2000), 5–15% of treated cows ( Roy and Twagiramungu 1996 and1999;

Seguin, 1997 and Dejarnette et al., 2001) and in 11-14% of treated cows

(Gabor et al., 2002). In Ovsynch protocol after the injection of PGF2on the

day 7 at times resulted inpremature estrus, incomplete luteal regression

associated with conception failure (Burke et al., 1996).

Similarly, Moreira et al. (2000a) demonstrated that the CL formed by

the first injection of GnRH had not regressed completely after the PGF2

injection. Therefore modifications have been employed to improve the

Ovsynch protocol (Lamb et al., 2001 and Martinez et al., 2000).

Alternatively, inclusion of an exogenous progesterone device such as

CIDR/PRID inserts between GnRH and PGF2 injection prevented

premature estrus with increased response to estrus synchronization in cyclical

as well as anoestrus cows (Kojima et al., 2000) with enhanced the pregnancy

rate (Martinez et al., 2002 and Steckler et al., 2002).Accordingly, Thatcher et

al. (1989) observed that buserelin administration at the time of CIDR insertion

causedovulation or partial luteinization of follicles and 3 to 4 days later a new

wave of follicles were recruited.Ryan et al. (1995) reported that intravaginal

insertion of a progestagen for 8 d, with buserelin treatment on the day of

progestagen insertion and PGF2 treatment on the day before progestagen

removal resulted in the best overall estrus detection rate and pregnancy

rate.Intravaginal progesterone insert (CIDR) during an Ovsynch protocol

enhanced the fertility rate in noncycling cows (Pursley et al., 2001).

13

Kim et al. (2003) have also tried insertion of CIDR progesterone

device combined with a capsule containing 10 mg estradiol benzoate as an

alternative to administration of the first GnRH treatment in the Ovsynch

protocol and stated that progesterone based TAI protocol would prevent

premature estrus and incomplete luteal regression with increased pregnancy

rate.

Savio et al. (1993) and Diskin et al. (2002) have stated that the size and

maturity of the follicle at the time of PGF2 treatment was an influencing

factor in synchrony of estrus and ovulation.Utt et al. (2003) concluded that

removal of CIDR after 9 days resulted in a larger dominant follicle compared

to removal after 7 days.

In the studies of Lane et al. (2001); Utt et al. (2003);Cavalieri et al.

(2004) and Hittinger et al. (2004) the diameter of the ovulatory follicle,

synchrony of the estrus, synchrony of ovulation and diameter of the corpus

luteum post ovulation were not influenced by the duration of the

progesterone-releasing insert (5, 7, 8, or 9 days).

Kawate et al.(2004) conducted experiments in suckled Japanese Black

cows were in control group were given GnRH (100µg) on day zero, PGF2α

(500µg) on day 7, GnRH on day 9 with AI on day 10 and cows of treatment

group were given CIDR for 7 days along with standard Ovsynch protocol.

Plasma progesterone concentrations were determined on day 0, 1, 7, 9, 10 and

17. The conception rate was greater in Ovsynch+CIDR group (72.5%) when

compared to Ovsynch group (47.7%).

14

Kim et al. (2005) reported that the treatment with GnRH in a CIDR

based FTAI protocol has induced synchronized follicular wave emergence

with a large preovulatory follicle and synchronous ovulation following a

second injection of GnRH with acceptable pregnancy rate in lactating Holstein

cows.

Amongst Ovsynch and Ovsynch+CIDR protocols,Ovsynch+CIDR

protocol had more conception rate of 67.7% when compared to Ovsynch

alone. Inclusion of CIDR in Ovsynch protocol prevented early maturation of

follicles by maintaining elevated blood progesterone concentrations until

PGF2α was administered (Sakase et al., 2005).

Kim et al. (2005) evaluated the efficacy of three protocols using

CIDR+P+G, G+CIDR+P+G and EB+CIDR+P+G for estrus synchronization

in lactating Holstein cows. They concluded that G+CIDR+P+G protocol

resulted in synchronous follicular wave emergence, large preovulatory

follicles and synchronous ovulation with higher pregnancy rates to TAI.

Stevenson et al.(2006) conducted studies with CIDR insert during the

Ovsynch protocol and recorded increased fertility in lactating cows having

low serum progesterone before PGF2α injection and concluded

thatprogesterone supplementation during an Ovsynch protocol would enhance

fertility in lactating dairy cows. They further opined that supplementation of

progesterone, pretreatment cycling status, and luteal status before PGF2α

injection altered the follicular diameters at the time of the second GnRH

injection, but were unrelated to pregnancy outcomes.

15

Sakase et al. (2007) demonstrated that Ovsynch+CIDR protocol

corrected premature follicular maturation and ovulation and precised the

timing of TAI in the beef cows by maintaining high blood progesterone level

until administration of PGF2.

Ambrose et al. (2008) observed that pregnancy rates (61.8%) were

higher in Holstein heifers, irrespective of season when Ovsynch protocol was

adopted in combination insertion of intravaginal progesterone releasing device

(CIDR) when compared to that of Ovsynch alone.CIDR (Progesterone

releasing device) and PGF2α program increased the occurrence of estrus,

pregnancy rates and overall conception rates were significantly high in

anoestrus non-descriptive cows than unsynchronized cattle (Vijayarajan and

Meenakshisundaram, 2013).

2.1.3 PRID with PG protocol

The retention rate of PRID was 100% and more than 80% of the

animals exhibited signs of estrus after removal of PRID in post-partum cows.

Maximum progesterone concentrations were attained within 24h of PRID

insertion which was 2.5 ng /ml and gradually decreased to 1ng/ml by day 12

the day of device removal. Conception rates to natural mating were 26% while

the same for fixed-time insemination at 60 and 84 h after PRID removal was

33% and it was concluded that use of intra vaginal progesterone device

without any hormonal supplementation yielded very low conception rates

(Rajamahendran et al., 1983).

16

In the absence of a corpus luteum, intravaginal progesterone (CIDR)

inserts consisting of a layer of silicone containing 10% progesterone by

weight (1.38g) molded over a T-shaped nylon spine acted as an artificial CL

by releasing sufficient progesterone to maintain plasma progesterone

concentrations >2 ng/ml for at least 7 d (Brown et al., 1988; Macmillan et al.,

1991; Van Cleeff et al., 1992 and Lucy et al., 2001) and an injection of

PGF2 the day before CIDR removal caused synchronization and improved

pregnancy rates (Lucy et al., 2001).While, Kyle et al. (1992) reported that

exposure to progesterone was a prerequisite to first postpartum behavioral

estrus in a large portion of cows. Anderson et al. (1996), Fike et al. (1997)

and Imwalleet al. (1998) also reported that progesterone in the

synchronization of estrus treatment regimen initiates estrous cycles in a

portion of anoestrus cows and peripubertal heifers andreduced the interval to

pregnancy.

McDougall et al.(1992) reported that progesterone pretreatment in

postpartum cows sensitized the receptors in the brain, which were then able to

respond to ovarian estrogen by eliciting the psychic phenomenon associated

with estrus.

Roche et al. (1999) reported that administration of the progestagen for

7 days before PGF2ensures that CL would regress in response to

PGF2becauseall cattle would have a CL aged about 7 days. They have also

stated that the progestagen will also delay the estrus incattle that naturally

undergo CL regression during the progestagen treatment period before

17

PGF2injection.The rationale for inclusion of the progesterone during the

period between GnRH and prostaglandin was that progesterone prevented

premature ovulation after spontaneous luteolysis during the treatment period

and a small proportion of cows whose dominant follicles failed to respond to

GnRH (Xu and Burton, 2000 and Xu et al., 2000).

Lane et al. (2000) conducted a study by decreasing the length of

treatment with PRID inserted for 7 days and injected PGF2α one day before

the device removal and observed that progesterone treatment improved the

estrus synchrony. They opined that pregnancy rates were high when short-

term progestagens were used along with a luteolytic agent when compared to

use of long-term progestagens. While, Stevenson et al. (2000) stated that

limitation of synchronization programs was the presence of either anoestrus

cows or prepubertal heifers in a breeding herd and about 60% of beef cows

may not be cycling at the initiation of a breeding season. On the other hand,

Lucy et al. (2001) have observed that administration of the CIDR for 7 days

with an injection of PGF2 on day 6 of insertion was an effective method for

estrus synchronization in cattle.

Progestagen administration accomplished two objectives: 1) it

suppressed estrus in cows and heifers that have spontaneously regressing CL

during the administration period and upon progesterone withdrawal these

cows and heifers expressed a synchronized estrus and 2) the CL in cows and

heifers that was within 1-5 d of the estrous cycle at initiation of progesterone

administration, continued to develop during the progesterone administration

18

period, so that it responded to PGF2 at the time of its administration. Thus,

all cows and heifers that are estrus/cycling are expected to have a

synchronized estrus in response to a program incorporating administration of

progesterone for 7 d with an injection of PGF2 near the end of progesterone

administration (Chenault et al., 2003).

The increased proportions of anoestrus suckled beef cows that ovulated

and formed a CL of normal lifespan after CIDR removal were 44% ( Fike et

al., 1997) and 41% for multiparous cows and 75% for primiparous cows

(Wheaton and Lamb, 2007).

Alnimer and Lubbadeh (2003) recorded an increased pregnancy rates

by adopting PRID in combination with Ovsynch protocol (65%) with TAI

compared to that of Ovsynch alone (30%) with AI at detected estrus. This

justifies the use of PRID along with Ovsynch to induce estrus, increase

pregnancy rates and decrease days open.

Chenault et al. (2003) reported that initiation of estrous cycles after

administration of CIDR inserts could have a major impact on the fertility as

measured by first service conception rate or first-service pregnancy rate at the

estrus following insert removal. Further, they have reported that retention

rates of CIDR inserts ranged from 90 to 96%, if loss rates were>5% the

producers should evaluate their insertion technique, housing and animal

density and should consider clipping the tails of the inserts after insertion into

the vagina. Since loss of a CIDR insert does not necessarily result in lack of a

synchronized estrus, those cows and heifers that have lost their inserts might

19

have a spontaneously regressed CL during the CIDR administration period

which might lead to occurrence of a pre-mature estrus instead of a

synchronized estrus during the first 3 to 4 d after the scheduled day for insert

removal.

Walsh et al. (2007) determined the occurrence and intensity of

vaginitis based on the amount of debris on the intra vaginal progesterone

device at removal. They recorded that 28% of the cows had evidence of mild

vaginitis in response to the intra vaginal device, whereas 6% of the cows had

copious debris associated with device at removal. They further reported that

the pregnancy rates were 43.8% after fixed artificial insemination when

treated with PRID and the same were 34.9% when treated with placebo intra

vaginal device. PRID has the efficacy to induce estrus in anoestrus cows and

decrease total days open. They concluded that vaginitis score did not show

any effect on the reproductive outcome.

Earlier reports cited that fertility depends on the lifespan of ovulatory

follicle and concluded that ovulatory estrus could be successfully induced by

using PRID for 15 days and bred at 48-72 h after its removal. It was observed

that short life spanned ovulatory follicle had better post-ovulation luteal

profile than long life spanned ovulatory follicles in true anoestrus buffalo

heifers (Jagir Singh et al., 2009).

Pacala et al. (2010) evaluated two protocols of Ovsynch+PRID (1.55g)

with that of Ovsynch alone where PRID was removed on day 8 and eCG was

given instead of GnRH as in Ovsynch. They opined that progestagen

20

treatments mimed the luteal phase of estrous cycle and thus recommended

their use for heat induction as they stimulated the hypothalamus-pituitary-

ovarian axis, while eCG at the end of progestagenic treatment stimulated the

terminal folliculogenesis and ovulation.

Ovarian inactivity during anoestrus was related to low level of pituitary

and gonadotrophic hormones, thus progesterone treatment in these animals

was effective for induction of estrus and also explains that

Progesterone+GnRH treatment was further more effective. Progesterone acted

as an artificial CL, while its withdrawal removedthe negative feedback on

hypothalamus and promoted estrus with follicular development (Pawar et al.,

2012).

From the later studies it was noticed that PRID-Delta (1.55g)

maintained greater circulating progesterone levels when compared to CIDR

(1.38g) where animals had come to estrus within 4 days of insertion with

potential benefits in fertility of dairy cows. The protocol included insertion of

PRID/CIDR on day 0, PGF2α (25mg) on day 6, PRID/CIDR removed on day

7 and the animals were inseminated 56 h after removal. Final cumulative

pregnancies per AI were greater in PRID-Delta group (Van Werven et al.,

2013).

Progesterone therapy along with other hormones was effective for

treatment of anoestrus cows, for better response an intravaginal progesterone

device was used along with other hormones which resulted in estrus induction

rates ranging from 80% to 100%. GnRH along with intravaginal device like

21

PRID induces ovulation if a mature follicle was present at the time of

inducing LH surge, while a single injection of GnRH was not effective in deep

anoestrus cases. For better results GnRH has been combined with drugs such

as phosphorous, estradiol and progesterone (Shams et al., 1991 and Rhodes et

al., 2003). It was further opined that PGF2α was effective between 6-16th day

of the cycle in the presence of active CL (Kumar et al., 2014).

Mozaffari et al. (2014) concluded that progesterone therapy was the

treatment of choice for inducing cyclicity in postpartum anoestrus cows with

low body condition score (<3 in1-5 score).

2.2 ESTRUS INDUCTION EFFICIENCY

2.2.1 Estrus Response

Ryan et al. (1995) reported that estrus detection rate in CIDR+PG

protocol was 85 % (8 days CIDR with PG one day before), but Chenault et al.

(2003) reported that the estrus was 58 % in cows on d 1, 2, and 3 after insert

removal following the CIDR+PG treatment.

Steckler et al. (1999) reported that estrus response was 94% with both

Ovsynch and Ovsynch +CIDR protocols.

Lucy et al. (2001) recorded that 59% of cows were in estrus within 3 d

across all locations in both cyclic and acyclic statuses in CIDR+PG protocol,

respectively.

Estrus was synchronized in only 48% of the cows treated on d 3 and in

100% of the cows when treatment began on d 9 of the estrous cycle with

progesterone treatment (Pratt et al., 1991).

22

Stevenson et al. (2004) stated that the proportion of cows that were

detected in estrus after 2nd GnRH in Ovsynch were 54% and addition of

CIDR insert had no effect on the incidence of estrus. But, Xu and Burton,

(2000) reported that estrus response was 92.2% when cows were treated with

GnRH agonist and CIDR followed by PGF2 injection on day 7 and device

removed on day 8 and 92.8% when device was removed on same day in a

total of 6 days.

Murugavel et al. (2010) observed that estrus induction in anoestrus

cows after CIDR+PG protocol was 81.82% with 63.64% ovulation rate

following induction of estrus.Cevik et al. (2010) reported that estrus response

in lactating dairy cows treated with Ovsynch and CIDR+PG protocol were

46.2 and 66.7%, respectively.

2.2.2 Duration of estrus

Sathiamoorthy et al. (2007) reported that estrus duration was 18.4±2.6

h in post partum nondescript cows in Ovsynch protocol and the same was

16.2±4.2 h in buffaloes.

Jyothi (2011)reported that the mean duration of estrus in postpartum

crossbred cows with Ovsynch,Ovsynch+CIDRand CIDR+PG protocols was

24.17±0.75 (range from 21 to 28), 23.33±1.18 h (range from 17 to 30) and

25.50±0.70 (range from 24 to 30), respectively and concluded that the

duration of estrus in CIDR+PG protocol was longest when compared with

other protocols.

23

2.2.3 Intensity of estrus

Callesen et al. (1986, 1987 and 1993) reported that animals that

belonged to weak estrus intensity group were expected to have poor oocyte

and/or follicular quality with reduced capacity for fertilization and embryonic

development.

Ravikumar et al. (2009) observed that the percent of animals showing

intense estrus was almost the similar in Ovsynch and Ovsynch+CIDR

treatments in subestrus buffaloes.

In CIDR+PG protocol the percent of anestrous cows exhibiting intense

estrus behavior were 63.64 and the percent of moderate and weak estrus

intensity was 18.18 and 18.18, respectively (Murugavel et al., 2010).

Jyothi (2011)observed that thepostpartum crossbred cows treated with

Ovsynch,Ovsynch+CIDRand CIDR+PG protocols exhibited intense estrus in

33.33 (4/12), 58.33 (7/12) and 66.67 (8/12) percent of cows and intermediate

estrus in 33.33 (4/12), 16.67 (2/12) and 33.33 (4/12) percent of cows,

respectively. Weak estrus was recorded in Ovsynch (33.33) (4/12)

andOvsynch+CIDR (25.00) (3/12) protocols.

2.3 SERUM PROGESTERONE ASSAY

Fonseca et al. (1983) showed a positive relationship between

concentration of progesterone at induction of luteolysis and conception rates.

Schmitt et al. (1996) indicated that the mean progesterone values were

in increasing trend from the day of treatment which might be due to the

formation of accessory CL by GnRH administration.

24

Pursley et al. (1997a) reported that evaluation of serum progesterone

concentration at each hormonal injection indicated that the first injection of

GnRH synchronized luteal function of lactating dairy cows but not of heifers

and CL regression occurred in 94% of cows in Ovsynch group (high

progesterone on the day of PGF2 injection and low progesterone 24 to 48 h

later). On the day of first GnRH injection, where 61.2% cows showed high

concentration of progesterone and this percentage has increased to 86% on the

day of PGF2 injection but the pregnancy rate was unaffected by the

concentration of progesterone.

Vasconcelos et al. (1999) observed that the stage of estrous cycle

affected serum progesterone concentration at first GnRH and at PGF2

treatment but not at second GnRH. Further, cows in the later estrous cycle

(days 10-12) were more likely to have low serum progesterone (<1ng/ml) at

PGF2 than those early in the cycle. Overall 20% of cows had a low

progesterone concentration before the injection of PGF2 and 93% had low

progesterone by the time of second GnRH. They have also showed that cows

having low progesterone concentrations at the time of the administration of

PGF2 in the Ovsynch protocol had significantly lower conception rate when

compared to that of those cows having high progesterone concentration.

Kawate et al. (2004) observed that plasma progesterone concentrations

were not significantly different between days 0 and 1 and significantly

increased from days 1 to 7 in Ovsynch group but the same progesterone

25

concentration were increased significantly from days 0 to 1 and did not

change significantly from days 1 to 7 in the Ovsynch+CIDR group.

Sakase et al. (2005) observed significantly higher plasma progesterone

concentrations in Ovsynch+CIDR than in Ovsynch protocol on day 7. Plasma

progesterone concentrations on day 1 were greater in the Ovsynch+CIDR

group than in the Ovsynch group and tended to be higher in the

Ovsynch+CIDR group than in the Ovsynch group on day 7 but were not

significantly different between the groups on days 9, 10, or 17 (Kawate et al.,

2004). Further, It has been suggested by Folman et al. (1990) that higher

blood progesterone concentrations during the luteal phase preceding the

insemination increased conception rates in dairy cows. It was also mentioned

that plasma progesterone concentrations in CIDRtreated cows remained

elevated for 7 days (to the time of CIDR removal), perhaps these elevated

progesterone concentrations increased the conception rate (Kawate et al.,

2004).

Stevenson et al. (2006) stated that cows having low progesterone at

PGF2 injection benefited from progesterone supplementation regardless of

pretreatment cyclic status (conception rate in Ovsynch+CIDR 36% vs

Ovsynch18%). They have also observed that the progesterone concentration in

blood samples collected just before the insert removal was 2.2±0.3 and

2.4±0.3 ng/ ml in Ovsynch and Ovsynch+CIDR, respectively. Progesterone

concentration in blood samples one hour after insert removal and before

PGF2 injection was 2.4±0.2 and 2.5±0.2 ng/ml in Ovsynch+CIDR and

26

Ovsynch groups, respectively. The serum progesterone concentration

approached basal levels in 83 to 99% cows in Ovsynch+CIDR and 79 to

100% in Ovsynch cows.

Sakase et al. (2007) reported that the plasma progesterone

concentrations decreased below 0.5ng/ml on day 5 in Ovsynch group whereas

this occurred on day 8 in Ovsynch+CIDR treated group. They have concluded

that the dominant follicle ovulated on day 10 in the Ovsynch group while it

ovulated on day 11 in Ovsynch+CIDRtreated group.

Sathiamoorthy et al. (2007) reported that the progesterone

concentrations on day 0, 6 and 9 were 3.84±0.57, 4.98±0.63 and 0.65±0.12

ng/ml, respectively in cows treated with Ovsynch protocol.

Jyothi (2011)recorded that the mean serum progesterone concentrations

on the day of treatment initiation i.e. on day 0 were 1.16±0.28 (range from

0.38 to 2.31), 1.98±0.34 (range from 0.07 to 3.08) and 2.29±0.26 (range from

1.28 to 3.28) ng/ml in Ovsynch,Ovsynch+CIDR and CIDR+PG groups,

respectively. It was also observed that the mean serum progesterone

concentration on the day of AI were lowest in Ovsynch protocol (0.95±0.05

ng/ml) (range from 0.77 to 1.17) when compared withOvsynch+CIDR

(0.96±0.17 ng/ml) (range from 0.04 to 1.46), CIDR+PG (1.00±0.16 ng/ml)

(range from 0.62 to 1.69) protocols.

27

2.4 EFFECT OF ESTRUS SYNCHRONIZATION PROTOCOLS ON

CONCEPTION RATE

2.4.1 Breeding at FTAI

Artificial inseminations impart new genetic material from progeny

tested bulls but it requires accurate estrus detection (Taponen, 2009). Lack of

an efficient and accurate method of estrus detection at field level was the

major limiting factor in the reproductive performance of lactating dairy cattle

(Mayne et al., 2002). The estrus periods in nearly half of the normal cycling

dairy cows in a herd could not be detected as reported by Rounsaville et al.

(1979) and Pankowski et al. (1995).

Larson and Ball, (1992) reported that pregnancy rates from fixed TAI

following synchronization with PGF2 were inconsistent particularly in

lactating cows as compared to heifers mainly due to changes in the time of

ovulation in relation to AI which resulted in variable estrous cycle length in

cows (Smith et al., 1998).

Pursley et al. (1995 and 1997) have developed TAI program with

Ovsynch that reduced the emphasis on detection of estrus because all cows

were inseminated at a specified time relative to hormonal injection.

Thatcher et al. (1993) reported that fixed time insemination program

could potentially revolutionize reproductive management in dairy animals

which eliminated the need of estrus detection.

28

Burke et al. (1996) and Stevenson et al. (1999) observed that the

pregnancy rates in cows receiving AI at observed estrus after GnRH–PGF2

were higher when compared to Ovsynch with FTAI program.

Chenault et al. (2003) concluded that estrus after CIDR insert removal

was distributed over a 3 to 4d interval making it unacceptable for fixed TAI.

Lamb et al. (2010) reported that TAI protocols reduced the annoying

factors associated with ovulation synchronization and AI on detected estrus

and opined that they could provide an fixed time insemination program

efficient and effective means for capturing selective genetic traits of economic

consequences. They have also stated that differences in the pasture and diet,

breed composition, body condition, postpartum interval, climate, and

geographic location would affect the success of TAI protocols. All these

hurdles have motivated the scientists to evolvenumerous estrous

synchronization protocols having TAI that suited the area specific

requirements (Borman et al., 2003 and Amer, 2008).

2.4.2 Conception Rate

The first service pregnancy rate was a valuable tool for evaluation of

fertility which at 60-70% was stated to be optimum in well managed herds

(Yildiz, 2010). Geary et al. (2001) reported that conception rates of TAI were

higher for cycling cows than for non-cycling cows. Burfening et al. (1978)

stated that breeding the heifers at 80 h after the second injection of PGF2

might be too late thus leading to reduced conception rates.

29

Hansel et al. (1961) and Zimbelman et al. (1970) reported that fertility

was considerably reduced by use of long term progestagens treatment (10 to

20 d) while short-term progestagen treatments (< 10 d) used in conjunction

with PGF2 did not reduce the pregnancy rates (Roche, 1976 and Heersche et

al., 1979).

However, Beal et al. (1988) reported that short term progestagen

treatment in cattle which was initiated later than 13d of the estrous cycle also

exhibited reduced fertility. This reduction in the fertility rate might be

associated with the ovulation of persistent follicles (Rajamahendran and

Taylor, 1991 and Schmitt et al., 1994) which might be overcome by

administering GnRH for recruitment of a new follicular wave (Macmillan and

Thatcher, 1991 and Twagiramungu et al., 1992).

Elsewhere it was reported that conception rates were 26% in natural

mating and the same at fixed time artificial insemination was 33% which was

higher than natural mating (Rajamahendran, 1991).

In CIDR+PG protocol (8 days CIDR with PG one day before) the

pregnancy rate to first AI was 46.6% (Ryan et al., 1995).In CIDR based

synchronization studies, Day and Macmillan (1996) and Kastelic et al. (2001)

proved that conception rates to fixed TAI were not different when PGF2 was

administered on Days 6 or 7 of a 7 day CIDR treatment regime. While, Xu et

al. (1996) and Ryan et al. (1995) reported a reduced conception rate to AI in

lactating dairy cows following 8d CIDR insert administration with

administration of PGF2 either 1 day or 2 day before insert removal.

30

Lucy et al. (2001) observed that beef cows and heifers were in estrus

within the first 3 d after removal of the CIDR insert with comparable

conception rate to contemporary cycling herd mates. They have also reported

46 and 71 percent pregnancy rate in anoestrus cows and heifers and 61 and 65

percent of first service conception rate in cyclic cows and heifers treated with

CIDR+PG protocol, respectively.

Many previous studies have evaluated the fertility of lactating dairy

cows following Ovsynch protocol and the fertility rates per AI varied from 27

to 39% (Pursley et al., 1997a; Pursley et al., 1997b; Pursley et al., 1998;

Burke et al., 1996; Vasconcelos et al., 1999 and Peters and Pursley, 2003).

Pursley et al. (1995) observed that when PGF2was administered 48 h

and 24 h prior to second GnRH conception rates were 55 and 46%,

respectively. They further observed that pregnancy rates varied in cows

inseminated at fixed time of 0, 8, 16, 24 and 32 h after the second injection of

GnRH in the Ovsynch protocol were 37, 40, 44, 40 and 32%, respectively.

Similarly, Fricke et al. (1998), Stevenson et al. (1996) and Klindworth et al.

(2001) reported 48.9, 41 and 35% conception rates, respectively with Ovsynch

protocol.

Burke et al. (1996) compared the effectiveness of TAI and AI at

detected estrus after Ovsynch protocol in lactating dairy cows and observed

that the conception rates were 30.5 and 29.0%, respectively.

Pursley et al. (1997a) reported that cows in the Ovsynch group that

were above 76 days postpartum had 43.4% pregnancy rate and cows that were

31

60-75 days postpartum had 26% pregnancy rate. They have also reported that

pregnancy rate was 38.9% following TAI after the Ovsynch treatment in

lactating dairy cows which was similar to that of cows bred following double

PG and inseminated 12 h after detection of estrus (37.8%).

In postpartum (43–57 days) lactating dairy cows, Momcilovic et al.

(1998) reported that conception rates with the Ovsynch protocol were 33%

compared to conception rates of only 6% in those inseminated after

spontaneous estrus.Burke et al. (1996), Britt and Gaska, (1998), Momcilovic

et al. (1998) and Stevenson et al. (1999) observed similar pregnancy rates

after TAI following with Ovsynch with that of AI after detected estrus.

Steckler et al. (1999) reported that pregnancy rates in Ovsynch and

Ovsynch+CIDR protocols at day 28 were 49 and 72%, respectively and at day

56 were 37 and 55%, respectively.

Vasconcelos et al. (1999) reported that ovulation after administration

of first GnRH was 87% while the same after second GnRH was 64% in

Ovsynch protocol.In lactating dairy cowsnon-cystic ovarian cows were given

GPG protocol with timed AI after 16 h of second GnRH injection had a

conception and pregnancy rates of 31.5% (Bartolome et al., 2000).

Whisnant et al. (2000) reported that estrus synchronization has become

a reproductive managemental tool for dairy producers where estrus

synchronization controls both the function of corpus luteum and the

development of follicles with viable oocytes which are critical for conception.

They observed that the conception and pregnancy rates from a series of

32

studies using Ovsynch protocol averaged 34.9 and 34.9%,respectively and the

same at breeding during detected estrus after PGF2α averaged 37.7% and

19.8%, respectively.

Fricke and Wiltbank (1999) and Bartolome et al. (2000) recorded that

Ovsynch followed by TAI performed 16-20 h after the second GnRH

treatment yielded 25% pregnancy rates in cows with occurrence of

synchronization of ovulation.

Xu and Burton (2000) reported that conception rate in Ovsynch+CIDR

group was 64.6% and the same was 56.5% when the device was removed one

day after PGF2 injection but the precision of estrus was less in former than

later.

Lamb et al. (2001) and Kawate et al. (2004) reported that pregnancy

rates were varied from 47.7 to 53% after the Ovsynch protocol in beef cows.

Dejarnette et al. (2001) observed that 20% of Ovsynch treated cows

were detected in estrus before 48 h after PGF2injection. High conception

rates of 49 and 72 percent have been reported by Pursley et al. (2001) and

Steckler et al. (2002) in Ovsynch and Ovsynch+CIDR treated postpartum

cows, respectively.

The pregnancy rates for a CIDRbased Ovsynch protocol in beef heifers

ranged from 55.9 to 70.6% (Martinez et al., 2002).

Kim et al. (2003) concluded that the pregnancy rate after TAI

following the inclusion of exogenous progesterone for 7 days and estrogens

on the initiation of treatment in Ovsynch protocol was higher than that after

33

TAI following the Ovsynch protocol (41.2 vs 20.6%) and opined that the

higher pregnancy rate might be due to a decreased incidence of premature

estrus and incomplete luteal regression.Pregnancy rates were recorded to be

65% in PRID+Ovsynch protocol and 30% in Ovsynch (Alnimer and

Lubbadeh, 2003).

Kawate et al. (2004) reported that the conception rate was 72.5 and

47.7% in the Ovsynch+CIDR and Ovsynch group, respectively indicating that

CIDR with the Ovsynch protocol had significantly improved the conception

rates in postpartum suckled Japanese Black beef cows.

Stevenson et al. (2004) reported that the pregnancy rate after Ovsynch

was 27% and in combination with CIDR it was 30.6%.

Kasimanickam et al. (2005) reported 23.9% of conception rate with

Ovsynch protocol in lactating dairy cows.While, Sathiamoorthy et al. (2007)

and Keskin et al. (2010) reported that pregnancy rates were 40 and 48.4%,

respectively in lactating dairy cows.

Sakase et al. (2005) reported that conception rate in Ovsynch and

Ovsynch+CIDR group was 48.6 and 67.7%, respectively in Japanese Black

beef cows.Ui-Hyug Kim et al. (2005) also reported that conception rates were

65% in GnRH + CIDR protocol.

Kasimanickam et al. (2006) reported that the pregnancy rates were

55.6% and 56.5% with Ovsynch+CIDR and in slightly modified

Ovsynch+CIDR protocol, where PGF2 administration and CIDR removal are

delayed by 12 h.

34

Stevenson et al. (2006) reported that conception rates after TAI in

Ovsynch and Ovsynch+CIDR protocols at day 28 were 40 and 50%,

respectively and at day 56 same were 33 and 38%, respectively.

Vasconcelos et al.(2006) concluded that fertility in cattle was

influenced by many factors which included cyclicity, energy balance, heat

stress, parity, level of milk production, diet and diseases.

Aali et al. (2008) reported that the cows which responded to ovulation

after Ovsynch treatment was 49% with an overall pregnancy rate of 31%.

Ambrose (2008) reported that pregnancy rates in Ovsynch+PRID were

61.8% while the same in Ovsynch was 55.6%.

Taponen, (2009) reported that the pregnancy rate was 51.5% in a

modified Ovsynch protocol in which animals without CL (20.7%) after 7 days

of initiation of protocol were rejected.

Murugavel et al. (2010) reported that first service pregnancy rate in

CIDR+PG protocol was 36.36% in anoestrus cows.

Cevik et al. (2010) reported that first and second service pregnancy rate

in lactating dairy cows treated was 76.9 and 53.3%, respectively with

Ovsynch and 100 and 71.4%, respectively with CIDR+PG protocol.

Ozturk et al. (2010) reported that pregnancy rates of cows having

small, medium, or large follicles at the day of second GnRH administration in

the Ovsynch group were 45.5, 28.1 and 5.3%, respectively showing

dramatically reduced pregnancy rates as follicle size increased particularly in

cows with follicles greater than 16 mm.

35

Yildiz, (2010) concluded that Ovsynch did not offer desired pregnancy

rate with FTAI and he had also reported that pregnancy rates for cows treated

with Ovsynch protocol, Ovsynch with post breeding infusion treatment and

control group were 25, 66.7 and 41.7%, respectively which were inseminated

after spontaneous estrus.

Pregnancy rates for Ovsynch+PRID was 39% and Ovsynch alone was

31.7% (Pacala et al., 2010).

Jyothi (2011)observed that thecows inseminated with frozen semen at

the fixed time the first service conception rate was 33.33 (4/12), 41.67 (5/12)

and 41.67 (5/12) percent in crossbred cows synchronized withOvsynch,

Ovsynch+CIDR and CIDR+PG protocols, respectively. While, the second

service conception rateswith Ovsynch,Ovsynch+CIDRand CIDR+PG groups

were16.67 (2/12), 16.67 (2/12) and 00.00 (0/12) percent, respectively.From

this study it was concluded that the overall conception rate in

Ovsynch,Ovsynch+CIDR andCIDR+PG, groups was 50.00 (6/12), 58.33

(7/12) and 41.67 (5/12) percent, respectively. It was concluded that the overall

conception rate in Ovsynch+CIDR was highest when compared to Ovsynch,

CIDR+PG. In another studyRaquel Rodrigues Costa Mello et al. (2013)

reported that pregnancy rate was 50% on using PRID alone.

CHAPTER-III

3. MATERIALS AND METHODS

The present study on “Synchronization of estrus and ovulation in

postpartum acyclic crossbred cows” was carried out at College of Veterinary

Science, Tirupati.

3.1 EXPERIMENTAL ANIMALS

Healthy crossbred cows, with 60 days postpartum interval maintained

under rural conditions were included in the present study. These animals were

fed mainly with paddy straw, dry fodders and concentrate mixture.

3.1.1 Selection of Animals

The crossbred cows were subjected to thorough clinical examination

including rectal palpation and animals with smooth inactive ovaries were

selected. These cows were given presynchronized mineral supplementation

and oral dewormer. Rectal palpations were done twice; spread apart at 12 days

was carried out to confirm acyclicity.

3.2 DESIGN OF THE STUDY

Twenty four cows were divided randomly into three uniform groups

and assigned to Group I (Ovsynch), Group II (Ovsynch+PRID) and Group III

(PRID+PG).

Cows in Group I were administered with 10µg of GnRH (Pregulate,

Virbac Animal Health India Pvt. Limited) intramuscularly on day 0, 500 µg of

cloprostenol sodium (Pregova, Virbac Animal Health India Pvt. Limited) on

day 7 and 10µg of GnRH on day 9, respectively.

36

37

Cows in Group II were administered intramuscularly with 10µg of

GnRH with simultaneous insertion of progesterone releasing intravaginal

device-PRID (TRIU-B, Progesterone Impregnated Intravaginal Device, Virbac

Animal Health India Pvt. Limited) on day 0 (Fig 1 & 2), 500 µg of

cloprostenol sodium intramuscularly at removal of PRID on day 7 and 10µg

of GnRH on day 9, respectively.

In Group III, cows were inserted with PRID and kept in situ for 7 days,

administered 500 µg of cloprostenol sodium at removal of PRID on day 7 and

10µg of GnRH on day 9, respectively. Fixed time artificial insemination (TAI)

was performed 16 to 24 hrs after the end of protocols in all the animals.

3.3 EVALUATION OF ESTRUS

The observations on estrus response, duration of estrus and intensity of

estrus were evaluated (Rao and Rao, 1981).


Blood sera were collected sterile by making use of vaccutainers from

all the animals for estimation of progesterone on day ‘0’ (day of initiation of

treatment) and at the time of AI and stored at -200C until progesterone assay.

Serum progesterone concentrations were determined by ELISA (enzyme

linked immune sorbent assay) based commercial diagnostic kit (Pathozyme

progesterone, Omega diagnostics Ltd., cotland, UK).

40

3.5 FERTILITY

Fertility in all the three groups, based on 60 day pregnancy rates by

rectal examination were determined for all the under the study (Noakes et al.,

2009). 3.6 STATISTICAL ANALYSIS

The results were tabulated and analyzed by using One-way ANOVA

and Chi-square which are standard statistical procedures adopted by SPSS

windows package.

CHAPTER-IV

4. RESULTS

The results of the study titled “Synchronization of estrus and ovulation

in postpartum acyclic crossbred cows” were presented in this chapter.


4.1.1 Estrus response

The estrus response after synchronization of postpartum acyclic

crossbred cows with Group I (Ovsynch), Group II(Ovsynch+PRID) andGroup

III(PRID+PG) protocols was 75.00 (6/8), 100 (8/8) and 87.50 (7/8) percent,

respectively (Table 1 and Fig.6). The highest estrus response is observed

inGroup IIprotocol synchronized cows followed by Group IIIand Group I.

4.1.2 Duration of estrus

The mean duration of estrus in postpartum acyclic crossbred cows with

Group I,Group IIand Group IIIprotocols was 50.62±8.39hrs (ranging from 0 to

72), 68.00±2.61 hrs (ranging from 56 to 72) and 58.63±9.36hrs (ranging from

0 to 72), respectively (Table 1 and Fig.8 ). The difference in the duration of

estrus among all groups was not significant (P>0.05) but duration of estrus in

Group II protocol was longest when compared with other protocols (Table 2).

4.1.3 Intensity of estrus

The postpartum acyclic crossbred cows treated with Group I, Group II

and Group IIIprotocols exhibited intense estrus in 25.00 (2/8), 62.50 (5/8) and

50.00 (4/8) percent of cows and intermediate estrus in 62.50 (5/8), 37.50(3/8)

and 37.50 (3/8) percent of cows, respectively (Table No. 2 and Fig. 7). None

41

42

of theacyclic crossbred cows were detected to be in weak estrus when treated

with Group II protocol, while 12.5 and 12.5 percent of cows were detected to

be in weak estrus when treated with Group I and Group III protocols,

respectively. Highest number of postpartum acyclic crossbred cows exhibited

intense estrus (62.5%) when synchronized with Group II protocol followed by

Group III (50.0%) and Group I (25.0%) protocols.

4.2 SERUM PROGESTERONEASSAY

The mean serum progesterone concentrations on treatment initiation

day i.e. on day 0 were 2.56±1.04ng/ml(ranging from 0.30 to 3.0),1.66±0.24

ng/ml (ranging from 0.7 to 2.6) and 1.43±0.32ng/ml(ranging from 0.3 to

27.4)in Group I, Group II and Group III groups respectively (Table 3).

Concentrations of the same at the time of AI in Group I,Group II and Group

III protocol was 7.60±0.87ng/ml (ranging from 3.8 to 11.4),1.86±0.28ng/ml

(ranging from 0.4 to 3.0) and 2.50±0.65ng/ml (range from 0.5 to 5.6),

respectively (Table 3 and Fig.9 and 10 ). Highest mean progesterone

concentration at AI injection was observed inGroup Ifollowed by Group

IIIand Group II.

The mean serum progesterone concentration on the day of initiation of

treatment were lowest in Group II protocol (1.66±0.24ng/ml)when compared

toGroup I(2.56±1.04ng/ml)followed byGroup III (4.67±3.25ng/ml) protocols.

The difference in the serum progesterone concentrations amongst all

the three synchronization protocols was non-significant (P>0.05), further a

non-significant difference (P>0.05) in the serum progesterone concentrations

43

recorded at day 0 and day of AI for all the three different protocols studied

(Table 3&4).

4.3 Fertility

4.3.1 First service conception rate

All the cows were inseminated with frozen semen at the specified time

of 16-24 hrs after administration of second GnRH. The first service

conception rate (%) was 50.00 (4/8),37.50 (3/8) and 50.00 (4/8) percent in

crossbred acyclic cows synchronized with Group I, Group II and Group III

protocols, respectively (Table 5). The first service conception rate was similar

in Group I and Group III protocols (50.00% each) while it was comparatively

lower in Group II protocol (37.50%).

4.3.2 Second service conception rate

Second service conception rate in Group I, Group II and Group III

groups was 00.00 (0/4), 20.00 (1/5) and 25.00 (1/4) percent, respectively

(Table5). The second service conception rate was slightly higher in Group

IIIprotocol in the present study.

4.3.3 Overall conception rate

The overall conception rate in Group I,Group II and Group III

protocols was50.00 (4/8), 50.00 (4/8) and 62.50 (5/8) percent, respectively

(Table 5 and Fig.11 ). The overall conception rate in Group III was marginally

high when compared to Group I and Group IIprotocols while the differences

were non-significant (P>0.05).

44

Table 1: Effect of synchronization on estrus induction

Groups Estrus

response %

Intensity of estrus % Duration of estrus (hrs) (Mean±SE) Weak Intermediate Intense

Group I 75.0 12.5 62.5 25.0 50.62±8.39NS

Group II 100.0 00.0 37.5 62.5 68.00±2.61NS

Group III 87.5 12.5 37.5 50.0 58.63±9.36NS

Chi-square value

2.28NS 1.09NS 1.35NS 2.35NS

NS: Non-significant

Table 2: Analysis of variance for duration of estrus

Sum of Squares

Degrees of

freedom

Mean Square F Sig

Between groups 1210.083 2 605.42 1.375NS 0.275

Within groups 9239.75 21 439.988

Total 10449.833 23

45

Fig.6: Comparison of estrus response

Fig7: Comparison of intensity of estrus

Fig8: Comparison of duration of estrus

0

20

40

60

80

100

120

Ovsynch Ovsynch + PRID

PRID + PG

Estrus response %

Ovsynch

Ovsynch + PRID

PRID + PG

0

10

20

30

40

50

60

70

ovsynch ovsynch+PRID PRID+PG

weak

intermediate

intense

0

10

20

30

40

50

60

70

80

Ovsynch Ovsynch + PRID PRID + PG

46

Table 3: Serum progesterone (Mean±SE) on day ‘0’ and on day of AI

NS: Non-significant

Table 4: Analysis of variance for means (±SE) of serum progesterone on day ‘0’ and day of AI

Day 0 Day of AI

df Mean Sum of Squares df Mean Sum of

Squares

Between Groups 2 19.130 2 68.780

Within Groups 21 31.370 21 27.333

Total 23 23

S No Groups Day 0 Day of AI

Mean±SE

1 Group I 2.56±1.04 7.60±0.87

2 Group II 1.66±0.24 1.86±0.28

3 Group III 1.43±0.32 2.50±0.65

4 F value 0.610NS 2.516NS

47

Fig 9: Comparison of mean serum progesterone concentrations (ng/ml) in various synchronization protocols on the day of initiation of treatment day ‘0’.

Fig 10: Comparison of mean serum progesterone concentrations (ng/ml) in various synchronization protocols on the day of AI.

0

1

2

3

4

5


Day of initiation of treatment ‘0’ day

0

1

2

3

4

5

6

7

8


Day of AI Mean ± SE

48

Table 5: Fertility response in crossbred acyclic cows synchronized with 3 different protocols

S No Conception rate (%) Group I Group II Group III Chi-square

value

1 First AI conception 50.00 (4/8) 37.50 (3/8) 50.00 (4/8)

2 Second AI conception 00.00 (0/4) 20.00 (1/5) 25.00 (1/4)

3 Overall conception 50.00 (4/8) 50.00 (4/8) 62.50 (5/8) 0.336NS

NS: Non-significant

Fig. 11 Comparison of conception rates

0

10

20

30

40

50

60

70

ovsynch ovsynch+PRID PRID+PG

ovsynch

ovsynch+PRID

PRID+PG

CHAPTER-V

5. DISCUSSION

In recent times, the fertility in lactating dairy cows was lower than the

desired conception rates and perusal of literature revealed that the conception

rates per AI have been decreased from 66% in 1951 to about 50% in 1975 and

at present it is about 40%. In order to enhance the lowered reproductive

efficiency scientists proposed estrus synchronization programs (Pursley et al.,

1997). Among the range of reproductive technologies available to programme

the reproductive management with the use of estrus and ovulation

synchronization and TAI protocols, three such protocols are compared in the

present study to understand the efficacy of these in inducing or synchronizing

the estrus and ovulation and enhancing the fertility among postpartum acyclic

crossbred cows.


5.1.1 Estrus Response

In the present study, estrus response in postpartum acyclic crossbred

cows after estrus synchronization was 75.00 (6/8), 100 (8/8) and 87.50 (7/8)

percent in Group I, Group II and Group III, respectively. The highest estrus

response was observed in Group II followed by Group III and Group I.

The estrus response recorded in Group II was the highest when

compared with Group I and Group III. The estrus response (87.50) observed

in Group III was similar that of Pratt et al. (1991). Whereas, Lucy et al.

(2001), Chenault et al. (2003), Murugavel et al. (2010) and Cevik et al. (2010)

49

50

recorded comparatively lowered estrus response (58 to 81.82%) in cows. The

highest estrus response attained in Group II in the present study might be

attributed to the progesterone priming sensitizing the endocrine axis to obtain

better follicular development (Anderson et al., 1996 and Noakes et al., 2009).

Estrus response observed in Group II was 100%. When the estrus

response was compared between Group I and Group II (75% vs 87.5%) there

was no significant difference between the responses, whereas no such

difference was noticed by Stevenson et al. (2004) with Ovsynch+CIDR

(54%). This difference in the estrus response might be due to the inclusion of

exogenous progesterone during the interval between GnRH and PGF2

administration which might have prevented premature estrus and thus had

increased the estrus response (Steckler et al., 2002). In addition induction of

luteolysis with PGF2 on day 7 of the treatment might have caused an

increase in the plasma concentration of estradiol leading to the enhanced

response (Taponen et al., 1999).

Similar percentage of estrus response (75%) in Group I in the present

study was in agreement with the findings of Stevenson et al. (2004) and Cevik

et al. (2010) who recorded 54 and 46.2%, respectively. While, Steckler et al.

(1999) reported higher estrus response (94%) with Ovsynch protocol as

compared to the findings in the present study. This lowered estrus response in

Group I might be due to presence of a small, less estrogenic dominant follicle

(Brantmeier et al., 1987) and also the stage of estrous cycle at the time of

initiation of the protocol in Ovsynch treated cows (Vasconcelos et al., 1999).

Further, during Ovsynch protocol peak concentrations of estrogen secreted by

51

the preovulatory follicle might have prematurely abrogated LH surge induced

by the second GnRH injection (Stevenson et al., 2004). The lowered response

in the present study might be attributed to the nutritional status of animals,

scarcity of fodder during summer, seasonal, breed and locational differences

which might have adversely affected the follicular growth and ovulation

(Dejarnette, 2004).

5.1.2 Duration of Estrus

The mean duration of estrus in this study in postpartum crossbred cows

with Group I, Group II and Group III was 50.62±8.39 hrs (0 to 72),

68.00±2.62 hrs (56 to 72) and 58.63±9.36 hrs (0 to 72), respectively with no

statistical significance between groups.

However, the mean duration of estrus in Group II (68.00±2.62 hrs) was

longer when compared to Group I and Group III in the present study and was

in line with Sathiamoorthy et al. (2007). Whereas, Jyothi (2011) reported

lesser duration with Ovsynch+CIDR when compared to the present study.

The mean duration of estrus 68.00±2.62 hrs in Group II in the present

study was longer (16.2±4.2 and 17.61±0.36 hrs) when compared to the reports

of Kim et al. (2005) and Sakase et al. (2005), respectively. The duration of

estrus in Group III was 58.63±9.36 hrs which was more or less similar to

Group I in the present study.

5.1.3 Intensity of Estrus

During the period of induced estrus all the cows have exhibited the

estrous symptoms but the intensity of estrus symptoms were at varying levels.

The percentage of crossbred cows those exhibited intense, intermediate and

52

weak estrus after synchronization were 12.5, 62.5 and 25.0; 00.0, 37.5 and

62.5 and 12.5, 37.5 and 50.0 respectively in Group I, Group II and Group III

in the present study.

The intense estrus (62.5%) observed in Group II was comparable with

that of Alnimer and Lubbadeh (2003) and Ravikumar et al. (2009). In

contrast, less intensity of estrus was recorded by Fike et al. (1997) in

Ovsynch+PRID protocol. The variations in the intensity of estrus recorded in

the present study, amongst the different protocols might be due to variations

in the methodology of estrus score, parity, breed and environment. To

conclude better estrus characteristics were observed in postpartum acyclic

cows in Group II might be due to the fact that progesterone pretreatment in

postpartum cows sensitized the receptors in the brain, which were then able to

respond to the ovarian estrogen by eliciting the psychic phenomenon

associated with estrus (McDougall et al., 1992).


The mean serum progesterone concentrations (ng/ml) on day 0 were

2.56±1.04 (0.30 to 7.6), 1.66±0.24 (0.7 to 2.6) and 4.67±3.25 (0.3 to 27.4) in

Group I, Group II and Group III, respectively while the same at the time of AI

were 7.60±0.87 (3.8 to 11.4), 1.86±0.28 (0.4 to 3.0) and 5.51±3.06 (0.5 to

26.6) in Group I, Group II and Group III, respectively. The difference in the

serum progesterone concentrations among protocols and among different days

of collection was non-significant.

53

In the present study no significant difference (P>0.05) was observed in

mean serum progesterone concentration between day 0 and on the day of AI.

On the contrary, Prakash et al. (1995) and Dugwekar et al. (2003) reported

lower serum progesterone values (0.58±0.14 ng/ml) at the time of AI when

compared to those recorded in the present study.

While, Kim et al. (2003) in Ovsynch protocol reported lower mean

serum progesterone concentration (0.96±0.1 ng/ml) on day of AI than the

present study. Similary, Deshmukh et al. (2010) recorded lower levels of

progesterone on day of AI with a mean value of 1.1±0.2 ng/ml which was less

than those recorded in the present study (7.60±0.87 ng/ml) at AI.

In parallel to the present study with Ovsynch, Vasconcelos et al. (1999)

reported a fall in progesterone concentration on day 9 (on the day of AI) with

concentrations 0.50±0.1, 0.40±0.1, 0.20±0 and 0.40±0.1 ng/ml on day 1-4,

day 5-9, day 10-16 and day 17-21, respectively which were less than those

recorded in the present study (7.60±0.87 ng/ml) at the time of AI.

In contrast, Kawate et al. (2004) observed a significant difference in

plasma progesterone concentration between the concentrations on treatment

initiation day and on day of administration of PGF2α. Sathiamoorthy et al.

(2007) also reported the progesterone concentration on day 0 and 9 as

3.84±0.57 and 0.65±0.12 ng/ml, respectively in Ovsynch protocol. Stevenson

et al., (2006) also reported basal concentration of progesterone in 79-100% of

Ovsynch treated cows.

54

In Group I, Group II and Group III no significant difference in the

concentrations of progesterone was observed on day ‘0’ and on the day of AI

which were similar in findings with Kawate et al. (2004). Apparently higher

serum progesterone concentrations on the day of AI were observed in Group I

might have caused ovulatory aberrations leading to reduced conception rate.

5.3 FERTILITY RESPONSE

In the present investigation, the first service conception rate in

postpartum crossbred cows synchronized in Group I, Group II and Group III

was 50.00, 37.50 and 50.00 percent, respectively. Similarly, same protocols

have resulted overall conception rate of 50.00, 50.00 and 62.50 percent,

respectively in postpartum acyclic crossbred cows with no significant

difference between groups.

The first service conception rate and overall conception rate (62.5%) in

postpartum crossbred cows in Group III was highest in the present study was

in support with the findings of Lucy et al. (2001) and Cevik et al. (2010). In

contrast, Murugavel et al. (2010) and Stevenson et al. (2004) reported lower

conception rates of 36.36 and 30.6% with CIDR+PG, respectively in cows

compared with those observed in the present study might be due to

administration of progesterone during late luteal phase of the estrous cycle

might have caused development of persistent dominant follicles in the absence

of a functional CL and subsequent ovulation of aged and unhealthy oocytes in

some of the cows (Ahmad et al., 1995; Revah and Butler, 1996; Roche et al.,

1999 and Lucy et al., 2001) and resulted in a poor quality embryos and

55

embryonic deaths with a reduction in a conception rate at synchronized estrus

(Mihm et al., 1994; Ahmad et al., 1995 and Chenault et al., 2003).

While, the higher conception rates in cows synchronized in Group III

(62.5%) than the Group II and Group I protocols observed in the present study

might be due to reasons that ovarian acyclicity during anoestrus was related to

low level of pituitary and gonadotrophic hormones and thus progesterone

treatment in these animals might be effective for induction of estrus and also

justified that Progesterone+GnRH treatment was further more effective as

progesterone would act as an artificial CL, while its withdrawal removed the

negative feedback on hypothalamus and promoted estrus with follicular

development leading to ovulation and thus conception on fixed time AI

(Anderson et al., 1996; Imwalle et al., 1998 and Pawar et al., 2012).

Ovsynch protocol in the present study has resulted in first service

conception rates as 50.00 percent and none of the remaining animals

conceived on second AI. The overall conception rate with Ovsynch (%) in

postpartum crossbred cows in the present study is in agreement with the

findings (47.7 to 53%) of Geary et al. (2001), Lamb et al. (2001), and

Klindworth et al., (2001), Kawate et al. (2004), Taponen (2009) and Keskin et

al. (2010), However, Pursley et al. (1995), Pursley et al. (1997), Burke et al.

(1996), Stevenson et al. (1996), Momcilovic et al. (1998), Fricke and

Wiltbank (1999), Bartolome et al. (2000), Kim et al. (2003), Kasimanikam et

al. (2005), Stevenson et al. (2006), Sathiamoorthy et al. (2007), Aali et al.

(2008) and Yildiz (2010) observed lower conception rate (20.6 to 41%) in

56

cows than the present study. This higher conception rates obtained with

Ovsynch might be due to reason that the first GnRH might have initiated the

growth of a new wave of follicles (Macmillan and Thatcher, 1991 and

Twagiramungu et al., 1994) followed by emergence and selection of a

dominant follicle that becomes preovulatory within 9 days of emergence and

the most fertile ova (Savio et al., 1993).

These variations observed by various authors in the conception rates

with Ovsynch protocol might be due to the difference in response to the first

GnRH administration and also cyclicity status of the animal at the time of

initiation of the treatment (Thatcher et al., 2002 and Bello et al., 2006). The

estrus stage at the time of first injection of GnRH in the Ovsynch protocol is

particularly critical because the proportion of follicles ovulating in response to

the first GnRH injection and the synchrony of ovulation following the second

GnRH injection may affect the pregnancy rate (Vasconcelos et al., 1999).

Premature follicular maturation before 2nd GnRH, ovulation before TAI

(Moreira et al., 2000a) and inability of 1st GnRH to turn over the dominant

follicle late in the estrous cycle leading to premature estrus (Geary et al.,

2000) might also have decreased the conception rate. Further, the dominant

follicle present at the time of second GnRH treatment might have failed to

express LH receptors leading to failure of ovulation suggesting that GnRH did

not induce ovulation due to asynchronous wave emergence and/or a small

dominant follicle without LH receptors (Colazu et al., 2004). Further, the

second GnRH which resulted in ovulation and the subsequent CL formed

57

might have produced lesser progesterone leading embryonic mortality

resulting in reduced conception rate (Moreira et al., 2000a). Lowered

conception rate observed in Ovsynch protocol along with higher levels of

serum progesterone at the time of AI might be suggestive of incomplete luteal

regression following the injection of PGF2 (Burke et al., 1996; Peters et al.,

1999; Moreira et al., 2000; Lamb et al., 2001and Kim et al., 2003).

To correct the above premature follicular maturation and ovulatory

problems supplementation of progesterone through PRID was attempted to

enhance the blood progesterone concentrations until PGF2 administration

and conception rate. Group II cows, in the present study the first service,

second service and overall conception rates in postpartum crossbred cows

were 37.00, 20.00 and 50.00 percent, respectively. These findings gained

support from the reports of Steckler et al. (1999) and Kasimanikam et al.

(2006) who have recorded conception rates of 55.0 and 55.6%, respectively.

However, Xu and Burton (2000), Pursley et al. (2001), Steckler et al. (2002),

Kawate et al. (2004) and Sakase et al. (2005) observed higher conception

rates (64.6 to 72.5%) than those recorded in the present study in crossbred

cows. In contrast, Kim et al. (2003) and Stevenson et al. (2006) reported

lower conception rates (41.2 and 38%).

The improved and higher conception rate observed in this study in

Group III when compared with previous studies might be due to the

synchrony between luteolysis, ovulation and TAI and the supplementation of

progesterone through PRID (Steckler et al., 2002 and Stevenson et al., 2006).

58

Further, the low progesterone concentration recorded in this study at the time

of AI expect in Ovsynch group might have caused marginal enhancement in

the conception rate (Lamb et al., 2001). The enhanced conception rates with

PRID+PG protocol might be due to increased concentrations of estradiol-17

beta in cows treated previously with progesterone that caused release of more

LH after GnRH resulting in increased ovulatory response and in turn

conception rates (Thompson et al., 1999). In addition, supplementation of

progesterone with GnRH would reduce the dominant follicle size at the time

of PGF2 treatment and improve the fertility, apparently due to the reduced

incidence of persistence of follicle (Martinez et al., 2001). The highest

conception rates in the present study also might be due to creation of a

congenial environment in the uterus since progesterone concentrations during

one estrous cycle affects the endometrial morphology in the subsequent cycle

and maintains the pregnancy effectively (Albalancy et al., 1997). Further,

Pacala et al. (2010) opined that progestagen treatment mimed the luteal phase

of the estrous cycle and thus recommended their use for heat induction as they

stimulated the hypothalamus-pituitary-ovarian axis.

It is concluded from the present study that supplementation of

exogenous progestagens like PRID to Ovsynch protocol had helped the cows

to exhibit better estrus parameters due to progesterone priming. Further, it was

opined that PRID+PG (Group III) protocol yielded apparently higher

conception rates compared to Ovsynch+PRID (Group II) and Ovsynch (Group

I) protocols. Under field conditions in the treatment of postpartum acyclic

59

crossbred cows estrus parameters had little significance when compared to

achieving optimum conception rate with a cost effective protocol like

PRID+PG (Group III) which required one dose less of GnRH hormone which

is economical to the farmers. However, systematic studies on a large sample

of cows are warranted before drawing concrete conclusions.

CHAPTER-V1

6. SUMMARY

The present study was done on 24 healthy postpartum acyclic crossbred

cows maintained under diverse managemental conditions to study the efficacy

of different synchronization protocols viz., Ovsynch (Group I),

Ovsynch+PRID (Group II) and PRID+PG (Group III) based on estrus

response, duration of estrus, intensity of estrus, serum progesterone

concentration and conception rate.

The estrus response in Group I, Group II and Group III was was 75.00

(6/8), 100 (8/8) and 87.50 (7/8) percent, respectively. The highest estrus

response is observed in Group II synchronized cows followed by Group III

and Group I.

The mean duration of estrus in postpartum acyclic crossbred cows in

Group I, Group II and Group III was 50.62±8.39 hrs, 68.00±2.61 hrs and

58.63±9.36 hrs, respectively. The difference in the duration of estrus among

all groups was not significant (P>0.05) but duration of estrus in Group II was

longest when compared with other protocols.

The postpartum acyclic crossbred cows treated in Group I, Group II

and Group III was exhibited intense estrus in 25.00, 62.50 and 50.00 percent

of cows and intermediate estrus in 62.50, 37.50 and 37.50 percent of cows,

respectively. None of the acyclic crossbred cows were detected to be in weak

estrus when treated with Ovsynch+PRID protocol, while 12.5 and 12.5

percent of cows were detected to be in weak estrus when treated with

60

61

Ovsynch and PRID+PG protocols, respectively. Highest number of

postpartum acyclic crossbred cows exhibited intense estrus (62.5%) when

synchronized with Ovsynch+PRID protocol followed by PRID+PG (50.0%)

and Ovsynch (25.0%) protocols.

The mean serum progesterone concentrations on treatment initiation

day i.e. on day 0 were 2.56±1.04 ng/ml, 1.66±0.24 ng/ml and 1.43±0.32 ng/ml

in in Group I, Group II and Group III, respectively. Concentrations of the

same at the time of AI were 7.60±0.87 ng/ml, 1.86±0.28 ng/ml and 2.50±0.65

ng/ml, respectively. Highest mean progesterone concentration at AI injection

was observed in Ovsynch followed by PRID+PG and Ovsynch+PRID.

The mean serum progesterone concentration on the day of initiation of

treatment were lowest in Ovsynch+PRID protocol (1.66±0.24 ng/ml) when

compared to Ovsynch (2.56±1.04 ng/ml) followed by PRID+PG (4.67±3.25

ng/ml) protocols.

The difference in the serum progesterone concentrations amongst all

the three synchronization protocols was non-significant (P>0.05), further a

non-significant difference (P>0.05) in the serum progesterone concentrations

recorded at day 0 and day of AI for all the three different protocols studied.

All the cows were inseminated with frozen semen at the specified time

of 16-24 hrs after administration of second GnRH. The first service

conception rate (%) was 50.00, 37.50 and 50.00 percent in crossbred acyclic

cows synchronized in Group I, Group II and Group III, respectively. The first

service conception rate was similar in Group I (Ovsynch) and Group III

62

(PRID+PG) (50.00% each) while it was comparatively lower in

Ovsynch+PRID protocol (37.50%). Second service conception rate in

Ovsynch, Ovsynch+PRID and PRID+PG groups was 00.00, 20.00 and 25.00

percent, respectively. The second service conception rate was slightly higher

in PRID+PG protocol in the present study. The overall conception rate in

Ovsynch, Ovsynch+PRID and PRID+PG protocols was 50.00, 50.00 and

62.50 percent, respectively.

From the present study it was concluded that Ovsynch+PRID protocol

was effective with regards to estrus parameters while the overall conception

rate in PRID+PG was marginally high when compared to Ovsynch and

Ovsynch+PRID protocols. However, the present study was targeted to achieve

synchronized estrus in postpartum acyclic cows with normal conception rate

by adopting fixed time artificial insemination. Under field conditions in the

treatment of postpartum acyclic crossbred cows estrus parameters had little

significance when compared to achieving optimum conception rate with a cost

effective protocol like PRID+PG (Group III) which required one dose less of

GnRH hormone which is economical to the farmers.

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synchronization of estrus and ovulation …...viii acknowledgments i thank the god almighty for...

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