STRATEGIES TO IMPROVE REPRODUCTIVE EFFICIENCY IN BEEF FEMALES AND

ENHANCE OFFSPRING POSTNATAL PERFORMANCE

By

VITOR RODRIGUES GOMES MERCADANTE

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2015

© 2015 Vitor Rodrigues Gomes Mercadante

To my wife Paula

4

ACKNOWLEDGMENTS

I am deeply thankful to Dr. G. Cliff Lamb for allowing me the opportunity to come to the

University of Florida. I joined his program first as an intern, then as a Master’s student, and

finally as a PhD candidate. Dr. Lamb sets an example as an animal scientist for his commitment

with quality research, scholarship in extension, and mentoring abilities that go beyond

professional and scientific training. He will always be a role model to me.

I would also like to thank the members of my committee. Dr. Nicolas DiLorenzo, who

has been deeply involved with my graduate training since the beginning, he helped develop my

interest for beef cattle nutrition, and became a dear friend. Dr. Owen Rae, who has helped to

keep my veterinarian training alive through the years. I am thankful for his passion for teaching,

he sets an example for teaching effectively. In addition, Dr. Geoff Dahl, for being always

available to discuss science, and share professional experiences and advices. I deeply admire his

commitment to excellence and ability to be scientifically productive.

My appreciation is extended to Dr. John Arthington, Dr. João Vendramini, Dr. William

Thatcher, Dr. José Dubeux, Dr. Peter Hansen, Dr. Alvin Warnick, Dr. Philipe Moriel, Dr. Alan

Ealy, Dr. Sally Johnson, Dr. Reinaldo Cooke, Dr. Ronaldo Cerri, Dr. Jamie Larson, Dr. Carl

Dahlen, and Dr. Jeff Stevenson for scientific collaboration, guidance and friendship.

My professional training began during vet school in Brazil, and it was while working

under Dr. Vasconcelos “Zequinha” at Conapec Jr. that I developed a passion for research and

extension. It was through Dr. Vasconcelos and Conapec Jr. that I had the opportunity to interact

with Dr. Lamb, and for that I will always be grateful. Zequinha’s ability and passion to shape

young professionals and nourish talents will always inspire me to be a better teacher and adviser.

Graduate school is a lot more fun with lab mates, and I have had the pleasure to work

with some amazing and talented fellow students from our lab and many others, in addition to the

5

many friends I made along the way. People that became like family to me. My special thanks go

to André Aguiar, Anna Denicol, Bianca Martins, Daniel Taylor, Darren D. Henry, Eduardo S.

Ribeiro, Erick Santos, Fábio S. Lima, Francine M. Ciriaco, Gláucio Lopes, Gleise Medeiros,

Guilherme Marquezini, João Bittar, Kalyn Waters, Leandro Greco, Luiz Felipe Ferraretto,

Marcelo Wallau, Marta Kohmann, Natalia Martinez Patino, Nicky Oosthuizen, Pedro Levy, Tera

and Seth Black. Many thanks go to the amazing team at the North Florida Research and

Education Center. Our amazing lab techs Don Jones, Tessa Schulmeister and Martin Ruiz-

Moreno. The incredible Tina Gwin and Gina Arnet. And the best beef cattle crew in Florida,

David Thomas, Mark, Pete, Cole, Butch and Olivia.

A special thanks to my amazing family; my parents, Guilherme e Ângela, for

unconditional love; my brothers, Rafael and João, for their friendship; my in-laws, Hans and

Waléria, and sister in-law Luíza, for unconditional support; my grandparents for inspiration; and

my wife Paula, who has been with me in this journey since the start, she is simply the best, the

love of my life.

I have been truly lucky throughout my life and career to have been always surrounded by

the best people one can imagine. Individuals that impacted my life and made me better in many

ways, and for that I will be eternally grateful.

6

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...............................................................................................................4

LIST OF TABLES ...........................................................................................................................8

LIST OF FIGURES .........................................................................................................................9

LIST OF ABBREVIATIONS ........................................................................................................11

ABSTRACT ...................................................................................................................................13

CHAPTER

1 INTRODUCTION ..................................................................................................................15

2 LITERATURE REVIEW .......................................................................................................19

Bovine Estrous Cycle .............................................................................................................19

Follicle dynamics ....................................................................................................................19 Ovulation ................................................................................................................................20 Corpus luteum formation ........................................................................................................21

Luteolysis ................................................................................................................................21 Control of the Estrous Cycle...................................................................................................22

Current Estrous Synchronization Protocols for Beef Females ...............................................24

Effects of Progesterone Concentration on Fertility ................................................................25

Strategies to Alter Concentration of Progesterone in TAI Protocols .....................................26 Fertilization .............................................................................................................................28

Early Embryo Development ...................................................................................................28 Blastocyst formation ...............................................................................................................29 Gastrulation and Conceptus Elongation .................................................................................30

Maternal Recognition of Pregnancy .......................................................................................31 Fetal Development ..................................................................................................................32 Placental Development ...........................................................................................................33 Pregnancy-Associated Glycoprotein ......................................................................................34

Pregnancy Maintenance and Loss ..........................................................................................36 Early embryonic loss .......................................................................................................37

Late embryonic loss .........................................................................................................38 Fetal Loss .........................................................................................................................38

Strategies to Improve Embryo Survival in Cattle ...................................................................39 Fetal Programming .................................................................................................................41

Organ structure ................................................................................................................41

Alterations in cell number ...............................................................................................42 Clonal selection ...............................................................................................................42 Epigenetics ......................................................................................................................43

Evidence of Fetal Programming in Ruminants .......................................................................44

7

Maternal Nutritional Status and Fetal Programming ..............................................................45

Strategies to Induce Fetal Programming in Ruminants ..........................................................46

3 EFFECTS OF ADMINISTRATION OF PROSTAGLANDIN F2α AT INITIATION OF

THE 7-d CO-SYNCH+CIDR OVULATION SYNCHRONIZATION PROTOCOL

FOR SUCKLED BEEF COWS AND REPLACEMENT BEEF HEIFERS ..........................51

Introduction .............................................................................................................................51 Materials and Methods ...........................................................................................................52

Animals and Treatments ..................................................................................................52 Blood Collection and Radioimmunoassay ......................................................................53

Ultrasonography ..............................................................................................................54 Statistical Analyses ..........................................................................................................55

Results.....................................................................................................................................56 Discussion ...............................................................................................................................59 Conclusion ..............................................................................................................................63

4 EFFECTS OF RECOMBINANT BOVINE SOMATOTROPIN ADMINISTRATION

AT BREEDING ON THE COW, CONCEPTUS AND SUBSEQUENT OFFSPRING

PERFORMANCE OF BEEF CATTLE..................................................................................76

Introduction .............................................................................................................................76 Materials and Methods ...........................................................................................................77

Animals and Treatments ..................................................................................................77

Diagnosis of Pregnancy and Embryo/Fetal Morphometry ..............................................78 Blood Collection and Analysis ........................................................................................78

Liver Biopsies Collection and Tissue Homogenization ..................................................79 RNA Extraction and Quantitative RT-PCR ....................................................................80

Statistical Analysis ..........................................................................................................81 Results and Discussion ...........................................................................................................81

Conclusion ..............................................................................................................................87

5 EFFECTS OF RECOMBINANT BOVINE SOMATOTROPIN ADMINISTRATION

AT BREEDING ON HORMONAL CONCENTRATION, PREGNANCY RATE AND

CONCEPTUS DEVELOPMENT OF REPLACEMENT BEEF HEIFERS ..........................99

Introduction .............................................................................................................................99 Materials and Methods .........................................................................................................100

Animals and Treatments ................................................................................................100

Diagnosis of Pregnancy and Embryo/Fetal Morphometry ............................................101 Blood Collection and Analysis ......................................................................................101

Statistical Analysis ........................................................................................................102 Results and Discussion .........................................................................................................103 Conclusion ............................................................................................................................108

LIST OF REFERENCES .............................................................................................................114

BIOGRAPHICAL SKETCH .......................................................................................................127

8

LIST OF TABLES

Table page

3-1 Characteristics of suckled beef cows by location enrolled in the CO-Synch+CIDR or

PG-CO-Synch+CIDR ovulation synchronization protocols. .............................................65

3-2 Characteristics of beef heifers by location enrolled in the CO-Synch+CIDR or PG-

CO-Synch+CIDR ovulation synchronization protocols ....................................................66

3-3 Pregnancy rates to fixed-timed AI in suckled beef cows after treatment with CO-

Synch+CIDR or PG-CO-Synch+CIDR .............................................................................67

3-4 Pregnancy rates to fixed-timed AI in replacement beef heifers after treatment with

Co-Synch+CIDR or PG-CO-Synch+CIDR .......................................................................68

3-5 Factors affecting pregnancy rates to fixed-timed AI in sucked beef cows at 3

locations1. ...........................................................................................................................69

3-6 Factors affecting pregnancy rates to fixed-timed AI in replacement beef heifers .............70

4-1 Nucleotide sequence of bovine-specific primers used in the quantitative real-time

reverse transcription PCR to determine the hepatic expression of target genes ................89

4-2 Fertility, gestation, fetal development and calf performance measurements from

suckled beef cows previously treated with bST .................................................................90

4-3 Pearson’s correlation coefficient among concentration of IGF-1, PSPB and fetal

measurements2 of suckled beef cows receiving an injection of bST around the time

of breeding1. .......................................................................................................................91

5-1 Body condition score, body weight, pregnancy rate and fetal development

measurements from replacement beef heifers treated with bST in Experiment 1 ...........109

5-2 Body condition score, body weight, pregnancy rate and fetal development

measurements from replacement beef heifers treated with bST in Experiment 2 ...........110

9

LIST OF FIGURES

Figure page

2-1 Beef Reproduction Task Force chart of recommended estrous synchronization and

TAI protocols for beef cows ..............................................................................................49

2-2 Beef Reproduction Task Force chart of recommended estrous synchronization and

TAI protocols for beef heifers............................................................................................50

3-1 Schematic of treatments. ....................................................................................................71

3-2 Concentrations of progesterone on d -10, -3 and 0 relative to fixed-timed AI of

suckled beef cows by treatment, Exp. 1... ..........................................................................72

3-3 Diameter of the largest ovarian follicle present on d -10 and -3 relative to fixed-

timed AI of suckled beef cows by treatment, Exp. 1... ......................................................73

3-4 Concentrations of progesterone on d -10, -3 and 0 relative to fixed-timed AI of

replacement beef heifers by treatment, Exp. 2... ................................................................74

3-5 Diameter of the largest ovarian follicle present on d -10 and -3 relative to fixed-

timed AI of replacement beef heifers by treatment, Exp. 2.. .............................................75

4-1 Experiment outline and schematic of treatments.. .............................................................92

4-2 Concentrations of insulin-like growth factor 1 (IGF-1) of beef cows on days relative

to fixed-timed AI (TAI) by treatment. ...............................................................................93

4-3 Concentrations of progesterone (P4) of pregnant beef cows on days relative to fixed-

timed AI (TAI) by treatment. .............................................................................................94

4-4 Concentration of pregnancy-specific protein B (PSPB) of pregnant beef cows on

days relative to fixed-timed AI (TAI) by treatment ...........................................................95

4-5 Expression of 2’-5’-oligoadenylate synthetase 1 (OAS1) mRNA on d 18 and 21 of

gestation on suckled beef cows treated with bST.. ............................................................96

4-6 Expression of Myxovirus 2 (MX2) mRNA on d 18 and 21 of gestation on suckled

beef cows treated with bST. ...............................................................................................97

4-7 Expression of hepatic insulin-like growth factor 1 (IGF1), insulin-like growth factor

2 (IGF2), insulin-like growth factor 1 receptor (IGFR1), and insulin-like binding

protein 3 (IGFBP3) mRNA of calves born to suckled beef cows treated with bST. .........98

5-1 Concentration of insulin-like growth factor 1 (IGF-1) of beef heifers on days relative

to fixed-timed AI (TAI) by treatment in Experiment 1 ...................................................111

10

5-2 Concentration of progesterone (P4) of pregnant beef heifers on days relative to fixed

timed AI (TAI) by treatment in Experiment 1. ................................................................112

5-3 Concentration of pregnancy-specific protein B (PSPB) of pregnant beef heifers on

days relative to fixed-timed AI (TAI) by treatment in Experiment 1. .............................113

11

LIST OF ABBREVIATIONS

AFC Antral follicle count

AI Artificial insemination

AMH Anti-Mullerian hormone

BCS Body condition score

BRTF Beef Reproduction Task Force

bST Bovine somatotropin

BW Body weight

CIDR Controlled internal drug release

CL Corpus luteum

CNL Crown-to-nose length

CRL Crown-to-rump length

Cyp1 Cyclophilin-1

DPP Days postpartum

E2 Estradiol

FAO Food and Agriculture Organization

FSH Follicle stimulating hormone

GH Growth hormone

GnRH Gonadotropin releasing hormone

hCG Human chorionic gonadotropin

ICM Inner cell mass

IFNT Interferon-tau

IGF-1 Insulin-like growth factor 1

IGF1 Insulin-like growth factor 1 gene

IGF2 Insulin-like growth factor 2 gene

12

IGFBP3 Insulin-like growth factor binding protein 3 gene

IGFR1 Insulin-like growth factor 1 receptor gene

ISG Interferon-stimulated genes

ISG15 Interferon-stimulated gene 15

IUGR Intrauterine growth retardation

LH Luteinizing hormone

MX1 Myxovirus resistance 1

MX2 Myxovirus resistance 2

OAS1 2’,5’-oligoadenylate synthetase

P4 Progesterone

PAG Pregnancy-associated glycoprotein

PBL Peripheral blood leukocytes

PE Primitive endoderm

PGE Prostaglandin E2

PGF Prostaglandin F2α

PSPB Pregnancy-specific protein B

RPS9 Ribosomal protein S9

RTP4 Receptor transporter protein 4

TAI Fixed-time artificial insemination

US Ultrasonography

ZP Zona pellucida

13

Abstract of Dissertation Presented to the Graduate School

of the University of Florida in Partial Fulfillment of the

Requirements for the Degree of Doctor of Philosophy

STRATEGIES TO IMPROVE REPRODUCTIVE EFFICIENCY IN BEEF FEMALES AND

ENHANCE OFFSPRING POSTNATAL PERFORMANCE

By

Vitor Rodrigues Gomes Mercadante

December 2015

Chair: G. C. Lamb

Major: Animal Sciences

To evaluate the effects of reducing concentrations of progesterone (P4) during an

ovulation synchronization protocol on pregnancy rates to fixed-timed artificial insemination

(TAI), beef cows and heifers were assigned to receive treatments: 7-d CO-Synch+CIDR (CO-

Synch+CIDR); or 7-d CO-Synch+CIDR with an additional injection of prostaglandin F2α (PGF)

at the initiation of the protocol (PG-CO-Synch+CIDR). Concentrations of P4 were greater at

CIDR removal for CO-Synch+CIDR than for PG-CO-Synch+CIDR for cows and heifers.

Follicle diameter at CIDR removal was greater for PG-CO-Synch+CIDR compared to CO-

Synch+CIDR for cows, but not heifers, and pregnancy rates to TAI were similar between

treatments. To determine the effects of administration of bovine somatotropin (bST; 325 mg) on

hormone concentration, conceptus development and postnatal offspring performance, suckled

beef cows were exposed to a TAI protocol, and randomly assigned to receive treatments: CTRL,

no bST; TAIbST, bST on d 0 (TAI); d14bST, bST on d 14; 2bST, bST on d 0 and 14.

Administration of bST at TAI increased plasma concentration of insulin-like growth factor 1

(IGF-1). The TAIbST treatment had increased mRNA expression of myxovirus 2 on d 21

compared to 2bST. However, fetal size, birth weight and postnatal performance did not differ

among treatments. In another experiment, Year 1, heifers were enrolled in a TAI protocol and

14

assigned to receive either no bST (CTRL), 325 mg at TAI (S-bST) or 14 d later (d14bST), or 650

mg at TAI (D-bST). Injection of bST increased concentrations of IGF-1, however, did not altered

fetal size. Heifers in D-bST had greater pregnancy rate to TAI compared to d14bST and CTRL.

Year 2, heifers were assigned to D-bST or CTRL treatments, and data from both years were

combined. Injection of bST increased concentration of IGF-1, but failed to increase fetal size,

and a treatment × year interaction was detected for pregnancy rate to TAI. In conclusion,

addition of PGF to the 7-d CO-Synch+CIDR decreased concentrations of P4, but failed to

increase TAI pregnancy rates. Administration of bST enhanced concentrations of IGF-1, but

failed to improve TAI pregnancy rates, fetal size, had no effect on calf birth weight and postnatal

performance.

15

CHAPTER 1

INTRODUCTION

It is estimated that the world’s population will increase by 34% (6.8 to 9.1 billion people)

over the next 40 years. Global agriculture productivity will need to increase by 70% and animal

protein by 50% to accommodate this increased population. Of the production increase, 80%

would need to come from increases in yields and cropping intensity, and only 20% from

expansion of land (Food and Agriculture Organization - FAO, 2009). Rising feed costs, global

competition, and societal concerns about energy policy and the environment have created new

economic challenges for the beef industry. In order to sustain adequate supply for the growing

protein demands all efforts should focus on increasing efficiency of beef production through

adoption of technology.

The economic success of cow-calf beef operations relies on the ability to produce one

live calf per cow per year. To achieve this goal, cow-calf producers need to overcome several

obstacles related to the cow, bull and the offspring including, ovulation and fertilization rates and

embryonic, fetal and postnatal survivals (Inskeep and Dailey, 2005). Over the last four decades

several advances in reproductive biotechnologies such as, artificial insemination (AI), estrus-

synchronization, and fixed-time AI (TAI) have helped producers improve genetic traits of their

cattle, tighten the breeding season and shorten the calving season leading to an increase in

overall profitability of cow-calf production systems (Lamb et al., 2010; Rodgers et al., 2012).

However, even with these advancements, reproductive failure and embryo mortality are still a

major cause for economic loss in beef production (Diskin and Morris, 2008). Recently, we

projected a loss of $6.25 per exposed cow for every 1% decrease in pregnancy rate, with an

estimated gross loss of $2.8 billion annually in the United States due to infertility of beef females

(Lamb et al., 2011). Therefore, the development of strategies that aims to reduce embryonic and

16

fetal mortality and increase the number of females pregnant during the breeding season are

needed and can greatly impact the success of beef production worldwide.

Utilization of estrus or ovulation synchronization and TAI has facilitated the widespread

utilization of AI with proven sires to desirable genetic characteristics (Lamb et al., 2010). In

addition, utilization of TAI protocols can also impact the economic viability of cow-calf systems

by enhancing weaning weights per cow exposed (Rodgers et al., 2012). The majority of these

TAI protocols depend largely on the use of exogenous progesterone (P4) supplemented by an

intravaginal controlled internal drug release (CIDR) insert, gonadotropin releasing hormone

(GnRH) induced ovulation, and prostaglandin F2α (PGF) to induce luteolysis (Larson et al., 2006;

Lamb et al., 2010). Reduced concentrations of P4 at the time of CIDR removal can increase

luteinizing hormone (LH) pulse frequency (Kojima et al., 1992) and enhance development and

growth rates of ovarian follicles (Fortune, 1994), both of which have improved pregnancy to TAI

in beef females (Lamb et al., 2001). The development of TAI protocols for beef females that

yield reduced concentrations of P4 at CIDR removal can, therefore, enhance fertility and

pregnancy success of beef cow-calf production systems.

Although, fertilization rates in lactating and nonlactating beef cows average 75% and

96%, respectively (Santos et al., 2004b), pregnancy rates of beef females exposed to TAI average

only between 45% and 60% (Lamb et al., 2010). It is estimated that 75 to 80% of the embryonic

loss occurs by d 20 of gestation (Inskeep and Dailey, 2005). Embryonic losses in cattle are

related to fertilization failure, incompetence of embryos originated from low quality oocytes and

suboptimal uterine conditions (Santos et al., 2004b). A key factor on embryonic and fetal

survival is the somatotropic axis, which is of extreme importance on conceptus growth and

development, by acting directly on the oocyte, endometrium, placenta, and embryo. The major

17

components of the somatotropic axis include somatotropin or growth hormone (GH) and insulin-

like growth factor 1 (IGF-1); which are involved in carbohydrate, protein and fat metabolism,

and cell proliferation and differentiation (Le Roith et al., 2001). Supplementation of exogenous

bovine somatotropin (bST) to lactating dairy cows enhanced post-TAI concentrations of GH and

IGF-1, generated larger embryo/fetus on days 34 and 48 respectively, increased pregnancy rates

on d 66 and reduced pregnancy loss (Ribeiro et al., 2013). Therefore, supplementation of GH and

IGF-1 during the peri- and pre-implantation periods via bST administration enhances conceptus

growth and improved embryonic survival and conception rates in cattle (Starbuck et al., 2006;

Ribeiro et al., 2013).

The trajectory of fetal growth is thought to be set at an early stage in development. Peri-

implantation alterations in maternal diet leading to changes in plasma concentrations of GH,

IGF-1 and P4 can lead to epigenetic changes in gene expression in the embryo leading to further

changes in fetal growth trajectory (Godfrey and Barker, 2000). Fetal programming is a key

concept during critical prenatal development stages that may have lasting impacts on postnatal

growth and adult function (Godfrey, 1998). For instance, the changes in muscle fiber types in

offspring of malnourished animals may alter the production and utilization of glucose with

consequences for adult glucose tolerance (Fowden et al., 2005). Studies on fetal programming

with sheep using supplementation with bST at breeding led to more efficient placenta, larger

birth weight lambs, and increased postnatal growth (Costine et al., 2005). In a similar study, a

single dose of bST at breeding increased birth weight and the lambs of bST treated ewes were

still heavier at 100 days postnatal compared with control lambs (Koch et al., 2010).

Further gains in production efficiency will require new insights into the physiological,

endocrine, and molecular mechanisms controlling various aspects of animal growth and

18

development. Recent findings on epigenetics and fetal programming of livestock animals have

created new opportunities to enhance animal performance and protein production, however

further research is needed to fully understand these processes and establish strategies to take

advantage of them. In addition, little information is available on the effects of bST administration

on fetal programming and subsequent offspring performance in cattle. Thus, supplementation of

beef females with bST during the pre- and peri-implantation period is a strategy that can

potentially enhance conceptus development, reduce embryonic loss and alter subsequent

offspring performance improving overall efficiency of beef production systems.

19

CHAPTER 2

LITERATURE REVIEW

Bovine Estrous Cycle

The estrous cycle consists of a series of events that ultimately leads to estrus and

ovulation. In the bovine, four primary organs are responsible for controlling the estrous cycle via

hormone synthesis, secretion, and cessation. These are the hypothalamus, hypophysis or pituitary

gland, ovary, and uterus. The length of the bovine estrous cycle ranges from 17 to 24 days,

averaging 21 days, and is categorized into 4 different stages: 1) estrus: from 25 to 30 hours after

acceptance of mount, characterized by great concentrations of estradiol (E2) produced by the

dominant follicle, stimulation of LH surge and subsequent ovulation; 2) metestrus: from 1 to 5

days after estrus, characterized by the transition from E2 dominance, with reducing

concentrations of E2, to P4 dominance, with increasing concentrations of P4 produced by the

corpus luteum (CL); 3) diestrus: from 5 to 15 days after estrus, characterized by P4 dominance,

with a fully developed CL secreting great concentrations of P4; 4) proestrus: from d 2 to 5 before

estrus, initiated by CL regression and characterized by the period of transition from P4

dominance to E2 dominance (Amstalden and Willians, 2015).

Follicle dynamics

In the cow, the estrous cycle is characterized by follicle growth waves, where only the

last wave will result in ovulation. After the development of the antral oocyte, the follicle requires

follicle stimulate hormone (FSH) and LH for growth and development. In every growth wave,

several antral follicles are recruited in a cohort. Only one follicle will acquire dominance, grow,

and will undergo atresia or ovulation depending in which follicular wave it is. A cohort of antral

follicles is recruited with the increase in concentrations of FSH. This hormone will promote the

proliferation and prevent atretic degeneration of the early antral follicles. The follicles will

20

continue to grow under FSH influence until they reach 3mm. By the time follicles are 5 mm they

produce estradiol and inhibin, which are FSH inhibitors. At this period one follicle will be more

developed than the others and will acquire dominance, whereas the remaining follicles will

undergo atresia (Aerts and Bols, 2010). The dominant follicle is the primary FSH inhibitor and

can optimize the utilization of low concentrations of FSH. Also, the dominant follicle undergoes

a transition in gonadotropin dependency from FSH to LH, enabling it to survive and mature

despite the low concentrations of FSH. Increased estradiol by the dominant follicle will

positively affect the pulsatility of LH leading to a surge. With the absence of a functional CL,

this surge will stimulate the ovulation process (Ginther et al., 2001).

Ovulation

Two important neurons (Kiss-1 and GnRH neurons) that regulate the estrous cycle are

present in the hypothalamus. Stimulation of Kiss-1 causes release of kisspeptin. Kisspeptin acts

on the GPR54 receptor expressed on the GnRH neuron stimulating secretion of GnRH into the

portal blood system of the brain, GnRH then reaches the anterior pituitary stimulating synthesis

and secretion of two gonadotropic hormones, FSH and LH, both of which are released into the

systemic circulation reaching the ovaries and stimulating follicle growth. Several follicles grow

together until one of them becomes dominant. The dominant follicle synthesizes and secretes E2

and inhibin. Both hormones are released into the blood stream reaching the hypothalamus and

hypophysis causing a negative feedback on GnRH, FSH, and LH secretion at early stage of

dominance (Atkins et al., 2008). The E2 secretion increases as the dominant follicle grows and

influences GnRH secretion patterns via Kiss-1 neuron. High concentration of E2 up-regulate

Kiss-1, increasing GnRH secretion and gonadotropin secretion inducing a surge of LH and

ovulation of the dominant follicle and expulsion of the oocyte into the uterus oviduct (Stumpf et

al., 1991).

21

Corpus luteum formation

Luteinizing hormone is responsible for luteinization of the granulosa and theca cells after

ovulation of a follicle (Farin et al., 1988). Luteinization is characterized by transformation of

granulosa and theca cells into luteal cells (Donaldson and Hansel, 1965). The LH surge drives

the transformation of the luteal cells and the acquisition of capacity to synthesize steroid

hormones such as P4 and E2 (Farin et al., 1988). The granulosa cells become large luteal cells

and are responsible for most P4 and E2 synthesis. The theca cells become small luteal cells,

which are less steroidogenic active and have no secretory granules (Niswender et al., 2000). The

CL is a highly vascularized heterogeneous tissue composed of cells such as pericytes, fibricytes,

nerves, smooth muscle, in addition to the steroidogenic small and large luteal cells (Farin et al.,

1988; Niswender et al., 2000).

Luteolysis

Around d 16 post-ovulation, if the oocyte had not been fertilized, the receptors of

oxytocin on the uterus endometrium wall are up-regulated (Wathes et al., 1995). The up-

regulation of the oxytocin receptors induces synthesis and release of PGF by the endometrium.

Initially there is an increase in blood flow followed by an immediately decrease, causing

luteolysis by decreasing nutrients and substrates for steroidogenesis, and luteotropic support for

the CL (Phariss et al., 1969). The deprivation of CL blood flow occurs prior to morphological

changes of the luteal cells (apoptosis) and also before a decline in LH receptors (Spicer et al.,

1981). Low concentrations of P4 and the lack of the negative feedback in the hypothalamus,

enable GnRH build up stimulating FSH and LH secretion. A new LH surge is secreted inducing

a new ovulation and initiation of a new estrous cycle.

22

Control of the Estrous Cycle

Advances in reproductive biotechnologies and enhanced understanding of the dynamics

of the estrous cycle have made possible the development of protocols to manipulate the estrous

cycle and control ovulation utilizing natural and/or artificially synthesized hormones, such as

GnRH, FSH, LH, PGF and progestins. Utilization of estrus or ovulation synchronization and TAI

has facilitated the widespread utilization of AI and can greatly impact the economic viability of

cow-calf systems by enhancing weaning weights (Rodgers et al., 2012). Implementation of TAI

programs by beef producers, however, depends largely on 2 key factors: 1) limited frequency of

handling cattle; and 2) elimination of detection of estrus by employing TAI (Lamb et al., 2010).

During the past decade TAI protocols have been developed that eliminate detecting estrus and

yield satisfactory pregnancy rates. The majority of these TAI protocols depend largely on the use

of exogenous P4, GnRH-induced ovulation, and luteolysis via administration of PGF (Larson et

al., 2006; Lamb et al., 2010).

A single injection of PGF to induce luteolysis followed by detection of estrus and AI in

heifers was one of the first attempts to synchronize estrus (Lauderdale et al., 1974). Conception

rates did not differ between control (21 days of detection of estrus) and treated heifers. However,

induction of luteolysis via injection of PGF can only be achieved during diestrus when a CL is

present (Wiltbank et al., 1995) and injection of PGF in females during metestrus failed to induce

luteolysis due to the refractoriness of the young CL during this phase of the estrous cycle

(Lauderdale et al., 1974). In addition, no estrus response would be observed in anestrus or

prepubertal females, because of the absence of a CL (Macmillan and Henderson, 1983).

Administration of GnRH to cows successfully increased concentrations of LH (Carter et

al., 1980; Zaied et al., 1980; Williams et al., 1982) and ovulation of the dominant follicle was

achieved within 24 to 32 hours after GnRH injection (Pursley et al., 1995). A combination of

23

GnRH injection followed by PGF injection 7 days later was utilized to synchronize the follicular

wave and induce luteolysis, respectively, allowing a concentration of estrus activity and

increased number of females exposed to AI (Pursley et al., 1995). A second injection of GnRH

48 hours after PGF injection was introduced in order to induce ovulation of the dominant follicle,

allowing TAI to be performed and excluding the necessity of detection of estrus. This TAI

protocol was named Ovsynch (Pursley et al., 1995). In the Ovsynch protocol, the first GnRH

injection is administered to induce ovulation of the dominant follcile, resetting the follicular

wave. However, the ovulation response to the first injection of GnRH was 31% in anestrous beef

cows (Geary et al., 2001b) and varies according to the day of the estrous cycle (Price and Webb,

1989; Roche et al. 1992; Vasconcelos et al., 1999; Atkins et al., 2008), diameter of the dominant

follicle (Roche et al., 1992), and stage of follicular development (Pursley et al. 1995; Roche et

al., 1992).When ovulation from the first GnRH fails, the second GnRH injection may cause the

dominant follicle to ovulate at unexpected intervals prior to AI impairing fertilization and

pregnancy success (Lamb et al., 2001; Kojima et al., 2003).

Later the inclusion of P4, supplemented by a CIDR device, to prevent ovulation prior to

PGF injection was extensively investigated (Lamb et al., 2001, 2006; Larson et al., 2006; Bridges

et al., 2008; Busch et al., 2008). Conception rates comparing protocols with or without

exogenous sources of P4 resulted in fertility that was improved when the device was applied (7-d

CO-Synch+CIDR protocol; Lamb et al., 2001, 2006; Larson et al., 2006). In addition, pregnancy

rates of anestrus cows synchronized with the CIDR was similar to cyclic cows (Stevenson et al.,

2000; Lamb et al., 2001; Busch et al., 2008). However, follicles that fail to ovulate to the first

GnRH in the 7-d CO-Synch+CIDR protocol (Lamb et al., 2006; Larson et al., 2006) may become

persistent during the 7 d period in which the CIDR is present, thereby reducing fertility to TAI.

24

The proestrus phase in the 7-d CO-Synch+CIDR protocol may be defined as the interval from

the administration of PGF to the second injection of GnRH, which may have a duration of 60 to

72 hours (Lamb et al., 2001; Larson et al., 2006; Bridges et al., 2008). Recently, in an attempt to

improve fertility, research focused on increasing the length of the proestrus in estrous

synchronization protocols from 66 hours (7-d CO-Synch+CIDR) to 72 hours (5-d CO-

Synch+CIDR; Bridges et al., 2008; Busch et al., 2008; Wilson et al., 2010).

Current Estrous Synchronization Protocols for Beef Females

Extensive research has been done and is still being conducted by several research groups

to enhance the understanding of physiological process involved in the estrous cycle and to

enhance fertility and pregnancy success of TAI protocols. In an effort to combine expertise in

reproductive physiology and estrous synchronization and encourage research cooperation across

the United States, the Beef Reproduction Task Force (BRTF) was formed in 2002. The Beef

Reproduction Task Force is a multi-state team of reproductive physiology experts from seven to

nine Universities across the United States (http://beefrepro.unl.edu/). The objectives of the

BRTF are:

Improve the understanding of the physiological processes of the estrous cycle, the procedures available to synchronize estrus and ovulation and the proper application of

these systems.

Improve the understanding of methods to assess male fertility and how it affects the success of AI programs.

Every year the BRTF releases an updated chart of recommended estrous synchronization

and TAI protocols that have been tested and are proven to be effective for beef cows and heifers,

including different protocols for Bos taurus and Bos indicus cattle (Figure 2-1, and Figure 2-2).

These charts are an excellent source of information and serve as a guideline for beef producers

and industry leaders in the United States.

http://beefrepro.unl.edu/

25

Effects of Progesterone Concentration on Fertility

Although oocytes and cumulus cells are responsive to P4, the effects of circulating

progesterone on follicular growth and oocyte competence in cattle are likely mediated by

changes in LH release rather than a direct action on the follicle. Increased concentrations of P4

has an inhibitory effect on the frequency of LH pulses released by the pituitary gland (Clarke,

1995; Nation et al., 2000; Endo et al., 2012); however, reduced concentrations of P4 can increase

LH pulse frequency (Kojima et al., 1992) and enhance development and growth rates of ovarian

follicles (Fortune, 1994). Accordingly, increasing concentrations of P4 in plasma has been

shown to reduce the growth rate and the maximal diameter of the ovulatory follicle in beef

(Callejas et al., 2006; Pfeifer et al., 2009) and lactating dairy cows (Cerri et al., 2011a, b). Both,

reduced concentrations of P4 and enhanced development and growth rates of ovarian follicles,

have been shown to improve pregnancy to TAI (Lamb et al., 2001).

It is possible that the effects of pre-ovulatory concentrations of P4 on subsequent

endometrial function are mediated by differences in E2 concentrations during proestrus. Cows

assigned to have reduced concentrations of P4 during follicular development often ovulate larger

follicles capable of producing larger amounts of E2 (Cerri et al., 2011a). A protocol to evaluate

the effects of different proestrus lengths on fertility responses in beef cows was developed

(Bridges et al., 2010). In a presynchronized estrous cycle, the dominant follicle from the first

wave was aspirated and cows were treated with PGF either 4.5 or 5.5 days later. All cows were

induced to ovulate the dominant follicle from the second wave with GnRH on d 6.75 after

follicle ablation; thereby, allowing for a similar period of dominance with differing lengths of

proestrus. Although the diameter of the ovulatory follicle was not affected by treatment, cows

with longer proestrus had greater estradiol concentrations and pregnancy per AI compared with

those with shorter proestrus (Bridges et al., 2010). The proportion of cows with short luteal

26

phases was greater for those with short proestrus (Bridges et al., 2010), which was associated

with reduced expression of estrogen receptor alpha and smaller protein abundance for nuclear P4

receptors in the endometrium on d 15.5 after induced ovulation (Bridges et al., 2012). These

results contradict those from Shaham-Albalancy et al. (2001), Cerri et al. (2011a), and Bisinotto

et al. (2010a) where lactating dairy cows with low P4 concentrations during follicle grow (i.e.

greater E2 concentrations during proestrus) had increased PGF release in response to oxytocin

and were more likely to have short reinsemination intervals.

Strategies to Alter Concentration of Progesterone in TAI Protocols

It has been suggested that a decrease in concentrations of P4 and an increase in E2 at the

initiation of synchronization protocol may be important in initiating an increase in LH release

and consequently ovulation (Grant et al., 2011). Elevated concentrations of P4 at the start of the

Ovsynch protocol are associated with increased pregnancy to TAI in Holstein dairy cows

(Stevenson et al., 2012). Presynchronization with PGF before initiating a TAI protocol in dairy

cows improved oocyte quality (Cerri et al., 2009) and pregnancy rates (Atkins et al., 2010). In

beef cows, presynchronization (PGF administered 3 d before initiating a 6-d CO-Synch+CIDR

TAI protocol) improved follicle turnover in response to the first GnRH and subsequent

pregnancy success compared with a 5-d CO-Synch+CIDR treatment (Perry et al., 2012). In

addition, heifers were more likely to display estrus and had better follicle turnover after the first

GnRH when presynchronized with PGF (Perry et al., 2012).

Recently, it has been reported that suckled beef cows with concentrations of P4 of ≥ 4

ng/mL at the initiation of a synchronization protocol had a 7.6% increase in pregnancy to TAI

than cows with concentrations of P4 of ≤ 0.5 ng/mL (Hill et al., 2014). In that same study,

pregnancy to TAI for cows with concentration of P4 at CIDR insertion of 0.50 to 3.99 ng/mL did

not differ from cows with concentration of P4 of ≤ 0.5 ng/mL. In contrast, beef cows with

27

reduced concentrations of P4 at the onset of TAI protocols have been shown to enhance

pregnancy to TAI success (Perry et al., 2012). It has been suggested that the probability of

pregnancy is more likely not only related to the concentration of P4 at the initiation of the TAI

protocol, but also related to developmental stage of the preovulatory follicle as well as the age of

the CL (Hill et al., 2014).

Several ovulation-synchronization protocols utilize GnRH at the time of CIDR insertion

to induce ovulation and reset follicular waves in order to improve pregnancy outcomes (Lamb et

al., 2010). Ovulation induced by GnRH is dependent on the stage of follicular maturity when

GnRH injection occurs (Bridges et al., 2010) and ovulation of follicles smaller than 11 mm in

diameter resulted in compromised pregnancy rates to TAI (Perry et al., 2005). In addition,

replacement beef heifers were more likely to become pregnant when follicles induced to ovulate

with GnRH ranged from 10.7 to 15.7 mm in diameter (Perry et al., 2007).

Oocyte Maturation

Oocyte maturation is the first of several elements that dictate the success of fertilization

and early embryonic development. In cattle, follicle formation occurs during early fetal

development within the first 30 to 60 d of gestation, starting with migration of primordial germ

cells to the genital ridge followed by differentiation into oogonia and primordial follicles within

90 to 140 d (Evans et al., 2012). Although new evidence on the existence of proliferative germ

cells that sustain oocyte and follicle production postnatal on mammals has emerged (Johnson et

al., 2004), these findings do not change the fact that exhaustion of the ovarian reserve occurs

with advanced chronological age (Skaznik-Wikiel et al., 2007). The number of antral follicles in

beef cows increases to 5 yrs of age and then begins to decline (Cushman et al., 2009). In cattle,

the number of follicles in the ovarian reserve is correlated with fertility, and can be assessed by

28

antral follicle count (AFC) independent of stage of the estrous cycle (Cushman et al., 2009), and

anti-Mullerian hormone (AMH) and FSH concentrations (Fortune et al., 2013).

Fertilization

Fertilization is an orchestrated process where female and male gametes fuse and generate

a new organism. Once spermatazoa reach the oviduct in the female reproductive tract, they

interact with a binder of sperm protein in the epithelium surface where they undergo biochemical

processes that provides them with the capacity to fertilize, a process known as capacitation

(Suarez, 2008; Sutovsky, 2009; Ikawa et al., 2010). After ovulation of the cumulus oocyte

complex, spermatozoa will swim through the cumulus cells. An acrosome reaction ensues once

the sperm reach the zona pellucida (ZP). The acrosome is a Golgi-derived organelle that covers

the tip of the sperm head, and its activation releases enzymes that degrade the acrosome and

cause an inner border of sperm membrane specific set of antigens to present themselves to the ZP

(Ikawa et al., 2010). Sperm antigens bind to glycoproteins in the ZP that triggers the completion

of the acrosome reaction leading to the ability of penetrating the ZP and fuse to the egg’s

membrane and posterior haploid nucleus fusion of egg and sperm (Wassarman and Litscher,

2008). Once the sperm succeed in binding and fusing with the oocyte membrane, cortical

granules within the oocyte are activated to prevent further spermatozoa penetration, or

polyspermy.

Early Embryo Development

After fertilization, the zygote undergoes a series of developmental steps before it attaches

to the uterine lumen, which occurs on or after d 19 of gestation in cattle (Ealy and Yang, 2009).

The 1-cell embryo cleaves to form multiple cells, termed blastomeres. After 2 to 3 cleavage

events (i.e. 8-16-cell stage), the embryo is transported from the oviduct to the uterus for further

development (Telford et al., 1990). At the 8-16 cell stage, the bovine embryonic genome begins

29

to be transcribed, this event involves degradation of the maternal RNA and proteins that was

accumulated during oocyte maturation, demethylation of the maternal and paternal DNA,

remodeling of the embryonic new DNA and, finally, activation of the embryonic genome by

transcription factors (Li et al., 2010).

Blastocyst formation

As additional cleavage divisions continue, the embryo will develop into a clump of

uniform cells known as the morula. Blastomeres then begin to compact and differentiate and

eventually form a blastocyst comprised of trophoblast cells along the outer border

(trophectoderm) and non-differentiated cells within the inner cell mass (ICM; Watson et al.,

1999). The ICM is a population of cells positioned inside of the embryo that will retain the

pluripotency of the cells. It will give rise to the entire fetus and extraembryonic tissues

(Zernicka-Goetz et al., 2009). In the bovine species, blastocysts are usually formed on d 7 of

gestation (Lindneri and Wright Jr., 1983). The trophectoderm is the outer cells layer that will

develop in the fetal part of the placenta once implantation occurs. It is the first differentiated cell

type of development (Duranthon et al., 2008). During the blastocyst formation the water has an

osmotic movement into the extracellular space of the embryo caused by the Na/K-ATPase

confined in the trophectoderm basolateral membrane and facilitated by basal and apical

molecular water channels called aquaporins (Watson et al., 1999).These events combined with

the establishment of a trophectoderm tight junctional seal makes possible the blastocoel cavity

formation (Duranthon et al., 2008).

The next stage in conceptus development is blastocyst hatching. This occurs on d 8-9 of

gestation (Rodríguez-Alvarez et al., 2009). This process consists in the loss of the ZP by rupture

and hatching after blastocyst growth (Spencer et al., 2004). This period also coincides with a

second cell fate decision, where the ICM cells in contact with the blastocoel cavity tend to

30

differentiate into primitive endoderm (PE). The PE is a monolayer of cells on the surface of the

ICM that will further differentiate into visceral and parietal endoderm that will form the

extraembryonic membranes (Gasperowicz and Natale, 2011). The inner ICM cells, or cells that

make up the epiblast, maintain their pluripotency and become progenitors of other cell types that

make up the fetus (Zernicka-Goetz et al., 2009; Gasperowicz and Natale, 2011).

Gastrulation and Conceptus Elongation

Around d 14 post-fertilization the bovine epiblast differentiates further as gastrulation

takes place. In this stage the pluripotent epiblast differentiates into three cell layers: endoderm,

mesoderm, and ectoderm. Each cell germ layer will give rise to specific tissues and organs in the

developing calf (Vejlsted et al., 2006). The ectoderm will differentiate into surface ectoderm and

the major part will transform in neural ectoderm in a cranial-caudal direction, beginning the

neurulation process. The mesoderm germ layer will differentiate into somites and

extraembryonic mesoderm. The inner layer of extraembryonic mesoderm will form an

extraembryonic membrane called the yolk sac with the endoderm, and the outer layer will form

the chorion with the trophectoderm. The chorion will rise up from around the embryo creating a

fluid-filled extraembryonic space to protect the embryo called amnion. The mesoderm also forms

the allantois that is the extraembryonic membrane that will vascularize the chorion and amnion

(Schlafer et al., 2000). The endoderm germ layer will differentiate into primitive guts and

portions of allantois (Vejlsted et al., 2006).

Concomitant with gastrulation the embryo starts the elongation process, which is the

lengthening and morphological transition from ovoid to filamentous stage. This expansion of the

trophoblast begins around d 13 to 15 of gestation in cattle. During this period the bovine

conceptus size increases more than 100 fold and can extend to the entire length of both uterine

horn (Blomberg et al., 2008). This extensive surface contact with the uterine lining increases

31

placental surface area that is important for feto-maternal communication and exchange of

nutrients essential for conceptus well-being (Blomberg et al., 2008).

Maternal Recognition of Pregnancy

Concomitant with the expansion of trophectoderm maternal recognition of pregnancy

occurs around d 16 of gestation in cattle. To enable maternal recognition the conceptus

trophectoderm has to produce and release interferon-tau (IFNT) in sufficient amounts, with a

surge at d 14-15 of gestation in cattle (Bazer et al., 2009). This period is coincident with

conceptus elongation, therefore the extensive trophectoderm mass is important not only for

conceptus attachment and placental formation, but also for large amounts of IFNT production.

The expression of IFNT rapidly decreases after d 21 of gestation in cattle, coincident with

trophectoderm attachment to the maternal uterus (Ealy and Yang, 2009).

In order to maintain the pregnancy IFNT will act as antiluteolytic factor. In cattle, IFNT

will down-regulate oxytocin receptors in the uterus endometrium, inhibiting the CL released

oxytocin to bind to its receptor that is up-regulated in the endometrium by the release of E2 by

the follicles (Mann and Lamming, 2001). This inhibition will prevent pulses of uterine PGF

release to occur, sustaining CL function (Demmers et al., 2001; Thatcher et al., 2001). In

addition, IFNT also acts as a luteotrophic factor by promoting endometrial production of

prostaglandin E2 (PGE) without impacting PGF production, therefore increasing the PGE/PGF

ratio. The luteotrophic and antiluteolytic actions of IFNT are indispensable for establishing and

maintaining pregnancy in cattle (Ealy and Yang, 2009).

The actions of IFNT are not restricted to the uterus, and once in the systemic circulation

IFNT induces expression of interferon-stimulated genes (ISG) in peripheral blood leukocytes

(PBL), including 2’,5’-oligoadenylate synthetase 1 (OAS1), beta2-microglobulin, interferon-

stimulated gene 15 (ISG15), myxovirus resistance 1/interferon-inducible protein p78 (MX1), and

32

myxovirus resistance 2 (MX2) (Gifford et al., 2007; Gifford et al., 2008). Systemic induction of

ISG in PBL in response to pregnancy occurs early in ruminants, even before the detection of

IFNT in the circulation. The expression of MX1 mRNA was elevated in PBL from d 15 through

30 after insemination in pregnant compared to bred, nonpregnant ewes (Yankeey et al., 2001).

Subsequently, PBL from pregnant cows exhibited increased expression of MX1 and ISG15 on d

18 and 20 of gestation and of MX2 on d 16, 18, and 20 of gestation compared with bred,

nonpregnant cows (Gifford et al., 2007). Activation of ISG leads to recirculation and

redistribution of immune cells in the endometrium, which may be important for tolerance of

conceptus alloantigens, conceptus implantation and survival in cattle (Ribeiro et al., 2013).

Fetal Development

After the conceptus is fully elongated the chorion will extend throughout the whole

uterine lumen at d 23 of gestation. At this period the allantois develops visibly with an initial

vasculature formation and the yolk sac which arises from the ventral part of the embryo becomes

prominent (Senger, 2003). At d 25-26 of gestation three brain vesicles, bud of forelimb and bud

hindlimb appear in cattle embryo (Assis Neto et al., 2010). Around d 30 of gestation the bundle

that comprises the allantois, yolk sac and ventral amnion wrapping them, becomes longer,

thereby forming the umbilical cord (Senger, 2003). Shortly thereafter the genital tubercle is

present, amniotic growth begins and is more distinct after d 40 of gestation, when embryo sexual

differentiation occurs (Assis Neto et al., 2010). After d 42 of gestation the nomenclature changes

from embryo to fetus with organogenesis. From d 50 to 70 of gestation the amnion does not

change in size in relation to the fetus. The umbilical cord becomes as long as the hind leg with

the umbilical vessels (allantoic arteries and veins) being evident. In most cases, the yolk sac

disappears completely by d 70 of gestation in cattle (Assis Neto et al., 2010).

33

Placental Development

The placenta is an remarkable organ with the sole purpose of supporting pregnancy to

term and provide nutrients and immune components for fetal development and birth (Schlafer et

al., 2000). The ruminant placenta is classified as cotyledonary and synepitheliochorial, because it

is formed by localized special round structures (cotyledons) where the fetal-maternal interaction

occurs. In the cow, cotyledon formation begins at four weeks of gestation. The extraembryonic

membrane, the chorioallantois, which has as its epithelial cell layer, the trophectoderm, attaches

to the uterus and starts to form cotyledons that form villous projections that interdigitate in the

irregular caruncle surface, enhancing the contact area between these two membranes. This

adhesion of fetal and maternal tissues, cotyledon and caruncle respectively, is called a

placentome (Wooding, 1992). The placentome is the functional unit of feto-maternal exchange

that enables supporting the increasing fetal metabolic demands. In the cow, the number of

placentomes varies between 70 to 120 during the whole gestation, they spread throughout the

whole uterus and appear in different sizes, with the largest placentomes closer to the fetus

(Schlafer et al., 2000). Development of the placenta, a process known as placentation, is diverse

among mammals but most mammals undergo the same series of events. These include

trophoblast apposition to the uterine lumen followed by trophoblast adhesion and eventually

trophoblast invasion (Schlafer et al., 2000). The apposition process is possible by P4

downregulation of mucin-1 in the endometrium. Expression of other endometrial proteins is

involved in the trophoblast adhesion process, such as glycosylated cell adhesion molecule 1,

galectin-15, osteopotin and integrins. Glycosylated cell adhesion molecule 1, galectin-15 and

osteopotin are adhesion proteins secreted by the epithelium that will mediate the adhesion

between trophoblast and endometrial cells. Integrins act as membrane receptors of maternal and

conceptus cells that will bind adhesion proteins (Spencer et al., 2004).

34

Once adhesion is completed the invasion phase begins. Binucleate cells form in the

trophectoderm and migrate to form a hybrid trinucleate cell with the fetus trophectoderm and

maternal endometrium (Bazer et al., 2009). Binucleate trophoblast cells, also called trophoblast

giant cells, are cells of the trophectoderm first seen at the beginning of implantation and continue

throughout pregnancy in all ruminants. The mature binucleate cells migrate from the chorionic

epithelium and through the apical trophectoderm tight junction; they fuse with uterine cells of the

maternal side of the placenta and form a feto-maternal syncytium. The second main function of

the binucleated cells is the endocrine role that they play during gestation. Migration and fusion of

these cells with maternal epithelial cells are important mechanisms for the purpose of delivering

binucleate cell granules contents into the maternal system (Wooding, 1982). These granules

contain hormones associated with pregnancy, such as placental lactogen, prolactin-related

protein-1, P4, E2 and pregnancy-associated glycoprotein (PAG; Wooding, 1982; Igwebuike,

2006).

Pregnancy-Associated Glycoprotein

Pregnancy-associated glycoproteins are a family of several glycoproteins which are

secreted by binucleated cells in early placentation and can be detected in the maternal circulation

throughout most of pregnancy. The first members of the PAG family of proteins were identified

when bovine cotyledons were homogenized and protein lysates were used to inoculate rabbits.

The antisera produced reacted with two pregnancy-specific antigens: pregnancy-specific protein

A and pregnancy-specific protein B (PSPB; Butler et al., 1982). Later, a different group of

researchers (Zoli et al., 1991) purified what turned out to be a similar antigen with the same

plasma profile during gestation. This molecule was named pregnancy-associated glycoprotein 1.

Eventually it was discovered that the PAG-1 molecule had very similar properties when

compared with PSPB (Green et al., 1998).

35

The PAG found in ruminants can be categorized into two main groups based on their site

of expression within the placenta. One group is referred to as the ‘ancient PAG’. Tracing the

evolutionary pathway reveals that the ‘ancient PAGs’ may have arisen more than 80 million

years ago, indicating that it was created with the appearance of the Artiodactyla family (Telugu

et al., 2009). These PAGs are expressed in both mononucleate and binucleated cells. Some of

these include PAG-2, -8, -10, -11, -12 and -13. Most of PAG in the ancient group contain

enzymatic activity (Telugu et al., 2009). The second PAG group is termed the ‘modern PAG’

which appears to have arisen 50 to 55 million years ago. This indicates that they may have first

been created when ruminants diverged from swine (Telugu et al., 2009). These PAG are

expressed primarily by binucleated cells and as such make up the majority of PAG found in

maternal blood during pregnancy and include PAG-1, -3 to -7, -9 and -14 to -21. It appears that

all of the modern PAG are inactive proteases and many of them may be able to bind certain

proteins they appear unable to cleave proteins (Green et al., 2000; Telugu et al., 2009).

It has become clear that there are probably more than 100 PAG genes and cattle have 22

distinct PAG cDNAs in the Genbank (Telugu et al., 2009). In addition, it has been shown that

PAG have a long half-life and are expressed differently throughout gestation (Green et al., 2000;

Szafranska et al., 2006). The PAG-1 or PSPB profile is interesting. Its plasma concentrations

increase rapidly between d 24 and 40 post-breeding, then decreases until d 60, before beginning

to increase again. At parturition there is a rapid surge in PSPB concentrations peaking around 10

d before parturition (Sasser et al., 1986). Recently, it was concluded that pregnancy diagnosis

can be made with accuracy using PAG concentration by d 28 post AI, as long as the voluntary

waiting period exceeded 60 days post-partum (Haugejorden et al., 2006). Several studies

reported that PAG plasma concentration measurement is an easy method of pregnancy diagnosis

36

with high accuracy, varying from 86 to 94.8% between d 30 to 35 of pregnancy (Humblot et al.,

1988; Szenci et al., 1997). Most assays determined an accuracy of 90-95% for diagnosing

pregnancy by d 27 of pregnancy when a polyclonal ELISA was used (Green et al., 2005; Silva et

al., 2007; Green et al., 2009; Thompson et al., 2010). It was also suggested that concentrations of

PAG may serve as a marker of placental function by reflecting early embryonic loss (Gábor et

al., 2007; López-Gatius et al., 2007; Thompson et al., 2010) and fetal well-being (Szenci et al.,

2003; Giordano et al., 2012), since PAG values rapidly decrease as soon as an embryo/fetal death

occurs. In addition, PAG concentration can be used as an indicator of the probability of dystocia

in ruminants (Dobson et al., 1993; Kindahl et al., 2002; Kornmatitsuk et al., 2002).

Pregnancy Maintenance and Loss

Although, fertilization rates in lactating and nonlactating beef cows average 75% and

96%, respectively (Santos et al., 2004b), pregnancy rates of beef females exposed to TAI average

only between 45% and 60% (Lamb et al., 2010). It is estimated that 75 to 80% of embryonic loss

occurs by d 20 of gestation (Inskeep and Dailey, 2005). Therefore is important to understand the

nature of these losses so that strategies to prevent and reduce pregnancy loss can be developed.

The Committee on Bovine Reproductive Nomenclature (1972) established definitions for the

various forms of pregnancy loss in cattle. The embryonic period of gestation extends from

conception to the end of the differentiation stage, at approximately 42 d of gestation in cattle, and

that the fetal period extends from gestation d 42 to the delivery of the calf. Therefore, pregnancy

loss is classified into embryonic loss and fetal loss. During the embryonic loss stage there is a

higher rate of pregnancy loss in cows, and it is subdivided into early embryonic death that

includes the period of conception to implantation (d 24 of gestation in cattle) and late embryonic

death, which occurs from d 24 to 42 of pregnancy (Santos et al., 2004b; López-Gatius et al.,

2007). Several factors are responsible for pregnancy loss in cattle, including bacteria, viruses,

37

fungi and protozoa that are potential infectious sources of conceptus loss, whereas toxins, genetic

defects and stress can be noninfectious inducers of pregnancy loss (Whitlock and Maxwell,

2008). The following review will focus on noninfectious factors that impact pregnancy failure in

cattle.

Early embryonic loss

A large portion of these losses occur during the first week of gestation and likely are

caused by issues relating fertilization failure, incompetence of embryos originated from low

quality oocytes and suboptimal uterine conditions (Lucy, 2001; Santos et al., 2004b), genetic

abnormalities of the zygote/embryo, and uterine insufficiency (King, 1991). Extended periods of

follicle dominance with larger persistent pre-ovulatory follicles reduced conception rates

compared with smaller pre-ovulatory follicles in beef cows (Breuel et al., 1993), due to the

exposure of the oocyte to high peak frequencies of LH inducing premature resumption of meiosis

(Revah and Butler, 1996), and by causing biochemical and morphological changes in the oocyte

reducing fertility (Mihm et al., 1994). Fortunately, normal fertility is resumed after the persistent

follicle regresses (Smith and Stevenson, 1995). In dairy cattle, only 65% of the fertilized zygotes

are considered viable at d 5 to 6 of gestation (Santos et al., 2004b).

Substantial pregnancy losses also occur in the second and third week of gestation.

Thatcher et al. (2001) estimated that 40% of all fertile mattings occur at this time. This is not

surprising given that IFNT signaling must occur at this time and conceptus- or uterine-related

issues that retard conceptus elongation and IFNT production can lead to luteolysis, return to

estrus and, consequently, pregnancy loss. Also, the epiblast is beginning to form germ cells and

undergo gastrulation during this period, and issues with this development will induce pregnancy

loss (Santos et al., 2004b; Ealy and Yang, 2009).

38

Late embryonic loss

Limited data are available on late embryonic death in beef cattle. Nonetheless, in an

analyses of several studies with dairy cattle, Santos et al. (2004) observed that late embryonic

loss after d 27 of gestation was variable, ranging from 3.2% in high producing dairy cows to as

great as 42.7% in high producing dairy cows under heat stress, but it normally remains close to

10% (Whitlock and Maxwell, 2008). Losses within this period usually are a result of failure in

the attachment of the developing placenta to the uterine wall. However, since many fetal

developmental events occur at this stage, such as organogenesis, it is possible that a fair amount

of this loss may be caused by lethal embryonic abnormalities (Stevenson, 2001).

Nutritional status of the cow, evidenced by body condition score (BCS), has great effect

on embryonic survival. Numerous studies document that increasing nutritional levels following

parturition have a positive effect on cyclic status, and conception and pregnancy rates in beef

cows (Wiltbank et al., 1964; Santos et al., 2004b; Inskeep and Dailey, 2005; Diskin and Morris,

2008; Lents et al., 2008). Periparturient diseases have been associated with reduced reproduction

success in cattle. Cows diagnosed with clinical and subclinical mastitis, retained placenta, milk

fever and subclinical and clinical metritis are more likely to suffer embryonic loss due to

disruption of uterine environment (Santos et al., 2004b).

Fetal Loss

Dairy and beef cows have a fetal loss rate of up to 11%, but in general fetal death remains

low and is of minor importance when compared to early embryonic loss. In heifers, fetal death

rate drops to 4.2% and 2.5% for dairy and beef, respectively (Santos et al., 2004b; Whitlock and

Maxwell, 2008). Approximately half of the losses after d 42 are caused by trauma or infections.

The remaining losses are largely unexplained. The cost of fetal losses increases dramatically with

an increase in gestation. In cattle, late embryonic death and fetal loss are not high, but consist of

39

substantial economic losses to producers, because it is often too late to rebreed the females

during the defined breeding season system (Diskin and Morris, 2008).

Strategies to Improve Embryo Survival in Cattle

The majority of strategies to improve embryo survival in cattle rely on increasing

concentrations of P4 either via exogenous supplementation or by the formation of an accessory

CL, and on improving embryonic development via nutritional intervention. Human chorionic

gonadotropin (hCG) is a glycoprotein hormone produced by the human embryo soon after

conception and by the syncytiotrophoblasts (trophoblasts cells that invade the uterine wall),

which stimulates P4 production and sustain pregnancy early in gestation (Pierce and Parsons,

1981). When administered to cattle, hCG increased blood concentrations of P4 by forming an

accessory CL resulting in enhanced pregnancy rates in cattle (Fricke et al., 1993; Schmitt et al.,

1996; Santos et al., 2001). Supplemental P4 significantly increased conception rates when

administered prior to d 6 after AI in lactating dairy cows (Mann and Lamming, 1999).

Furthermore, the benefits of supplemental P4 were more clearly evident when utilized in

lactating cows of lower fertility, such as cows with conception rates of less than 50%. Therefore,

timing of administration of supplemental P4 is critical (d 4-5 of gestation) probably because it

alters the secretory activity of the endometrium, thus influencing embryonic growth (Garret et

al., 1998; Geisert et al., 1992).

The effects of bST administration on reproductive function and conceptus development

of cattle have also been investigated. Long term treatment of 500 mg of bST at 14-d intervals

improved fertility of dairy cows subjected to synchronized ovulation (Moreira et al., 2000;

Moreira et al., 2001; Santos et al., 2004a). However, it appears that benefits of bST

administration on reproduction are dose dependent and high doses of bST can have detrimental

effects on establishment and maintenance of pregnancy in cattle (Bilby et al., 2004; Rivera et al.,

40

2010). A single injection of 500 mg of bST at insemination improved conception rates of

lactating dairy cows, but had no effect on dairy heifers or beef cows (Starbuck et al., 2006),

animals that typically have greater concentrations of IGF-1 than lactating dairy cows (Bilby et

al., 1999; Starbuck et al., 2006). It has been shown that bST interacts with the granulosa cells and

stimulates the oxidative activity of ooplasmic mitochondria decreasing the cytoplasmic calcium

contents, influencing nuclear and cytoplasmic maturation of oocytes, increasing the quality of

bovine oocytes and subsequent development to the blastocyst stage after in vitro fertilization

(Kuzmina et al., 2007). In addition, bST treatment increased concentrations of P4 in cows during

the estrous cycle and increased luteal weights post insemination (Lucy et al., 1994; Lucy et al.,

1995).

Transcript expression of the GH receptor is correlated to IGF-1 transcript expression in

the uterus of lactating dairy cows (Rhoads et al., 2008), and systemic increases in GH and IGF-1

due to bST treatment also increases uterine IGF-1, which could aid conceptus nourishment and

growth (Ribeiro et al., 2013). Additionally, bST decreased production of PGF by in vitro cultured

bovine endometrial cells which, if true in vivo could potentially aid luteolysis blockage during

the time of maternal recognition (Badinga et al., 2002). Embryonic survival depends largely on

the crosstalk between trophectoderm and endometrial cells for establishment of conceptus

nourishment and block of luteolytic cascade via IFNT production by the trophoblast (Bilby et al.,

2006). Lactating dairy cows receiving two low doses of bST (325 mg on d 0 and 14 relative to

TAI) had greater post-TAI concentrations of GH and IGF-1, larger embryo/fetus on d 34 and 48

respectively, enhanced expression of ISG on d 18 relative to TAI, increased pregnancy rates on d

66 and reduced pregnancy loss compared with control cows and cows receiving one low dose of

bST at TAI (Ribeiro et al., 2013). Therefore, supplementation of GH and IGF-1 during the peri-

41

and pre-implantation periods via bST administration enhances conceptus growth and,

consequently increases concentration of IFNT and ISG expression leading to decreased

concentration of PGF, maintenance of the CL and improved embryonic survival and conception

rates in cattle (Starbuck et al., 2006; Ribeiro et al., 2013).

Fetal Programming

Developmental programming, also termed “fetal programming”, “the barker hypothesis”,

or “developmental origins of health and disease” is the concept that insults during critical

prenatal development stages may have lasting impacts on postnatal growth and adult function

(Godfrey, 1998). The trajectory of fetal growth is thought to be set at an early stage in

development. Peri-implantation alterations in maternal diet leading to changes in plasma

concentrations of GH, IGF-1, insulin and P4 can lead to epigenetic changes in gene expression in

the embryo leading to further changes in fetal growth trajectory (Godfrey and Barker, 2000).

Waterland and Garza (1999) proposed a list of potential mechanisms by which pre and perinatal

nutrition may persistently affect an organism’s structure or function, including: induced

variations in organ structure, alterations in cell number, clonal selection, and epigenetics.

Organ structure

Morphologic alterations that occur during organogenesis may affect the ability of

individual cells to generate and respond to external signals within the organism. For example,

nutrition-induced alterations on organ vascularization may affect the cellular responses to blood-

borne nutrients or hormonal signals. During limited periods of organogenesis, the fate of cells

depends on externally-derived signals from adjacent and distant cells. Consequently, it is

reasonable to postulate that local concentrations of nutrients and metabolites may modulate the

end result of organogenesis (Waterland and Garza, 1999).

42

Alterations in cell number

During development, organ mass increases either by increasing the number of cells

(hyperplasia) or cell size (hypertrophy). However, different tissues experience diverse, limited

periods of hyperplastic and hypertrophic growth. Cell growth rate is nutrient-dependent, and

hence, nutritional deprivation or surplus, during critical periods of cell division, may lead to

permanent changes in cell number, regardless of subsequent nutrient surplus (Waterland and

Garza, 1999). For instance, offspring born from ewes fed 50% of their total digestible nutrients

requirements from d 28 to 78 of gestation had lesser secondary muscle fibers compared to

offspring born from nutrient-unrestricted ewes (Zhu et al., 2004). The number of muscle fibers is

determined during the prenatal muscle development, and does not increase during the postnatal

life. Thus, prenatal nutrition has profound effects on muscle growth and development during the

later postnatal life (Zhu et al., 2004).

Clonal selection

Cellular proliferation of all organs involves the proliferation of a finite population of

founder cells. As cell proliferation proceeds, the early genetic and epigenetic modifications that

occur within individual cells distinguish them from others in subpopulations of rapidly dividing

cells. Thus, the nutrient environment may induce an incorrect base pairing during DNA

replication, and result in subtle effects on cellular metabolism that may be transmitted to

daughter cells (Waterland and Garza, 1999; Fenech, 2010). Vitamins and minerals serve as

cofactors for enzymes and protein structures involved in DNA synthesis, repair and maintenance

of genome integrity (Neibergs and Johnson, 2012). Hence, suboptimal intake of vitamins and

minerals may permanently damage the DNA and alter the genomic stability (Fenech, 2010).

43

Epigenetics

The epigenetic process is a genetic modification that cannot be explained by changes in

DNA sequence (Riggs and Porter, 1996), and likely occurs during periods of genome

reprogramming, such as embryogenesis and gestation (Jirtle and Skinner, 2007). Epigenetic

mechanisms induced by dietary modifications include methylation, histone modifications, non-

coding small ribonucleic acid, and chromatin-associated proteins (Neibergs and Johnson, 2012).

Methylation of DNA and histone modifications are the major contributors to chromatin

modification, and are the main epigenetic mechanism by which tissue-specific gene expression

patterns are established and maintained (Thiagalingam et al., 2003).

Methylation of DNA molecules is highly correlated with gene expression, and consists of

DNA methyltransferases adding methyl groups at cytosine-purine-guanine islands that are often

associated with the promoter region of genes (Simmons, 2011). Hypomethylation at the promoter

regions of DNA enhances mRNA transcription through chromatin remodeling, whereas

hypermethylation is associated with suppressed mRNA transcription (Simmons, 2011). The

methylation pattern varies among cells in different tissues (i.e. oocytes and sperm DNA are less

methylated compared to cells in somatic tissues, such as muscle), and is maintained during DNA

replication, which allows the specific methylation pattern to be transmitted to progeny cells

(Waterland and Garza, 1999).

In eukaryotic nuclei, DNA is packaged with histone proteins and other DNA-binding

proteins in a highly compact configuration, called chromatin. The chromatin structure is highly

correlated with gene expression (Riggs and Porter, 1996), and can be presented in an open and

active (euchromatin) or closed and inactive configuration (heterochromatin). The primary unit of

chromatin structure is the nucleosome, which consists of approximately 146 base pairs of DNA

wrapped around a histone octamer (Thiagalingam et al., 2003), which serves as a target for

44

methylation, acetylation, ubiquitination and phosphorylation of lysine residues (Fenech, 2010).

Histone acetylation is associated with regions of open chromatin configuration (Turner, 1991),

and may serve as an epigenetic marker to identify DNA regions that should be maintained in a

transcriptional active configuration (Waterland and Garza, 1999).

Evidence of Fetal Programming in Ruminants

Growth and development of the fetus are complex biological events influenced by

genetic, epigenetic, maternal maturity, as well as environmental and other factors. These factors

affect the size and functional capacity of the placenta, uteroplacental transfer of nutrients and

oxygen from mother to fetus, conceptus nutrient availability, the fetal endocrine milieu, and

metabolic pathways (Bell and Ehrhardt, 2002; Fowden et al. 2005; Reynolds et al., 2005). The

most common model of fetal programming studied is intrauterine growth retardation (IUGR),

that can be defined as impaired growth and development of the mammalian embryo or its organs

during pregnancy (Wu et al., 2006). Animal studies using the IUGR model, reviewed by Fowden

et al. (2005), have shown that the pattern of intrauterine growth influences the postnatal function

of a wide range of endocrine glands and hormone axes including the hypothalamic-pituitary-

adrenal axis, the hypothalamic-pituitary-gonad axis, the adrenomedullary system, the renin-

angiotensin system, the endocrine pancreas, the somatotropic axis and the hormones regulating

appetite and food intake, such as leptin. The adverse impact of IUGR on health, growth and

reproductive performance of ruminants has been extensively studied and there is compelling

evidence that the intrauterine environment may alter expression of the fetal genome with lifelong

consequences including, reduced insulin secretion, insulin resistance, dyslipidemia,

cardiovascular dysfunction, lesser