© 2015 vitor rodrigues gomes mercadante · 2016. 2. 25. · vitor rodrigues gomes mercadante...
TRANSCRIPT
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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
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© 2015 Vitor Rodrigues Gomes Mercadante
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To my wife Paula
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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
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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.
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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
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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
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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
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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
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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
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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
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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
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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
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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.
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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
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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
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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
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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.
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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
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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).
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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.
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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
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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.
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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/
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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
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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
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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
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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
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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
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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
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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
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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).
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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).
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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).
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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
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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,
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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).
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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
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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.,
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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-
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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).
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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).
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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
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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