K. G. Pohler

Follicular determinants of pregnancy establishment and maintenance

Synchronization of dominant follicle development and control of ovulation/oocyte retrieval are commonly used assisted reproductive technologies in both cattle and humans. The final maturation of the dominant follicle is intimately tied to the final maturation of the oocyte, preovulatory secretion of estradiol, preparation of follicular cells for luteinization, postovulatory secretion of progesterone and endocrine control of the oviductal and uterine environment for gamete and embryo development. The physiological maturity of a dominant/ovulatory follicle can affect the establishment and maintenance of pregnancy. Premature induction of the ovulatory process can reduce pregnancy rates and increase late embryonic/fetal mortality in cattle, which is likely mediated through inadequate oocyte competence and a compromised maternal environment. Oocyte competence increases with follicular maturity and is dependent upon acquisition of a complete complement of mRNA transcripts and establishment of the appropriate epigenetic marking of the oocyte genome before the preovulatory gonadotropin surge. Preovulatory secretion of estradiol is a reflection of follicular maturity and affects the oocyte, follicular cells, oviduct and uterus. The corpus luteum is a continuation of follicular maturation and rate of progesterone secretion following ovulation is linked to fertility. Advancements in our understanding of how the follicular microenvironment affects pregnancy establishment and maintenance will improve the efficiency of assisted reproductive technologies in all species. The purpose of this review is to discuss how follicular microenvironment, oocyte competence, preovulatory secretion of estradiol and postovulatory secretion of progesterone can affect pregnancy establishment and embryo/fetal survival, with an emphasis on cattle.

Preovulatory estradiol and the establishment and maintenance of pregnancy in suckled beef cows.

In postpartum beef cows, GnRH-induced ovulation of small dominant follicles decreased pregnancy rates and increased late embryonic/fetal mortality. In Exp. 1, single ovulation reciprocal embryo transfer (ET) was used to examine the relationship between preovulatory serum concentrations of estradiol at GnRH-induced ovulation in donor and recipient cows and establishment and maintenance of pregnancy. Suckled beef cows (n = 1,164) were administered GnRH (GnRH1, 100 μg) on d -9 (GnRH1), PGF(2α) on d -2, and GnRH2 (GnRH2, 100 μg) on d 0 (CO-Synch protocol) either with (donors; n = 810) or without (recipients; n = 354) AI. Single embryos (n = 394) or oocytes (n = 45) were recovered from the donor cows (d 7; ET) and all live embryos were transferred into recipients. Serum concentration of estradiol at GnRH2 was positively correlated with follicle size at GnRH2 (r = 0.45, P < 0.01) and progesterone at ET (r =0.34, P < 0.01). Donor cows with greater estradiol at GnRH2 were more likely to yield an embryo than an unfertilized oocyte (P < 0.01). Donor and recipient cows were retrospectively divided into 4 groups [low estradiol (<8.4 pg/mL) or high estradiol (≥8.4 pg/mL)] based on serum concentration of estradiol at GnRH2. Pregnancy rate at d 27 for low-low (n = 78), low-high (n = 80), high-low (n = 91), and high-high (n = 101) groups (donor-recipient, respectively) was 45, 65, 43, and 61% respectively (P < 0.02). Because recipient cows with greater estradiol concentration at GnRH2 had greater pregnancy rates in Exp. 1, the objective of Exp. 2 was to evaluate the effect of estradiol supplementation on pregnancy rate. Ovulation was synchronized in suckled beef cows (n = 600) using the CO-Synch protocol with the insertion of a controlled internal drug release (CIDR; intravaginal progesterone supplement) from d -9 until d -2. Approximately one-half of the cows (n = 297) received an injection of estradiol cypionate (ECP; 0.5 mg intramuscularly) 24 h before AI. Compared with the no treatment (Control) cows, ECP treatment increased (P < 0.01) pregnancy rates of cows induced to ovulate smaller dominant follicles (<12.2 mm). In conclusion, GnRH-induced ovulation of small dominant follicles was associated with reduced serum estradiol, fertilization rate (donor cows), and pregnancy establishment (recipient cows). Furthermore, ECP supplementation during the preovulatory period increased pregnancy rates in cows induced to ovulate smaller dominant follicles.

Placental PAGs: gene origins, expression patterns, and use as markers of pregnancy

Pregnancy-associated glycoproteins (PAGs) are abundantly expressed products of the placenta of species within the Cetartiodactyla order (even-toed ungulates). They are restricted to this order and they are particularly numerous in the Bovidae. The PAGs exhibit a range of temporal and spatial expression patterns by the placental trophoblasts and probably represent a group of related proteins that perform a range of distinct functions in the epitheliochorial and synepitheliochorial placental forms. This review presents an overview of the origins of the PAGs, a summary of PAG expression patterns, and their use as markers of pregnancy status. Speculations about their putative role(s) in pregnancy are also presented.

The WNT signaling antagonist Dickkopf-1 directs lineage commitment and promotes survival of the preimplantation embryo

Successful embryonic development is dependent on factors secreted by the reproductive tract. Dickkopf‐1 (DKK1), an antagonist of the wingless‐related mouse mammary tumor virus (WNT) signaling pathway, is one endometrial secretory protein potentially involved in maternal‐embryo communication. The purpose of this study was to investigate the roles of DKK1 in embryo cell fate decisions and competence to establish pregnancy. Using in vitro‐produced bovine embryos, we demonstrate that exposure of embryos to DKK1 during the period of morula to blastocyst transition (between d 5 and 8 of development) promotes the first 2 cell fate decisions leading to increased differentiation of cells toward the trophectoderm and hypoblast lineages compared with that for control embryos treated with vehicle. Moreover, treatment of embryos with DKK1 or colony‐stimulating factor 2 (CSF2; an endometrial cytokine known to improve embryo development and pregnancy establishment) between d 5 and 7 of development improves embryo survival after transfer to recipients. Pregnancy success at d 32 of gestation was 27% for cows receiving control embryos treated with vehicle, 41% for cows receiving embryos treated with DKK1, and 39% for cows receiving embryos treated with CSF2. These novel findings represent the first evidence of a role for maternally derived WNT regulators during this period and could lead to improvements in assisted reproductive technologies.—Denicol, A. C., Block, J., Kelley, D. E., Pohler, K. G., Dobbs, K. B., Mortensen, C. J., Ortega, M. S., Hansen, P. J. The WNT signaling antagonist Dickkopf‐1 directs lineage commitment and promotes survival of the preim‐plantation embryo. FASEB J. 28, 3975‐3986 (2014). www.fasebj.org

Circulating concentrations of bovine pregnancy-associated glycoproteins and late embryonic mortality in lactating dairy herds

The objectives of these experiments were as follows: (1) to determine the association between circulating concentrations of pregnancy-associated glycoproteins (PAG) and late embryonic mortality (EM) in lactating dairy cattle following fixed-time artificial insemination (TAI) on d 0 or timed embryo transfer (TET) on d 7, (2) to identify a circulating concentration of PAG on d 31 below which late EM would be likely to occur, and (3) to identify when during gestation (d 31-59) late EM is occurring. Cows were diagnosed pregnant on d 31 of gestation based on presence of a fetal heartbeat and reconfirmed to be pregnant on d 59 of gestation. Late EM occurred when a cow had a viable embryo on d 31 of gestation but not on d 59 following TAI or TET. Only pregnant cows on d 31 were included in the analysis (TAI-maintained, n=413; TAI-EM, n=77; TET-maintained, n=238; TET-EM, n=47). Cows that were pregnant at d 31 of gestation and maintained the pregnancy until d 59 had significantly higher circulating concentrations of PAG at d 31 of gestation compared with cows that experienced late EM between d 31 and 59 of gestation in both TAI and TET. To conduct a more stringent test of the effectiveness of a single circulating PAG concentration (d 31) to predict EM, a receiver-operating characteristic curve was generated to identify a PAG concentration on d 31 that would predict EM with ≥95% accuracy in cows that received TAI or TET. Based on positive and negative predicative value analysis, a circulating concentration of PAG below 1.4 ng/mL (TAI; minimal detectable level 0.28 ng/mL) and 1.85 ng/mL (TET) was 95% accurate in predicting EM (between d 31 and 59) at d 31 of gestation, respectively. Following TET, embryonic loss was tracked by Doppler ultrasound, progesterone, and PAG from d 24 to 59 of gestation, with more than 50% of the loss occurring between d 31 and 38 of gestation. In summary, circulating concentrations of PAG on d 31 of gestation may provide a good marker for predicting EM between d 31 and 59 of gestation, and the data suggest that this model could help predict which cows will undergo late EM.

Triennial Reproduction Symposium: influence of follicular characteristics at ovulation on early embryonic survival.

Reproductive failure in livestock can result from failure to fertilize the oocyte or embryonic loss during gestation. Although fertilization failure occurs, embryonic mortality represents a greater contribution to reproductive failure. Reproductive success varies among species and production goals but is measured as a binomial trait (i.e., pregnancy), derived by the success or failure of multiple biological steps. This review focuses primarily on follicular characteristics affecting oocyte quality, fertilization, and embryonic health that lead to pregnancy establishment in beef cattle. When estrous cycles are manipulated with assisted reproductive technologies and ovulation is induced, duration of proestrus (i.e., interval from induced luteolysis to induced ovulation), ovulatory follicle growth rate, and ovulatory follicle size are factors that affect the maturation of the follicle and oocyte at induced ovulation. The most critical maturational component of the ovulatory follicle is the production of sufficient estradiol to prepare follicular cells for luteinization and progesterone synthesis and prepare the uterus for pregnancy. The exact roles of estradiol in oocyte maturation remain unclear, but cows that have lesser serum concentrations of estradiol have decreased fertilization rates and decreased embryo survival on d 7 after induced ovulation. When length of proestrus is held constant, perhaps the most practical follicular measure of fertility is ovulatory follicle size because it is an easily measured attribute of the follicle that is highly associated with its ability to produce estradiol.

Circulating bovine pregnancy associated glycoproteins are associated with late embryonic/fetal survival but not ovulatory follicle size in suckled beef cows1

The objective was to examine the relationship between ovulatory follicle size and embryo and fetal survival by using circulating concentrations of bovine pregnancy associated glycoproteins (bPAG) to detect the presence of an embryo or fetus and monitor placental function. Before examining the relationship between bPAG, ovulatory follicle size, and embryo and fetal survival, the half-life of bPAG was determined in Exp. 1. The half-life of bPAG after PGF2α-induced abortion on d 32 to 36 postinsemination was 35.8 ± 21.9 h (mean ± SD; range 7.1 to 78.5 h). In Exp. 2, suckled beef cows (n = 91) were treated with the CO-Synch protocol (GnRH on d -9, PGF2α on d -2, and GnRH and AI 48 h later [d 0]) and classified into 1 of 2 ovulatory follicle size groups: 1) small follicle (<12.5 mm; n = 25) or 2) large follicle (≥ 12.5 mm; n = 66). The first increase (P < 0.0001) in serum bPAG occurred in pregnant cows on d 24 after insemination and circulating bPAG decreased before a decrease in progesterone in 3 of 4 cows that lost an embryo or fetus. Pattern of secretion of bPAG in serum from d 24 to 60 after insemination (d 0) was affected by day (P < 0.0001), but not ovulatory follicle size. In Exp. 3, suckled beef cows (n = 1164) were administered the CO-Synch protocol either with (donor cows; n = 810) or without (recipient cows; n = 354) AI on d 0. Single embryos (n = 394) or oocytes (n = 45) were recovered from the donor cows [d 7; embryo transfer (ET)] and all live embryos were transferred into recipients the same day. Cows were classified on d 0 as having a small (<12.5 mm) or large (≥ 12.5 mm) ovulatory follicle, and randomly chosen as donors or recipients to remove confounding effects of ovulatory follicle size on fertility. Serum concentration of bPAG at d 28 was not affected by ovulatory follicle size (P = 0.85), embryo stage at ET (P = 0.75), embryo quality at ET (P = 0.64), estradiol at GnRH2 (P = 0.62) or serum progesterone at ET (d7; P = 0.14). Compared with cows that maintained pregnancy (n = 176), cows that exhibited late embryonic or fetal mortality (n = 19) after d 28 had decreased (P < 0.05) concentrations of bPAG on d 28. In summary, there was no relationship between serum bPAG and ovulatory follicle size or embryo stage or quality at ET; however, cows that lost an embryo after d 28 had reduced concentrations of bPAG on d 28 compared with cows that maintained pregnancy.

Effect of progesterone concentrations, follicle diameter, timing of artificial insemination, and ovulatory stimulus on pregnancy rate to synchronized artificial insemination in postpubertal Nellore heifers

Two experiments were designed to evaluate the effects of treatments with low versus high serum progesterone (P4) concentrations on factors associated with pregnancy success in postpubertal Nellore heifers submitted to either conventional or fixed timed artificial insemination (FTAI). Heifers were synchronized with a new controlled internal drug release device (CIDR; 1.9 g of P4 [CIDR1]) or a CIDR previously used for 18 days (CIDR3) plus 2 mg of estradiol (E2) benzoate on Day 0 and 12.5 mg of prostaglandin F2α on Day 7. In experiment 1 (n = 723), CIDR were removed on Day 7 or 9 and heifers were inseminated after estrus detection. In experiment 2 (n = 1083), CIDR were all removed on Day 9 and FTAI was performed either 48 hours later in heifers that received E2 cypionate (ECP) on Day 9 (0.5 mg; E48) or 54 or 72 hours later in conjunction with administration of GnRH (100 μg; G54 or G72). Synchronization with CIDR1 resulted in greater serum P4 concentrations and smaller follicle diameters on Days 7 and 9 in both experiments. In experiment 1, treatment with CIDR for 9 days decreased the interval from CIDR removal to estrus (Day 7, 3.76 ± 0.08 days vs. Day 9, 2.90 ± 0.07; P < 0.01) and improved conception (Day 7, 57.1% vs. Day 9, 65.8%; P = 0.05) and pregnancy rates (Day 7, 37.6% vs. Day 9, 45.3%; P = 0.04). In experiment 2, treatment with ECP improved (P < 0.01) the proportion of heifers in estrus (E48, 40.9%(a); G54, 17.1%(c); and G72, 32.0%(b)), but the pregnancy rate was not affected (P = 0.64) by treatments (E48, 38.8%; G54, 35.5%; G72, 37.5%). Synchronization with CIDR3 increased follicle diameter at FTAI (CIDR1, 11.07 ± 0.10 vs. CIDR3, 11.61 ± 0.10 mm; P < 0.01), ovulation rate (CIDR1, 82.8% vs. CIDR3, 88.0%; P < 0.01) and did not affect conception (CIDR1, 42.2 vs. CIDR3, 45.1%; P = 0.38) or pregnancy rates (CIDR1, 34.7 vs. CIDR3, 39.4%; P = 0.11). In conclusion, length of treatment with P4 affected the fertility of heifers bred based on estrus detection. When the heifers were submitted to FTAI protocol, follicle diameter at FTAI (≤10.7 mm, 23.6%; 10.8-15.7 mm, 51.5%; ≥15.8 mm, 30.0%; P < 0.01) was the main factor that affected conception and pregnancy rates.

Physiology and Endocrinology of Puberty in Heifers

When it comes to development of beef heifers, the importance of maximizing the proportion of heifers that are born early in the calving season cannot be overemphasized. Because early onset of puberty is of critical importance to heifer development, the purpose of this article was to describe the physiological and endocrine maturation that control the onset of puberty in beef cattle. These concepts will be helpful in understanding the physiological and endocrine changes that take place in a heifer as she approaches puberty and in understanding how specific estrous synchronization products are able to induce and synchronize estrus and ovulation.

Predicting Embryo Presence and Viability.

Pregnancy establishment, followed by birth of live offspring, is essential to all mammals. The biological processes leading up to pregnancy establishment, maintenance, and birth are complex and dependent on the coordinated timing of a series of events at the molecular, cellular, and physiological level. The ability to ovulate a competent oocyte, which is capable of undergoing fertilization, is only the initial step in achieving a successful pregnancy. Once fertilization has occurred and early embryonic development is initiated, early pregnancy detection is critical to provide proper prenatal care (humans) or appropriate management (domestic livestock). However, the simple presence of an embryo, early in gestation, does not guarantee the birth of a live offspring. Pregnancy loss (embryonic mortality, spontaneous abortions, etc.) has been well documented in all mammals, especially in humans and domestic livestock species, and is a major cause of reproductive loss. It has been estimated that only about 25-30% of all fertilized oocytes in humans result in birth of a live offspring; however, identifying the embryos that will not survive to parturition has not been an easy task. Therefore, investigators have focused the identification of products in maternal circulation that permit the detection of an embryo and assessment of its well-being. This review will focus on the advances in predicting embryonic presence and viability, in vivo.