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.

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.

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.

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.

Reproductive inefficiency in cattle has major impacts on overall productivity of cattle operations, increasing cost of production, and impacting the sustainability of the cattle enterprise. Decreased reproductive success and associated disease states have been correlated with the presence of specific microbes and microbial community profiles, yet details of the relationship between microbial communities and host physiology are not well known. The present study profiles and compares the microbial communities in the bovine uterus and vagina using 16S rRNA sequencing of the V1–V3 hypervariable region at the time of artificial insemination. Significant differences (p < 0.05) between the vaginal and uterine communities were observed at the level of α-diversity metrics, including Chao1, Shannon’s Diversity Index, and observed OTU. Greater clustering of vaginal OTU was apparent in principal coordinate analysis compared to uterine OTU, despite greater diversity in the vaginal community in both weighted and unweighted UniFrac distance matrices (p < 0.05). There was a significantly greater relative abundance of unassigned taxa in the uterus (p = 0.008), otherwise there were few differences between the overall community profiles. Both vaginal and uterine communities were dominated by Firmicutes, although the relative abundance of rRNA sequences corresponding to species in this phylum was significantly (p = 0.007) lower in the uterine community. Additional differences were observed at the genus level, specifically in abundances within Clostridium (p = 0.009), Anaerofustis (p = 0.018), Atopobium (p = 0.035), Oscillospira (p = 0.035), 5-7N15 (p = 0.035), Mycoplasma (p = 0.035), Odoribacter (p = 0.042), and within the families Clostridiaceae (p = 0.006), Alcaligenaceae (p = 0.021), and Ruminococcaceae (p = 0.021). Overall, the comparison revealed differences and commonalities among bovine reproductive organs, which may be influenced by host physiology. The increased abundance of unassigned taxa found in the uterus may play a significant biological role in the reproductive status of the animal. The study represents an initial dataset for comparing bacterial communities prior to establishment of pregnancy.