Immunohistochemical examination of the utero-placental interface of cows on days 21, 31, 40, and 67 of gestation.

What we understand about early stages of placentation in cattle is based on an elegant series of electron microscopic images that provide exquisite detail, but limited appreciation for the microanatomy across the utero-placental interface. In order to achieve a global perspective on the histology of bovine placentation during critical early stages of gestation, i.e., days 21, 31, 40, and 67, we performed immunohistochemistry to detect cell-specific expression of pregnancy-associated glycoprotein (PAG), cytokeratin, epithelial (E)-cadherin, and serine hydroxymethyltransferase 2 (SHMT2) at the intact utero-placental interface. Key findings from the immunohistochemical analyses are that there are: (1) PAG-positive cells with a single nucleus within the uterine luminal epithelial (LE) cells; (2) PAG-positive cells with two nuclei in the LE; (3) PAG-positive syncytial cells with more than three nuclei in the LE; (4) LE cells that are dissociated from one another and dissociated from the basement membrane in regions of syncytialization within the LE layer; (5) replacement of the mononuclear LE with a multi-layer thick population of PAG-positive cells invading into the uterine stroma of caruncles, but not into the stroma of intercaruncular endometrium; and (6) PAG-, E-cadherin- and SHMT2-positive mononuclear cells at the leading edge of developing cotyledonary villi that eventually represent the majority of the epithelial surface separating caruncular stroma from cotyledonary stroma. Finally, the utero-placental interface of ruminants is not always uniform across a single cross-section of a site of placentation which allows different conclusions to be made depending on the part of the utero-placental interface being examined.

Evaluating the use of luteal color Doppler ultrasonography and pregnancy-associated glycoproteins to diagnose pregnancy and predict pregnancy loss in Bos taurus beef replacement heifers.

The objective of this study was to evaluate the use of corpus luteum (CL) color Doppler (CD) ultrasonography and pregnancy-associated glycoproteins (PAG) for early pregnancy diagnosis and examine their ability to predict late embryonic/early fetal mortality (LEM) in Bos taurus beef replacement heifers. Beef heifers (n = 178) were exposed to a 7-d CO-Synch + CIDR protocol followed by fixed-time artificial insemination (day 0). On days 20 and 22, B-mode and CD ultrasonography were performed to evaluate CL morphometries and blood perfusion, respectively. Heifers were considered nonpregnant when CL area was <2 cm2 or estimated luteal blood perfusion was ≤30% of the total luteal area. Blood samples were collected on days 25 and 29 to estimate circulating concentrations of PAG. Conventional ultrasonography on days 29 and 94 was utilized to determine pregnancy status and considered the gold standard method for pregnancy diagnosis. Pregnant heifers had greater (P < 0.01) CL diameter, area, volume, and blood perfusion when compared with nonpregnant heifers on days 20 and 22. Accuracy of CD on days 20 and 22, and PAG on days 25 and 29 were 91%, 94%, 96%, and 98%, respectively. No false-negative results were observed for CD on both days 20 and 22 (negative predicted value = 100%) and false-positive results represented 8% and 6% of the diagnoses. Heifers that experienced LEM between days 29 and 94 of gestation had decreased luteal (P = 0.02) volume on day 20 and tended (P = 0.07) to have decreased concentrations of PAG on day 29 compared with heifers that maintained pregnancy. However, both CD and PAG failed to predict embryonic mortality. In conclusion, CD successfully detected most nonpregnant replacement heifers as early as day 20 of gestation, while resulting in no false negative diagnoses. Both CD and PAG failed to predict LEM in the present study.

Identifying nonpregnant females early after breeding allows cattle producers to rapidly rebreed females that failed to conceive after their first artificial insemination and consequently increases reproductive efficiency. Additionally, embryonic and fetal mortality during early gestation are the main drivers of pregnancy failure and represents a significant economic burden to both the beef and dairy industries. This study evaluated the use of color Doppler (CD) ultrasonography of specific ovarian structures and blood concentrations of pregnancy-associated glycoproteins (PAG) as potential methods to diagnose pregnancy earlier than industry-standard techniques. Moreover, the present study investigated the use of CD and blood PAG as predictor of pregnancy loss. Data generated here indicates that CD and blood PAG can accurately detect pregnancy as early as 20 and 25 d after insemination, respectively. In the present study, heifers that experienced pregnancy loss between days 29 and 94 of gestation tended to have decreased plasma concentrations of PAG during early pregnancy. Heifers experiencing pregnancy loss between days 29 and 94 of gestation were also more likely to have a luteal cavity on day 20 of gestation and had decreased luteal volume on the same day. However, results reported herein indicate that both CD and blood concentrations of PAG failed to predict pregnancy loss in replacement heifers.

Actions of DKK1 on the preimplantation bovine embryo to affect pregnancy establishment, placental function, and postnatal phenotype†.

One mechanism by which the maternal environment regulates the early embryo is by secretion of cell-signaling molecules. One of these is dickkopf WNT signaling pathway inhibitor 1. Objectives were to (A) resolve discrepancies in the literature regarding effects of dickkopf WNT signaling pathway inhibitor 1 in the bovine embryo on development of trophectoderm and competence to establish pregnancy after embryo transfer and (B) determine whether there are long-term consequences of dickkopf WNT signaling pathway inhibitor 1 on placental function and postnatal phenotype. Embryos produced in vitro were cultured with vehicle or 100 ng/mL recombinant human dickkopf WNT signaling pathway inhibitor 1 from Days 5 to 7.5 of development (i.e., the morula and blastocyst stages of development). dickkopf WNT signaling pathway inhibitor 1 increased the number of cells positive for the trophectoderm marker CDX2 at Day 7.5 of development while having no effect on number of cells positive for the inner cell mass marker SOX2. There was no effect of dickkopf WNT signaling pathway inhibitor 1 on pregnancy or calving rate after transfer of blastocysts produced with Y-sorted semen to either lactating dairy cows or suckling beef cows. Treatment with dickkopf WNT signaling pathway inhibitor 1 at the morula-to-blastocyst stages programmed placental function, as measured by an effect of dickkopf WNT signaling pathway inhibitor 1 on plasma concentrations of pregnancy associated glycoproteins and placental lactogen at Day 160 of gestation (although not on other days examined). dickkopf WNT signaling pathway inhibitor 1 treatment also resulted in calves that were heavier at birth as compared to calves derived from control embryos. After birth, dickkopf WNT signaling pathway inhibitor 1 calves grew slower than controls. Results confirm that dickkopf WNT signaling pathway inhibitor 1 alters the developmental program of the bovine embryo to affect both prenatal and postnatal phenotypes.

Effects of prolonging the interval from progestin removal to prostaglandin F2α injection from 16 to 17 d in a long-term estrus synchronization protocol in beef heifers.

To determine effects of delaying the injection of prostaglandin F2α (PGF) and fixed-time artificial insemination (TAI) in the 14-d CIDR-PG protocol, 1,049 Angus heifers at six locations were enrolled in a completely randomized design. Within location heifers were randomly assigned to one of two treatment groups: 1) PG16 (n = 518), heifers received a controlled internal drug release (CIDR) insert on d 0 for 14 d, a 25-mg injection of PGF 16 d after CIDR removal (d 30), and a 100-µg injection of gonadotropin-releasing hormone concurrent with TAI 66 ± 2 h later; or 2) PG17 (n = 531), heifers were treated the same as PG16, however, PGF was administered 17 d after CIDR removal (d 31), and heifers were TAI 66 ± 2 h later. Estrus detection patches were applied to a subset (n = 482) of heifers at the time of PGF administration and were examined for activation at TAI. Dominant follicle diameter was determined via transrectal ultrasonography at PGF administration and TAI in a subset of heifers (n = 116). Transrectal ultrasonography was performed to determine pregnancy rates to TAI (PR/AI) between 30 and 45 d after TAI. Estrus expression prior to TAI differed by treatment where PG17 heifers had greater (P < 0.01) expression of estrus than PG16 heifers (57.8 ± 6.1% vs. 43.4 ± 6.1%, respectively). Nevertheless, dominant follicle diameters at PGF and at TAI were similar (P ≥ 0.59) between PG16 and PG17 heifers. In addition, PR/AI did not differ (P = 0.29) between PG16 and PG17 treatments (50.5 ± 3.2% vs. 45.7 ± 3.1%, respectively). Results of this experiment indicate that delaying the injection of PGF and TAI in the 14-d CIDR-PG protocol increased estrus expression prior to TAI yet did not improve fertility in beef heifers.