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Graphical Abstract Summary: We investigated the effect of increasing circulating progesterone (P4) on timing of embryonic attachment as measured by daily pregnancy-associated glycoproteins (PAG) concentrations. Further, to investigate the underlying cause of pregnancy loss, the timing of corpus luteum (CL) regression and embryonic death was determined by daily P4 and PAG in dairy cows undergoing pregnancy loss. Until d 33, PAG was similar, regardless of elevated P4 concentrations, but it was greater on d 47 and d 61 of pregnancy in cows with greater P4. Pregnancy loss between d 20 and 33 could be initiated by either CL regression (P4 decrease, constant PAG; 53% of cows) or embryonic death (PAG decrease, constant P4; 47% of cows).

The objective of this work was to determine if specific circulating microRNA (miRNA) differed due to pregnancy status in heifers. Blood samples were collected from heifers 21 days after receiving an in vitro-produced embryo. Pregnancy status was diagnosed 21 days after embryo transfer, equivalent to day 28 of gestation, with rectal ultrasonography. Blood samples from 10 pregnant and 10 nonpregnant heifers were then evaluated for miRNA expression. There were five different miRNAs quantified using delta-delta Ct and qPCR methodology. These miRNAs had previously been associated with early pregnancy in cattle. The miRNA Let-7d-5p was decreased in nonpregnant as compared to pregnant females (p<0.05). There were no changes in 16-5p, 16-1-3p, 16-2-3p, and 26a-5p associated with pregnancy (p>0.05). Results demonstrate an opportunity to identify and study the differential expression of miRNAs from the blood of pregnant cows. The Let-7d-5p miRNA is a potential early pregnancy marker and is critical to better understand the early relationships of the cellular and molecular interactions of the cow and embryo.

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Bovine twin birth is associated with detriments including increased embryo/fetal losses, malpresentation and dystocia. Incidence of these is lessened in bilateral compared to unilateral twin pregnancy. This study was undertaken to assess use of follicular ablation by aspiration to create bilateral twin pregnancies in females with genetic potential for ~3.5 ovulations per cycle (Trio allele carriers). In Experiment 1, carriers (n=30) and non-carriers (n=10) were synchronized for ovulation and timed artificial insemination (TAI). Follicles (>5 mm) in excess of one per ovary were aspirated ~16h preceding TAI. Follicle count for females with follicles on only one ovary was reduced to two. Blood was sampled 2 weeks post-TAI to assess progesterone (P4) concentrations; embryo count was determined by ultrasound 6 weeks post-TAI. Circulating P4 concentration post-TAI was significantly (p& 0.001) associated with both genotype and subsequent pregnancy status (pregnant non-carriers, 7.06±0.68ng/mL; pregnant carriers, 5.54±0.55ng/mL; non-pregnant non-carriers 5.22±1.05ng/mL; non-pregnant carriers, 3.13±0.42ng/mL). Experiment 2 was undertaken to offset negative effects of follicular aspiration on subsequent P4 concentration observed in Experiment 1. Carriers (n=38) and non-carriers (n=32) were submitted to TAI and follicle ablation as described for Experiment 1. Additionally, accessory corpora lutea (CL) were induced in carriers by administration of human chorionic gonadotropin (carriers) at d6 post-TAI. Consequently, P4 concentration post-TAI was significantly (p& 0.05) associated with subsequent pregnancy status (pregnant, 8.48±0.61ng/mL; non-pregnant, 6.70±0.63 ng/mL) but not with genotype (carrier, 8.01±0.59 ng/mL; non-carrier, 7.17±0.64 ng/mL). Embryo number was greater in carriers (Exp1, 1.64±0.81; Exp2, 1.45±0.09) vs non-carriers (1.00±0.00, both Experiments). Single, twin, and triplet pregnancies occurred in carriers in Experiment 1 whereas multiples in Experiment 2 were limited to twin pregnancies. Genotype effects on pregnancy rate were not significant (p >0.10) in either experiment. Results suggest that follicular ablation to create bilateral twin pregnancies in Trio carriers is feasible but requires induction of accessory CL to offset the negative effects of follicular aspiration on subsequent P4 concentration and associated fertility outcomes.

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Studying selection of multiple dominant follicles (DF) in monovulatory species can advance our understanding of mechanisms regulating selection of single or multiple DF. Carriers of the bovine high fecundity Trio allele select multiple DF whereas half-sib noncarriers select a single DF. This study compared follicle selection during endogenous gonadotropin pulses vs during ablation of pulses with Acyline (GnRH-antagonist) and LH action replaced with nonpulsatile hCG treatment in Trio carriers (n = 28) vs noncarriers (n = 32). On D1.5 (D0 = ovulation), heifers were randomized: 1) Control, untreated; 2) Acyline, two i.m. doses (D1.5 and D3) of 3 μg/kg; 3) hCG, single i.m. dose of 50 IU hCG on D1.5 followed by daily doses of 100 IU; and 4) Acyline+hCG. Treatments with nonpulsatile hCG were designed to replace LH action in heifers treated with Acyline. Acyline treatment resulted in cessation of follicle growth on D3 with smaller (P < 0.0001) maximum follicle diameter in Trio carriers (6.6 ± 0.2 mm) than noncarriers (8.7 ± 0.4 mm). Replacement of LH action (hCG) reestablished follicle diameter deviation and maximum diameter of DF in both genotypes (8.9 ± 0.3 mm and 13.1 ± 0.5 mm; P < 0.0001). Circulating FSH was greater in Acyline-treated than in controls. Finally, Acyline+hCG decreased (P < 0.0001) the number of DF from 2.7 ± 0.2 to 1.3 ± 0.2 in Trio carriers, with most heifers having only one DF. This demonstrates the necessity for LH in acquisition of dominance in Trio carriers (~6.5 mm) and noncarriers (~8.5 mm) and provides evidence for a role of GnRH-induced FSH/LH pulses in selection of multiple DF in Trio carriers and possibly other physiologic situations with increased ovulation rate.