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Previous research demonstrated that acute treatment with GnRH antagonist, Acyline, allowed follicle growth until ~8.5 mm and no dominant follicle was selected. This study evaluated whether deficient LH was the underlying mechanism for Acyline effects by replacing LH action, using human chorionic gonadotropin (hCG), during Acyline treatment. Holstein heifers (n = 24) during first follicular wave were evaluated by ultrasound and randomized into one of three treatments: Control (saline treatments), Acyline (5 µg/kg Acyline), or Acyline+hCG (Acyline plus 50 IU of hCG at start then 100 IU every 12 h). Pulses of LH were present in Control heifers (9 Pulses/10 h) but not during Acyline treatment. Data were normalized to the transition to diameter deviation (day 0; F1 ~7.5 mm). Diameter deviation of the largest (F1) and the second largest (F2) follicle was not observed in Acyline-treated heifers, whereas control heifers had decreased growth of F2 at F1 ~7.5 mm, indicating deviation. Selection of a single dominant follicle was restored by providing LH activity in Acyline+hCG heifers, as evidenced by F1 and F2 deviation, continued growth of F1, and elevated circulating estradiol. Separation of F1 and F2 occurred 12 h (~7.0 mm) earlier in Acyline+hCG heifers than Controls. Circulating FSH was greater in Acyline than Controls, but lower in Acyline+hCG than Controls after day 1.5. In conclusion, dominant follicle selection and growth after follicle deviation is due to LH action as shown by inhibition of this process during ablation of GnRH-stimulated LH pulses with Acyline and restoration of it after replacement of LH action by hCG treatment.

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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.

Initiation of luteolysis in ruminants is variable due to ill-defined mechanisms. Cycles of two follicular waves are shorter and have earlier luteolysis than three-wave cycles. This study validated a cytobrush technique for evaluating dynamics of endometrial gene expression and associated changes in mRNA with timing of luteolysis, based on circulating progesterone and ultrasound-determined changes in blood flow and volume of corpus luteum (CL). On day 8 (ovulation = day 0), Holstein heifers were randomized into two groups: cytobrush group (n = 9) had an endometrial sample collected every 48 h from day 8 until end of luteolysis (CL blood flow ≤ 20%) and control group was sampled only once either before (day 12; n = 4) or at the end of luteolysis (n = 5). Concentrations of progesterone, CL blood flow, CL volume, and the frequency of two and three-wave cycles were similar between groups. Endometrial mRNA for progesterone receptors and estradiol receptors 1 and 2 was greater on day 8 and decreased thereafter similarly in two and three-wave cycles. Oxytocin receptor mRNA increased earlier in two vs three-wave cycles (day 14 vs 18), and the increase was associated with the onset of luteolysis. In conclusion, the cytobrush technique allowed in vivo collection of multiple endometrial samples during the estrous cycle. Endometrial mRNA expression of steroid receptors did not explain the variability in timing of onset of luteolysis in heifers while the later onset of luteolysis in three-wave cycles was associated with later up-regulation of oxytocin receptor mRNA.

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.