In vitro derived embryos produced from mares treated with intravenous clomiphene citrate

Abstract

Optimizing the number of follicles available for ovum pick-up and in vitro embryo production (OPU-IVP) would increase the efficiency of OPU-IVP sessions. In other species, clomiphene citrate (CC) has been credited with enhancing follicular recruitment through augmented GnRH pulsatility. Our goal was to describe the number of follicles, reproductive hormone secretion patterns, and oocyte developmental potential in mares treated with CC and subjected to OPU-IVP. Six light-breed mares underwent follicle ablation (Day 0) to induce a new follicular wave, followed by administration of 250mcg of cloprostenol (PGF2-α) on Day 6. On Days 10-14, a five-day protocol of 0.5% intravenous CC (Annaji.et.al. AAPS PharmSciTech. 2023; 24:48) was administered at a rate of 2grams/mare/once daily; all mares subsequently underwent OPU on Day 17. Collected oocytes were shipped counter-to-counter to a laboratory for fertilization via intracytoplasmic sperm injection (ICSI). Ovarian follicle counts were performed via transrectal ultrasonography immediately prior to each daily administration of CC (Days 10-14) and on the day of OPU (Day 17); the total number of follicles, in addition to the diameter of the largest follicle (mm) were recorded. Serum samples were collected to quantify and characterize systemic reproductive hormone patterns (i.e., LH, FSH, AMH, and P4); sample collection started on the day of ablation (Day 0) and concluded one week following OPU (Day 24).CORR and PRINQUAL procedures of SAS version 9.4 (SAS Institute, Cary, North Carolina) were utilized to evaluate correlation amongst hormones. The development rate of embryos originating from CC treated mares were compared to development rates of commercial embryos produced during the same time period at the ICSI laboratory. Parametric (T-test) and nonparametric (NPAR1WAY) procedures (SAS) were utilized to compare embryo development rates. For all statistical analyses, significance level was set at p≤0.05. Upon completion of CC treatment, the mean (+/- SEM) diameter of the largest follicle and the mean (+/- SEM) total number of follicles was 40 mm (+/- 5.1 mm) and 11.3 (+/- 1.2), respectively. Significant negative correlations were found between LH and FSH (p=0.007), FSH and the largest follicle (p<0.001), and FSH and total follicle number (p=0.01). Conversely, significant positive correlations were found between LH and the largest follicle (p=0.003), AMH and total follicle number (p=0.01), and AMH and P4 (p=0.008). The overall embryo development rate was 50% for CC treated mares (15/30 injected oocytes) and 36.8% (14/38 injected oocytes) for commercial embryos (p=0.56). Although experimental control mares were not utilized in this pilot study, our results warrant continued investigation to identify the impact(s) of intravenous CC administration on follicular dynamics, OPU-IVP and foal production.