185 Effect of equine chorionic gonadotropin administration timing in fixed-time artificial insemination protocols on heifer’s corpus luteum steroidogenic cells

Administering equine chorionic gonadotrophin (eCG) two days before the intravaginal progesterone-releasing device (IVD) withdrawal in fixed-time AI (FTAI) protocol in cows resulted in a tendency to develop a larger corpus luteum (CL) with greater blood flow between Day 1 and 7 after ovulation (see our previous study, Vieytes et al. 2022 Reprod. Fertil. Dev. 35, 220). Thus, we hypothesise that a larger CL area with greater blood flow is associated with histological changes after CL maturation. This study aimed to determine the effect of eCG administration on Day 5 or 7 of FTAI protocol on the number of small luteal cells (SLC), large luteal cells (LLC), and the CL area occupied by SLC and LLC, as well as the total area occupied by all steroidogenic cells in the heifer’s CL 9 days after ovulation. Twenty heifers were synchronized with the same FTAI protocol (Day 0, 2 mg IM of oestradiol benzoate and IVD insertion, 750 mg of progesterone; Day 7, IVD withdrawal and 150 mg IM of D-cloprostenol). The eCG treatment (400 IU) was performed on Day 5 (T5, n = 7) or Day 7 (T7, n = 7), and a control group (TC, n = 6) did not receive eCG. Ovulation was monitored daily by ultrasonography. On Day 9 after ovulation, CL (n = 23) were enucleated by surgical colpotomy; one heifer of each group had double ovulation and both CL were included in the experiment. After collection, the CLs were rinsed in PBS, fixed in 4% paraformaldehyde, and processed by classical histology. Sections with 5 µm thickness were placed on glass slides and stained with PAS-hematoxylin. All slides were scanned using Motic Easy Scan One, and Pathomation PMA Start (v2.1) was used to select 10 random areas of interest to quantify and determine the area of large (LLC) and small luteal cells (SLC) in each CL. ImageJ2 (2.9.0/1.53t) software was used to count the total number of the SLC and LLC from the images captured in PMA Start using a scale of 40×, and the area was measured (µm²). Data did not present normal distribution; differences among groups were analysed by the Kruskal–Wallis test and subsequent Tukey test. Values are presented as means ± s.e.m. and P < 0.05. The CLs in T5 had a high quantity of both LLC and SLC (24.5 ± 1.9, 74.4 ± 0.7) than T7 (18.0 ± 0.7, 66.2 ± 2.2) or TC (16.1 ± 0.6, 67.6 ± 2.4); T5 had a higher CL percentage area occupied by LLC and SLC (26.9 ± 0.8, 21.2 ± 0.6) than T7 (16.3 ± 0.7, 13.9 ± 0.5) or TC (14.5 ± 0.5, 10.3 ± 0.4), and T5 had a greater total percentage area of CL occupied by steroidogenic cells (LLC+SLC; 48.4 ± 1.1) than T7 (30.2 ± 0.7) or TC (24.8 ± 0.5). In conclusion, administering eCG 2 days before the IVD withdrawal in an FTAI protocol generates a CL with a greater number of steroidogenic cells and greater area occupied by these cells.