123 QUANTIFICATION, MORPHOLOGY AND VIABILITY OF EQUINE PREANTRAL FOLLICLES OBTAINED VIA BIOPSY PICKUP METHOD

Abstract

To study early follicular development in the equine species, a biopsy pickup (BPU) method was tested to determine the feasibility of retrieving preantral follicles from mare ovaries in vivo. The hypotheses tested in this study were that (1) preantral follicle quantity, viability and morphology do not differ according to stage of the oestrous cycle; (2) rate of atresia increases with follicle class; and (3) the BPU method provides sufficient material for studies on the early stages of folliculogenesis in mares. The transvaginal, ultrasound-guided BPU device was a 48-cm-long automated, spring-loaded device with an inner trocar point plunger containing a 15-mm specimen notch surrounded by an outer 16 G cutting needle (US Biopsy, Franklin, IN, USA). A total of 33 ovarian biopsy procedures were performed on 19 mares during the breeding season. Mares were 5 to 21 years of age and biopsies were performed during oestrus, diestrus, or both as confirmed by transrectal ultrasonography. Follicles were isolated mechanically using a tissue chopper and were analysed for either viability (live vs dead using trypan blue dye to analyse cell membrane integrity) or morphological normality (intact oocyte surrounded by granulosa cells that are well organised in 1 or more layers). Statistical analyses were performed using Fisher's exact test, paired t-test and one-way ANOVA. A total of 256 biopsy attempts were made, resulting in 185 successful tissue sample collections (72% success rate). The mean weight of ovarian tissue collected per procedure was 25 mg. An average of 18.8 ± 1.9 (mean ± s.e.m.) follicles were isolated per biopsy procedure. Overall, 620 preantral follicles were collected and isolated (95% primordial and 5% primary). Primordial and primary follicles had an average diameter of 31.3 ± 6.2 μm and 41.1 ± 6.6 μm, respectively. Viability rate was higher (P < 0.001) for primordial follicles (91%) when compared with primary follicles (50%), with an overall follicle viability rate of 88%. Primordial follicles tended (P < 0.06) to have a higher rate of morphological normality (96%) when compared with primary follicles (80%). The total number of follicles isolated, amount of tissue harvested and number of follicles per milligram of tissue did not differ (P > 0.05) according to reproductive status. Younger mares (ages 5 to 7) had more (P < 0.05) follicles isolated per procedure than older mares (ages 14 to 21). The length of the interovulatory interval was not affected (P > 0.05) by the biopsy procedure. No adverse effects were observed in cyclicity or general health following any procedure. The results supported the hypotheses tested. In conclusion, the BPU method provides material for the study of early follicular development in the equine species. In the future, the BPU method might be a feasible technique to obtain preantral follicles for in vitro culture to enable the use of numerous oocytes present within the equine ovary to preserve genetic material or for large-scale embryo production.