169 Use of stored zonae pellucidae from young and old mares to study sperm–oocyte binding

Abstract

Natural and standard IVF requires sperm binding to the zona pellucida (ZP); however, difficulties in obtaining equine ZP limit research in this area. In a previous study, an in vitro assay was used to assess ZP sperm binding for oocytes collected from old and young mares’ dominant follicles, with similar numbers of sperm bound per ZP (means of 45.5 and 58.1 sperm per ZP; P = 0.12) (Lupole et al. 2010 Anim. Reprod. Sci. 121, 252-253). We hypothesised that ZP from uncleaved equine oocytes after intracytoplasmic sperm injection (ICSI) could be used to study ZP sperm binding. For this study, oocytes from mares >20 years (old, n = 13 oocytes) and 4 to 12 years (young, n = 16 oocytes) were collected by transvaginal, ultrasound-guided aspirations and injected with a single sperm. Only oocytes that failed to cleave into at least 2 cells within 2 days after ICSI were used. The oocytes were stored in hyperosmotic salt solution at 4°C for 2 years. The sperm binding assay was performed similar to the previous study (Lupole et al. 2010). Oocytes were removed from salt solution and placed into TALP and incubated for 1 h at 38.5°C in 6% CO₂ and air. Following incubation, oocytes were moved to a 45-µL drop of TALP. Frozen-thawed sperm (100,000 total sperm) from one stallion of good fertility were added and incubated for 2 h. Oocytes were then washed through 4 drops of TALP using a pipette tip with a 175-µm inner diameter to remove loosely bound sperm. Oocytes and tightly bound sperm were stained in 4’6-diamidino-2-phenyindole (DAPI; 10 µg mL⁻¹) for 15 min. The oocytes were secured on a glass slide under a coverslip that was supported by a mix of paraffin and petroleum jelly and viewed at 400× magnification using an epifluorescence microscope (Nikon Eclipse E800, Nikon, Tokyo, Japan). The number of tightly bound sperm was recorded. Sperm counts for age groups were compared using Student’s t-test, with data presented as mean ± s.e.m. Sperm binding was not different between groups (59.2 ± 16.9 for old mares and 36.8 ± 10.6 for young mares; P = 0.25). As previously observed with oocytes that had not been injected with sperm and minimal time in salt storage, we found no significant effect of mare age on sperm binding to the ZP. In addition, numbers of sperm bound to the ZP were similar between projects. Our findings suggest that equine oocytes that fail to cleave after ICSI could provide a valuable source of ZP for research. Further work is ongoing to determine the effect of time that oocytes are stored in salt solution on sperm binding.