371 INTRACYTOPLASMIC SPERM INJECTION OF EQUINE OOCYTES USING AIR-DRIED SPERM OR SPERM STORED IN A HIGH OSMOLARITY MEDIUM

Abstract

The generally accepted method of long-term sperm preservation is freezing in liquid nitrogen. However, it is not always available. Other techniques have shown to preserve sperm for a short period of time that can be used for intracytoplasmic sperm injection (ICSI). Mouse offspring have been produced after ICSI with sperm stored in a high osmolarity medium. Also, human embryos were obtained by ICSI with air-dried sperm. Recently, equine blastocysts have been obtained by ICSI with lyophilized sperm. In our study, cleavage rate was evaluated after ICSI of equine oocytes using air-dried sperm or sperm stored in a high osmolarity medium. Oocytes were obtained from slaughterhouse ovaries by scraping individual follicles and transported in a portable incubator at 38°C for 15 h in TCM-199 buffered with HEPES and supplemented with glutamine, sodium pyruvate, LH (Bioniche Animal Health, Inc., Beltville, Ontario, Canada), FSH (Bioniche Animal Health, Inc.), epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), and 10% FBS (GIBCO, Grand Island, NY, USA). On arrival, they were cultured for 6 more hours in 5% CO₂ in air at 38°C in microdroplets of the same medium, without HEPES. Sperm was collected from 2 stallions of proven fertility. The ejaculate was diluted in Kenney extender, centrifuged, and the pellet was resuspended in HEPES-TALP (H-TALP). Three sperm treatments were used: (C) Control: ejaculated motile sperm processed as described above; (1) Air-dried sperm: obtained from spreading sperm on a sterile slide and drying it for 10 min in a laminar flow chamber; (2) Sperm in a high osmolarity medium: ejaculated motile sperm resuspended in H-TALP with high osmolarity (800 mOsmol). Samples from groups 1 and 2 were stored at 5°C for 2 to 3 days before being used for sperm injection. Only oocytes with an intact cytoplasm and a visible polar body were selected for injection and randomly assigned to each experimental group. Each MII oocyte was injected with 1 sperm cell and activated in Ionomicin for 10 min and DMAP for 3 h. Injected oocytes were cultured in DMEM : F10 1 : 1 with 10% FBS at 38°C in 7% O₂ and 5% CO₂ for 48 h. The number of cleaved embryos was recorded. Data was analyzed by chi-square test. A total of 135 MII oocytes were injected. The cleavage rate in group 1 was significantly lower than in the control group (31/71, 43.66% vs. 28/38, 73.68%) (P < 0.05). No differences were observed between group (2) and control (19/29, 73.07% vs. 28/38, 73.68%) (P > 0.05). This is the first report of equine oocytes fertilized by ICSI with air-dried sperm or with sperm kept in high osmolarity medium. These simple sperm preservation techniques might be an alternative option when liquid nitrogen is not available. Further studies will determine if it is possible to obtain pregnancies or even healthy offspring.