261 FOALS PRODUCED AFTER ICSI USING FROZEN, SEX-SORTED, REFROZEN SPERM

Abstract

Obtaining adequate numbers of sex-selected sperm for uterine inseminations can be difficult, especially if fresh semen is not available or sperm numbers are limited. Intracytoplasmic sperm injection (ICSI) requires minimal sperm numbers and can be used for the assisted fertilization of oocytes. The objectives of this study were to: (1) thaw, sex-sort, and refreeze semen, and (2) determine if cleavage rates of oocytes were similar after ICSI using frozen v. frozen, thawed, sex-sorted, and refrozen sperm. The final aim was to produce foals by ICSI using sex-sorted sperm. Light-horse mares between 3 and 15 years were used as oocyte donors. When a follicle 30 to 35 mm and endometrial edema was imaged using ultrasound, deslorelin (1.5 mg, i.m., Franck’s Pharmacy, Ocala, FL, USA) was administered to induce follicular maturation. Between 20 and 24 h after deslorelin, oocytes from preovulatory follicles were collected by transvaginal, ultrasound-guided follicular aspirations. Oocytes were cultured for 16 to 18 h in TCM 199 with 10% FCS, 0.2 mm pyruvate at 38.5°C and in an atmosphere of 6% CO₂. Semen from a single ejaculate of two stallions was frozen for the experiment. Some of the frozen semen (control) was thawed and used for ICSI. Sperm from other straws were thawed, sex-sorted by flow cytometry, treated with cholesterol-loaded cyclodextrin, and refrozen in a skim milk-egg yolk diluent containing 0.52 m dimethyl formamide before thawing for ICSI. Sperm were thawed by cutting a thin section of a straw under liquid nitrogen; the straw section was dropped directly into medium at 38.5°C. Sperm were incubated for 10 min, before 1 μL of supernatant was removed and placed into a 5 μL drop of medium with 5% polyvinylpyrrolidone. From the droplet, sperm with progressive motility and normal morphology by visual inspection were selected for ICSI. Injected oocytes were placed in DMEM/F12 with 10% FCS for 48 h (±2 h) before assessment of cleavage. Numbers of cleaved per injected oocytes were compared by Fisher’s Exact Test and were lower (P < 0.001) for sex-sorted, refrozen sperm than for frozen control sperm (6/20, 30% and 15/18, 83%, respectively). At the completion of the project, three additional oocytes were injected with X-bearing sperm. The injected oocytes cleaved and developed into embryos under culture conditions [DMEM/F12 (Sigma-Aldrich, St. Louis, MO, USA) with 10% FCS at 38.5°C and an atmosphere of 5% CO₂, 5% O₂ and 90% N₂]. The resulting morula, blastocyst, and expanded blastocyst were transferred nonsurgically into recipients’ uteri. Pregnancies were established in recipients receiving the morula and expanded blastocyst, and two fillies were born in July 2008. Both foals appeared normal at birth; however, one foal became septic and was euthanized before 2 weeks of age. In this study, frozen, thawed, sex-sorted, and refrozen sperm were successfully incorporated into an ICSI program to produce early-stage embryos and sex-selected foals.