203 EQUINE EMBRYO IN VITRO DEVELOPMENT AFTER INTRACYTOPLASMIC SPERM INJECTION FOLLOWED BY CHEMICAL ACTIVATION

Abstract

Intracytoplasmic sperm injection (ICSI) is an alternative method for producing in vitro-fertilized embryos in horses. Some authors have suggested that using the piezo drill to inject the spermatozoon is required to obtain acceptable blastocyst rates after ICSI. In order to avoid the use of this equipment, the aim of our study was to evaluate 4 different chemical activation protocols and their effect on embryo development. Cumulus–oocyte complexes were recovered from ovaries of slaughtered mares. The maturation medium was DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 1 μL mL^–1 of insulin-transferrin-selenium, 1 mM sodium pyruvate, 100 mM cysteamine and 0.1 mg mL^–1 of FSH at 39°C in a humidified atmosphere of 6.5% CO₂ in air for 24 h. The ICSI was carried out in 20-μL droplets of TALP-HEPES with a 9-μm pipette, using frozen-thawed spermatozoa from 1 stallion. Spermatozoa were held separate in 100-μL droplets of Modified Whittens. Motile spermatozoa were aspirated and transferred to a 5-μL drop of 7% (v/v) polyvinylpyrrolidone, where 1 sperm was immobilized by swiping the injection pipette across its tail; then, the sperm was injected into the oocyte. All injected oocytes were subjected to 8.7 μM ionomycin for 4 min, followed by 1 of 3 further activation treatments: (1) 4-h culture in 1 mM 6-DMAP and 10 mg mL^–1 of cycloheximide, starting 3 h after ionomycin; (2) 5-h culture in 10 mg mL^–1 of cycloheximide, starting 10 min after ionomycin; (3) An extra incubation with 5 mM ionomycin for 4 min, starting 3 h after ionomycin. Some injected oocytes were left without a further activation protocol (group 4). After activation, injected oocytes were cultured in 100-μL droplets of DMEM/F12 with 5% of FBS at 39°C in a humidified atmosphere of 5% O₂, 5% CO₂ and 90% N₂. Cleavage (48 h after activation) and blastocyst formation (7–8 days) of all experimental groups were assessed. Culture medium was renewed on Day 3 with fresh DMEM/F12 with 5% of FBS. At Day 9, the zona pellucida of some blastocysts was removed and the blastocysts were maintained in culture until Day 15. Blastocyst growth was determined every 24 h. Statistical differences (using chi-square analysis) were observed in cleavage with treatments 1 and 3 when compared to the other groups (1: 30/52, 58%; 2: 8/40, 20%; 3: 9/25, 36%; and 4: 10/38, 26%). There was no difference on blastocyst rates based on injected oocytes (1: 5/52, 9.6%; 2: 2/40, 5%; 3: 1/25, 4%; and 4: 2/38, 5.3%). On Day 7, blastocyst quality did not differ among treatments and on Day 15, blastocysts from groups 3 and 4 reached 1130 μm and 4300 μm, respectively. Despite the difference observed in cleavage, this work suggests that equine blastocysts could be obtained with all of the activation protocols, without the use of the piezo drill. Further studies are required to assess the effect of chemical activation on in vivo development of produced blastocysts to confirm that they are not parthenogenetic.

We are grateful to Mr. Willem Melchior, La Vanguardia Polo Club for some financial support and encouragement to undertake this project.