Validación de un protocolo de inyección intracitoplasmática “ICSI” de espermatozoides en la especie equina

Abstract

Intracytoplasmic sperm injection (ICSI) in oocytes is an advanced reproductive biotechnology that has shown excellent results in the equine species. This technical project aimed to validate the heterologous ICSI technique by microinjecting equine spermatozoa into bovine oocytes (in metaphase II). Simultaneously, conventional bovine in vitro fertilization (IVF) was performed as a reference procedure to compare with ICSI. The efficiency of both protocols, ICSI and IVF, was evaluated through cleavage rates. The project followed a workflow based on in vitro embryo production (IVP) with the following steps: 1) in vitro maturation (IVM) of bovine oocytes; 2a) homologous IVF using bovine spermatozoa; 2b) heterologous ICSI using equine spermatozoa; and 3) in vitro culture (IVC). Prior to performing ICSI, pneumatic microinjectors, three-dimensional micromanipulators, needles, clamps, and the holding pipette were carefully adjusted. Equine spermatozoa, previously thawed and selected, were immobilized in a PBS solution containing 10 % PVP and subsequently aspirated into the ICSI needle. The injection of spermatozoa into denuded oocytes was performed using the holding pipette. After microinjection, the oocytes were chemically activated with ionomycin (10 µM) for 5 minutes. At day 5 of in vitro culture, cleavage rates of embryos obtained through IVF and ICSI were evaluated using epifluorescence microscopy. Results showed that heterologous ICSI (n = 47) achieved a cleavage rate of 26.67 %, while homologous IVF (n = 22) achieved a rate of 52.38 %. In conclusion, the heterologous ICSI protocol using bovine oocytes and equine spermatozoa was validated, achieving successful cleavage rates. Future studies are recommended to focus on homologous ICSI procedures in the equine species to evaluate their efficiency.