Production of live foals from sperm-injected oocytes harvested from pregnant mares.

This research article explores the use of sperm injection techniques in the fertilization of equine (horse) oocytes (egg cells). It investigates two key objectives: achieving fertilization through sperm injection, and determining the benefits of low-glucose culture media for developing early-stage equine embryos.

Context and Aims

• The study acknowledges challenges in successful in vitro fertilization in horses such as difficulty in gathering good quality oocytes, alterations in the zona pellucida (outer layer of the egg) during the maturation process in vitro, and inadequate preparation of equine sperm cells.
• Similar studies indicate that high glucose concentration in culture media can hinder embryonic development in vitro, an issue that might also affect equine embryos. Therefore, part of the study investigates the benefits of culture media containing low glucose concentrations for developing early stage equine embryos.
• The main objectives were to carry out fertilization through sperm injection and to assess the effect of media with low glucose concentration on embryo development.

Experiment 1

• In the first experiment, matured oocytes from pregnant mares were subjected to intracytoplasmic sperm injection (ICSI) or subzonal sperm injection (SUZI). Three control treatments were also carried out simultaneously.
• The results revealed better cleavage rates with ICSI (39%) compared to SUZI (6%) highlighting the effectiveness of ICSI method.
• However, an embryo produced through ICSI and transferred to a recipient mare was lost between days 16 and 18 post-ICSI, pointing to a potential issue with the early embryonic development in vitro.

Experiment 2

• In the second experiment, oocytes were subjected to ICSI and then cultured for 48 hours in two different media: TCM-199 and P-1(TM) (glucose- and phosphate-free), both supplemented with 15% fetal bovine serum.
• The experiment showed different cleavage rates in both media (47% in TCM-199 and 63% in P-1(TM)), hinting that media without glucose might be more beneficial for embryo cleavage.
• Four grade 1 embryos from this process were surgically transferred into the oviducts of four recipient mares after 48-hours from ICSI. Of these, three mares showed pregnancies by day 16 post-ICSI, verified through ultrasonography.
• Two of these pregnancies proceeded to term, resulting in the birth of two healthy fillies hence illustrating the potential of this method.