60 VITRIFICATION AND CONVENTIONAL CRYOPRESERVATION OF EQUINE EMBRYOS

Abstract

Vitrification and conventional cryopreservation are effective methods of preserving equine embryos smaller than 300 μm in diameter. This study was designed to compare pregnancy rates using these methods to cryopreserve embryos of similar size. Sport horse mares approximately 2–20 years old were flushed nonsurgically between Days 6.5 and 7 post-ovulation with 2 L of lactated-Ringers solution (Braun, Melsungen, Germany). Thirty-one embryos were collected, washed 4 times with 1 mL of ViGro® holding medium (Bioniche Animal Health, Bogart, GA), graded for quality, measured for diameter, and blocked into 2 groups (<200 μm, 200 to 300 μm). Embryos were either vitrified with a commercial equine vitrification kit (Bioniche Animal Health) according to the manufacturer’s instructions in 0.25-mL straws or subjected to a slow cooling method. For vitrification, embryos were sequentially transferred to 2 wells containing 0.5 mL of 2 Syngro®-based vitrification solutions (VS1 and VS2) and held for 5 min each. Embryos were incubated in a third vitrification solution (VS3) for 45 s during which time they were loaded into straws. Straws were held in liquid nitrogen-cooled air for 1 min before submersion in liquid nitrogen. For slow freezing, embryos were consecutively placed into 0.5 mL of the following Syngro®-based solutions for 5 min each: 1.8 m glycerol, 1.8 m glycerol + 1.8 m ethylene glycol (EG), and 0.9 m glycerol + 0.9 m EG + 0.5 m galactose. Embryos were loaded into 0.25-mL straws, placed in a chamber pre-cooled to –6°C, and held for 10 min. Straws were seeded after 2 min. The temperature was lowered to –32°C at a rate of 0.5°C min–1. Embryos were then plunged into liquid nitrogen within 3 min of reaching –32°C. For warming vitrified embryos, straws were held in air for 10 s followed by submersion into a 35°C water bath for 20 s. Straws were flicked 5 times to mix the diluent solution with the VS3-containing embryos, which were transferred within 7 min of being thawed. For thawing conventionally frozen embryos, straws were held in air for 10 s followed by submersion into a 35°C water bath for 30 s. Contents of the straw were immediately expelled into a Petri dish, and the embryos were transferred immediately to 0.5 mL of 1.2 m glycerol +1.2 m EG + 0.5 m galactose and held for 5 min. This was followed by a 5-min incubation in 0.5 mL of each of the following solutions: 0.6 m glycerol + 0.6 m EG + 0.5 m galactose, 0.25 m glycerol + 0.25 m EG + 0.5 m galactose, and 0.5 m galactose. After exposure to the last solution, embryos were transferred to Syngro®, loaded into a straw, and immediately transferred into 2-year-old virgin recipients 6 days after ovulation as detected by rectal palpation and ultrasonography. Of the 21 embryos <200 μm collected, 11 were vitrified and 10 frozen slowly. Three of these 11 vitrified embryos and 7 of 10 slow-frozen embryos resulted in Day 16 pregnancies (27 and 70%, respectively). None of the embryos >200 μm resulted in pregnancies in either the vitrification (n = 5) or slow-freeze treatments (n = 4).