88 ESTABLISHMENT OF PREGNANCIES AFTER VITRIFICATION OF EQUINE EMBRYOS

Abstract

Vitrification of equine embryos has been limited, with only one study reporting transfer of vitrified embryos into recipients (1994 Theriogenology 42, 483–488). The objective of this study was to vitrify equine embryos in different stages of development based on a protocol that had been successful in sheep (Naitanas et al., 1996 Theriogenology 46, 813–824) and buffalo (2001 Theriogenology 55, 307). Embryos, Days 6.5 to 7.5, were assigned to a 3 (stage) × 2 (media) factorial design (n = 3 per group) based on diameter and developmental stage [(1) morulae + very early blastocysts; (2) blastocysts <300 μm; (3) blastocysts >300 μm] and base media [(1) PBS supplemented with 0.3 mM pyruvate, 3.3 mM glucose and 10% FCS or (2) HCDM (Olson SE and Seidel GE Jr. 2000 J. Anim. Sci. 78, 152–157) with Eagle’s essential and nonessential amino acids, 2 mM glucose, 3 mg mL⁻¹ BSA and 20% FCS]. Embryos were placed sequentially in 200 μL of 1.4 M glycerol for 5 min, 200 μL of 1.4 M glycerol + 3.6 M ethylene glycol for 5 min, and 30 μL of 3.4 M glycerol + 4.6 M ethylene glycol. Within 30 s, the 30-μL drop containing the embryo was loaded into the center of a 0.25-mL straw separated by air from columns (30 μL) of the same solution. Straw ends were loaded with columns (60 μL) of 0.5 M galactose in base medium. The straw was heat-sealed and exposed to liquid-nitrogen vapor for 1 min before being plunged into liquid nitrogen. To warm embryos, straws were held in air for 10 s and then immersed in 20°C water for 10 s. Embryos were transferred into 200 μL of 0.25 M galactose solution for 5 min, and then placed in PBS or HCDM. Within 10 min, embryos (n = 3 per recipient), grouped for stage and media, were transferred nonsurgically into the uteri of 6 recipients that had ovulated 6 days earlier. Ultrasound exams were done at 4, 6, 8 and 10 days after transfer. Three morulae or very early blastocysts and one blastocyst <300 μm, all vitrified in PBS, formed embryonic vesicles. Two of 3 vesicles, in the same recipient, were manually removed on Day 16. The remaining vesicle and the single vesicle, from a blastocyst <300 μm, had normal development until Day 38 when pregnancies were terminated. No pregnancies were observed on Day 16 after use of HCDM. Because numbers of embryos were low, it is not clear if HCDM had a detrimental effect (P > 0.1). Embryos were transferred in October, at the end of the breeding season. Therefore, quality of available recipients could have affected pregnancy rates. No pregnancies were observed from expanded blastocysts. Potentially, the embryonic capsule, present at this stage, impeded diffusion of cryoprotectants. These preliminary results demonstrate the potential for vitrification of small equine embryos.