215 COMPARISON BETWEEN CONVENTIONAL DIRECT TRANSFER FREEZING AND VITRIFICATION FOR THE CRYOPRESERVATION OF IN VIVO EMBRYOS FROM BRAHMAN CATTLE

Abstract

There is a need to develop an efficient cryopreservation technique for Brahman cattle embryos that would lead to improved success in the propagation of this breed. The objective of this study was to compare the post-thaw pregnancy rates in recipient cows after nonsurgical transfer of Brahman in vivo-derived embryos frozen in ethylene glycol (EG) and by a new vitrification (VT) method. Prior to the initial in vivo study, hatching rates were recorded 72 h post-thaw on 3 treatment groups (No treatment: 43/55, 78%; EG: 39/83, 47%; and VT: 33/103, 32%) of IVF embryos to determine if development could be achieved. This warranted Phase I: In vivo embryos were produced by multiple ovulation and nonsurgical embryo collections from Brahman cows 7 days post-estrus and AI. Embryos in morula/blastocyst stage of development were graded and maintained in holding medium (Vigro; Bioniche Animal Health, Inc., Belleville, Ontario, Canada) after collection, until randomly allocated for EG or VT protocols. Vitrification was performed using experimental media provided by a commercial company (V1: Vigro + 5 M EG; V2: Vigro + 7 M EG + 0.5 M Galactose + Ficoll; and Diluent: Vigro + 0.5 M Galactose) and by direct plunging into LN₂ (Walker et al. 2005 Reprod. Fertil. Dev. 17, 153). Freezing was performed at 0.5°C min^-1 from -6°C to -32°C, using a commercially available medium (Vigro Freeze Plus; Bioniche). All embryos were packaged in sterile 0.25-mL plastic straws to allow for direct transfer (DT). Embryos were stored in LN₂, and later thawed and nonsurgically transferred to synchronized recipient cows on Day 7 of their cycle. EG straws were air-thawed 5 s and then thawed in 30°C H₂O for 10 s. VT straws were air-thawed 10 s and then thawed in 37°C H₂O for 20 s prior to shaking them down to mix columns. Pregnancy results evaluated by ultrasound on Day 35 for the presence of a fetus from 4 replicates were 27% for EG (3/11) and 8% for VT (1/13) embryos. Problems with VT straws cracking during air thawing led to a second trial (Phase II) which changed the direct plunging technique for VT to a vapor freeze of 1 to 15 min prior to plunging into LN₂. Pregnancy results from 16 transfers were 38% for EG (3/8) and 50% for VT (4/8) embryos, with no straws cracking. Although the number of observations is small, the results obtained showed that VT-DT is satisfactory for producing pregnancies comparable to EG-DT. Further research is required to confirm the results obtained in this preliminary study, and to test whether this method will allow the successful production of pregnancies under different environmental and field conditions.