47 COMPARISON BETWEEN CRYOLOOP AND OPEN PULLED STRAW VITRIFICATION METHODS FOR BOS INDICUS BLASTOCYSTS

Abstract

A major obstacle of large-scale commercial application of bovine in vitro fertilization is the lack of a suitable cryopreservation method for supernumerary embryos produced. The traditional slow-freezing method has proven to be effective for embryos of a wide range of mammalian species; however, the formation of intracellular ice is still a challenge and the efficiency needs to be improved. Over the past decade, several advances have taken place in vitrification technologies, such that it can provide high efficiency with better pregnancy outcome due to its high cooling rates and the lack of crystals formed inside the cells. Most vitrification methods have been evaluated in Bos taurus cattle but more still remains to be investigated in Bos indicus races predominant in the tropics. There are several vitrification protocols and holders, including CryoLoop, open pulled straw (OPS), MS Grids, and Cryotop, among others. The CryoLoop method uses a nylon loop attached to a metal Cryovial lid were blastocysts are placed on an equilibration solution film. CryoLoop cooling rates are approximately 20.000°C min^–1 and have shown very good results in humans. The OPS is a well-known support for bovine blastocysts; the embryos are taken by capillarity into the OPS and use a 1- to 2-μL drop of final equilibration solution. Cooling rates using this method are approximately 2.000°C min^–1. The aim of this work was to prove CryoLoop and OPS vitrification methods in Bos indicus blastocyst and compare re-expansion and hatching rates 24 h after warming. Ovaries were collected from a local slaughterhouse and cumulus-oocyte complexes (COC) were treated for the standard IVF method. A total of 60 blastocysts were vitrified in CryoLoops and 68 blastocysts in OPS (within 4 repeats). For CryoLoops, groups of 2 blastocysts were placed in a solution of 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 3 min, and then were placed in a solution of 15% EG, 15% DMSO, 10 mg mL^–1 of Ficoll 70, and 0.65 M sucrose for 20 s, and rapidly were put into the nylon loop and taken to the LN. For OPS, groups of 2 to 3 blastocysts were placed in a solution of 10% EG and 10% DMSO for 1 min, and then were placed in a solution of 20% EG and 20% DMSO for 20 s, and rapidly were taken by capillarity into the OPS and taken to the LN. Thawing was the same for both treatments; vitrified blastocysts were taken out from the LN and rapidly put into a solution of 0.3 M sucrose for 2 min and then put into a solution of 0.2 M sucrose for 3 min, were washed twice in TCM199 supplemented with 10% FCS, and cultured for 24 h in CR1aa media. Data were analysed using the R language. Media comparison for proportions was done using a chi-squared test. No significant difference was observed in re-expansion or hatching rates between CryoLoop and OPS supports (P = 0.01 for both); however, the CryoLoop method showed more efficiency than OPS in re-expansion rate (65 v. 44.4%, respectively) and hatching rate (30.8 v. 20%, respectively). In all cases, the CryoLoop method showed much better outcomes. The results indicate that vitrification in CryoLoops is a suitable method for cryopreservation of Bos indicus blastocysts.