Cryopreservation and Sexing of <i>In Vivo</i>- and <i>In Vitro</i>-Produced Bovine Embryos for Their Practical Use

Abstract

Abstract. My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GLTip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of 0.1-1.0% deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-PCR and purification of DNA product before standard sexing PCR of biopsy samples from Day 3-4 in vitro-derived embryos allowed accurate sex determination, and Day-7 blastocysts developed from Day 3-4 embryos were cryopreserved by GLTip vitrification without a loss of their viability. Thus the field application of bovine embryo transfer is in part supported by improvements of technologies in embryo cryopreservation and sex predetermination.

A deterministic simulation study of embryo marker-assisted selection for age at first calving in Nellore (Bos indicus) beef cattle

“…There are various reports of livestock pregnancy rates of 30% to 60% when using biopsied embryos, similar to the rates obtained with intact embryos and considered sufficient for commercial applications (Agca et al, 1998, Chrenek et al 2001, Garcia, 2001, Lopes et al 2001, Park et al 2001, Chen et al 2002, Tominaga, 2004, Tominaga and Hamada, 2004.…”

Conception rate after sex determination and cryopreservation of D7 bovine embryos:

“…The embryos that reached the blastocyst stage earlier were predominantly males and these male embryos had better morphology and subsequently hatched at a greater rate than female embryos. PCR analysis for sexing of in vivo produced D 7 embryos indicated that male embryos developed faster and were graded higher than female embryos (Tominaga, 2004). The sex ratio was significantly shifted to the male IVP embryos also in more recent publications (Kimura et al, 2008;Alonso et al, 2009).…”

“…The freeze-thawingbiopsy protocol rendered again, with both cryoprotectants, satisfactory pregnancy rates (about 50%). Also the ultra-rapid vitrification, using the gel-loading tip (Tominaga, 2004), is acceptable for embryos that underwent the biopsy and method is available to practical use in the field.…”

Efecto del ácido linoleico conjugado sobre la proporción de sexos y calidad de embriones bovinos producidos in vitro

“…Dentro de los suplementos, se sabe que las moléculas que modifican los ácidos grasos en la membrana afectan diferentes funciones estructurales, celulares e incluso vías de señalización (Spector y Yorek 1985, Talukdar y col 2005. Se ha informado que el uso de PUFA como el ácido linoleico (C18:2n-6) (Tominaga 2004), ácido araquidónico (C20:4n-6) y eicosapentaenoico (C20:5n-3) (Marques y col 2007) en los medios de cultivo han demostrado proporcionar mayor fluidez y versatilidad a las membranas (Zeron y col 2002, Spector y Yorek 1985, lo que estaría relacionado con una mejor calidad embrionaria y mayor criotolerancia (Marei y col 2010).…”