Abstract
Abstract. The investigation aimed to compare the effect of fresh and frozen-thawed X and Y fractions of flow-cytometrically sorted bovine spermatozoa on in vitro fertilization of bovine in vitro matured oocytes and subsequent blastocyst development. Sperm cells sorted in MoFloSX cytometer were used either for IVF or frozen and stored in liquid nitrogen. Immature oocytes recovered from ovaries of slaughtered animals and matured in vitro in TCM-199 containing 20% estrus cow serum and additional granulosa cells were fertilized in vitro with fresh or frozen-thawed fractions of sorted sperm. Simultaneously, control, fresh or frozen/thawed sperm was used for IVF. A total number of 2712 IVM oocytes were fertilized with sorted and control sperm of 6 bulls. Embryo cleavage rates were significantly affected by bull (P<0.0001), sperm sexing (P<0.0001) and sperm freezing (P<0.01). Blastocysts development was affected by sperm freezing (P<0.04) and sperm sexing (P<0.01). The significant differences were shown between unsorted and sorted sperm, however no differences in embryo cleavage rates and blastocysts rates were observed between X- and Y-sperm fractions, both fresh and frozen/ thawed. There were significant differences in cleavage rates among fresh, control sperm (52.7%), X fraction (26.8%) and Y fraction (24.7%). Similar differences in cleavage rates were shown for frozen/thawed control sperm (52.8%), X fraction (33.9%) and Y fraction (26.2%). The female blastocysts were frozen for further transfer, while the hatched male blastocysts were analysed by PCR revealing 76.2% accuracy. The results suggest that there were significant differences in cleavage rates and blastocyst rates due to sperm sorting in comparison to unsorted sperm and no differences between effectiveness of X and Y fractions of spermatozoa.
Highlights
Gender preselection in mammals may be carried out both by segregation of spermatozoa or by transfer of embryos with predetermined sex
Several authors dealing with in vitro embryo production in cattle (MILLER and HUNTER, 1987; AOYAGI et al, 1988; EYESTONE and FIRST, 1989; GALLI and LAZZARI, 1996; KĄTSKA et al, 1996; CHAMBERLAND et al, 2001; GALLI et al, 2003; KĄTSKA-KSIĄŻKIEWICZ et al, 2005) have described the variability among bulls in the ability of their sperm to become capacitated, fertilize oocytes and subsequently produce competent embryos developing to the blastocyst stage
Even in the case of a severe bull selection, the developmental capacity of in vitro produced embryos is still lower in comparison to their in vivo counterparts (LEIBFRIEDRUTLEDGE et al, 1987; LONERGAN et al, 2003; GALLI et al, 2003) and may be numerically further reduced when sorted sperm is used for fertilization (CRAN et al, 1993; 1994; LU et al, 1999; ZHANG et al, 2003; 2005)
Summary
Gender preselection in mammals may be carried out both by segregation of spermatozoa or by transfer of embryos with predetermined sex. In development of IVF procedure for sexed bovine sperm it seems interesting to determine the effect of sperm fraction on developmental competence of the in vitro produced embryos It has been shown in IVF experiments that male embryos develop more rapidly and with higher efficiency in comparison to female embryos (AVERY et al, 1991; DOMINKO and FIRST, 1993; WAYDA et al, 1995). The objective for these experiments was to compare the effectiveness of using fresh and/or frozen/thawed X- and Y-bearing fractions of flow-cytometrically sorted bovine spermatozoa for in vitro fertilization of bovine in vitro mature oocytes and subsequent blastocyst development
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