In vitro production of bovine embryos using flow-cytometrically sexed sperm

Abstract

The investigation aimed to compare the effect of fresh and frozen-thawed X and Y fractions of flowcytometrically 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.

Sex determination in bisected bovine embryos and conception rate after the transfer of female demi-embryos

“…Sperm sexing and separation into X-and Y-bearing fractions for purposes of insemination or in vitro production of embryos (IVP) also seems to be a promising procedure in sex determination. However, actual publications show that the procedure for the capacitation of flow cytometry sorted sperm might be successfully applied for fresh sperm in IVP programs (Katska-Ksiazkiewicz et al, 2005) but the sorted frozen/thawed fractions of sperm poorly survived after the capacitation treatment (Katska-Ksiazkiewicz et al, 2006). Moreover, only few spermatozoa could be recovered following Percoll's separation.…”

Semen Bovino Sexado Congelado-Descongelado en Producción de Embriones In vitro

“…Coinciden los resultados en el desarrollo embrionario al utilizar el semen del toro 1, en el toro 2 no hay diferencias ni en el clivaje, ni en el desarrollo embrionario. Kastska-Ksiaz kiewicz et al (2006), utilizando SX, semen sexado Y (SY) y NS (congelado-decongelado), reportan diferencias significativas en la tasa de clivaje del NS respecto al semen sexado (X e Y), sin embargo el desarrollo embrionario a blastocistos fue similar para el NS y el SX, lo cual coincide con el desarrollo embrionario en el toro 2. Puglisi et al (2006), estudiando el comportamiento en la FIV de SX y NS provenientes de diferentes toros, reportan una menor tasa de clivaje para el SX, no encontrando diferencias significativas en el desarrollo embrionario.…”

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

“…However, recent results show that the capacitation of sorted freezethawed fractions of sperm and the developmental capacity of IVP embryos with desired sex is very low (Zhang et al, 2005;Katska-Ksiazkiewicz et al, 2006;Blondin et al, 2009). The pregnancy rates with low doses of sexed sperm drop about 10-40% compared to conventional doses of unsexed sperm (Andersson et al, 2006;Schenk et al, 2006Schenk et al, , 2009Seidel Jr., 2007;DeJarnette et al, 2009).…”