Flow cytometric sorting of sperm: Influence on fertilization and embryo/fetal development in the rabbit

Abstract

Viable, intact rabbit sperm, prepared, processed, and flow cytometrically sorted, were used in this study to determine the influence of flow sorting on fertilization and embryo development. In experiment I, flow-sorted or control (unstained and unsorted) sperm were surgically inseminated into the uterine horn of hormonally primed does (10 to 12 does per time point). At 42 hr postsurgical insemination, flushed embryos were assessed for development. Fetal development was determined at day 7, day 14, and day 21 post-surgical insemination. Embryos resulting from does surgically inseminated with control sperm at 42 hr post-insemination were observed to be at the early morula stage of development (> 16 cell), whereas embryos from does inseminated with flow sorted sperm were at the 8- to 16-cell stage. No difference was observed between treatments at day 7, 14, or 21, however, there was a significant decrease in fetus number per doe inseminated with flow-sorted sperm over time. In experiment II, mature oocytes were flushed from the oviducts of superovulated does and coincubated in vitro (IVF) with flow-sorted or control rabbit sperm. Oocytes observed at 6 hr post-coincubation exhibited swollen sperm heads in the cytoplasm, demonstrating that fertilization had occurred (2 PN + T). There was a higher percentage of fertilized oocytes by 8 hr post-coincubation for both control (31%) and flow-sorted sperm (31%) when used for IVF. By 10 and 12 hr post-coincubation, little difference was observed in the number of fertilized oocytes between sperm treatments (52% and 66% for control vs. 57 and 54% for flow-sorted, respectively). These studies demonstrate that flow-sorted sperm are capable of fertilizing mature oocytes under in vitro conditions. In addition they show that flow sorting may not negatively influence fertilization events, but likely interferes during early embryonic and fetal development.

Production of Lambs After the Transfer of Fresh and Cryopreserved in vitro Produced Embryos from Prepubertal Lamb Oocytes and Unsorted and Sex‐sorted Frozen‐thawed Spermatozoa

“…However, success rates were clearly below what may be considered commercially acceptable, and there is considerable scope for improvement in some areas of the whole procedure. Oocyte fertilisation did not differ between unsorted, X-and Y-spermatozoa, in agreement with previous studies examining the IVF of IVM-adult ewe (Hollinshead et al 2003a) and in vivo matured rabbit (McNutt and Johnson 1996) oocytes, although potential subtle differences in oocyte fertilisation may not have been observed because of the relatively low numbers of oocytes examined in the present study.…”

“…Previous studies have observed that during the in vitro culture of cattle IVF-blastocysts, those derived from sorted spermatozoa displayed a 12-h delay in development compared to unsorted spermatozoa (Cran et al 1994). Delays in embryonic development after the AI of rabbits with sorted spermatozoa have also been observed by McNutt and Johnson (1996). However, there was no significant difference in development rate for blastocysts derived from sorted and unsorted frozenthawed spermatozoa in the present study (Fig.…”

Electrophoretic Gel Analysis of Hoechst 33342 Stained and Flow Cytometrically Sorted Bovine Sperm Membrane Proteins

“…The process of staining and flow-sorting may influence sperm function, although how this may occur is not clear. Sorted H042 stained sperm have a fertilization rate slightly lower than normal and embryogenesis is somewhat retarded (Johnson et al 1989;McNutt and Johnson 1996), viable offspring have, however, been obtained (Johnson et al 1989;Johnson 1991;Cran et al 1993;Cran et al 1995). This study has given indication that modifications to the sperm surface membranes are occurring but it is not clear if these modifications influence events associated with or leading to fertilization or to some other event associated with fertilization.…”

“…This study has given indication that modifications to the sperm surface membranes are occurring but it is not clear if these modifications influence events associated with or leading to fertilization or to some other event associated with fertilization. The results obtained in an earlier study (McNutt and Johnson 1996) indicated reduced fertility due to embryo mortality; whether or not modification to the sperm surface is due to sorting is unknown.…”

Flow cytometric sperm sorting: Effects of varying laser power on embryo development in swine*

“…With respect to fertilization, our data indicate that sperm cells sorted at 25 mW may be at a disadvantage after oviductal insemination compared to nonsorted and 125 mW all-sort spermatozoa. In previous studies, using standard or high speed sorter sperm cell sorting, no effect on fertilization rate was found in rabbits (McNutt and Johnson, 1996), cattle (Cran et al, 1993;Schenk et al, 1999), or in swine when in vitro fertilization conditions were optimized to accommodate sorted spermatozoa (Abeydeera et al, 1998;Rath et al, 1999). When spermatozoa from the 25 mW all-sort were used for fertilization, compared to nonsorted sperma-tozoa, the population of embryos shifted more toward 3±4 cell stages and away from 5±9 cell stages and the percentage of fragmenting embryos was increased.…”