In vitro fertilisation with frozen–thawed bovine sperm sexed by flow cytometry and validated for accuracy by real-time PCR

Abstract

The methodologies used for cytometric sorting of fresh spermatozoa never allowed a clear resolution of sexual chromosomes of frozen-thawed semen. To devise a novel method for the production of bovine predefined sexed embryos using frozen-thawed semen, sorting efficiency of different protocols was studied using a new quantitative real-time PCR method to verify the purity of sexed semen. To this aim, after Percoll separation, frozen-thawed samples were stained at different temperatures and concentrations of Hoechst 33342 using a short-incubation time. The concentration of Hoechst 33342 of 500 mg/ml at a temperature of 37 8C provided good and stable fluorescence signals. Preventing the sperm clustering by adding 0.6% BSA in the 90% Percoll fraction led to X-bearing sperms purity of 91G2%. Thereafter, sorted sperms were used for in vitro fertilisation (IVF). Despite the lower cleavage rates reported in the sorted groups when compared with the control groups (40 vs 48%, P!0.01), blastocyst formation in the sorted and control groups was not different (27 vs 24% of the cleaved respectively). The PCR analysis of 30 blastocysts confirmed 26 embryos to be correctly sexed (87%). Transfer of two embryos per recipient into six synchronised heifers resulted in four pregnancies. Two abortions occurred at day 60, while two pregnancies went to term delivering two female calves. In conclusion, high purity and repeatability of sorting was obtained with frozen-thawed bull semen that was subsequently used for IVF giving rise to viable embryos and offspring. In addition, real-time PCR revealed to be an optimal support for these studies, providing a rapid and reliable estimation of flowcytometric efficiency.

In vitro and in vivo quality of bovine embryos in vitro produced with sex-sorted sperm

“…In vitro fertilization with sexed sperm has been approached with different sperm gradient separation techniques [6], modifying heparin concentrations, adding penicillamine, hipotaurine, and epinephrine (PHE) [7], or by means of new sperm cryopreservation procedures [8]. However, after fertilization with sex-sorted spermatozoa, lower fertility rates have been observed both in vivo [9 -13] (after AI) and in vitro [14 -16].…”

“…Moreover, ultrastructural studies showed that blastocysts produced with flow-cytometrically sexsorted spermatozoa have a lower quality than their nonsorted counterparts [19]. Puglisi and coworkers [6] described a minor tolerance of the sexed frozen-thawed sperm to the stress, making the manipulation procedures important during IVF. Although these kinetic and morphologic alterations may be responsible for the compromised development observed in embryos produced with sex-sorted spermatozoa, we could hypothesize that embryo development could be improved by using an optimized postfertilization culture.…”

Comparative validation using quantitative real-time PCR (qPCR) and conventional PCR of bovine semen centrifuged in continuous density gradient

“…This can be explained by the effect of some components found in Percoll, such as silica and PVP, which may have interfered with the DNA extraction process or amplification by qPCR, despite successive washes. There have been reports that Percoll at 80% concentration produced great variability during sexing sperm by flow cytometry (Stap et al, 1998;Puglisi et al, 2006). In the present study, centrifuged sperm were recovered in a fraction containing 90% Percoll, which theoretically could have affected qPCR results, as seen in sexing by flow cytometry.…”

“…However, OptiPrep was not effective to separate X sperm. The qPCR has recently been used as a reliable technique for the quantification of X and Y sperm cells in semen samples, especially to validate techniques for sexing sperm (Joerg et al, 2004;Parati et al, 2006;Puglisi et al, 2006).…”

“…Furthermore, it was possible to quantify the proportion of X and Y sperm in semen samples before centrifugation without any adjustments by using linear regression, as described in other studies (Parati et al, 2006;Puglisi et al, 2006).…”