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

Istituto Sperimentale Italiano Lazzaro Spallanzani, Viale Forlanini 23, 20134 Milan, Italy, ¹ Laboratorio di Tecnologie della Riproduzione, Istituto Sperimentale Italiano Lazzaro Spallanzani, CIZ srl, via Porcellasco 7/f, 26100 Cremona, Italy and ² Dipartimento Clinico Veterinario, Università di Bologna, Via Tolara di sopra 50, 40064 Ozzano Emilia (Bologna), Italy

Correspondence should be addressed to R Puglisi who is now at Istituto Sperimentale Italiano Lazzaro Spallanzani, Loc. La Quercia, 26027 Rivolta d’Adda (Cremona), Italy; Email: isils{at}tin.it

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 µg/ml at a temperature of 37 °C 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 91±2%. 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 repeatabilityof sorting was obtained with frozen–thawed bull semen that was subsequently used for IVF giving rise toviable 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.