Effect of quality of sperm chromatin structure on in -vitro production of cattle embryos

Abstract

Abstract. Bull effect on results of in vitro embryo production has been well documented. The aim of the present study was to find the relationship between quality of bull sperm chromatin and its effect on in vitro embryo production. Bovine in vitro matured oocytes were fertilized in vitro using capacitated spermatozoa (freshly ejaculated or frozen-thawed) of 12 bulls. Semen was simultaneously processed according to the sperm chromatin structure assay (SCSA) method and was analysed by flow cytometry. At least 3 replications of IVP with the same semen sample were done. The percentage of spermatozoa with abnormal chromatin ranged from 0.4% to 23.8%. All bulls used for the experiment were divided into three groups showing minimal (0.82% ± 6.82%), low (1.70% ± 15.82%) and high (18.16% ± 53.59%) percentages of spermatozoa with abnormal chromatin structure. Both cleavage rates and embryo development to the blastocyst stage were correlated significantly with sperm chromatin abnormalities and resulted in 23.1, 17.7 and 12.2% of blastocysts respectively for sperm with minimal, low and high percentages of chromatin abnormalities. The SCSA method may be used as a practical indicator of suitability of bull ejaculate for IVP purposes.

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

“…Discussion An attractive feature of IVF is that fewer sexed sperm are needed than for artificial insemination. In our preliminary trials aiming to develop the procedure for capacitation of flow cytometrically sorted sperm (data not shown) it has been found that standard capacitation protocol used in our laboratory for frozen/thawed sperm (KĄTSKA-KSIĄŻKIEWICZ et al, 2005) might be successfully applied for fresh sperm, both sorted and unsorted. However, the sorted and then frozen/thawed fractions of sperm poorly survived after the capacitation treatment and, moreover, only few sperm could be recovered following Percoll's separation.…”

“…However, the applied modification limiting preparation of these fractions only to a centrifugation of thawed sperm did not allow removing dead spermatozoa that had been damaged following the cryoconservation procedure, which might exert a detrimental effect on fertilization results. 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. In terms of factors affecting the outcome of IVF, bull variability has been the main source of difficulty in the work.…”

“…Reanalysis revealed a satisfactory purity of sorted fractions oscillating between 81 to 82 %. The standard protocol of sperm capacitation (KĄTSKA-KSIĄŻKIEWICZ et al, 2005) was applied for both control, fresh and frozen/thawed sperm and for fresh fractions of sexed sperm. Briefly, -sperm was separated on a discontinuous Percoll (Pharmacia, Uppsala, Sweden) density gradient (1 ml 45% Percoll over 1 ml 90% Percoll) by centrifugation for 30 min at 300 x g at room temperature, washed in calcium ion-free TALP medium (PARRISH et al, 1988) and pelleted by centrifugation at 100 x g for 10 min.…”

“…The only successful method of sexing spermatozoa is quantifying sperm DNA-binding dye, followed by flow cytometry and cell sorting. The previous experiments on bovine IVF (MILLER and HUNTER, 1987;AOYAGI et al, 1988;EYESTONE and FIRST, 1989;KĄTSKA et al, 1996;CHAMBERLAND et al, 2001;KĄTSKA-KSIĄŻKIEWICZ et al, 2005) have shown that in terms of factors affecting the outcome of the procedure bull variability has appeared to be the main source of differentiation in the efficiency of this technology. When sexed sperm is used the efficiency of IVF may be affected not only by bull effect but by differentiated susceptibility of bull spermatozoa to Hoechst 3342 staining as well (SMORĄG et al, 1999).…”

“…When sexed sperm is used the efficiency of IVF may be affected not only by bull effect but by differentiated susceptibility of bull spermatozoa to Hoechst 3342 staining as well (SMORĄG et al, 1999). The differentiated susceptibility of spermatozoa to the vital staining might be conditioned not only by specific bull effect but by differentiated sensitivity of sperm chromatin to denaturation as well (BOCHENEK et al, 2001;KĄTSKA-KSIĄŻKIEWICZ et al, 2005). 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.…”