Effects of bovine spermatozoa preparation on embryonic development in vitro

The aim of our research was to examine the ability of density gradient preparation BoviPure(® )and swim up method on bull sperm separation and in vitro embryo production (IVP) systems. Frozen/thawed semen from six Simmental bulls was pooled and treated using both methods. The sperm motility, concentration, membrane activity, membrane integrity and acrosomal status were evaluated and compared before and after sperm processing using BoviPure(® )and swim up methods. We also evaluated and compared cleavage rates, embryo yield and quality between the methods. There were significant differences (P < 0.05) between the sperm characteristics before and after BoviPure(®), but not after swim up method. However, there were significant differences for sperm results among those two mentioned methods. A total of 641 oocytes were matured and fertilized in vitro and cultured in SOFaaBSA. The percentage of cleavage (Day 2) and the percentage of hatched embryos (Day 9) were similar for both methods. However, embryo production rate (Day 7) was significantly higher using BoviPure(® )method (P < 0.05). Also, total cell number and embryo differential staining (inner cell mass and trophectoderm cells) of Day 7 morulas and blastocysts showed that BoviPure(® )treated sperm displayed higher quality embryos compared to swim up method (P < 0.05). Our results indicate that BoviPure(® )method has an enhanced capacity in sperm selection for in vitro embryo production when compared with swim up method. So, we concluded that BoviPure(® )could be considered as a better alternative to swim up method for separating bull spermatozoa from frozen/thawed semen for IVP of bovine embryos

Optimization of IVF pregnancy outcomes with donor spermatozoa

Purpose: To identify risk factors for suboptimal IVF outcomes using insemination with donor spermatozoa and to define a lower threshold that may signal a conversion to fertilization by ICSI rather than insemination. Method: Retrospective, age-matched, case-control study of women undergoing non-donor oocyte IVF cycles using either freshly ejaculated (N = 138) or cryopreserved donor spermatozoa (N = 69). Associations between method of fertilization, semen sample parameters, and pregnancy rates were analyzed. Results: In vitro fertilization of oocytes with donor spermatozoa by insemination results in equivalent fertilization and pregnancy rates compared to those of freshly ejaculated spermatozoa from men with normal semen analyses when the post-processing motility is greater than or equal to 88%. IVF by insemination with donor spermatozoa when the post-processing motility is less than 88% is associated with a 5-fold reduction in pregnancy rates when compared to those of donor spermatozoa above this motility threshold. When the post-processing donor spermatozoa motility is low, fertilization by ICSI is associated with significantly higher pregnancy rates compared to those of insemination. Conclusion: While ICSI does not need to be categorically instituted when using donor spermatozoa in IVF, patients should be counseled that conversion from insemination to ICSI may be recommended based on low post-processing motility

Treatment of human sperm with serine protease during density gradient centrifugation

PURPOSE: Seminal pathogens can bind specifically or non-specifically to spermatozoa, rendering semen decontamination procedures ineffective, whereby vertical or horizontal transmission of the infection could occur. Serine proteases have been demonstrated to effectively inactivate viruses and to break pathogen-sperm bonds. However, the addition of a protease to density gradient layers during semen processing could negatively impact on sperm parameters. This study investigated the effect of the addition of a recombinant, human-sequence protease (rhProtease) on sperm parameters during density gradient centrifugation. METHODS: (i) Pooled semen samples (n = 9) were split and processed by density gradient centrifugation, with the top density layers supplemented, or non-supplemented with rhProtease at three different concentrations (diluted 2, 10 and 20 times). Sperm parameters were then analysed by flow cytometry and computer-assisted semen analyses. (ii) Semen samples (n = 5) were split and similarly processed using PureSperm® Pro, with rhProtease in the 40 % density gradient layer, or standard PureSperm® not supplemented with rhProtease (Nidacon, International) respectively. The Hemizona assay was then utilized to compare sperm-zona binding post processing. RESULTS: Evaluation of sperm parameters indicated that rhProtease did not, at any of the tested concentrations, have an impact on (i) mitochondrial membrane potential, vitality, motility, or (ii) zona binding potential. CONCLUSION: We report that the addition of rhProtease to density gradients is a non-detrimental approach that could improve the effectiveness of semen processing for the elimination of seminal pathogens, and benefit assisted reproduction outcome