Experiments to improve the quality of sex-sorted fresh and frozen porcine spermatozoa

Experiments to improve the quality of sex-sorted fresh and frozen porcine spermatozoaFor several reasons the demand for sex sorted porcine spermatozoa has been expressed. This includes advantages for herd management, improved production efficiency, foreseeable restrictions of male castration and gene preservation programmes. Due to the high costs for the sex-sorting process and the reduced storage time in liquid state, freezing of sex-sorted spermatozoa in liquid nitrogen would be very advantageous if the post thawing sperm quality would be acceptable. Therefore, the ultimate goal of the present study was to adapt existing freezing methods for porcine spermatozoa to the post sort processing of sexed spermatozoa. In two chapters five trials were conducted, in order to improve the quality of frozen- and sex-sorted spermatozoa. In presence of Sodium Pyruvate, the addition of the ROS scavengers Catalase and Mercaptoethanol, and the Vitamin E analogue Trolox to cooling and freezing media of boar semen was tested. Frozen spermatozoa were incubated for up to six hours after thawing and motility, morphological status, membrane integrity, and acrosomal status via triple staining in a flow cytometer (SYTO-17/FITC-PNA/PI) were recorded. In a second trial the influence of individual freezing curves for four boars were tested. Frozen spermatozoa were incubated at 38°C for up to six hours after thawing and motility as well as the morphological status were recorded. Also membrane integrity and acrosome status via triple staining in a flowcytometer (SYTO-17/FITC-PNA/PI) were recorded 0 and 2 hours after thawing. In the second chapter the addition of Pyruvate, Catalase and Mercaptoethanol to media for fresh semen preservation and to cooling and freezing media for cryopreservation were tested. It was also tested if sex-sorted and frozen-thawed porcine spermatozoa can be used for surgical insemination into the tip of the uterine horn. Initially fresh and sex-sorted semen were stored for up to five days with or without the addition of Pyruvate, Catalase and Mercaptoethanol to the storage media. Semen samples were put in a thermo resistance test at 38°C for two hours after storage at 15°C for 0, 24 and 120 hours and the motility as well as the morphological status after 120 hours were recorded. In a subsequent trial two different concentrations of Pyruvate, Catalase and Mercaptoethanol were added to cooling and freezing media in two different sperm concentrations (40 and 80 x 106 sperm per ml), and motility as well as the morphological status were recorded in a thermo resistance test. Also the membrane integrity and acrosome status were recorded employing a flow cytometrical analysis with a triple staining protocol (SYTO-17/FITC-PNA/PI) 0, 3 and 6 hours after thawing. Finally, six gilts were inseminated surgically into the ampoule of the oviduct with four million sex-sorted spermatozoa frozen and thawed with addition of Pyruvate, Catalase and Mercaptoethanol. Gilts were slaughtered 48 hours later and the embryos were reflushed from the genital tract. In summary the following results were obtained. Directly after thawing fresh spermatozoa frozen in the presence of antioxidants showed a significantly (P≤0.05) improved motility compared to spermatozoa frozen without additives. The percentage of normal apical ridges and the gross morphology did not differ significantly between the treatment groups. After flow cytometrical analysis of membrane integrity and acrosome status no significant differences could be detected among treatment groups, but the percentage of viable spermatozoa decreased and the percentage of acrosome damaged spermatozoa increased in eleven of twelve sperm subgroups within one respective treatment group during incubation time. After the evaluation of different freezing curves for individual animals significant differences could only be detected for one boar directly after thawing. The spermatozoa of this boar showed a higher (P≤0.05) motility after being frozen with a short, instead with a long freezing curve. The analysis of the morphological status showed no significant differences in all boars between the different freezing curves. The evaluation of the viability and acrosome reaction showed that the percentage of viable, acrosome reacted spermatozoa and the percentage of membrane damaged and acrosome reacted spermatozoa of one boar were significantly lower in the sample frozen following the medium timed protocol than following the short and long protocol. Significant differences were also recorded in part of the treatment groups of all boars, were the percentage of viable spermatozoa decreased and the percentage of acrosomal damages increased after two hours of incubation at 38°C. Motility of fresh non-sorted and sex-sorted sperm samples decreased during storage at 15°C as well as during incubation at 38°C in all treatment groups. The sperm motility was higher in the non-sorted groups than in the sex-sorted groups, although the difference was only statistically significant (P≤0.05) after 120h storage at 15°C and 15min incubation at 38°C. The addition of antioxidants had a slightly beneficial effect on the motility of sex-sorted spermatozoa, but the difference was not statistically significant. The percentage of acrosomal damaged spermatozoa was significantly (P≤0.05) higher in the sex-sorted spermatozoa than in the fresh semen samples, but did not differ between the samples stored with or without additives.In the subsequent experiment 0h after thawing only the sex-sorted samples frozen and thawed in sperm concentration of 80 x 106/ml without addition of Pyruvate, Catalase and Mercaptoethanol showed a significant (P≤0.05) lower motility than all other groups. All other groups showed no significant differences. However, the non-sorted semen samples showed an elevated motility when compared with sex-sorted samples, but from 1h after thawing up until 6h no statistically significant differences could be measured in the sperm motility between all treatment groups. No significant differences in the morphological status of the spermatozoa could be detected between all treatment groups. Directly after thawing the non-sorted sperm frozen without antioxidants showed a significantly (P≤0.05) higher portion of viable sperm with intact acrosomes than the sex-sorted samples frozen without additives and 80 x 106 sperm per ml. Further incubation did not reveal significant differences in acrosomes. In all treatment groups the percentage of vital and acrosome intact spermatozoa decreased significantly during incubation for six hours. The last trial proved that sex-sorted porcine spermatozoa frozen with antioxidants and Pyruvate can successfully be used to fertilize oocytes in vivo after surgical insemination. From six gilts 70 embryos were reflushed. From these embryos 52.5% were fertilized and 27.1% developed to blastocysts stages. From these results it can be concluded that the addition of Pyruvate, and the antioxidants Catalase and Mercaptoethanol have only limited effect on motility of fresh, sex-sorted and frozen porcine spermatozoa and almost no effect on sperm morphology. Antioxidants did not significantly improve the quality of sex-sorted semen after either fresh or frozen storage. There may be other damages during flowcytometrical sorting that can not be prevented by the addition of the ROS scavengers.