Artificial insemination (AI) with sex-sorted bottlenose dolphin spermatozoa provides female calves for obtaining more cohesive social groups and optimum genetic management of captive populations. However, distance of animals to the sorting facility represents a limit to the procedure. Although one bottlenose dolphin calf has been born using spermatozoa from frozen-thawed, sorted and recryopreserved spermatozoa, critical evaluation of the steps involved in this process is required to maximize its efficiency for future AIs and expansion of the technology to other species. Two experiments were designed to determine the efficiency of the sorting process and the quality of frozen-thawed bottlenose dolphin spermatozoa during sorting and recryopreservation. In experiment 1, the effect of two washing media (with and without 4 percent egg yolk, v/v) following density gradient centrifugation (DGC) on sperm recovery rate and in vitro characteristics of cryopreserved spermatozoa was examined. In experiment 2, cryopreserved semen was used to compare the effects of two recryopreservation methods (conventional straw freezing and directional freezing) on in vitro sperm characteristics of control (non-sorted) and sorted spermatozoa. Egg yolk supplementation of the washing medium in experiment 1 did not influence (P > 0.05) the sperm recovery rate, however, sperm motility parameters and viability were improved (P < 0.05). For Experiment 2, motility parameters and viability were influenced by stage of sex-sorting process, sperm type (non-sorted and sorted) and freezing method (P < 0.05). After recryopreservation, sorted spermatozoa frozen with the directional freezing method maintained higher (P < 0.05) motility parameters over the 24 h incubation period compared to spermatozoa frozen using straws. Quality of sperm DNA of nonsorted spermatozoa, as assessed by the SCSA, remained unchanged throughout the process. However, a possible interaction between Hoechst 33342 and acridine orange was observed in sorted samples. After recryopreservation, viability of sorted spermatozoa was higher (P < 0.05) than that of non-sorted spermatozoa across all time points. The percentages of viable spermatozoa determined by light (eosin-nigrosin) and fluorescence microscopy (propidium iodide) techniques were correlated (R^2=0.79, P < 0.001). Collective results indicate that bottlenose dolphin spermatozoa undergoing cryopreservation, sorting and recryopreservation are of adequate quality for use in AI.

För att bedöma mäns spermiekvalitet och därmed pars chanser till en graviditet har standardparametrar tidigare använts som fertilitetsindikator, men har visat sig vara en mindre tillförlitlig metod. Därför har en ny metod som bygger på flödescytometri och kallas Sperm Chromatin Structure Assay (SCSA) utvecklats som fokuserar på spermiernas arvsmassa, och som då har visat sig vara en mycket mer tillförlitlig metod. Syftet med denna studie var att jämföra DNA Fragmentations Index (DFI) -värde mellan svenska och danska män och se om en signifikant skillnad finns. För att uppfylla detta späddes och färgades spermaproven in och analyserades med SCSA, som därefter gav hur stor andel av spermiernas DNA som är defekt, vilket presenterades som ett procentuellt DFI-värde. Resultatet visade en signifikant skillnad av medianvärdet mellan svenska och danska män med avseende på DFI, där svenska män hade högre medianvärde än de danska männen. Detta i kombination med tidigare forskning som gjordes mellan svenska och danska män och som visade att svenska män hade bättre kvalitet än danska män med avseende på standardparametrarna, stödjer tidigare forskning som visar på att det ej finns ett samband mellan standardparametrar och DFI. Dock hade ytterligare forskning av skillnaderna mellan svenska och danska män behövts, för att validera fynden i denna studie. / To assess men's sperm quality and thus couples' chances of pregnancy, standard parameters have previously been used as a fertility indicator, but have proven to be a less reliable method. Therefore, a new method based on flow cytometry called Sperm Chromatin Structure Assay (SCSA) has been developed which focuses on the sperm's genetic mass, and which has been shown to be a much more reliable method. The purpose of this study was to compare the DNA Fragmentation Index (DFI) value between Swedish and Danish men and see if a significant difference exists. To fulfill this, the sperm samples were diluted and stained and analyzed with SCSA, which then gave the ratio of the sperm's DNA that is defective, which was presented as a percentage DFI value. The result showed a significant difference in the median value between Swedish and Danish men with regard to DFI, where Swedish men had a higher median value than the Danish men. This, in combination with previous research that has been done between Swedish and Danish men which showed that Swedish men had better quality than Danish men in regards to the standard parameters, supports previous research that shows that there is no connection between standard parameters and DFI. However, further research into the differences between Swedish and Danish men is needed to validate the findings in this study.