Sammendrag
Fertility in the cattle breeding industry is an important trait to select for, but has been neglected for many years, resulting in a worldwide decline of fertility in most breeds. The Norwegian Red breed on the other hand shows that it is possible to increase the fertility within 50 years, provided good selection practices. Even though the Norwegian Red bulls are known for their higher fertility in general, there are still bulls within this breed that produce semen with lower fertilizing potential than others. Lower fertility causes higher costs for the breeding industry and the end-user, the farmer. At the semen production facility, semen doses for sale and for artificial insemination are proved or discarded according to the sperm quality after freeze-thawing, most often assessed with total motility. Even doses with similar total motility show differences in pregnancy outcome, highlighting the importance of additional knowledge on sperm quality markers.
Flow cytometric analysis is a diverse tool that can be used for a simultaneous analysis of multiple sperm quality parameters by using several fluorescent dyes. Oxidative stress (OS) is shown to influence the quality of the sperm sample and could therefore be a useful parameter to analyse in relation to fertility. OS is induced by reactive oxygen species (ROS) that also serves as an important molecule for several biological process. Though, an excess of these molecules can lead to cell damage and overall lower sperm quality.
The goal of this thesis was to identify bull sperm health markers for flow cytometric analysis that link OS to bull fertility. To accomplish this, fluorescent dyes for different sperm parameters had to be carefully selected. First, a viability stain had to be identified that could be combined with several other dyes for OS. For this latter aim, a single dye was tested that could both identify sperm cell and asses the viability of the sample. This is reported in Paper 1, where Hoechst 33342 (HOE) was used in combination with propidium iodine (PI) and SYTO™60 (S60). Results showed that HOE could identify sperm cells from non-cell debris the same way as S60 does. In addition, there was a strong correlation between PI-defined and HOE-defined sperm viability, concluding that HOE can be used as a single dye to identify sperm cells in a semen sample and gives an indication of the viability of the sample. Using only HOE for viability assessment opened up fluorescent channels in flow cytometry that were used in Paper 2.
In Paper 2, sperm health was studied in function of OS and in relation to fertility. Sperm samples were stained with CM-H2DCFDA (CM) and MitoSOX™ (MS) to measure OS, together with MitoTracker™ (MT), a mitochondrial activity marker, and HOE for viability assessment. Samples were stressed by incubation at a physiological temperature over time and by centrifugation combined with chemical induction to induce OS. Sperm cell analysis was performed after three different incubation times, post-thaw (T0), one hour (T1) and four hours (T4), and chemical induction was achieved with tert-butyl hydroperoxide (TBH) and menadione (Men) for one hour, and PBS as a control (C). All samples were analysed by flow cytometry and had available motility and fertility data. Results showed that oxidative stress and mitochondrial activity were related to motility. Interestingly, the size of LiveCM and LiveMT population was bigger in spermatozoa from bulls with higher fertility than from bulls with lower fertility. Moreover, there were a higher proportion of the DeadMS spermatozoa in bulls with lower fertility compared to higher fertility. In conclusion, the results showed that there is a relationship between OS and the fertilizing potential of a bull sperm sample, and that OS can be an indicator for sperm health.