Optimering av reproduktiv effektivitet hos mjölkkor genom förbättring av spermiekvaliteten inför artificiell insemination

The purpose of the proposed project is to improve sperm quality in bull semen doses for artificial insemination (AI), in an effort to reverse the trend towards declining fertility seen in dairy cattle worldwide. The content of reactive oxygen species (ROS) will be analysed in semen samples from bulls of high and low fertility, and the effect of selecting robust spermatozoa by colloid centrifugation on their ROS-content will be investigated. In addition, the potential beneficial effect of adding heat shock proteins to bull spermatozoa on their quality during freezing and storage will be assessed. These proteins are considered to be involved in sperm binding to oviductal epithelial cells and therefore may be associated with fertility. The results could lead to improvements in bull semen handling resulting in improved sperm quality in sperm doses for AI.

Projektets mål är att förbättra spermiekvaliteten i doser av tjurspermier för artificiell inseminering (AI) för att undvika ytterligare sänkning av fertiliteten hos mjölkkor. Ämnet är mycket relevant för mjölkavel, i synnerhet för mjölkproducenter, eftersom fertiliteten hos ffa Holsteinrasen (SLB) har visat sig sjunka världen över. Innehållet av syre (”reactive oxygen species”, ROS) i spermier från låg- respektive högfertila tjurar undersöks samt effekten av kolloidcentrifugering på ROS-innehållet. Tillsättning av ”heat shock proteins” till tjurspermier bedöms för sin potentiella effekt på spermiekvalitet under frysning och frysförvaring. Dessa proteiner är inblandade i spermiebindning till epitel i äggledare och betraktas därför som viktiga för fertilitet. En förbättring av spermiehanteringen kan förbättra spermiekvaliteten i doser för AI.

Projektets resultat visar att spermiekvaliteten påverkas av många mer externa faktorer än tidigare rapporterats, särskilt typ och ras av tjur, spädningsvätska, insamlingssäsong mm. Fertilitet kan också påverkas, åtminstone vid in vitro fertilisering. Analys av spermiernas kvalitet eller indikatorer på fertilitet bör anpassas beroende på vilken typ eller ras av tjur som utvärderas istället för att använda generiska trösklar som vanligtvis görs nu. Effekterna av säsongen bör övervägas vid beredning av sperma doser för artificiell insemination eller åtgärder tas för att modifiera extrema klimatförhållanden i tjurens miljö. Det är viktigt att förbättra spermiekvaliteten genom kolloidcentrifugering, speciellt med Single Layer Centrifugation (SLC). Detta förfarande är enkelt och effektivt, och är praktiskt för användning vid spermasamlingsstationer. Emellertid är inte alla spädningsvätskor lämpliga för användning med SLC. Därför måste det rekommenderade protokollet följas exakt.

Background: An alarming trend in reduced fertility has been observed in dairy cattle worldwide in recent decades, with high yielding breeds apparently being more affected than lower yielding ones. Sperm preparation techniques, such as colloid centrifugation, that select the best quality spermatozoa could help to reduce harmful ROS-production and aid cryosurvival, thus contributing to improved cow fertility.

Reactive oxygen species (ROS) are produced as byproducts of metabolism; the more metabolically active the spermatozoa are, the more ROS they produce. Differences in bull fertility may be due to differences in ROS production, or to differences in antioxidants in the seminal plasma. Therefore, investigating ROS-production in bull spermatozoa may help to unravel some of the intricacies of bull fertility.

The proposed plan was to measure the content of reactive oxygen species (ROS) in semen samples from bulls of high and low fertility. In addition, the effect of selecting robust spermatozoa by colloid centrifugation on their ROS-content would be investigated. Finally, the potential beneficial effect of adding heat shock proteins to bull spermatozoa on their survival during freezing and cryostorage would be assessed.

Results
There were differences in ROS production between bulls although a number of extraneous factors were found to influence sperm quality evaluation which may have masked a fertility effect. The following factors were found to affect sperm quality:
1. Season: The effect of season on sperm quality was most pronounced in Sweden compared to bulls in Spain and also in Thailand. This finding tends to suggest that bulls can adapt to climatic conditions such as warm temperatures or high humidity, but extreme changes in climate occurring over a few months do have an effect on sperm quality.
2. Beef versus dairy breeds: Sperm quality was evaluated in beef and dairy bulls. Beef bulls produced less superoxide than dairy bulls but more hydrogen peroxide. Beef bull spermatozoa were less metabolically active than dairy bull spermatozoa and showed more DNA damage; however, there were more live spermatozoa with unreacted acrosomes in semen from beef bulls.
3. Breed of bull: within dairy bulls there is an effect of breed on sperm quality, with different parameters of sperm quality being linked to fertility for Holstein and Swedish Red breeds. Therefore, the same methods cannot be used to evaluate sperm quality in the different breeds.
The conclusion from these observations was that either assays of sperm quality have to be selected for each type or breed of bull or, alternatively, better methods of evaluating sperm quality are needed.
A new model for determining sperm quality was suggested, using a combination of factors including metabolic activity and production of reactive oxygen species.
In addition, fertility-associated proteins in seminal plasma from bulls of high and lower fertility were analysed by Fast Protein Liquid Chromatography (FPLC). Differences in the amounts of non-heparin binding, phosphorylcholine binding and heparin-binding proteins were found between bulls of different fertility. Therefore, quantification of seminal plasma proteins may provide an indicator of a bull´s potential fertility.

The effect of SLC on bull sperm quality was investigated, with particular reference to ROS production. Some aspects of sperm quality were improved by SLC, e.g. the proportion of spermatozoa with high mitochondrial membrane potential was increased, DNA integrity is better, and ROS-production is different between selected and non-selected sperm samples. Further studies are underway to determine whether fertility is also improved in the SLC samples. This procedure is practical for field use and could be adopted easily by semen processing stations.

In contrast to previous reports, addition of heat shock protein did not improve bull sperm cryosurvival in our hands.

Relevance to the Cattle Breeding Industry
i. Assays of sperm quality, or indicators of fertility, should be adjusted according to the type of bull semen being evaluated.
ii. Measures should be taken to mediate extreme climatic conditions. Bulls appear to be more affected by fluctuations in temperature and humidity than by continuous extreme conditions per se
iii. Sperm quality can be improved by Single Layer Centrifugation.