A study of functional markers in raw and processed bovine sperm and their potential uses for fertility prediction and process refinement

The extensive assessment of bull’s reproductive potential prior to breeding is highly important and includes examination of general physical soundness, external and internal genitalia and semen quality. Breeding success depends on the efficient use of bulls with high breeding value but simultaneously semen quality imposes restrictions on the use of these bulls in AI. Several techniques have been devised to assess quality of either fresh or frozen-thawed semen. Among a variety of traditional parameters sperm concentration, sperm raw and post-thaw motility and sperm morphology are commonly used for routine semen assessment in the laboratory. In this study, we investigated differences in sperm metabolic activity relative to their motility that may reflect better the fertility of bulls from their non-return rates (NRRs). To investigate the relationship between mid-piece length and fertility of bovine spermatozoa, sperm biometry was performed on ejaculates obtained from 34 bulls representing six breeds: Holstein (yearlings and mature), Friesian, Belgian Blue, Aberdeen Angus, Charolais and Limousin. Significant differences (P<0.01) between ejaculates were found in 9/34 bulls, as well as differences (P<0.001) between individual bulls within the same breed. The average mid-piece length for Aberdeen Angus was 13. 35μm, for Belgian Blues and Limousin around 13. 8μm, and for Charolais 13. 68μm: for dairy breeds (Holstein and Friesian) it was about 13. 4μm. The mean value of mid-piece length for breed was compared with their 49 day non-return rate; a negative correlation was found in the dairy breeds, while in bulls from beef breeds this correlation was positive but very low: the small numbers of bulls involved prevented meaningful statistical relationships being established. To differentiate live and dead sperm and non-sperm-specific particles, a flow cytometry method was developed by labelling sperm with JC-1 and propidium iodide (PI) dyes and to determine maximum mitochondrial membrane potential (ΔΨm) at minimum incubation. This method entailed setting regional and logical gates to exclude dead sperm and other non-cellular components from live sperm present within an ejaculate. It was confirmed that spermatozoa of both fresh and frozen-thawed semen exhibited maximum high:low ΔΨm ratio after 40 min incubation. Flow cytometric dot plots of analyses of fresh and frozen-thawed spermatozoa incubated with JC-1 could identify a unified sperm population of membrane-intact cells, each population characterised by both low and high ΔΨm but to varying degrees suggesting that this flow cytometric method simplifies the determination of mitochondrial membrane potential using JC-1. This method serves two purposes: using this method, one could able to evaluate sperm ΔΨm as well as the proportion of live:dead. Changes in mitochondrial structure and integrity appear to be an important component associated with sperm motility and reduced fertility. The ΔΨm was assessed using JC-1 and PI in the presence of glycolytic and respiratory inhibitors. Mean high ΔΨm was significantly greater for control compared to the treatments in fresh and frozen-thawed semen. In samples treated with valinomycin (VAL) and iodoacetamide (IAM) ΔΨm was lowered significantly. The proportion of sperm with a high ratio of high:low ΔΨm was higher in control and 2-deoxy-D-glucose (DOG) treated samples representing more active mitochondria: in samples treated with VAL and IAM the ratio was reduced, representing loss in activity of mitochondria. Cryopreservation significantly decreases high:low ΔΨm ratio in control suggesting that lower mitochondrial activity may be associated with oxidative stress produced by reduced antioxidant levels due to the freeze/thaw cycle. The relationship between ZO2 (µl oxygen consumed /108 spermatozoa/hr) and mitochondrial function was assessed in fresh and frozen-thawed semen. Sperm oxygen consumption was greater in fresh compared to frozen-thawed semen. Insignificant positive correlations existed between ZO2 and ratio of high:low ΔΨm in fresh (r=0.82) and frozen-thawed (r= 0.49) semen suggesting that the ΔΨm measured in this way by flow cytometry can be used as an indicator of ZO2. Finally, the metabolic pathways by which spermatozoa produce energy to support their motility were investigated in fresh and frozen-thawed semen diluted in media containing glycolytic and respiratory inhibitors. Total and progressive motilities were not significantly different in sperm incubated with DOG and VAL but decreased significantly with IAM compared to control. This indicates that sperm can maintain a similar degree of motility when generating their energy exclusively from either glycolysis or mitochondrial activity. IAM significantly lowered sperm motility as well as mitochondrial activity (as described above) and was found to be an inhibitor of both glycolysis and respiration possibly linked with either modification of mitochondrial cysteine and/or glutathione levels. Sperm are considered in a state of hyperactivation/capacitation when their amplitude of lateral head displacement (ALH) increases and path straightness (STR) and linearity (LIN) decrease. In the present study higher ALH and lower STR and LIN were observed when spermatozoa were dependent on mitochondrial energy (DOG), whereas these estimates were reversed when they were on glycolytic energy (VAL) indicating that sperm hyperactivation and capacitation are associated with mitochondrial function. There was a positive correlation of sperm progressive motility, ZO2 and high:low ΔΨm ratio with bull NNRs suggesting that these sperm characteristics may be useful for predicting bull fertility. Furthermore sperm mid-piece length was significantly correlated with sperm average curvilinear velocity and amplitude of lateral head displacement. Since the mitochondria are localized on the sperm mid-piece, it is likely that its energy may contribute in high sperm velocity and also hyperactivation that helps sperm disengagement from oviduct epithelium and positioning at the site of fertilization.