The role of antibiotics (AB) in semen extenders as a potential contribution to the global antimicrobial resistance threat is emerging. Here, we establish an AB-free hypothermic preservation strategy for boar semen and investigate its impact on sperm function, microbial load and fertility after artificial insemination (AI). Spermatozoa (12 boars) preserved in AB-free AndroStar Premium extender at 5 °C maintained high motility, membrane integrity, and a low DNA-fragmentation index throughout 72 h storage and results did not significantly differ from controls stored at 17 °C in extender containing AB (p = 0.072). Likewise, kinetic response of spermatoza to the capacitation stimulus bicarbonate during 180 min incubation in Tyrode’s medium did not differ from 17 °C-controls. In a competitive sperm oviduct binding assay, binding indices did not differ between semen stored for 72 h AB-free at 5 °C and 17 °C-controls (n = 6 boars). Bacterial load < 10³ CFU/ml after 72 h was measured in 88.9% of samples stored at 5 °C AB-free compared to 97.2% in 17 °C-controls (n = 36 semen pools, 23 boars). Fertility traits of 817 females did not differ significantly between the two semen groups (p > 0.05). In conclusion, a hypothermic semen preservation strategy is presented which offers antibiotic-free storage of boar semen doses.

In vitro short-term storage of boar semen for up to 72 h before insemination negatively affects fertility, but this often remains undetected during semen quality assessment. One important sperm function is the ability to form the functional sperm reservoir in the oviduct. In the present study, we used the modified oviductal explant assay to study sperm binding to oviductal epithelium in vitro in diluted boar semen stored for 24 or 72 h. First, we determined the kinetics of in vitro sperm binding to oviductal epithelium in relation to co-incubation time of sperm and oviductal tissue pieces. Then, we studied how the binding of sperm to oviductal epithelium was affected by in vitro semen storage and by differences among individual boars. Sperm binding after different incubation times was significantly higher when semen was stored 24 h than after 72-h storage (P < 0.05), and peaked at 30–90 min of incubation. Sperm binding differed between boars (n = 44), and was negatively correlated to the percentage of sperm with cytoplasmic droplets (R = −0.51, P < 0.001). There were no significant changes in motility, acrosome integrity and propidium iodide stainability during the 72-h storage period. However, sperm-binding indices were significantly lower after 72 h in vitro storage than after 24-h storage in sperm from boars with normal semen quality (P < 0.05); in contrast, the binding capacity of sperm from boars with higher percentages of morphologically altered sperm remained at a low level. The sperm-binding capacity of sperm from four of the five boars with known subfertility was lower than the mean binding index minus one standard deviation of the boar population studied here. It is concluded that changes in the plasma membrane associated with in vitro ageing reduce the ability of stored boar sperm to bind to the oviductal epithelium. This study shows the potential of sperm–oviduct binding as a tool to assess both male fertility and changes in sperm function associated with in vitro ageing. Abstract In vitro short-term storage of boar semen for up to 72 h before insemination negatively affects fertility, but this often remains undetected during semen quality assessment. One important sperm function is the ability to form the functional sperm reservoir in the oviduct. In the present study, we used the modified oviductal explant assay to study sperm binding to oviductal epithelium in vitro in diluted boar semen stored for 24 or 72 h. First, we determined the kinetics of in vitro sperm binding to oviductal epithelium in relation to co-incubation time of sperm and oviductal tissue pieces. Then, we studied how the binding of sperm to oviductal epithelium was affected by in vitro semen storage and by differences among individual boars. Sperm binding after different incubation times was significantly higher when semen was stored 24 h than after 72-h storage (P < 0.05), and peaked at 30–90 min of incubation. Sperm binding differed between boars (n = 44), and was negatively correlated to the percentage of sperm with cytoplasmic droplets (R = −0.51, P < 0.001). There were no significant changes in motility, acrosome integrity and propidium iodide stainability during the 72-h storage period. However, sperm-binding indices were significantly lower after 72 h in vitro storage than after 24-h storage in sperm from boars with normal semen quality (P < 0.05); in contrast, the binding capacity of sperm from boars with higher percentages of morphologically altered sperm remained at a low level. The sperm-binding capacity of sperm from four of the five boars with known subfertility was lower than the mean binding index minus one standard deviation of the boar population studied here. It is concluded that changes in the plasma membrane associated with in vitro ageing reduce the ability of stored boar sperm to bind to the oviductal epithelium. This study shows the potential of sperm–oviduct binding as a tool to assess both male fertility and changes in sperm function associated with in vitro ageing.

Fertility is a convenient but meaningless term unless the outcome measure is stipulated and accounts for dependence of male fertility on the female population. We describe outcome measures and detail the impacts of the physiological status of each female and her external environment, as well as management imposed by humans. We explain the dominant role of the female reproductive tract as a series of hurdles for sperm seeking an ovum. Each spermatozoon in an ejaculate is unique, although usually most are morphologically similar. Semen seemingly contains three subpopulations of sperm, based on fate within a female and role in hampering the success of the ultimate winning spermatozoon; we define these subpopulations. The numerical size of each subpopulation placed into a female determines the shape of the dose-response curve leading to possible live young. Heterospermic artificial insemination provides far greater sensitivity to detect differences, partly because the female environment is identical for each comparison.