To date sperm-oviduct interactions have largely been investigated under in vitro conditions. Therefore we set out to characterize the behaviour of bovine spermatozoa within the sperm reservoir under near in vivo conditions and in real-time using a novel live cell imaging technology and a newly established fluorescent sperm binding assay. Sperm structure and tubal reactions after sperm binding were analysed using scanning and transmission electron microscopy and histochemistry. As a model to specify the impact of stress on sperm-oviduct interactions, frozen-thawed conventional and sex-sorted spermatozoa from the same bulls (n = 7) were co-incubated with oviducts obtained from cows immediately after slaughter. Our studies revealed that within the oviductal sperm reservoir agile (bound at a tangential angle of about 30°, actively beating undulating tail), lagging (bound at a lower angle, reduced tail movement), immotile (absence of tail movement) and hyperactivated (whip-like movement of tail) spermatozoa occur, the prevalence of which changes in a time-dependent pattern. After formation of the sperm reservoir, tubal ciliary beat frequency is significantly increased (p = 0.022) and the epithelial cells show increased activity of endoplasmic reticula. After sex sorting, spermatozoa occasionally display abnormal movement patterns characterized by a 360° rotating head and tail. Sperm binding in the oviduct is significantly reduced (p = 0.008) following sexing. Sex-sorted spermatozoa reveal deformations in the head, sharp bends in the tail and a significantly increased prevalence of damaged mitochondria (p < 0.001). Our results imply that the oviductal cells specifically react to the binding of spermatozoa, maintaining sperm survival within the tubal reservoir. The sex-sorting process, which is associated with mechanical, chemical and time stress, impacts sperm binding to the oviduct and mitochondrial integrity affecting sperm motility and function.

In the three decades since its inception, the sex-sorting technology has progressed significantly. However, field studies report conflicting findings regarding reproductive outcomes. Therefore, we conducted this meta-analysis of all trials published between 1999 and 2021. Non-return rates after 24 or 60 d (NRR 24/60), pregnancy, calving, abortion, and stillbirth rates were compared after AI with sex-sorted vs non-sorted sperm. Additionally, the impact of recent developments in the sex-sorting technology was assessed. Of 860 studies found, 45 studies (72 trials) provided extractable data and were included. Overall, the results of this meta-analysis provided evidence that the NRR 24/60 was diminished by 13%, pregnancy rates were reduced by 23% (25% cows, 21% heifers) and calving rates were reduced by 24% when using sex-sorted sperm. Enhancing the dosage to 4 million sex-sorted sperm/straw (including recent improvements, high vs low dose) as well as using fresh sex-sorted sperm (sorted vs non-sorted) increased pregnancy rate ratios by 7 percentage points. The refinement of the sex-sorting technology after 2015 resulted in a lowered reduction of pregnancy and calving rate of 19% and 23%, respectively. Whereas abortion rates were similar, the stillbirth of male calves was increased by 6.3%.

Sperm binding within the oviductal sperm reservoir plays an important role for reproductive success by enabling sperm survival and maintaining fertilizing capacity. To date, numerous in vitro technologies have been established to measure sperm binding capacity to cultured oviductal cells or oviductal explants. However, these methods do not accurately represent the microenvironment and complex multi-molecular nature of the oviduct. In this paper, we describe a novel protocol for assessing sperm binding capacity in the tubal sperm reservoir using an ex vivo oviduct culture in the bovine model. This protocol includes the staining of frozen-thawed bovine spermatozoa with the DNA-binding dye Hoechst 33342, the co-incubation of stained sperm in closed segments of the oviduct and the visualization and quantification of bound spermatozoa by fluorescence microscopy. By generating overlays of multiple Z-stacks of randomly selected regions of interest (ROIs), spermatozoa bound in the sperm reservoir can be visualized and quantified within the 3D arrangement of the oviductal folds. This method, which is applicable to multiple species, can be used to assess individual sperm binding capacity in males for prognostic purposes as well as to assess the impact of diseases and medications on the formation of the sperm reservoir in the oviduct in humans and animals.