Sperm functionality is differentially regulated by porcine oviductal extracellular vesicles from the distinct phases of the estrous cycle.

Context Extracellular vesicles (EVs) derived from the oviductal fluid (oEVs) play a critical role in various reproductive processes, including sperm capacitation, fertilisation, and early embryo development. Aims To characterise porcine oEVs (poEVs) from different stages of the estrous cycle (late follicular, LF; early luteal, EL; mid luteal, ML; late luteal, LL) and investigate their impact on sperm functionality. Methods poEVs were isolated, characterised, and labelled to assess their binding to boar spermatozoa. The effects of poEVs on sperm motility, viability, acrosomal status, protein kinase A phosphorylation (pPKAs), tyrosine phosphorylation (Tyr-P), and in in vitro fertility were analysed. Key results poEVs were observed as round or cup-shaped membrane-surrounded vesicles. Statistical analysis showed that poEVs did not significantly differ in size, quantity, or protein concentration among phases of the estrous cycle. However, LF poEVs demonstrated a higher affinity for binding to sperm. Treatment with EL, ML, and LL poEVs resulted in a decrease in sperm progressive motility and total motility. Moreover, pPKA levels were reduced in presence of LF, EL, and ML poEVs, while Tyr-P levels did not differ between groups. LF poEVs also reduced sperm penetration rate and the number of spermatozoa per penetrated oocyte (P Conclusions poEVs from different stages of the estrous cycle play a modulatory role in sperm functionality by interacting with spermatozoa, affecting motility and capacitation, and participating in sperm-oocyte interaction. Implications The differential effects of LF and LL poEVs suggest the potential use of poEVs as additives in IVF systems to regulate sperm-oocyte interaction.

Implications of boar sperm kinematics and rheotaxis for fertility after preservation.

Artificial insemination (AI) is the single most important assisted reproductive technique devised to facilitate the genetic improvement of livestock. In the swine industry, it has broadly replaced natural service over the last number of decades which has been made possible by the high pregnancy rates and litter sizes obtainable with semen extended, up to, and sometimes beyond 5 d. Central to achieving good reproductive performance is the ability of boar studs to monitor semen quality, the basis of which has long been the assessment of sperm motility by subjective and, more recently, by more objective computerised systems. In this review, the literature on the relationship between sperm motility and kinematic parameters and field fertility is summarised. We discuss how this relationship is dependent on factors such as the viscosity of the media and the use of standard operating procedures. Emerging evidence is discussed regarding the importance of sperm rheotaxis and thigmotaxis as long-distance sperm guidance mechanisms, which enable motile functional spermatozoa to avoid the backflow of fluid, mucus and semen from the sow's uterus in the hours post AI, facilitating the establishment of sperm reservoirs in the oviducts. The literature on the use of microfluidics in studying sperm rheotaxis in vitro is also summarised, and we discuss how these systems, when combined with techniques such as lensless microscopy, have the potential to offer more physiological assessments of the swimming patterns of boar spermatozoa. Finally, possible future avenues of further investigation are proposed.

Progesterone induces the release of bull spermatozoa from oviductal epithelial cells.

The mechanism that causes the detachment of spermatozoa from the oviductal reservoir around the time of ovulation remains to be elucidated. Because the cumulus cells of the bovine oocyte are known to secrete progesterone (P4), and P4 has been shown to act upon cation channels of spermatozoa (CatSper) in human spermatozoa, it was hypothesised that P4 could induce hyperactivation due to an influx of extracellular calcium, and this would facilitate detachment of spermatozoa from oviductal epithelial cells. Therefore, this study aimed to investigate the role and mechanism of action of P4 in the release of spermatozoa from bovine oviduct epithelial cells (BOEC). Initial dose-response assessments on sperm hyperactivation determined the optimum concentration of P4 (10 nM), mibefradil (a non-specific Ca2+ channel antagonist; 5µM), NNC 55-0396 dihydrochloride (NNC; a CatSper antagonist; 2µM), mifepristone (a classical and membrane P4 receptor antagonist; 400nM) and AG205 (a membrane P4 receptor antagonist; 10µM). BOEC explants were incubated with frozen-thawed bovine spermatozoa for 30min, following which loosely bound spermatozoa were removed. Two experiments were completed. In Experiment 1, BOECs were treated for 30min with either no treatment, P4, NNC, mibefradil, P4+mibefradil, P4+NNC, P4+mibefradil+NNC or P4+EGTA. In Experiment 2, BOECs were treated for 30min with either no treatment, P4, mifepristone, AG205, mifepristone+AG205, P4+mifepristone, P4+AG205 or P4+mifepristone+AG205. The number of spermatozoa remaining bound per millimetre squared of BOEC explant was determined. Progesterone stimulated the release of bound spermatozoa from BOEC explants, whereas NNC, mibefradil and EGTA inhibited this release. The release of spermatozoa by P4 was inhibited in the presence of both mifepristone and AG205, whereas the combination of both had the greatest inhibitory action on P4 release of spermatozoa. These findings suggest the presence of a P4 membrane receptor on bovine spermatozoa and that P4-induced release of spermatozoa from BOECs is likely mediated by extracellular Ca2+.

Recombinant ß-defensin 126 promotes bull sperm binding to bovine oviductal epithelia.

Primate ß-defensin 126 regulates the ability of spermatozoa to bind to oviductal epithelial cells invitro. Bovine ß-defensin 126 (BBD126) exhibits preferential expression in the cauda epididymis of the bull, but there have been few studies on its functional role in cattle. The aim of the present study was to examine the role of BBD126 in bull sperm binding to bovine oviductal epithelial cell (BOEC) explants. BBD126 has been shown to be highly resistant to the standard methods of dissociation used in other species and, as a result, corpus epididymal spermatozoa, which have not been exposed to the protein, were used to study the functional role of BBD126. Corpus epididymal spermatozoa were incubated with recombinant (r) BBD126 in the absence or presence of anti-BBD126 antibody. Addition of rBBD126 significantly enhanced the ability of epididymal spermatozoa to bind to BOEC explants (P<0.05). Anti-BBD126 antibody blocked the BBD126-mediated increase in sperm binding capacity. Ejaculated spermatozoa, which are coated with native BBD126 protein but also a large number of seminal plasma proteins invivo, were incubated with rBBD126 in the absence or presence of the anti-BBD126 antibody. Addition of rBBD126 significantly enhanced the ability of ejaculated spermatozoa to bind to BOEC explants (P<0.05), whereas rBBD126 also reduced corpus sperm agglutination (P<0.05). These results suggest that, similar to the role of its analogue in the macaque, spermatozoa with more BBD126 in their acrosome may represent spermatozoa with more oviduct binding capacity.

Application of reproductive biotechnology for the recovery of endangered breeds: birth of the first calf of Murciana-Levantina bovine breed derived by OPU, in vitro production and embryo vitrification.

In a conservation project, reproductive biotechnology was implemented for the recovery and conservation of an endangered bovine breed in Spain. The breed Murciana-Levantina, declared to be worthy of special protection status (http://www.fao.org/newsroom/en/news/2006/1000464/index.html), is of great interest because of its hardness, longevity, docility and disease resistance. This contribution describes the birth of the first calf of this breed obtained by reproductive biotechnology, using ultrasound-guided punction and aspiration of ovarian follicles, in vitro embryo production, vitrification of embryos by a cryotop device and, finally, the transfer of cryopreserved embryos to recipient heifers of a commercial dairy herd.