Spermatozoa produced during winter are superior in terms of phenotypic characteristics and oviduct explants binding ability in the water buffalo (Bubalus bubalis).

Although reduced reproductive efficiency during summer has been well documented in buffaloes, the reason for the same is yet to be understood. The present study was conducted to identify the subtle differences in sperm phenotypic characteristics (motility, membrane integrity, acrosome reaction and lipid peroxidation status), oviduct binding ability and expression of fertility-associated genes (AK 1, ATP5D, CatSper 1, Cytochrome P450 aromatase, SPP1 and PEBP1) between winter and summer seasons in buffaloes. Cryopreserved spermatozoa from 6 Murrah buffalo bulls (3 ejaculates/bull/season) were utilized for the study. Real-time quantitative PCR was performed for assessing the expression patterns of select fertility-associated genes. The proportion of motile and membrane intact spermatozoa was significantly higher (p < .05) in winter as compared to summer ejaculates. The proportion of moribund and lipid peroxidized spermatozoa was significantly lower (p < .05) in winter ejaculates as compared to summer. The sperm-oviduct binding index was significantly lower (p < .01) when spermatozoa from summer ejaculates were used as compared to winter ejaculates. The expression of fertility-associated genes did not differ significantly between the two seasons except for PEPB1; the transcriptional abundance of PEPB1 was significantly (p < .05) lower in summer as compared to winter season. It was inferred that buffalo spermatozoa produced during winter season were superior in terms of cryotolerance, membrane and acrosome integrity, lipid peroxidation status and the ability to bind with oviduct explants.

Equilibration in freezing extender alters in vitro sperm-oviduct binding in the domestic cat (Felis catus).

For the preservation of endangered felid species, epididymal sperm may be received from valuable individuals after castration or death and they need to be cryopreserved for storage. However, pregnancy rates with epididymal or cryopreserved sperm are lower than with ejaculated and non-frozen semen even if insemination is surgically performed into the oviduct. To investigate whether equilibration, the first step of the cryopreservation procedure, has an impact on sperm-oviduct binding, we generated oviduct epithelial cell vesicles from isthmus segments of preovulatory domestic cats. Binding assays were performed with epididymal sperm in a cell culture medium (M199) without supplements, or after cooling to 15 °C in a freezing extender (TestG), supplemented with glycerol and the water-soluble fraction of hen's egg yolk mainly comprising LDL. The sperm-oviduct binding was assessed both quantitatively and qualitatively (head or tail binding of sperm with active or inactive mitochondria). Most of the bound sperm prepared in M199 had active mitochondria and were attached to the vesicles by their heads. In equilibrated samples, the proportion of bound sperm with active mitochondria and the proportion of head-bound spermatozoa were reduced. The total motility of the sperm after 1 h of incubation in the absence or presence of vesicles were also affected by the preparation (higher in equilibrated) and the incubation (lower in co-incubated), while mitochondrial activity was influenced just by the preparation. Obviously, LDL has a beneficial effect on sperm motility, but we suggest that it interferes with the molecular sperm-oviduct crosstalk and causes a reduced binding of "good" sperm.

Anandamide exerts a suppressive effect on sperm binding to oviduct explants through CB1 receptors in the water buffalo (Bubalus bubalis).

An endocannabinoid system comprising of Anandamide (AEA) and its receptor has been shown to play a role in sperm acquisition of fertilizing potential and sperm-oviduct interaction. In the present study, we assessed the effect of sperm pre-treatment with AEA or co-incubation of sperm-oviduct explants with AEA in the presence or absence of CB1 receptor antagonist (SR141716A) on sperm-oviduct binding in the water buffalo. Cryopreserved spermatozoa from 3 Murrah buffalo bulls (3 ejaculates from each bull) were utilized for the study. Oviduct explants were prepared by overnight culture of epithelial cells in TCM- 199 and washed spermatozoa were added to the oviduct explants and incubated for 1h. Then, sperm-oviduct explants were stained with a fluorescent stain (JC-1) and sperm binding index (BI - No. of bound spermatozoa/unit area of oviduct explants) was assessed. The results indicate that BI decreased significantly (P<0.05) when spermatozoa were either pre-treated with AEA (14.16±0.87) or sperm-oviduct explants were co-incubated with AEA (16.27±0.86) at 1nM concentration compared to the control group (29.12±2.17), however such effect was not observed when AEA was used at 1µM concentration. Incorporation of SR141716A in the incubation medium inhibited the suppressive effect of AEA on BI. It was concluded that AEA, at 1nM concentration, decreased the number of spermatozoa bound to the oviduct explants and the suppressive effect of AEA on sperm-oviduct binding was inhibited by CB1 receptor antagonist suggesting that the effect of AEA was mediated through CB1 receptor in the water buffalo.

Spermatozoa with high mitochondrial membrane potential and low tyrosine phosphorylation preferentially bind to oviduct explants in the water buffalo (Bubalus bubalis).

Although it is understood that spermatozoa are subjected to selection processes to form a functional sperm reservoir in the oviduct, the mechanism remains obscure. With the aim to understand the sperm selection process in the oviduct, in the present in vitro study, we analyzed mitochondrial membrane potential and tyrosine phosphorylation status in oviduct-explants bound and unbound spermatozoa. Frozen semen from Murrah buffalo bulls (n=10) used under progeny testing programme were utilized for the study. Oviduct explants were prepared by overnight culture of epithelial cells in TCM- 199 and washed spermatozoa were added to the oviduct explants and incubated for 4h. Mitochondrial membrane potential (MMP) and tyrosine phosphorylation status of bound and unbound spermatozoa were assessed at 1h and 4h of incubation. The proportion of spermatozoa with high MMP was significantly higher (P<0.001) among the bound spermatozoa (range 84.67-96.56%) compared to unbound (range 8.70-21.03%) spermatozoa. The proportion of tyrosine phosphorylated spermatozoa was significantly higher (P<0.001) among unbound population as compared to bound population. The proportion of spermatozoa displaying tyrosine phosphorylation at acrosomal area was significantly (P<0.05) lower in bound sperm population compared to unbound population. It was inferred that spermatozoa with high MMP and low tyrosine phosphorylation were preferred for oviduct-explants binding in the buffalo.

Oviduct binding ability of porcine spermatozoa develops in the epididymis and can be advanced by incubation with caudal fluid.

The sperm reservoir is formed when spermatozoa bind to the epithelium of the uterotubal junction and caudal isthmus of the oviduct. It is an important mechanism that helps synchronize the meeting of gametes by regulating untimely capacitation and polyspermic fertilization. This study investigated the influence of epididymal maturation and caudal fluid on the ability of spermatozoa to bind to oviduct epithelium using a model porcine oviduct explant assay. Spermatozoa from the rete testis, middle caput (E2-E3), middle corpus (E6), and cauda (E8) of Large White or Large White × Landrace boars aged 10 to 14 months were diluted in modified Androhep solution and incubated with porcine oviduct explants. Results reported in this study support our hypothesis that testicular spermatozoa need to pass through the regions of the epididymis to acquire the ability to bind to the oviduct. There was a sequential increase in the number of spermatozoa that bound to oviduct explants from the rete testis to caudal epididymis. Binding of caudal spermatozoa to isthmic explants was the highest (15.0 ± 1.2 spermatozoa per 1.25 mm(2), mean ± standard error of the mean; P ≤ 0.05) and lowest by spermatozoa from the rete testis (2.0 ± 0.3 per 1.25 mm(2)), and higher to isthmus from sows compared to gilts (35.8 ± 6.7 per 1.25 mm(2) vs. 14.8 ± 3.0 per 1.25 mm(2); P ≤ 0.05). Binding of ejaculated spermatozoa to porcine isthmus was higher than that for caudal spermatozoa (26.3 ± 1.4 per 1.25 mm(2) vs. 15.0 ± 0.8 per 1.25 mm(2); P ≤ 0.05) and higher to porcine than to bovine isthmus (26.3 ± 2.3 per 1.25 mm(2) vs. 18.8 ± 1.9 per 1.25 mm(2); P ≤ 0.05). Incubation of spermatozoa from the caput and corpus in caudal fluid increased the ability of spermatozoa to bind to the oviduct epithelium (P ≤ 0.05). In conclusion, the capacity of testicular spermatozoa to bind to the oviduct epithelium increases during their maturation in the epididymis and can be advanced by components of the caudal fluid.