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Hyaluronan (HA) is involved in several important steps of sperm storage and of fertilization. This study investigates the presence and concentration of HA in oviductal fluid (ODF), together with the localization of HA and the presence of hyaluronan-binding proteins (HABPs) in the oviductal epithelium of normally cycling dairy heifers and cows. The concentration and amount of HA in ODF, collected over the course of several oestrous cycles via catheters placed in the isthmic and ampullar tubal segments, were measured using an ELISA. The concentration and amount of HA in ODF did not vary significantly between these anatomical regions, nor between the stages of the oestrous cycle (p > 0.05), although the amount of HA seemed to peak during oestrous. The most HA per day (2.9 ± 0.64 µg, least square mean ± SEM) was produced on the day of ovulation, whereas the lowest amount (1.25 ± 0.68 µg) was produced 4 days before ovulation. To investigate the localization of HA, tissue samples were retrieved at well-defined stages of the oestrous cycle and from corresponding regions of the oviduct. Sections and protein extracts from the tissue samples were studied histochemically using biotinylated HABP and immunoblotted with fluorescein isothiocyanate (FITC)-HA, respectively. Presence of HA labelling in the oviductal epithelium was restricted to the sperm reservoir, a localization that seemed to be cycle-independent. The immunoblotting of samples from the lining epithelium revealed seven bands of HABPs. We confirm that the bovine oviduct produces HA and its binding proteins, and that HA is mainly localized to the epithelium of the sperm reservoir.

Sperm capacitation is crucial for fertilization. However, debate continues on exactly how, where and when capacitation is elicited in the bovine female genital tract. In this study we used merocyanine-540 and the chlortetracycline (CTC) assay to test how capacitation of bull spermatozoa is affected in vitro by exposure to oviductal fluid (ODF) collected in vivo, various glycosaminoglycans (GAGs) or bicarbonate. Following different durations of exposure, spermatozoa were stained with CTC or merocyanine-540, and evaluated with epifluorescent light microscopy or flow cytometry, respectively. Incubation time did not significantly affect capacitation. Exposure (30-120 min) to ODF capacitated (p < 0.05) bull spermatozoa as measured by either merocyanine-540 or CTC. Hyaluronan was the only GAG that induced a significant increase in B-pattern spermatozoa (capacitated; p = 0.012) compared with controls. Dermatan sulphate also induced capacitation (merocyanine-540 high fluorescence; p = 0.035). Exposure to bicarbonate-enriched media also yielded an increase in merocyanine-540 high fluorescence (p < 0.0001). When bicarbonate was added to the other treatments (ODF or GAGs) an equal increase in merocyanine-540 high fluorescence was noted (p < 0.0001), compared with before addition of bicarbonate and independent of the treatment before exposure. There was no significant difference in the number of B-pattern spermatozoa when bicarbonate was added, but an significant increase in spermatozoa with an acrosome-reacted (AR)-pattern (p < 0.0001) was observed. Exposure of spermatozoa to solubilized zonae pellucidae significantly increased the AR-pattern spermatozoa (p = 0.016). In conclusion, ODF was more potent in inducing capacitation of bull spermatozoa than the individual GAGs. Our results also indicate that bicarbonate is an effector of bull sperm capacitation.

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BackgroundThe purpose of this study was to validate a commercially available enzyme-linked immunosorbent assay (ELISA) developed for measuring free cortisol in human saliva and total cortisol concentration in diluted human serum, for its applicability in measuring cortisol concentration in pig saliva. Collection of saliva is less stressful than e.g. blood sampling, and is a non-invasive method.FindingsSaliva was collected by allowing sows to chew on cotton swabs held by forceps. Thereafter, the swabs were centrifuged to retrieve the saliva. The ELISA was performed according to instructions provided by the manufacturer. To validate the ELISA, determination of the intra-assay coefficient of variation (CV), inter-assay CV, recovery, linearity and parallelism was performed. The intra-assay CV was below 10% and inter-assay CV below 15% for samples of high, medium and low cortisol concentrations. The mean recovery was 117% and the linearity and parallelism showed an r2-value of 0.994 and 0.993, respectively. For biological assessment of induced social stress, two saliva samples were collected in the morning from 6 primiparous and 21 multiparous sows. One sample was collected when the sows were individually housed in a farrowing pen and a second sample was collected when the sows were group housed. The primiparous sows had a significant higher cortisol concentration compared to the multiparous sows when group housed.ConclusionThe results obtained in this validation study indicate that the ELISA is suitable for measuring cortisol concentration in porcine saliva.

Following insemination, spermatozoa are retained in the utero-tubal junction and isthmic region of the oviduct, where essential steps of capacitation are coordinated. Although a majority of the spermatozoa is exposed to similar conditions in the oviduct, the speed of the response varies depending on the individual male and the state of the spermatozoa. The present study evaluated individual boar variations in terms of the ability of spermatozoa to undergo tyrosine phosphorylation in response to isthmic oviductal fluid (ODF). Cryopreserved spermatozoa from four boars were incubated with pre- and post-ovulatory ODF for 6 hr. Sperm kinematics, global protein tyrosine phosphorylation, and dynamics of different phosphorylation patterns were analyzed at hourly interval. The percentage of phosphorylated spermatozoa in the pre-ovulatory ODF-treated group was significantly (P < 0.001) higher than in the other treatment groups. Motility, velocity, and protein tyrosine phosphorylation in spermatozoa in response to ODF and control media also showed differences between boars. Spermatozoa from all four boars showed strong expression of a 19-kDa phosphoprotein while spermatozoa from two boars showed additionally strong expression of a 32-kDa phosphoprotein when incubated with pre-ovulatory ODF. While phosphorylation of proteins in the acrosome and the equatorial segment of the sperm were noticed at an early stage during incubation with ODF, tail phosphorylation appeared at a later stage of capacitation. The results indicate individual variation between boars in terms of sperm proteins, including different phosphorylation patterns, in response to ODF, which might be related to fertility.

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