**4. Discussion**

This manuscript reports, for the first time, changes in oxytocin concentrations in saliva associated with the ejaculation process in a livestock species. To the best of our knowledge, increases in oxytocin levels after ejaculation have only been reported in cerebrospinal fluid of rats [27], in blood of rabbits [28] and blood and saliva of humans [17,18,21]. All these studies reported an increase of oxytocin levels associated with ejaculation and support our hypothesis that oxytocin would also increase in pig saliva.

Specific studies addressing how oxytocin reaches saliva in pigs have not been reported. However, mammals share essential oxytocin system characteristics, such as production of oxytocin in the

hypothalamus and peripheral and central release [29]. Oxytocin is synthesized in the hypothalamic supraoptic and paraventricular nuclei as a neurohormone and is released through the posterior pituitary gland into the bloodstream [30]. In humans, oxytocin reaches the salivary glands via blood circulation through active transport mechanisms [31]. However, the dynamic of oxytocin in both fluids (blood and saliva) could be different, since saliva concentrations of oxytocin do not reflect peripherally circulating oxytocin after exogenous administration in humans [32]. Martin et al. [33] found that oxytocin concentrations in human saliva are more correlated with those in cerebrospinal fluid, and therefore with central oxytocin, than with those of blood plasma.

In this study, two assays were used for measurement of salivary oxytocin, namely monoclonal and polyclonal assays. The results evidenced that the polyclonal assay detects changes of higher magnitude in salivary oxytocin concentrations, especially at 2 h after ejaculate collection. This could be related to the ability of polyclonal assays to detect different metabolites of the oxytocin molecule together with oxytocin bound to other proteins [24] that could remain 2 h after ejaculation. Differences between both assays have also been described in a previous study measuring oxytocin in lactating sows [24]. Together, these results would make polyclonal assays the assay of choice for measuring changes in salivary oxytocin in breeding boars. Accordingly, the discussion will be focused on the results achieved using the polyclonal assay.

The increased salivary oxytocin concentration during ejaculation found in our study reflect its involvement in the ejaculation process [9,34]. Oxytocin contributes to the modulation of sexual behavior [35] and is also involved in the control of male fertility, promoting the transport of sperm through the regulation of basal contractility of smooth muscle in the cauda epididymis [34], as well as stimulation of contraction of the prostate during ejaculation [36]. In addition to its physiological role in the mechanisms related to ejaculation, the increase in oxytocin during ejaculate collection time could also be associated with positive emotions experienced by boars during ejaculation. Oxytocin may represent a link between the social and physical feelings and emotions and sexual behavior [5,37], being released through sensory stimuli, such as touch, warmth and odor [20,38,39]. In addition, oxytocin has become a central component of the mechanisms mediating the well-being and anti-stress effects of positive social interactions in humans [40,41] and animals [6,42].

Interestingly, not all boars showed increased oxytocin concentrations during ejaculation time, as it increased in only 20 of the 33 breeding boars under study. There are several factors that can influence the sexual behavior of boars at ejaculation, such as genetic factors [43]. In this study, more Pietrain than Duroc boars showed increased oxytocin concentrations at T0. The weaker libido of Duroc boars compared with those of others breeds [44,45], as well as the lower total number of sperm and percentage of live sperm in the ejaculates compared with those of Pietrain boars [46], could explain the lower or non-existent increase in salivary oxytocin concentrations at ejaculation time. Furthermore, the age of boars is another influencing factor, as younger boars showed the highest salivary oxytocin concentrations during the ejaculate collection, while the older boars did not show changes between times. We could not find information in the literature to explain differences in salivary oxytocin levels related to age. However, previous studies in boars found that those aged 18 to 24 months had a higher percentage of motile sperm than those aged more than 30 months, which also had higher percentages of sperm abnormalities, such as simple bent tails [47]. In line with this, pregnan<sup>t</sup> women of the first child showed higher serum oxytocin levels compared to pregnan<sup>t</sup> women who already had children [48].

Libido was another factor influencing the salivary oxytocin levels. Boars with high libido showed the highest oxytocin concentrations in saliva at ejaculation time, while boars with moderate or low libido did not show changes in oxytocin concentrations between times. Previous studies in humans found that blood oxytocin was positively related with intensity of sexual behavior and couple interaction [49]. Therefore, it could be postulated that increased libido in boars could be caused by a higher oxytocin synthesis. However, it could also be to the contrary, i.e., increased libido induces increased synthesis of oxytocin. More studies are needed to clarify this issue.
