**1. Introduction**

Oxytocin is a neuropeptide hormone that is synthesized in the supraoptic and paraventricular nucleus of the hypothalamus [1]. This hormone has an important role in some physiological functions, such as labor and lactation [2]. In addition, oxytocin inhibits the secretion of glucocorticoids, the hormones associated with anxiety and stress [3]. Moreover, it is increasingly studied in the field of positive emotions and welfare in humans [4,5] and animals [6].

Oxytocin plays an important facilitating role during both male and female reproductive behavior in mammals [7,8]. In male reproduction, oxytocin is thought to be associated with ejaculation by increasing sperm number and contracting smooth muscle of reproductive ducts [9]. Intravenous or intraperitoneal injection of oxytocin contributes to ejaculation in different species, such as rats, rabbits and bulls [10–12]. Similarly, intra-testicular injection of oxytocin increased basal testosterone level in mice [13]. The addition of oxytocin to boar semen artificial insemination (AI) doses improves both sperm transport to the oviduct and conception rates [14–16], but it has no influence on the quality of sperm [15]. In humans, there is an increase in plasma oxytocin levels during, and at 5 min, after ejaculation [17,18].

In pigs, the use of saliva samples for laboratory analysis is a suitable non-invasive and non-stressful alternative to blood. Oxytocin can be measured in pig saliva, it changes throughout lactation period in nursing sows [19] and it is associated to some behaviours [20]. In addition, oxytocin in saliva samples increases in men after sexual self-stimulation [21]. However, to the best of our knowledge, there are no studies about possible changes of oxytocin in saliva related to the ejaculation process in boars. It could be postulated that oxytocin could increase during the ejaculate collection time, possibly due to two main reasons: (1) the role of the oxytocin in the ejaculation, increasing contractility of reproductive smooth muscle [22], and (2) the possible emotional status associated with ejaculation [23].

The purpose of this study was to evaluate possible changes in the salivary oxytocin concentrations during ejaculate collection time in breeding boars. In addition, the influence of breed, age and libido of breeding boars in salivary oxytocin levels was also be evaluated. To accomplish these goals, two new methods previously developed and validated for oxytocin measurement in pig saliva were used, one based in the use of a monoclonal antibody that measures free oxytocin [19], and the other using a polyclonal antibody that measures oxytocin linked to protein [24].

#### **2. Materials and Methods**

#### *2.1. Animals and Ejaculate Collection*

A total of 33 mature and fertile breeding boars of three different breeds (2 Landrace, 13 Duroc and 18 Pietrain boars) were included in the study. Boars were housed in a farm located in southern Spain. The boars were housed in individual pens in a building with a controlled environment (16 h of light per day and 18–24 ◦C), with free access to water and fed with commercial feedstuff twice a day. The boars were included in artificial insemination (AI) programs with a regular ejaculate collection of two ejaculates per week. For ejaculate collection, the boars were moved from their pens to another individual pen containing a dummy sow. Once free mounted to the dummy, entire ejaculate was collected using the semi-automatic procedure called Collectis® (IMV technologies, L'Aigle, France).

#### *2.2. Saliva Collection and Oxytocin Measurement*

Saliva samples were collected using Salivette tubes (Sarstedt, Aktiengesellschaft & Co. D−51588 Nümbrecht, Germany) that contained a sponge. For sampling, the boars chewed the sponge, which was clipped to a metal rod, until the sponge was moist. The sponges were placed in test Salivette tubes and refrigerated until arrival at the laboratory. The tubes were then centrifuged at 3500 rpm at 4 ◦C for 10 min. Finally, saliva samples were stored at −80 ◦C until analysis. The research protocols were approved by the Bioethical Commission of Murcia University according to the European Council Directives regarding the protection of animals used for experimental purposes (approval number, 235/2016; approval date, 25/04/2016).

#### *2.3. Measurement of Salivary Oxytocin Concentration*

Saliva samples were thawed at room temperature and oxytocin concentrations were measured using two different methods previously developed and validated for pig saliva [19,24]. One method was a direct competition assay based on AlphaLISA (PerkinElmer Inc., Waltham, MA, USA) technology using monoclonal antibody against oxytocin, while the other consisted of an indirect competition assay based on AlphaLISA technology using polyclonal antibody against oxytocin. Although these assays have been previously described [19,24], in brief, AlphaLISA monoclonal method was performed as follows: 15 μL of sample diluted 1:2 with AlphaLISA buffer and 15 μL of acceptor beads were added to the well and after 90 min, 10 μL of biotinylated oxytocin was added and incubated for 60 min. Then, 10 μL of donor beads was added and after 30 min in the dark, the fluorescence intensity was measured using the Enspire Multimode Plate Reader (PerkinElmer Inc.). In the case of the AlphaLISA polyclonal method, 10 μL of sample diluted 1:2 with AlphaLISA buffer and 10 μL of anti-oxytocin polyclonal antibody were added to the well and after 45 min, 10 μL of protein G acceptor beads was added and incubated for 45 min. Then, 10 μL of biotinylated oxytocin was added and after 45 min, 10 μL of donor beads was added and incubated in the dark for 30 min. Subsequently, the fluorescence intensity was measured using the Enspire Multimode Plate Reader (Perkin Elmer Inc., USA). The accuracy of assays had a correlation coefficient which ranged between 0.96 and 0.99 in the case of the monoclonal method and between 0.98 and 0.99 in the case of the polyclonal method. The intra-assay coefficients of variation were between 5.02–6.07% in the case of the monoclonal method and 4.36–11.37% in the case of the polyclonal method, while the inter-assay coefficients of variation were between 4.60–17.70% in the case of the monoclonal method and 8.52–13.38% in the case of the polyclonal method. The sensitivity of the assays was 112.95 pg/mL and 58.40 pg/mL in the cases of the monoclonal and polyclonal method, respectively.
