**3. Results**

The salivary oxytocin concentrations measured with monoclonal and polyclonal assays showed a low correlation (R = 0.323, *p* < 0.05). As such, the results for each assay are presented separately.

#### *3.1. Oxytocin Concentrations Measured with the Monoclonal Assay*

Salivary oxytocin concentrations were significantly higher (*p* < 0.05) at T0 (1077.0 pg/mL; 25−75th percentile: 527.3–2555.0 pg/mL) than at DB time (775.6 pg/mL; 25–75th percentile: 512.8–1494.0 pg/mL) but there were no significant di fferences (*p* > 0.05) when compared with T2 (802.6 pg/mL; 25–75th percentile: 386.5–1821.0 pg/mL). Interestingly, not all boars showed the same pattern of variation. While some boars showed increased concentrations of oxytocin at T0 compared to DB, other boars showed the opposite behavior (decreased concentrations at T0). Therefore, changes in salivary oxytocin concentrations were evaluated separately in each of the two boar groups. Oxytocin concentrations in the group that showed an increase at T0 (*n* = 21) were higher (*p* < 0.01) at T0 (1767.0 pg/mL; 25–75th percentile: 737.7–3342.0 pg/mL) than at DB (653.4 pg/mL; 25–75th percentile: 413.3–1322.0 pg/mL) and T2 (771.2 pg/mL; 25–75th percentile: 336.5–1821.0 pg/mL). Oxytocin concentrations in the other group, which showed a decrease at T0 (*n* = 12), were lower (*p* < 0.05) at T0 (574.8 pg/mL; 25–75th percentile: 393.2–1069.0 pg/mL) than at DB (1219.0 pg/mL; 25–75th percentile: 550.6–1981.0 pg/mL) and T2 (1022.0 pg/mL; 25–75th percentile: 477.2–1829.0 pg/mL). These results are shown in Figure 1.

**Figure 1.** Changes in salivary oxytocin concentrations measured with AlphaLISA monoclonal assay at di fferent times: the day before ejaculate collection (DB), immediately after starting the ejaculation (T0) and two hours after ejaculation (T2). Data for all 33 boars (**a**), data for the 21 boars showing increased oxytocin concentration at ejaculation time (**b**) and data for the 12 boars that did not show increased oxytocin concentrations at ejaculation time (**c**). The plots show medians (line within box), 25th and 75th percentiles (boxes), min and max values (whiskers) and individual data points. Asterisks indicate di fferences between times (\*\*\*\* *p* ≤ 0.0001; \*\* *p* ≤ 0.01; \* *p* ≤ 0.05).

When pigs were classified by age (boars aged 12 to 24 months, aged 24 to 36 months, and aged more than 36 months), no significant di fferences in salivary oxytocin between times were seen within each group (Figure 2). However, when the pigs were classified according to their libido, boars with a libido intensity of 3 had higher (*p* < 0.05) oxytocin concentrations at T0 (1250.0 pg/mL; 25–75th percentile: 511.9–2599.0 pg/mL) than DB (834.9 pg/mL; 25–75th percentile: 357.8–1565.0 pg/mL) and did not show significant changes at T2 (749.5 pg/mL; 25–75th percentile: 397.6–1815.0 pg/mL), whereas boars with libido intensity values of 1 and 2 showed similar salivary oxytocin concentrations across the three collection times (Figure 3).

The frequency of individuals having increases in oxytocin concentrations at T0 was di fferent depending on the breed. It was found that around 83.4% of Pietrain boars showed higher oxytocin concentrations at T0 than at DB (*p* < 0.01) and T2 (*p* < 0.05). However, only 46.1% of Duroc boars showed higher oxytocin concentrations at T0 but without di fferences between DB and T0. The two Landrace boars did not show significant changes at the di fferent times.

**Figure 2.** Changes in salivary oxytocin concentrations measured with AlphaLISA monoclonal assay at di fferent times: the day before ejaculate collection (DB), immediately after starting the ejaculation (T0) and two hours after ejaculation (T2). Figure (**a**) shows the data for boars aged 12–24 months and (**b**,**<sup>c</sup>**) those for boars aged 24–36 and aged more than 36 months, respectively. The plots show medians (line within box), 25th and 75th percentiles (boxes), min and max values (whiskers) and individual data points.

**Figure 3.** Changes in salivary oxytocin concentrations measured with AlphaLISA monoclonal assay at different times: the day before ejaculate collection (DB), immediately after starting the ejaculation (T0) and two hours after ejaculation (T2). Figure (**a**) shows the data for boars with libido intensity of 3 and (**b**,**<sup>c</sup>**) those with libido intensity of 2 and 1, respectively. The plots show medians (line within box), 25th and 75th percentiles (boxes), min and max values (whiskers) and individual data points. Asterisk indicate differences between times (\* *p* ≤ 0.05).

#### *3.2. Oxytocin Concentration Measured with the Polyclonal Assay*

Salivary oxytocin concentrations were significantly higher (*p* < 0.05) at T0 (25.8 ng/mL; 25–75th percentile: 7.2–76.7 ng/mL) and T2 (20.3 ng/mL; 25–75th percentile: 11.9–46.5 ng/mL) than at DB time (13.3 ng/mL; 25–75th percentile: 6.1–24.5 ng/mL). Regarding the different pattern of variation, changes in oxytocin concentrations were evaluated separately in each of the two boar groups depending on the increase or decrease at T0. Oxytocin concentrations in the group that showed an increase at T0 (*n* = 20) were higher (*p* < 0.05) at T0 (57.2 ng/mL; 25–75th percentile: 19.9–80.0 ng/mL) and T2 (28.4 ng/mL; 25–75th percentile: 12.4–73.7 ng/mL) than DB (8.7 ng/mL; 25–75th percentile: 5.8–24.8 ng/mL). Oxytocin concentrations in the group that showed a decrease at T0 (*n* = 13) were higher (*p* < 0.05) at DB (13.8 ng/mL; 25–75th percentile: 8.8–23.2 ng/mL) and T2 (15.1 ng/mL; 25–75th percentile: 7.8–24.7 ng/mL) than T0 (6.9 ng/mL; 25–75th percentile: 3.6–11.5 ng/mL). These results are shown in Figure 4.

When pigs were classified by age (boars aged 12 to 24 months, aged 24 to 36 months, and aged more than 36 months), significant differences between times were seen in boars aged 12 to 24 months and in boars aged 24 to 36 months (Figure 5). Oxytocin concentrations observed in boars aged 12 to 24 months were higher (*p* < 0.05) at T0 (67.5 ng/mL; 25–75th percentile: 10.4–108.4 ng/mL) than at DB (13.3 ng/mL; 25–75th percentile: 5.3–39.3 ng/mL). Oxytocin concentrations in boars aged 24 to 36 months were higher (*p* < 0.01) at T2 (32.9 ng/mL; 25–75th percentile: 12.7–79.7 ng/mL) than DB (13.9 ng/mL; 25–75th percentile: 6.1–22.3 ng/mL). Boars aged more than 36 months showed similar salivary oxytocin concentrations at the three collection times. When the pigs were classified according to their libido, boars with a libido intensity of 3 showed higher (*p* < 0.05) oxytocin concentrations at T0 (25.8 ng/mL; 25–75th percentile: 6.9–77.4 ng/mL) and at T2 (24.5 ng/mL; 25–75th percentile: 11.9–56.9 ng/mL) than DB (9.8 ng/mL; 25–75th percentile: 5.9–24.2 ng/mL). Boars with libido intensity 2 and 1 showed similar salivary oxytocin concentrations at the three collection times (Figure 6).

**Figure 4.** Changes in salivary oxytocin concentrations measured with AlphaLISA polyclonal assay at different times: the day before ejaculate collection (DB), immediately after starting the ejaculation (T0) and two hours after ejaculation (T2). Data for all 33 boars (**a**), data for the 20 boars showing increased oxytocin concentration at ejaculation time (**b**), and data for the 13 boars that did not show increased oxytocin concentrations at ejaculation time (**c**). The plots show medians (line within box), 25th and 75th percentiles (boxes), min and max values (whiskers) and individual data points. Asterisks indicate differences between times (\*\*\*\* *p* ≤ 0.0001; \* *p* ≤ 0.05).

**Figure 5.** Changes in salivary oxytocin concentrations measured with AlphaLISA polyclonal assay at different times: the day before ejaculate collection (DB), immediately after starting the ejaculation (T0),

and two hours after ejaculation (T2). Figure (**a**) shows the data for boars aged 12–24 months and (**b**) and (**c**) those for boars aged 24–36 and aged more than 36 months, respectively. The plots show medians (line within box), 25th and 75th percentiles (boxes), min and max values (whiskers) and individual data points. Asterisks indicate differences between times (\*\* *p* ≤ 0.01; \* *p* ≤ 0.05).

**Figure 6.** Changes in salivary oxytocin concentrations measured with AlphaLISA polyclonal method at different times: the day before ejaculate collection (DB), immediately after starting the ejaculation (T0) and two hours after ejaculation (T2). Figure (**a**) shows the data for boars with libido intensity of 3 and (**b**,**<sup>c</sup>**) those with libido intensity of 2 and 1, respectively. The plots show medians (line within box), 25th and 75th percentiles (boxes), min and max values (whiskers) and individual data points. Asterisk indicate differences between times (\* *p* ≤ 0.05).

The frequency of individuals having increased oxytocin concentrations at T0 was different depending on the breed. We found that 72.2% of Pietrain boars showed higher oxytocin concentrations at T0 than at DB (*p* < 0.01). However, 46.1% of Duroc boars showed an increase at T0 but without differences between DB and T0, although oxytocin concentrations at T2 were higher (*p* < 0.05) than at DB. The two Landrace boars did not show significant changes at the different times.
