Boars used in this study were Sire Line PIC 337 and 415 (PIC^®, Pig Improvement Company, Pasig City, Philippines), Large White (La Botica Genética Porcina S.A., Buenos Aires, Argentina); Pietrain (Cooperativa Marcos Juarez, Cordoba, Argentina); and Landrance (Topigs Norsvin, Buenos Aires, Argentina). A total of 250 seminal doses from 13 boars were used (please, see Supplementary Table 1 for the distribution of seminal doses used). For AI, 250 sows Large White/Landrace, with average parity of 2.1 were used. No nulliparous sows were included. Ethical approval of the research involving animals was given by the Comité Institucional para el Cuidado y Uso de Animales de Laboratorio (CICUAL-FCByF), Universidad Nacional de Rosario (File N° 6060/316).

Semen samples were collected from adult fertile boars by the glove-hand method by seminal doses producer company, Medax (Chañar Ladeado, Santa Fe, Argentina). The frequency of collection was every 7–10 days per boar. All the samples conformed to normal seminal parameters for pig, such as motility > 80% (by CASA, please see complete description in the next section), total sperm in the ejaculate determined by dilution and counting in Neubauer chamber > 20 × 10⁹, and morphological defects [by Wells-Awa staining (21)] < 15%. Sperm-rich fractions of ejaculates were diluted 20 to 30-fold to reach a concentration of 3 × 10⁷ sperm/ml in Sus extender: consisting of monosaccharide energy substrate, ionic salts, Tris (hydroxymethyl) ethane, broad-spectrum antibiotic and 4% EDTA disodium salt (C.A.S. registry 6381-92-6) (Medi Nova, Reggio Emilia, Italy), then aliquots of each seminal dose were supplemented with 0 (control), 0.5, 1, 2, and 3 mM ZnCl₂ (Merck KGaA, Darmstadt, Germany), and conserved at 16°C until use (up to 3 days). For in vitro analyzes, doses were divided into five aliquots and supplemented with ZnCl₂ in the mentioned concentrations and samples of these aliquots were taken for analysis every 24 h for 3 days. Eight individual doses from eight individual boars were tested for in vitro analyzes.

Considering the reported mean value of zinc concentration in seminal plasma of boars is 1,927.5 μg/dL (2.96 mM of Zn⁺²) (22); we used the following final concentrations: 0.5, 1, 2, and 3 mM of ZnCl₂ to extenders. Doses were maintained at 16°C upon dilution and samples were taken for analysis every 24 h for 3 days.

Viability (n = 8, eight individual seminal doses from eight different boars) was measured using the eosin exclusion test (23). Briefly, 5 μl of sperm suspension were mixed with 5 μl of eosin-nigrosin solution and smeared in a slide.

Acrosome integrity (n = 8, eight individual seminal doses from eight different boars) was evaluated by Wells-Awa staining according to characterizations for acrosome alterations in previous works (24). Sperm were observed at magnification of 100× by bright field microscopy. Acrosome intact sperm were considered to be those having a thickened blue-green region at the apex of the head, a blue-green cap covering the anterior two-thirds of the head and a pink color in the posterior one-third of the head. Cytoplasmic droplets and flagellum morphology were also observed. For viability, morphological defects and acrosome integrity analyses at least 200 sperm were counted.

Sperm trajectories were examined using computer-assisted semen analysis (CASA) system (IVOS I Sperm Analyzer, Hamilton Thorne) (n = 8, eight individual seminal doses from eight different boars). Aliquots of 8 μl of seminal doses containing 3 × 10⁷ sperm/ml were placed between slides and 18 × 18 mm coverslips. As variations using slides and coverslips instead of commercial chambers are expected, we compared Hamilton Thorne system and the Proiser using ISAS^® D4C20 chamber (Supplementary Table 2). No differences were founded between the two methods. Thirty frames were acquired at 60 Hz. The software settings for the IVOS I were: minimum contrast 18, minimum cell size (pix) 7, cell size (pix) 9, cell intensity 125, slow-static cells with average path velocity (VAP) cut-off (l m/s) 20, and straight-line velocity (VSL) cut-off (l m/s) 5, minimum static intensity gates 0.5, maximum static intensity gates 2.5, minimum static size gates 0.65, maximum static size gates 2.6, minimum elongation gates 20 and maximum elongation gates 85 according to Vyt et al. (25). The following parameters of sperm motility were measured: mean path velocity (VAP, μm/s), curvilinear velocity (VCL, μm/s), straight-line velocity (VSL, μm/s), linearity (LIN, %), amplitude of lateral head displacement (ALH, μm), and straightness (STR, %). Sperm with velocities < 15 μm/s. were considered slow, between 16 and 35 μm/s were understood as medium speed sperm and those with a velocity >35 μm/s were considered rapid sperm.

Mitochondria and DNA integrity were evaluated simultaneously on the same samples (n = 8, 8 individual seminal doses from 8 different boars) by analysis of cytochrome oxidase C activity (26) and sperm chromatin dispersion (27), respectively. Briefly, 100 μl of seminal doses containing 3 × 10⁶ spermatozoa were incubated for 30 min at 37°C with 100 μl of extender solution containing 100 μg of 3,3'-diaminobenzidine (DAB, D-5637 Sigma, Buenos Aires, Argentina). After incubation, 30 μl of suspension were mixed with 70 μl of pre-warmed 1% low-melting agarose at 37°C. Fifteen microliters of suspension were pipetted onto slides pre-coated with 0.65% agarose pre-cooled at 4°C, and covered. The preparation was incubated for 8 min at 4°C, coverslips were removed and sperm incubated for 8 min with acid lysis solution (0.084 N HCl) at 22°C. After washing out the acid lysis solution, samples were incubated for 25 min with lysis solution (0.4 M Tris–HCl pH 7.5, 2 M NaCl, 1% SDS, 0.05 M EDTA, 5% β-mercaptoethanol) at 22°C. Slides were washed for 5 min, dehydrated in sequential 70, 90 and 100% ethanol baths, and stained with Wright solution for 15 min with gentle agitation. Samples were observed using an Olympus microscope BX40 (Japan) at 400× magnification. For evaluation of mitochondria integrity, spermatozoa were classified according to Fariello et al. (28) as follows: pattern I (100% of the midpiece stained), pattern II (>50% of the midpiece stained), pattern III (<50% of the midpiece stained), and pattern IV (no staining of the midpiece) (Supplementary Figure 1A). According to this classification, sperm with pattern I maintained complete mitochondrial activity; with pattern II, lose some mitochondrial activity without impairing cellular motility and fertilizing capacity; with pattern III, presented extensive loss of mitochondrial activity; and with pattern IV were either dead or had minimal energy production. To test DNA fragmentation, we looked at the halos of diffusion of 200 of Wright-stained spermatozoa. Cells with large, medium or small halos of diffusion corresponded to sperm nuclei containing fragmented DNA. Sperm nuclei without DNA fragmentation did not show halos. As control for DNA damage, we incubated aliquots of sperm during 1, 2, and 3 days with 300 μM of H₂O₂, but we did not observe effects as described for human sperm (29). Instead, we had to store sperm at−80°C in order to produce damage to the DNA (Supplementary Figure 1B). At least 200 individual sperm were counted.

ROS determination was performed according to Benjamin et al. (30) (n = 8, eight individual seminal doses from eight different boars). This method measured a variety of intra and extra-cellular ROS, including O2•-, H₂O₂, and OH^•. The results were expressed as relative light units (RLU) per 1.2 × 10⁶ sperm. Cells supplemented with H₂O₂ were used as positive control for oxidative damage.

Artificial insemination was performed as previously reported (31) in a local farm located in Monte Maíz, Córdoba, Argentina (GPS coordinates: −33.206561, −62.600330). In order to have an approach beyond laboratory conditions and not to skew the observations, we set up the working conditions with the less modifications possible to farm daily operations. Therefore, in our work we tried to explore the effect of zinc in a consuetudinary context instead of laboratory conditions. The AI were performed by day 3 of storage in order to have information from laboratory examinations. Thus, viability, acrosomal integrity and motility were assessed before AI. As neither of these parameters were compromised, then AI was carried out. Personnel who performed AI didn't know about the seminal doses with zinc added. Lactation in the farm is 21-days length. The trial was conducted between November 15th and July 20th 2017. Briefly, estrus was checked daily in the presence of a mature teaser boar. Occurrence of estrus was defined by the standing reflex in front of a boar and back pressure-test. Inseminations were done using spiral catheters (Magapor^®, Zaragoza, Spain) and 3 × 10⁹ sperm in 100 mL doses were deposited in sow's cervix. All sows were inseminated in standing heat in the presence of a boar. Eighty-six sows chosen at random were allocated to be inseminated with doses supplemented with 2 mM ZnCl₂ and 164 were inseminated with untreated doses as control. Seminal doses from 13 boars were used. Each individual ejaculate was divided in parts; one part was diluted with extender supplemented with 2 mM ZnCl₂ and the other with extender without supplementation, and both were used for AI. The distribution of ejaculates per boar used in AI is presented in Supplementary Table 1.

A protector effect on mitochondria integrity and a higher percentage of progressive and rapid sperm in doses supplemented with 2 mM ZnCl₂ was observed. Also, the percentage of straightness and linearity was higher in doses supplemented with 2 mM ZnCl2 by day 3 of storage. Considering that the kinematic parameters mentioned are a key factor for AI, we performed AI with seminal doses supplemented with 2 mM ZnCl₂.

For viability, motility, acrosome and DNA integrity, and ROS levels, data were analyzed for normality by Shapiro-Wilks's test (32) and Bartlett's test to homogeneity of variances. Then, one-way analysis of variance (ANOVA) for normally distributed data was applied. When F-test results were significant in ANOVA, individual means were further tested by Tukey's multiple range test. Kruskal-Wallis test was applied for data without normal distribution. Assessment of sperm parameters was done using eight biological replicates. Data for progressive motility, VAP, VSL, VCL, ALH, BFC, STR, and acrosome integrity did not show normal distribution. Data regarding mitochondria integrity were analyzed by generalized linear mixed models contemplating interaction between days and treatment. To each variable, a mixed general linear model was adjusted for longitudinal data considering fixed effect for time and treatment, and random effect for boars. Through residuals analysis of the model and Goodness-of-Fit test by Bayesian information criterion (BIC) and Akaike information criterion (AIC), variability intra- and inter-individual were determinate. There was no interaction between time and treatment. Also, variance heterogeneity was considered. For AI trial, the farrowing rate was analyzed by logistic regression model and data regarding to numbers of piglets by Poisson regression. The statistical model that includes the boar factor and corrections made for unequal distribution of semen doses was applied. A random effect for the boar was included to account for the variability and correlation produced by the use of different donors.

Addition of ZnCl₂ to the extender did not affect the cell viability based on membrane integrity, regardless of zinc concentration or time elapsed after dilution (Figure 1). However, all doses supplemented with ZnCl₂ showed a higher percentage of sperm with unaltered acrosomes compared to the control without added zinc (Figure 2).

Table 1 shows the quantification of the sperm motility measured by examination of the trajectories of individual cells. Sperm with slow and medium speeds displayed significant differences between treatments and different kinematic parameters changing through the days in response to added ZnCl₂. However, the percentage of progressive and rapid sperm in the samples were mainly conserved over time in doses supplemented with 2 mM ZnCl₂, except for day 3 when a higher percentage of rapid sperm was observed in doses supplemented with 1 mM ZnCl₂.

While percentage of motile sperm did not show significant differences between treatments, VAP, VCL, VSL, LIN, ALH, and STR changed after Day 1 (Table 2). By day 2, VAP, VSL and ALH showed the highest values, while BCF showed the lowest values in doses supplemented with 2 and 3 mM of ZnCl₂. At this time, VCL was higher in treatments at all concentrations of ZnCl₂. By day 3, VAP, VSL, VCL, showed the higher values at 0.5 and 1 mM of ZnCl₂, and ALH did not show differences with the control. Zinc-supplemented samples displayed the highest values of LIN at 2 mM ZnCl₂, and the lowest value of BCF by day 3. These results indicate that addition of ZnCl₂ to the extender affects the kinetic parameters of the sperm.

Figure 3 shows the percentage of sperm displaying different patterns of staining of the midpiece (I to IV), from 100 % staining (full mitochondrial activity) to no staining (dead cells or cells with no mitochondrial activity) (Supplementary Figure 1A). A decrease in the number of cells showing pattern I and II was observed at all tested times after dilution, with an associated increase of cells with pattern III. But the increase in sperm displaying pattern III (cells that present extensive loss of mitochondrial activity) in the control was significantly higher than those of samples containing added ZnCl₂ (at all concentrations). An interaction effect between day and treatment was observed for pattern III (p = 0.0149). The number of cells displaying pattern IV was not affected by either time or treatment.

Addition of ZnCl₂ resulted in no significant differences in DNA fragmentation (see Supplementary Figure 1B), regardless the length of storage (Figure 4).

Table 3 shows the levels of ROS in sperm in the presence and absence of added ZnCl₂, and over time, expressed as relative units per 1.2 × 10⁶ spermatozoa. Results show that in all tested conditions, the addition of ZnCl₂ causes no effect on ROS production, at all tested times.

From a total of 250 sows inseminated in the trial, 204 became pregnant. The farrowing rate using doses supplemented with zinc was 85.7%, while the farrowing rate without zinc supplementation was 79.5%. No statistically differences between the control and the ZnCl₂-supplemented sperm were observed in farrowing rate (Table 4), number and distribution of total number of piglets, live piglets, stillborn and mummified fetuses (Table 5).