Swine artificial insemination (AI) is performed using semen preserved in extender (1). Semen samples to be employed are subjected to several quality tests in order to maximize the production of doses for AI. Subjective semen evaluations have been replaced with objective analysis in effort to improve the assessment of fertilizing potential (2). Objective assessments provide the precision and accuracy required to obtain reliability in the estimation of quality variables (3) and would contribute to reducing technician mistakes (4). To this end, computer-assisted semen analysis (CASA) represents a valuable resource (5).

CASA technology has different modules for analyses, such as CASA-Mot (motility and kinematics) and CASA-Morph (morphometry) (6). Using CASA-Mot, spermatozoa can be classified according to their velocity as rapid, medium, slow, and static and, moreover allow for a detailed analysis of kinematic variables (7). CASA-Morph assesses morphology by using individual dimensions (length, width, area, perimeter) and shape (ellipticity, rugosity, elongation, and regularity) of the sperm head (8).

Seminal production depends on multiple factors, such as genetic improvement, reproduction techniques, health, nutrition, and handling (9). In boars, nutrition is of the utmost importance because an inadequate balance in the diet affects the libido and sperm quality (10). For this reason, supplementation must take into account season (11), age of sire (10), and weight and activity of sire (12) in order to supply the necessary mineral, vitamin, and protein requirements (13). Restrictions or deficiencies in some nutrients (14) entail a nutritional imbalance that could influence the libido and the seminal quality of the ejaculate of the reproductive boar (10).

Reactive oxygen species (ROS) are believed to be important for the normal sperm function, including processes underlying cell viability and preparation for fertilization (such as capacitation, hyperactivation, and the acrosome reaction) (15). However, sperm are susceptible to peroxidative damage due to an imbalance between ROS production and the capacity of antioxidant systems (15). The increase in ROS causes damage to the mitochondria; therefore, sperm with defective mitochondria would produce ATP inefficiently (16). In addition, an excess of ROS can also generate errors during sperm production (spermatogenesis) leading to a premature release of sperm from the germinal epithelium (17). Antioxidants can be classified into enzymatic and non-enzymatic (18). Enzymes, which are responsible for protecting sperm in the epididymis, include glutathione peroxidase (GPx), phospholipid hydroperoxide glutathione peroxidase (PHGPx), superoxide dismutase (SOD), glutathione reductase (GR) and catalase (CAT) (19). The main non-enzymatic antioxidants are vitamins A, C, and E (19).

Vitamin A is known to be necessary for the normal process of spermatogenesis (20), with retinoic acid being an alternative metabolite of vitamin A; it controls the differentiation of spermatogonia and adhesion characteristics of spermatids (14). Vitamins E and C are the most important non-enzymatic antioxidants in nutritional supplementation (21). Vitamin E includes a group of fat-soluble compounds, tocopherols, and tocotrienols, that act as antioxidants against oxidative stress (16, 22). This is because vitamin E captures free radicals, stabilizing the sperm membrane with the formation of less harmful complexes (23). High supplementation with vitamin D (2,000–4,000 Ul·Kg⁻¹), is positively associated with seminal quality (24).

Seminal quality has been linked to the presence of vitamins as supplementation (24). On the other hand, deficiency of vitamins, such as vitamin E, impacts directly ATP concentration (25), sperm production, and quality of ejaculates through the swimming patterns and morphometric characteristics (26). Because of the important role of vitamins, the present study was undertaken to examine the effect of season dietary supplementation of fat-soluble vitamins on semen quality, paying particular attention to sperm morphology and kinematics.

The use and care of animals in experimental treatments complied with the Costa Rica Institute of Technology animal welfare guidelines. Ethical approval has been given by the Committee of Centro de Investigación y Desarrollo en Agricultura Sostenible para el Trópico Húmedo at the Costa Rica Institute of Technology (CIDASTH-ITCR) according to Section 08/2020, article 1.0, DAGSC-100-2020.

The volume of semen in boar ejaculates of the FVD1 group was higher than those in the FVD12 and COD groups. Other authors reported in boars (Duroc × Pietrain), subjected to a diet similar to the control diet of the present work, a mean ejaculate volume of 245.10 ± 3.43 ml (11), which is similar to the mean value obtained in the present study for the control treatment (COD = 239.07 ± 4.15 ml). In another study, it was demonstrated that supplementation with fat-soluble vitamins improved semen production in boars (40), but supplementation with vitamin D alone did not have significant effects (24). In other species, it has been reported that supplementation with minerals and vitamins does not have any effect (p > 0.05) on the ejaculate volume (41). Some studies have indicated that the higher the volume of the ejaculate, the lower the sperm concentration (42). These findings are similar to those described in this work, in which we found reduced sperm concentration in the FVD1 (p < 0.05) group. The higher sperm concentration in the COD and FVD12 groups could be due to compensations in sperm production; compensation can be a useful strategy to correct suboptimal handling conditions (43). In other species, an increase in sperm concentration of the ejaculate has been observed after supplementation with 0.35 and 0.70 ppm of Zn in comparison to animals that did not receive supplementation (44).

Morphoanomalies have relevance semen quality (45), in particular those that are the result of alterations during spermatogenesis. The data obtained in this study showed that, regardless of treatment, the most common morphological abnormality in ejaculates was distal cytoplasmatic droplets. The percentage of distal cytoplasmic droplets was lower (p < 0.05) in boars supplemented with FVD1. This type of abnormality can arise from the very rapid passage of sperm through the epididymis (46), compromising sperm maturation (47). This could relate also to the compensation of seminal production, with adequate fat-soluble vitamin supplementation providing conditions to produce ejaculates with high-quality spermatozoa, whereas inadequate nutrition may force sires to use immature sperm. The FVD1 treatment led to a limited percentage of abnormalities since the percentage of normal spermatozoa was >90%. It has been pointed out that it is important to maintain a threshold of 90% of normal sperm in the boar ejaculate (45).

In this study, supplementation with vitamins in the FVD1 group improved the percentage of normal cells, but the other treatments (COD and FVD12) did not show differences (p > 0.05) between them. Other studies have shown that supplementation with Zn (48) and Se (49) is necessary for normal sperm development, due to their role as cofactors of many enzymes (50). In another study examining supplementation with Se and vitamin E, a decrease in abnormal sperm was obtained with the addition of 0.5 mg of Se plus 60 mg of vitamin E per kg of feed (26). This could indicate that Se supports populations of Sertoli cells, which aid in the maturation of spermatids (14, 25). Vitamin E is an antioxidant soluble in lipids of the cell membrane, which interrupts lipid peroxidation and enhances the activity of some antioxidant enzymes that control free radicals generated during the activity of some enzymes (51). On the other hand, some authors have not reported differences (p > 0.05) in the percentage of normal spermatozoa when comparing treatments with supplementation with inorganic Se vs. control groups without supplementation (52).

In relation to sperm head morphometry, differences were observed after supplementation in the FVD1 treatment group. The sperm from this treatment had longer heads (8.93 ± 0.01 μm), with a greater area (36.91 ± 0.10 μm²) and perimeter (24.73 ± 0.03 μm), but they were narrower (4.55 ± 0.01 μm). Some authors indicated that the greater the volume of ejaculate, the larger the sperm head area (42), and with higher sperm concentration, the sperm tended to be longer and with narrower heads (53). The above could differ from the data obtained in the analysis of sperm head morphometry in this study since, although the concentration between treatments showed significant differences (p < 0.05), in the FVD12 treatment this value was higher (259.64 ± 4.18 × 10⁶ ml⁻¹) than in the other treatments. In addition, in the FVD1 treatment, head length (8.93 ± 0.01 μm) and width (4.55 ± 0.01 μm) were higher than in FVD12, which contrasts with the values described by previous authors. The shape of the head can affect sperm movement (54, 55) since those with an elongated head move better than those with a more rounded head (55). This could explain why, in the present work, the group with the highest mean value of sperm head length presented a better value of sperm motility.

In this study, the percentage of total motility improved with supplementation in treatment FVD1, although there were no differences (p > 0.05) with regards to treatment FVD12. In a studyon boars under tropical conditions, a standard diet was administered to crossbred animals (Duroc × Pietrain) and sperm values for fast, medium, and slow movement patterns were 47.34 ± 1.51%, 22.82 ± 0.77%, and 7.42 ± 0.59%, respectively (56). These results are lower than those reported in the present work for the treatment where there was 100% fat-soluble vitamin supplementation (FVD1), with a value of 53% of the proportion of fast sperm, indicating that there is a positive effect of the supplementation on the sperm motility patterns. Trace elements are cofactors of enzymes that act as antioxidants (57), which protect sperm cells from damage caused by ROS (58). The decrease in sperm motility can be due to inappropriate use of ATP (59) and/or damage to the membrane integrity induced by ROS (60). Some authors have shown that Se helps improve sperm motility as it acts as a cofactor for the antioxidant enzyme GPx (61), and antioxidants are the most important defense against oxidative stress in the cell (51). Other studies in avian species showed that fat-soluble vitamin supplementation improves semen quality and quantity, especially sperm viability and motility (62). In our work, we determine that fat-soluble vitamin supplementation improves the total and progressive motilities, swimming parameters, and semen doses and that long term omission may result in adverse effects.

Regarding kinematic variables of velocity (VCL, VSL, VAP) and progressiveness (LIN), the supplementation with fat-soluble vitamins presented differences (p < 0.05) in relation to the control treatment, however, there were no differences (p > 0.05) between the treatments that received fat-soluble vitamin supplementation (FVD1 and FVD12). In a study by Lin et al. (24), an increase in curvilinear and rectilinear velocity was recorded with a supplement of 2,000 IU of vitamin D per kg of feed, compared to another treatment that consisted of a supplement with 200 IU of vitamin D per kg of feed.

When comparing boars within treatment, it was possible to observe differences between boars for all treatments of the experiment, even when they presented the same breed composition. These differences between boars can be explained by considering the ejaculate of the same boar as a heterogeneous population (29), or by the different subpopulations within each ejaculate (63) that could generate significant variability in motile sperm and kinematic patterns (64). These differences could be related to factors such as age (43), genotype (65), nutrition (22), temperature (11), and health status (66). The results obtained in this work thus show that the supplementation of fat-soluble vitamins influenced the kinematic patterns of boar spermatozoa. This suggests that reproductive management of males could include fat-soluble vitamin supplementation in boars subjected to continuous rhythms of semen collection.

A comparison of ejaculates obtained at the beginning (week 1) and end (week 32) of treatments revealed some changes in sperm parameters. The percentages of total and progressive motility showed an increase after both treatments with little change in the control (COD). An effect of fat-soluble vitamin supplementation was evident after 60–75 days of treatment, which could relate to the duration of spermatogenesis and the time of epididymal transit. Variables such as semen volume, sperm concentration, or the number of seminal doses showed a decrease in the treatment groups, and this could be explained by the frequency of semen collections, which is a well-known effect (67, 68), as well as the physical exertion that males experience over time, especially because some boars may be sensitive to increases in temperature and the observation that there may be a negative effect on spermatogenesis with temperatures above 30°C (10, 69).

Overall, studies on vitamin supplementation in relation to ejaculate quality parameters have been very scarce and many of them have only addressed the study of macroscopic characteristics (volume and concentration) of semen and few have focused on parameters such as motility, kinematics, morphology, and morphometry through CASA systems interacting with nutritional aspects. It is necessary to continue work in which the effect of the interaction between nutrition and male reproduction through CASA technology is studied, emphasizing those factors that influence the quality of ejaculates in relation to fertility.

A relevant effect of fat-soluble vitamin supplementation was observed on the semen quality parameters of the boar. The restriction of vitamins causes alterations in sperm cell formation processes that increase the percentage of sperm with abnormal morphology and limits the motility and, in general, the kinematic patterns of the sperm. Furthermore, by improving motility and kinematic variables with fat-soluble vitamin supplementation, the possibility of higher fertility increases because of ejaculates with more functional spermatozoa. Furthermore, fat-soluble vitamin supplementation will likely result in seminal doses with fewer spermatozoa for an equal level of success.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Costa Rica Institute of Technology.

ER and AV: conceptualization, writing—review and editing, and visualization. JC-C and AV: methodology and investigation. JC-C: software. AV, FS, and VB: validation. AV: formal analysis, resources, data curation, supervision, project administration, and funding acquisition. JC-C, ER, VB, and AV: writing—original draft preparation. All authors listed have made a substantial, direct, and intellectual contribution to the work and agreed to the published version of the manuscript.

This work was supported by Fundación para el Fomento y Promoción de la Investigación y Transferencia de Tecnología Agropecuaria de Costa Rica (FITTACORI) and Costa Rica Institute of Technology [Vice-Chancellor's office of Research and Extension; VIE (Vicerrectoría de Investigación y Extensión); Project-VIE-5402-2151-1015]. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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