The aim of this field-based longitudinal study was to evaluate whether oral nutraceutical supplementation is associated with favourable changes in semen output, total motile sperm output and sperm morphology in breeding stallions monitored under routine reproductive-management conditions. The biological rationale for the study was that stallion spermatozoa are highly dependent on adequate metabolic support, membrane stability and antioxidant protection, and that oral nutraceutical supplementation may therefore support sperm production and sperm function over time. A repeated-ejaculate design was used because a single ejaculate does not necessarily reflect the reproductive profile of a stallion under field conditions. Serial assessment was considered more informative for the practical evaluation of semen output, sperm motility, sperm concentration and sperm morphology in breeding animals.
The study was conducted under field conditions in Paraná, Brazil, and involved clinically healthy Mangalarga Marchador stallions maintained under consistent reproductive and nutritional management. The same stallions were evaluated during a pre-supplementation phase and again after a 60-day supplementation period. During supplementation, each stallion received 80 g per day of a commercial oral nutraceutical product with the morning concentrate ration. The formulation included vitamin, mineral, amino-acid and yeast-derived components intended to support antioxidant protection, cellular metabolism and structural sperm integrity. The 60-day period was selected because it is compatible with the duration of spermatogenesis and epididymal maturation in the stallion, thereby allowing the post-supplementation phase to reflect sustained exposure to nutrients potentially relevant to sperm production and semen quality.
A total of 60 ejaculates were evaluated across the pre- and post-supplementation phases. Semen was collected with an artificial vagina after teasing with an oestrous mare. After collection, the gel fraction and visible contaminants were removed, and samples were processed promptly under standardised laboratory conditions. The semen examination followed conventional field-applicable equine andrology procedures and included ejaculate volume, total motility, vigour score, sperm concentration, total sperm output per ejaculate, total motile sperm output per ejaculate and sperm morphology. Total motility was recorded as the percentage of motile spermatozoa, vigour was scored on a 1-to-5 scale, and sperm concentration was determined with a Neubauer haemocytometer. Productive semen endpoints were calculated from ejaculate volume, sperm concentration and total motility. For morphology, stained smears were prepared and 200 spermatozoa per sample were classified as normal, with major defects, or with minor defects. In parallel, libido during collection was assessed on a modified 0-to-10 field scale adapted from a previously described stallion libido-scoring system.
The descriptive analysis demonstrated a favourable post-supplementation seminal profile (Table 1). Total motility increased from 80.7 ± 6.9% to 84.7 ± 5.1%, corresponding to a relative increase of 5.0%. Vigour score increased from 3.4 ± 0.5 to 3.9 ± 0.3, which represented an increase of 14.7%. Sperm concentration increased from 165.3 ± 60.6 to 193.7 ± 56.8 ×10⁶ spermatozoa/mL, corresponding to a 17.2% increase. Particularly relevant changes were observed in productive semen endpoints: total sperm output per ejaculate increased from 5.3 ± 1.5 to 7.1 ± 1.6 ×10⁹ spermatozoa, a relative increase of 33.8%, and total motile sperm output per ejaculate increased from 4.3 ± 1.2 to 6.1 ± 1.4 ×10⁹ motile spermatozoa, a relative increase of 40.7%. Major sperm defects decreased from 8.4 ± 5.5% to 4.9 ± 3.7%, corresponding to a reduction of 41.8%, whereas minor defects also declined numerically. Libido score and ejaculate volume showed only small numerical changes. These descriptive data are summarised in Table 1, and the relative changes in selected semen endpoints are visualised in Figure 1.
Inferential analysis was performed with mixed-effects models in order to account for repeated ejaculates obtained from the same animals (Table 2). Experimental phase, post-supplementation versus pre-supplementation, was fitted as the fixed effect, and stallion was included as the random effect. This modelling approach confirmed significant post-supplementation increases in total motility, vigour score, sperm concentration, total sperm output per ejaculate and total motile sperm output per ejaculate. The estimated post-supplementation increase was +4.00 percentage points for total motility (P = 0.004), +0.51 units for vigour score (P < 0.001), +28.40 ×10⁶ spermatozoa/mL for sperm concentration (P = 0.003), +1.81 ×10⁹ spermatozoa for total sperm output (P < 0.001) and +1.75 ×10⁹ motile spermatozoa for total motile sperm output (P < 0.001). Major sperm defects decreased significantly by −3.50 percentage points (P < 0.001). In contrast, libido score and ejaculate volume were not significantly altered, and the reduction in minor defects did not reach the predefined significance threshold.
To support the biological interpretation of variables measured on different scales, a complementary magnitude-based analysis was carried out (Table 3, Figure 2). This approach ranked semen endpoints according to the standardised magnitude of post-supplementation change. The strongest standardised response was observed for total motile sperm output per ejaculate (+1.30), followed by vigour score (+1.27), total sperm output per ejaculate (+1.15) and the reduction in major sperm defects (−0.75). Total motility and sperm concentration showed smaller but still relevant positive magnitudes, whereas ejaculate volume and libido score showed only small shifts. This ranking was consistent with both the descriptive analysis and the mixed-effects models, indicating that the most important biological changes were concentrated in effective sperm output, motility-related performance and morphologically relevant sperm defects.
From a clinical and field-andrology perspective, these results are meaningful because they are not restricted to an isolated semen descriptor. Instead, the post-supplementation response involved a coherent group of endpoints directly relevant to breeding-stallion management. In practical semen-use decisions, sperm concentration, total sperm output and especially total motile sperm output are more informative than a simple change in semen volume, because they express the pool of usable spermatozoa available per collection. Likewise, a reduction in major sperm defects suggests an improvement in the structural quality of the sperm population. The favourable direction of change observed in the present study is biologically plausible in light of current knowledge showing that stallion spermatozoa depend strongly on the interaction between mitochondrial metabolism, redox balance and membrane preservation. Nutraceutical components such as antioxidants, trace minerals, vitamins, amino acids and yeast-derived factors may support these pathways, thereby contributing to sperm function and structural stability under breeding conditions.
The longitudinal repeated-ejaculate design is another important element of the study. Serial monitoring reduces the interpretative weight of a single ejaculate and provides a more robust field-level picture of seminal change over time. This is especially relevant when assessing nutritional support strategies, because the expected response may develop progressively rather than appear as an abrupt one-time effect. In the present study, the repeated monitoring design allowed the post-supplementation seminal pattern to be compared with the previous profile of the same animals under comparable management conditions. This strengthens the practical relevance of the findings for routine equine reproduction.
In conclusion, the study showed that 60 days of oral nutraceutical supplementation were associated with favourable longitudinal changes in semen quality and effective semen output in stallions under routine field conditions. The most relevant responses were increases in sperm concentration, total sperm output, total motile sperm output, total motility and vigour score, together with a reduction in major sperm defects, while libido score and ejaculate volume were not materially altered. Taken together, the results support repeated-ejaculate monitoring as a useful approach for field-based semen assessment and suggest that oral nutraceutical supplementation may serve as a supportive management strategy for improving effective semen output and morphological sperm quality in breeding stallions (Table 1-3; Figure 1-2).
