For bulls, heat stress has primarily been linked to the inability to regulate body temperature as well as testicular dysfunction (Gloria et al., 2021). Thermal stress furthermore affects spermatogenesis, which can limit sperm quality and fertility potential (Corte Pause et al., 2022); (Freitas et al., 2020); (Netherton et al., 2022). The inability to regulate body temperature can provoke reactive oxygen species production, which can exceed the natural antioxidant capacity, resulting in cellular oxidative stress (Fraczek et al., 2022); (Hamilton et al., 2016); (Wang et al., 2025). Likewise, these conditions have been shown to decrease sperm concentration, motility, and integrity (Freitas et al., 2020), as well as cellular DNA chromatin (Fraczek et al., 2022), microRNA (Celeghini et al., 2024), mitochondrial activity (Serrano-Jara et al., 2026), and plasma antioxidant capacity in animal ejaculates (Garcia-Oliveros et al., 2022); (Sabés-Alsina et al., 2019). For this reason, the possibility of using bull ejaculates under these conditions for conservation and artificial insemination programs could be risky (Landaeta-Hernández et al., 2020). Therefore, it is necessary to study the effect of environmental factors such as temperature and humidity on animal stress, which can limit reproductive performance (Morrell, 2020).

The temperature-humidity index (THI) is a proven tool to calculate animal stress(Dunn et al., 2014); (Habeeb et al., 2020); (Popović et al., 2024) because it directly relates to bull sperm function (Landaeta-Hernández et al., 2020); (Llamas-Luceño et al., 2020); (Savvulidi et al., 2025). In the tropics, Bos indicus dominates breeding programs, as it can adapt to climatic factors better than Bos taurus (Alves et al., 2025). Within B. indicus breeds, nonetheless, there may be individual variations that compromise reproductive efficiency (Netherton et al., 2022). Because these climatic adaptation variations might affect reproductive performance, their study may still improve animal management and selection programs (Morrell, 2020); (Poclín-Rojas et al., 2025).

High THI can impact bull reproductive behavior; there is no consensus, however, on how high THI must go to define an animal as under stress (Savvulidi et al., 2025). Some researchers have used THI values greater than 72 points as the threshold (Popović et al., 2024). Other studies of zebu breeds indicate that this value can rise above 74 points (Díaz et al., 2020). For buffalo, heat stress is defined as values greater than 68 (Umar et al., 2021). A recent study has proposed a categorical heat stress scale for B. indicus bulls under tropical conditions, where an animal is beginning to suffer heat stress with THI under 76 points, on alert from 76.2–79.2, in danger from 79.2–84.1, and in an emergency at greater than 84.1 points (Alves et al., 2025). Despite extensive research on the relationship between THI and semen quality, it is necessary to evaluate heat stress in Costa Rica’s humid environment for the predominant Brahman breed (Vargas-Leitón et al., 2025), especially given changing climatic conditions.

This study used season and THI as complementary indicators of the thermal environment rather than interchangeable variables. Season reflects the overall climatic context, including concurrent changes in rainfall, solar radiation, and other environmental conditions that may affect thermoregulation and spermatogenesis (Amini & Behnam, 2026), whereas THI provides a quantitative estimate of combined heat and humidity load. Thus, season was treated as a broader ecological framework, while THI was used as a specific climatic proxy to examine whether variation in heat load was associated with changes in semen quality (Boonkum et al., 2025); (Popović et al., 2024). We hypothesized that, within a consistently warm tropical environment, variation in season and THI would be associated mainly with changes in sperm motility and kinematic traits rather than viability or morphology. The study then evaluated the effect of season and THI on Brahman semen quality under tropical conditions.

Table 1 shows the effect of season on sperm TM, PM, viability, and morphology. A significant effect of season was found on TM, which was higher during the transition. A similar effect was determined for PM, which was lower during the dry and rainy seasons (p<0.05). Sperm viability did not differ significantly among seasons. Season was significantly associated with sperm morphology, with the rainy season showing a higher percentage of normal sperm morphology and a correspondingly lower percentage of abnormal sperm than the dry and transitional seasons (p<0.05). THI behavior was analyzed for each season. Mean THI increased significantly from the dry to the rainy season (p<0.05). The mean during the dry season was 79.51±0.03 points, the transitional 80.17±0.10 points, and the rainy season was 80.64±0.08 points.

In the continuous THI mixed model, fitted at the ejaculate level with bull as a random effect, THI showed significant positive associations with TM (β = 9.674, SE = 4.864, p = 0.047) and PM (β = 9.365, SE = 4.767, p = 0.049). In contrast, THI was not significantly associated with VCL (β = 13.302, SE = 17.830, p = 0.456), VSL (β = 1.605, SE = 10.686, p = 0.881), VAP (β = 11.089, SE = 11.395, p = 0.330), LIN (β = -1.383, SE = 2.433, p = 0.570), STR (β = -3.309, SE = 1.971, p = 0.093), WOB (β = 1.571, SE = 1.633, p = 0.336), viability (β = -0.172, SE = 3.918, p = 0.965), or normal morphology (β = 2.941, SE = 4.281, p = 0.492) (Table 2).

Analysis of sperm kinematic variables across different seasons revealed a significant effect (p<0.05). Sperm had higher velocities during the dry season (Table 3). Regarding sperm kinematic patterns, the variables LIN, STR, and WOB were significantly higher during the dry season (p<0.05).

Figure 2 reports the behavior of relative humidity, temperature, and THI in each ejaculate analyzed. The temperature during the study remained between 26 °C and 28 °C, while the relative humidity ranged from 80% to 94%. THI fluctuated from 79.44 to 81.01. Based on THI data for the 39 ejaculates, two cohorts emerged: ≤80.1 (n = 20 ejaculates) and >80.1 (n = 19 ejaculates). No significant differences between THI cohorts were detected for TM, PM, viability, or normal sperm morphology (p>0.05) (Figure 3).

Table 4 shows the effect of THI on sperm cell kinematic variables. Comparing kinematic variables between THI cohorts revealed significant differences (p<0.05) in VCL, VAP, LIN, STR, and WOB. No significant effect of the THI cohort was detected for VSL (p>0.05). LIN and WOB were significantly greater in the ≤80.1 cohort, whereas STR was higher in the >80.1 cohort (p<0.05).

The interaction between season and THI was also explored. It was not significant for TM, PM, viability, and normal sperm morphology (p>0.05). On the other hand, when analyzing kinematic sperm variables, a non-significant effect on their behavior was also seen (p>0.05).

The effect of tropical environmental conditions on bull semen quality has been previously documented (Chacón et al., 2022); (Poclín-Rojas et al., 2025); (Sevilla et al., 2025). Our results indicate that season was associated with variation in several semen-quality traits in Brahman bulls. Total and progressive motility were highest during the transitional season, whereas sperm kinematic variables were generally higher during the dry season. In contrast, the percentage of morphologically normal sperm was highest during the rainy season, while sperm viability did not differ significantly among seasons. Therefore, seasonal variation was not restricted to sperm motility and kinematics but also involved sperm morphology. These findings partially support our initial hypothesis, as viability remained relatively stable across seasons, whereas morphology showed significant seasonal variation. Importantly, the seasonal variation observed in sperm morphology should not be interpreted as a direct THI-associated response. Normal morphology was not significantly associated with THI in the continuous analysis and did not differ significantly between the two THI cohorts. This distinction suggests that the seasonal pattern in sperm morphology may reflect broader seasonal conditions not fully captured by THI alone, which integrates temperature and relative humidity but does not account for other potentially relevant environmental factors. This pattern is consistent with previous reports in bulls under tropical conditions (Alves et al., 2025). It has been found that although some B. indicus animals exhibit adaptation to tropical conditions, a marked individual effect on semen production and quality can be observed (Alves et al., 2025); (Koivisto et al., 2009); (Netherton et al., 2022). It has been shown, however, that even when animals are under the same exposure scenarios to adverse weather conditions, heat stress can manifest differently (Arias & Mader, 2023); (Morrell, 2020). High temperatures alter spermatogenesis and morphology and motility during the spermatozoan journey through the epididymis (Boe‐Hansen et al., 2020); (Freitas et al., 2020). These effects that occur during cell formation, development, and maturation relate to the presence of oxidative stress resulting from an imbalance between antioxidant capacity and free radical production (Hamilton et al., 2016); (Morrell, 2020); (Sabés-Alsina et al., 2019).

Previous studies have shown that these conditions can be more influential on testicles according to the blood irrigation capacity via the pampiniform plexus, which varies between subspecies and individuals (Brito et al., 2004). For spermatogenesis to occur normally, the animal needs to maintain the testicular temperature 2–6 °C below that of the rest of the body (Kastelic et al., 2021). Failure to maintain such homeostasis, therefore, can alter semen quality, including variations in seminal plasma composition (Vince et al., 2018). This can also cause problems when the sperm and egg recognize each other, resulting in decreased potential fertility of the male (Llamas-Luceño et al., 2020). On the other hand, heat can also affect normal mitochondrial functioning, specifically by altering oxidation of the electron transport chain complexes responsible for normal ATP production (Belhadj Slimen et al., 2016); (Capela et al., 2022); (Nichi et al., 2006).

Our results generally reveal THI values higher than the 76-point threshold traditionally reported in the literature as heat stress (Alves et al., 2025); (Rodriguez-Venegas et al., 2023). Overall, temperatures and relative humidity were relatively similar across the 39 ejaculates analyzed. By observing their behavior during the 60 days prior to each semen collection, we gathered as much information as possible related to the hypothetical conditions during spermatogenesis. Study results show that, regardless of season, THI values were consistently high and varied within a limited interval, which restricted our ability to compare thermoneutral versus clearly heat-stressed conditions. Because THI integrates temperature and relative humidity, the persistently high humidity observed during the study may have contributed to the relatively narrow THI range despite modest variation in ambient temperature (Dunn et al., 2014).

In the tropics, particular conditions of high relative humidity have also been noted as associated with higher THI values during the summer (Kumar et al., 2019). Our results show that, despite the effect of season, the relative humidity during the study was almost constant and considerably high, ranging from 80%–94%. Other studies in Zebu breeds have demonstrated a trend whereby, as the THI increases, the quality of sperm decreases under tropical conditions (Alves et al., 2025). This has been observed with variables such as sperm morphology, motility, volume, and ejaculate concentration(Freitas et al., 2020); (Garcia-Oliveros et al., 2022); (Seifi-Jamadi et al., 2019). The differences from previous studies may reflect breed-specific adaptation, the narrow THI variation here, exposure quantification variations, and the modest sample size, rather than a true reversal of the general heat-stress pattern. One possible explanation is that unmeasured nocturnal cooling may have partially mitigated the impact of daytime heat load (Freitas et al., 2020); however, this remains speculative because nighttime thermal indices were not analyzed in the present study.

We also explored sperm quality characteristics in both cohorts. Our results show that within the THI cohorts, no significant differences were observed in TM, PM, viability, normal sperm morphology, and VSL, while there were significant variations in VCL, VAP, LIN, STR, and WOB. These differences suggest changes in sperm motile patterns rather than uniform improvement or deterioration in semen quality. Previous studies have shown that sperm motility and velocity tend to decrease with higher THI (Landaeta-Hernández et al., 2020); (Rahbar et al., 2025). This phenomenon could be related to the role of Ca²⁺ ions in the sperm flagellum (Abd El-Emam et al., 2023). During their movement, high thermal stress can prematurely activate the sperm cell (Swain et al., 2025). Because all ejaculates were collected under consistently high THI conditions, corresponding to a heat-stress range previously proposed for B. indicus bulls under tropical conditions (Alves et al., 2025), and because the THI range was narrow, these findings reflect variation within a generally heat-challenged tropical environment, rather than contrasts between thermoneutral and severely heat-stressed conditions. These findings further suggest that higher THI was associated with altered sperm motility patterns rather than a uniform increase in all kinematic traits. A recent study has shown that these heat-induced effects can alter the function of Ca²⁺/calmodulin-dependent protein kinase II, which has been linked to early sperm acrosome reaction and fertility problems (Santos et al., 2025). Previous studies conducted in European bull breeds have reported reductions in membrane integrity and sperm morphology with increasing THI (Gloria et al., 2021). In the present study, however, neither viability nor normal morphology was significantly associated with continuous THI or differed significantly between THI cohorts. Therefore, no clear THI-related pattern could be established for these traits under the relatively narrow and consistently high THI range evaluated. In this context, although breeds with some degree of adaptability to tropical conditions are already used, selection for animals with greater reproductive resilience to heat stress warrants further investigation (Capela et al., 2022); (Livernois et al., 2018); (Slayi & Jaja, 2025). Because semen-quality traits may have a heritable component, selection for heat-resilient reproductive phenotypes warrants further investigation (Vanselow et al., 2024).

In the continuous THI analysis, thermal load was not consistently associated with semen quality traits. Although TM and PM showed statistically significant positive associations with THI, no significant relationships were found for the remaining kinematic variables, viability, or normal morphology. This pattern indicates that, under the relatively homogeneous and generally high THI conditions recorded in this study, continuous THI variation had limited explanatory value for most semen traits.

For this reason, indicators of thermal comfort, such as THI, provide more information for decision makers (Mader et al., 2006); (Souto et al., 2022), whether for natural mating or germplasm conservation and reproductive biotechnologies. One limitation of this work, however, is the need to complement conventional semen analysis with more complex techniques such as omics science, which provide a broader view of what might be happening inside the sperm cell (Celeghini et al., 2024); (Chitkara et al., 2025); (Kumar et al., 2024).

This study indicates that season is associated with variation in sperm motility, kinematic patterns, and morphology in Brahman bulls raised under humid tropical conditions. Total and progressive motility were highest during the transitional season, whereas the percentage of morphologically normal sperm was highest during the rainy season; sperm viability remained relatively stable across seasons. Continuous THI analysis showed positive associations with total and progressive motility; however, these findings should be interpreted cautiously because they occurred within a narrow and consistently high THI range, without confirmation by the categorical THI-cohort comparison. Moreover, neither viability nor normal morphology was significantly associated with continuous THI, indicating that the seasonal variation observed in morphology should not be attributed directly to THI. The results do not suggest a beneficial effect of heat stress, but rather highlight the complexity of interpreting semen quality responses under tropical conditions in adapted B. indicus bulls. The modest sample size, high interindividual variability, and limited environmental contrast influence the final interpretation. Further studies integrating larger datasets, fertility outcomes, and cellular or molecular indicators of sperm function are needed before these traits can be used to select breeding bulls under humid tropical conditions.

The authors declare that there is no conflict of interest.

The authors thank the Costa Rica Institute of Technology for financing this study. This work was part of the research project VIE-2151083, Optimización de la conservación y búsqueda de parámetros de la fertilidad en espermatozoides de animales de interés productivo and Postgraduate Office of ITCR. The APC was funded by the Costa Rica Institute of Technology´s Vice-Chancellor’s office of Research and Extension. I.A.-Z. and F.S. thank the Postgraduate Office of the Costa Rica Institute of Technology.

During the writing process, none of the authors used generative AI or AI-assisted technologies.