In emerging nations like India, pig production is becoming more and more crucial to supplying the rising need for animal protein (Cao and Li, 2013). A crucial reproductive technique for genetic enhancement, illness prevention and effective use of exceptional breeding boars is artificial insemination (AI) (Das et al., 2022). In contrast to cattle, AI in pigs usually uses liquid-stored semen instead of frozen semen since spermatozoa of boar are naturally sensitive to cryopreservation (Zuidema et al., 2021). Boar sperm are particularly vulnerable to oxidative stress and cold shock due to their plasma membrane’s high polyunsaturated fatty acid content, low cholesterol-to-phospholipid ratio and weak antioxidant defense systems (Castro et al., 2025). Beltsville Thawing Solution (BTS), one of several extenders designed for liquid boar semen preservation, is frequently used for short-term storage because of its ease of use, buffering capabilities and potential to preserve sperm viability for up to three to five days at 15 to 18°C (Paul et al., 2024). However, a number of internal and external factors, such as breed, age, nutrition, extender composition, storage temperature and most importantly environmental conditions, affect how well liquid semen is preserved. One of the most significant environmental elements influencing male pig reproductive success is seasonal fluctuation (Hagan and Etim, 2019). Seasonal variations in room based temperature, relative humidity and temperature-humidity index (THI) can have a significant impact on spermatogenesis, sperm maturation and seminal plasma composition in tropical and subtropical regions. Reduced semen production, sperm concentration, motility and an increase in sperm abnormalities have all been linked to heat stress during the warmer months (Rahman et al., 2018). Additionally, seasonal stress may have an impact on spermatozoa’s metabolic activity and membrane stability, which could decrease their capacity to survive in liquid storage (Rizkallah et al., 2022). While seasonal impacts on fresh boar semen features have been recorded in a number of research, little is known about how season affects CASA based sperm movement during liquid storage, especially in Meghalaya’s Indian climate. Meghalaya literally translates to “Abode of Clouds.” The state is connected to clouds and rain, as the name implies (Sahoo, 2021). Meghalaya typically has a fairly humid climate. The north-east winter winds and the south-west monsoon have a direct impact on it. The southern Garo hills receive the most rainfall, while the middle and northern regions receive less. With an average annual rainfall of 12670 mm, the Cherrapunji-Mawsynram region experiences the most extreme precipitation. The state’s average rainfall is 12000 mm (Lyngdoh and Purkayastha, 2024). Springtime brings with it an increase in temperature, which peaks in May and June during the summer monsoon (Sahoo, 2021). The winter is quite severe with minimum temperature coming down to as low as 2°C in the Khasi Hills (Lyngdoh and Purkayastha, 2024). April and May are the warmest months and January is the coldest month (Lyngdoh and Purkayastha, 2024). All year long, the weather is nice and devoid of pollution. Meghalaya has a pleasant climate throughout the year due to the North-East winter winds. Meghalaya experiences four distinct seasons: (i) spring, which lasts from March to April; (ii) summer (monsoon), which lasts from May to September; (iii) autumn, which lasts from October to November; and (iv) winter, which lasts from December to February. The highest and lowest temperatures reached during summer are 20°C and 15°C while during winter these are 16°C and 4°C respectively (Tanti et al., 2017). In October and November months the climate is cool. After November, the winter sets in and continues up to the end of February. Rainfall starts by the third week of May and continues up to September end and sometimes up till middle of October. Computer-assisted analysis of semen provides objective and detailed examination of sperm motion characteristics, which are closely associated with fertilizing ability (Amann and Waberski, 2014). Therefore, enhancing AI programs in pigs requires an awareness of seasonal fluctuations in CASA derived movement attributes during semen preservation. This current research work was therefore undertaken to evaluate the impact of season on the liquid preservation of semen from Hampshire crossbred boars diluted in BTS extender and stored at 18°C.

The experiment was set at the ICAR RC for North Eastern Hill (NEH) Region, Umiam, Meghalaya, India, during March 2025 to February 2026. The Institute’s Animal Ethics Committee approved the experimental plan for doing this research work. Based on prevailing climatic conditions, the year was divided into three seasons: pre-monsoon (March to May), monsoon (June to September) and post-monsoon (October to February) (Choudhury et al., 2012). These seasons are characterized by distinct variations in ambient temperature and humidity. Sexually mature Hampshire crossbred boars maintained under uniform management, housing and feeding conditions were cast-off for the study. Boars were clinically healthy and regularly used for semen collection. Standard management practices were followed throughout the experimental period to minimize non-seasonal variations. The gloved-hand method was used to gather semen. Semen samples were assessed right away following collection. For additional processing, only ejaculates with ≥70% initial motility was chosen. To reach the required sperm concentration, Beltsville Thawing Solution (BTS) dilutor was used on a subset of ejaculates. Samples of extended semen were kept at 18°C after being gradually cooled. At 0 (fresh extended semen), 3 and 5 days after preservation, the quality of the semen was examined.
       
The motion characteristics of pig spermatozoa were investigated using a computer-assisted sperm motility analysis (CASA) system. Accordingly, a phase contrast microscope that had been preheated to 37°C was used to examine 2 mL of each ejaculate on a slide. Total motility (TMOT,%), progressive motility (PMOT,%), curvilinear velocity (VCL, µm/s), which is the time-averaged velocity along the actual sperm trajectory; average path velocity (VAP, µm/s), which is the velocity along a smoothed movement path; and straight-line velocity (VSL, µm/s), which is the net distance travelled from the start to the end of the track per unit time, were among the computer-assisted sperm motion characteristics. Beat cross frequency (BCF, Hz), which shows the rate of lateral head displacement across the mean path, linearity (LIN, %), which is the ratio of VSL to VCL; straightness (STR, %), which is derived from the ratio of VSL to VAP; and wobble (WOB, %), which represents the oscillation head of the sperm during forward progression, were additional kinematic indices that were recorded (Rajashri et al., 2018).

The impact of season, preservation duration and their interaction were assessed using factorial analysis of variance (ANOVA). Mean differences were compared using the appropriate post-hoc tests. Pearson’s correlation coefficient (r) was used to determine the relationship between TM and PM and a number of kinematic factors. Differences were considered significant at p<0.05 and extremely significant at p<0.01.

The impact of season and duration of liquid preservation on sperm motility and kinematic parameters of boar semen stored in BTS extender at 18°C are presented in Table 1. At day 0 of preservation, total motility (TM) and progressive motility (PM) were significantly higher (p<0.01) during the pre-monsoon season compared to the monsoon and post-monsoon seasons. These findings indicate superior initial semen quality during the pre-monsoon period, which is consistent with earlier reports describing favorable semen characteristics under moderate environmental conditions (Colenbrander and Kemp, 1990; Petrocelli et al., 2015; Hensel et al., 2023).


       
In all seasons, there was a substantial (p<0.01) decrease in TM and PM as storage duration increased. This research results closely agreement with reported by Laskar et al. (2025). At day 3, pre-monsoon semen maintained significantly higher motility than monsoon and post-monsoon semen, suggesting enhanced resistance to storage-induced stress. However, by day 5 of preservation, TM and PM declined drastically in all seasons and seasonal differences became non-significant, indicating that prolonged liquid storage adversely affects sperm motility irrespective of season. Similar time-dependent reductions in boar sperm movement during liquid storage have been stated previously (Dziekońska et al., 2009; Fraser et al., 2003; Katiyar et al., 2024).
       
Season and storage duration had a substantial impact on velocity characteristics such as curvilinear velocity (VCL), average path velocity (VAP) and straight-line velocity (VSL). At every stage of preservation, semen obtained during the pre-monsoon season consistently showed higher velocity values, indicating improved sperm vitality and metabolic competence. On the other hand, post-monsoon semen displayed noticeably reduced velocity metrics, especially on days 3 and 5, indicating impaired sperm energy metabolism during unfavourable seasonal conditions. Increased oxidative stress and changed sperm membrane dynamics brought on by high humidity and temperature fluctuations may be the cause of the more noticeable decrease in velocity metrics both during and after the monsoon season (Khoi et al., 2021; Ibanescu et al., 2015; Basumatary et al., 2024).
       
As storage time increased, trajectory-related metrics like linear (LIN), straightness metric (STR) and wobbling (WOB) likewise dramatically decreased. Seasonal variations in these values were negligible at day 0, suggesting similar starting patterns of sperm migration. In contrast to prior to and monsoon semen, post-monsoon semen showed considerably lower LIN, STR, as well as WOB values at days 3 and 5 (p<0.01), indicating compromised sperm trajectory and decreased directional movement during prolonged storage. Boars and other livestock species have shown comparable seasonal effects on CASA-derived kinematic parameters. (Ibanescu et al., 2015; Fernandez-Novo et al., 2021).
       
In every season, the beat cross frequencies (BCF) dramatically dropped with storage time. However, for the course of the storage period, pre-monsoon semen retained relatively greater BCF values, indicating improved flagellar activity and prolonged motility. Reduced ATP generation and mitochondrial efficiency in spermatozoa have been linked to diminishing BCF during liquid storage (Dziekońska et al., 2009; Fraser et al., 2003).
       
Correlation analysis revealed significant and season-specific associations between motility attributes and kinematics of spermatozoa (Table 2 and 3). During the monsoon and post-monsoon seasons, TM and PM showed strong positive correlations with LIN, STR and WOB at day 3, emphasizing the importance of sperm trajectory characteristics in maintaining motility during intermediate storage periods. These findings suggest that, under environmentally stressful seasons, sperm movement linearity and path stability become critical determinants of motility retention. Similar associations between CASA-derived kinematic parameters and sperm motility during liquid storage have been reported by (Khoi et al., 2021; Colenbrander and Kemp, 1990).




       
All things considered, the current investigation unequivocally shows that seasonal variation has a major impact on the liquid keeping potential of boar semen kept in BTS extender at 18°C. The pre-monsoon season’s superior semen quality may be explained by the comparatively mild weather, which lowers oxidative damage, thermal stress and spermatogenic efficiency. On the other hand, the rainy season and post-monsoon seasons, which are marked by high humidity and temperature swings, may cause oxidative and biological stress, which could result in altered metabolic activity, decreased motility and compromised sperm membrane integrity (Khoi et al., 2021; Hensel et al., 2023).
               
Previous results on liquid survival of pig semen are supported by the current study’s steady fall in CASA-derived mobility and kinematic properties with increasing storage period (Fraser et al., 2003; Dziekońska et al., 2009). The importance of CASA-based kinematic examination as a reliable method for forecasting the storage capabilities and fertilizing capacity of liquid-stored boar semen is further highlighted by the substantial correlations between TM, PM and important velocity and trajectory parameters.

The quality and liquid storage ability of a standard Hampshire crossbred boar semen that is dissolved in BTS extender at 18°C are found to be significantly impacted by season. It was discovered that the pre-monsoon era was the best for preserving the motion of sperm and kinematic characteristics for up to three days. Strong correlations were found between CASA-derived kinematic parameters and TM and PM, suggesting that they could be trustworthy markers of semen quality during liquid retention.

The authors acknowledge the facilities provided by the ICAR Research Complex for NEH Region, Umiam, Meghalaya and All India Coordinated Research Project on Pig for conducting the present study.

The authors declare that they have no conflicts of interest related to the publication of this article.