Seasonal effect on semen quality of Limousine and Simmental bulls

This study aimed to determine the effect of breed and season on the semen quality of AI bulls. The material used was n= 78,648 records of ejaculated semen from 85 Limousine and 88 Simmental bulls collected between period 2010 and 2020. Semen qualities evaluated in the study were: ejaculate volume (Vol), pH, individual sperm motility (ISM), sperm concentration (SC)


INTRODUCTION
Artificial Insemination (AI) is the first generation of biotechnology that plays an important role in improving the genetic quality of livestock, especially cattle. AI can maximize the use of superior bull and reduce the possibility of the spread of infectious reproductive diseases (Konenda et al., 2020). The success of artificial insemination depends on the semen quality, which has been affected by genetic and environmental factors (Brito et al., 2002;Isnaini et al., 2021). The quality of semen could be different between individuals as well as between breeds of bulls (Söderquist et al., 1991).
Frozen semen of Limousine and Simmental Bull is the most widely used in Indonesia. In one year, more than three million doses of frozen semen were distributed throughout the regions in Indonesia. Currently, almost Limousine and Simmental Bull used for the production of frozen semen are imported from a subtropical country. That can be the inhibiting factor for reproductive performance and impact the sustainability of frozen semen production due to seasonal and climatic differences between the origin. In temperate regions, photoperiod (time interval between sunset and sunrise) is the most responsible factor affecting seasonal variation in semen quality (Snoj et al., 2013). While in the tropical region, since the photoperiod is almost similar throughout the year, other factors such as temperature and relative humidity probably become contributors to the semen quality variation (Auvigne et al., 2010).
Many factors can affect semen quality. It becomes a challenge for artificial insemination centers to produce frozen semen in a sustainable manner with a sufficient amount to fulfill the national demand. Therefore, to answer these challenges this study aims to determine the effect of season, breed, and their interaction on the semen quality of AI Bulls.

Data Collections
Semen quality data of fresh and frozen semen (n= 78,648) was recorded from 173 bulls at Lembang artificial insemination center of Indonesia. The collection periods were from 2010 to 2020. The detail of the data used in the study is presented in Table 1. The seasonal data was car-ried out from the Indonesian Agency for Meteorological Climatological and Geophysics. The data consist of temperature, humidity, precipitation, and sunshine duration. Seasonal data are presented in Table 2. Breeds categories were Limousine (n=37,901) and Simmental (n=40,747). Seasons were categorized based on the precipitation: dry season (rainfall <100 mm/ month; n=26,152 ejaculate); humid season (rainfall 100-200 mm/month; n=14,595 ejaculate) and rainy season (rainfall > 200; n=36,501 ejaculate). Semen quality observed in this study were ejaculate volume of semen (Vol), pH, individual sperm motility (ISM), sperm concentration (SC), and post-thawing motility (PTM).

Data Analysis
The data have been analyzed with the MIXED procedure of SAS On Demand for Academic (SAS, 2021). Breed, season, and their interaction were designed as fixed effects and bull as a random effect. The linear mixed model was as follows: where y ijklm is the observation of semen quality, B i the ith fixed effect of breed, S j the jth fixed effect of season, BS ijk the kth effects of interactions between ith of breed with jth effect of season, a l is the random effect of animal, and e ijklm the random residual of y ijklm . Tukey-Kramer multiple comparisons were used with a significant level 5%.

RESULTS AND DISCUSSION
The analysis of variance showed that breed, season, and their interaction have significantly affected sperm quality except for pH. The result agreed with a previous study reported for crossbred (Karan Fries) bulls in India (Bhakat et al., 2014). pH was not affected by genetics and environment, it could be influenced by the protein involved in sperm. Kasimanickam et al. (2019) stated that sperm is a complex biological fluid containing proteins, amino acids, enzymes, fructose, lipids, major minerals, and other carbohydrates.
The average of Simmental bulls was slightly higher than that of Limousine bulls. Further, the same result showed significant differences in each season (Table 3). The opposite result by Isnaini et al. (2019) reported that Vol of Limousine bulls was higher than Simmental bulls. Genetic, environmental, and management previously reported affected Vol of bulls (Mathevon et al., 1998;Fuerst-Waltl et al., 2006). Both Simental and Limousine Bulls indicated higher Vol (6.68 ± 2.09 and 6.77 ± 1.98, respectively) in the humid season than in other seasons. In contrast, Prastowo et al. (2019) reported that the season had not affected Vol of Bali bulls. Bulls produce a lot more Vol in the humid season could be due to heat stress.
The ISM average of Simental and Limousine bulls was similar. Breed had not significant but season has statistically significant on ISM (Table 4). The result agreed with the previous studies stating that season significantly affects ISM of AI bulls (Ax et al., 2000;Brito et al., 2002;Tiwari et al., 2012;Perumal et al., 2017;Konenda et al., 2020). The highest ISM 64.94 ± 1.58 for Limousine and 64.49 ± 1.48 for Simmental was shown in the rainy season. The results indicated even though the bulls produce more ejaculate volume in the humid season, the semen had not better ISM. Heat stress makes the ISM on the semen decrease significantly. Sperm kinematics, viability, acrosome integrity, and reactive oxygen species were affected by season (Sabes-Alsina et al., 2019;Morrell, 2020). In the subtropical condition, ISM of semen collected in winter or springs had better membrane integrity (Valeanu et al., 2015). Heat stress reduces motility through the mechanism of increasing gene expression from heat shock protein (HSP) (Cheng et al.. 2016). Increasing temperature has an impact on the process of spermatogenesis.
The SC was significantly affected by breed and season. Simmental bulls have higher SC than Limousine bulls all over the seasons. The results agreed with a previous study by Sumeidiana et al. (2007) reported the average SC was 1,788 and 1,388 x106/ml, respectively for Simmental and Limousine bulls. Seasons significant effect on Values within a column without a common superscript are different (P < 0.05). x,y Values within a row without a common superscript are different (P < 0.05).
SC with the lowest concentration found in the dry season and the highest concentration in the humid season (Table 5). The same results were for Limousine and Simmental bulls. In line with the studies reported the effect of season on SC in Limousine by Konenda et al. (2020), Ongole grade cattle by Suretno et al. (2018), andIsnaini et al. (2019) Simmental by Nichi et al. (2006). The increase of SC in the humid season has been shown directly proportional to the increase of Vol.
The last evaluation on sperm quality is PTM, PTM was also affected by breed and sea-son. The PTM of Simmental was higher than that of Limousine. The highest PTM was shown in the humid season for both breeds (Table 6). Murphy et al. (2018) reported that PTM of Holstein Friesian was higher in winter. The result of the study could be due to the difference in freezerthawing protocols among seasons.

CONCLUSION
Breeds and seasons have shown favorable interaction affected sperm quality. The increase in volume and concentration have not proportion- Values within a column without a common superscript are different (P < 0.05). x,y Values within a row without a common superscript are different (P < 0.05).
Values within a column without a common superscript are different (P < 0.05). x,y Values within a row without a common superscript are different (P < 0.05). Values within a column without a common superscript are different (P < 0.05).
x,y Values within a row without a common superscript are different (P < 0.05).
ally affected to individual sperm motility. Need a focused study about the effect freezer-thawing process on post-thawing motility.