This is an open-access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) P-ISSN: 2304-3075; E-ISSN: 2305-4360 International Journal of Veterinary Science www.ijvets.com; editor@ijvets.com Research Article https://doi.org/10.47278/journal.ijvs/2024.209 Season and Breed Effect in Fresh Semen Parameters at a Bull Semen Production Center in Peru Deysi Dipaz-Berrocal †, Silvia León†, Deyanira Figueroa *, Rolando Mamani , Juan Ramirez, Wuesley Yusmein Alvarez-García , Richard Estrada and Carlos Quilcate Dirección de Desarrollo Tecnológico Agrario, Instituto Nacional de Innovación Agraria (INIA), Lima, Perú †These authors contributed equally to this work and shared the first authorship. *Corresponding author: deyanirafigueroa66@gmail.com Article History: 24-507 Received: 20-May-24 Revised: 25-Jun-24 Accepted: 26-Jun-24 Online First: 14-Jul-24 ABSTRACT In the present study, the effect of breed, season, and their interaction on semen volume, sperm concentration, and motility was tested. Nine bulls were tested: three Braunvieh, three Simmental (B. taurus), and three Gyr (B. indicus). These bulls were managed equally in a cold semi-arid climate, divided into hot (from November to April) and cold (from May to October) for maximum and minimum temperature and relative humidity. Semen was collected twice a week for 24 consecutive months. MANOVA by ranks was used to determine significant effects; breed had a significant effect (P<0.001) on semen parameters, and seasonal variations were not significant (P>0.05). However, there was a significant effect (P<0.001) for the interaction between breed and season. To evaluate differences between breeds, we performed Tukey's post-hoc test; all pairwise comparisons (Braunvieh-Gyr, Braunvieh-Simmental and Gyr-Simmental) showed significant differences (P<0.001). Nonparametric one-way analysis showed significantly higher B. taurus breeds’ sperm concentration and B. indicus breed’s semen volume. For interaction between breed and season, Gyr reported higher sperm volume than B. taurus breeds in both seasons (P>0.05), but B. taurus breeds had higher sperm concentration in the cold season. The findings will help management make better decisions in semen collection, especially during peak season. Key words: Bulls, Semen, Breed, Season, Effect INTRODUCTION center is to ensure the sufficient quantity and quality of semen supply. Artificial insemination (AI) is a highly used The assessment of fertility post-artificial insemination biotechnology for genetic improvement, allowing necessitates a meticulous evaluation of sperm quality producers to use superior sires with many females (Morrell et al. 2017), involving its ability to migrate, (Murtina et al. 2012; Parkinson and Morrell 2019). AI is penetrate the ovum, and activate it for embryo formation designed to facilitate fertilization control and has been the (Sabés-Alsina et al. 2019). When assessing sperm quality most widespread, least costly and easily accessible to in bulls, critical factors to consider include sperm count, Peruvian farmers. To support the implementation of AI in motility, morphology, viability, acrosome integrity, Peruvian regions, a Genetic Nucleus for semen concentration in seminal plasma, DNA integrity, and production was established in Huaral – Lima, Peru (128 seminal fluid characteristics (Sankhi et al. 2018; Kumaresan masl; 11°31′18″ S and 77°14′06″ W). In Peru, Creole et al. 2020). These parameters collectively serve as cattle are the most predominant cattle population indicators of the reproductive capacity of a bull, influencing (64.03%) (Instituto Nacional de Estadística e Informática its potential for successful fertilization. A high sperm 2012) and the breeding strategy of small farmers is to concentration, progressive motility, normal morphology, crossbreed these animals with specialized breeds such as and intact acrosomes indicate good - quality sperm. Simmental, Gyr and others (Rivas et al. 2007), taking Additionally, assessing viability, DNA integrity, and advantage of the heterosis effect (Williams and Anderson seminal fluid characteristics provides comprehensive 2019). That is why the main objective of the production insights into the overall health and fertility potential of the Cite This Article as: Dipaz-Berrocal D, León S, Figueroa D, Mamani R, Ramirez J, Alvarez-García WY, Estrada R and Quilcate C, 2024. Season and breed effect in fresh semen parameters at a bull semen production center in Peru. International Journal of Veterinary Science x(x): xxxx. https://doi.org/10.47278/journal.ijvs/2024.209 1 Int J Vet Sci, 2024, x(x): xxx. sperm (Allouche et al. 2017; Morrell et al. 2017; Narud et Animal management and semen collection al. 2022). Regular evaluation of these parameters through Bulls (from 4 to 6 years old) of the breeds Braunvieh semen analysis is essential in cattle breeding programs to (BU), Gyr (GI) and Simmental (SM) were kept under ensure optimal reproductive performance and successful uniform feeding and management conditions during each artificial insemination or natural mating. season. Bulls were housed in indoor stalls, fed twice daily, Sperm motility has traditionally been used as an and had ad libitum access to water and feed. Semen indicator of fertility (Foote 2003), but bull sperm samples were collected from these nine bulls (three BU, characteristics and morphology are influenced by a three GI, and three SM) through an artificial vagina twice multifaceted interplay of genetic, environmental, and a week, and the temperature of the artificial vagina was management factors (Brito et al. 2002; Koivisto et al. 2009; varied for each bull (ranging from 56 to 62°C). All bulls Snoj et al. 2013; Malama et al. 2017). Since were sexually stimulated using a teaser bull or cow, and spermatogenesis is the intricate process of sperm semen collection was performed by well-trained technicians using a standard protocol with a standard production that occurs within the seminiferous tubules of checklist. The method of stimulation and collection was the testis, and various factors such as nutrition and similar for all collections and remained constant from year environmental conditions can influence the efficiency of to year. spermatogenesis in bulls, this highlights the importance of optimal conditions for maintaining robust reproductive Assessment of sperm parameters performance in livestock breeding programs (Staub and The ejaculate samples from each bull were carefully Johnson 2018; Harrison et al. 2022). labeled, and the sperm concentration was measured using a The season can impact semen quality, but there is no photometer (Minitube®). Furthermore, the volume was consensus between studies. Researchers found out in measured with a micropipette of 1000mL and each sample northern Spain that Holstein bull sperm quality was was pre-diluted with commercial diluent OptiXcell ((IMV affected by season, with better values during spring than in Technologies, France), according to the concentration in winter (Sabés‐Alsina et al. 2017), but a study in Brazil with 1:1 (< 500 sperm/mL) or 1:2 (501 - 1200) or 1:3 (>1300). different breeds showed no effect of ambient temperature, The pre-diluted samples were then transported to the humidity or season on sperm production and semen quality laboratory (15 minutes from the farm) at 28°C, and the (Brito et al. 2002), on the other hand, Simmental bulls in individual motility of the spermatozoa was measured by Brazil reported a higher sperm defects during summer than phase-contrast microscopy and expressed as the proportion in winter (Nichi et al. 2006). It is essential to know the of motile spermatozoa (percentage). Immediately, the effect of each of these factors to ensure the success of AI. dilution is completed to obtain the final concentration There has been previous research on the seasonal (20x106spz/0.25mL) to continue the cryopreservation fluctuations in spermatozoa morphology (Koivisto et al. process of the semen. 2009), ejaculate volume and sperm concentration in the semen of bulls (Wildeus and Hammond 1993) but there is Semen cryopreservation disagreement on the results due to scrotal thermoregulation Samples with sperm motility that were more and heat dissipation mechanisms of bulls (Netherton et al. outstanding than 60% were considered for the 2022; Capela et al. 2022). Therefore, the present study cryopreservation process. Any ejaculation that did not meet aimed to determine seasonal and genotypic effects on this criterion was excluded from the production of genetic material, but the data were still included in this study. After sperm motility, sperm concentration and volume by final dilution, samples were incubated at 5°C for five hours. monitoring bulls in an AI center for 24 months. Then, each sample per bull and consecutive ejaculate number was routinely filled and sealed into straws (0.25cc) MATERIALS AND METHODS using an automatic semen filling and sealing machine (Minitube). The straws were placed in a programmable Ethical approval freezer (from 5 to -100°C), and then the straws were rapidly The semen sample collection from the cattle was immersed in liquid nitrogen. Three straws from each conducted in accordance with Peruvian National Law No. ejaculate of each bull were thawed at 37°C for 45s in a 30407, “Animal Protection and Welfare.” water bath. The thawed samples were immediately evaluated by standard microscopy, and only those with Season motility more outstanding than 35% were stored. This work was done in the Central Genetic Nucleus for Bovine, located in the Agricultural Research Station Statistical analysis Donoso (EEA Donoso in Spanish), Huaral - Lima, Peru. All semen variables (semen volume, sperm Data collected from May 2021 to April 2023 were concentration, and motility) and factors (breed, season, and categorized according to two seasons of collection: Hot breed-season interaction) were analyzed with R software (November, December, January, February, March, April) version 4.3 (R Core Team 2021), using the Rank MANOVA and Cold (May, June, July, August, September, and method of the rank MANOVA package (Dobler et al. 2017), October). This division in the season was made considering and multiple means were compared using the Tukey test of the mean temperature and relative humidity recorded by the the same package. Also, a linear discriminant analysis National Service of Meteorology and Hydrology of Peru (LDA) was done with the ggplot package (Wickhan 2016) (SENAMHI in Spanish); data was collected from the to have a graphic representation of the results. Also, we used closest weather station to the Central Genetic Nucleus. a nonparametric one-way analysis of variance to address 2 Int J Vet Sci, 2024, x(x): xxx. potential differences between the significant effects found season and humidity varies from 71.7-98% in cold season in the first analysis, and posthoc analysis was performed and from 80.2-98.3% in hot season. using Dwass-Steel-Chritchlow-Figner pairwise comparisons; these last analysis was done with the JVM R Sperm parameters package (Selker et al. 2023). Table 1 summarizes ejaculate characteristics of Braunvieh (BU), Gyr (GI) and Simmental (SM) for each RESULTS season. The mean ejaculate volume ranged from 3.6 to 7.26mL and 3.79 to 6.24mL in cold and hot seasons, Season respectively. Mean sperm concentration varied between From May 2021 to April 2023, the closest weather 967.18 to 1304.97 x 106⁄mL and 1109.3 to 1181.4 x 106⁄mL station to the EEA Donoso, reported the mean temperature in cold and hot seasons, respectively. Mean sperm motility (Fig. 1) and humidity (Fig. 2). Mean temperature varies varied from 54.2 to 60.3% and 57.5 to 61.1% in cold and from 13.2-22°C in cold season and from 14.8-27°C in hot hot seasons. Table 1: Summary of the ejaculate characteristics of the three breeds for each season. Season Breed Sperm concentration (106 /mL) Semen volume (mL) Sperm motility (%) Cold BU 1304.97±335.5 3.6±1.3 58.6±11.2 GI 967.18±366.24 7.26±3.7 54.2±11.9 SM 1181.4±375.6 4.59±1.7 60.3±12.8 Hot BU 1175.16±380.6 3.79±1.6 57.5±11.9 GI 1109.3±341.0 6.24±2.6 61.1±10.0 SM 1208.4±338.9 4.42±2 60.4±10.4 Fig. 1: Mean temperature from May 2021 to April 2023, data were separated into hot and cold seasons. Fig. 2: As described before, data on relative humidity from May 2021 to April 2023 were separated into hot and cold seasons. 3 Int J Vet Sci, 2024, x(x): xxx. Statistical analysis Table 4: Differences between breeds for each semen Multivariate analysis of variance characteristics This study evaluated the effect of breed, seasonal χ² df P Value variations and their interaction (Table 2) in semen quality Sperm concentration 17.23485 2 P<0.0001809*** in BU, GI and SM, where the null hypothesis (H ) means Semen volume 109.1618 2 P<0.0000001*** 0 that all mean vectors are equal and the alternative Sperm motility 5.119954 2 P<0.0773065 hypothesis means that at least one mean vector is different ***highly significant (P<0.001). from the rest. Breed had a significant (P<0.001) effect on the semen parameters. By obtaining a P<0.001, the null hypothesis (H0) is rejected, meaning there are significant differences between the three breeds. Therefore, it is assumed that at least one vector of means is different from the rest. Also, a significant (P<0.001) effect exists on the interaction between breed and season. However, seasonal variations are not significant (P>0.05), and the null hypothesis (H0) is not rejected, which means that there are no significant differences between the two seasons studied. Table 2: Effect of breed, seasonal variations and their interaction. Statistic test P value Breed 81.219 P<0.001*** Season 10.267 0.054 Breed:Season 150.531 P<0.001*** ***highly significant (P<0.001). Tukey post-hoc analysis The effect of breed, seasonal variations and their interaction were evaluated, as the breed effect was Fig. 3: Linear discriminant analysis of breed's effect. significant; since we do not know which treatment this different mean corresponds or which treatments are different from each other, we proceed to perform the Tukey post-hoc test (Table 3), all pairwise comparisons showed significant differences (P<0.001). Table 3: Pairwise comparisons between breeds. Statistic test P value GI – BU 62.998 P<0.001*** SM – BU 72.373 P<0.001*** SM – GI 85.785 P<0.001*** ***highly significant (P<0.001). Linear discriminant analysis (LDA) LDA was performed to maximize the separation between groups. The effect of the breed (Fig. 3) shows that the groups are not very far apart. However, the rankMANOVA method is usually susceptible to small changes (Dobler et al. 2020), so this slight separation is already detected as a significant difference. Although the season was not significant, we performed the LDA (Fig. 4) to support our results. In this case, the Fig. 4: Linear discriminant analysis of season’s effect. groups are much closer to each other than in the previous case, which is why the rank MANOVA did not detect significant differences in BU-GI (P=0.0005795) and GI- significant differences. SM (P=0.0001929) for sperm concentration and BU-GI (P<0.0000001), BU-SM (P=0.0000075) and GI-SM Nonparametric one-way analysis of variance (P<0.0000001) for semen volume, however, there was no Nonparametric one-way analysis of variance revealed evidence of a significant difference in sperm concentration significant differences between breeds for sperm between BU-SM (P=0.9763773) (Table 5). concentration (P=0.0001809) and semen volume (P<0.0000001) but no significant differences were Pairwise comparisons between the interaction of determined for motility (P=0.0773065) (Table 4). breeds and season, showed significant differences in all of the pairwise in cold season for semen volume and only in Dwass-Steel-Chritchlow-Figner pairwise comparisons BU-GI and GI-SM pairwise for sperm motility, however post-hoc analysis there was no evidence of a significant difference in hot Post hoc analysis with Dwass-Steel-Chritchlow- season for any pairwise for sperm concentration and Figner pairwise comparisons between breeds showed motility (Table 6). 4 Int J Vet Sci, 2024, x(x): xxx. Table 5: Pairwise comparisons between breeds Sperm concentration Semen volume Sperm motility W P value W P value W P value BU - GI -5.26426 P<0.0005795*** 14.120908 P<0.0000001*** 1.192111 P<0.6765349 BU - SM -1.92821 P<0.9763773 6.658369 P<0.0000075*** 3.187561 P<0.0624943 GI - SM 5.21325 P<0.0001929*** -10.22086 P<0.0000001*** 1.252697 P<0.6493779 ***highly significant (P<0.001). Table 6: Pairwise comparisons between the interaction of breeds and season Sperm concentration Semen volume Sperm motility W P value W P value W P value Cold BU - GI -5.4374 P<0.001686** 9.068784 P<0.0000001*** -2.81251 P<0.348697 BU - SM -1.32915 P<0.936316 4.875442 P<0.0074886** 1.400486 P<0.921435 GI - SM 4.97026 P<0.005893** -7.01666 P<0.0000104*** 4.27442 P<0.098119 Hot BU - GI -2.26967 P<0.595356 10.51378 P<0.0000001*** 3.672223 P<0.174508 BU - SM 1.42599 P<0.915481 4.61322 P<0.0141200* 2.80376 P<0.352406 GI - SM 3.49019 P<0.133827 -7.39775 P<0.0000025*** -0.40953 P<0.9997279 *significant (P<0.05), ** significant (P<0.01), ***highly significant (P<0.001). DISCUSSION determined in B. indicus, possibly due to the tolerance to heat stress attributed to B. indicus breeds (Riley et al. Consider the present work as the first report that 2012), which may be because our study was on a temperate evaluates the effect of breed, seasonal variation and their climate. For the Simmental breed, Novianti et al. (2020) interaction on seminal volume, sperm concentration and reported that compared to Indonesian indigenous breeds, motility in the Central Genetic Nucleus - EEA Donoso. In Simmental had lower sperm motility with Peru, artificial insemination was introduced in the 1940s 65.959%±4.45%, which is higher than the mean sperm and today it is highly applied in different parts of the motility value in the present study. To sum up, BU and SM country, especially in dairy cattle, used strategically to had significantly higher sperm concentration than GI on accelerate genetic improvement and to maximize pairwise comparison, while semen volume was the reproductive efficiency, breeders must be aware of the opposite, GI had significantly higher values than BU and limits and capabilities of a bull's reproductive ability. The SM. Conversely, sperm motility differences were not quality of semen in bulls can be influenced by both breed significant for any pairwise comparison. characteristics and seasonal variations, as well as the Seasonal fluctuations, can include changes in interaction between them (Brito et al. 2002; Koivisto et al. temperature, photoperiod, and nutritional status, can 2009; Sabés‐Alsina et al. 2017). Different breeds exhibit impact reproductive hormone levels and semen quality, varying levels of fertility and semen quality, with genetic additionally, heat stress, common in hotter months, can factors playing a significant role (Wildeus and Hammond further reduce semen quality (Capela et al. 2022; 1993; Mathevon et al. 1998; de Lucio et al. 2014). In this Netherton et al. 2022). In our study, only temperature was study, the breed had a significant effect in sperm considered and two seasons (hot and cold) were concentration and ejaculate volume, Braunvieh (BU), Gyr evaluated. The effect of seasonal variation was not (GI) and Simmental (SM) individuals were tested. significant, but if we look at the P-value (0.054), it is very There were significant differences between SM and close to 0.05, which is the cut-off point, meaning it is very BU compared to GI in sperm concentration and ejaculate close to being significant. Despite this result, there is no volume, this may be because SM and BU are B. taurus consensus about the seasonal effect in bull sperm breeds while GI is a B. indicus breed, also, non-significant characteristics; some studies show seasonal effects difference in sperm concentration may be explained by the (Söderquist et al. 1996; Vilakazi and Webb 2004; Nichi same reason. A previous study had reported higher sperm et al. 2006; Murphy et al. 2018; Nongbua et al. 2020) and concentration in B. indicus bulls than in B. taurus bulls but others do not (Mathevon et al. 1998; Brito et al. 2002). smaller ejaculate volumes (Brito et al. 2002), on the Other factors, such as local adaptation of cattle, individual contrary, our results pointed that BU and SM sperm thermoregulation, and genotype x environment concentrations were higher than GI, but GI had higher interactions, may be attributed to these differences. Brito ejaculate volume than BU and SM. The previous study was et al. (2002) in a study conducted in Brazil, found that done in Brazil, where B. indicus bulls show a higher there was no significant effect of ambient temperature or performance because of their better thermoregulatory humidity on sperm production and semen quality; they capabilities, also it was done with a majority of other breeds mentioned that this is consistent with another study (Holstein, Red Angus, Aberdeen Angus, Limousin and (Everett and Bean 1982) conducted in temperate Nelore), while it only had one GI and two SM included, environments, both studies have temperatures and this could explain the differences found in our study. humidity similar to ours. In addition, the seasonal effects Sperm motility was not significantly affected by could be attributed to temperature and humidity, the length breeds, similar to Wildeus and Hammond (1993), Rekwot of the day and management (Fuerst-Waltl et al. 2006), et al. (1987) and Brito et al. (2002) studies which reported variations are expressed differently in different locations that no significant differences were found between B. according to latitude. It would also be interesting to taurus breeds and for Zebu cattle, but in other study evaluate the spermatozoa's morphology, which has been (Wildeus and Hammond 1993) higher motility was reported to have an effect at higher temperatures (Seifi- 5 Int J Vet Sci, 2024, x(x): xxx. Jamadi et al. 2020). Our results might be because bulls take Conclusion around 60 days to complete spermatogenesis (Staub and In conclusion, sperm concentration, semen volume Johnson 2018), therefore, changes in ejaculate and sperm motility of Braunvieh, Gyr and Simmental bulls characteristics brought on by unfavorable circumstances, were studied, results showed a significant effect of breed like variations in testicular temperature, would not be and breed x season interaction on sperm concentration and immediately noticed. Therefore, it depends on the time the semen volume. These results will help to improve the assessment of sperm quality is done, and the impact of technical aspects of management decisions at Central climate on sperm quality could go unnoticed. Genetic Nucleus. Our results underline the necessity of Our study's effect on the interaction between breed conducting regular evaluations of all the bulls, to increase and season was significant; previous work has obtained seminal characteristics data. Further studies are needed to similar results (Chacón et al 2002; Teixeira et al. 2011; investigate other factors affecting semen characteristics, Landaeta-Hernández et al. 2020; Llamas-Luceño et al. add pre- and post-freezing data, and provide fertility data. 2020), but studies had a different methodology, some of them use a temperature-humidity index (Llamas-Luceño et Author contributions al. 2020) or monthly temperatures (Teixeira et al. 2011) Conceptualization, D. D-B., S.L. and C.Q.; while others use month-established seasons (Landaeta- methodology, D. D-B. and S.L.; data collection Hernández et al. 2020), in our case, temperature-humidity (laboratory), D. D-B. and S.L.; data collection (field), R.M. index did not seem to be ideal because our humidity levels and J.R.; animal management, R.M. and J.R.; software, do not fluctuate as in other regions. Our findings showed D.F.; General revision of the manuscript, W.Y.A-G.; lower sperm motility of Bos Taurus bulls (BU and SM) Review and editing in English, W.Y.A-G. and D.F.; formal during the hot season; this could be due to lower heat analysis, R.E. and D.F.; writing—original draft tolerance of these animals in contrast to the innate preparation, D.F. and D. D-B.; supervision, W.Y.A-G. and tolerance of Gyr, however our results did not find these C.Q.; project administration, C.Q.; funding acquisition, differences to be significant. As mentioned above, the C.Q. All authors have read and agreed to the published seasonal effects could not only be attributed to temperature version of the manuscript. and humidity; high temperatures and poor pasture quality have been reported to affect sperm volume (Koivisto et al. Funding 2009), unlike our study, where all bulls were adequately This research was funded by the following research fed with the same diet. In cold season, BU and SM project: "Mejoramiento de la disponibilidad de material presented significantly higher sperm concentration than genético de ganado bovino con alto valor a nivel nacional Gyr, this could be because Gyr is a tropical cattle, on the contrary, Gyr had significantly higher semen volume than 7 departamentos" ("Improving the availability of high- the Bos taurus breeds, Koivisto et al. (2009) also reported value cattle genetic material at national level 7 significantly higher values for Bos indicus breeds in all departments") of the Ministry of Agrarian Development four seasons of their study. On the other hand, in hot and Irrigation (MIDAGRI) of the Peruvian Government, season, only Gyr reported significantly higher semen with grant numbers CUI 2432072. volume than BU and SM, the reason for this might be that these breeds don't tolerate heat as well as Gyr does Acknowledgment (Hansen 2004). In a study (Freitas et al. 2020), there was The authors thank the Promeg Nacional team for a significant effect of the temperature-humidity index in supporting the logistic activities in the laboratory. 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