Comparison of seminal characteristics of Aberdeen Angus, Holstein and Nelore bulls before and after cryopreservation

The aim of this study was to evaluate parameters indicative of sperm quality of fresh and post-thawed semen of Aberdeen Angus, Holstein and Nelore bulls. Thirty-nine bulls were used: Aberdeen Angus (n=13), Holstein (n=13) and Nelore (n=13). The ejaculate collects were performed twice a week using artificial vagina, totaling 792 semen collections, 307 for Aberdeen Angus, 225 for Holstein and 260 for Nelore bulls. After collection, fresh semen was evaluated and semen freezing was performed. After freezing, the batches were thawed and progressive motility was determined. The analysis of fresh semen showed that there was no difference (P = 0.053) between the Aberdeen Angus and Nelore breeds, while ejaculates from Holstein bulls showed a statistical difference (P = 0.024). As well, a difference (P<0.001) was identified in the sperm concentration of the three breeds. In the samples evaluated after thawing, a statistical difference was observed between Holstein and Nelore breeds (P<0.001), while the values of the Research, Society and Development, v. 10, n. 16, e408101623382, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i16.23382 2 Angus breed were similar to the other two breeds. The difference in motility of fresh and post-thawing semen showed that Nelore and Angus bulls showed greater variation in values between the analyzes (26.0±8.9% and 25.3±8.4%, respectively) showing a significant difference (P<0.001) in relation to Holstein bulls (20.6±9.3%) that obtained the smallest difference. The analysis of fresh and post-thawing semen did not show any significant difference (P=0.13) between breeds. In conclusion, the semen cryopreservation process causes a decrease in the physical parameters of the semen and these quality losses suffer interference according to the breeds.


Introduction
Reproductive biotechnologies are used to accelerate production, increase reproductive efficiency, and promote genetic improvement in the herd. In Brazil, artificial insemination (AI) has been standing out, both in the beef herd and in the dairy herd, reflecting an increase of 30.1% in sales and 32% in the production of doses in the year 2020 (ASBIA, 2020).
The use of frozen bovine semen represents the main reproductive biotechnique for animal genetic improvement (Freitas et al., 2009;Leite et al., 2011). However, the cryopreservation process can cause damage to sperm, which reflect a decrease of approximately 50% of sperm motility (Celeghini et al., 2007;Thomas et al., 1997). Although the negative effects on sperm after thawing are notable, such as decreased motility due to changes in mitochondrial function, damage to the plasma membrane and acrosome (Layek et al., 2016;Ntenka et al., 2016), sperm cryopreservation has the advantage of storage for indefinite periods and providing worldwide distribution (Layek et al., 2016). In order to obtain good results in the use of artificial insemination, rigorous quality processes are necessary, from the management of breeding bulls to the stages of collection and cryopreservation of the semen, taking into account that the process of the semen cryopreservation causes a drop in sperm quality (Abud et al., 2014;García-Álvarez et al., 2014). Furthermore, it is essential to select breeding bulls capable of producing cryopreservation-resistant ejaculates and maintaining high sperm viability and fertilizing capacity after thawing (Ntenka et al., 2016;Queiroz et al., 2015;Ram et al., 2017).
However, not all bulls are able to produce frozen semen, since numerous intrinsic variables (age, bull's health, libido) and extrinsic (season, diet, management) are involved in this process. (El-Harairy et al., 2011;Queiroz et al., 2015). Another important factor to be considered is the difference in semen quality between the bovine breeds and the respective aptitudes of the animals, which even lacking scientific studies, it is speculated that dairy bulls produce ejaculates with higher quality compared to beef bulls (Morrell et al., 2018).
Thus, this study aimed to evaluate and compare the sperm characteristics of fresh and cryopreserved semen between Aberdeen Angus, Holstein and Nelore bulls breeds.

Ethical aspects
This study was approved by the Research Ethics Committee under number 04/2016. All procedures followed federal law No. 11,794 of October 8, 2008.

Location and animals
The study was conducted at Central Bela Vista, located in Botucatu, State of São Paulo, in September and October 2020. Retrospective data from andrological examinations and semen freezing of 39 bulls of the breeds were analyzed; Nelore (n=13), Holstein (n=13) and Aberdeen Angus (n=13). All bulls were managed in individual paddocks and received a diet based on corn silage, Tifton hay (Cynodon spp.), soybean meal, minerals twice a day and water ad libitum.

Collection, freezing and semen analysis
Semen collections were performed on alternate days, twice a week using the artificial vagina method (IMV Technologies, France), totaling two jumps per day. A total of 792 semen collections were carried out, 307 for Aberdeen Angus bulls, 225 for Holstein bulls and 260 for Nelore bulls. The samples were identified with the name of the bull and the respective jump and sent to the andrology laboratory, located in the same center. In the laboratory, the samples remained in a water bath at 35°-37°C until macroscopic analysis, to classify the appearance and volume of the ejaculate. Two aliquots of semen were removed for concentration and sperm morphology analysis. Then, the tube with the ejaculate was weighed on a precision scale to measure the volume and based on the weight, add an antibiotic solution with the intention of avoiding external contamination. This antibiotic solution was made daily and consisted of 0.375 ml of Tylan® 200 (Elanco, USA), 3.75 ml of Pangram® (Virbac, France), 4.5 ml of Linco Spectin (Zoetis, Brazil) and 6.375 ml of ultrapure water, totaling 15 ml of antibiotic solution, aliquots of which were used according to the ejaculate, in the proportion of 0.02 ml of antibiotic for each ml of semen. To determine the sperm concentration, a dilution was made with 0.27 ml of semen in 9.90 ml of a solution based on sodium phosphate, sodium hydroxide and octylphenol ethoxylate (Reagent s100®, ChemoMetec, Denmark). This solution promotes a lysis of the sperm cell membrane making the nuclei susceptible to staining. After that, an aliquot of 0.06 ml was deposited in an SP1-Cassette™ chamber (ChemoMetec, Denmark) which has inside a fluorescent material, propidium iodide (PI), which in contact with the sample dissolves and stains sperm nuclei. Then the SP1-Cassette™ was placed in the Core Counter® SP-100™ equipment (ChemoMetech, Denmark) which read and calculated the concentration of the ejaculate. Research, Society and Development, v. 10, n. 16, e408101623382, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i16.23382 For morphological analysis, dilution was performed with an aliquot of 0.1 ml of semen in 0.5 ml of formalin-saline solution. After dilution, a drop of this sample was placed on a slide and covered with a cover slip, and 100 cells of each ejaculate were evaluated. The reading of the slide was performed under a phase contrast microscope (Olympus® BX53, Japan) with a magnification of 100x. Sperm defects were classified as major and minor, and only samples with less than 30% of total defects were cryopreserved (Bloom, 1973).
Sperm motility was measured subjectively, ranging from 0 to 100%, and force of rectilinear and progressive movement, rated from 1 to 5, after evaluating 5 fields (CBRA, 2013). For the evaluation, a drop of semen and a drop of freezing medium were deposited on a slide and covered with a pre-warmed cover slip at 37°C, being visualized under an optical microscope (Olympus® BX53, Japan) with 10x magnification. Only samples that showed motility above 60% and a minimum vigor of 3 were selected for cryopreservation (CBRA, 2013).
After the evaluations, the last semen dilution was carried out with the Optixcell 2® diluent, according to the volume and concentration of the ejaculate. After dilution, the semen was submitted to a cooling curve for 3 hours, at a temperature of 4º C, in which it remained until the straws were filled by an system automated IMV-MRS4® (IMV Technologies, France). Semen freezing was performed in a Digitcool® equipment (IMV Technologies, France) with a freezing curve of 12.4 oC/min until reaching a temperature of -145°C.
After undergoing the refrigeration and freezing process, one match of each bull jump was evaluated. Sperm parameters were determined using computerized semen analysis (CASA-Computer Assisted Sperm Analysis, Hamilton Thorne II, USA).
For analysis, the semen batches were thawed in a water bath at 35º C to 37º C for 30 seconds, then the slides were prepared (Leja®, IMV Technologies, France) and placed for reading. In this analysis, samples should have a minimum of 60% viable cells, 30% progressive motility and vigor ≥ 3.

Statistical analysis
Initially, the parameters evaluated before semen freezing were compared between breeds. Motility and vigor were compared before and after freezing, as well as the difference between them. Data were subjected to analysis of variance and compared by Tukey test with 95% confidence interval using Minitab 19 software.

Results
Regarding the sperm characteristics evaluated in fresh semen, it was possible to observe that for the motility parameter there was no significant difference between the bulls breeds (P = 0.053). The vigor values showed similarities between the Aberdeen Angus and Nelore breeds, while in the ejaculates of Holstein bulls there was a statistical difference (P= 0.024); (Table 1).
When evaluating the sperm concentration, it was observed that this parameter was relevant, showing a difference between the three evaluated breeds (P<0.001). Three jumps of Angus bulls and seven jumps of Holstein and Nelore bulls were excluded from the analysis for presenting inadequate motility after cryopreservation with values between zero and 23%. The post-thawing semen parameters are summarized in Table 2. It was possible to observe a difference for the sperm motility variable between the Holstein and Nelore breeds, with the averages for the Holstein breed being higher than the means for the Nelore breed bulls (P<0.001). On the other hand, it was possible to observe that there was no difference in the values of spermatic vigor between the evaluated breeds (P = 0.516). Regarding the percentage difference in motility in the analysis of fresh and post-thawed semen (Figure 1) The difference in vigor values between fresh and post-thawing semen analysis (Figure 2) did not show any difference (P= 0.13) between the breeds. The mean variation of vigor between the breeds was: Aberdeen Angus bulls 1.0±0.5%. Holstein bulls 0.9±0.4% and Nellore bulls 1.0±0.3.

Discussion
It is known that sperm motility is considered the most reliable method for selecting the ejaculate, since this parameter has shown a positive correlation with in vivo and in vitro conception rates (Bergstein et al., 2014;Zhang et al., 1998). In this study, sperm motility was also used as a criterion for selecting ejaculates for freezing.
Data from this study show that there was no significant difference in fresh semen for the motility parameter between races. Similar results were also found by Brito et al. (2002), who evaluated the effect of seasonality on the semen production of B. taurus and B. indicus over a period of two consecutive years and concluded that factors such as temperature, humidity and seasonality did not significantly affect semen production, as well as quality . Motility did not show statistical difference between genetic groups, in the first (B. indicus 58.8% versus B. taurus 60%) and second year (B. indicus 58% versus B. taurus 59.3%). As for Chacur et al. (2012), who, when comparing the seminal quality of zebu and taurine bulls in the four seasons, did not identify motility differences between the subspecies.
On the other hand, a study by Koivisto et al. (2009) can be deduced that sperm motility is of great relevance in the fertility of cows submitted to artificial insemination, being considered as a good parameter in the selection of semen samples for use in biotechniques applied to animal reproduction.
Although the results of the present study are not in agreement with some data already published, it is believed that Holstein bulls showed a lower motility difference and a supposedly better semen viability after cryopreservation is due to cellular factors associated with the breed. Morrell et al. (2018) reported indices such as sperm plasma membrane integrity to be higher in dairy bulls compared to beef bulls (46±8% versus 40±11%, respectively), as well as superiority in relation to mitochondrial potential in which the proportion of sperm with high mitochondrial activity was 52±12% in dairy bulls and 36±10% in beef bulls.

Conclusion
The semen cryopreservation process causes a decrease in the physical parameters of the semen and these quality losses, suffer interference according to the breeds, genetic groups evaluated and the system in which the animal is inserted.
However, further studies are needed in order to improve gamete cryopreservation protocols and, in this way, increase the quality of the ejaculate and have greater success in pregnancy rates.