Introduction
Brazil is the world’s second-largest producer of beef with a herd of 218.2 million animals. Notably, Brazil has become the world’s largest beef exporter, exporting 2.69 million tons of bovine carcass weight, a trade worth more than US$8.4 billion in 2021 (IBGE, 2017; De Nadai Fernandes et al., Reference De Nadai Fernandes, Sarriés, Bacchi, Mazola, Gonzaga and Sarriés2020; Zu Ermgassen et al., Reference Zu Ermgassen, Godar, Lathuillière, Löfgren, Gardner, Vasconcelos and Meyfroidt2020; ABIEC, 2021). Roughly 80% of the Brazilian herd is composed of Zebu cattle (Bos taurus indicus), mainly the Nellore breed. The other 20% are European breeds (Bos taurus taurus), especially the Angus breed and more recently the Wagyu breed (Rodrigues et al., Reference Rodrigues, Chizzotti, Vital, Baracat-Pereira, Barros, Busato, Gomes, Ladeira and Martins2017; Rezende-de-Souza et al., Reference Rezende-de-Souza, Cardello, de Paula, Ribeiro, Calkins and Pflanzer2021).
Consumers have become more careful with the quality of food in general, especially with the quality of meat and how it can directly influence health. In this sense, in Brazil, the high demand for special meats has directed enterprises, such as slaughterhouses, meat shops, or restaurants, to produce high-quality meat. The increased intramuscular or marbling fat content is correlated to juiciness, tenderness, and meat flavour. Wagyu adds value from the marbling process as Wagyu has a high propensity to accumulate intramuscular fat. Furthermore, the fatty acid composition of lipids in meat is also important for human health and, therefore, this subject has been extensively studied in recent years (Radunz et al., Reference Radunz, Loerch, Lowe, Fluharty and Zerby2009; Ladeira et al., Reference Ladeira, Schoonmaker, Swanson, Duckett, Gionbelli, Rodrigues and Teixeira2018; Connolly et al., Reference Connolly, Dona, Hamblin, D’Occhio and González2020; Facioli et al., Reference Facioli, De Marchi, Marques, Michelon, Zanella, Caires, Reeves and Zanella2020).
Animal reproduction is fundamental for addressing the growing demand for access to animal proteins. Therefore, this leads farmers to be as efficient as possible in the breeding and management of livestock (Yánez-Ortiz et al., Reference Yánez-Ortiz, Catalán, Rodríguez-Gil, Miró and Yeste2022). To increase reproductive capacity, bulls must produce morphologically normal and large numbers of sperm (Teixeira et al., Reference Teixeira, Coelho, Tomich, Pacheco Rodrigues, Camρos, Machado, Gualberto Barbosa da Silva, Monteiro and Ribeiro Pereira2019). In this context, a detailed breeding soundness evaluation (BSE) of the bull should be carried out to assess the sire’s reproductive health (Silva et al., Reference Silva, Pedrosa, Silva, Eler, Guimarães and Albuquerque2011; Romanello et al., Reference Romanello, de Brito Lourenço Junior, Barioni Junior, Brandão, Marcondes, Pezzopane, de Andrade Pantoja, Botta, Giro, Moura, do Nascimento Barreto and Garcia2018; Rodrigues et al., Reference Rodrigues, Rossi, Vrisman, Taira, Souza, Zorzetto, Bastos, de Paz, de Lima, Monteiro and Franco Oliveira2020; Leite et al., Reference Leite, de Agostini Losano, de Souza Ramos Angrimani, Sousa, de Miranda Alves, Cavallin, Kawai, Cortada, Zuge and Nichi2021).
Nonetheless, Wagyu bulls have a higher libido, higher levels of testosterone and sperm production when compared with other taurine and indicine breeds (Smith-Thomas, pers. commun.; Sosa et al., Reference Sosa, Senger and Reeves2002; Tatman et al., Reference Tatman, Chase, Wilson, Neuendorff, Lewis, Brown and Randel2022). The purpose of this study was to evaluate whether there were differences in the hormone and sperm characteristics of Wagyu bulls compared with the same characteristics of subspecies Bos indicus and Bos taurus sires. Wagyu bulls are known to have a highly exacerbated libido, as shown by the intense sexual interest of young calves. Therefore we believe that Wagyu male animals have specialized Sertoli and Leydig cells that are directly involved with the sexual precocity in this breed as mature animals have a small scrotal circumference that can affect sperm production (Sosa et al., Reference Sosa, Senger and Reeves2002).
Materials and methods
Experimental procedures adopted by this study are in agreement with the Principles of Ethics in Animal Research adopted by the Commission of Bioethics, Federal University of ABC (UFABC, protocol # 8993100516). This study was partially supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
Experimental design
Before starting the experiment, we performed a power analysis using PROC POWER (SAS System for Windows 9.3) to determine the minimal number of experimental units for each group. We considered only the variable motility. A result above 0.8 indicated that five experimental units per group would be adequate for the experiment, ensuring a high probability of observing the effect of treatments. Therefore, the experimental design was performed with five animals per group; each breed was considered as an experimental group: Angus, Nellore, and Wagyu.
Semen batches from Nellore and Angus bulls were donated from a commercial artificial insemination centre in Brazil (Seleon Biotechnology, Itatinga, SP, Brazil). Semen batches from Wagyu bulls were donated from a breeding herd located in the state of Rio Grande do Sul, Brazil. Data regarding sperm analysis before cryopreservation were not available for this study. However, all samples from all three breeds were diluted to a concentration of 25 million sperm cells/straw for cryopreservation. The cryopreservation procedure was performed according to local standard protocols and both used egg yolk-based extenders.
All semen samples were submitted to evaluate sperm motility using computer-assisted sperm analysis (CASA), acrosome integrity (Pisum sativum agglutinin; FITC-PSA), plasma membrane integrity (propidium iodide; PI), mitochondrial membrane potential (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide; JC-1), and chromatin resistance using flow cytometry and oxidative stress by thiobarbituric acid reactive substances assay (TBARS). Blood samples were used to evaluate testosterone and luteinizing hormone (LH).
Semen preparation
Semen straws were thawed at 37ºC for 30 s. To remove the extender and recover sperm cells, 250 µl of semen were centrifuged at room temperature for 6 min at 9000 g in FertTALP warmed at 38.5°C (Parrish et al., Reference Parrish, Susko-Parrish, Winer and First1988) with no capacitation inductors. Sperm cells were recovered and washed again in FertTALP medium for 3 min at 9000 g. The sperm samples were then evaluated for acrosome integrity, membrane integrity, mitochondrial membrane potential, sperm chromatin status, and lipid peroxidation.
Assessment of sperm kinetics
Sperm motility was evaluated using CASA (Hamilton-Thorne, HTR Ceros 12.3, USA) as described by Leite et al. (Reference Leite, Losano, Kawai, Rui, Nagai, Castiglioni, Siqueira, D’Avila Assumpção, Baruselli and Nichi2022) elsewhere. Briefly, 7 µl of each sample were placed between a Leja counting chamber (Orange Medical, Brussels, Belgium) previously heated to 37°C. A minimum of five randomly selected microscopic fields per sample was analyzed. The following variables were considered: total motility (MOT; %), progressive motility (PROG; %), average path velocity (VAP; µm/s), straight-line velocity (VSL; µm/s), curvilinear velocity (VCL; µm/s), amplitude of lateral head displacement (ALH; µm), beat cross-frequency (BCF; Hz), straightness (STR, VSL/VAP; %), and linearity (LIN; %). Sperm cells were also classified based on velocity: (1) rapid (RAP, VAP > 50 µm/s, %); (2) medium (MED, 30 µm/s < VAP < 50 µm/s, %); slow (SLOW, VAP < 30 µm/s or VSL < 15 µm/s, %); or (4) static (STATIC, %). The setup for sperm kinetic analysis was 60 Hz frames/s for image acquisition, with a minimum of 80 cells per field, minimum cell size of 5 pixels, 70 for cell intensity, path velocity of 50 µm/s and STR of 70% for progressive cells; and VAP and VSL cutoff at 30 and 15 µm/s for slow cells.
Acrosome integrity, membrane integrity, mitochondrial membrane potential, and sperm chromatin status evaluation
Acrosome integrity, membrane integrity, mitochondrial membrane potential, and sperm chromatin status were evaluated as described by Siqueira et al. (Reference Siqueira, de Castro, de Assis, Bicudo, Mendes, Nichi, Visintin and Assumpção2018). Evaluations were performed using flow cytometry (Guava EasyCyte™, Guava® Technologies, Hayward, CA, USA) equipped with a 20 mW 488 nm argon excitation laser. Probes were purchased from Sigma and Molecular Probes.
Cytometry data were analyzed using the FlowJo software (version 10.2 Flow Cytometry Analysis Software–Tree Star Inc., Ashland, Oregon, USA). In total, 20,000 events per sample were analyzed. Cells were identified and selected excluding debris, probes particles and non-single sperm events, applying a gate on forward scatter (FSC) versus green fluorescence dot plot (log mode) around the single sperm events to acrosome, membrane, and mitochondrial analysis. For chromatin analysis, the single sperm events were selected by applying a gate on forward scatter (FSC) versus red fluorescence dot plot (log mode).
Simultaneously, a staining protocol was performed by incubating 187,500 cells for 5 min with 6 μM of propidium iodide (PI) associated with 5 μg of fluorescein-conjugated Pisum sativum (FITC-PSA). PI emits red fluorescence for damaged plasma membrane, while FITC-PSA emits green fluorescence for damaged acrosome. Mitochondrial membrane potential was analyzed by incubating 187,500 cells for 5 min with 1 μM of tetraethylbenzimidazolycarbocyanine iodide (JC-1) that emits green fluorescence for low mitochondrial potential and red/orange fluorescence for high mitochondrial potential.
As a positive control for damaged acrosome and membrane, and lack of mitochondrial membrane potential, a sample was prepared as described by Siqueira et al. (Reference Siqueira, de Castro, de Assis, Bicudo, Mendes, Nichi, Visintin and Assumpção2018). Briefly, a sperm sample was submitted to freezing/thawing cycles (1 min in liquid nitrogen/1 min in a water bath at 60°C, five times). As a negative control, one sample was prepared as described above for experimental samples.
Chromatin sperm analysis was performed according to Simões et al. (Reference Simões, Feitosa, Siqueira, Nichi, Paula-Lopes, Marques, Peres, Barnabe, Visintin and Assumpção2013) with some modifications. Briefly, 375,000 sperm cells were added to 50 μl TNE buffer (10 mM Tris−HCl, 0.15 M NaCl, 1 mM EDTA disodium pH 7.4) and 100 μl of acid detergent solution [0.08 M HCl, 0.15 M NaCl, 0.1% (v/v) Triton X-100, pH 1.4]. After 30 s, 300 μl of acridine orange solution (6 μg/ml in 0.1 M citric acid, 0.2 M Na2HPO4, 1 mM de EDTA, 0.15 M NaCl, pH 6.0) were added, and an evaluation was performed using flow cytometry at 3–5 min following acridine orange solution addition. As a control of chromatin damage, a sample was incubated with HCl (1.2 M in acid detergent solution, pH 0.1) for 1 min. As a control of chromatin resistance, a sample was prepared as described above for the experimental samples.
To calculate the determination coefficient of cytometry analyses, a positive (100%) and a negative control (0%) for each analysis were evaluated individually and mixed in different proportions: 1:3 (25%); 1:1 (50%); 3:1 (75%). Negative and positive thresholds and gates for each analysis were set up to achieve the highest determination coefficient on controls and proportions analysis. All traits analysis reached coefficients higher than 0.94. Compensation parameters and gates applied were held for all samples. Dot plots showing rectangular gates applied to select single sperm events and histograms showing bisector gates applied to separate negative and positive events are presented as Supplementary Material (Figures S1–S4). We presented graphs of negative and positive controls, along with their respective mixtures from each probe analyzed.
Sperm lipid peroxidation assessment
Sperm susceptibility to lipid peroxidation was evaluated by the TBARS assay. The TBARS reaction evaluates malondialdehyde (MDA) concentrations as products of lipid peroxidation. Thiobarbituric acid reacts with MDA to produce a pink-coloured complex.
This analysis was performed as described by Ohkawa et al. (Reference Ohkawa, Ohishi and Yagi1979) and adapted by Nichi et al. (Reference Nichi, Bols, Züge, Barnabe, Goovaerts, Barnabe and Cortada2006). In summary, lipid peroxidation was induced by adding ferrous sulfate (100 μl, 4 mM) and sodium ascorbate (100 μl, 20 mM) to 400 μl of the sperm suspension (2.5 × 106 sperms/ml). The mixture was then incubated for 90 min at 37°C. Subsequently, ice-cold 10% trichloroacetic acid (TCA; ratio 2:1) were added. Samples were then centrifuged (18000 g, 10 min, 5°C) to precipitate the protein. The supernatant was mixed with 1% thiobarbituric acid (TBA, ratio 1:1) in a cryotube vial. Samples were placed into a boiling water bath (100°C) for 15 min and then immediately cooled in an ice bath (0°C) to stop the chemical reaction. The TBARS were then quantified using a spectrophotometer (UV–vis spectrophotometer, Ultrospec 3300 Pro, Biochrom Ltd, Cambridge, UK) at a wavelength of 532 nm. The lipid peroxidation index was described as nanograms of TBARS/106 sperm.
Blood samples and hormonal assay
Blood samples (10 ml) were collected from a jugular vein into centrifuge vacuum tubes containing heparin. Blood tubes were centrifuged at 3000 g for 20 min. Plasma samples were stored individually at −20°C until further analysis.
Plasma LH was evaluated as previously described by Bolt and Rollins (Reference Bolt and Rollins1983) and Bolt et al. (Reference Bolt, Scott and Kiracofe1990). Plasma LH concentrations were measured by double-antibody radioimmunoassay (RIA) using purified bovine LH standards. A highly purified LH (AFP8614B; National Hormone and Pituitary Programme) was used for both the iodinated tracer and reference standard preparation. The intra-assay coefficients of variation (high and low) and the sensitivity for LH were 9.15%, 5.97% and 0.0373 ng/ml, respectively. Testosterone concentration was determined by an RIA using a commercial kit RIA Testosterone, direct (Beckman Coulter, Prague, Czech Republic) according to the manufacturer’s instructions.
Statistical analysis
Statistical Analysis System 9.3 (SAS Institute, Cary, NC) was used to analyze the dependent variables. All data were tested for normality of residues and homogeneity of variances. Variables that did not comply with these statistical premises were transformed (logarithmic transformation was used to evaluate the chromatin susceptibility; testosterone quantification; percentage of sperm with damaged acrosome and intact membrane; percentage of sperm with static movement; and intracellular lipid peroxidation; square root transformation was used to evaluate the percentage of sperm with damaged plasma and acrosome membranes; and the inverse of the data was used to evaluate the percentage of sperm with medium movement). The general linear models (GLM) procedure was used to evaluate the breed effect (Angus, Nellore, and Wagyu). Comparisons of means were performed using the Duncan Test. A 5% significance level was used to reject the null hypothesis. The results are presented as mean ± standard error of the mean (SEM).
Results
Assessment of sperm kinetics
Unfortunately, two Wagyu bulls presented difficulties during semen collection compromising the sperm processing, therefore the Wagyu group was formed with only three experimental units.
The results corresponding to sperm kinetics from Angus, Nellore, and Wagyu bulls are shown in Figure 1. Sperm from Nellore bulls showed greater amplitude of lateral head displacement (ALH) when compared with Wagyu and Angus bulls (P = 0.016; Figure 1a). However, Wagyu bulls showed no significant difference for the same parameter when compared with Angus bulls. Considering the variable STR, sperm from Wagyu bulls showed higher straightness (STR) cells when compared with Nellore bulls (P = 0.032; Figure 1b). However, Angus bulls showed no significant difference for the same parameter when compared with Nellore and Wagyu bulls.
Mean ± standard error of the mean (SEM) of sperm kinetic analysis by computer-assisted sperm analysis (CASA). (a) Amplitude of lateral head displacement (ALH; µm). (b) Straightness (STR, VSL/VAP; %). (c) Linearity (LIN; %). (d) Total motility (MOT; %). (e) Medium movement (MEDIUM; %). (f) Static sperm (STATIC; %) in semen samples of Angus (n = 5), Nellore (n = 5) and Wagyu (n = 3) bull. Different superscript letters represent significant statistical differences (P < 0.05).
When evaluating linearity (LIN), Nellore bulls showed cells with lower LIN than Wagyu and Angus bulls (P = 0.016; Figure 1c). Angus and Wagyu sires did not show significant differences for LIN. Still, Wagyu bulls showed sperm with lower total motility (MOT) when compared with Nellore and Angus bulls (P = 0.012; Figure 1d). Conversely, Angus and Nellore bulls showed no significant difference for MOT. Wagyu sires showed sperm cells with lower medium movement (MEDIUM) than Angus and Nellore bulls (P = 0.004; Figure 1e). However, Angus bulls showed no significant difference for the same parameter when compared with Nellore bulls. Finally, sperm from Wagyu bulls had a higher percentage of static sperm (STATIC) when compared with Nellore and Angus bulls (P = 0.022; Figure 1f). Angus bulls showed no significant difference for the same parameter when compared with Nellore bulls.
Acrosome integrity, membrane integrity, mitochondrial membrane potential, and sperm chromatin status evaluation
The results corresponding to acrosome integrity, plasma membrane integrity, mitochondrial membrane potential, and sperm chromatin status evaluations from Wagyu, Nellore, and Angus bulls are shown on Table 1. Spermatozoa from the three bull breeds (Nellore, Wagyu and Angus) showed no significant difference in terms of sperm chromatin structure assay (SCSA) quality (P = 0.903), high mitochondrial membrane potential (HIGH JC-1; P = 0.753). Nellore, Wagyu, and Angus bulls did not show significant differences in terms of sperm cells with damaged acrosome and intact plasma membrane (FITC–PI+/−; P = 0.900); number of sperm cells with damaged acrosome and plasma membrane (FITC–PI+/+ ; P = 0.146); number of sperm cells with intact acrosome and damaged plasma membrane (FITC–PI−/+; P = 0.860); and amount of sperm cells with intact acrosome and plasma membrane (FITC–PI−/−; P = 0.625).
Mean ± standard error of the mean (SEM) and probability (P) of sperm chromatin structure (SCSA), mitochondrial membrane potential (JC-1), acrosome and plasma membrane integrities (FITC–PI) and sperm lipid peroxidation status evaluated by induced thiobarbituric acid reactive substances (TBARS) assay among Angus (n = 5), Nellore (n = 5) and Wagyu (n = 3) bulls
SCSA+ = intact sperm chromatin; JC-1 HIGH = high mitochondrial membrane potential; FITC–PI (+/−) = damaged acrosome and intact plasma membrane; FITC–PI (+/+) = damaged plasma and acrosome membranes; FITC–PI (−/+) = intact acrosome and damaged plasma membrane; FITC–PI (−/−) = intact plasma and acrosome membranes; TBARS = induced thiobarbituric acid reactive substances (ng TBARS/106 sperm).
Sperm lipid peroxidation assessment
The results corresponding to sperm cell lipid peroxidation status from Wagyu, Nellore, and Angus sires are shown in Table 1. Spermatozoa from the three bull breeds (Nellore, Wagyu, and Angus) showed no significant difference (P = 0.646) in terms of sperm lipid peroxidation status.
Hormonal assay
The results corresponding to serum testosterone and LH concentrations from Wagyu, Nellore, and Angus sires are shown in Table 2. Spermatozoa from the three bull breeds (Nellore, Wagyu and Angus) showed no significant difference (P = 0.384) in terms of serum testosterone and LH concentrations.
Mean ± standard error of the mean (SEM) and probability (P) of serum testosterone and luteinizing hormone (LH) concentrations among Angus (n = 5), Nellore (n = 5) and Wagyu (n = 3) bulls
Discussion
Sire fertility is extremely important for reproduction in several species, including cattle. After the introduction of artificial insemination (AI) practice and increasing worldwide export of semen straws, sperm analysis became even more substantial (Amann and Waberski, Reference Amann and Waberski2014; Nagy et al., Reference Nagy, Polichronopoulos, Gáspárdy, Solti and Cseh2015; Leite et al., Reference Leite, Losano, Kawai, Rui, Nagai, Castiglioni, Siqueira, D’Avila Assumpção, Baruselli and Nichi2022; O’Meara et al., Reference O’Meara, Henrotte, Kupisiewicz, Latour, Broekhuijse, Camus, Gavin-Plagne and Sellem2022). Motility is one of the most important seminal parameters associated with the sperm fertilizing ability and it may indicate spermatozoa viability and structural integrity (Kathiravan et al., Reference Kathiravan, Kalatharan, Karthikeya, Rengarajan and Kadirvel2011; Nagy et al., Reference Nagy, Polichronopoulos, Gáspárdy, Solti and Cseh2015; Singh et al., Reference Singh, Kumar and Bisla2021; Leite et al., Reference Leite, Losano, Kawai, Rui, Nagai, Castiglioni, Siqueira, D’Avila Assumpção, Baruselli and Nichi2022). Conventional semen analysis (semen volume, colour, density, viscosity, and sperm motility, vigour, concentration and morphology) is an important and also a practical and low-cost evaluation to identify sperm quality. However, as sperm cells are highly specialized cells, conventional analysis is not sufficient to evaluate sperm function and fertilization ability (Perumal et al., Reference Perumal, Srivastava, Ghosh and Baruah2014; Nagy et al., Reference Nagy, Polichronopoulos, Gáspárdy, Solti and Cseh2015; Leite et al., Reference Leite, Losano, Kawai, Rui, Nagai, Castiglioni, Siqueira, D’Avila Assumpção, Baruselli and Nichi2022; O’Meara et al., Reference O’Meara, Henrotte, Kupisiewicz, Latour, Broekhuijse, Camus, Gavin-Plagne and Sellem2022). There is a large variation among laboratories and even within each or between technicians. According to Brazil et al. (Reference Brazil, Swan, Tollner, Treece, Drobnis, Wang, Redmon and Overstreet2004) the variation coefficient among laboratories is ∼23–73% for sperm concentration, 9–37% for sperm motility and 25–87% for sperm morphology evaluation.
To overcome the operator dependency of conventional semen analysis, technologies have been developed. CASA is a computer system equipped with a high-resolution camera and microscope that allows a more accurate sperm motion evaluation. Moreover, CASA allows the reduction in bias compared with visual evaluations (Kathiravan et al., Reference Kathiravan, Kalatharan, Karthikeya, Rengarajan and Kadirvel2011; Amann and Waberski, Reference Amann and Waberski2014; Nagy et al., Reference Nagy, Polichronopoulos, Gáspárdy, Solti and Cseh2015; Valverde et al., Reference Valverde, Barquero and Soler2020; Fernandez-Novo et al., Reference Fernandez-Novo, Santos-Lopez, Barrajon-Masa, Mozas, de Mercado, Caceres, Garrafa, Gonzalez-Martin, Perez-Villalobos, Oliet, Astiz and Perez-Garnelo2021). The results for sperm kinetics analyses in the present study indicated that the Nellore bulls showed the highest head displacement amplitude (ALH) compared with Angus and Wagyu sires. Still, Wagyu and Angus bulls, despite showing lower ALH values, did not show a significant difference between them. The sperm cells with higher ALH indicated a higher ability to penetrate cervical mucus and fuse with the zona pellucida of the oocyte. It is believed that the higher the ALH, the greater the chance of fertilizing the female gamete (Perumal et al., Reference Perumal, Srivastava, Ghosh and Baruah2014).
The STR and LIN parameters are related to the straightness of the sperm’s movement and ability to migrate in cervical mucus (Goovaerts et al., Reference Goovaerts, Hoflack, Van Soom, Dewulf, Nichi, de Kruif and Bols2006; Robayo et al., Reference Robayo, Montenegro, Valdés and Cox2008; Kathiravan et al., Reference Kathiravan, Kalatharan, Karthikeya, Rengarajan and Kadirvel2011; Perumal et al., Reference Perumal, Srivastava, Ghosh and Baruah2014). The results of sperm kinetics analysis in the present study indicated that the Wagyu sires showed higher values for both parameters when compared with Angus and Nellore bulls. However, the STR from Wagyu bulls sperm cells was not significantly different from Angus bulls. Conversely, the STR of Angus bulls was not significantly different from Nellore bulls. Regarding linearity (LIN), the Angus bulls had the best results when compared with Nellore bulls, but did not differ significantly when compared with the LIN of the Wagyu bulls. High ALH and low STR values are characteristic of hyperactive spermatozoa. Conversely, high values of LIN and STR indicated a progressive movement pattern of sperm cells (Ratnawati and Luthfi, Reference Ratnawati and Luthfi2020). Considering all this together, it seems that sperm cells from Nellore sires that showed higher ALH and lower STR and LIN had started the capacitation process before Angus and Wagyu. Still, Wagyu spermatozoa are more capable of swimming in a straight line.
Considering total motility (MOT) and not motile (STATIC) cells, it was observed that Wagyu sires showed lower MOT than Angus and Nellore bulls and a higher number of STATIC cells. Despite having a straighter pattern than Nellore and Angus bulls, Wagyu sperm were slower. Some might think that these characteristics of sperm kinetics in Wagyu sires may be worrying, as the bovine oocyte after being ovulated remains viable in the fallopian tube for an average of 6–8 h (Miller, Reference Miller2018). In other words, fertilization of aged oocytes produces poorer quality embryos. Notwithstanding, another possible hypothesis that could explain lower motility is the sperm freezing procedure. According to Siqueira et al. (Reference Siqueira, de Castro, de Assis, Bicudo, Mendes, Nichi, Visintin and Assumpção2018) and Kathiravan et al. (Reference Kathiravan, Kalatharan, Karthikeya, Rengarajan and Kadirvel2011), sperm cells that survive suboptimal conditions during the cryopreservation process are more resistant to in vitro conditions, showing higher longevity after thawing, indicating that those spermatozoa may be more prone to fertilize the oocyte, even though they were less motile. Yet, sperm motion characteristics seem to be of minor importance, as the uterine contractions may be of greater consequence (Kunz et al., Reference Kunz, Beil, Deiniger, Einspanier, Mall and Leyendecker1997) or when considering IVP conditions in which samples are submitted to motile sperm selection techniques as density gradient centrifugation overcoming lower initial motility.
Differences in sperm kinetics evaluation among sires may be due to differences in the genetic group of animals (Hoflack et al., Reference Hoflack, Opsomer, Rijsselaere, Van Soom, Maes, de Kruif and Duchateau2007; Kathiravan et al., Reference Kathiravan, Kalatharan, Edwin and Veerapandian2008; Morrell et al., Reference Morrell, Valeanu, Lundeheim and Johannisson2018). Detailed studies, however, must be conducted to more accurately determine the associations among values for sperm kinetics and those for fertility variables. Conversely, a higher ALH and lower STR and LIN as observed in Nellore bulls may not be good sperm traits to be considered as biomarkers. Sperm hyperactivation alters sperm swimming patterns, increasing ALH and decreasing LIN values (Shi and Roldan, Reference Shi and Roldan1995; Sansegundo et al., Reference Sansegundo, Tourmente and Roldan2022). It seems reasonable to hypothesize that higher hyperactivated sperm cells as seen in Nellore bulls in the present study may be related to a premature event that leads to higher energy consumption and ATP decrease more rapidly. This situation could diminish fertilization rates in Nellore sires when compared with Angus and Wagyu. However, more studies are needed to confirm this hypothesis.
As fertilization is a complex and multistep process, it is unlikely that only one sperm attribute could be considered a fertility biomarker (Oliveira et al., Reference Oliveira, de Arruda, de Andrade, Celeghini, Reeb, Martins, dos Santos, Beletti, Peres, Monteiro and Hossepian de Lima2013). Therefore, it is important to evaluate a wide range of sperm features to identify potential biomarkers for bull fertility (Utt, Reference Utt2016). In addition to sperm motility, the evaluation of sperm function attributes such as acrosomal and plasma membrane integrity, chromatin status and mitochondrial activity remains one of the keystones in semen laboratory analysis (Simões et al., Reference Simões, Feitosa, Siqueira, Nichi, Paula-Lopes, Marques, Peres, Barnabe, Visintin and Assumpção2013; Malama et al., Reference Malama, Zeron, Janett, Siuda, Roth and Bollwein2017; Bernecic et al., Reference Bernecic, Donnellan, O’Callaghan, Kupisiewicz, O’Meara, Weldon, Lonergan, Kenny and Fair2021). IVP is a useful tool to compare in vitro fertility of samples from bulls and breeds under certain conditions. Nevertheless, until now, no single assay has been able to accurately predict bull fertility.
In Angus sires it was shown that 82% of in vitro fertilization rates may vary according to sperm features such as sperm morphology, acrosome and plasma membrane integrities (Tartaglione and Ritta, Reference Tartaglione and Ritta2004). In Holstein Friesian bulls, Bernecic et al. (Reference Bernecic, Donnellan, O’Callaghan, Kupisiewicz, O’Meara, Weldon, Lonergan, Kenny and Fair2021) developed a linear and logistic predictive model to evaluate bull fertility. According to the authors, 47% of bull fertility variation is due to plasma membrane changes and 90% to acrosome damage. Oliveira et al. (Reference Oliveira, Arruda, Thomé, Maturana Filho, Oliveira, Guimarães, Nichi, Silva and Celeghini2014) showed that semen of Nellore sires with intact plasma and acrosome membranes and high mitochondrial potential resulted in increased pregnancy rates when compared with spermatozoa with membrane damage and diminished mitochondrial potential. The role of mitochondria in several intracellular processes related to sperm metabolism, cell signalling and sperm apoptosis has made the assessment of mitochondrial function a promising tool for the assessment of sperm fertilization capacity (Malama et al., Reference Malama, Zeron, Janett, Siuda, Roth and Bollwein2017; Leite et al., Reference Leite, Losano, Kawai, Rui, Nagai, Castiglioni, Siqueira, D’Avila Assumpção, Baruselli and Nichi2022).
With respect to sperm chromatin integrity, several studies have already shown that bull fertility and embryo development can be negatively affected when sperm DNA is damaged (Simões et al., Reference Simões, Feitosa, Siqueira, Nichi, Paula-Lopes, Marques, Peres, Barnabe, Visintin and Assumpção2013; Dogan et al., Reference Dogan, Vargovic, Oliveira, Belser, Kaya, Moura, Sutovsky, Parrish, Topper and Memili2015; Kumaresan et al., Reference Kumaresan, Johannisson, Al-Essawe and Morrell2017; Narud et al., Reference Narud, Klinkenberg, Khezri, Zeremichael, Stenseth, Nordborg, Haukaas, Morrell, Heringstad, Myromslien and Kommisrud2020). Nonetheless, in the present study, semen functional parameters were not different among sires. Even with no apparent impairment of sperm function, Wagyu bulls may have lower fertility as they have slower sperm cells and therefore may produce poorer quality embryos. This fact can decrease the pregnancy rate, which increases the cost of breeding. Given the considerable proportion of variation in bull fertility that yet remains not explained, it would be of interest to carry out a further study focusing on sperm molecular-based characterization and also explore other sperm features to identify possible cellular structures that may be related to the decline in bull fertility.
Reactive oxygen species (ROS) mediate essential intracellular signalling cascades needed for sperm physiological function, namely sperm flagellar hyperactivation, capacitation, and acrosome reaction, sperm oocyte interactions, signalling pathways involved in the fertilization, implantation and early embryo development (Agarwal and SenGupta, Reference Agarwal and SenGupta2020; Dutta et al., Reference Dutta, Henkel, Sengupta, Agarwal, Parekattil, Esteves and Agarwal2020; Alyethodi et al., Reference Alyethodi, Sirohi, Karthik, Tyagi, Perumal, Singh, Sharma and Kundu2021; Upadhyay et al., Reference Upadhyay, Ramesh, Dewry, Yadav and Ponraj2022). However, oxidative stress (imbalance of ROS not mitigated by the antioxidant mechanisms) may reduce sperm fertilizing ability due to toxicity (Ugur et al., Reference Ugur, Saber Abdelrahman, Evans, Gilmore, Hitit, Arifiantini, Purwantara, Kaya and Memili2019; Ribas-Maynou et al., Reference Ribas-Maynou, Yeste and Salas-Huetos2020; Saraf et al., Reference Saraf, Kumaresan, Sinha and Datta2021). Mitochondrial impairment and sperm DNA fragmentation are positively correlated with oxidative stress (Simões et al., Reference Simões, Feitosa, Siqueira, Nichi, Paula-Lopes, Marques, Peres, Barnabe, Visintin and Assumpção2013; Leite et al., Reference Leite, Losano, Kawai, Rui, Nagai, Castiglioni, Siqueira, D’Avila Assumpção, Baruselli and Nichi2022). It is known that frozen–thawed semen has an increased lipid peroxidation rate, once sperm cells are rich in polyunsaturated fatty acids and lack antioxidant enzyme defences (Amaral et al., Reference Amaral, Lourenço, Marques and Ramalho-Santos2013; Castiglioni et al., Reference Castiglioni, Siqueira, Bicudo, de Almeida, Hamilton, de Castro, Mendes, Nichi, Losano, Visitin and Assumpção2021; Pintus and Ros-Santaella, Reference Pintus and Ros-Santaella2021). Lipid peroxidation is considered a specific indicator of oxidative stress in sperm cells (Gallo et al., Reference Gallo, Esposito, Tosti and Boni2021). Indeed, there is damage caused by oxidative stress to the nature and amount of ROS involved and also on the duration of ROS exposure and extracellular factors, namely temperature, oxygen tension and the surrounding environment (Agarwal et al., Reference Agarwal, Makker and Sharma2008; Pintus and Ros-Santaella, Reference Pintus and Ros-Santaella2021).
In the present study, semen lipid peroxidation was not different among sires. Taken together with the results discussed above (sperm kinetics and sperm function analysis) it was possible to infer that the amount of ROS was not able (or sufficient) to trigger mitochondrial dysfunction, leading to altered respiratory chain activity, plasma membrane degradation, and thereby decreasing cell motility. The same was true for sperm DNA damage, as chromatin is the last cell structure to be affected by oxidative stress. It has been demonstrated that when sperm are exposed to low levels of oxidative stress, blastocyst formation is decreased, but not the cleavage rate. Conversely, when exposure to oxidative stress is severe, it is able to reduce cleavage and blastocyst rates, embryo quality and also compromise sperm DNA integrity (de Assis et al., Reference de Assis, Castro, Siqueira, Delgado, Hamilton, Goissis, Mendes, Nichi, Visintin and Assumpção2015; de Castro et al., Reference de Castro, de Assis, Siqueira, Hamilton, Mendes, Losano, Nichi, Visintin and Assumpção2016; Simões et al., Reference Simões, Feitosa, Siqueira, Nichi, Paula-Lopes, Marques, Peres, Barnabe, Visintin and Assumpção2013).
The relationship between testosterone and LH can be used to measure Leydig cell development (Teerds and Huhtaniemi, Reference Teerds and Huhtaniemi2015). Both are released in response to GnRH during puberty. Luteinizing hormone together with FSH is able to modulate testosterone synthesis in Leydig cells (Anand-Ivell et al., Reference Anand-Ivell, Byrne, Arnecke, Fair, Lonergan, Kenny and Ivell2019; Kowalczyk et al., Reference Kowalczyk, Gałęska, Czerniawska-Piątkowska, Szul and Hebda2021). Conversely, testosterone is an important hormone for spermatogenesis and is responsible for secondary sexual traits and libido (Kowalczyk et al., Reference Kowalczyk, Gałęska, Czerniawska-Piątkowska, Szul and Hebda2021). Also, testosterone is an important hormone in male fertility as its concentration in seminal plasma shows a positive correlation with sperm concentration, sperm motility, and other sperm traits (Souza et al., Reference Souza, Andrade, Celeghini, Negrão and Arruda2011).
In the present study, testosterone and LH serum concentration were not different among sires. Nonetheless, our results showed a higher testosterone serum concentration in Nellore bulls than found in a previous studies. Santos et al. (Reference Santos, Torres, Ruas, Guimarães and Silva Filho2004) observed an average of 4.04 ng/ml for the same bull breed. A similar result was shown in a study conducted by Amorim et al. (Reference Amorim, Kawamoto, Torres, Guimarães, Silva Filho, Oliveira, Carvalho and Fonseca2015) that found a serum testosterone concentration with an average of 5.41 ng/ml. In another study, Chacur et al. (Reference Chacur, Mizusaki, Filho, Oba and Ramos2013) evaluated seasonal effects on semen and testosterone concentration in Zebu (Nellore; Bos indicus) and taurine (Simental; Bos taurus) bulls. They showed that there was a difference between breeds when considering testosterone serum concentrations, demonstrating a higher adaptability of Nellore sires to the field conditions. Their results showed that Nellore had 2.34–4.3 ng/ml of serum testosterone and Simental had 5.84–9.01 ng/ml of serum testosterone. Interestingly, in the present study the mean testosterone serum concentration for Bos taurus (Angus bulls) was lower than for the data found by Chacur et al. (Reference Chacur, Mizusaki, Filho, Oba and Ramos2013). Despite differences between testosterone serum concentration in the present study and in previous literature, we showed that there was no difference for other sperm traits, except for sperm kinetics as discussed above.
One possible reason for the results present in this study is that the bulls were not grouped according to fertility or other traits, as the goal was to evaluate whether there were differences in the hormone and sperm characteristics of Wagyu bulls compared with the same characteristics of subspecies Bos indicus and Bos taurus sires. Moreover, the age of the bull could be an aspect that influenced the results as the mean ages of the Angus, Nellore, and Wagyu sires were 44.8, 39.0 and 12 months, respectively.
Bos indicus cattle are more resistant to higher temperature and humidity than Bos taurus. Indeed, as a consequence, its productivity and precocity are diminished. In general, Nellore bulls have slower testicular development and reach puberty later than Bos taurus (Nogueira, Reference Nogueira2004). According to Reis et al. (Reference Reis, Ramos, Camargos and Oba2016), Nellore puberty lasts from 18 to 26 months of age. In a study conducted by Brito et al. (Reference Brito, Silva, Unanian, Dode, Barbosa and Kastelic2004), age at puberty ranged from 17.6 to 22.4 months in Zebu (B. indicus) cattle. The seminal traits at this age were slightly poor, as mentioned above. Only after 26 months of age, with an increasing level of LH and testosterone, Nellore bulls reached sexual maturation. At this time, bulls exhibit an increased seminal quality (Reis et al., Reference Reis, Ramos, Camargos and Oba2016). Sexual maturity in B. taurus is achieved earlier than in B. indicus (Nogueira, Reference Nogueira2004; Brito, Reference Brito and Hopper2021). Fields et al. (Reference Fields, Hentges and Cornelisse1982) found an average age at puberty of 15.7 months for the Angus breed. In agreement with these results, Brito et al. (Reference Brito, Barth, Wilde and Kastelic2012) reported that Angus bulls reached puberty at 12.6 months of age and sexual maturity at 15.4 months old. Also, Tatman and co-workers (2022) observed that Angus bulls reached puberty at 12 months old. Considering Wagyu sires, to the best of our knowledge, there are scarce data available from Wagyu sire puberty. Tatman et al. (Reference Tatman, Chase, Wilson, Neuendorff, Lewis, Brown and Randel2022) found that Wagyu cattle were the youngest breed at puberty (10.7 months old). Casas et al. (Reference Casas, Lunstra, Cundiff and Ford2007) showed that the age at puberty was 10.06 months for Wagyu sires.
It is possible that we were not able to show significant differences between sires from different breeds considering sperm traits and sexual hormone levels because Nellore and Angus were older and probably more sexually mature than Wagyu sires. It is noteworthy that the presented data were based on a limited number of bulls. Perhaps, increasing the number of bulls and selecting animals with similar ages might allow additional significant relationships to be revealed. Once the present study was not able to find differences among Wagyu, Nellore, and Angus sires and some studies had pointed out that even Wagyu sires had a smaller scrotal circumference, had a higher libido and higher sperm production, therefore more studies are needed to determine what makes Wagyu different from other sires considering reproductive traits.
In conclusion, the results from the present study indicated that Wagyu sires had lower total motility when compared with Nellore and Angus bulls. This difference could be an effect of the high percentage of sperm with no motility observed in Wagyu semen. However, even failing the breeding soundness exams, because scrotal circumferences were too small, the Wagyu breed did not differ from those breeds when considering plasma and acrosome membranes integrity, mitochondrial potential, chromatin resistance, sperm lipid peroxidation or hormone (LH and testosterone) concentrations.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0967199423000278
Data availability
The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.
Acknowledgements
We would like to thank Seleon Biotechnology (Itatinga, SP, Brazil) and Agropecuária Zanella (Paim Filho, RS, Brazil) for providing bull semen straws for our research.
Competing interests
The authors declare that they have no competing interests.
