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Seasonal variation in semen quality in Bos indicus and Bos taurus bulls raised under tropical conditions
Theriogenology 66 (2006) 822–828 www.journals.elsevierhealth.com/periodicals/the
Seasonal variation in semen quality in Bos indicus and Bos taurus bulls raised under tropical conditions M. Nichi a,b,*, P.E.J. Bols a, R.M. Zu¨ge c, V.H. Barnabe b, I.G.F. Goovaerts a, R.C. Barnabe b, C.N.M. Cortada b a
University of Antwerp, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Laboratory of Veterinary Physiology, Universiteitsplein 1, Gebouw U, B-2610 Wilrijk, Belgium b University of Sa˜o Paulo (USP), Faculty of Veterinary Medicine and Animal Science (FMVZ), Department of Animal Reproduction (VRA), Av. Prof. Orlando Marques de Paiva, n887, CEP 05508-900, Sa˜o Paulo, SP, Brazil c Parana´ Institute of Technology (Tecpar), Av. Algacyr Munhoz Mader, n83775, CEP 81350-010, Curitiba, Parana´, Brazil Received 15 August 2005; accepted 23 January 2006
Abstract In the present study, we tested the hypothesis that Bos taurus taurus bulls have greater reactive oxygen species (ROS) and lower activity of antioxidant enzymes in their semen than Bos taurus indicus bulls. Sixteen Simmental bulls (B. t. taurus) and 11 Nelore bulls (B. t. indicus) were managed extensively in a tropical environment. Semen was collected twice annually (summer and winter) for 2 consecutive years. Simmental bulls had significantly higher percentages of major sperm defects during the summer than the winter (20.3 3.1% versus 12.2 2.4%, respectively; mean S.E.M.). There was an interaction of breed and season for minor sperm defects (P = 0.037; highest in Nelore bulls in the summer) and an effect of season on total defects (P = 0.066; higher in summer). To evaluate oxidative damage, malondialdehyde (lipid-peroxidation metabolite) concentrations were indirectly measured by semen concentrations of thiobarbituric acid reactive substances (TBARS); these were higher in summer than in winter (728.1 79.3 ng/mL versus 423.8 72.6 ng/mL, respectively; P = 0.01). Glutathione peroxidase/redutase (GPx) activity in semen was higher in Simmental versus Nelore bulls (741.6 62.1 versus 510.2 62.8; P < 0.01). However, superoxide dismutase (SOD), another antioxidant enzyme, was not significantly affected by breed or season. There were correlations between TBARS and sperm primary defects during the summer for both Simmental and Nelore bulls (r = 0.59, P = 0.021 and r = 0.40, P = 0.034, respectively), and between SOD and primary defects during summer for Simmental bulls only (r = 0.51, P = 0.041). In conclusion, there was a higher level of lipid peroxidation (ROS) in semen of Simmental versus Nelore bulls; apparently the higher GPx activity in Simmental bulls was insufficient to avoid damage that occurred concurrent with increased ROS production during the summer. # 2006 Elsevier Inc. All rights reserved. Keywords: Reactive oxygen species; Semen antioxidants; Heat stress; Bulls
1. Introduction Bull fertility is extremely important for efficient production of beef cattle bred by natural service under
* Corresponding author. Tel.: +32 3 820 23 95; fax: +32 3 820 24 33. E-mail address: [email protected] (M. Nichi). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.01.056
extensive production systems. Under tropical conditions, European bulls (Bos taurus taurus) have lower fertility than Zebu bulls (Bos taurus indicus) [1–3]. To function normally, bovine testicular temperature must not exceed 33–34.5 8C [4,5]. Under moderate ambient temperatures, the bovine testes are on the brink of hypoxia [5]; increased ambient temperatures increase testicular temperatures, metabolic rates, and oxygen
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requirements. However, in the absence of increased blood flow, the testicular parenchyma becomes hypoxic [6]. Hypoxia probably increases production of reactive oxygen species (ROS) through the ischemia-reperfusion mechanism [7,8]. Thus, one hypothesis to explain the decreased fertility of European bulls, compared to Zebu bulls, when raised under tropical conditions, would be a higher index of testicular oxidative stress. Oxidative stress is caused by reactive oxygen species (ROS) that may cause structural damage to biomolecules, DNA, lipids, carbohydrates and proteins, as well as other cellular components. In humans, ROS were identified as a cause of infertility [9–11]. The ROS are free radicals, i.e. atoms or molecules with one or more unpaired electrons [12]. Among ROS, the most important are the hydroxyl radical (OHS), the superoxide anion (O2 ), hydrogen peroxide (H2O2), and nitric oxide (NO). Both O2 and H2O2 are primary ROS; the latter is generated through enzymatic or nonenzymatic dismutation of the former [13]. Several antioxidants counteract the effects of ROS. Human semen antioxidant enzymes (superoxide dismutase – SOD, glutathione peroxidase/redutase – GPx and catalase), as well as other antioxidants, including albumin, glutathione, pyruvate, taurine, hypotaurine, and vitamins E and C [14], have been indentified. Furthermore, spermatozoa have only a limited capacity for oxidative stress resistance; they are highly dependent on seminal plasma for antioxidant protection. The susceptibility of human spermatozoa to oxidative stress may be due to their relative lack of cytoplasm, limiting the available quantity of enzymatic antioxidant [15]. Another important factor is the abundance of polyunsaturated fatty acids (PUFA) in sperm plasma membranes, providing the required fluidity to engage in the membrane fusion events associated with fertilization [16,17]. Unfortunately, the presence of double bonds in these molecules makes them vulnerable to free radical attacks and the initiation of a lipid-peroxidation cascade [15]. Because oxidative stress corresponds to an imbalance between the ROS production and scavenger systems, oxidative stress can be assessed through the direct quantification of ROS, antioxidants, and by the measurement of oxidative stress end products. Measurement of tiobarbituric acid reactive substances (TBARS) is the most frequently used method. The rationale behind TBARS measurements is to indirectly quantify oxidative stress through the measurement of malondialdehyde (MDA) concentration, an end product of lipid peroxidation. The objective of the present study was to determine production of ROS and activity of antioxidant enzymes
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in semen of Bos taurus and Bos indicus bulls under tropical conditions. We tested the hypothesis that B. t. taurus bulls have greater ROS and lower activity of antioxidant enzymes in their semen than B. t. indicus bulls. 2. Materials and methods 2.1. Bulls Sixteen Simmental (B. t. taurus) and 11 Nelore (B. t. indicus) bulls, from 3 to 4 years of age, were used for this study. These bulls were maintained under an extensive management system, on pasture composed of brachiaria (Brachiaria sp.), on a farm near Dourados, Mato Grosso do Sul, Brazil (approximately 228 south, 548 west and 458 m above sea level). This region is characterized by tropical, hot and humid weather in the summer, and a moderate winter climate, with minimal, maximal and average temperatures averaging 10, 35 and 24 8C, respectively, and an average annual rainfall of approximately 1500 mm (with the majority of rainfall during summer). 2.2. Semen collection For each bull, semen was collected by electroejaculation on four occassions, in August 2000 (winter), February 2001 (summer), July 2001 (winter) and February 2002 (summer). Semen was collected in a prewarmed, graded, conical plastic tube, protected (by a polystyrene cover) from light, cold shock, and rapid temperature changes. 2.3. Experimental design The experiment was performed as a 2 2 2 factorial design; the factors were collection year (first versus second), breed (Simmental versus Nelore), and season (winter versus summer). 2.4. Semen evaluation The proportion of progressively motile spermatozoa was assessed immediately following semen collection. A small drop of semen was placed on a pre-warmed slide, covered with a cover slip, and examined with a bright-field microscope (400 magnification) with a heated stage. The proportion of spermatozoa that were progressively motile was estimated in increments of 5%. Immediately after semen collection, an aliquot was fixed in buffered isotonic formal-saline (1:200) for
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subsequent analysis of sperm morphology. Under phase-contrast microscopy (magnification, 1250), at least 200 spermatozoa/ejaculate were examined in random fields [18]. Sperm defects were categorized as either minor or major and their sum as total defects [19]. For correlation analyses, sperm abnormatilities were classified as primary or secondary defects [20], in order to establish whether the effects of reactive oxygen species on the sperm cells occurred during spermatogenesis (primary) or sperm maturation (secondary).
as the MDA extinction coefficient [23]. The lipidperoxidation index was described as nanograms of TBARS/mL of semen.
2.5. ROS and antioxidant activity
2.5.2.1. Catalase (E.C.1.11.1.6). Catalase activity was assessed through measurement of hydrogen peroxide consumption. The reaction solution contained 10 mL of seminal plasma added to 90 mL of Tris(hydroxymethyl)amino-methane/EDTA buffer solution (50 and 250 mM, respectively) and 900 mL of H2O2 (9.0 mM). The reaction was allowed to take place at pH 8.0, 30 8C, for 8 min [24], and then the enzymatic activity was measured using a spectrophotometer (wavelength, 230 nm). The absorbance was measured every 5 s, and the curve of H2O2 consumption was compared to a blank. Calculations used 0.071 M 1 cm 1 as the extinction coefficient for hydrogen peroxide (H2O2) [24].
Immediately after collection, aliquots of semen (approximately 2 mL) were placed in cryotubes and frozen in liquid nitrogen (without any cryoprotectant) for all subsequent biochemical analyses. Frozen samples were thawed (by placing the tube into water at 37 8C for approximately 15 s), immediately refrozen, and then thawed again. For most cells, this procedure is sufficient to rupture cellular membranes and release intracellular enzymatic antioxidant contents [21]. Thereafter, the samples were centrifuged (10,000 g, 15 min, 4 8C) to obtain the supernatant containing the enzymes (free of remaining intact spermatozoa and cellular debris). All chemical reagents, unless otherwise noted, were obtained from Sigma–Aldrich (St. Louis, MO, USA). 2.5.1. Thiobarbituric acid reactive substances – TBARS These measurements were made in accordance with a protocol first described by Ohkawa et al. [22]. The method is based on the reaction of two molecules of thiobarbituric acid with one molecule of malondialdehyde, at high temperatures and low pH, resulting in a pink chromogen that can be quantified with a spectrophotometer. To precipitate proteins, 500 mL of seminal plasma and 1000 mL of a 10% solution (v:v) of trichloroacetic acid (TCA 10%) were mixed and centrifuged (18,000 g for 15 min at 15 8C). After centrifugation, 500 mL of the supernatant and 500 mL of 1% (v:v) thiobarbituric acid (TBA, 1%), in 0.05N sodium hydroxide in glass tubes were placed into a boiling water bath (100 8C) for 10 min, and subsequently cooled in an ice bath (0 8C) 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. Results were compared to a standard curve previously prepared with a standard solution of malondiadehyde. The TBARS concentration was determined using the value of 1.56 105 M 1 cm 1
2.5.2. Enzymatic antioxidant activity Enzymatic antioxidant activity was evaluated by measuring the activity of catalase, superoxide dismutase, and glutathione peroxidase enzymes. All determinations were performed by spectrophotometric analysis.
2.5.2.2. Superoxide dismutase (SOD; E.C.1.15.1.1). The SOD activity was measured indirectly, through the reduction of cytochrome C by the superoxide anion (O2 ). The xanthine–xanthine oxidase system continuously generated O2 (that reduced cytochrome C). The SOD present in the sample competed with the cytochrome C by converting the superoxide free radical to H2O2 and O2 , thereby slowing the rate of cytochrome C reduction [25]. During the assay, absorbance was determined every 5 min in a spectrophotometer fitted with a temperature regulator maintained at 25 8C (absorbance was measured every 5 s). The assay mixture consisted of 10 mL of seminal plasma, 835 mL of a solution containing cytochrome C (1 mM) and xanthine (50 mM), and 155 mL of xanthine oxidase diluted in sodium phosphate/EDTA buffer (50 and 100 mM, respectively, pH 7.8). The concentration of xanthine oxidase was calculated to generate the optimum amount of O2 with a consequent reduction of cytochrome C that was calculated as the rate of cytochrome C reduction of 0.025 units of absorbance/ min (at 550 nm of wavelength); the basis of this calculation is that 1 unit of total SOD activity corresponded to 50% of this value. Therefore, SOD activity in the sample decreased the rate of cytochrome reduction when compared to the blank.
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2 . 5 . 2 . 3 . G l u t a t h i o n e p e ro x i d a s e ( G S H - P X ) (E.C.1.11.1.9). The enzymatic activity of GSPH-Px was determined through a modification of the method of Beutler [26], performed by Homem de Bittencourt [27]. This method is based on measuring the consumption of NADPH; the reaction between a hydroperoxide and reduced glutathione (GSH) that is catalyzed by the GSPH-Px together with the enzyme glutathione reductase (GSSGr), is induced. This reaction causes the conversion of glutathione disulfide (GSSH – glutathione oxidized) to GSH, which in turn consumes NADPH (measured with a spectrophotometer). The assay mixture consisted of NADPH (0.12 mM, 1 mL), GSH (1 mM, 100 mL), GSSGr (0.25 U/mL, 20 mL), and sodium azide (0.25 mM, 20 mL). A volume of 100 mL of seminal plasma was used. The spectrophotometer cell was brought up to a volume of 1.9 mL with phosphate buffer 143 mM, EDTA 6.3 mM, (pH 7.5), that was also used to dissolve the NADPH. The GSH was dissolved in 5% metaphosphoric acid. Sodium azide was used to inhibit the action of catalase. This reaction was initiated with the addition of 1.2 mM of tert-butyl hydro peroxide (TBHP, 100 mL), and the consumption of NADPH was detected at a wavelength of 340 nm, for 10 min at 37 8C (measurements performed every 5 s). The results of GSH-Px were expressed as units of GSH-Px/mL of semen, and calculations used 6.22 mM 1 cm 1 as the extinction coefficient of NADPH [26]. 2.6. Statistical analysis All data were evaluated using SAS System for Windows (SAS Institute Inc., Cary, NC, USA). The effects of year (first versus second), breeds (Simmental versus Nelore) and seasons (winter versus summer), as well as the interactions between those factors, were determined by PROC GLM. Due to the absence of any interactions involving collection year, the analysis was simplified to a 2 2 factorial (with breed and season). Differences between treatments were analyzed using parametric (GLM procedure for each factor separately or LSD when combining factors) and non-parametric (Wilcoxon) tests, according to the residue normality (Gaussian distribution) and variance homogeneity. Percentage of major and total sperm defects and glutathione levels were log transformed. Sperm motility and superoxide levels did not have randomly distributed residuals and no transformation was effective; therefore, they were analyzed with non-parametric procedures. A probability value of P < 0.05 was considered significant. Results were reported as untransformed
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means S.E.M. Pearson and Spearman correlations were used to calculate the relationship between sperm characteristics and oxidative stress-related measurements for parametric and non-parametric variables, respectively, and for each of the four groups (breed/ season) separately. 3. Results There was no significant interaction between year of collection and the other factors for any of the variables studied. Therefore, the analysis was simplified to a 2 2 factorial, with breed and season as main effects. 3.1. Sperm motility and morphology There was no significant main effect or interaction for sperm motility (Table 1). For major defects, there was an effect of breed (P = 0.0275) and a tendency (P = 0.0754) for a breed by season interaction; Simmental bulls had higher percentages of major sperm defects during the summer than the winter (20.3 3.1% versus 12.2 2.4%, respectively; mean S.E.M.; Fig. 1), and in the summer, they had higher percentages of major defects than the Nelore bulls (20.3 3.1% versus 10.1 2.9%). There was a breed by season interaction for minor defects (P = 0.0374); during the summer, Nelore bulls had more minor sperm defects than Simmental bulls (17.3 3.2% versus 10.4 1.4%). Total defects tended (P = 0.066) to be higher in summer than winter. 3.2. ROS and antioxidant activity There was a higher concentration of TBARS during the summer versus winter (728.1 79.3 and Table 1 Probability values for main effects of breed (Nelore vs. Simmental) and season (winter vs. summer) and their interaction, on sperm motility and morphology and oxidative-stress-related measurements in semen samples of extensively raised bulls collected in consecutive winters and summers
Motility Major defects Minor defects Total defects TBARS SOD GPx
Breed
Season
Breed season
0.3760 0.0275 0.5484 0.4297 0.2560 0.7543 0.0097
0.5831 0.2618 0.5185 0.0664 0.0100 0.2283 0.2958
0.1720 0.0754 0.0374 0.9844 0.1569 0.9537 0.9543
TBARS, thiobarbituric acid rective substances (ng/mL); SOD, superoxide dismutase (U/mL); GPx, glutathione peroxidase (U/mL).
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Fig. 1. Effect of breed and season on the mean (S.E.M.) percentage of major, minor and total sperm (top of column) defects, in semen collected from extensively raised bulls.
Fig. 2. Effect of breeds (Nelore vs. Simmental) and seasons (winter vs. summer) on the seminal concentration of tiobarbituric acid reactive substances (TBARS), and on the seminal activity of the enzymatic antioxidant glutathione peroxidase (GPx) in semen samples collected from extensively raised bulls. abDifferent superscripts indicate differences between seasons in thiobarbituric acid reactive substances (TBARS) concentrations (P = 0.01). ABDifferent superscripts indicate differences between breeds in glutathione peroxidase/redutase (GPx) activity (P = 0.0097).
423.8 72.6 ng/mL, respectively; P = 0.01; Fig. 2). There were positive correlations between TBARS concentration and primary defects during the summer for both Simmental and Nelore bulls (r = 0.59, P = 0.021 and r = 0.40, P = 0.034). Catalase activity was not detected in any of the samples. There was no significant main effect or interaction for SOD activity, but it was negatively correlated with primary defects in Simmental bulls during the summer (r = 0.51, P = 0.041). For GPx activity, there was only an effect of breed; Simmental bulls had greater activity than Nelore bulls (741.5 62.1 versus 510.3 62.8, P = 0.01). 4. Discussion There was no significant effect of breed on ROS in semen; the only effect of breed on antioxidant
enzyme activity was that GPx was significantly higher in semen from Simmental versus Nelore bulls. Therefore, the hypothesis that B. t. taurus bulls have greater ROS and lower activity of antioxidant enzymes in their semen than B. t. indicus bulls was not supported. Literature regarding oxidative stress characteristics in bull semen is very limited; the objective of most experiments was to evaluate the effects of prooxidants or antioxidants on the concentrations of TBARS, or on semen quality. That semen concentrations of TBARS were higher in summer than winter suggested there was more lipid peroxidation in bull semen during the summer months, presumably due to increased production in the testis. Furthermore, the increased incidence of major sperm defects and the significant correlations between TBARS and primary defects in the summer (for both breeds) suggested an association between lipid peroxidation and sperm quality. Similarly, there was a high correlation (r = 0.64, P < 0.05) between TBARS concentrations and the percentage of sperm head defects in human semen [28]. The SOD activity in the semen of Nelore and Simmental bulls (43.2 4.8 and 46.0 5.1 U/mL, respectively) were similar to that previously reported in semen of poultry [29] and human [30]. In the latter, there were higher activities of SOD in infertile nonazoospermic men (compared to fertile and infertile azoospermic men). The authors suggested that in one or more tissues with SOD expression (testicle, epididymis, prostate, and seminal vesicle), there was higher expression of SOD in response to impaired spermatogenesis. The negative correlation between SOD and primary sperm defects in Simmental bulls during summer (r = 0.51, P = 0.041) suggested a protective effect of SOD activity on semen quality. The higher concentrations of TBARS found in the Simmental bulls during the summer were apparently related to higher levels of ROS, and not due to a lower antioxidant capacity. In fact, the higher GPx activity in Simmental versus Nelore bulls could have been in response to the higher levels of ROS in the B. taurus bulls, particularly in the summer. However, these higher levels of GPx were apparently unable to counteract the oxidative stress, resulting in higher TBARS concentrations. Perhaps B. indicus bulls have higher resistance to heat-induced lipid peroxidation, resulting in lower ROS production and smaller increases in the number of defective sperm in the summer. The routine semen analyses in the present experiment were similar to previous results comparing B.
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taurus and B. indicus bulls experiencing heat stress [2,31,32]. The increase in total number of defective sperm during the summer was consistent with the wellknown effects of increased temperature on semen quality. This effect is extremely important, especially in tropical regions, were breeding takes place during the summer. The greater increase in major defects in the summer in Simmental versus Nelore bulls was consistent with previous reports. Kumi-Diaka et al. [2], studying bulls raised in the north of Nigeria, and Chaco´n et al. [31], studying bulls from Costa Rica, both reported higher percentages of sperm head and intermediary piece defects during the summer in B. taurus bulls compared to B. indicus bulls. Fructose concentrations in seminal plasma could have affected concentrations of TBARS in semen. This carbohydrate, an important source of energy for sperm cells, can react with thiobarbituric acid, falsely increasing TBARS concentrations [33]. However, fructose concentrations in bull semen were significantly lower during the hot season [1,34]. Therefore, the increased TBARS concentrations during the summer were apparently not due to higher fructose concentrations. In the present experiment, no catalase activity was detected in any sample, consistent with a previous failure to detect this enzyme in bovine sperm after percoll gradient selection [35]. Catalase activity in sperm remains a matter of debate, presumably owing to varying degress of sample purity. The presence of catalase in human semen samples may have been due to contamination by neutrophils (that possess a high quantity of this enzyme) [16], consistent with the variable results in human semen samples when the presence of neutrophils was not considered. It was reported that human sperm are especially vulnerable to lipid peroxidation (caused by H2O2), due to low endogenous concentrations of catalase and GPx [36]. In conclusion, despite higher GPx activity in Simmental versus Nelore bulls in summer, lipid peroxidation was greater in Simmental bulls, consistent with their higher percentages of defective sperm. Acknowledgements The authors thank the Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and the Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) for financial support. The authors also thank M. Julian (JustMe Editing, Storrs, CT) for editing and critical reading of the manuscript.
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Seasonal variation in semen quality in Bos indicus and Bos taurus bulls raised under tropical conditions M. Nichi a,b,*, P.E.J. Bols a, R.M. Zu¨ge c, V.H. Barnabe b, I.G.F. Goovaerts a, R.C. Barnabe b, C.N.M. Cortada b a
University of Antwerp, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Laboratory of Veterinary Physiology, Universiteitsplein 1, Gebouw U, B-2610 Wilrijk, Belgium b University of Sa˜o Paulo (USP), Faculty of Veterinary Medicine and Animal Science (FMVZ), Department of Animal Reproduction (VRA), Av. Prof. Orlando Marques de Paiva, n887, CEP 05508-900, Sa˜o Paulo, SP, Brazil c Parana´ Institute of Technology (Tecpar), Av. Algacyr Munhoz Mader, n83775, CEP 81350-010, Curitiba, Parana´, Brazil Received 15 August 2005; accepted 23 January 2006
Abstract In the present study, we tested the hypothesis that Bos taurus taurus bulls have greater reactive oxygen species (ROS) and lower activity of antioxidant enzymes in their semen than Bos taurus indicus bulls. Sixteen Simmental bulls (B. t. taurus) and 11 Nelore bulls (B. t. indicus) were managed extensively in a tropical environment. Semen was collected twice annually (summer and winter) for 2 consecutive years. Simmental bulls had significantly higher percentages of major sperm defects during the summer than the winter (20.3 3.1% versus 12.2 2.4%, respectively; mean S.E.M.). There was an interaction of breed and season for minor sperm defects (P = 0.037; highest in Nelore bulls in the summer) and an effect of season on total defects (P = 0.066; higher in summer). To evaluate oxidative damage, malondialdehyde (lipid-peroxidation metabolite) concentrations were indirectly measured by semen concentrations of thiobarbituric acid reactive substances (TBARS); these were higher in summer than in winter (728.1 79.3 ng/mL versus 423.8 72.6 ng/mL, respectively; P = 0.01). Glutathione peroxidase/redutase (GPx) activity in semen was higher in Simmental versus Nelore bulls (741.6 62.1 versus 510.2 62.8; P < 0.01). However, superoxide dismutase (SOD), another antioxidant enzyme, was not significantly affected by breed or season. There were correlations between TBARS and sperm primary defects during the summer for both Simmental and Nelore bulls (r = 0.59, P = 0.021 and r = 0.40, P = 0.034, respectively), and between SOD and primary defects during summer for Simmental bulls only (r = 0.51, P = 0.041). In conclusion, there was a higher level of lipid peroxidation (ROS) in semen of Simmental versus Nelore bulls; apparently the higher GPx activity in Simmental bulls was insufficient to avoid damage that occurred concurrent with increased ROS production during the summer. # 2006 Elsevier Inc. All rights reserved. Keywords: Reactive oxygen species; Semen antioxidants; Heat stress; Bulls
1. Introduction Bull fertility is extremely important for efficient production of beef cattle bred by natural service under
* Corresponding author. Tel.: +32 3 820 23 95; fax: +32 3 820 24 33. E-mail address: [email protected] (M. Nichi). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.01.056
extensive production systems. Under tropical conditions, European bulls (Bos taurus taurus) have lower fertility than Zebu bulls (Bos taurus indicus) [1–3]. To function normally, bovine testicular temperature must not exceed 33–34.5 8C [4,5]. Under moderate ambient temperatures, the bovine testes are on the brink of hypoxia [5]; increased ambient temperatures increase testicular temperatures, metabolic rates, and oxygen
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requirements. However, in the absence of increased blood flow, the testicular parenchyma becomes hypoxic [6]. Hypoxia probably increases production of reactive oxygen species (ROS) through the ischemia-reperfusion mechanism [7,8]. Thus, one hypothesis to explain the decreased fertility of European bulls, compared to Zebu bulls, when raised under tropical conditions, would be a higher index of testicular oxidative stress. Oxidative stress is caused by reactive oxygen species (ROS) that may cause structural damage to biomolecules, DNA, lipids, carbohydrates and proteins, as well as other cellular components. In humans, ROS were identified as a cause of infertility [9–11]. The ROS are free radicals, i.e. atoms or molecules with one or more unpaired electrons [12]. Among ROS, the most important are the hydroxyl radical (OHS), the superoxide anion (O2 ), hydrogen peroxide (H2O2), and nitric oxide (NO). Both O2 and H2O2 are primary ROS; the latter is generated through enzymatic or nonenzymatic dismutation of the former [13]. Several antioxidants counteract the effects of ROS. Human semen antioxidant enzymes (superoxide dismutase – SOD, glutathione peroxidase/redutase – GPx and catalase), as well as other antioxidants, including albumin, glutathione, pyruvate, taurine, hypotaurine, and vitamins E and C [14], have been indentified. Furthermore, spermatozoa have only a limited capacity for oxidative stress resistance; they are highly dependent on seminal plasma for antioxidant protection. The susceptibility of human spermatozoa to oxidative stress may be due to their relative lack of cytoplasm, limiting the available quantity of enzymatic antioxidant [15]. Another important factor is the abundance of polyunsaturated fatty acids (PUFA) in sperm plasma membranes, providing the required fluidity to engage in the membrane fusion events associated with fertilization [16,17]. Unfortunately, the presence of double bonds in these molecules makes them vulnerable to free radical attacks and the initiation of a lipid-peroxidation cascade [15]. Because oxidative stress corresponds to an imbalance between the ROS production and scavenger systems, oxidative stress can be assessed through the direct quantification of ROS, antioxidants, and by the measurement of oxidative stress end products. Measurement of tiobarbituric acid reactive substances (TBARS) is the most frequently used method. The rationale behind TBARS measurements is to indirectly quantify oxidative stress through the measurement of malondialdehyde (MDA) concentration, an end product of lipid peroxidation. The objective of the present study was to determine production of ROS and activity of antioxidant enzymes
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in semen of Bos taurus and Bos indicus bulls under tropical conditions. We tested the hypothesis that B. t. taurus bulls have greater ROS and lower activity of antioxidant enzymes in their semen than B. t. indicus bulls. 2. Materials and methods 2.1. Bulls Sixteen Simmental (B. t. taurus) and 11 Nelore (B. t. indicus) bulls, from 3 to 4 years of age, were used for this study. These bulls were maintained under an extensive management system, on pasture composed of brachiaria (Brachiaria sp.), on a farm near Dourados, Mato Grosso do Sul, Brazil (approximately 228 south, 548 west and 458 m above sea level). This region is characterized by tropical, hot and humid weather in the summer, and a moderate winter climate, with minimal, maximal and average temperatures averaging 10, 35 and 24 8C, respectively, and an average annual rainfall of approximately 1500 mm (with the majority of rainfall during summer). 2.2. Semen collection For each bull, semen was collected by electroejaculation on four occassions, in August 2000 (winter), February 2001 (summer), July 2001 (winter) and February 2002 (summer). Semen was collected in a prewarmed, graded, conical plastic tube, protected (by a polystyrene cover) from light, cold shock, and rapid temperature changes. 2.3. Experimental design The experiment was performed as a 2 2 2 factorial design; the factors were collection year (first versus second), breed (Simmental versus Nelore), and season (winter versus summer). 2.4. Semen evaluation The proportion of progressively motile spermatozoa was assessed immediately following semen collection. A small drop of semen was placed on a pre-warmed slide, covered with a cover slip, and examined with a bright-field microscope (400 magnification) with a heated stage. The proportion of spermatozoa that were progressively motile was estimated in increments of 5%. Immediately after semen collection, an aliquot was fixed in buffered isotonic formal-saline (1:200) for
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subsequent analysis of sperm morphology. Under phase-contrast microscopy (magnification, 1250), at least 200 spermatozoa/ejaculate were examined in random fields [18]. Sperm defects were categorized as either minor or major and their sum as total defects [19]. For correlation analyses, sperm abnormatilities were classified as primary or secondary defects [20], in order to establish whether the effects of reactive oxygen species on the sperm cells occurred during spermatogenesis (primary) or sperm maturation (secondary).
as the MDA extinction coefficient [23]. The lipidperoxidation index was described as nanograms of TBARS/mL of semen.
2.5. ROS and antioxidant activity
2.5.2.1. Catalase (E.C.1.11.1.6). Catalase activity was assessed through measurement of hydrogen peroxide consumption. The reaction solution contained 10 mL of seminal plasma added to 90 mL of Tris(hydroxymethyl)amino-methane/EDTA buffer solution (50 and 250 mM, respectively) and 900 mL of H2O2 (9.0 mM). The reaction was allowed to take place at pH 8.0, 30 8C, for 8 min [24], and then the enzymatic activity was measured using a spectrophotometer (wavelength, 230 nm). The absorbance was measured every 5 s, and the curve of H2O2 consumption was compared to a blank. Calculations used 0.071 M 1 cm 1 as the extinction coefficient for hydrogen peroxide (H2O2) [24].
Immediately after collection, aliquots of semen (approximately 2 mL) were placed in cryotubes and frozen in liquid nitrogen (without any cryoprotectant) for all subsequent biochemical analyses. Frozen samples were thawed (by placing the tube into water at 37 8C for approximately 15 s), immediately refrozen, and then thawed again. For most cells, this procedure is sufficient to rupture cellular membranes and release intracellular enzymatic antioxidant contents [21]. Thereafter, the samples were centrifuged (10,000 g, 15 min, 4 8C) to obtain the supernatant containing the enzymes (free of remaining intact spermatozoa and cellular debris). All chemical reagents, unless otherwise noted, were obtained from Sigma–Aldrich (St. Louis, MO, USA). 2.5.1. Thiobarbituric acid reactive substances – TBARS These measurements were made in accordance with a protocol first described by Ohkawa et al. [22]. The method is based on the reaction of two molecules of thiobarbituric acid with one molecule of malondialdehyde, at high temperatures and low pH, resulting in a pink chromogen that can be quantified with a spectrophotometer. To precipitate proteins, 500 mL of seminal plasma and 1000 mL of a 10% solution (v:v) of trichloroacetic acid (TCA 10%) were mixed and centrifuged (18,000 g for 15 min at 15 8C). After centrifugation, 500 mL of the supernatant and 500 mL of 1% (v:v) thiobarbituric acid (TBA, 1%), in 0.05N sodium hydroxide in glass tubes were placed into a boiling water bath (100 8C) for 10 min, and subsequently cooled in an ice bath (0 8C) 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. Results were compared to a standard curve previously prepared with a standard solution of malondiadehyde. The TBARS concentration was determined using the value of 1.56 105 M 1 cm 1
2.5.2. Enzymatic antioxidant activity Enzymatic antioxidant activity was evaluated by measuring the activity of catalase, superoxide dismutase, and glutathione peroxidase enzymes. All determinations were performed by spectrophotometric analysis.
2.5.2.2. Superoxide dismutase (SOD; E.C.1.15.1.1). The SOD activity was measured indirectly, through the reduction of cytochrome C by the superoxide anion (O2 ). The xanthine–xanthine oxidase system continuously generated O2 (that reduced cytochrome C). The SOD present in the sample competed with the cytochrome C by converting the superoxide free radical to H2O2 and O2 , thereby slowing the rate of cytochrome C reduction [25]. During the assay, absorbance was determined every 5 min in a spectrophotometer fitted with a temperature regulator maintained at 25 8C (absorbance was measured every 5 s). The assay mixture consisted of 10 mL of seminal plasma, 835 mL of a solution containing cytochrome C (1 mM) and xanthine (50 mM), and 155 mL of xanthine oxidase diluted in sodium phosphate/EDTA buffer (50 and 100 mM, respectively, pH 7.8). The concentration of xanthine oxidase was calculated to generate the optimum amount of O2 with a consequent reduction of cytochrome C that was calculated as the rate of cytochrome C reduction of 0.025 units of absorbance/ min (at 550 nm of wavelength); the basis of this calculation is that 1 unit of total SOD activity corresponded to 50% of this value. Therefore, SOD activity in the sample decreased the rate of cytochrome reduction when compared to the blank.
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2 . 5 . 2 . 3 . G l u t a t h i o n e p e ro x i d a s e ( G S H - P X ) (E.C.1.11.1.9). The enzymatic activity of GSPH-Px was determined through a modification of the method of Beutler [26], performed by Homem de Bittencourt [27]. This method is based on measuring the consumption of NADPH; the reaction between a hydroperoxide and reduced glutathione (GSH) that is catalyzed by the GSPH-Px together with the enzyme glutathione reductase (GSSGr), is induced. This reaction causes the conversion of glutathione disulfide (GSSH – glutathione oxidized) to GSH, which in turn consumes NADPH (measured with a spectrophotometer). The assay mixture consisted of NADPH (0.12 mM, 1 mL), GSH (1 mM, 100 mL), GSSGr (0.25 U/mL, 20 mL), and sodium azide (0.25 mM, 20 mL). A volume of 100 mL of seminal plasma was used. The spectrophotometer cell was brought up to a volume of 1.9 mL with phosphate buffer 143 mM, EDTA 6.3 mM, (pH 7.5), that was also used to dissolve the NADPH. The GSH was dissolved in 5% metaphosphoric acid. Sodium azide was used to inhibit the action of catalase. This reaction was initiated with the addition of 1.2 mM of tert-butyl hydro peroxide (TBHP, 100 mL), and the consumption of NADPH was detected at a wavelength of 340 nm, for 10 min at 37 8C (measurements performed every 5 s). The results of GSH-Px were expressed as units of GSH-Px/mL of semen, and calculations used 6.22 mM 1 cm 1 as the extinction coefficient of NADPH [26]. 2.6. Statistical analysis All data were evaluated using SAS System for Windows (SAS Institute Inc., Cary, NC, USA). The effects of year (first versus second), breeds (Simmental versus Nelore) and seasons (winter versus summer), as well as the interactions between those factors, were determined by PROC GLM. Due to the absence of any interactions involving collection year, the analysis was simplified to a 2 2 factorial (with breed and season). Differences between treatments were analyzed using parametric (GLM procedure for each factor separately or LSD when combining factors) and non-parametric (Wilcoxon) tests, according to the residue normality (Gaussian distribution) and variance homogeneity. Percentage of major and total sperm defects and glutathione levels were log transformed. Sperm motility and superoxide levels did not have randomly distributed residuals and no transformation was effective; therefore, they were analyzed with non-parametric procedures. A probability value of P < 0.05 was considered significant. Results were reported as untransformed
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means S.E.M. Pearson and Spearman correlations were used to calculate the relationship between sperm characteristics and oxidative stress-related measurements for parametric and non-parametric variables, respectively, and for each of the four groups (breed/ season) separately. 3. Results There was no significant interaction between year of collection and the other factors for any of the variables studied. Therefore, the analysis was simplified to a 2 2 factorial, with breed and season as main effects. 3.1. Sperm motility and morphology There was no significant main effect or interaction for sperm motility (Table 1). For major defects, there was an effect of breed (P = 0.0275) and a tendency (P = 0.0754) for a breed by season interaction; Simmental bulls had higher percentages of major sperm defects during the summer than the winter (20.3 3.1% versus 12.2 2.4%, respectively; mean S.E.M.; Fig. 1), and in the summer, they had higher percentages of major defects than the Nelore bulls (20.3 3.1% versus 10.1 2.9%). There was a breed by season interaction for minor defects (P = 0.0374); during the summer, Nelore bulls had more minor sperm defects than Simmental bulls (17.3 3.2% versus 10.4 1.4%). Total defects tended (P = 0.066) to be higher in summer than winter. 3.2. ROS and antioxidant activity There was a higher concentration of TBARS during the summer versus winter (728.1 79.3 and Table 1 Probability values for main effects of breed (Nelore vs. Simmental) and season (winter vs. summer) and their interaction, on sperm motility and morphology and oxidative-stress-related measurements in semen samples of extensively raised bulls collected in consecutive winters and summers
Motility Major defects Minor defects Total defects TBARS SOD GPx
Breed
Season
Breed season
0.3760 0.0275 0.5484 0.4297 0.2560 0.7543 0.0097
0.5831 0.2618 0.5185 0.0664 0.0100 0.2283 0.2958
0.1720 0.0754 0.0374 0.9844 0.1569 0.9537 0.9543
TBARS, thiobarbituric acid rective substances (ng/mL); SOD, superoxide dismutase (U/mL); GPx, glutathione peroxidase (U/mL).
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Fig. 1. Effect of breed and season on the mean (S.E.M.) percentage of major, minor and total sperm (top of column) defects, in semen collected from extensively raised bulls.
Fig. 2. Effect of breeds (Nelore vs. Simmental) and seasons (winter vs. summer) on the seminal concentration of tiobarbituric acid reactive substances (TBARS), and on the seminal activity of the enzymatic antioxidant glutathione peroxidase (GPx) in semen samples collected from extensively raised bulls. abDifferent superscripts indicate differences between seasons in thiobarbituric acid reactive substances (TBARS) concentrations (P = 0.01). ABDifferent superscripts indicate differences between breeds in glutathione peroxidase/redutase (GPx) activity (P = 0.0097).
423.8 72.6 ng/mL, respectively; P = 0.01; Fig. 2). There were positive correlations between TBARS concentration and primary defects during the summer for both Simmental and Nelore bulls (r = 0.59, P = 0.021 and r = 0.40, P = 0.034). Catalase activity was not detected in any of the samples. There was no significant main effect or interaction for SOD activity, but it was negatively correlated with primary defects in Simmental bulls during the summer (r = 0.51, P = 0.041). For GPx activity, there was only an effect of breed; Simmental bulls had greater activity than Nelore bulls (741.5 62.1 versus 510.3 62.8, P = 0.01). 4. Discussion There was no significant effect of breed on ROS in semen; the only effect of breed on antioxidant
enzyme activity was that GPx was significantly higher in semen from Simmental versus Nelore bulls. Therefore, the hypothesis that B. t. taurus bulls have greater ROS and lower activity of antioxidant enzymes in their semen than B. t. indicus bulls was not supported. Literature regarding oxidative stress characteristics in bull semen is very limited; the objective of most experiments was to evaluate the effects of prooxidants or antioxidants on the concentrations of TBARS, or on semen quality. That semen concentrations of TBARS were higher in summer than winter suggested there was more lipid peroxidation in bull semen during the summer months, presumably due to increased production in the testis. Furthermore, the increased incidence of major sperm defects and the significant correlations between TBARS and primary defects in the summer (for both breeds) suggested an association between lipid peroxidation and sperm quality. Similarly, there was a high correlation (r = 0.64, P < 0.05) between TBARS concentrations and the percentage of sperm head defects in human semen [28]. The SOD activity in the semen of Nelore and Simmental bulls (43.2 4.8 and 46.0 5.1 U/mL, respectively) were similar to that previously reported in semen of poultry [29] and human [30]. In the latter, there were higher activities of SOD in infertile nonazoospermic men (compared to fertile and infertile azoospermic men). The authors suggested that in one or more tissues with SOD expression (testicle, epididymis, prostate, and seminal vesicle), there was higher expression of SOD in response to impaired spermatogenesis. The negative correlation between SOD and primary sperm defects in Simmental bulls during summer (r = 0.51, P = 0.041) suggested a protective effect of SOD activity on semen quality. The higher concentrations of TBARS found in the Simmental bulls during the summer were apparently related to higher levels of ROS, and not due to a lower antioxidant capacity. In fact, the higher GPx activity in Simmental versus Nelore bulls could have been in response to the higher levels of ROS in the B. taurus bulls, particularly in the summer. However, these higher levels of GPx were apparently unable to counteract the oxidative stress, resulting in higher TBARS concentrations. Perhaps B. indicus bulls have higher resistance to heat-induced lipid peroxidation, resulting in lower ROS production and smaller increases in the number of defective sperm in the summer. The routine semen analyses in the present experiment were similar to previous results comparing B.
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taurus and B. indicus bulls experiencing heat stress [2,31,32]. The increase in total number of defective sperm during the summer was consistent with the wellknown effects of increased temperature on semen quality. This effect is extremely important, especially in tropical regions, were breeding takes place during the summer. The greater increase in major defects in the summer in Simmental versus Nelore bulls was consistent with previous reports. Kumi-Diaka et al. [2], studying bulls raised in the north of Nigeria, and Chaco´n et al. [31], studying bulls from Costa Rica, both reported higher percentages of sperm head and intermediary piece defects during the summer in B. taurus bulls compared to B. indicus bulls. Fructose concentrations in seminal plasma could have affected concentrations of TBARS in semen. This carbohydrate, an important source of energy for sperm cells, can react with thiobarbituric acid, falsely increasing TBARS concentrations [33]. However, fructose concentrations in bull semen were significantly lower during the hot season [1,34]. Therefore, the increased TBARS concentrations during the summer were apparently not due to higher fructose concentrations. In the present experiment, no catalase activity was detected in any sample, consistent with a previous failure to detect this enzyme in bovine sperm after percoll gradient selection [35]. Catalase activity in sperm remains a matter of debate, presumably owing to varying degress of sample purity. The presence of catalase in human semen samples may have been due to contamination by neutrophils (that possess a high quantity of this enzyme) [16], consistent with the variable results in human semen samples when the presence of neutrophils was not considered. It was reported that human sperm are especially vulnerable to lipid peroxidation (caused by H2O2), due to low endogenous concentrations of catalase and GPx [36]. In conclusion, despite higher GPx activity in Simmental versus Nelore bulls in summer, lipid peroxidation was greater in Simmental bulls, consistent with their higher percentages of defective sperm. Acknowledgements The authors thank the Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and the Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) for financial support. The authors also thank M. Julian (JustMe Editing, Storrs, CT) for editing and critical reading of the manuscript.
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