Introduction
Binding of sperm to the zona pellucida (ZP) is one of the key events in the process of fertilization. In the ampulla of the oviduct, sperm penetrate the cumulus oophorus and bind to the ZP, which is the last barrier that sperm have to overcome before fertilizing the oocyte (Coutinho da Silva et al., Reference Coutinho da Silva, Seidel, Squires, Graham and Carnevale2012). Sperm–ZP binding dysfunction is associated with male infertility because it produces a failed or sub-optimal sperm fertilizing capacity (Overstreet et al., Reference Overstreet, Yanagimachi, Katz, Hayashi and Hanson1980; Oehninger et al., Reference Oehninger, Mahony, Ozgür, Kolm, Kruger and Franken1997). For those reasons, ZP-binding assays are used to estimate sperm fertilizing capacity in several species including bovine (Ivanova and Mollova, Reference Ivanova and Mollova1993; Fazeli et al., Reference Fazeli, Steenweg, Bevers Mvan den Broek, Bracher, Parlevliet and Colenbrander1995; Zhang et al., Reference Zhang, Larsson, Lundeheim, Håård and Rodriguez-Martinez1999; Coutinho da Silva et al., Reference Coutinho da Silva, Seidel, Squires, Graham and Carnevale2012).
Small physiological concentrations of reactive oxygen species (ROS) are implicated in the control of normal sperm function, such as the ability of spermatozoa to bind to ZP (Aitken et al., Reference Aitken, Jones and Robertson2012). However, excessive ROS production induces DNA damage, impairs sperm motility and membrane permeability and may eventually induce sperm cell death (Barroso et al., Reference Barroso, Morshedi and Oehninger2000; Gil-Guzman et al., Reference Gil-Guzman, Ollero, Lopez, Sharma, Alvarez, Thomas and Agarwal2001; Aitken and Baker, Reference Aitken and Baker2004; Shamsi et al., Reference Shamsi, Kumar and Dada2008; Aitken et al., Reference Aitken, Baker and Nixon2015). Copper (Cu), manganese (Mn), selenium (Se) and zinc (Zn) are present in all mammalian tissues and protect cells against ROS-induced damage (Underwood and Suttle, Reference Underwood and Suttle1999; Chihuailaf et al., Reference Chihuailaf, Contreras and Wittwer2002). Cu, Mn, Se and Zn improve sperm quality parameters (Lapointe et al., Reference Lapointe, Ahmad, Buhr and Sirard1996; Ursini et al., Reference Ursini, Heim, Kiess and Flohé1999; Colagar et al., Reference Colagar, Marzony and Chaichi2009; Anchordoquy et al., Reference Anchordoquy, Anchordoquy, Pascua, Nikoloff, Peral-García and Furnus2017), but very little information is known about their effects on sperm–ZP binding.
The objective of this study was to determine the effect of trace minerals Cu, Mn, Se and Zn on binding of bovine spermatozoa to the ZP. Moreover, sperm viability, sperm membrane integrity, acrosomal status (AS), total antioxidant capacity (TAC) and sperm lipid peroxidation were evaluated.
Materials and methods
Reagents and media
Reagents for culture media and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (CAS 57360-69-7) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Standards as water solutions of copper sulphate, zinc sulphate, manganese sulphate and sodium selenite were purchased from Merck (Tokyo, Japan). The in vitro fertilization (IVF) medium used was TALP (Parrish et al., Reference Parrish, Susko-Parrish, Leibfried-Rutledge, Critser, Eyestone and First1986) supplemented with 6 mg/ml bovine serum albumin-fatty acid free (BSA-FAF), 20 µM penicillamine, 10 µM hypotaurine and 100 µg/ml heparin sulphate.
Oocyte recovery
Bovine ovaries obtained from slaughterhouse were transported within 3 h to the laboratory in NaCl solution (9 g/l NaCl) with streptomycin and penicillin at 37°C. Ovaries were used regardless of the stage of the oestrus cycle of the donor. Cumulus−oocyte complexes (COC) were aspirated from 3−8 mm follicles, using an 18-G needle connected to a sterile syringe. Only cumulus-intact complexes with evenly granulated cytoplasm were selected, using a low power (×20–30 magnification) stereomicroscope.
Semen preparation
In all experiments, frozen semen from a fertile bull, tested both in vivo and in vitro, was used. Straws containing 40×106 spermatozoa were thawed in a 37°C water bath and washed in a discontinuous Percoll gradient prepared by depositing 2 ml 90% Percoll under 2 ml 45% Percoll in a 15-ml centrifuge tube. Semen samples were deposited on the top of the Percoll gradient and centrifuged for 20 min at 500 g. The pellet was removed and resuspended in 300 µl of HEPES-TALP solution and centrifuged at 300 g for 10 min. For the binding assay, hypo-osmotic swelling test (HOST) and AS supernatant were discarded and spermatozoa were resuspended in IVF medium, counted in a haemocytometer chamber and further diluted. The final sperm concentration in IVF was 2×106 sperm/ml The incubation was conducted at 38.5°C in 5% CO2 in air with saturated humidity. For the MTT assay, the final sperm concentration in IVF medium was 10×106 sperm/ml and 40×10 6 sperm/ml for TAC and the thiobarbituric acid-reactive substances (TBARS) assay.
Binding assays
Before each experiment, immature COCs were denuded by vortexing with 0.1% (w/v) hyaluronidase in HEPES-TALP, washed twice and incubated in droplets of 40 µl IVF medium for at least 2 h at 38.5°C in an atmosphere of 5% CO2 in air. After incubation, oocytes were co-incubated with 2×106 spermatozoa/ml at 38.5°C in an atmosphere of 5% CO2 in air for 2 h. Afterwards, the oocytes were washed five times in phosphate-buffered saline (PBS) to remove loosely bound spermatozoa and then fixed and stained with Hoechst 33342. The number of spermatozoa bound to each egg was determined by observation at ×400 magnification under an epifluorescence microscope Olympus BX40 (Olympus, Tokyo, Japan) equipped with a 365 nm excitation filter, a 400 nm barrier filter and a 400 nm emission filter.
MTT reduction assay
MTT (3[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide) is a yellow dye that is converted to purple formazan by the succinate dehydrogenase system of active mitochondria (Slater et al., Reference Slater, Sawyer and Sträuli1963). Therefore, the amount of formazan formed can be determined spectrophotometrically and serves as an estimate of the number of mitochondria and hence the number of living cells in the sample (Denizot and Lang, Reference Denizot and Lang1986). The MTT assay was performed according to the method of Mosmann (Reference Mosmann1983). For each sample, six wells of a 96-well microplate were used. In total, 100 μl sperm suspension (described above) plus 10 μl MTT stock solution (5 mg MTT/ml PBS) were placed in each well. According to Aziz (Reference Aziz2006), the optical density of samples should be measured immediately after 1 and 2 h incubation at 37°C using a spectrophotometer (Biotek Instruments Inc., Bedfordshire, UK) at a wavelength of 550 nm. MTT reduction rate (optical density) for each sample was calculated by calculating the difference between the first and second readings and the first and third readings of the spectrophotometer, for the reduction rate after 1 h and 2 h incubation, respectively. Results for mitochondrial activity were expressed by normalizing the data in relation to the Control, in which mitochondrial activity was considered to be 100%.
The hypo-osmotic swelling test
HOST was used to evaluate functional sperm membrane integrity (Revell and Mrode, Reference Revell and Mrode1994). The test was performed by incubation of 25 µl semen with 200 µl HOST solution (100 mOsm/l, 57.6 mM fructose and 19.2 mM sodium citrate) for 30 min at room temperature (RT, 20°C). A wet mount was made using a 10 µl drop of homogenized mixture placed directly on a microscopic slide and covered with a coverslip. In total, 200 spermatozoa were counted in at least five different microscopic fields. The percentages of spermatozoa with swollen and curved tails were recorded.
Acrosomal status
The AS was assessed using Pisum sativum agglutinin conjugated to fluorescein isothiocyanate (PSA−FITC; Sigma Chemical Company, St Louis, MO, USA), as described earlier (Mendoza et al., Reference Mendoza, Carreras, Moos and Tesarik1992). Briefly, sperm smears were fixed in methanol for 30 s after air drying and then stained using 50 mg/ml PSA−FITC in PBS for 30 min in a humidified chamber (HC) at RT. The slides were washed with distilled water and mounted. In total, 200 spermatozoa per sample were counted using an Olympus BX40 epifluorescence microscope (Olympus, Tokyo, Japan) using excitation wavelengths of 450–490 nm and a magnification of ×1000. The acrosomal region of the acrosome-intact spermatozoa was PSA−FITC positive and labelled green, while the acrosome-reacted spermatozoa retained only an equatorial labelled band with little or no labelling of the anterior head region.
Measurement of lipid peroxidation
Lipid peroxidation levels were measured using the TBARS method. The TBARS concentration in sperm suspension (described above) was measured spectrophotometrically and expressed as the malondialdehyde (MDA) level. The TBARS concentration was expressed as nmol MDA/106 sperm using tetramethoxypropane (TMP) as a standard. An aliquot of 100 µl of sperm suspension was mixed with 100 µl 8.1% SDS solution and 750 µl 20% acetic acid solution. After adding 750 µl 0.8% TBA solution and 2 ml distilled water to this mixture, it was heated for 1 h in a 95°C oven. The mixture was then cooled at RT and centrifuged at 4220 g for 15 min, after which the absorbance of the supernatant was measured at 532 nm using a spectrophotometer. The value was subsequently determined based on comparison with a TMP standard curve.
Total antioxidant status
Total antioxidant capacity was measured using a colorimetric method using the Randox total antioxidant status kit (cat no. NX2332, Randox Laboratories, Ltd, Crumlin, UK) with slight modifications. Briefly, 20 µl of sperm suspension (described above) was added to 1 ml of the chromogen, ABTS (2,2′-azino-di-[3-ethylbenzthiazoline sulphonate]). In total, 20 μl Trolox (6-hydroxyl-2,5,7,8-tetramethylchroman-2-carboxylic acid) at a concentration of 2.27 mmol/l was used as standard, whereas 20 μl deionized water was used as the blank. Chromogen (1 ml) was added to standard and blank samples. The absorbance was measured 3 min after substrate addition at 600 nm with a spectrophotometer. Results were expressed as mmol/l. Measurements in duplicate were used to calculate intra-assay variability.
Experimental design
Experiment 1: Effect of Cu, Mn, Se and Zn on the sperm–ZP binding
In Experiment 1, immature denuded oocytes and sperm were co-incubated for 2 h (as described above) in IVF medium supplemented with:
(i) 0, 2, 5 or 6 ng/ml Mn (n=86 COC). The concentrations used are according to Kincaid (Reference Kincaid1999) and Underwood and Suttle (Reference Underwood and Suttle1999) classifications for Mn status in cattle (Experiment 1a);
(ii) 0, 10, 50 or 100 ng/ml Se (n=120 COC). Concentration used were according the classification for Se status in cattle reported by Kincaid (Reference Kincaid1999) and Underwood and Suttle (Reference Underwood and Suttle1999) (Experiment 1b);
(iii) Cu+Mn+Se+Zn. Copper (0.4 µg/ml Cu; Anchordoquy et al., Reference Anchordoquy, Anchordoquy, Pascua, Nikoloff, Peral-García and Furnus2017); and Zn (0.8 µg/ml Zn; unpublished data) were established previously in our laboratory.
Mn and Se concentrations depended on the results of Experiments 1(a) and 1(b), respectively. A Control group without mineral supplementation was also analyzed (n=354 COC) (Experiment 1c). All experiments were performed with COC obtained in separate batches of ovaries from 3 different days. The number of sperm bound to ZP was expressed as mean±standard error of the mean (SEM).
Experiment 2: Effect of Cu, Mn, Se and Zn on sperm viability by MTT assay
In Experiment 2, the viabilities of sperm incubated for 1 or 2 h in IVF medium supplemented with Cu, Mn, Se, Zn, Cu+Mn+Se+Zn, or without supplement (Control) were investigated by MTT reduction assay (described above). In each replicate, 12 semen samples were pooled. Results were expressed as the percentage of mitochondrial activity and MTT reduction rate (mean±SEM) from three independent replicates.
Experiment 3: Effect of Cu, Mn, Se and Zn on functional sperm membrane integrity
In Experiment 3, the functional sperm membrane integrity of sperm incubated for 0, 1 or 2 h in IVF medium supplemented with: Cu, Mn, Se, Zn, Cu+Mn+Se+Zn, or without supplement (Control) was investigated using HOST (described above). In each replicate, three semen samples were pooled. Results were expressed as the percentage of HOST positive sperm from three independent replicates.
Experiment 4: Effect of Cu, Mn, Se and Zn on acrosomal status
In Experiment 4, acrosomal status of sperm incubated for 1 or 2 h in IVF medium supplemented with Cu, Mn, Se, Zn, Cu+Mn+Se+Zn, or without supplement (Control) was investigated by PSA−FITC staining (described above). In each replicate, three semen samples were pooled. Results were expressed as the percentage from three independent replicates.
Experiment 5: Effect of Cu, Mn, Se and Zn on sperm lipid peroxidation
In Experiment 5, lipid peroxidation status of sperm incubated for 2 h in IVF medium supplemented with Cu, Mn, Se, Zn, Cu+Mn+Se+Zn, or without supplement (Control) was investigated using the TBARS method (described above). In each replicate, 12 semen samples were pooled. Results were expressed as the mean±SEM from three independent replicates.
Experiment 6: Effect of Cu, Mn, Se and Zn on sperm total antioxidant capacity
In Experiment 6, TAC of sperm incubated for 2 h in IVF medium supplemented with Cu, Mn, Se, Zn, Cu+Mn+Se+Zn, or without supplement (Control) was investigated by total antioxidant status kit (described above). In each replicate, 12 semen samples were pooled. Results were expressed as the mean±SEM from three independent replicates.
Statistical analysis
A completely randomized block design was used. The statistical model included the random effects of block (n=3) and the fixed effect of treatment [0 vs 2 vs 5 vs 6 ng/ml Mn in Experiment 1(a); 0 vs 10 vs 50 vs 100 ng/ml Se in Experiment 1(b); and Control vs Cu vs Mn vs Se vs Zn vs Cu+Mn+Se+Zn in Experiments 1(c), 2, 3, 4, 5 and 6]. Variables such as number of sperm bound to ZP and MTT were analyzed with linear models using the MIXED procedure of SAS (SAS Institute, Cary, NC, USA). HOST and AS were analyzed by logistic regression using the GENMOD procedure (SAS Institute). TBARS and TAC were analyzed using the GLIMIX procedure (SAS Institute) with gamma distribution. Statistical significance was set at P<0.05, while a trend for statistical significance was set between P>0.05 and ≤0.10.
Results
Experiment 1: Effect of Cu, Mn, Se and Zn on sperm–ZP binding
In Experiment 1(a), there were no significant differences in number of sperm bound to ZP when Mn was added to IVF medium (P>0.05; Fig. 1A). Manganese at a concentration of 5 ng/ml showed the highest number of ZPs and consequently this concentration was chosen for the next experiments. In Experiment 1(b), considerably more sperm bound to ZP were observed when Se was added to IVF medium at all concentrations studied (P<0.01), but the difference was highest when using 50 and 100 ng/ml Se (Fig. 1B). For the next experiments 100 ng/ml Se was used. In Experiment 1(c), sperm bound to ZP was higher when Cu, Se or Zn were added to IVF medium (P<0.01), but there were no differences among the Control, Mn and Cu+Mn+Se+Zn samples (P>0.05; Fig. 1C).
Effect of trace minerals Cu, Mn, Se and Zn on the sperm–ZP binding. The number of sperm bound to the zona pellucida (ZP) is expressed as mean±standard error of the mean (SEM) (three replicates on different days). The figure shows the number of sperm bound to ZP after 2 h of incubation in IVF medium supplemented with 0 (Control), 2, 5 or 6 ng/ml Mn (A); 0 (Control), 10, 50 or 100 ng/ml Se (B); and Cu (0.4 µg/ml Cu), Mn (5 ng/ml Mn), Se (100 ng/ml Se), Zn (0.8 µg/ml Zn), Cu+Mn+Se+Zn, or without supplement (Control; C). a,b,cBars with different letters differ statistically (P<0.05).
Experiment 2: Effect of Cu, Mn, Se and Zn on sperm viability by MTT assay
In Experiment 2, spermatozoa incubated with Cu, Mn or Se showed a significant increase in mitochondrial activity after 1 h of incubation (P<0.05), but there were no differences among Control, Zn and Cu+Mn+Se+Zn (P>0.05). However, after 2 h of incubation the spermatozoa mitochondrial activity was significantly higher only for Mn addition to IVF medium with respect to the Control (P<0.05; Table 1).
Effect of trace minerals Cu, Mn, Se and Zn on sperm viability based on MTT assay
Mitochondrial activity is expressed as percentages and reduction rate as mean±standard error of the mean (SEM) (three replicates on different days). For mitochondrial activity, data were normalized to measurements from Control cultures, which were as considered 100%. Viability based on MTT assay of sperm cultured in IVF medium supplemented with Cu (0.4 µg/ml Cu), Mn (5 ng/ml Mn), Se (100 ng/ml Se), Zn (0.8 µg/ml Zn), Cu+Mn+Se+Zn, or without supplement (Control) were evaluated after 1 or 2 h of incubation. a,b,cValues with different superscripts within a column differ significantly (P<0.05).
Experiment 3: Effect of Cu, Mn, Se and Zn on the functional sperm membrane integrity
In Experiment 3, percentage of HOST positive sperm at 0 and 1 h were similar among all treatments (P>0.05). However, functional membrane integrity was increased after 2 h of sperm incubation with Cu compared with the Control (P<0.05). Moreover, Se supplementation after 2 h of incubation tended to increase HOST positive sperm with respect to the Control (P=0.08; Table 2).
Effect of trace minerals Cu, Mn, Se and Zn on functional sperm membrane integrity
HOST, hypo-osmotic swelling test (functional sperm membrane integrity) is expressed as percentages (three replicates on different days). HOST of sperm cultured in IVF medium supplemented with Cu (0.4 µg/ml Cu), Mn (5 ng/ml Mn), Se (100 ng/ml Se), Zn (0.8 µg/ml Zn), Cu+Mn+Se+Zn, or without supplement (Control) were evaluated after 0, 1 or 2 h of incubation. a,b,cValues with different superscripts within a column differ significantly (P<0.05). After 2 h of incubation, Se supplementation tended to increase the HOST positive sperm compared with the Control (P=0.08).
Experiment 4: Effect of Cu, Mn, Se and Zn on acrosomal status
In Experiment 4, percentages of sperm with intact acrosome at 0 h were higher with the addition of Cu, Mn, or Se to IVF medium with respect to the Control (P<0.05). However, after 1 h of incubation, acrosome integrity were significantly higher in sperm treated with Zn or Cu+Mn+Se+Zn (P<0.05). After 2 h of incubation, Se and Zn supplementation tended to increase the percentage of acrosome-intact sperm compared with the Control (P=0.08; Table 3).
Effect of trace minerals Cu, Mn, Se and Zn on acrosomal status
Acrosome integrity is expressed as percentages (three replicates on different days). Acrosomal status of sperm cultured in IVF medium supplemented with Cu (0.4 µg/ml Cu), Mn (5 ng/ml Mn), Se (100 ng/ml Se), Zn (0.8 µg/ml Zn), Cu+Mn+Se+Zn, or without supplement (Control) were evaluated after 0, 1 or 2 h of incubation. a,b,cValues with different superscripts within a column differ significantly (P<0.05). After 2 h of incubation, Se and Zn supplementation tended to increase the percentage of acrosome-intact sperm compared with the Control (P=0.08).
Experiment 5: Effect of Cu, Mn, Se and Zn on lipid peroxidation
In Experiment 5, after 2 h of incubation, lipid peroxidation expressed by MDA level was significantly higher in sperm treated with Cu or Cu+Mn+Se+Zn in comparison with the Control (P<0.05), but there were no differences among Control, Mn and Zn (P>0.05). Moreover, Se supplementation tended to increase MDA levels respect to Control (P=0.07; Fig. 2).
Effect of trace minerals Cu, Mn, Se and Zn on lipid peroxidation. Lipid peroxidation is expressed as malondialdehyde (MDA) level. Data are expressed as mean±standard error of the mean (SEM) (three replicates on different days). Lipid peroxidation of sperm cultured in IVF medium supplemented with Cu (0.4 µg/ml Cu), Mn (5 ng/ml Mn), Se (100 ng/ml Se), Zn (0.8 µg/ml Zn), Cu+Mn+Se+Zn, or without supplement (Control) was evaluated after 2 h of incubation. a,b,c,dBars with different letters differ statistically (P<0.05). Bars with (*) tended to be different (P=0.07).
Experiment 6: Effect of Cu, Mn, Se and Zn on sperm total antioxidant capacity
In Experiment 6, after 2 h of incubation the mean TAC of sperm treated with Cu, Mn, Zn or Cu+Mn+Se+Zn were significantly lower than the Control (P<0.05), but there was no difference between Control and Se (P>0.05; Fig. 3).
Effect of trace minerals Cu, Mn, Se and Zn on sperm total antioxidant capacity. Total antioxidant capacity is expressed as mmol/l. Data are expressed as mean±standard error of the mean (SEM) (three replicates on different days). Total antioxidant capacity of sperm cultured in IVF medium supplemented with Cu (0.4 µg/ml Cu), Mn (5 ng/ml Mn), Se (100 ng/ml Se), Zn (0.8 µg/ml Zn), Cu+Mn+Se+Zn, or without supplement (Control) was evaluated after 2 h of incubation. a,bBars with different letters differ statistically (P<0.05).
Discussion
The objective of this study was to determine the effect of trace minerals Cu, Mn, Se and Zn on sperm–ZP binding. Our results demonstrated that: (a) Cu, Se and Zn added to IVF medium bound more sperm to ZP; (b) Cu, Mn and Se augmented sperm viability; (c) Cu improved functional membrane integrity; (d) Cu and Mn increased intact acrosome sperm percentages; (e) Cu and Cu+Mn+Se+Zn intensified lipid peroxidation; and (f) Cu, Mn, Zn and Cu+Mn+Se+Zn diminished TAC in sperm.
The ZP surrounds mammalian oocytes, which is a permeable extracellular glycoprotein matrix (Wassarman, Reference Wassarman1990; Sinowatz et al., Reference Sinowatz, Töpfer‐Petersen, Kölle and Palma2001; Wassarman and Litscher, Reference Wassarman and Litscher2008). The ability of spermatozoa to bind ZP is achieved after processes of spermatogenesis, epididymal maturation and capacitation (Reid et al., Reference Reid, Redgrove, Aitken and Nixon2011). This binding is mediated by a number of putative ZP sperm receptor, including glycosyl enzymes, such as β-1,4-galactosyltransferase, fucosyltransferase-5 and α-d-mannosidase, which requires a metal ion as a cofactor, preferentially Mn (Shur and Neely, Reference Shur and Neely1988; Cornwall et al., Reference Cornwall, Tulsiani and Orgebin-Crist1991; Miller et al., Reference Miller, Macek and Shur1992; Gong et al., Reference Gong, Dubois, Miller and Shur1995; Chiu et al., Reference Chiu, Chung, Koistinen and Yeung2007). In the present study, Cu, Se and Zn supplementation increased the number of sperm bound to ZP; for Mn, there was some increase but not significant. This finding is in disagreement with Liu et al. (Reference Liu, Sie, Liu, Agresta and Baker2009) who reported that supplementation of culture media with Zn had no effect on spermatozoa–ZP binding. However, the Zn concentration used by Liu and colleagues (Reference Liu, Sie, Liu, Agresta and Baker2009) was 400 times higher than that used in the present study. The improvement in sperm–ZP binding with Cu addition to IVF medium is in agreement with our previous work (Anchordoquy et al., Reference Anchordoquy, Anchordoquy, Pascua, Nikoloff, Peral-García and Furnus2017). To the best of our knowledge, this is the first time that the effects of Se, Mn or the combination of Cu+Mn+Se+Zn on sperm–ZP binding have been evaluated.
To determine whether the increase in sperm–ZP binding observed in this study was due to greater sperm viability, an MTT assay was performed. Therefore, after 1 h of incubation, an increase on sperm viability was observed with Cu, Mn and Se supplementation, whereas after 2 h, only Mn showed an improvement. Se has a favourable influence on sperm viability providing protection against oxidative stress (Ahsan et al., Reference Ahsan, Kamran, Raza and Iqbal2014). Moreover, dietary supplementation with Se improves sperm viability in rams (Marai et al., Reference Marai, El-Darawany, Ismail and Abdel-Hafez2009) and buffalos (El-Sharawy et al., Reference El-Sharawy, Eid, Darwish, Abdel-Razek, Islam, Kubota, Yamauchi and El-Shamaa2017). Rise in bovine sperm viability has also been observed after 3 h of incubation with Cu and after 6 h with Mn (Lapointe et al., Reference Lapointe, Ahmad, Buhr and Sirard1996; Anchordoquy et al, Reference Anchordoquy, Anchordoquy, Pascua, Nikoloff, Peral-García and Furnus2017). Chia and colleagues (Reference Chia, Ong, Chua, Ho and Tay2000) demonstrated a relationship between Zn concentration in seminal plasma and sperm viability. However, the addition of Zn sulphate to semen extender in concentrations similar to those used in this study had a detrimental effect on the viability and membrane integrity of buffalo spermatozoa (Dorostkar et al., Reference Dorostkar, Alavi Shoushtari and Khaki2014).
The integrity and functional activity of the sperm membrane are key factors that influence zona-binding ability and fertilization of mammalian spermatozoa (Varghese et al., Reference Varghese, Sinha and Bhattacharyya2005). In the present study, HOST showed an increase in functional membrane integrity when sperm were incubated with Cu and Se. This finding is in agreement with a previous study in which we observed an increase in the percentage of HOST positive sperm with Cu supplementation to IVF medium (Anchordoquy et al., Reference Anchordoquy, Anchordoquy, Pascua, Nikoloff, Peral-García and Furnus2017). For Se, addition of this mineral to semen extender increased functional membrane integrity of frozen–thawed buffalo sperm (Dorostkar et al., Reference Dorostkar, Alavi-Shoushtari and Mokarizadeh2012). Recently, Reis et al. (Reference Reis, Ramos, Camargos and Oba2014) showed that membrane integrity of Nellore sperm was increased by dietary Mn supplementation. In vitro, Mn supplementation of bovine semen during cryopreservation showed a protective effect, growing the percentage of HOS-positive spermatozoa (Cheema et al., Reference Cheema, Bansal and Bilaspuri2009). However, in the present study, Mn addition to IVF medium did not modify the functional integrity of the sperm’s plasma membrane.
Only motile acrosome-intact sperm bind to the ZP (Hoodbhoy and Dean, Reference Hoodbhoy and Dean2004). In the present study, Cu, Mn or Se supplementation increased percentages of acrosome-intact sperm at 0 h, suggesting a protective effect of these minerals against spontaneous acrosome reaction (AR). Although, after 1 h of incubation Zn and Cu+Mn+Se+Zn increased the percentages of intact acrosomes, after 2 h that improvement was only maintained by Zn and Se treatments. These results are consistent with that observed in a previous study in which Cu supplementation did not modify AS after 3 or 6 h of incubation (Anchordoquy et al., Reference Anchordoquy, Anchordoquy, Pascua, Nikoloff, Peral-García and Furnus2017). In addition, Roblero et al. (Reference Roblero, Guadarrama, Lopez and Zegers-Hochschild1996) found that AR was not affected when sperm were incubated for 5 h in medium containing 1 µg/dl to 1 mg/dl Cu. With respect to Se, Marai et al. (Reference Marai, El-Darawany, Ismail and Abdel-Hafez2009) reported that dietary supplementation with sodium selenite improved semen quality, decreasing acrosome damage. The relationship between Zn and AR has already been studied. Michailov et al. (Reference Michailov, Ickowicz and Breitbart2014) showed that Zn stimulated AR in capacitated sperm through the epidermal growth factor receptor. In the present study, Zn maintained the percentages of acrosome-intact sperm during the 2 h of incubation.
Oxidative stress is a major contributor to defective sperm function including the competence for fertilization. These effects include LPO, ending in cytotoxic aldehydes generation such as MDA and 4-hydroxynonenal (Jones et al., Reference Jones, Mann and Sherins1979; Nair et al., Reference Nair, Brar, Ahuja, Sangha and Chaudhary2006; Kasimanickam et al., Reference Kasimanickam, Kasimanickam, Thatcher, Nebel and Cassell2007; Aitken and Curry, Reference Aitken and Curry2011). However, abundant evidence suggests that ROS produced by mammalian sperm play a physiologically role promoting capacitation process through redox regulation of tyrosine phosphorylation (Baumber et al., Reference Baumber, Sabeur, Vo and Ball2003; Ecroyd et al., Reference Ecroyd, Jones and Aitken2003; Rivlin et al., Reference Rivlin, Mendel, Rubinstein, Etkovitz and Breitbart2004; Roy and Atreja, Reference Roy and Atreja2008; Basim et al., Reference Basim, Mackenzie-Bell and Buhr2009; Gonçalves et al., Reference Gonçalves, Barretto, Arruda, Perri and Mingoti2010). Mammalian spermatozoa including bovine have the capacity to generate ROS, mainly hydrogen peroxide (Tosic and Walton, Reference Tosic and Walton1946). Mitochondria in the sperm midpiece are the major source of oxygen metabolites (Storey, Reference Storey2008). Transition metals such as Fe, Cu, Pb, or Cd have been described to be among several causes of mitochondrial ROS generation (Jones et al., Reference Jones, Mann and Sherins1979; Kiziler et al., Reference Kiziler, Aydemir, Onaran, Alici, Ozkara, Gulyasar and Akyolcu2007). In the present study, MDA production was increased when Cu, Se and Cu+Mn+Se+Zn were added to IVF medium, but not with Mn or Zn supplementation. These results are in agreement with Kaushik and colleagues (Reference Kaushik, Mittal and Kalla2015) who demonstrated that Mn is a strong ion inhibitor of LPO in human semen that is far superior compared with Zn. The Mn inhibitory role on LPO has been described in vivo and in vitro (Aitken, Reference Aitken1997). Manganese is required for mitochondrial superoxide dismutase synthesis (Sikka, Reference Sikka1996) and has an important role as an antioxidant, regulating peroxyl radicals (Coassin et al., Reference Coassin, Ursini and Bindoli1992). Gavella and Lipovac (Reference Gavella and Lipovac1998) studied the inhibitory effect of Zn on superoxide anion generation in human spermatozoa, demonstrating that Zn ions themselves are not able to act as inducers of LPO. This is in agreement with the results obtained in this study. Conversely, an interesting fact is that the metals that produced greater LPO such as Cu and Se are those that increased the number of sperm bound to ZP. These results are consistent with that observed by Aitken et al. (Reference Aitken, Clarkson and Fishel1989) and Kodama et al. (Reference Kodama, Kuribayashi and Gagnon1996) who demonstrated that the induction of mild lipid peroxidation increases sperm–zona binding and sperm fertilizing capacity. This unpredicted peroxidative effect is an unexpected positive, although the mechanisms involved are not yet clarified (Aitken et al., Reference Aitken, Jones and Robertson2012).
Transitions metals such as Mn, Cu and Zn are involved in essential biological processes, as they are cofactors of metalloproteins, many of these with antioxidant activity (Underwood and Suttle, Reference Underwood and Suttle1999; Chihuailaf et al., Reference Chihuailaf, Contreras and Wittwer2002). Although essential in trace amounts, at higher levels these metals may have pro-oxidant effect (Underwood and Suttle, Reference Underwood and Suttle1999; Chihuailaf et al., Reference Chihuailaf, Contreras and Wittwer2002). The decrease in TAC observed after supplementation of IVF medium with Cu, Mn or Zn suggested that these trace minerals might increase the spermatic levels of ROS.
Although spermatozoa from a single bull were used in this study, the results were similar when frozen semen from other bulls were used (unpublished observations). In a previous study using another bull, we observed comparable results with Cu supplementation (Anchordoquy et al., Reference Anchordoquy, Anchordoquy, Pascua, Nikoloff, Peral-García and Furnus2017).
In conclusion, the results from the present study showed that the presence of Cu, Se and Zn in the IVF medium increased the number of spermatozoa bound to ZP. This may, at least in part, be due to an increase in: (i) the viability and functional membrane integrity of sperm (Cu and Se); (ii) the number of spermatozoa with intact acrosome (Se and Zn); and (iii) lipid peroxidation (Cu and Se). We inferred that Cu, Se and Zn might play an important role in sperm–ZP binding, highlighting the importance of these minerals in the fertilization process.
Acknowledgements
We are grateful to Centro de Inseminación Artificial La Elisa S.A. (CIALE) for providing bovine frozen semen. Thanks are also due to A. Di Maggio for manuscript correction and edition.
Financial support
This work was supported by Grant PICT 2015–2154 from Agencia Nacional de Promoción Científica y Tecnológica de la República Argentina (MINCyT).
Conflicts of interest
The authors declare that there are no conflicts of interest.
Ethical standards
Not applicable.
