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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Effect of manganese supplementation on the membrane integrity and the mitochondrial potential of the sperm of grazing Nelore bulls
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L.S.L.S. Reis a,b,∗ , A.A. Ramos c , A.S. Camargos b , E. Oba b,1 a
Department of Animal Nutrition and Food Science, University of Santo Amaro, São Paulo, SP, Brazil Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science, São Paulo State University, Botucatu, SP, Brazil c Department of Animal Production, School of Veterinary Medicine and Animal Science, São Paulo State University, Botucatu, SP, Brazil b
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a b s t r a c t
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Article history: Received 17 January 2013 Received in revised form 24 June 2014 Accepted 30 June 2014 Available online xxx
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Keywords: Manganese Spermatozoa Integrity of sperm membrane and acrosome Cattle Mineral salt
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1. Introduction
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The effect of dietary manganese (Mn2+ ) supplementation on the reproductive performance of Nelore bulls was evaluated by assessment of sperm membrane integrity. Sixty Nelore bulls (Bos taurus indicus) aged 18–20 mo were randomly divided into four groups (n = 15) receiving dietary Mn2+ supplementation at 540, 1300, 3800 and 6300 mg/kg (treatments TC , T1300 , T3800 and T6300 , respectively). The diets were changed for the groups every 70 d. Semen samples were obtained 15 and 56 d after the diet change, which corresponded to the period of adjustment to the new diet and the time required for a complete spermatogenesis cycle, respectively. Sperm integrity was assessed by detection of: intact (IMe) or damaged (DMe) membranes, intact (IA) or damaged (DA) acrosomes, and high (HM) or low (LM) mitochondrial membrane potentials. Only bulls from the TC treatment showed a significant increase in the production of intact sperm [IMe/IA/LM] and decrease in the production of sperm with damaged acrosome [IMe/DA/LM] or completely damaged sperm [DMe/DA/LM] (P < 0.05). The Mn2+ concentrations in the semen were positively correlated with the incidence of sperm with IMe, DA, and LM and negatively correlated with number of sperm with DMe, IA, and LM. Therefore, dietary Mn2+ supplementation for Nelore bulls must be limited to 540 mg of Mn2+ /kg given that higher doses are detrimental to the integrity of the plasma and acrosomal sperm membranes. © 2014 Published by Elsevier B.V.
Manganese (Mn) has an essential role in the physiological processes of cattle, protein synthesis and processing (Szentmihályi et al., 2006), carbohydrate metabolism (Underwood and Sutlle, 2004; Szentmihályi
∗ Corresponding author at: Rua Padre Davi 190, Assis CEP 19800-220, SP, Brazil. Tel.: +55 18 9776 2550. E-mail address: [email protected] (L.S.L.S. Reis). 1 CNPq researcher.
et al., 2006), and lipid metabolism (Jenkins and Kramer, 1991; Underwood and Sutlle, 2004; Szentmihályi et al., 2006; Hansen et al., 2006). Thus, this mineral can be correlated with the integrity of the sperm plasma membrane and is involved in the metabolism of the main organic components that compose this membrane, which include phospholipids and integral and peripheral membrane proteins (Alberts et al., 2010). Moreover, Mn is also involved in the protection of the plasma membrane of the head, acrosome, and mitochondria against free radicals (hydroperoxides), which are formed by decomposing metalloenzymes and are highly oxidative and
http://dx.doi.org/10.1016/j.anireprosci.2014.06.033 0378-4320/© 2014 Published by Elsevier B.V.
Please cite this article in press as: Reis, L.S.L.S., et al., Effect of manganese supplementation on the membrane integrity and the mitochondrial potential of the sperm of grazing Nelore bulls. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.06.033
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damaging to membranes, proteins, and DNA (Underwood and Sutlle, 2004; Hansen et al., 2006; Cheema et al., 2009; Ansenberger-Fricano et al., 2012). The NRC (2000) recommends that beef cattle receive a daily intake of 40 mg of Mn during their reproductive periods. However, in many countries, Mn2+ is naturally present at concentrations that exceed the NRCrecommended dose by 30-fold (2000). In Brazil, there are reports of concentrations of up to 1.305 mg Mn2+ /kg DM (Wunsch et al., 2005), and New Zealand reports concentrations of 400 mg Mn2+ /kg DM (Grace, 1973). Moreover, some manufacturers continue to add increased amounts of Mn2+ to mineral supplements, and these can reach up to 2000 ppm in Brazil. This is of concern because the effects of these diets with excessive concentrations of Mn2+ on the integrity of the plasma membrane of the head, acrosome, and mitochondria of bull sperm are not well elucidated. To establish suitable Mn2+ supplementation for the diets of Nelore bulls that do not negatively impact their reproductive performance, the present study tested the effect of dietary supplementation with different amounts of Mn2+ on the sperm membrane, the acrosomal integrity, and the mitochondrial membrane potential.
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2. Materials and methods
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The procedures used in this study were approved by the Ethics Committee on Animal Use of the School of Veterinary Medicine and Animal Science of São Paulo State University, Botucatu, SP, Brazil. 2.1. Experimental design
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The duration of the experiment, which was performed in Lutécia, SP, Brazil, was 280 d. The altitude of the study area was 602 m, and the climate of the study area was tropical and characterized by a rainy season (October–April) and a dry season (May–September), a mean annual precipitation of 1300 mm, a relative air humidity of 64%, and a mean temperature of 25 ◦ C. Using a 4 × 4 randomized block (Latin square) design, we divided the cattle into four groups (n = 15) with a similar live weight (G1 = 264.5 ± 22.5 kg; G2 = 261.5 ± 23.4 kg; G3 = 268.5 ± 19.6 kg, and G4 = 267.9 ± 22.5 kg); the four groups were fed four experimental diets. At the onset of the experiment, the 60 Nelore bulls (Bos taurus indicus) were 18–20 mo of age. Every 70 d, the experimental groups were transferred to another paddock and fed a different experimental diet; thus, all of the bulls were fed all the diets by the end of the study. The sperm quality was assessed at the end of each treatment period.
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2.2. Experimental diets
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The bulls grazed on Brachiaria decumbens (Table 1) in an extended grazing system and received a supplemental protein mineral mixture with different amounts of Mn2+ ad libitum. This protein mineral supplement was used to meet the nutritional protein requirements. The protein sources were urea, ammonium sulfate, and soybean meal.
The mineral mixture was prepared based on the results of a pilot project, which evaluated the use of a basic mixture of mineral salt. Based on the daily consumption, Mn2+ was added such that each animal consumed specific daily quantities of Mn2+ . The base diet of the control group did not meet the nutritional Mn2+ requirements recommended by the NRC (2000) because, in Brazil, this mineral is present in the environment greater amounts than the recommended dietary intake (Wunsch et al., 2005). Thus, to observe the effects of manganese on the membrane integrity and the acrosomal and mitochondrial potential of sperm, four mineral supplements were formulated. The mixture (1 kg) contained 402 mg of copper, 8000 mg of sulfur, 20 mg of iron, 2.8 mg of selenium, 1400 mg of zinc, 150 mg of fluoride, 400 g of crude protein, and 337.5 g of protein equivalent. In addition, the experimental diets contained 540 mg of Mn2+ (control treatment, TC ), which was present in the other sources of mineral elements and/or soybean meal included in the mixture, 1300 mg of Mn2+ (T1300 ), 3800 mg of Mn2+ (T3800 ), and 6300 mg (T6300 ) of Mn2+ per kilogram of mineral mixture. The diets were offered in the paddocks in 13-cmlong feeders for each animal. A water trough was placed 50 m from the feeder, as recommended by the Brazilian Association of Mineral Supplement Industries (2003). The consumption of the mineral mixtures was determined weekly throughout the experiment. At the onset of the study period, the mixture was weighed and placed into the trough; the mineral mix was then weighed 7 d later. By subtracting the second weight from the first weight, the average consumption was determined for each batch of bulls. After this batch average consumption was divided by the number of bulls in the lot, the average daily consumption of each supplement was determined. As a result, the consumption of the additives was found to be 369 g/bull/day. 2.3. Semen collection and evaluation The semen samples were collected by electroejaculation twice during each study period (season). Because these animals are generally bred using natural mating rather than AI, these bulls were not adapted to semen collection with an artificial vagina. The first collection (Day 0) was performed 15 d after the cattle had been moved to a new paddock, i.e., a new Mn2+ treatment. However, because the aim was to initiate a new spermatogenesis cycle, this semen was not evaluated. After 56 d (Day 56), i.e., at the end of the season, semen samples were collected again to determine the integrity and functionality of the sperm. The motility, sperm vigor, turmoil, sperm membrane and acrosome integrity and mitochondrial activity were determined after the seeds on the farm were harvested. Thus, all the equipments needed to perform these analyses in sperm were brought to the farm. The sperm membrane and acrosome integrity were assessed by fluorescence microscopy using a combination of fluorescent probes, as recommended by Celeghini et al. (2007): propidium iodide, Hoechst 33342, fluorescein
Please cite this article in press as: Reis, L.S.L.S., et al., Effect of manganese supplementation on the membrane integrity and the mitochondrial potential of the sperm of grazing Nelore bulls. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.06.033
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Table 1 Cattle lot rotation among pastures and chemical composition of Brachiaria decumbens. Season
Paddocka
Group
Mn2+ (ppm)
DM
CP
NDT
CF
NDF
ADF
EE
MM
Summer/10
I II III IV
G1 G2 G3 G4
29.3 28.4 27.2 29.4
7.2 9.2 9.6 7.6
60.3 59.6 58.4 64.0
29.6 26.5 22.1 25.6
74.2 72.9 74.9 68.4
34.8 39.7 32.7 41.9
1.5 1.9 1.9 1.6
6.8 7.4 7.2 5.6
195.0 141.0 100.0 160.0
Fall/10
I II III IV
G4 G3 G2 G1
40.1 42.4 63.5 57.1
5.3 5.3 4.8 4.4
59.4 59.1 60.4 61.3
31.9 32.2 29.4 29.3
77.2 76.1 76.9 75.4
41.8 43.4 47.1 46.7
1.7 1.4 1.6 1.4
6.6 6.6 6.7 5.7
216.0 219.0 117.0 157.0
Winter/10
I II III IV
G3 G4 G1 G2
65.2 78.0 78.7 60.4
5.2 4.4 3.9 4.2
59.0 60.7 61.1 60.0
30.6 30.0 29.4 31.7
78.8 80.7 78.5 77.4
44.1 49.1 48.8 44.0
1.4 1.1 2.4 1.5
7.5 5.9 5.8 5.6
235.0 265.0 183.0 153.0
Spring/10
I II III IV
G2 G1 G4 G3
46.1 40.2 40.3 42.8
8.5 6.7 7.9 7.5
64.5 62.4 63.2 63.9
23.2 28.0 25.9 25.0
67.3 72.0 70.8 72.5
30.6 41.2 37.8 37.6
1.6 1.5 1.3 1.6
7.4 6.2 6.9 6.6
160.0 156.0 112.0 127.0
Values expressed as %DM
a Paddocks I, II, III and IV used of manganese supplementation, in the control group (TC ) had 540 mg of Mn2+ that was present in other sources of mineral elements and added 1300 mg (T1300 ), 3800 mg (T3800 ) and 6300 mg (T6300 ) of Mn2+ per kilogram of mineral mixture. DM, dry matter; CP, crude protein; NDT, total digestible nutrients; CF, crude fiber; NDF, neutral detergent fiber; ADF, acid detergent fiber; EE, ether extract; MM, mineral matter in forage samples.
155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172
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isothiocyanate-Pisum sativum iodide, and 5,5 ,6,6 tetrachloro-1,1,3,3 -tetraethylbenzimidazolcarbocyanine. The mitochondrial activity was also measured due to its relationship with sperm motility. The sperm were classified into eight integrity categories depending on whether they exhibited intact (IMe) or damaged membranes (DMe), intact (IA) or damaged acrosomes (DA), and high (HM) or low (LM) mitochondrial activities. To determine sperm motility, a drop of fresh semen was placed between a slide and a coverslip and pre-heated to 37 ◦ C using a board heater. The percentage of sperm with rectilinear movement was then visually estimated under an optical microscope equipped with a platinum heater. The progressive velocity of the sperm in uniform motion was assessed using scores in the range of 0–5, where 0 indicates the absence of progressive movement and 5 refers to rapidly moving sperm with a vigorous forward movement (Colégio Brasileiro de Reproduc¸ão Animal, 1992). 2.4. Forage analysis
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The four paddocks used in the experiment had a similar topography and were covered with B. decumbens. At the beginning of the experiment, samples of this forage grass were cut at the grazing height to determine the concentration of Mn2+ in the grass through flame atomic absorption spectrometry. Official AOAC methods were adopted to determine the dry matter (DM), crude protein (CP), total digestible nutrients (TDN), crude fiber (CF), neutral detergent fiber (NDF), acid detergent fiber (ADF), ether extract (EE), and mineral matter (MM) in the forage samples.
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2.5. Data analyses
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The experimental design was based on a 4 × 4 Latin square, i.e., four treatments and four experimental
periods, which coincided with the seasons (spring, summer, autumn, and winter). The four treatments included the TC treatment, i.e., control treatment, which consisted of the protein mixture mineral without additional Mn2+ in its composition, the T1300 group, which consisted of the protein mineral mixture with 1300 mg Mn2+ /kg of treatment, the T3800 group, which consisted of the protein mineral mixture with 3800 mg Mn2+ /kg of treatment, and the T6300 group, which consisted of the protein mineral mixture containing 6300 mg Mn2+ /kg of treatment. It can be observed that all of the groups consumed each of the four mineral mixes during an experimental period of 56 d, i.e., at the end of the experiment, all of the groups had consumed all of the mineral treatments. Each of the four groups consisted of 15 Nelore bulls that were approximately 18–20 mo of age. The body weight of the bulls was similar between the groups (264.5 ± G1 = 22.5 kg, 261.5 ± G2 = 23.4 kg, 268.5 ± G3 = 19.6 kg, and 267.9 ± G4 = 22.5 kg). The animals grazed in pastures filled with B. decumbens and were rotated through the pastures and treatments every 70 d for a total of 280 d, as required by the experimental design. Given the importance of the project, the experimental design took into account the periods of the year (summer 2010, fall 2010, winter 2010, and spring 2010) as a covariate and the initial weight of the animals in accordance with the following mathematical model: Y ijkl = + bPI + Pi + Rj + Tk + εijkl , where Yijkl is the set of variables used to study the effect of the initial weight of the ith season or period, jth repetition, and kth treatment; is the average variable studied in the experiment; bPI represents the regression coefficient of the effect of the initial weight; Pi is the effect of the ith period or season for P = 1, 2, 3, and 4; Rj refers to the effect of the jth repetition for R = 1, 2, 3, and 4; Tk effect of kth treatment for
Please cite this article in press as: Reis, L.S.L.S., et al., Effect of manganese supplementation on the membrane integrity and the mitochondrial potential of the sperm of grazing Nelore bulls. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.06.033
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Fig. 1. Effect of dietary treatment (TC , T1300 , T3800 and T6300 ) on the frequency (mean + SD) of Nelore bull sperm rather with intact membranes (data pooled within each treatment, n = 60 per season). Spermatozoa were classified into eight integrity categories considering the combination of intact (IMe) or damaged membranes (DMe), intact (IA) or damaged acrosomes (DA) and high (HM) or low (LM) mitochondrial activity. Manganese supplementation, in the control group (TC ) had 540 mg of Mn2+ that was present in other sources of mineral elements and added 1300 mg (T1300 ), 3800 mg (T3800 ) and 6300 mg (T6300 ) of Mn2+ /kg of mineral mixture. Different letters in a same category indicate statistical differences between treatments (P < 0.05) and ns = non-significant (P > 0.05).
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T = 1, 2, 3 and 4, and εijklm is the experimental error, which was assumed to be normal and independently distributed, i.e., NID ( = 0, e2 = 1). The sperm integrity data were square root transformed (X + 1) prior to their analysis using an ANOVA. The diet was set as the variation source, and the animal weight was used as a covariate. Tukey’s multiple comparisons test was performed to determine the significance of the differences between means. Pearson’s correlation was used to test the association between the sperm membrane integrity, the sperm functionality (motility, vigor, and wave motion), and the Mn2+ concentration in the semen. The alpha error was set to 0.05.
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3. Results and discussion
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Findings in the present study show that supplementation with a large dose of Mn2+ compromises the integrity and functionality of the sperm of Nelore bulls. However, this improvement in the semen quality is offset by Mn2+ consumption even at the least amount tested. Compared with other studies, the Mn2+ concentrations in the forage samples were evaluated, and the magnitude of the Mn2+ fluctuations across the seasons was minimal (Table 1). Other studies have reported much greater Mn2+ concentrations in forage grasses. For example, Tebaldi et al. (2000) found a concentration in the range of 104.25–379.90 mg Mn/kg DM in forage grasses during the dry season and a concentration range of 57.85–591.10 mg/kg DM during the rainy season. Wunsch et al. (2005) described an annual variation in the range
of 166–1305 mg Mn/kg DM. In addition, the minimal pasture quality differences detected between the paddocks and across the seasons were counteracted by the cattle rotation. The control treatment (TC ) provided approximately 150 mg of Mn2+ to each bull, which is 3.75-fold greater than the daily dose of 40 mg recommended by the NRC (2000). Thus, the bulls that received the T6300 treatment consumed 1800 mg of Mn2+ /animal/day, which is 45-fold higher than the recommended amount. Despite this increase, the evaluation of the effect of dietary Mn2+ amounts on the sperm integrity (Fig. 1) showed that the sperm of the bulls that received the TC and the T6300 treatments had the greatest frequency of intact sperm [IMe, IA, LM] and the least number of sperm with acrosome damage [IMe, DA, LM]. In contrast, all of the groups that received a dietary supplementation of Mn2+ had the greatest incidence of damaged sperm [DMe, DA, LM] or sperm with damaged acrosomes [IMe, DA, LM] (Fig. 1). These important results indicate that the intermediate amount of Mn2+ , which were used in the T1300 and the T3800 treatments, compromised the membrane of the sperm of Nelore cattle. However, greater supplementation amounts, such as that tested with the T6300 treatment, cannot be considered harmful to the sperm integrity because the intact sperm [IMe, IA, LM] count in the group that received the T6300 treatment was similar to that observed in the group that received the TC treatment, which was considered to be the most desirable amount for supplementation (P > 0.05). The amount of Mn2+ supplementation used in the T6300 treatment, however, should not be recommended because its use resulted
Please cite this article in press as: Reis, L.S.L.S., et al., Effect of manganese supplementation on the membrane integrity and the mitochondrial potential of the sperm of grazing Nelore bulls. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.06.033
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Table 2 Pearson’s correlation coefficients among sperm membrane integrity and functionality (motility, vigor and wave motion), semen Mn2+ levels of Nelore bulls. Spermatozoa category
n
IMe, IA, HM IMe, IA, LM IMe, DA, HM IMe, DA, LM DMe, IA, HM DMe, IA, LM DMe, DA, HM DMe, DA, LM
Coefficient of correlation
281 281 281 281 281 281 281 281
Sperm motility
Sperm vigor
Wave motion
Semen Mn2+
−0.02 0.24** 0.05 0.31** 0.02 −0.14* −0.12* −0.26**
−0.03 0.23** 0.09 0.22** 0.03 −0.09 −0.11* −0.22**
0.01 0.27** 0.10 0.21** 0.01 −0.18** −0.07 −0.19**
−0.01 0.06 −0.02 −0.34** −0.01 0.13* 0.01 0.02
Intact (IMe) or damaged membranes (DMe), intact (IA) or damaged acrosomes (DA) and high (HM) or low (LM) mitochondrial activity. * Significant at P < 0.05. ** Significant at P < 0.01.
282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297
in a greater number of completely damaged sperm [DMe, DA, LM; Fig. 1]. McDowell (1992) reported that animals deficient in Mn2+ can have sperm abnormalities in the functioning and structure of mitochondria, but it has not been established whether a larger dose of Mn2+ can also interfere with the function of this organelle. As shown in Fig. 1, there were no sperm with a greater mitochondrial potential (HM) in the semen of bulls during the trial period. This finding indicates that diets with a larger amounts of Mn2+ can also induce changes in the mitochondria of sperm, which resulted in the bulls having sperm with a low mitochondrial potential (LM). Interestingly, the amounts of dietary Mn2+ were positively correlated with sperm displaying intact membranes and damaged acrosomes, i.e., the [IMe, DA, LM]
category, and negatively correlated with sperm with damaged membranes and intact acrosomes, i.e., the [DMe, IA, LM] category (Table 2). Therefore, to maximize the production of sperm with both membrane and acrosome integrity, the mean Mn2+ concentration in the semen must be balanced. In the present study, this was achieved with the TC treatment, which indicates that a dose of 2.11 ± 1.39 mg Mn2+ /L of semen (150 mg Mn/day) promoted sperm integrity. Significant interactions (P > 0.05) were found between the treatments with Mn2+ and the experimental groups (Table 3). These show that the integrity of the plasma membrane and the acrosomal and mitochondrial potential are dependent on the concentration of Mn2+ in the diet, which is associated with the spermatogenic response of bulls toward these treatments.
Table 3
Q3 Interaction among the effects of supplementation with manganese and experimental groups on the plasma membrane integrity, acrosomal and mitochondrial potential of spermatozoa from Nelore bulls. Interaction
Frequency (%)a
Treatment with Mn and experimental groups
IMe, IA, HM
IMe, IA, LM**
IMe, DA, HM
IMe, DA, LM**
DMe, IA, HM
DMe, IA, LM**
DMe, DA, HM
DMe, DA, LM**
TC T1300 T3800 T6300
G1
1.00 1.00 1.14 1.00
± ± ± ±
0.00 0.00 0.37 0.00
6.92 2.41 1.23 3.85
± ± ± ±
1.37 0.67 0.37 2.96
1.00 1.09 1.00 1.00
± ± ± ±
0.00 0.45 0.00 0.00
1.56 6.13 1.03 1.00
± ± ± ±
0.98 2.20 0.11 0.00
1.00 1.00 1.00 1.00
± ± ± ±
0.00 0.00 0.00 0.00
6.53 5.14 8.82 6.93
± ± ± ±
0.99 1.85 0.68 1.74
1.00 1.00 1.05 1.00
± ± ± ±
0.00 0.00 0.19 0.00
2.06 3.26 4.58 5.06
± ± ± ±
1.83 1.06 1.24 1.79
TC T1300 T3800 T6300
G2
1.00 1.10 1.00 1.09
± ± ± ±
0.00 0.36 0.00 0.27
4.67 2.29 2.99 2.79
± ± ± ±
3.38 0.79 0.81 1.31
1.00 1.00 1.00 1.00
± ± ± ±
0.00 0.00 0.00 0.00
1.00 5.97 6.37 2.31
± ± ± ±
0.00 1.50 0.80 1.17
1.00 1.00 1.00 1.03
± ± ± ±
0.00 0.00 0.00 0.10
7.28 6.75 6.50 7.48
± ± ± ±
2.31 1.03 0.95 1.04
1.00 1.00 1.00 1.00
± ± ± ±
0.00 0.00 0.00 0.00
2.91 3.60 3.11 5.33
± ± ± ±
1.70 1.01 0.82 1.59
TC T1300 T3800 T6300
G3
1.00 1.00 1.00 1.00
± ± ± ±
0.00 0.00 0.00 0.00
1.32 1.00 4.99 6.30
± ± ± ±
0.58 0.00 2.45 0.70
1.00 1.00 1.00 1.00
± ± ± ±
0.00 0.00 0.00 0.00
1.24 1.00 3.64 3.95
± ± ± ±
0.45 0.00 2.95 0.82
1.00 1.00 1.00 1.00
± ± ± ±
0.00 0.00 0.00 0.00
8.76 8.57 5.15 6.27
± ± ± ±
0.92 1.33 1.37 0.37
1.00 1.00 1.00 1.00
± ± ± ±
0.00 0.00 0.00 0.00
4.43 4.87 4.15 2.80
± ± ± ±
1.82 1.89 1.68 0.57
G4
1.00 1.00 1.00 1.00
± ± ± ±
0.00 0.00 0.00 0.00
1.94 3.95 1.00 3.05
± ± ± ±
0.46 3.35 0.00 1.34
1.00 1.00 1.00 1.00
± ± ± ±
0.00 0.00 0.00 0.00
7.22 1.10 1.00 5.33
± ± ± ±
0.62 0.28 0.00 1.37
1.00 1.00 1.00 1.00
± ± ± ±
0.00 0.00 0.00 0.00
5.99 7.02 7.48 5.89
± ± ± ±
1.60 2.09 2.54 1.59
1.00 1.00 1.53 1.09
± ± ± ±
0.00 0.00 2.06 0.33
2.90 4.46 5.35 4.10
± ± ± ±
0.94 1.72 2.52 1.24
TC T1300 T3800 T6300
a Adjusted means for extracting the square root (X + 1) da [IMe, IA, HM], [IMe, IA, LM], [IMe, DA, HM], [IMe, DA, LM], [DMe, IA, HM], [DMe, IA, HM], [DMe, DA, HM] and [DMe, DA, LM]. Plasma membrane intact (IMe) or injured (IMe), acrosome intact (IA) or injured (DA) and high (HM) or low mitochondrial potential (LM). *Interaction significant (P < 0.05) between manganese supplementation (control group (TC ) had 540 mg of Mn2+ that was present in other sources of mineral elements and added 1300 mg (T1300 ), 3800 mg (T3800 ) and 6300 mg (T6300 ) of Mn2+ /kg of mineral mixture) and groups of bulls. ** Interaction significant (P < 0.01) between manganese supplementation (control group (TC ) had 540 mg of Mn2+ that was present in other sources of mineral elements and added 1300 mg (T1300 ), 3800 mg (T3800 ) and 6300 mg (T6300 ) of Mn2+ /kg of mineral mixture) and groups of bulls.
Please cite this article in press as: Reis, L.S.L.S., et al., Effect of manganese supplementation on the membrane integrity and the mitochondrial potential of the sperm of grazing Nelore bulls. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.06.033
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In fact, the plasma and acrosomal membranes of the sperm may have been injured during spermatogenesis because such lesions mainly occur during the spermatogenic process, as indicated by Garner and Hafez (2004). Despite the fact that supplemental Mn2+ alters the physiological processes that occur during spermatogenesis in bulls, data in Table 3 indicate the supplementation control (TC ) exhibited a 50% efficiency in the stimulation of the production of intact sperm [DMe, IA, LM], as observed in the G1 and G2 groups. However, this supplementation resulted in a greater efficiency of 75% for the production of totally damaged sperm [DMe, DA, LM], as was observed in groups G1 , G2 , and G4 , and sperm with an injured acrosomal membrane [DMe, DA, LM], as was observed in groups G1 , G2 , and G3 . Therefore, these results confirm the findings presented in Fig. 1, which demonstrate that the TC treatment was beneficial to the integrity of both the plasma and the acrosomal membranes of the bull sperm. Furthermore, these results show that the addition of larger amounts of Mn2+ to mineral mixes, as is performed by most companies that produce mineral supplements, is harmful to the sperm membrane. The semen produced by the bulls that received the TC treatment had a greater quality and greater proportion of intact sperm [IMe, IA, LM; Fig. 1]. This sperm category is directly associated with sperm motility, vigor, and wave motion (Table 2), which are important because vigorous sperm more efficiently reach the oviduct and fertilize oocytes and thus enhance fertilization rates. In contrast, sperm with damaged membranes [DMe, IA, LM/DMe, DA, HM/DMe, DA, LM], which were mainly found in the bulls that received Mn2+ supplementation, were negatively associated with the functional variables (motility, vigor, and wave motion). The number of severely damaged sperm in the semen obtained from bulls that were receiving Mn2+ supplementation (Fig. 1) indicates the decreased fertilization potential. This finding corroborates the results reported by McDowell (1992), who demonstrated that dairy cattle in a pasture that contained less than 200 ppm Mn2+ exhibited the greatest number of services per pregnancy and had a lesser birth rate of calves. These results were somewhat expected because the amounts of Mn2+ fed to these animals were 9.1- to 44.5-fold greater than those recommended by the NRC (2000). According to the results, a daily large dose of Mn2+ is detrimental to the plasma and acrosomal membrane integrity of the sperm of Nelore bulls.
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Conflict of interest
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Acknowledgements The authors would like to thank Fundac¸ão de Amparo a Q2 Pesquisa do Estado de São Paulo for sponsoring the study and TORTUGA Companhia Zootécnica Agrária for the support provided. References Alberts, A., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P., 2010. Estrutura da membrane. In: Alberts, A., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P. (Eds.), Biologia molecular da célula. Artmed, Porto Alegre, Brazil, pp. 617–650. Ansenberger-Fricano, K., Ganini, D., Mao, M., Chatterjee, S., Dallas, S., Mason, R.P., Stadler, K., Santos, J.H., Bonini, M.G., 2012. The peroxidase activity of mitochondrial superoxide dismutase. Free Radic. Biol. Med. 12, 1080–1085. Associac¸ão Brasileira das Indústrias de Suplementos Minerais (ASBRAM), 2003. Guia prático para a correta suplementac¸ão pecuária. Contatocom, Sorocaba, Brazil. Celeghini, E.C.C., Arruda, R.P., Andrade, A.F.C., Nascimento, J., Raphael, C.F., 2007. Practical techniques for bovine spermatozoa simultaneous fluorimetric assessment of plasma, acrosomal and mitochondrial membranes. Reprod. Domest. Anim. 42, 479–488. Cheema, R.S., Bansal, A.K., Bilaspuri, G.S., 2009. Manganese provides antioxidant protection for spermatozoa cryopreservation that may offer new consideration for clinical fertility. Oxid. Med. Cell. Longev. 2, 152–159. Colégio Brasileiro de Reproduc¸ão Animal (CBRA), 1992. Procedimentos para exame andrológico e avaliac¸ão de semen animal. Belo Horizonte, Brazil. Garner, D.L., Hafez, E.S.E., 2004. Espermatozóides e plasma seminal. In: Hafez, E.S.E., Hafez, B. (Eds.), Reproduc¸ão Animal. , sétima ed. Manole, Barueri, Brazil, pp. 97–110. Grace, N.D., 1973. Effect of high dietary Mn levels on the growth rate and the level of mineral elements in the plasma and soft tissues of sheep. N. Z. J. Agric. Res. 16, 177–180. Hansen, S.L., Spears, J.W., Lloyd, K.E., Whisnant, C.S., 2006. Growth, reproductive performance, and manganese status of heifers fed varying concentrations of manganese. J. Anim. Sci. 8, 3375–3380. Jenkins, K.J., Kramer, J.K.G., 1991. Effect of excess dietary manganese on lipid composition of calf blood plasma, heart and liver. J. Dairy Sci. 74, 3944–3948. McDowell, L.R., 1992. Manganese. In: McDowell, L.R. (Ed.), Minerals in Animal and Human Nutritional. Academic Press, San Diego, New York, pp. 246–264. National Research Council (NRC), 2000. Minerals. In: Nutrient Requirements of Beef Cattle. Natl. Acad. Press, Washington, DC, pp. 54–74. Szentmihályi, K., Vinkler, P., Fodor, J., Balla, J., Lakatos, B., 2006. The role of manganese in the human organism. Orv. Hetil. 147, 2027–2030. Tebaldi, F.L.H., Coelho da Silva, J.F., Maldonado Vasquez, H., Thiebaut, J.T.L., 2000. Composic¸ão mineral das pastagens das regiões norte e nordeste do Estado do Rio de Janeiro. 2. Manganês, ferro, zinco, cobre, cobalto, molibdênio e chumbo. Rev. Bras. Zootec. 29, 616–629. Underwood, E.J., Sutlle, N.F., 2004. Manganese. In: Underwood, E.J., Sutlle, N.F. (Eds.), The Mineral Nutrition of Livestock. , third ed. CAB International, Wallingford, Oxfordshire, pp. 397–419. Wunsch, C., Barcellos, J.O.J., Prates, E.R., Grecellé, R.A., Costa, E.C., 2005. Microminerais para bovinos de corte nas pastagens nativas dos Campos de Cima da Serra, RS, Brasil. Cienc. Rural 35, 903–908.
No conflicts of interest between authors.
Please cite this article in press as: Reis, L.S.L.S., et al., Effect of manganese supplementation on the membrane integrity and the mitochondrial potential of the sperm of grazing Nelore bulls. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.06.033
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Effect of manganese supplementation on the membrane integrity and the mitochondrial potential of the sperm of grazing Nelore bulls
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L.S.L.S. Reis a,b,∗ , A.A. Ramos c , A.S. Camargos b , E. Oba b,1 a
Department of Animal Nutrition and Food Science, University of Santo Amaro, São Paulo, SP, Brazil Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science, São Paulo State University, Botucatu, SP, Brazil c Department of Animal Production, School of Veterinary Medicine and Animal Science, São Paulo State University, Botucatu, SP, Brazil b
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Article history: Received 17 January 2013 Received in revised form 24 June 2014 Accepted 30 June 2014 Available online xxx
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Keywords: Manganese Spermatozoa Integrity of sperm membrane and acrosome Cattle Mineral salt
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1. Introduction
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The effect of dietary manganese (Mn2+ ) supplementation on the reproductive performance of Nelore bulls was evaluated by assessment of sperm membrane integrity. Sixty Nelore bulls (Bos taurus indicus) aged 18–20 mo were randomly divided into four groups (n = 15) receiving dietary Mn2+ supplementation at 540, 1300, 3800 and 6300 mg/kg (treatments TC , T1300 , T3800 and T6300 , respectively). The diets were changed for the groups every 70 d. Semen samples were obtained 15 and 56 d after the diet change, which corresponded to the period of adjustment to the new diet and the time required for a complete spermatogenesis cycle, respectively. Sperm integrity was assessed by detection of: intact (IMe) or damaged (DMe) membranes, intact (IA) or damaged (DA) acrosomes, and high (HM) or low (LM) mitochondrial membrane potentials. Only bulls from the TC treatment showed a significant increase in the production of intact sperm [IMe/IA/LM] and decrease in the production of sperm with damaged acrosome [IMe/DA/LM] or completely damaged sperm [DMe/DA/LM] (P < 0.05). The Mn2+ concentrations in the semen were positively correlated with the incidence of sperm with IMe, DA, and LM and negatively correlated with number of sperm with DMe, IA, and LM. Therefore, dietary Mn2+ supplementation for Nelore bulls must be limited to 540 mg of Mn2+ /kg given that higher doses are detrimental to the integrity of the plasma and acrosomal sperm membranes. © 2014 Published by Elsevier B.V.
Manganese (Mn) has an essential role in the physiological processes of cattle, protein synthesis and processing (Szentmihályi et al., 2006), carbohydrate metabolism (Underwood and Sutlle, 2004; Szentmihályi
∗ Corresponding author at: Rua Padre Davi 190, Assis CEP 19800-220, SP, Brazil. Tel.: +55 18 9776 2550. E-mail address: [email protected] (L.S.L.S. Reis). 1 CNPq researcher.
et al., 2006), and lipid metabolism (Jenkins and Kramer, 1991; Underwood and Sutlle, 2004; Szentmihályi et al., 2006; Hansen et al., 2006). Thus, this mineral can be correlated with the integrity of the sperm plasma membrane and is involved in the metabolism of the main organic components that compose this membrane, which include phospholipids and integral and peripheral membrane proteins (Alberts et al., 2010). Moreover, Mn is also involved in the protection of the plasma membrane of the head, acrosome, and mitochondria against free radicals (hydroperoxides), which are formed by decomposing metalloenzymes and are highly oxidative and
http://dx.doi.org/10.1016/j.anireprosci.2014.06.033 0378-4320/© 2014 Published by Elsevier B.V.
Please cite this article in press as: Reis, L.S.L.S., et al., Effect of manganese supplementation on the membrane integrity and the mitochondrial potential of the sperm of grazing Nelore bulls. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.06.033
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damaging to membranes, proteins, and DNA (Underwood and Sutlle, 2004; Hansen et al., 2006; Cheema et al., 2009; Ansenberger-Fricano et al., 2012). The NRC (2000) recommends that beef cattle receive a daily intake of 40 mg of Mn during their reproductive periods. However, in many countries, Mn2+ is naturally present at concentrations that exceed the NRCrecommended dose by 30-fold (2000). In Brazil, there are reports of concentrations of up to 1.305 mg Mn2+ /kg DM (Wunsch et al., 2005), and New Zealand reports concentrations of 400 mg Mn2+ /kg DM (Grace, 1973). Moreover, some manufacturers continue to add increased amounts of Mn2+ to mineral supplements, and these can reach up to 2000 ppm in Brazil. This is of concern because the effects of these diets with excessive concentrations of Mn2+ on the integrity of the plasma membrane of the head, acrosome, and mitochondria of bull sperm are not well elucidated. To establish suitable Mn2+ supplementation for the diets of Nelore bulls that do not negatively impact their reproductive performance, the present study tested the effect of dietary supplementation with different amounts of Mn2+ on the sperm membrane, the acrosomal integrity, and the mitochondrial membrane potential.
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2. Materials and methods
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The procedures used in this study were approved by the Ethics Committee on Animal Use of the School of Veterinary Medicine and Animal Science of São Paulo State University, Botucatu, SP, Brazil. 2.1. Experimental design
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The duration of the experiment, which was performed in Lutécia, SP, Brazil, was 280 d. The altitude of the study area was 602 m, and the climate of the study area was tropical and characterized by a rainy season (October–April) and a dry season (May–September), a mean annual precipitation of 1300 mm, a relative air humidity of 64%, and a mean temperature of 25 ◦ C. Using a 4 × 4 randomized block (Latin square) design, we divided the cattle into four groups (n = 15) with a similar live weight (G1 = 264.5 ± 22.5 kg; G2 = 261.5 ± 23.4 kg; G3 = 268.5 ± 19.6 kg, and G4 = 267.9 ± 22.5 kg); the four groups were fed four experimental diets. At the onset of the experiment, the 60 Nelore bulls (Bos taurus indicus) were 18–20 mo of age. Every 70 d, the experimental groups were transferred to another paddock and fed a different experimental diet; thus, all of the bulls were fed all the diets by the end of the study. The sperm quality was assessed at the end of each treatment period.
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2.2. Experimental diets
71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87
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The bulls grazed on Brachiaria decumbens (Table 1) in an extended grazing system and received a supplemental protein mineral mixture with different amounts of Mn2+ ad libitum. This protein mineral supplement was used to meet the nutritional protein requirements. The protein sources were urea, ammonium sulfate, and soybean meal.
The mineral mixture was prepared based on the results of a pilot project, which evaluated the use of a basic mixture of mineral salt. Based on the daily consumption, Mn2+ was added such that each animal consumed specific daily quantities of Mn2+ . The base diet of the control group did not meet the nutritional Mn2+ requirements recommended by the NRC (2000) because, in Brazil, this mineral is present in the environment greater amounts than the recommended dietary intake (Wunsch et al., 2005). Thus, to observe the effects of manganese on the membrane integrity and the acrosomal and mitochondrial potential of sperm, four mineral supplements were formulated. The mixture (1 kg) contained 402 mg of copper, 8000 mg of sulfur, 20 mg of iron, 2.8 mg of selenium, 1400 mg of zinc, 150 mg of fluoride, 400 g of crude protein, and 337.5 g of protein equivalent. In addition, the experimental diets contained 540 mg of Mn2+ (control treatment, TC ), which was present in the other sources of mineral elements and/or soybean meal included in the mixture, 1300 mg of Mn2+ (T1300 ), 3800 mg of Mn2+ (T3800 ), and 6300 mg (T6300 ) of Mn2+ per kilogram of mineral mixture. The diets were offered in the paddocks in 13-cmlong feeders for each animal. A water trough was placed 50 m from the feeder, as recommended by the Brazilian Association of Mineral Supplement Industries (2003). The consumption of the mineral mixtures was determined weekly throughout the experiment. At the onset of the study period, the mixture was weighed and placed into the trough; the mineral mix was then weighed 7 d later. By subtracting the second weight from the first weight, the average consumption was determined for each batch of bulls. After this batch average consumption was divided by the number of bulls in the lot, the average daily consumption of each supplement was determined. As a result, the consumption of the additives was found to be 369 g/bull/day. 2.3. Semen collection and evaluation The semen samples were collected by electroejaculation twice during each study period (season). Because these animals are generally bred using natural mating rather than AI, these bulls were not adapted to semen collection with an artificial vagina. The first collection (Day 0) was performed 15 d after the cattle had been moved to a new paddock, i.e., a new Mn2+ treatment. However, because the aim was to initiate a new spermatogenesis cycle, this semen was not evaluated. After 56 d (Day 56), i.e., at the end of the season, semen samples were collected again to determine the integrity and functionality of the sperm. The motility, sperm vigor, turmoil, sperm membrane and acrosome integrity and mitochondrial activity were determined after the seeds on the farm were harvested. Thus, all the equipments needed to perform these analyses in sperm were brought to the farm. The sperm membrane and acrosome integrity were assessed by fluorescence microscopy using a combination of fluorescent probes, as recommended by Celeghini et al. (2007): propidium iodide, Hoechst 33342, fluorescein
Please cite this article in press as: Reis, L.S.L.S., et al., Effect of manganese supplementation on the membrane integrity and the mitochondrial potential of the sperm of grazing Nelore bulls. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.06.033
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Table 1 Cattle lot rotation among pastures and chemical composition of Brachiaria decumbens. Season
Paddocka
Group
Mn2+ (ppm)
DM
CP
NDT
CF
NDF
ADF
EE
MM
Summer/10
I II III IV
G1 G2 G3 G4
29.3 28.4 27.2 29.4
7.2 9.2 9.6 7.6
60.3 59.6 58.4 64.0
29.6 26.5 22.1 25.6
74.2 72.9 74.9 68.4
34.8 39.7 32.7 41.9
1.5 1.9 1.9 1.6
6.8 7.4 7.2 5.6
195.0 141.0 100.0 160.0
Fall/10
I II III IV
G4 G3 G2 G1
40.1 42.4 63.5 57.1
5.3 5.3 4.8 4.4
59.4 59.1 60.4 61.3
31.9 32.2 29.4 29.3
77.2 76.1 76.9 75.4
41.8 43.4 47.1 46.7
1.7 1.4 1.6 1.4
6.6 6.6 6.7 5.7
216.0 219.0 117.0 157.0
Winter/10
I II III IV
G3 G4 G1 G2
65.2 78.0 78.7 60.4
5.2 4.4 3.9 4.2
59.0 60.7 61.1 60.0
30.6 30.0 29.4 31.7
78.8 80.7 78.5 77.4
44.1 49.1 48.8 44.0
1.4 1.1 2.4 1.5
7.5 5.9 5.8 5.6
235.0 265.0 183.0 153.0
Spring/10
I II III IV
G2 G1 G4 G3
46.1 40.2 40.3 42.8
8.5 6.7 7.9 7.5
64.5 62.4 63.2 63.9
23.2 28.0 25.9 25.0
67.3 72.0 70.8 72.5
30.6 41.2 37.8 37.6
1.6 1.5 1.3 1.6
7.4 6.2 6.9 6.6
160.0 156.0 112.0 127.0
Values expressed as %DM
a Paddocks I, II, III and IV used of manganese supplementation, in the control group (TC ) had 540 mg of Mn2+ that was present in other sources of mineral elements and added 1300 mg (T1300 ), 3800 mg (T3800 ) and 6300 mg (T6300 ) of Mn2+ per kilogram of mineral mixture. DM, dry matter; CP, crude protein; NDT, total digestible nutrients; CF, crude fiber; NDF, neutral detergent fiber; ADF, acid detergent fiber; EE, ether extract; MM, mineral matter in forage samples.
155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172
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isothiocyanate-Pisum sativum iodide, and 5,5 ,6,6 tetrachloro-1,1,3,3 -tetraethylbenzimidazolcarbocyanine. The mitochondrial activity was also measured due to its relationship with sperm motility. The sperm were classified into eight integrity categories depending on whether they exhibited intact (IMe) or damaged membranes (DMe), intact (IA) or damaged acrosomes (DA), and high (HM) or low (LM) mitochondrial activities. To determine sperm motility, a drop of fresh semen was placed between a slide and a coverslip and pre-heated to 37 ◦ C using a board heater. The percentage of sperm with rectilinear movement was then visually estimated under an optical microscope equipped with a platinum heater. The progressive velocity of the sperm in uniform motion was assessed using scores in the range of 0–5, where 0 indicates the absence of progressive movement and 5 refers to rapidly moving sperm with a vigorous forward movement (Colégio Brasileiro de Reproduc¸ão Animal, 1992). 2.4. Forage analysis
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The four paddocks used in the experiment had a similar topography and were covered with B. decumbens. At the beginning of the experiment, samples of this forage grass were cut at the grazing height to determine the concentration of Mn2+ in the grass through flame atomic absorption spectrometry. Official AOAC methods were adopted to determine the dry matter (DM), crude protein (CP), total digestible nutrients (TDN), crude fiber (CF), neutral detergent fiber (NDF), acid detergent fiber (ADF), ether extract (EE), and mineral matter (MM) in the forage samples.
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2.5. Data analyses
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The experimental design was based on a 4 × 4 Latin square, i.e., four treatments and four experimental
periods, which coincided with the seasons (spring, summer, autumn, and winter). The four treatments included the TC treatment, i.e., control treatment, which consisted of the protein mixture mineral without additional Mn2+ in its composition, the T1300 group, which consisted of the protein mineral mixture with 1300 mg Mn2+ /kg of treatment, the T3800 group, which consisted of the protein mineral mixture with 3800 mg Mn2+ /kg of treatment, and the T6300 group, which consisted of the protein mineral mixture containing 6300 mg Mn2+ /kg of treatment. It can be observed that all of the groups consumed each of the four mineral mixes during an experimental period of 56 d, i.e., at the end of the experiment, all of the groups had consumed all of the mineral treatments. Each of the four groups consisted of 15 Nelore bulls that were approximately 18–20 mo of age. The body weight of the bulls was similar between the groups (264.5 ± G1 = 22.5 kg, 261.5 ± G2 = 23.4 kg, 268.5 ± G3 = 19.6 kg, and 267.9 ± G4 = 22.5 kg). The animals grazed in pastures filled with B. decumbens and were rotated through the pastures and treatments every 70 d for a total of 280 d, as required by the experimental design. Given the importance of the project, the experimental design took into account the periods of the year (summer 2010, fall 2010, winter 2010, and spring 2010) as a covariate and the initial weight of the animals in accordance with the following mathematical model: Y ijkl = + bPI + Pi + Rj + Tk + εijkl , where Yijkl is the set of variables used to study the effect of the initial weight of the ith season or period, jth repetition, and kth treatment; is the average variable studied in the experiment; bPI represents the regression coefficient of the effect of the initial weight; Pi is the effect of the ith period or season for P = 1, 2, 3, and 4; Rj refers to the effect of the jth repetition for R = 1, 2, 3, and 4; Tk effect of kth treatment for
Please cite this article in press as: Reis, L.S.L.S., et al., Effect of manganese supplementation on the membrane integrity and the mitochondrial potential of the sperm of grazing Nelore bulls. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.06.033
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Fig. 1. Effect of dietary treatment (TC , T1300 , T3800 and T6300 ) on the frequency (mean + SD) of Nelore bull sperm rather with intact membranes (data pooled within each treatment, n = 60 per season). Spermatozoa were classified into eight integrity categories considering the combination of intact (IMe) or damaged membranes (DMe), intact (IA) or damaged acrosomes (DA) and high (HM) or low (LM) mitochondrial activity. Manganese supplementation, in the control group (TC ) had 540 mg of Mn2+ that was present in other sources of mineral elements and added 1300 mg (T1300 ), 3800 mg (T3800 ) and 6300 mg (T6300 ) of Mn2+ /kg of mineral mixture. Different letters in a same category indicate statistical differences between treatments (P < 0.05) and ns = non-significant (P > 0.05).
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T = 1, 2, 3 and 4, and εijklm is the experimental error, which was assumed to be normal and independently distributed, i.e., NID ( = 0, e2 = 1). The sperm integrity data were square root transformed (X + 1) prior to their analysis using an ANOVA. The diet was set as the variation source, and the animal weight was used as a covariate. Tukey’s multiple comparisons test was performed to determine the significance of the differences between means. Pearson’s correlation was used to test the association between the sperm membrane integrity, the sperm functionality (motility, vigor, and wave motion), and the Mn2+ concentration in the semen. The alpha error was set to 0.05.
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3. Results and discussion
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Findings in the present study show that supplementation with a large dose of Mn2+ compromises the integrity and functionality of the sperm of Nelore bulls. However, this improvement in the semen quality is offset by Mn2+ consumption even at the least amount tested. Compared with other studies, the Mn2+ concentrations in the forage samples were evaluated, and the magnitude of the Mn2+ fluctuations across the seasons was minimal (Table 1). Other studies have reported much greater Mn2+ concentrations in forage grasses. For example, Tebaldi et al. (2000) found a concentration in the range of 104.25–379.90 mg Mn/kg DM in forage grasses during the dry season and a concentration range of 57.85–591.10 mg/kg DM during the rainy season. Wunsch et al. (2005) described an annual variation in the range
of 166–1305 mg Mn/kg DM. In addition, the minimal pasture quality differences detected between the paddocks and across the seasons were counteracted by the cattle rotation. The control treatment (TC ) provided approximately 150 mg of Mn2+ to each bull, which is 3.75-fold greater than the daily dose of 40 mg recommended by the NRC (2000). Thus, the bulls that received the T6300 treatment consumed 1800 mg of Mn2+ /animal/day, which is 45-fold higher than the recommended amount. Despite this increase, the evaluation of the effect of dietary Mn2+ amounts on the sperm integrity (Fig. 1) showed that the sperm of the bulls that received the TC and the T6300 treatments had the greatest frequency of intact sperm [IMe, IA, LM] and the least number of sperm with acrosome damage [IMe, DA, LM]. In contrast, all of the groups that received a dietary supplementation of Mn2+ had the greatest incidence of damaged sperm [DMe, DA, LM] or sperm with damaged acrosomes [IMe, DA, LM] (Fig. 1). These important results indicate that the intermediate amount of Mn2+ , which were used in the T1300 and the T3800 treatments, compromised the membrane of the sperm of Nelore cattle. However, greater supplementation amounts, such as that tested with the T6300 treatment, cannot be considered harmful to the sperm integrity because the intact sperm [IMe, IA, LM] count in the group that received the T6300 treatment was similar to that observed in the group that received the TC treatment, which was considered to be the most desirable amount for supplementation (P > 0.05). The amount of Mn2+ supplementation used in the T6300 treatment, however, should not be recommended because its use resulted
Please cite this article in press as: Reis, L.S.L.S., et al., Effect of manganese supplementation on the membrane integrity and the mitochondrial potential of the sperm of grazing Nelore bulls. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.06.033
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Table 2 Pearson’s correlation coefficients among sperm membrane integrity and functionality (motility, vigor and wave motion), semen Mn2+ levels of Nelore bulls. Spermatozoa category
n
IMe, IA, HM IMe, IA, LM IMe, DA, HM IMe, DA, LM DMe, IA, HM DMe, IA, LM DMe, DA, HM DMe, DA, LM
Coefficient of correlation
281 281 281 281 281 281 281 281
Sperm motility
Sperm vigor
Wave motion
Semen Mn2+
−0.02 0.24** 0.05 0.31** 0.02 −0.14* −0.12* −0.26**
−0.03 0.23** 0.09 0.22** 0.03 −0.09 −0.11* −0.22**
0.01 0.27** 0.10 0.21** 0.01 −0.18** −0.07 −0.19**
−0.01 0.06 −0.02 −0.34** −0.01 0.13* 0.01 0.02
Intact (IMe) or damaged membranes (DMe), intact (IA) or damaged acrosomes (DA) and high (HM) or low (LM) mitochondrial activity. * Significant at P < 0.05. ** Significant at P < 0.01.
282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297
in a greater number of completely damaged sperm [DMe, DA, LM; Fig. 1]. McDowell (1992) reported that animals deficient in Mn2+ can have sperm abnormalities in the functioning and structure of mitochondria, but it has not been established whether a larger dose of Mn2+ can also interfere with the function of this organelle. As shown in Fig. 1, there were no sperm with a greater mitochondrial potential (HM) in the semen of bulls during the trial period. This finding indicates that diets with a larger amounts of Mn2+ can also induce changes in the mitochondria of sperm, which resulted in the bulls having sperm with a low mitochondrial potential (LM). Interestingly, the amounts of dietary Mn2+ were positively correlated with sperm displaying intact membranes and damaged acrosomes, i.e., the [IMe, DA, LM]
category, and negatively correlated with sperm with damaged membranes and intact acrosomes, i.e., the [DMe, IA, LM] category (Table 2). Therefore, to maximize the production of sperm with both membrane and acrosome integrity, the mean Mn2+ concentration in the semen must be balanced. In the present study, this was achieved with the TC treatment, which indicates that a dose of 2.11 ± 1.39 mg Mn2+ /L of semen (150 mg Mn/day) promoted sperm integrity. Significant interactions (P > 0.05) were found between the treatments with Mn2+ and the experimental groups (Table 3). These show that the integrity of the plasma membrane and the acrosomal and mitochondrial potential are dependent on the concentration of Mn2+ in the diet, which is associated with the spermatogenic response of bulls toward these treatments.
Table 3
Q3 Interaction among the effects of supplementation with manganese and experimental groups on the plasma membrane integrity, acrosomal and mitochondrial potential of spermatozoa from Nelore bulls. Interaction
Frequency (%)a
Treatment with Mn and experimental groups
IMe, IA, HM
IMe, IA, LM**
IMe, DA, HM
IMe, DA, LM**
DMe, IA, HM
DMe, IA, LM**
DMe, DA, HM
DMe, DA, LM**
TC T1300 T3800 T6300
G1
1.00 1.00 1.14 1.00
± ± ± ±
0.00 0.00 0.37 0.00
6.92 2.41 1.23 3.85
± ± ± ±
1.37 0.67 0.37 2.96
1.00 1.09 1.00 1.00
± ± ± ±
0.00 0.45 0.00 0.00
1.56 6.13 1.03 1.00
± ± ± ±
0.98 2.20 0.11 0.00
1.00 1.00 1.00 1.00
± ± ± ±
0.00 0.00 0.00 0.00
6.53 5.14 8.82 6.93
± ± ± ±
0.99 1.85 0.68 1.74
1.00 1.00 1.05 1.00
± ± ± ±
0.00 0.00 0.19 0.00
2.06 3.26 4.58 5.06
± ± ± ±
1.83 1.06 1.24 1.79
TC T1300 T3800 T6300
G2
1.00 1.10 1.00 1.09
± ± ± ±
0.00 0.36 0.00 0.27
4.67 2.29 2.99 2.79
± ± ± ±
3.38 0.79 0.81 1.31
1.00 1.00 1.00 1.00
± ± ± ±
0.00 0.00 0.00 0.00
1.00 5.97 6.37 2.31
± ± ± ±
0.00 1.50 0.80 1.17
1.00 1.00 1.00 1.03
± ± ± ±
0.00 0.00 0.00 0.10
7.28 6.75 6.50 7.48
± ± ± ±
2.31 1.03 0.95 1.04
1.00 1.00 1.00 1.00
± ± ± ±
0.00 0.00 0.00 0.00
2.91 3.60 3.11 5.33
± ± ± ±
1.70 1.01 0.82 1.59
TC T1300 T3800 T6300
G3
1.00 1.00 1.00 1.00
± ± ± ±
0.00 0.00 0.00 0.00
1.32 1.00 4.99 6.30
± ± ± ±
0.58 0.00 2.45 0.70
1.00 1.00 1.00 1.00
± ± ± ±
0.00 0.00 0.00 0.00
1.24 1.00 3.64 3.95
± ± ± ±
0.45 0.00 2.95 0.82
1.00 1.00 1.00 1.00
± ± ± ±
0.00 0.00 0.00 0.00
8.76 8.57 5.15 6.27
± ± ± ±
0.92 1.33 1.37 0.37
1.00 1.00 1.00 1.00
± ± ± ±
0.00 0.00 0.00 0.00
4.43 4.87 4.15 2.80
± ± ± ±
1.82 1.89 1.68 0.57
G4
1.00 1.00 1.00 1.00
± ± ± ±
0.00 0.00 0.00 0.00
1.94 3.95 1.00 3.05
± ± ± ±
0.46 3.35 0.00 1.34
1.00 1.00 1.00 1.00
± ± ± ±
0.00 0.00 0.00 0.00
7.22 1.10 1.00 5.33
± ± ± ±
0.62 0.28 0.00 1.37
1.00 1.00 1.00 1.00
± ± ± ±
0.00 0.00 0.00 0.00
5.99 7.02 7.48 5.89
± ± ± ±
1.60 2.09 2.54 1.59
1.00 1.00 1.53 1.09
± ± ± ±
0.00 0.00 2.06 0.33
2.90 4.46 5.35 4.10
± ± ± ±
0.94 1.72 2.52 1.24
TC T1300 T3800 T6300
a Adjusted means for extracting the square root (X + 1) da [IMe, IA, HM], [IMe, IA, LM], [IMe, DA, HM], [IMe, DA, LM], [DMe, IA, HM], [DMe, IA, HM], [DMe, DA, HM] and [DMe, DA, LM]. Plasma membrane intact (IMe) or injured (IMe), acrosome intact (IA) or injured (DA) and high (HM) or low mitochondrial potential (LM). *Interaction significant (P < 0.05) between manganese supplementation (control group (TC ) had 540 mg of Mn2+ that was present in other sources of mineral elements and added 1300 mg (T1300 ), 3800 mg (T3800 ) and 6300 mg (T6300 ) of Mn2+ /kg of mineral mixture) and groups of bulls. ** Interaction significant (P < 0.01) between manganese supplementation (control group (TC ) had 540 mg of Mn2+ that was present in other sources of mineral elements and added 1300 mg (T1300 ), 3800 mg (T3800 ) and 6300 mg (T6300 ) of Mn2+ /kg of mineral mixture) and groups of bulls.
Please cite this article in press as: Reis, L.S.L.S., et al., Effect of manganese supplementation on the membrane integrity and the mitochondrial potential of the sperm of grazing Nelore bulls. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.06.033
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In fact, the plasma and acrosomal membranes of the sperm may have been injured during spermatogenesis because such lesions mainly occur during the spermatogenic process, as indicated by Garner and Hafez (2004). Despite the fact that supplemental Mn2+ alters the physiological processes that occur during spermatogenesis in bulls, data in Table 3 indicate the supplementation control (TC ) exhibited a 50% efficiency in the stimulation of the production of intact sperm [DMe, IA, LM], as observed in the G1 and G2 groups. However, this supplementation resulted in a greater efficiency of 75% for the production of totally damaged sperm [DMe, DA, LM], as was observed in groups G1 , G2 , and G4 , and sperm with an injured acrosomal membrane [DMe, DA, LM], as was observed in groups G1 , G2 , and G3 . Therefore, these results confirm the findings presented in Fig. 1, which demonstrate that the TC treatment was beneficial to the integrity of both the plasma and the acrosomal membranes of the bull sperm. Furthermore, these results show that the addition of larger amounts of Mn2+ to mineral mixes, as is performed by most companies that produce mineral supplements, is harmful to the sperm membrane. The semen produced by the bulls that received the TC treatment had a greater quality and greater proportion of intact sperm [IMe, IA, LM; Fig. 1]. This sperm category is directly associated with sperm motility, vigor, and wave motion (Table 2), which are important because vigorous sperm more efficiently reach the oviduct and fertilize oocytes and thus enhance fertilization rates. In contrast, sperm with damaged membranes [DMe, IA, LM/DMe, DA, HM/DMe, DA, LM], which were mainly found in the bulls that received Mn2+ supplementation, were negatively associated with the functional variables (motility, vigor, and wave motion). The number of severely damaged sperm in the semen obtained from bulls that were receiving Mn2+ supplementation (Fig. 1) indicates the decreased fertilization potential. This finding corroborates the results reported by McDowell (1992), who demonstrated that dairy cattle in a pasture that contained less than 200 ppm Mn2+ exhibited the greatest number of services per pregnancy and had a lesser birth rate of calves. These results were somewhat expected because the amounts of Mn2+ fed to these animals were 9.1- to 44.5-fold greater than those recommended by the NRC (2000). According to the results, a daily large dose of Mn2+ is detrimental to the plasma and acrosomal membrane integrity of the sperm of Nelore bulls.
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Conflict of interest
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No conflicts of interest between authors.
Please cite this article in press as: Reis, L.S.L.S., et al., Effect of manganese supplementation on the membrane integrity and the mitochondrial potential of the sperm of grazing Nelore bulls. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.06.033
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