- Email: [email protected]
Organic selenium increases PHGPx, but does not affect quality sperm in raw boar semen
Livestock Science 164 (2014) 175–178
Contents lists available at ScienceDirect
Livestock Science journal homepage: www.elsevier.com/locate/livsci
Short communication
Organic selenium increases PHGPx, but does not affect quality sperm in raw boar semen S.M.M.K. Martins a,b, A.F.C. De Andrade a,b, F.G. Zaffalon a, L.J. Parazzi b, F.F. Bressan c, S.M.P. Pugine d, M.P. Melo d, M.R. Chiaratti e, C.T. Marino b, E.R. Afonso b, A.S. Moretti b, R.P. Arruda a,n a
Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Pirassununga, SP, Brazil Department of Animal Nutrition and Production, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Pirassununga, SP, Brazil c Department of Veterinary Medicine, School of Animal Science and Food Engineering, University of Sao Paulo, Pirassununga, SP, Brazil d Department of Basic Sciences, School of Animal Science and Food Engineering, University of Sao Paulo, Pirassununga, SP, Brazil e Department of Genetics and Evolution, Federal University of Sao Carlos, São Carlos, SP, Brazil b
a r t i c l e i n f o
abstract
Article history: Received 6 August 2013 Received in revised form 4 January 2014 Accepted 23 February 2014
This study assessed the effects of feeding organic selenium (Se) on sperm morphology, motility, membrane integrity and lipidic peroxidation, also on sperm ATP and phospholipid hydroperoxide glutathione peroxidase (PHGPx) in raw semen of mature boars. Twelve boars were divided into three groups: Control, 0.3 ppm sodium selenite, Inorganic, 0.5 ppm sodium selenite and Organic, 0.5 ppm Se yeast for 11 weeks. Organic diet presented a higher total sperm number (Po 0.05) when compared to the inorganic treatment; however, no difference was observed regarding volume, sperm concentration, number of cells with plasma and/or acrosomal membrane integrity, membrane mitochondrial potential, ATP assay and Se concentration in seminal and blood content. Also, no difference was observed on motility parameters, sperm morphology and membrane peroxidation, however, PHGPx was influenced (P o 0.05) in organic treatment. Thus organic Se feeding influenced none of the analyzed seminal characteristics in raw semen, except for PHGPx and total number of sperm. & 2014 Elsevier B.V. All rights reserved.
Keywords: ATP sperm Membrane integrity Morphology sperm Motility sperm
1. Introduction The role of Se in male fertility is associated with selenoprotein P and PHGPx in spermiogenesis. PHGPx is one of the major selenoproteins in spermatids, and in its mitochondrial form plays a major role in suppressing the effects of reactive oxygen species (ROS) (Arai et al., 1999).
n Correspondence to: Departamento de Reprodução Animal, Faculdade de Medicina Veterinária e Zootecnia, Avenida Duque de Caxias Norte, 225-Campus da USP, 13635-900 Pirassununga, SP, Brazil. Tel.: þ 55 19 3565 4221; fax: þ55 19 3565 4060. E-mail address: [email protected] (R.P. Arruda).
http://dx.doi.org/10.1016/j.livsci.2014.02.018 1871-1413 & 2014 Elsevier B.V. All rights reserved.
Boars fed with diets containing low quantities of Se also present structural abnormalities in the mitochondria of spermatozoa, lower ATP concentrations and lower glutathione peroxidase activities (Marin-Guzman et al., 1997, 2000b). Concerning the key role of Se in sperm development and functionality, studies have been conducted to investigate the effects of Se feeding on reproductive characteristics of boars mostly based on testing different concentrations (Marin-Guzman et al., 1997, 2000a, 2000b). Although Lopez et al. (2010) and Speight et al. (2012) investigated the effect of different sources of Se, these authors did not measure their effect on PHGPx and ATP sperm. Therefore, this experiment was aimed to assess the
176
S.M.M.K. Martins et al. / Livestock Science 164 (2014) 175–178
possible beneficial effects of feeding organic Se in boars on PHGPx, membrane integrity, motility, ATP, morphology and membrane peroxidation in raw sperm. 2. Materials and methods
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1) (153 μM, Molecular Probes) or (3) PI (0.5 mg/mL, Sigma-Aldrich ) C11-BODIPY581/591 association (1 mg/mL, Molecular Probes). The analyses were performed in a FACSAria (BD) flow cytometer, controlled by BD FACSDiva 6.0 software (BD).
2.1. Semen collection and treatments Twelve boars (Agroceres PIC) with approximately 18 months of age and 257 kg body weight. Animals were housed in individual pens of 9.76 m2/boar, in a concrete floor pen within the same building during all experimental period and before the start of the trial, they received a commercial feed containing inorganic Se (0.3 ppm, 2.0 kg/ boar/day). Prior to the experiment, two independent clinical examinations accompanied by morphological and physical (motility and vigor) analysis of seminal characteristics were performed on each animal in accordance with CBRA (Colégio Brasileiro de Reprodução Animal) (CBRA, 1998) guidelines. The results of two ejaculates were used to prepare an index determined for each animal. The index calculation formulae Score of the boar¼(3 Sperm major defects)þ(2 Sperm minor defects)þ(1 Progressive motility inverse). Thus, the boars were assigned blocked by quality semen. The animals were distributed into one of three treatments: control (CON, 0.3 ppm sodium selenite; inorganic (INO, 0.5 ppm sodium selenite); and organic (ORG, 0.5 ppm Se as Sel-Plex, Alltech), fed 2.6 kg/boar/day (NRC, 1998). Six samples ( 7, 1st, 3rd, 5th, 7th and 9th weeks) were collected for evaluation of Se concentration on blood plasma and seminal plasma; 6 samples (0, 2nd, 4th, 6th, 8th and 10th weeks) for evaluation of semen characteristics.
2.4.1. Simultaneous evaluation of plasma and acrosomal membranes in raw semen These probes was to stain cells that had damaged plasma membranes (PI positive) as demonstrated by De Andrade et al. (2007) and those with damaged acrosomal membranes (FITC-PSA (þ)). For sperm membrane integrity, only percentages of cells with intact plasma and acrosomal membranes (IPIA) were used. 2.4.2. Evaluation of lipidic membrane peroxidation Viable sperm (PI negative, C11-BODIPY581 591-1 positive) was detected and analyzed by the mean intensity of fluorescence emission (IPERMI) (De Andrade et al., 2012). 2.4.3. Evaluation of membrane mitochondrial potential in raw semen This analysis generated dot plots identifying three subpopulations: sperm with low (LMP), medium (MMP), and high (HMP) mitochondrial membrane potentials. It is emphasized that for the statistical analysis, we considered the sum of the cells with mitochondrial membrane potential (WMP¼HMP þMMP) (De Andrade et al., 2011). 2.5. ATP assay ATP bioluminescent somatic cells assay kit (FLASC, Sigma-Aldrich) was used for ATP measurement following manufacturer's instructions. Luminescence was read in FluoStar Optima (BMG LABTECH) equipment (Rieger, 1997).
2.2. Assessment of volume, sperm concentration, total number of sperm and morphology
2.6. Measurement of PHGPx
The volume was determined based on weight to semen. Sperm concentration analysis was performed in a Neubauer chamber after 1:100 dilution in formaldehydephosphate buffered saline. Total number of sperm (TNS) was calculated by multiplication sperm concentration and volume and progressive motile. To assess morphology, the semen was diluted and fixed in formaldehyde-PBS.
The PHGPx evaluation for boar semen was adapted from Garolla et al. (2005). The activity was calculated by the computer-aided evaluation of the kinetic slope from 3 min (37 1C, 340 nm, Beckman DU800 spectrophotometer) was used for calibration, using home-made phosphatidylcholine hydroperoxide as substrate according to Maiorino et al. (1990).
2.3. Computer-assisted sperm analysis motility
2.7. Se concentration in circulating blood and semen
For sperm motility assessment, the CASA system (HTMIVOS-Ultimate) was pre-adjusted for swine semen analysis. 2 mL of each sample (25 106 spermatozoa/mL) were dispensed on 20 mm standard count analysis chambers (Celeghini et al., 2008).
Se concentration was measured every two weeks in blood and seminal plasma during the semen collections by the fluorometric method of Olson et al. (1975) using 2,3 diaminonaphthalene as the complexing reagent. 2.8. Statistical analysis
2.4. Flow cytometry analysis Semen aliquots were subdivided and stained with: (1) propidium iodide (PI, 0.5 mg/mL, Sigma-Aldrich) and fluorescein isothiocyanate conjugated with Pisum sativum Agglutinin (FITC-PSA-100 mg/mL, Sigma-Aldrich) association; or (2)
Data were analyzed using the MIXED procedure (SAS, 2002) according to a blocked design containing treatments as main factors. Each animal was considered as one experimental unit. Treatments were evaluated using orthogonal contrast to analyze treatment effects; contrast 1 (C1)
S.M.M.K. Martins et al. / Livestock Science 164 (2014) 175–178
177
referred to the effect of supplementation and contrast 2 (C2) referred to the effect of source. Differences were considered significant when Po0.05 and all results were expressed as means7standard error of the mean (SEM). Effect of time was analyzed using the REPEATED procedure in MIXED of SAS.
Concerning IA cells, a tendency to week treatment was observed (P ¼0.0684). IPIA and IP varied (P o0.05) during the 11 weeks. A slight augment in IP cells was observed after second week, followed by a decline.
3. Results
Analyses of both membrane mitochondrial potential and ATP are considered different assays to evaluate mitochondrial function. Mitochondrial function was not influenced by Se supplementation regardless of source. Both characteristics presented mean values closely related between treatments, and only an effect of week was observed (Po0.05).
No interaction was observed between treatments and weeks for all variables analyzed. Thus, the main-effect analysis could be performed. 3.1. Effects of Se supplementation on volume, TNS, concentration and morphology sperm No difference was observed among the three groups regarding volume, but the comparisons between weeks showed that the three first weeks differed from the three last weeks (P o0.05). This effect was observed in almost all boars and the range for each groups were CON (160.00–600.00 mL), INO (130.00–450.00 mL) and ORG (190.00–600.00 mL). No difference was observed between treatments or in week effect (P ¼0.0542) for sperm concentration, however a higher value was observed in ORG group. Along the 11 weeks the TNS increased gradually from 181.04 109 sperm/ejaculate to 299.38 109 sperm/ ejaculate. ORG group presented a higher (P o0.05) TNS compared to INO group (83.26 and 61.61 109 sperm/ ejaculate, respectively). No difference was found between CON and INO groups. The effect of week on dietary feeding with organic Se did not affect sperm morphology. Boars supplemented with organic Se showed a higher percentage of normal cells and diminished percentages of proximal cytoplasmic droplet, head and midpiece alterations, in the latter a tendency (P¼0.0897) concerning the interaction was observed. 3.2. Effects of selenium supplementation in sperm motility parameters obtained by the CASA Motility parameters of raw semen were not influenced by Se supplementation in the diet. The mean7SEM to total motility observed in all groups were within of expected 90.5471.01, 89.72 71.23 and 91.0671.07, CON, INO and ORG groups, respectively. Throughout the weeks, however, progressive motility was diminished (P o0.05) until week 4, followed by a recovery, the results observed were 61.72 74.72, 55.6273.65, 48.03 74.49, 60.9673.48, 58.6374.06 and 63.5473.08, weeks 0, 2, 4, 6, 8 and 10, respectively. Beat frequency (BCF, Hz) presented higher values on week 3 of treatment. It was observed that the first three week presented a higher frequency when compared to the later three. 3.3. Effects of Se supplementation on plasma and acrosomal membranes Analysis of raw semen indicated that the diet provided to the boars did not increase the number of IPIA, IP or IA, regardless of supplementation and the source of Se used.
3.4. Effects of Se supplementation on mitochondrial function
3.5. Effects of Se supplementation on PHGPx and lipidic membrane peroxidation The PHGPx measured on spermatozoa was influenced (P o0.05) by the organic Se corresponding to 47.54 mU/mg protein (141.27 vs. 188.81 mU/mg protein, INO vs. ORG, respectively). An effect of week (P o0.05) was observed. PHGPx decreased from first to second week and then an increase of values was observed in the following weeks, maintained until the fifth week. A reduction in the first three weeks was observed (P o0.05, 255.50, 121.00 and 97.08 u.a., week 0, week 2, week 4, respectively). However not significant, animals of ORG group showed a reduction in spermatozoa lipid peroxidation. 3.6. Effects of supplementation on Se concentration in seminal and blood plasma Blood concentration of Se was higher along weeks (P o0.05). Mean concentration of blood Se from ORG treatment was superior (P 40.05) when compared to the other treatments. Seminal plasma concentration of Se was also not influenced by supplementation; however, ORG group presented higher values (P 40.05) when compared to the other treatments. 4. Discussion This study demonstrates that organic Se feeding increased PHGPx in raw boar semen although it did not significantly influence Se concentration in seminal and blood content, seminal volume, sperm concentration, sperm morphology, plasma and acrosomal membrane integrity, membrane peroxidation, mitochondrial function, or sperm motility parameters. Although volume and concentration did not differ between treatments, there were numerically higher in ORG group, resulting in a higher TNS, which is in disagreement with Speight et al. (2012). Boars from ORG group produced 8.89 and 7.22 semen doses more than CON and INO boars, respectively. The semen doses were composed of 3 109 spermatozoa/100 mL. Studies on the motility of raw semen from boars fed with similar inorganic Se source led to an improvement in motility (Marin-Guzman et al., 1997, 2000a), but no differences were observed in our results. PHGPx constitutes more
178
S.M.M.K. Martins et al. / Livestock Science 164 (2014) 175–178
than half of the mitochondrial capsule of mature spermatozoa. This capsule embeds the mitochondrial helix and is therefore responsible for the integrity of mature spermatozoa (Ursini et al., 1999) which could enhance boar sperm quality by increasing sperm motility and reducing abnormal morphology, but it was not observed. In our study no difference was observed regarding the morphology of sperm, however a higher percentage of normal cells and a diminished percentage of head alterations, midpiece and proximal protoplasmic drop on supplemented animals were observed, in disagreement with the study by Jacyno and Kawecka (2002). It was expected that organic Se supplementation, would increase PGHPx, which in turn would exert its anti-oxidant function, leading to a reduction in ROS formation and enhancing preservation of the integrity of the sperm cell (Ursini et al., 1999). The results from this study indicate that organic Se had no effect (P4 0.05) on preserving the integrity of sperm cells. Nevertheless, Se concentration in the seminal plasma and GPx activities tended to be higher when Se supplemented the boars’ diet (Marin-Guzman et al., 1997). In contrast with the findings of Marin-Guzman et al. (2000b), ATP concentration was not influenced by either feeding or Se source utilized. Such unexpected result is reenforced by a diminished percentage of midpiece alterations and higher PHGPx on ORG group, what would be essential for a higher ATP concentration on semen. Results reported by Marin-Guzman et al. (2000b) regarding Se deficiency, which led to the occurrence of midpiece alterations, decrease on ATP and motility, but the similar results were not obtained in our study. In conclusion, organic Se feeding increased PHGPx and number of semen doses, but did not influence remaining of the analyzed seminal characteristics in raw semen boars. Author contributions All listed authors have made substantial contributions; Simone Martins, Rubens Arruda and Aníbal Moretti made the research design; Simone Martins, André Andrade, Fabiane Zaffalon, Larissa Parazzi, Fabiana Bressan, Silvana Pugine, Mariza Melo, Marcos Chiaratti, Carolina Marino, contributed in the acquisition, analysis and interpretation of data; Simone Martins, André Andrade, Esther Afonso, Aníbal Moretti and Rubens Arruda drafting the paper and revising it critically. Conflict of interest statement None. Acknowledgments This research was supported by FAPESP (Grant numbers 07/55613-6 and 08/55059-1), Alltech Inc., Vet life
Veterinary Products and Agroceres Multimix Nutrição Animal Ltda. References Arai, M., Imai, H., Koumura, T., Yoshida, M., Emoto, K., Umeda, M., Chiba, N., Nakagawa, Y., 1999. Mitochondrial phospholipid hydroperoxide glutathione peroxidase plays a major role in preventing oxidative injury to cells. J. Biol. Chem. 274, 4924–4933. CBRA Colégio Brasileiro de Reprodução Animal, 1998. Manual para exame andrológico e avaliação de sêmen animal. CBRA, Belo Horizonte. Celeghini, E.C.C., Arruda, R.P., Andrade, A.F.C., Nascimento, J., Raphael, C.F., Rodrigues, P.H.M., 2008. Effects that bovine sperm cryopreservation using two different extenders has on sperm membranes and chromatin. Anim. Reprod. Sci. 104, 119–131. De Andrade, A.F.C., Arruda, R.P., Celeghini, E.C.C., Nascimento, J., Martins, S.M.M.K., Raphael, C.F., Moretti, A.S., 2007. Fluorescent stain method for the simultaneous determination of mitochondrial potential and integrity of plasma and acrosomal membranes in boar sperm. Reprod. Domest. Anim. 42, 190–194. De Andrade, A.F.C., Zaffalon, F.G., Celeghini, E.C.C., Nascimento, J., Tarragó, O.F.B., Martins, S.M.M.K., Alonso, M.A., Arruda, R.P., 2011. Addition of seminal plasma to post-thawing equine semen: what is the effect on sperm cell viability? Reprod. Domest. Anim. 46, 682–686. De Andrade, A.F.C., Zaffalon, F.G., Celeghini, E.C.C., Nascimento, J., Bressan, F.F., Martins, S.M.M.K., Arruda, R.P., 2012. Post-thaw seminal plasma addition reduces tyrosine phosphorylation present in the cryopreserved equine sperm surface, but does not reduce lipid peroxidation. Theriogenology 77, 1866–1872. Garolla, A., Maiorino, M., Roverato, A., Roveri, A., Ursini, F., Foresta, C., 2005. Oral carnitine supplementation increases sperm motility in asthenozoospermic men with normal sperm phospholipid hydroperoxide gluthatione peroxidase levels. Fertil. Steril. 83, 355–361. Jacyno, E., Kawecka, M., 2002. Influence of inorganic Se þ vitamin E and organic Se þ vitamin E on reproductive performance of young boars. Agric. Food Sci. Finland, 175–184. Lopez, A., Rijsselare, T., Van-Soom, A., Leroy, J.L.M.R., De Clercq, J.B.P., Bols, P.E.J., Maes, D., 2010. Effect of organic selenium in the diet on sperm quality of boars. Reprod. Domest. Anim. 45, 297–2305. Maiorino, M., Gregolin, C., Ursini, F., 1990. Phospholipid hydroperoxide glutathione peroxidase. Methods Enzymol. 186, 448–457. Marin-Guzman, J., Mahan, D.C., Chung, Y.K., Pate, J.L., Pope, W.F., 1997. Effects of dietary selenium and vitamin E on boar performance and tissue responses, semen quality, and subsequent fertilization rates in mature gilts. J. Anim. Sci. 75, 2994–3003. Marin-Guzman, J., Mahan, D.C., Pate, J.L., 2000a. Effect of dietary selenium and vitamin E on spermatogenic development in boars. J. Anim. Sci. 78, 1537–1543. Marin-Guzman, J., Mahan, D.C., Whitmoyer, R., 2000b. Effect of dietary selenium and vitamin E on the ultrastructure and ATP concentration of boar spermatozoa, and the efficacy of added sodium selenite in extended semen on sperm motility. J. Anim. Sci. 78, 1544–1550. NRC. Nutrient Requirements of Swine, 1998. 10th revised edition. National Academy Press, Washington, DC. Olson, O.E., Palmer, L.S., Cary, E.L., 1975. Modification of the official fluorimetric method for selenium in plants. J. AOAC 58, 117–121. Rieger, D., 1997. Batch analysis of the ATP content of bovine sperm, oocytes, and early embryos using a scintillation counter to measure the chemiluminescence produced by the luciferin–luciferase reaction. Anal. Biochem. 246, 67–70. SAS, 2002. Statistical Analisys System: Software. Version 9.0. SAS Institute, Cary. Speight, S.M., Estienne, M.J., Harper, A.F., Crawford, R.J., Knight, J.W., Whitaker, B.D., 2012. Effects of dietary supplementation with on organic source of selenium on characteristics of semen quality and in vitro fertility in boars. J. Anim. Sci. 90, 761–770. Ursini, F., Heim, S., Kiess, M., Maiorino, M., Roveri, A., Wissing, J., Flohé, L., 1999. Dual function of the selenoprotein phgpx during sperm maturation. Science 285, 1393–1396.
Contents lists available at ScienceDirect
Livestock Science journal homepage: www.elsevier.com/locate/livsci
Short communication
Organic selenium increases PHGPx, but does not affect quality sperm in raw boar semen S.M.M.K. Martins a,b, A.F.C. De Andrade a,b, F.G. Zaffalon a, L.J. Parazzi b, F.F. Bressan c, S.M.P. Pugine d, M.P. Melo d, M.R. Chiaratti e, C.T. Marino b, E.R. Afonso b, A.S. Moretti b, R.P. Arruda a,n a
Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Pirassununga, SP, Brazil Department of Animal Nutrition and Production, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Pirassununga, SP, Brazil c Department of Veterinary Medicine, School of Animal Science and Food Engineering, University of Sao Paulo, Pirassununga, SP, Brazil d Department of Basic Sciences, School of Animal Science and Food Engineering, University of Sao Paulo, Pirassununga, SP, Brazil e Department of Genetics and Evolution, Federal University of Sao Carlos, São Carlos, SP, Brazil b
a r t i c l e i n f o
abstract
Article history: Received 6 August 2013 Received in revised form 4 January 2014 Accepted 23 February 2014
This study assessed the effects of feeding organic selenium (Se) on sperm morphology, motility, membrane integrity and lipidic peroxidation, also on sperm ATP and phospholipid hydroperoxide glutathione peroxidase (PHGPx) in raw semen of mature boars. Twelve boars were divided into three groups: Control, 0.3 ppm sodium selenite, Inorganic, 0.5 ppm sodium selenite and Organic, 0.5 ppm Se yeast for 11 weeks. Organic diet presented a higher total sperm number (Po 0.05) when compared to the inorganic treatment; however, no difference was observed regarding volume, sperm concentration, number of cells with plasma and/or acrosomal membrane integrity, membrane mitochondrial potential, ATP assay and Se concentration in seminal and blood content. Also, no difference was observed on motility parameters, sperm morphology and membrane peroxidation, however, PHGPx was influenced (P o 0.05) in organic treatment. Thus organic Se feeding influenced none of the analyzed seminal characteristics in raw semen, except for PHGPx and total number of sperm. & 2014 Elsevier B.V. All rights reserved.
Keywords: ATP sperm Membrane integrity Morphology sperm Motility sperm
1. Introduction The role of Se in male fertility is associated with selenoprotein P and PHGPx in spermiogenesis. PHGPx is one of the major selenoproteins in spermatids, and in its mitochondrial form plays a major role in suppressing the effects of reactive oxygen species (ROS) (Arai et al., 1999).
n Correspondence to: Departamento de Reprodução Animal, Faculdade de Medicina Veterinária e Zootecnia, Avenida Duque de Caxias Norte, 225-Campus da USP, 13635-900 Pirassununga, SP, Brazil. Tel.: þ 55 19 3565 4221; fax: þ55 19 3565 4060. E-mail address: [email protected] (R.P. Arruda).
http://dx.doi.org/10.1016/j.livsci.2014.02.018 1871-1413 & 2014 Elsevier B.V. All rights reserved.
Boars fed with diets containing low quantities of Se also present structural abnormalities in the mitochondria of spermatozoa, lower ATP concentrations and lower glutathione peroxidase activities (Marin-Guzman et al., 1997, 2000b). Concerning the key role of Se in sperm development and functionality, studies have been conducted to investigate the effects of Se feeding on reproductive characteristics of boars mostly based on testing different concentrations (Marin-Guzman et al., 1997, 2000a, 2000b). Although Lopez et al. (2010) and Speight et al. (2012) investigated the effect of different sources of Se, these authors did not measure their effect on PHGPx and ATP sperm. Therefore, this experiment was aimed to assess the
176
S.M.M.K. Martins et al. / Livestock Science 164 (2014) 175–178
possible beneficial effects of feeding organic Se in boars on PHGPx, membrane integrity, motility, ATP, morphology and membrane peroxidation in raw sperm. 2. Materials and methods
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1) (153 μM, Molecular Probes) or (3) PI (0.5 mg/mL, Sigma-Aldrich ) C11-BODIPY581/591 association (1 mg/mL, Molecular Probes). The analyses were performed in a FACSAria (BD) flow cytometer, controlled by BD FACSDiva 6.0 software (BD).
2.1. Semen collection and treatments Twelve boars (Agroceres PIC) with approximately 18 months of age and 257 kg body weight. Animals were housed in individual pens of 9.76 m2/boar, in a concrete floor pen within the same building during all experimental period and before the start of the trial, they received a commercial feed containing inorganic Se (0.3 ppm, 2.0 kg/ boar/day). Prior to the experiment, two independent clinical examinations accompanied by morphological and physical (motility and vigor) analysis of seminal characteristics were performed on each animal in accordance with CBRA (Colégio Brasileiro de Reprodução Animal) (CBRA, 1998) guidelines. The results of two ejaculates were used to prepare an index determined for each animal. The index calculation formulae Score of the boar¼(3 Sperm major defects)þ(2 Sperm minor defects)þ(1 Progressive motility inverse). Thus, the boars were assigned blocked by quality semen. The animals were distributed into one of three treatments: control (CON, 0.3 ppm sodium selenite; inorganic (INO, 0.5 ppm sodium selenite); and organic (ORG, 0.5 ppm Se as Sel-Plex, Alltech), fed 2.6 kg/boar/day (NRC, 1998). Six samples ( 7, 1st, 3rd, 5th, 7th and 9th weeks) were collected for evaluation of Se concentration on blood plasma and seminal plasma; 6 samples (0, 2nd, 4th, 6th, 8th and 10th weeks) for evaluation of semen characteristics.
2.4.1. Simultaneous evaluation of plasma and acrosomal membranes in raw semen These probes was to stain cells that had damaged plasma membranes (PI positive) as demonstrated by De Andrade et al. (2007) and those with damaged acrosomal membranes (FITC-PSA (þ)). For sperm membrane integrity, only percentages of cells with intact plasma and acrosomal membranes (IPIA) were used. 2.4.2. Evaluation of lipidic membrane peroxidation Viable sperm (PI negative, C11-BODIPY581 591-1 positive) was detected and analyzed by the mean intensity of fluorescence emission (IPERMI) (De Andrade et al., 2012). 2.4.3. Evaluation of membrane mitochondrial potential in raw semen This analysis generated dot plots identifying three subpopulations: sperm with low (LMP), medium (MMP), and high (HMP) mitochondrial membrane potentials. It is emphasized that for the statistical analysis, we considered the sum of the cells with mitochondrial membrane potential (WMP¼HMP þMMP) (De Andrade et al., 2011). 2.5. ATP assay ATP bioluminescent somatic cells assay kit (FLASC, Sigma-Aldrich) was used for ATP measurement following manufacturer's instructions. Luminescence was read in FluoStar Optima (BMG LABTECH) equipment (Rieger, 1997).
2.2. Assessment of volume, sperm concentration, total number of sperm and morphology
2.6. Measurement of PHGPx
The volume was determined based on weight to semen. Sperm concentration analysis was performed in a Neubauer chamber after 1:100 dilution in formaldehydephosphate buffered saline. Total number of sperm (TNS) was calculated by multiplication sperm concentration and volume and progressive motile. To assess morphology, the semen was diluted and fixed in formaldehyde-PBS.
The PHGPx evaluation for boar semen was adapted from Garolla et al. (2005). The activity was calculated by the computer-aided evaluation of the kinetic slope from 3 min (37 1C, 340 nm, Beckman DU800 spectrophotometer) was used for calibration, using home-made phosphatidylcholine hydroperoxide as substrate according to Maiorino et al. (1990).
2.3. Computer-assisted sperm analysis motility
2.7. Se concentration in circulating blood and semen
For sperm motility assessment, the CASA system (HTMIVOS-Ultimate) was pre-adjusted for swine semen analysis. 2 mL of each sample (25 106 spermatozoa/mL) were dispensed on 20 mm standard count analysis chambers (Celeghini et al., 2008).
Se concentration was measured every two weeks in blood and seminal plasma during the semen collections by the fluorometric method of Olson et al. (1975) using 2,3 diaminonaphthalene as the complexing reagent. 2.8. Statistical analysis
2.4. Flow cytometry analysis Semen aliquots were subdivided and stained with: (1) propidium iodide (PI, 0.5 mg/mL, Sigma-Aldrich) and fluorescein isothiocyanate conjugated with Pisum sativum Agglutinin (FITC-PSA-100 mg/mL, Sigma-Aldrich) association; or (2)
Data were analyzed using the MIXED procedure (SAS, 2002) according to a blocked design containing treatments as main factors. Each animal was considered as one experimental unit. Treatments were evaluated using orthogonal contrast to analyze treatment effects; contrast 1 (C1)
S.M.M.K. Martins et al. / Livestock Science 164 (2014) 175–178
177
referred to the effect of supplementation and contrast 2 (C2) referred to the effect of source. Differences were considered significant when Po0.05 and all results were expressed as means7standard error of the mean (SEM). Effect of time was analyzed using the REPEATED procedure in MIXED of SAS.
Concerning IA cells, a tendency to week treatment was observed (P ¼0.0684). IPIA and IP varied (P o0.05) during the 11 weeks. A slight augment in IP cells was observed after second week, followed by a decline.
3. Results
Analyses of both membrane mitochondrial potential and ATP are considered different assays to evaluate mitochondrial function. Mitochondrial function was not influenced by Se supplementation regardless of source. Both characteristics presented mean values closely related between treatments, and only an effect of week was observed (Po0.05).
No interaction was observed between treatments and weeks for all variables analyzed. Thus, the main-effect analysis could be performed. 3.1. Effects of Se supplementation on volume, TNS, concentration and morphology sperm No difference was observed among the three groups regarding volume, but the comparisons between weeks showed that the three first weeks differed from the three last weeks (P o0.05). This effect was observed in almost all boars and the range for each groups were CON (160.00–600.00 mL), INO (130.00–450.00 mL) and ORG (190.00–600.00 mL). No difference was observed between treatments or in week effect (P ¼0.0542) for sperm concentration, however a higher value was observed in ORG group. Along the 11 weeks the TNS increased gradually from 181.04 109 sperm/ejaculate to 299.38 109 sperm/ ejaculate. ORG group presented a higher (P o0.05) TNS compared to INO group (83.26 and 61.61 109 sperm/ ejaculate, respectively). No difference was found between CON and INO groups. The effect of week on dietary feeding with organic Se did not affect sperm morphology. Boars supplemented with organic Se showed a higher percentage of normal cells and diminished percentages of proximal cytoplasmic droplet, head and midpiece alterations, in the latter a tendency (P¼0.0897) concerning the interaction was observed. 3.2. Effects of selenium supplementation in sperm motility parameters obtained by the CASA Motility parameters of raw semen were not influenced by Se supplementation in the diet. The mean7SEM to total motility observed in all groups were within of expected 90.5471.01, 89.72 71.23 and 91.0671.07, CON, INO and ORG groups, respectively. Throughout the weeks, however, progressive motility was diminished (P o0.05) until week 4, followed by a recovery, the results observed were 61.72 74.72, 55.6273.65, 48.03 74.49, 60.9673.48, 58.6374.06 and 63.5473.08, weeks 0, 2, 4, 6, 8 and 10, respectively. Beat frequency (BCF, Hz) presented higher values on week 3 of treatment. It was observed that the first three week presented a higher frequency when compared to the later three. 3.3. Effects of Se supplementation on plasma and acrosomal membranes Analysis of raw semen indicated that the diet provided to the boars did not increase the number of IPIA, IP or IA, regardless of supplementation and the source of Se used.
3.4. Effects of Se supplementation on mitochondrial function
3.5. Effects of Se supplementation on PHGPx and lipidic membrane peroxidation The PHGPx measured on spermatozoa was influenced (P o0.05) by the organic Se corresponding to 47.54 mU/mg protein (141.27 vs. 188.81 mU/mg protein, INO vs. ORG, respectively). An effect of week (P o0.05) was observed. PHGPx decreased from first to second week and then an increase of values was observed in the following weeks, maintained until the fifth week. A reduction in the first three weeks was observed (P o0.05, 255.50, 121.00 and 97.08 u.a., week 0, week 2, week 4, respectively). However not significant, animals of ORG group showed a reduction in spermatozoa lipid peroxidation. 3.6. Effects of supplementation on Se concentration in seminal and blood plasma Blood concentration of Se was higher along weeks (P o0.05). Mean concentration of blood Se from ORG treatment was superior (P 40.05) when compared to the other treatments. Seminal plasma concentration of Se was also not influenced by supplementation; however, ORG group presented higher values (P 40.05) when compared to the other treatments. 4. Discussion This study demonstrates that organic Se feeding increased PHGPx in raw boar semen although it did not significantly influence Se concentration in seminal and blood content, seminal volume, sperm concentration, sperm morphology, plasma and acrosomal membrane integrity, membrane peroxidation, mitochondrial function, or sperm motility parameters. Although volume and concentration did not differ between treatments, there were numerically higher in ORG group, resulting in a higher TNS, which is in disagreement with Speight et al. (2012). Boars from ORG group produced 8.89 and 7.22 semen doses more than CON and INO boars, respectively. The semen doses were composed of 3 109 spermatozoa/100 mL. Studies on the motility of raw semen from boars fed with similar inorganic Se source led to an improvement in motility (Marin-Guzman et al., 1997, 2000a), but no differences were observed in our results. PHGPx constitutes more
178
S.M.M.K. Martins et al. / Livestock Science 164 (2014) 175–178
than half of the mitochondrial capsule of mature spermatozoa. This capsule embeds the mitochondrial helix and is therefore responsible for the integrity of mature spermatozoa (Ursini et al., 1999) which could enhance boar sperm quality by increasing sperm motility and reducing abnormal morphology, but it was not observed. In our study no difference was observed regarding the morphology of sperm, however a higher percentage of normal cells and a diminished percentage of head alterations, midpiece and proximal protoplasmic drop on supplemented animals were observed, in disagreement with the study by Jacyno and Kawecka (2002). It was expected that organic Se supplementation, would increase PGHPx, which in turn would exert its anti-oxidant function, leading to a reduction in ROS formation and enhancing preservation of the integrity of the sperm cell (Ursini et al., 1999). The results from this study indicate that organic Se had no effect (P4 0.05) on preserving the integrity of sperm cells. Nevertheless, Se concentration in the seminal plasma and GPx activities tended to be higher when Se supplemented the boars’ diet (Marin-Guzman et al., 1997). In contrast with the findings of Marin-Guzman et al. (2000b), ATP concentration was not influenced by either feeding or Se source utilized. Such unexpected result is reenforced by a diminished percentage of midpiece alterations and higher PHGPx on ORG group, what would be essential for a higher ATP concentration on semen. Results reported by Marin-Guzman et al. (2000b) regarding Se deficiency, which led to the occurrence of midpiece alterations, decrease on ATP and motility, but the similar results were not obtained in our study. In conclusion, organic Se feeding increased PHGPx and number of semen doses, but did not influence remaining of the analyzed seminal characteristics in raw semen boars. Author contributions All listed authors have made substantial contributions; Simone Martins, Rubens Arruda and Aníbal Moretti made the research design; Simone Martins, André Andrade, Fabiane Zaffalon, Larissa Parazzi, Fabiana Bressan, Silvana Pugine, Mariza Melo, Marcos Chiaratti, Carolina Marino, contributed in the acquisition, analysis and interpretation of data; Simone Martins, André Andrade, Esther Afonso, Aníbal Moretti and Rubens Arruda drafting the paper and revising it critically. Conflict of interest statement None. Acknowledgments This research was supported by FAPESP (Grant numbers 07/55613-6 and 08/55059-1), Alltech Inc., Vet life
Veterinary Products and Agroceres Multimix Nutrição Animal Ltda. References Arai, M., Imai, H., Koumura, T., Yoshida, M., Emoto, K., Umeda, M., Chiba, N., Nakagawa, Y., 1999. Mitochondrial phospholipid hydroperoxide glutathione peroxidase plays a major role in preventing oxidative injury to cells. J. Biol. Chem. 274, 4924–4933. CBRA Colégio Brasileiro de Reprodução Animal, 1998. Manual para exame andrológico e avaliação de sêmen animal. CBRA, Belo Horizonte. Celeghini, E.C.C., Arruda, R.P., Andrade, A.F.C., Nascimento, J., Raphael, C.F., Rodrigues, P.H.M., 2008. Effects that bovine sperm cryopreservation using two different extenders has on sperm membranes and chromatin. Anim. Reprod. Sci. 104, 119–131. De Andrade, A.F.C., Arruda, R.P., Celeghini, E.C.C., Nascimento, J., Martins, S.M.M.K., Raphael, C.F., Moretti, A.S., 2007. Fluorescent stain method for the simultaneous determination of mitochondrial potential and integrity of plasma and acrosomal membranes in boar sperm. Reprod. Domest. Anim. 42, 190–194. De Andrade, A.F.C., Zaffalon, F.G., Celeghini, E.C.C., Nascimento, J., Tarragó, O.F.B., Martins, S.M.M.K., Alonso, M.A., Arruda, R.P., 2011. Addition of seminal plasma to post-thawing equine semen: what is the effect on sperm cell viability? Reprod. Domest. Anim. 46, 682–686. De Andrade, A.F.C., Zaffalon, F.G., Celeghini, E.C.C., Nascimento, J., Bressan, F.F., Martins, S.M.M.K., Arruda, R.P., 2012. Post-thaw seminal plasma addition reduces tyrosine phosphorylation present in the cryopreserved equine sperm surface, but does not reduce lipid peroxidation. Theriogenology 77, 1866–1872. Garolla, A., Maiorino, M., Roverato, A., Roveri, A., Ursini, F., Foresta, C., 2005. Oral carnitine supplementation increases sperm motility in asthenozoospermic men with normal sperm phospholipid hydroperoxide gluthatione peroxidase levels. Fertil. Steril. 83, 355–361. Jacyno, E., Kawecka, M., 2002. Influence of inorganic Se þ vitamin E and organic Se þ vitamin E on reproductive performance of young boars. Agric. Food Sci. Finland, 175–184. Lopez, A., Rijsselare, T., Van-Soom, A., Leroy, J.L.M.R., De Clercq, J.B.P., Bols, P.E.J., Maes, D., 2010. Effect of organic selenium in the diet on sperm quality of boars. Reprod. Domest. Anim. 45, 297–2305. Maiorino, M., Gregolin, C., Ursini, F., 1990. Phospholipid hydroperoxide glutathione peroxidase. Methods Enzymol. 186, 448–457. Marin-Guzman, J., Mahan, D.C., Chung, Y.K., Pate, J.L., Pope, W.F., 1997. Effects of dietary selenium and vitamin E on boar performance and tissue responses, semen quality, and subsequent fertilization rates in mature gilts. J. Anim. Sci. 75, 2994–3003. Marin-Guzman, J., Mahan, D.C., Pate, J.L., 2000a. Effect of dietary selenium and vitamin E on spermatogenic development in boars. J. Anim. Sci. 78, 1537–1543. Marin-Guzman, J., Mahan, D.C., Whitmoyer, R., 2000b. Effect of dietary selenium and vitamin E on the ultrastructure and ATP concentration of boar spermatozoa, and the efficacy of added sodium selenite in extended semen on sperm motility. J. Anim. Sci. 78, 1544–1550. NRC. Nutrient Requirements of Swine, 1998. 10th revised edition. National Academy Press, Washington, DC. Olson, O.E., Palmer, L.S., Cary, E.L., 1975. Modification of the official fluorimetric method for selenium in plants. J. AOAC 58, 117–121. Rieger, D., 1997. Batch analysis of the ATP content of bovine sperm, oocytes, and early embryos using a scintillation counter to measure the chemiluminescence produced by the luciferin–luciferase reaction. Anal. Biochem. 246, 67–70. SAS, 2002. Statistical Analisys System: Software. Version 9.0. SAS Institute, Cary. Speight, S.M., Estienne, M.J., Harper, A.F., Crawford, R.J., Knight, J.W., Whitaker, B.D., 2012. Effects of dietary supplementation with on organic source of selenium on characteristics of semen quality and in vitro fertility in boars. J. Anim. Sci. 90, 761–770. Ursini, F., Heim, S., Kiess, M., Maiorino, M., Roveri, A., Wissing, J., Flohé, L., 1999. Dual function of the selenoprotein phgpx during sperm maturation. Science 285, 1393–1396.