Sperm filtration as an alternative technique for seminal plasma separation in boars
DOI:
https://doi.org/10.5433/1679-0359.2021v42n4p2439Palabras clave:
Hyperactivity, Sperm-filter, Semen, Lipid peroxidation, Boar.Resumen
The present study aimed to evaluate the filtration for separating seminal plasma of boars’ ejaculate by means of sperm viability and the occurrence of hyperactivation and lipid peroxidation in fresh semen and after cooling for up to 96 hours. The ejaculate of eight healthy boars of different breeds was collected and the gelatinous portion was separated and discarded. In the laboratory, the semen was fractioned into three aliquots (groups G1, G2 and G3) as follows: G1: semen with plasma diluted in BTS (TOTAL BTS); G2: semen centrifuged at 600G/10’ (BTS CEN); and G3: semen filtered with the Sperm-filter® following dilution of the retained cells with BTS (BTS FIL). The analyses were performed at three moments: with fresh samples (D0) and after 48 (D2) and 96 hours (D4) of cooling at 17ºC. The kinetic evaluation was performed using the CASA system, which provided data for the classification of sperm hyperactivity. For lipid stress analysis, the TBARS (thiobarbituric acid reactive substance) test was performed. The variance analysis test was conducted to compare the results between the groups and moments analyzed. The results showed better total motility values (%) for G1 at D0 (67.9, P= 0.001), D2 (36.6, P= 0.004) and D4 (26.1, P= 0.003). The occurrence of hyperactivity was observed in G2 and G3 at moments D2 and D4. In addition, TBARS showed higher peroxidation levels for G1 at D0 (8.1 mM MDA/ml, P= 0.01), D2 (7.4 mM MDA/ml, P= 0.02), and D4 (6.41mMol MDA/ml, P= 0.008) when compared to G2 and G3. Since the filtration method did not demonstrate any damage to the sperm viability, the study concluded that sperm filtration is an accessible and valid tool to replace centrifugation.Citas
Aitken, R. J., & Clarkson, J. S. (1988). Significance of Reactive Oxygen Species and Antioxidants in Defining the Efficacy of Sperm Preparation Techniques. Journal of Andrology, 9(6), 367-376. doi: 10. 1002/j.1939-4640.1988.tb01067.x
Albuquerque, R. S., Morais, R., Reis, A. N., Miranda, M. S., Dias, E. A. R., Monteiro, F. M., … Gimenes, L. U. (2017). Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation. Reproduction in Domestic Animals, 52(5), 905-910. doi: doi.org/10.1111/rda.12992
Alkmin, D. V, Perez-patiño, C., Barranco, I., Parrilla, I., Vazquez, J. M., Martinez, E. A., … Roca, J. (2014). Cryobiology Boar sperm cryosurvival is better after exposure to seminal plasma from selected fractions than to those from entire ejaculate q. Cryobiology, 69(2), 203-210. doi: 10.1016/j.cryobiol.2014.07.004
Alvarenga, M A, Melo, C. M., Magalhães, L. C. O., & Papa, F. O. (2010). A new method to concentrate equine sperm. Animal Reproduction Science, 121(S), 186-187. doi: 10.1016/j.anireprosci.2010.04.142
Alvarenga, Marco Antonio, Papa, F. O., Carmo, M. T., Kievitsbosch, T., Castro Chaves, M. M. B., & Ramires Neto, C. (2012). Methods of Concentrating Stallion Semen. Journal of Equine Veterinary Science, 32(8), 424-429. doi: 10.1016/j.jevs.2012.06.003
Awda, B. J., Mackenzie-Bell, M., & Buhr, M. M. (2009). Reactive Oxygen Species and Boar Sperm Function1. Biology of Reproduction, 81(3), 553–561. https://doi.org/10.1095/biolreprod.109.076471
Barreiros, A. L. B. S., David, J. M., & David, J. P. (2006). Estresse oxidativo: Relação entre geração de espécies reativas e defesa do organismo. Quimica Nova, 29(1), 113-123. doi: 10.1590/s0100-40422006 000100021
Bergstein-Galan, T. G., Weiss, R. R., Bertol, M. A. F., Abreu, A. C. M. R., Busato, E., Kozicki, L. E., & Bicudo, S. D. (2017). Quality and fertility of frozen ovine spermatozoa from epididymides stored at room temperature (18–25 °C) for up to 48 h post mortem. Theriogenology, 96(17), 69-75. doi: 10.10 16/j.theriogenology.2017.04.001
Bianchi, I., Calderam, K., Maschio, É. F., Madeira, E. M., da Rosa Ulguim, R., Corcini, C. D., … Corrêa, M. N. (2008). Evaluation of amides and centrifugation temperature in boar semen cryopreservation. Theriogenology, 69(5), 632-638. doi: 10.1016/j.theriogenology.2007.11.012
Campanholi, S. P., Monteiro, F. M., Ribeiro Dias, E. A., Mercadante, M. E. Z., de Paz, C. C. P., Dell’Aqua Junior, J. A., … Garcia, J. M. (2017). Effect of seminal plasma removal before cryopreservation of bovine semen obtained by electroejaculation on semen quality and in vitro fertility. Theriogenology, 89(16), 114-121. doi: 10.1016/j.theriogenology.2016.10.008
CBRA. (2013). Manual para exame andrológico e avaliação de sêmen animal. (3rd ed.). Belo Horizonte: Colégio Brasileiro de Reprodução Animal.
Gadea, J., Martínez-Miró, S., Decuadro-Hansen, G., & Matás, C. (2006). 92 Evaluation of Boar Sperm Functionality After a Cushioned Centrifugation Technique. Reproduction, Fertility and Development, 18(2), 154. doi: 10.1071/rdv18n2ab92
Gómez-Fernández, J., Gómez-Izquierdo, E., Tomás, C., Mocé, E., & Mercado, E. de. (2012). Effect of different monosaccharides and disaccharides on boar sperm quality after cryopreservation. Animal Reproduction Science, 133(1-2), 109-116. doi: 10.1016/j.anireprosci.2012.06.010
Hallap, T., Ha, M., Larsson, B., & Rodriguez-martinez, H. (2004). Does cleansing of frozen-thawed bull semen before assessment provide samples that relate better to potential fertility ? Theriogenology, 62 (04), 702-713. doi: 10.1016/j.theriogenology.2003.11.017
Hancock, G. L. (1959). The morphologic characteristics of spermatozoa and fertility. Internacional Journal of Fertility, 4, 347-359.
Hurtado de Llera, A., Gil, M. C., Bragado, M. J., Martin-Hidalgo, D., Garcia-Marin, L. J., & Perez, C. J. (2018). Boar sperm hyperactivated motility is induced by temperature via an intracellular calcium-dependent pathway. Reproduction, Fertility and Development, 30(11), 1462. doi: 10.1071/rd17549
Juyena, N. S., & Stelletta, C. (2012). Seminal Plasma : An Essential Attribute to Spermatozoa. 33(4), 536-551. doi: 10.2164/jandrol.110.012583
Larentis, G. R., Camozzato, G. C., Bastos, H. B. A., Gregory, R. M., & Mattos, R. C. (2018). Equine Sperm Selection by Synthetic Membrane Filter. Journal of Equine Veterinary Science, 63 (17), 69-73. doi: 10. 1016/j.jevs.2018.01.013
Li, J., Roca, J., Pérez-patiño, C., Barranco, I., & Martinez, E. A. (2018). Is boar sperm freezability more intrinsically linked to spermatozoa than to the surrounding seminal plasma ? Animal Reproduction Science, 195(5), 30-37. doi: 10.1016/j.anireprosci.2018.05.002
Luna, C., Yeste, M., Rivera Del Alamo, M. M., Domingo, J., Casao, A., Rodriguez-Gil, J. E., … Muiño-Blanco, T. (2017). Effect of seminal plasma proteins on the motile sperm subpopulations in ram ejaculates. Reproduction, Fertility and Development, 29(2), 394-405. doi: 10.1071/RD15231
Menegat, M. B., Mellagi, A. P. G., Bortolin, R. C., Menezes, T. A., Vargas, A. R., Bernardi, M. L., … Bortolozzo, F. P. (2017). Sperm quality and oxidative status as affected by homogenization of liquid-stored boar semen diluted in short- and long-term extenders. Animal Reproduction Science, 179(17), 67-79. doi: 10.1016/j.anireprosci.2017.02.003
Muiño, R., Rivera, M. M., Rigau, T., Rodriguez-Gil, J. E., & Peña, A. I. (2008). Effect of different thawing rates on post-thaw sperm viability, kinematic parameters and motile sperm subpopulations structure of bull semen. Animal Reproduction Science, 109(1-4), 50-64. doi: 10.1016/j.anireprosci.2007.11.028
Nogueira, B. G., Bitencourt, J. L., Sampaio, B. F. B., Bender, E. S. C., Costa e Silva, E. V., & Zúccari, C. E. S. N. (2014). Peroxidação lipídica e agentes antioxidantes no sêmen de mamíferos. Revista Electronica de Veterinaria, 15(1), 1-15. Recuperado de https://www.veterinaria.org/revistas/redvet/n010114
Okazaki, T., Abe, S., Yoshida, S., & Shimada, M. (2009). Seminal plasma damages sperm during cryopreservation , but its presence during thawing improves semen quality and conception rates in boars with poor post-thaw semen quality. Theriogenology, 71(9), 491-498. doi: 10.1016/j. theriogenology.2008.08.014
Pavaneli, A. P. P., Passarelli, M. da S., de Freitas, F. V., Ravagnani, G. M., Torres, M. A., Martins, S. M. M. K., … de Andrade, A. F. C. (2019). Removal of seminal plasma prior to liquid storage of boar spermatozoa: A practice that can improve their fertilizing ability. Theriogenology, 125(19), 79-86. doi: 10.1016/j.theriogenology.2018.10.020
Pedrazzi, C., Monteiro, G. A., Ramires Neto, C., Soares, R. F., Papa, F. O., Dell’aqua, J. A., & Alvarenga, M. A. (2012). Effect of removing seminal plasma using a sperm filter on the viability of refrigerated stallion semen. Journal of Equine Veterinary Science, 33(1), 40-43. doi: 10.1016/j. jevs.2012.04.008
Perez-patiño, C., Barranco, I., Parrilla, I., Valero, M. L., Martinez, E. A., Rodriguez-martinez, H., & Roca, J. (2016). Characterization of the porcine seminal plasma proteome comparing ejaculate portions. Journal of Proteomics, 142(16), 15-23. doi: 10.1016/j.jprot.2016.04.026
Placer, Z. A., Cushman, L. L., & Johnson, B. C. (1966). Estimation of product of lipid peroxidation (malonyl dialdehyde) in biochemical systems. Analytical Biochemistry, 16(2), 359-364. doi: 10.1016/0003-2697 (66)90167-9
Ramires, C., Augusto, G., Fatima, R., Pedrazzi, C., Antonio, J., Ozanan, F., & Antonio, M. (2013). Effect of removing seminal plasma using a sperm filter on the viability of refrigerated stallion semen. Journal of Equine Veterinary Science, 33(1), 40-43. doi: 10.1016/j.jevs.2012.04.008
Ribeiro, C. D. V., Moroni, J. L., & Menegat, M. B. (2016). Redalyc. The influence of air contact on the quality of extended boar semen. Acta Scientiae Veterinariae, 44(1), 1-7. doi: 10.22456/1679-9216.81 300
Saravia, F., Wallgren, M., Johannisson, A., Calvete, J. J., Sanz, L., & Pen, F. J. (2009). Exposure to the seminal plasma of different portions of the boar ejaculate modulates the survival of spermatozoa cryopreserved in MiniFlatPacks. Theriogenology, 71(9), 662-675. doi: 10.1016/j.theriogenology.2008 .09.037
Sobolewski, P., Kandel, J., & Eckmann, D. M. (2012). Air bubble contact with endothelial cells causes a calcium-independent loss in mitochondrial membrane potential. Plos One, 7(10), 1-8. doi: 10.1371/ journal.pone.0047254
Souza, F. C., Melanda, C. A. A., Zandonadi, F., Morotti, F., Martins, M. I. M., Silva, C. A., & Seneda, M. M. (2013). Análise do movimento espermático suíno submetido a refrigeração por até 96 horas em diferentes meios de diluição. 22° Encontro Anual de Iniciação Científica (EAIC) e 3° Encontro Anual de Iniciação Tecnológica e Inovação (EAITI), Londrina, PR, Brasil 22, 3.
Wasilewska, K., & Fraser, L. (2017). Boar variability in sperm cryo-tolerance after cooling of semen in di ff erent long-term extenders at various temperatures. Animal Reproduction Science, 185(8), 161-173. doi: 10.1016/j.anireprosci.2017.08.016
Yeste, M. (2016). Theriogenology Sperm cryopreservation update : Cryodamage, markers, and factors affecting the sperm freezability in pigs. Theriogenology, 85(1), 47-64. doi: 10.1016/j.theriogenology. 2015.09.047
Albuquerque, R. S., Morais, R., Reis, A. N., Miranda, M. S., Dias, E. A. R., Monteiro, F. M., … Gimenes, L. U. (2017). Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation. Reproduction in Domestic Animals, 52(5), 905-910. doi: doi.org/10.1111/rda.12992
Alkmin, D. V, Perez-patiño, C., Barranco, I., Parrilla, I., Vazquez, J. M., Martinez, E. A., … Roca, J. (2014). Cryobiology Boar sperm cryosurvival is better after exposure to seminal plasma from selected fractions than to those from entire ejaculate q. Cryobiology, 69(2), 203-210. doi: 10.1016/j.cryobiol.2014.07.004
Alvarenga, M A, Melo, C. M., Magalhães, L. C. O., & Papa, F. O. (2010). A new method to concentrate equine sperm. Animal Reproduction Science, 121(S), 186-187. doi: 10.1016/j.anireprosci.2010.04.142
Alvarenga, Marco Antonio, Papa, F. O., Carmo, M. T., Kievitsbosch, T., Castro Chaves, M. M. B., & Ramires Neto, C. (2012). Methods of Concentrating Stallion Semen. Journal of Equine Veterinary Science, 32(8), 424-429. doi: 10.1016/j.jevs.2012.06.003
Awda, B. J., Mackenzie-Bell, M., & Buhr, M. M. (2009). Reactive Oxygen Species and Boar Sperm Function1. Biology of Reproduction, 81(3), 553–561. https://doi.org/10.1095/biolreprod.109.076471
Barreiros, A. L. B. S., David, J. M., & David, J. P. (2006). Estresse oxidativo: Relação entre geração de espécies reativas e defesa do organismo. Quimica Nova, 29(1), 113-123. doi: 10.1590/s0100-40422006 000100021
Bergstein-Galan, T. G., Weiss, R. R., Bertol, M. A. F., Abreu, A. C. M. R., Busato, E., Kozicki, L. E., & Bicudo, S. D. (2017). Quality and fertility of frozen ovine spermatozoa from epididymides stored at room temperature (18–25 °C) for up to 48 h post mortem. Theriogenology, 96(17), 69-75. doi: 10.10 16/j.theriogenology.2017.04.001
Bianchi, I., Calderam, K., Maschio, É. F., Madeira, E. M., da Rosa Ulguim, R., Corcini, C. D., … Corrêa, M. N. (2008). Evaluation of amides and centrifugation temperature in boar semen cryopreservation. Theriogenology, 69(5), 632-638. doi: 10.1016/j.theriogenology.2007.11.012
Campanholi, S. P., Monteiro, F. M., Ribeiro Dias, E. A., Mercadante, M. E. Z., de Paz, C. C. P., Dell’Aqua Junior, J. A., … Garcia, J. M. (2017). Effect of seminal plasma removal before cryopreservation of bovine semen obtained by electroejaculation on semen quality and in vitro fertility. Theriogenology, 89(16), 114-121. doi: 10.1016/j.theriogenology.2016.10.008
CBRA. (2013). Manual para exame andrológico e avaliação de sêmen animal. (3rd ed.). Belo Horizonte: Colégio Brasileiro de Reprodução Animal.
Gadea, J., Martínez-Miró, S., Decuadro-Hansen, G., & Matás, C. (2006). 92 Evaluation of Boar Sperm Functionality After a Cushioned Centrifugation Technique. Reproduction, Fertility and Development, 18(2), 154. doi: 10.1071/rdv18n2ab92
Gómez-Fernández, J., Gómez-Izquierdo, E., Tomás, C., Mocé, E., & Mercado, E. de. (2012). Effect of different monosaccharides and disaccharides on boar sperm quality after cryopreservation. Animal Reproduction Science, 133(1-2), 109-116. doi: 10.1016/j.anireprosci.2012.06.010
Hallap, T., Ha, M., Larsson, B., & Rodriguez-martinez, H. (2004). Does cleansing of frozen-thawed bull semen before assessment provide samples that relate better to potential fertility ? Theriogenology, 62 (04), 702-713. doi: 10.1016/j.theriogenology.2003.11.017
Hancock, G. L. (1959). The morphologic characteristics of spermatozoa and fertility. Internacional Journal of Fertility, 4, 347-359.
Hurtado de Llera, A., Gil, M. C., Bragado, M. J., Martin-Hidalgo, D., Garcia-Marin, L. J., & Perez, C. J. (2018). Boar sperm hyperactivated motility is induced by temperature via an intracellular calcium-dependent pathway. Reproduction, Fertility and Development, 30(11), 1462. doi: 10.1071/rd17549
Juyena, N. S., & Stelletta, C. (2012). Seminal Plasma : An Essential Attribute to Spermatozoa. 33(4), 536-551. doi: 10.2164/jandrol.110.012583
Larentis, G. R., Camozzato, G. C., Bastos, H. B. A., Gregory, R. M., & Mattos, R. C. (2018). Equine Sperm Selection by Synthetic Membrane Filter. Journal of Equine Veterinary Science, 63 (17), 69-73. doi: 10. 1016/j.jevs.2018.01.013
Li, J., Roca, J., Pérez-patiño, C., Barranco, I., & Martinez, E. A. (2018). Is boar sperm freezability more intrinsically linked to spermatozoa than to the surrounding seminal plasma ? Animal Reproduction Science, 195(5), 30-37. doi: 10.1016/j.anireprosci.2018.05.002
Luna, C., Yeste, M., Rivera Del Alamo, M. M., Domingo, J., Casao, A., Rodriguez-Gil, J. E., … Muiño-Blanco, T. (2017). Effect of seminal plasma proteins on the motile sperm subpopulations in ram ejaculates. Reproduction, Fertility and Development, 29(2), 394-405. doi: 10.1071/RD15231
Menegat, M. B., Mellagi, A. P. G., Bortolin, R. C., Menezes, T. A., Vargas, A. R., Bernardi, M. L., … Bortolozzo, F. P. (2017). Sperm quality and oxidative status as affected by homogenization of liquid-stored boar semen diluted in short- and long-term extenders. Animal Reproduction Science, 179(17), 67-79. doi: 10.1016/j.anireprosci.2017.02.003
Muiño, R., Rivera, M. M., Rigau, T., Rodriguez-Gil, J. E., & Peña, A. I. (2008). Effect of different thawing rates on post-thaw sperm viability, kinematic parameters and motile sperm subpopulations structure of bull semen. Animal Reproduction Science, 109(1-4), 50-64. doi: 10.1016/j.anireprosci.2007.11.028
Nogueira, B. G., Bitencourt, J. L., Sampaio, B. F. B., Bender, E. S. C., Costa e Silva, E. V., & Zúccari, C. E. S. N. (2014). Peroxidação lipídica e agentes antioxidantes no sêmen de mamíferos. Revista Electronica de Veterinaria, 15(1), 1-15. Recuperado de https://www.veterinaria.org/revistas/redvet/n010114
Okazaki, T., Abe, S., Yoshida, S., & Shimada, M. (2009). Seminal plasma damages sperm during cryopreservation , but its presence during thawing improves semen quality and conception rates in boars with poor post-thaw semen quality. Theriogenology, 71(9), 491-498. doi: 10.1016/j. theriogenology.2008.08.014
Pavaneli, A. P. P., Passarelli, M. da S., de Freitas, F. V., Ravagnani, G. M., Torres, M. A., Martins, S. M. M. K., … de Andrade, A. F. C. (2019). Removal of seminal plasma prior to liquid storage of boar spermatozoa: A practice that can improve their fertilizing ability. Theriogenology, 125(19), 79-86. doi: 10.1016/j.theriogenology.2018.10.020
Pedrazzi, C., Monteiro, G. A., Ramires Neto, C., Soares, R. F., Papa, F. O., Dell’aqua, J. A., & Alvarenga, M. A. (2012). Effect of removing seminal plasma using a sperm filter on the viability of refrigerated stallion semen. Journal of Equine Veterinary Science, 33(1), 40-43. doi: 10.1016/j. jevs.2012.04.008
Perez-patiño, C., Barranco, I., Parrilla, I., Valero, M. L., Martinez, E. A., Rodriguez-martinez, H., & Roca, J. (2016). Characterization of the porcine seminal plasma proteome comparing ejaculate portions. Journal of Proteomics, 142(16), 15-23. doi: 10.1016/j.jprot.2016.04.026
Placer, Z. A., Cushman, L. L., & Johnson, B. C. (1966). Estimation of product of lipid peroxidation (malonyl dialdehyde) in biochemical systems. Analytical Biochemistry, 16(2), 359-364. doi: 10.1016/0003-2697 (66)90167-9
Ramires, C., Augusto, G., Fatima, R., Pedrazzi, C., Antonio, J., Ozanan, F., & Antonio, M. (2013). Effect of removing seminal plasma using a sperm filter on the viability of refrigerated stallion semen. Journal of Equine Veterinary Science, 33(1), 40-43. doi: 10.1016/j.jevs.2012.04.008
Ribeiro, C. D. V., Moroni, J. L., & Menegat, M. B. (2016). Redalyc. The influence of air contact on the quality of extended boar semen. Acta Scientiae Veterinariae, 44(1), 1-7. doi: 10.22456/1679-9216.81 300
Saravia, F., Wallgren, M., Johannisson, A., Calvete, J. J., Sanz, L., & Pen, F. J. (2009). Exposure to the seminal plasma of different portions of the boar ejaculate modulates the survival of spermatozoa cryopreserved in MiniFlatPacks. Theriogenology, 71(9), 662-675. doi: 10.1016/j.theriogenology.2008 .09.037
Sobolewski, P., Kandel, J., & Eckmann, D. M. (2012). Air bubble contact with endothelial cells causes a calcium-independent loss in mitochondrial membrane potential. Plos One, 7(10), 1-8. doi: 10.1371/ journal.pone.0047254
Souza, F. C., Melanda, C. A. A., Zandonadi, F., Morotti, F., Martins, M. I. M., Silva, C. A., & Seneda, M. M. (2013). Análise do movimento espermático suíno submetido a refrigeração por até 96 horas em diferentes meios de diluição. 22° Encontro Anual de Iniciação Científica (EAIC) e 3° Encontro Anual de Iniciação Tecnológica e Inovação (EAITI), Londrina, PR, Brasil 22, 3.
Wasilewska, K., & Fraser, L. (2017). Boar variability in sperm cryo-tolerance after cooling of semen in di ff erent long-term extenders at various temperatures. Animal Reproduction Science, 185(8), 161-173. doi: 10.1016/j.anireprosci.2017.08.016
Yeste, M. (2016). Theriogenology Sperm cryopreservation update : Cryodamage, markers, and factors affecting the sperm freezability in pigs. Theriogenology, 85(1), 47-64. doi: 10.1016/j.theriogenology. 2015.09.047
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2021-05-20
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Melanda, C. A. A., Almeida, A. B. M. de, Hidalgo, M. M. T., Souza, A. K., Trautwein, L. G. C., Haddad Neta, J., … Martins, M. I. M. (2021). Sperm filtration as an alternative technique for seminal plasma separation in boars. Semina: Ciências Agrárias, 42(4), 2439–2452. https://doi.org/10.5433/1679-0359.2021v42n4p2439
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