Tila de Alcantara Menezes1, Ivan Cunha Bustamante-Filho2, Aline Fernanda Lopes Paschoal1, Pedro Ferrari Dalberto3,4, Cristiano Valim Bizarro3,4, Mari Lourdes Bernardi5, Rafael da Rosa Ulguim1, Fernando Pandolfo Bortolozzo1, Ana Paula Gonçalves Mellagi1. 1. Setor de Suínos, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil. 2. Laboratório de Biotecnologia, Universidade do Vale do Taquari - Univates, Lajeado, Brazil. 3. Centro de Pesquisas em Biologia Molecular e Funcional, Instituto Nacional de Ciência e Tecnologia em Tuberculose, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Brazil. 4. Programa de Pós-Graduação em Biologia Celular e Molecular, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Brazil. 5. Departamento de Zootecnia, Faculdade de Agronomia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.
Abstract
BACKGROUND: Hypothermic storage at 5°C has been investigated as an alternative to promote the prudent use of antibiotics for boar artificial insemination doses. However, this temperature is challenging for some ejaculates or boars. OBJECTIVE: The present study aimed to identify putative biomarkers for semen resistance to hypothermic storage at 5°C by comparing the seminal plasma proteomes of boars with high and low seminal resistance to preservation at 5°C. MATERIALS AND METHODS: From an initial group of 34 boars, 15 were selected based on the following criteria: ejaculate with ≤20% abnormal spermatozoa and at least 70% progressive motility at 120 hours of storage at 17°C. Then, based on the response to semen hypothermic storage at 5°C, boars were classified into two categories: high resistance-progressive motility of >75% in the three collections (n = 3); and low resistance-progressive motility of <75% in the three collections (n = 3). Seminal plasma proteins were analyzed in pools, and differential proteomics was performed using Multidimensional Protein Identification Technology. RESULTS: Progressive motility was lower at 120 hours of storage in low resistance, compared to high resistance boars (P < .05). Acrosome and plasma membrane integrity were not affected by the boar category, storage time, or their interaction (P ≥ .104). Sixty-five proteins were considered for differential proteomics. Among the differentially expressed and exclusive proteins, the identification of proteins such cathepsin B, legumain, and cystatin B suggests significant changes in key enzymes (eg, metalloproteinases) involved in spermatogenesis, sperm integrity, and fertilizing potential. DISCUSSION AND CONCLUSION: Differences in the seminal plasma suggest that proteins involved in the proteolytic activation of metalloproteinases and proteins related to immune response modulation could disrupt key cellular pathways during spermatogenesis and epididymal maturation, resulting in altered resistance to chilling injury. Further in vivo studies focusing on the immunological crosstalk between epithelial cells and gametes might explain how the immune regulators influence sperm resistance to hipothermic storage.
BACKGROUND:Hypothermic storage at 5°C has been investigated as an alternative to promote the prudent use of antibiotics for boar artificial insemination doses. However, this temperature is challenging for some ejaculates or boars. OBJECTIVE: The present study aimed to identify putative biomarkers for semen resistance to hypothermic storage at 5°C by comparing the seminal plasma proteomes of boars with high and low seminal resistance to preservation at 5°C. MATERIALS AND METHODS: From an initial group of 34 boars, 15 were selected based on the following criteria: ejaculate with ≤20% abnormal spermatozoa and at least 70% progressive motility at 120 hours of storage at 17°C. Then, based on the response to semen hypothermic storage at 5°C, boars were classified into two categories: high resistance-progressive motility of >75% in the three collections (n = 3); and low resistance-progressive motility of <75% in the three collections (n = 3). Seminal plasma proteins were analyzed in pools, and differential proteomics was performed using Multidimensional Protein Identification Technology. RESULTS: Progressive motility was lower at 120 hours of storage in low resistance, compared to high resistance boars (P < .05). Acrosome and plasma membrane integrity were not affected by the boar category, storage time, or their interaction (P ≥ .104). Sixty-five proteins were considered for differential proteomics. Among the differentially expressed and exclusive proteins, the identification of proteins such cathepsin B, legumain, and cystatin B suggests significant changes in key enzymes (eg, metalloproteinases) involved in spermatogenesis, sperm integrity, and fertilizing potential. DISCUSSION AND CONCLUSION: Differences in the seminal plasma suggest that proteins involved in the proteolytic activation of metalloproteinases and proteins related to immune response modulation could disrupt key cellular pathways during spermatogenesis and epididymal maturation, resulting in altered resistance to chilling injury. Further in vivo studies focusing on the immunological crosstalk between epithelial cells and gametes might explain how the immune regulators influence sperm resistance to hipothermic storage.