Differential seminal plasma proteome signatures of boars with high and low resistance to hypothermic semen preservation at 5°C

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.

Bovine seminal plasma osteopontin: Structural modelling, recombinant expression and its relationship with semen quality

“…Right-Detailed view of the OPN (cyan) interaction with CD44 (green); and the alpha (green) and beta (yellow) subunits of α5β1 and αVβ3 Current in silico approaches for the study of protein-protein interaction were used to improve our understanding on how OPN interacts with membrane proteins. Together with structural modelling, these tools have been successfully used to better understand the function seminal plasma proteins such β-defensins (Weber et al, 2018), protein disulphide isomerase (van der Linden et al, 2020) and cathepsin B (Menezes et al, 2020). The first step was to generate a reliable tertiary structure for OPN using different protein modelling servers.…”

Sperm freezing damage: the role of regulated cell death

“…Furthermore, the surviving subpopulations of sperm demonstrate functional impairments upon thawing, compromising their ability to perform normal physiological functions [ 13 , 25 – 28 ]. Interestingly, fresh semen (with a total motility of over 70–80%) that is considered of sufficient quality for freezing also has a mortality rate of approximately 20% after ejaculation and has significant individual [ 29 , 30 ] and seasonal differences in freezability [ 31 , 32 ]. In addition, the freezability of semen is not only related to the sperm itself but also to the seminal plasma [ 33 ].…”

Update on artificial insemination: Semen, techniques, and sow fertility

“…Despite these promising findings, hypothermic storage still needs further investigation. Just one boar semen extender (Androstar ® Premium) has been successfully demonstrated as suitable for 5°C storage so far, and inappropriate cooling curves (Paschoal et al, 2020), individual resistance of boar to 5°C storage (Jäkel et al, 2021;Menezes, Bustamante-Filho, et al, 2020) have been reported to impair the application of hypothermic preservation.…”

“…Indeed, seminal plasma proteins such as cathepsin B, spermadhesin PSP‐I, epididymal secretory protein E1 precursor, granulin precursor, legumain, and AWN were associated with a reduction in resistance to liquid storage at 17°C (De Lazari et al, 2019; Höfner et al, 2020). In addition, our group recently suggested the proteins cathepsin B, legumain, and cystatin B as candidate markers for semen resistance to liquid preservation at 5°C (Menezes, Bustamante‐Filho, et al, 2020). From a practical point of view, the dilution rate of semen doses may vary among boar studs.…”

“…Despite these promising findings, hypothermic storage still needs further investigation. Just one boar semen extender (Androstar® Premium) has been successfully demonstrated as suitable for 5°C storage so far, and inappropriate cooling curves (Paschoal et al, 2020), individual resistance of boar to 5°C storage (Jäkel et al, 2021; Menezes, Bustamante‐Filho, et al, 2020) have been reported to impair the application of hypothermic preservation. Considering that bacterial growth can be minimized but not prevented in doses extended with antibiotic‐free Androstar® Premium and stored at 5°C (Menezes, Mellagi, et al, 2020), strict hygiene during semen collection and processing is still needed.…”