Antibiotic-free extended boar semen preserved under low temperature maintains acceptable in-vitro sperm quality and reduces bacterial load.

This study aimed to assess the sperm quality and number of colony-forming units (CFU mL-1) in extended boar semen stored at low temperatures with or without antibiotics. Normospermic ejaculates (n = 34) were diluted in split samples with Androstar® Premium with or without antibiotics (ampicillin and apramycin sulfate). The extended semen doses were stored for 120 h under three storage temperatures (5, 10, and 17 °C). Variables were analyzed as repeated measures using the GLIMMIX procedure, in a factorial design. The extended semen doses under low-temperature storage (5 and 10 °C) had total motility above 75% throughout the storage. The interaction antibiotic × temperature was significant for total (P = 0.004) and progressive motility (P = 0.005). In extended boar semen doses with antibiotics, the total and progressive motility increased as the storage temperature increased (80.2%, 84.5%, and 89.1%; 70.5%, 76.0%, and 82.9% for total and progressive motility at 5, 10, and 17 °C, respectively; P < 0.05). In extended semen doses without antibiotics, the total and progressive motility were lower when stored at 5 °C than at 10 °C and 17 °C (81.8%, 85.4% and 86.6% and 71.9%, 76.7%, 78.9% for total and progressive motility at 5, 10, and 17 °C, respectively; P < 0.05). After the thermoresistance test, total and progressive motility of doses with antibiotics were higher at 17 °C than 5 °C (P < 0.05); however, they were not affected (P > 0.05) by storage temperature in extended semen doses without antibiotics. The number of CFU mL-1 was lower in extended semen doses without antibiotics stored at 5 and 10 °C than at 17 °C (P < 0.05); however, in extended semen doses with antibiotics, no effect of storage temperature was observed (P > 0.05). The bacterial load was greater in extended semen without antibiotics than with antibiotics, regardless of the storage temperature (P < 0.05). The acrosome and sperm membrane integrity were not influenced (P > 0.05) by using antibiotics. A higher percentage of normal acrosomes was observed as the storage temperature increased (93.6%, 94.3%, and 96.8% at 5, 10, and 17 °C, respectively; P < 0.0001). The membrane integrity was higher (P < 0.0001) in extended semen doses stored at 17 °C than at 10 or 5 °C. The pH rose throughout the storage in all the treatments, except in extended semen doses stored at 17 °C without antibiotics, in which a decrease in the pH occurred at 120 h (P < 0.05). Although the sperm quality being negatively affected by low temperatures, the storage of extended boar semen doses at 5 °C is possible since the sperm viability in vitro was maintained for up to 5 days, fulfilling the requirements of semen quality to be used in artificial insemination. Nevertheless, the use of extended semen doses without antibiotics requires the optimization of hygiene procedures during semen dose processing.

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

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.

Role of insulin-like growth factor-I and follicular fluid from ovarian follicles with different diameters on porcine oocyte maturation and fertilization in vitro.

The objective of this study was to determine the effects of insulin-like growth factor-I (IGF-I) (0, 60, 120, 180, and 240 ng/mL) and follicular fluid (FF) derived from 2 to 5 and 6 to 10 mm diameter follicles (SpFFs and LpFFs, respectively) added during in vitro maturation (IVM) of porcine oocytes on nuclear maturation and IVF. Cumulus-oocyte complexes (COCs) were matured in NCSU-37 medium supplemented with SpFFs or LpFFs and various IGF-I concentrations. The COCs were cultured for 44 hours, and then fertilized in vitro. Maturation and IVF results were recorded 18 hours after insemination. The IVM (%) was higher (P < 0.05) in the COCs matured in LpFFs than with SpFFs when 0 (90.0 ± 6.9 vs. 76.3 ± 10.7) or 60 ng/mL IGF-I (92.0 ± 8.1 vs. 81.8 ± 10.2) was added. In SpFFs media, there was a quadratic relationship (P < 0.01) between IGF-I concentration and IVM (peak results at IGF-I = 129 ng/mL). However, when the COCs were matured with LpFFs, there was a decreasing linear effect between IGF-I concentration and IVM. At all concentrations of IGF-I, the percentage of degenerated oocytes was higher in COCs matured in SpFFs than in LpFFs. Penetration (%) did not differ (P > 0.05) between COCs matured with SpFFs or LpFFs when 60 (66.8 ± 9.4 vs. 72.7 ± 11.3) or 180 ng/mL of IGF-I (75.7 ± 10.4 vs. 73.8 ± 13.2) were used. Monospermy (%) was similar between SpFFs and LpFFs only with addition of 120 ng/mL IGF-I. The IVF performance (%) did not differ between COCs matured with SpFFs or LpFFs when IGF-I concentrations of 120 (28.5 ± 8.8 vs. 38.5 ± 8.3) and 180 ng/mL (24.3 ± 10.2 vs. 30.12 ± 8.2) were used. There was no effect of IGF-I concentration or of FF type on the number of penetrated sperm per oocyte and on male pronuclear formation. For COCs matured with SpFFs, there was a quadratic relationship between IGF-I concentration and penetration, monospermy, and IVF performance (peak results at IGF-I = 179, 122, and 135 ng/mL, respectively). Thus, on the basis of the observed quadratic relationships, we inferred that when using SpFFs, the addition of IGF-I (122-179 ng/mL) to the IVM medium produced results similar to those obtained with LpFFs without adding IGF-I. In conclusion, the addition of IGF-I to the IVM medium supplemented with SpFFs increased maturation and improved IVF results. Alternatively, IGF-I had no effect on IVM or IVF when used with LpFFs.