The association of resveratrol and AFPI did not enhance the cryoresistance of ram sperm

Abstract

Cryoprotectants are required to reduce damage caused to the cells due to low temperatures during the cryopreservation. Antifreeze proteins (AFP) have a well-known role in cell membrane protection, while resveratrol is a potent antioxidant. This study assessed the effect of the association of resveratrol concentrations and AFP I in a ram semen extender. Pooled semen of four rams was allocated into six treatments in a factorial arrangement: (CONT, only the semen extender); only AFP I (ANT: 0.1 µg/mL of AFP I), only resveratrol, one treatment with two levels (10 µM/mL or 50 µM/mL of resveratrol); and two treatments with the interactions, with one AFP I and one of the two levels of resveratrol (0.1 µg/mL of AFP I with 10 µM/mL resveratrol; 0.1 µg/mL of AFP I with 50 µM/mL resveratrol). No interaction between factors was observed on sperm kinetics, plasma membrane integrity, hypo-osmotic test, and mitochondrial activity parameters. There was a high probability (P = 0.06) of reducing sperm cells with functional membrane percentage in the hypo-osmotic test and increasing the percentage of sperm with high mitochondrial activity (P = 0.07) was observed in AFP presence. An interaction of AFP and resveratrol was observed in non-capacitated sperm (P = 0.009), acrosomal reaction (P = 0.034), and sperm binding (P = 0.04). In conclusion, the association of resveratrol and AFP did not improve the quality of frozen-thawed semen and even promoted deleterious effects compared to their single addition in the semen extender. The supplementation of 50 µM/mL of resveratrol improved the outcomes of frozen-thawed ram sperm, being a potential cryoprotectant.

Keywords: antioxidant; cryoprotectants; sheep; slow-freezing; spermatozoa.

Figure 1. Experimental design scheme: CONT) control containing only extender; ANT) added of AFPI; R10) 10 µM/mL resveratrol; R50) 50 µM/mL resveratrol; AR10) AFP I with 10 µM/mL resveratrol; AR50) AFP I with 50 µM/mL resveratrol. The concentration of AFP I was: 0.1 µg/mL.

Figure 2. Interaction of (A) capacitated sperm, (B) non-capacitated sperm, and (C) acrosome-reacted sperm of frozen-thawed ram semen with or without the association of AFP I and different concentrations of resveratrol, immediately after thawing. Within a column or row, values with different superscripts differ significantly (P < 0.05). A,B differs between the absence or presence of AFP I (0.1 μg/mL). ^a,bdiffers among resveratrol concentrations (0, 10, or 50 μM/mL).

Figure 3. Interaction between the association of AFP I and resveratrol concentrations in semen extender on frozen-thawed ram sperm bound to egg perivitelline membrane test, immediately after thawing. *represents the high probability (P = 0.071) of sperm bound in a single addiction of 0.1 µg/mL of AFP I in interaction analysis; **represents the high probability (P = 0.058) of sperm bound in a single addiction of 50 µM/mL of resveratrol in the interaction analysis.

Figure 4. A diagrammatic figure summarizing the main hypothesis explaining why the association between antifreeze protein I (AFP I) and resveratrol did not promote benefits for ram sperm cryopreservation.