Breed-Specific Responses of Rabbit Semen to Chilling Storage: Sperm Quality, Acrosome Status, and Oxidative Stress Biomarkers

Abstract

Artificial insemination (AI) in rabbits depends largely on chilled semen storage, but the physiological responses to chilling and associated biochemical changes in seminal plasma (SP) remain poorly understood, particularly across breeds. This study aimed to compare the semen preservation capacity of Algerian local population (LAP) and New Zealand White (NZW) rabbits and to explore the relationship between SP oxidative stress biomarkers and sperm traits during 72 h of chilled storage at 5 °C. Semen pools (nine/breed) were evaluated at 0, 4, 24, 48, and 72 h for motility, viability, and acrosome status. Oxidative stress markers were also assessed in the SP, including malondialdehyde (MDA), reactive oxygen metabolites (ROMs), superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT). LAP sperm showed higher motility (p < 0.001) and viability (p < 0.05), particularly between 4 h and 48 h, and exhibited a lower rate of acrosome reaction (p < 0.001) from 48 h to 72 h. Lower SOD and higher CAT activity in LAP (p < 0.001), correlated with MDA and acrosome status, respectively, may reflect a more balanced antioxidant response. Lipid peroxidation did not appear to be the main factor driving sperm deterioration (p > 0.05). These results demonstrate that LAP rabbits exhibit better resilience to chilled storage compared to NZW and highlight the potential value of CAT and SOD activities as indicators of sperm resilience during chilled storage. Further studies are required to validate and extend these findings, with the aim of improving semen preservation strategies.

Keywords: acrosome status; antioxidant enzymes; chilled semen storage; oxidative stress; rabbit; seminal plasma; sperm quality.

Figure 1

Effect of chilled storage (0, 4, 24, 48, and 72 h) on sperm motility (a) and viability (b) of LAP and NZW rabbits. Data are shown as the mean ± SEM. Different lowercase letters (a, b, c, and d) indicate significant differences (p < 0.05) between storage time points within the same breed. Asterisks indicate significant differences between breeds at the same time point (* p < 0.05 and ** p < 0.01). p-values from GLMM analysis for breed and storage time are displayed directly on each plot.

Figure 2

Effect of chilled storage (0, 4, 24, 48, and 72 h) on acrosome status ((a): un-capacitated acrosome (IN); (b): capacitated acrosome (CP); and (c): capacitated acrosome-reacted (AR)) of LAP and NZW rabbit sperm. Data are shown as the mean ± SEM. Different lowercase letters (a, b, c, and d) indicate significant differences (p < 0.05) between storage time points within the same breed. Asterisks indicate significant differences between breeds at the same time point (* p < 0.05, ** p < 0.01, and *** p < 0.001). p-values from GLMM analysis for breed and storage time are displayed directly on each plot.

Figure 3

Effect of chilled storage (0, 4, 24, 48, and 72 h) on MDA (a) and ROMs (b) levels and SOD (c), GPX (d), and CAT (e) activities in seminal plasma of LAP and NZW rabbits. Data are shown as the mean ± SEM. Asterisks indicate significant differences between breeds at the same time point (* p < 0.05, ** p < 0.01, and *** p < 0.001). p-values from GLMM analysis for breed and storage time are displayed directly on each plot.

Figure 4

Heat map showing the correlations between sperm quality parameters (sperm motility and viability), acrosome status (IN, CP, and AR frequencies), and OS biomarkers in SP (MDA, ROMs, SOD, GPX, and CAT activities) of LAP and NZW rabbits. The colors on the scale (1 to −1) indicate whether the correlation is positive (blue) or negative (orange). (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001.