Optimizing thyroxine supplementation for enhanced rabbit buck semen cryopreservation: impacts on oxidative status, apoptosis-related gene expression, and fertility

Abstract

Introduction This study evaluated the effects of thyroxine (T4) supplementation on the quality, functional characteristics, antioxidant status, apoptosis-related sperm-associated transcript abundance, and fertility of cryopreserved rabbit buck semen. Materials and methods Semen was collected weekly from 15 rabbit bucks over 12 weeks. Ejaculates meeting the predefined quality criteria were pooled within each weekly collection, and each weekly pool represented an independent biological replicate ( n = 12). Each pooled sample was divided into seven equal aliquots and assigned to a control extender without T4 or extenders supplemented with T4 at 0.25, 0.5, 0.75, 1.0, 1.25, or 1.5 μg/dL (T0.25, T0.5, T0.75, T1, T1.25, and T1.5, respectively). Result and discussion Moderate T4 supplementation, particularly at 0.75 and 1.0 μg/dL, improved several sperm-quality and functional parameters compared with the control and higher-dose treatments ( p < 0.05). The T0.5, T0.75, and T1 groups showed improved antioxidant status, mitochondrial membrane potential, DNA and membrane integrity, together with lower malondialdehyde levels. Flow-cytometric analysis also demonstrated higher proportions of viable sperm and lower proportions of late-apoptotic and necrotic sperm in these groups. qRT-PCR analysis revealed lower relative abundance of Bax and Caspase-3 transcripts and higher Bcl-2 transcript abundance with moderate T4 supplementation, resulting in a lower Bax/Bcl-2 transcript ratio. These findings represent differences in sperm-associated transcript abundance and should not be interpreted as evidence of active transcriptional regulation in mature spermatozoa. Fertility outcomes were also improved, with pregnancy rates of 93.33 and 83.33% in the T0.75 and T1 groups, respectively, compared with 73.33% in the control group. Litter size was also higher in the T0.25, T0.5, T0.75, and T1 groups ( p < 0.05). Overall, moderate T4 supplementation, particularly at 0.75–1.0 μg/dL, was associated with improved post-thaw sperm quality, antioxidant status, mitochondrial functional status, and fertility in rabbits. However, the mechanisms underlying these effects require further investigation.