Cryopreservation of Ram Semen: A Review on the Roles of Emerging Synthetic and Natural Additives

Cryopreservation techniques and protocols for ram semen, particularly those with a focus on semen extender additives, have gained popularity recently, as many scientists are looking for ways to mitigate cryodamage and improve post-thaw sperm quality. The purpose of this structured narrative review was to examine studies published in 2019–2024 on both synthetic and natural additives, to separate evidence obtained in rams from comparative evidence in other species, and to analyze current trends and potential gaps for future research. Cryopreservation is an indispensable aspect in livestock production and conservation of genes using artificial insemination. Nevertheless, the freezing and thawing processes result in DNA fragmentation, oxidative stress and low sperm motility, which are detrimental to reproductive success. Conventional extenders that contain glycerol or egg yolk have been used extensively to support cell membranes and prevent the formation of ice crystals. Nevertheless, the fertility rates obtained from frozen–thawed ram semen have been poor, which has led to the investigation of other substances. Natural additives like plant polyphenols, vitamins, fatty acids and sugars have shown great potential to decrease free radicals and enhance sperm integrity. Synthetic additives, including Trolox, MitoQ, and Mito-TEMPO, were also found to enhance the antioxidant capacity. Over the past few years, some trends have emerged, including an increased interest in plant-based antioxidants and nanoparticle-based additives. Nevertheless, several research gaps were observed, including the need for further optimization of the additive dose ratios, availability of studies on the stability of these additives when used long-term, and the compatibility of extender formulations across breeds and species. This review synthesizes recent developments in ram semen cryopreservation additives; for most additives, the evidence is presented in a single study with sperm-quality endpoints. Few studies measured in vivo fertility. It emphasizes the necessity of shifting from descriptive additive studies to mechanism-focused protocols validated across breeds to enhance the scalability and reliability of reproductive biotechnologies.