Additives used as stimulative and cryoprotective agents in bovine semen cryopreservation: a systematic review

Cryopreservation of bovine semen is critical for artificial insemination and genetic preservation, yet a significant remaining challenge is damage to sperm cells during the freezing and thawing process. Recent studies have led to the development of additives designed to shield sperm cells from the detrimental effects of cryopreservation. This systematic review evaluates the effectiveness of cryoprotective and stimulative additives in enhancing the quality of post-thaw bovine semen. A thorough literature search was conducted using Google Scholar, Science Direct, and PubMed databases to identify studies on stimulative and cryoprotective additives used in the cryopreservation of bull semen. The search was conducted using the key terms ‘stimulants bull semen cryopreservation’, ‘cryoprotective additives bull semen cryopreservation’, and ‘additives used in bovine semen cryopreservation’. A total of 57 studies were evaluated, identifying glutathione (2.5–4.0 mM), trehalose (30–100 mM), and melatonin (1–2 mM) as optimal additives for preserving sperm motility, membrane integrity, and reducing oxidative stress. Stimulants such as pentoxifylline (3.60–7.18 mM) improved motility by increasing cyclic adenosine monophosphate levels. Natural additives, including moringa extract and honey, showed similar effectiveness to synthetic additives, with cost and sustainability advantages. Additives such as glutathione, trehalose, and sericin have demonstrated significant effectiveness in preserving sperm membrane integrity and reducing oxidative stress. Critical gaps include dose-dependent toxicity of crocin and melatonin (> 3 mM). Combinatorial approaches such as glutathione + selenium nanoparticles are recommended to mitigate cryodamage. In conclusion, this review emphasizes optimizing additive selection and concentrations based on their specific roles and interactions. These findings guide the development of cost-effective extenders for improving cattle breeding programs globally.