Artificial insemination (AI) is a key reproductive biotechnology for genetic improvement in sheep. However, its efficiency remains lower and more variable than in most other livestock species. This review critically synthesizes the historical foundations of sheep AI, including methodological principles established by the Soviet school, and evaluates how these concepts have been further developed and adapted to contemporary reproductive biology. Particular emphasis is placed on estrous synchronization protocols, semen processing and cryopreservation, and insemination techniques. We highlight how anatomical constraints of the ovine cervix, seasonal reproductive physiology, and species-specific characteristics of ram sperm collectively limit fertility outcomes, especially when frozen–thawed semen is used. Comparative analysis of cervical, transcervical, and laparoscopic insemination methods indicates that laparoscopic AI remains the most reliable approach, although recent advances in catheter design and semen handling have improved the feasibility of less invasive techniques. This review further discusses emerging approaches, including sperm sex-sorting, alternative recovery methods, and early-stage spermatogonial stem cell–based technologies, emphasizing both their potential applications and current limitations. Overall, the available evidence suggests that future progress in sheep AI will depend on the integrated optimization of hormonal synchronization, semen preservation, and insemination strategies, rather than on isolated technical innovations.

In modern livestock, the demand for sperm sex selection technologies is high, as the ability to deliberately produce offspring of a specific sex offers significant economic advantages. Traditionally, sperm sorting is performed using Fluorescence-Activated Cell Sorting. However, the flow cytometric method is expensive, technically complex, and associated with reduced sperm viability. An alternative promising method involves the use of Toll-like TLR7/8 receptors for the selective binding of spermatozoa of a particular sex. It was discovered previously that the activation of TLR7/8 by its ligand(s) selectively inhibits the motility of X-bearing sperm without affecting the motility of Y-bearing sperm. The swim-up technique, which separates sperm based on sex chromosome type by isolating fractions enriched in either X- or Y-bearing gametes due to differences in their motility, can be used with this method. Sperm sex sorting via the TLR7/8 activation is cheap, technically non-complex, and does not affect sperm viability negatively. The goal of this review is to provide an overview of the TLR7/8-dependent sperm sorting method. Further, we discuss why the method of sperm sorting via TLR7/8 activation is successfully implemented in some animal species (such as murine, caprine, ovine, and bovine) but fails in others, like swine and canine.

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.