Extender development for optimal cryopreservation of buck sperm to increase reproductive efficiency of goats

Abstract

Preservation of sperm significantly contributes to the advancement of assisted reproductive technologies, genetic conservation and improvement efforts, and precision breeding of livestock. This review distills knowledge from the existing information and emerging patterns in the field of buck sperm cryopreservation. The primary focus is on the challenges and opportunities associated with improving extender formulations and freezing techniques in order to enhance the vitality of sperm after thawing and to increase the potential for conception. This review assesses the efficacy and limitations of conventional extenders derived from egg yolk or soybean lecithin, and the adverse impacts of seminal plasma enzymes on sperm quality during the processes of chilling and cryopreservation. Significant progress has been made in the fields of molecular biology namely lipidomics, proteomics, metabolomics, DNA methylation providing valuable knowledge regarding the unique reactions of sperm to cryopreservation. The utilization of the "omics" technologies has shown intricate molecular transformation that occur in sperm during freezing and thawing. Moreover, detection of molecular biomarkers that indicate the quality of sperm and their ability to withstand freezing provides opportunities to choose the best sperm samples for cryopreservation. This, in turn, enhances the results of artificial insemination and genetic conservation endeavors. This review emphasizes the necessity for adopting a comprehensive approach that combines molecular and cellular knowledge with practical methods in the field of sperm cryopreservation to ensure production of goats as major food animals in the global scale.

Keywords: antioxidants; cryopreservation; extender; omics; sperm; sustainable goat production.

Figure 1

Sperm cryopreservation protocol. The cryopreservation process of semen typically begins with the collection of semen using an artificial vagina. However, electroejaculation may be employed in cases where elite male animals are unable to mount due to conditions such as lameness, shyness, or limb abnormalities. The collected ejaculate is then evaluated for key quality parameters such as motility and morphology. Following this assessment, semen is diluted with appropriate extenders. The diluted semen is loaded into 0.25 or 0.5 mL straws and cooled to 5°C, followed by an equilibration at this temperature for ~3 h. During the freezing stage, temperature reduction occurs in distinct phases to minimize cellular damage and osmotic stress. Initially, the straws are cooled to −5 to −7°C to induce extracellular ice nucleation, preventing intracellular ice formation. This is followed by a controlled cooling phase, during which the temperature is gradually reduced at a rate of ~1°C per min until reaching −35°C. This step allows the cells to adapt to the temperature decrease, minimizing intracellular crystallization and membrane damage. The final phase involves rapid cooling to −196°C, which transitions the sperm cells into a vitrified state, halting all metabolic activities and preserving cellular integrity (204). Two main methods are employed during the freezing stage of cryopreservation. The first, conventional method involves placing the straws in a styrofoam box, 4–8 cm above the liquid nitrogen surface (with a nitrogen level of 5 cm) to be frozen in the nitrogen vapor for 15 min (Method A). The second method utilizes automatic freezing systems. These systems offer a wide range of temperatures, from +40°C to −180°C, and operate with precise cooling rates between 0.01°C and 60°C/min (Method B). Both methods aim to ensure the gradual adaptation of sperm cells to temperature changes, thus preserving their viability and motility. Following cryopreservation, the straws are transferred into liquid nitrogen (−196°C) for long-term storage.

Figure 2

Sperm evaluation. (A) CASA (Computer-Assisted Sperm Analysis). (B) Acrosomal Membrane Integrity, B1: Intact acrosomal membrane, B2: Damaged acrosomal membrane. (C) Plasma Membrane Integrity, C1: Intact plasma membrane, C2: Damaged plasma membrane. (D) Mitochondrial membrane potential, D1: High mitochondrial membrane potential, D2: Low mitochondrial membrane potential, D3: No mitochondrial membrane potential. (E) Immunolocalization of Histones in Bovine Sperm Cells, E1: Negative control sperm cell incubated with only the secondary antibody, E2: Sperm cell with a medium level of histone fluorescence, E3: Sperm cell with a high level of histone fluorescence. (F) Reactive Oxygen Species (H₂O₂) and Viability, F1: Viable sperm without ROS, F2: Dead sperm with ROS, F3: Viable sperm with ROS. (G) Mitochondrial $O_2^{-}$ generation, G1: Live cells without stain, G2: Dead cells with green stain, G3: positive mitochondrial $O_2^{-}$ generation, G4: Dead and positive mitochondrial $O_2^{-}$ generation. (H) Proteomics. (I) Metabolomics.