Effect of Coconut Milk in Extender on Ram Sperm Quality Post Freezing and Thawing Process

Sperm cryopreservation is a highly effective technique utilized in reproductive procedures, particularly in artificial insemination, as it enables the transfer of superior genes to the subsequent generation of animals. However, the exposure of sperm cells to low temperatures during the freezing process can have detrimental effects on their integrity, morphology, and viability (Gangwar et al., 2020). To counteract these negative impacts, cryoprotectants are added to the sperm extender. The primary purpose of incorporating cryoprotectants is to fulfill the energy requirements of the sperm, shield them from the adverse consequences of temperature fluctuations, mitigate the physical and chemical stresses associated with cooling, freezing, and thawing, and create an environment conducive to enhancing sperm survival (Barbas and Mascarenhas, 2009). In recent years, plant-based extenders have emerged as a viable alternative to animal-based extenders for semen preservation in various species. Coconut milk, in particular, has garnered attention due to its rich composition of fatty acids (including polyunsaturated fatty acids), amino acids, sugars, antioxidants, vitamins, and minerals. These components not only provide essential nutrients for maintaining cell function but also serve as a suitable medium for sperm extension and culture (dos Reis et al., 2023; Vasconcelos et al., 2009; Yong et al., 2009). Consequently, this research aims to explore the impact of coconut milk on the quality of ram sperm following cryopreservation. Twice a week, semen samples were obtained from five Lori-Bakhtiari rams using an artificial vagina. The collected semen from these five rams was then combined and divided into six different treatments. These treatments consisted of five variations with varying levels of coconut milk (5%, 10%, 15%, 20%, and 25%) and one treatment with egg yolk (15%). Various parameters such as sperm velocity, membrane integrity and activity, morphology, lipid peroxidation rate, and sperm DNA fragmentation were assessed both before and after the process of cryopreservation. The obtained data was subsequently analyzed using the SAS statistical software. Prior to cryopreservation, the results indicated that, with the exception of the BCF parameter, there were no disparities in the various aspects of sperm kinetics, viability, functionality, and morphology. However, a notable decline in sperm motility parameters was observed after thawing across all treatments. This detrimental impact following the freezing process was more pronounced (P<0.05) when using the extender containing coconut milk compared to the extender containing egg yolk. Consequently, in terms of other motility parameters, the egg yolk treatment exhibited significantly higher values (P<0.05) than the other treatments, except for VCL (94.36±10.41%) and BCF (5.83±0.70 Hz). The findings of Wojtusik et al. (2018) were in line with the results obtained in this study. It was observed that when coconut water and coconut milk were used as substitutes for egg yolk in rhinoceros sperm extenders, there was a decrease in both sperm motility and viability. Similarly, the use of powdered coconut water as an extender for cryopreservation of cat sperm resulted in a significant reduction in sperm movement quality compared to a tris extender (de Sousa Barbosa et al. 2020). Furthermore, when comparing different alternatives to egg yolk in the extender of rhinoceros sperm, such as soy lecithin (1 and 2%), coconut water (20%), and coconut milk (20%), it was found that the extenders containing coconut milk or water led to a loss of sperm mobility and viability. However, the extenders containing soy lecithin showed more promise as substitutes for egg yolk, as they maintained sperm viability, morphology, and acrosome integrity better than coconut milk and water (Wojtusik et al., 2018). In contrast to the findings of this investigation, the utilization of extenders comprising powdered coconut water for the cryopreservation of sperm in various animal species, including dogs, boars, horses, goats, and fish, resulted in well-preserved sperm morphological and motility parameters following the freeze-thaw process. Furthermore, studies have demonstrated that freezing goat sperm with coconut milk, bull sperm with coconut oil, and equine and boar sperm with coconut water led to enhanced sperm viability and quality compared to control groups. The ineffectiveness of the extender may be attributed to the presence of salts and ions, such as calcium, potassium, and sodium, in coconut water, which could potentially interact with the channel proteins of the sperm cell plasma membrane. It is important to note that the precise mechanism by which coconut products in the extender maintain sperm characteristics remains unknown. Previous research by Toniolli et al. (1996) suggested that coconut water may serve as a protective agent for sperm due to its content of 3-indoleacetic acid. Furthermore, the replacement of glycerol with dimethylformamide in the boar sperm extender resulted in better preservation of sperm quality after the freezing and thawing process compared to other treatments (Silva et al. 2015). This suggests that each ingredient present in an extender could have a significant impact on enhancing extender efficiency. It is worth noting that coconut milk contains high levels of polyunsaturated fatty acids, which can make sperm more vulnerable to oxidative stress (dos Reis et al., 2023; Vasconcelos et al., 2009). However, no significant differences were observed in the lipid peroxidation indicator and the DNA fragmentation index among the various extenders used in this study. This could indicate that the extenders had similar antioxidant properties. The variations in the reported findings may be attributed to differences in the combinations and quantities of products used in the extenders, the type of base extender employed, variations in semen combinations and the sensitivity of sperm in different animal species, or discrepancies in the coconut combinations utilized (dos Reis et al., 2023). This research suggests that substituting egg yolk with coconut milk entirely in a ram sperm extender does not mitigate the negative consequences of cryopreservation. Additional investigations are necessary to determine the specific composition of the extender incorporating coconut-based ingredients for the cryopreservation of ram sperm.