Increasing the freezeability of bovine sperm by adding skimmed cow's milk and seminal plasma to the composition of a Tris-based cryoprotective medium

Introduction: The decrease in the sperm number in each dose of artificial insemination (AI) has led to the deterioration of sperm quality in different species. Decreasing sperm quality could be a major obstacle to the commercial application of flow-sorted sperm for gender selection. The reduction in the viability of cryopreserved spermatozoa at high dilution rates (low number of sperm/dose) has been attributed to the lack of useful components of the seminal plasma in the final freezing medium. Although the effect of adding or removing seminal plasma or its proteins on sperm activity has been widely investigated, the results of these studies are contradictory. Factors such as the final dilution rate and the extenders used in each work may influence the results. On the other hand, a milk-based extender was reported to be better than a Tris-citrate-fructose extender for a percentage of uncapacitated and linear motile spermatozoa after freezing-thawing of ovine semen. However, milk-based diluents interfere with the evaluation of sperm kinematics by the computer-assisted sperm analysis (CASA) systems. It is speculated that by adding milk to the composition of Tris diluents while taking advantage of the positive effects of milk, problems related to sperm evaluation disorders by CASA will not occur. Therefore, this study aimed to investigate the impacts of adding bovine skim milk and seminal plasma into the formulation of a Tris-citrate-egg yolk LDL extender on the kinematics and viability of frozen-thawed bull sperm when sub-optimal sperm numbers were packed in straws. Materials and methods: Semen samples were collected from four bulls once a week for four consecutive weeks. The ejaculates collected on the same day were pooled provided that the semen concentration and progressive motility in each ejaculate were ≥2×109 cells/mL and ˃70%, respectively. The semen pool was divided into four equal aliquots and diluted with experimental extenders to obtain a final sperm concentration of 15×106 spermatozoa per mL. The base extender [made by Tris buffer (80% v/v), liquid ammonium sulfate-insoluble yolk fraction (20%), and glycerol (7% v/v)] was supplemented with 0, 5, and 10% (v/v) milk, and 0, 2 and 5% bovine seminal plasma. Therefore, the experiment was conducted in a completely randomized design with a factorial arrangement of 3×3. Diluted semen samples were frozen and stored in liquid nitrogen after being cooled and packed in 0.5 mL straws. The percentage of live cells in thawed samples was determined after Eosin-nigrosin staining and motility and kinematic parameters were analyzed by CASA. Data were subjected to analysis of variance using the general linear model of SAS software (version 9.2). Significant differences among treatment means were evaluated using the Tukey test. The statistical significance level was set at 0.05. Results and discussion: The results indicated that the addition of milk at both 5 and 10% led to an increase in progressive motility, straightness, and sperm velocity parameters (Average path velocity, curvilinear velocity, and straight-line velocity). Adding 5% seminal plasma to the basal extender formulation increased the motility indexes (total and progressive motility), velocity indexes, and directional indices of cryopreserved spermatozoa. Among the parameters reflecting sperm wobble characteristics, wobble and amplitude of literal head increased; however, beat cross frequency and mean angular displacement decreased in the freeze-thawed spermatozoa diluted in the extenders containing 5% seminal plasma compared to those in the control straws. The average concentrations of both factors (5% milk and 2% seminal plasma), when present together in the base composition of the diluent, have shown a better result in terms of percentage sperm viability than when present alone. Conclusions: According to the results, adding 5% seminal plasma to the extender formulation increased the kinematic parameters of frozen-thawed sperms. However, it should be noted that these increases do not seem to be desirable in sperm doses for artificial insemination but could be acceptable for in vitro fertilization (IVF). However, the 2% level of seminal plasma reduced the mean angular displacement parameter without affecting the velocity indices, which may indicate an improvement in sperm fertility. The addition of 2% semen plus 5% milk in bull semen diluent has resulted in better sperm viability. In addition, the 5% proportion of milk improved many parameters of sperm motility and kinematics, as did the 10% proportion. Therefore, the addition of 2% bovine seminal plasma plus 5% skimmed cow's milk to a Tris-LDL-citrate extender may positively affect the viability of frozen-thawed bull sperm and kinematic values ​​associated with fertility when semen is diluted in small amounts/doses.