Optimizing Bull Semen Cryopreservation Media Using Multivariate Statistics Approaches

SIMPLE SUMMARY: The dairy cattle industry relies heavily on artificial insemination facilitated by cryopreservation of bull semen. Cryopreservation causes a number of injuries to sperm reducing fertility and decreasing economic value. Protective extenders are media made up of a number of compounds. Most studies evaluate the impact of changing the concentration of one or two compounds at the same time. Here we use multivariate statistical methods on a large experimental dataset over twelve different compounds to identify key classes of compounds, key components of those classes, and the sensitivities of post-thaw viability on concentrations of these compounds. These statistical models and this methodology point towards improved cryopreservation protocols an improved understanding of the complex interactions among extender components. ABSTRACT: Cryo-injury reduces post-thaw semen quality. Extender components play a protective role, but existing experimental approaches do not elucidate interactions among extender components, semen samples, and post-thaw quality. To identify optimal concentrations for 12 extender ingredients, we ran 122 experiments with an I-optimal completely random design using a large dataset from our previous study. We obtained a maximum predicted total motility of 70.56% from an I-optimal design and 73.75% from a Monte Carlo simulation. Individual bull variations were significant and interacted with extenders independently. 67% of bulls reliably preferred extender formulations to reach maximum motility. Multifactor analysis suggests that some antioxidants may offer superior protection over others. Partial least squares path modeling (PLS-PM) found the highest positive loadings for glutathione in the antioxidant class, glycerol in the CPA class, and fructose in the basic compounds class. The optimal ranges for milk, water, and ethylene glycol were extremely narrow. Egg yolk, cholesterol-loaded cyclodextrin, and nerve growth factor had medium-loading impacts. PLS-PM showed that CPA, osmoregulators, and basic components were the most efficient contributors to motility, while the antioxidant and extracellular protectant classes had less efficiency. Thus, ingredients, concentrations, and interactions of extender compounds are critical to extender formulation, especially when using multiple compounds with the same function.