[Objetive] The objective of the present study was to characterize different sperm subpopulations based on morphometric parameters of frozen-thawed semen in two bovine subspecies using a CASA system. [Methodology] The experiment was carried out at the Costa Rica Institute of Technology from May to December 2023. Spermatozoa from 10 bulls (five animals of each species Bos taurus and Bos indicus) were evaluated after thawing of the semen doses by an ISAS^®v1, computer-assisted sperm analysis (CASA)-Morph system. Sub-populations of head morphometric spermatozoa were characterized using multivariate procedures such as principal components (PCs) analysis and clustering methods (k-means model) [Results] The ejaculate of male exhibits significant heterogeneity and comprises diverse sperm subpopulations with differing morphometric patterns. Three different sperm subpopulations were identified from three PCs: t head size, head shape, and symmetry of the sperm head and the degree to which it was pyriform. The proportions of the different sperm subpopulations varied in the two-bovine subspecies; Bos taurus and Bos indicus. Results indicated that subpopulations SP1, SP2, and SP3 were different for PC criteria and these differences were relevant. The variability in sperm morphometry assessments underscores the need to standardize semen evaluation protocols [Conclusions] These findings highlight the importance of knowing the diversity in sperm morphometry between subspecies to both improve in vitro evaluation tests and to distinguish subspecies.

The cryopreservation of gametes and embryos is an important element of biodiversity conservation. One species in need of conservation is the honey bee Apis mellifera L. Changing environmental factors, especially the anthropogenic factor, have led to a reduction in the numbers of this insect species. In this study, we provide an example of the creation of a biobank of honey bee drone sperm. For sperm cryopreservation, drones of the most common subspecies of honey bees common in Russia were selected. These were the dark forest bee, Apis mellifera mellifera from the Republic of Bashkortostan, with three subspecies (A. m. carnica, A. m. carpatica, and A. m. caucasica) from the southern regions of Russia, as well as two breeding stocks, the Far Eastern bee and Prioksky bee. For subspecies identification, morphometric and genetic methods were used. The subspecies of the studied samples were confirmed by the analysis of the tRNAleu-COII locus of mitochondrial DNA and nine microsatellite markers of nuclear DNA. It was shown that bees of the Prioksky breeding stock belong to the subspecies A. m. caucasica based on phylogenetic analysis, and the Far Eastern breeding stock is a stable hybrid, descending on the maternal line from the evolutionary lineage C or O. The results of the morphometric analysis are consistent with the results of the genetic analysis. For the cryopreservation of sperm, we used a honey diluent. As a result, the viability of frozen–thawed sperm decreased by 20.3% compared to fresh sperm, and overall motility decreased by 25-fold. The measurement of the sperm concentration in the spermatheca of artificially inseminated queens showed that it varied from 0.22 to 4.4 million/μL. Therefore, the use of honey in sperm cryopreservation has great potential.