Multicolor flow cytometric analysis of cryopreserved bovine sperm: A tool for the evaluation of bull fertility

The study aimed at the analysis of the functional status of cryopreserved bovine sperm using multicolor flow cytometry. The value of sperm functional traits as predictors of bull fertility was further evaluated through a retrospective fertility study. For this purpose, 20 Holstein-Friesian bulls serving as mature sperm donors in an artificial insemination (AI) center were selected based on their annual 56-d non-return rate (%) after at least 1,000 AI, and were accordingly classified as high (HF; n_HF = 10 bulls) or low fertility bulls (LF; n_LF = 10 bulls). Four to 5 cryopreserved ejaculates per bull (91 ejaculates in total) were examined immediately after thawing (0 h) and after a 3-h incubation at 38°C (3 h). A panel of 5 fluorochromes including calcein violet, propidium iodide, pycoerythrin-conjugated lectin of Arachis hypogea, Fluo-4, and cyanine dye DiIC₁(5) was configured by means of a 3-laser flow cytometer, to simultaneously assess sperm esterase activity, plasma membrane integrity, acrosomal status, intracellular Ca²⁺ levels, and mitochondrial membrane potential, respectively. The % relative size of 18 sperm sub-populations showing 2 or more of a combination of the following features was determined: high esterase activity (C_pos), intact plasma membrane (PI_neg), unstained acrosome (PNA_neg), low intracellular Ca²⁺ levels (F_neg), and high mitochondrial membrane potential (M_pos). In both fertility groups, M_pos cells comprised more than 90 and 84% of PI_negPNA_neg sperm at 0 and 3 h, respectively. The percentage of C_posPI_negPNA_negF_negM_pos sperm did not differ between HF and LF ejaculates; however, the percentage of F_neg cells within the PI_negPNA_neg and PI_negM_pos sperm populations at 0 h was higher in the HF than in the LF bulls. Applying the random forest ensemble learning method, approximately two-thirds of ejaculates could be correctly assigned to their fertility group. The fraction of F_neg sperm within the PI_negM_pos population at 0 h was the most important fertility predictor among the 18 defined sperm populations. In conclusion, multicolor flow cytometry offered an insight into the functional heterogeneity of cryopreserved bovine sperm. Indeed, the ability of viable sperm to retain low Ca²⁺ levels differed between bulls of diverse fertility. A classifier based on selected sperm populations assessed through multicolor flow cytometry could contribute to the prognosis of bull fertility after AI.