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

摘要

The study aimed at the analysis of the functionalstatus of cryopreserved bovine sperm using multicolorflow cytometry. The value of sperm functional traitsas predictors of bull fertility was further evaluatedthrough a retrospective fertility study. For this purpose,20 Holstein-Friesian bulls serving as mature spermdonors in an artificial insemination (AI) center wereselected based on their annual 56-d non-return rate (%)after at least 1,000 AI, and were accordingly classifiedas high (HF; n_(HF) = 10 bulls) or low fertility bulls (LF;n_(LF) = 10 bulls). Four to 5 cryopreserved ejaculates perbull (91 ejaculates in total) were examined immediatelyafter 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 ofArachis hypogea, Fluo-4, and cyanine dye DiIC_1(5) wasconfigured by means of a 3-laser flow cytometer, tosimultaneously assess sperm esterase activity, plasmamembrane integrity, acrosomal status, intracellular Ca~(2+)levels, and mitochondrial membrane potential, respectively.The % relative size of 18 sperm sub-populationsshowing 2 or more of a combination of the followingfeatures was determined: high esterase activity (C_(pos)),intact plasma membrane (PI_(neg)), unstained acrosome(PNA_(neg)), low intracellular Ca~(2+) levels (F_(neg)), and highmitochondrial membrane potential (M_(pos)). In both fertilitygroups, M_(pos) cells comprised more than 90 and84% of PI_(neg) PNA_(neg) sperm at 0 and 3 h, respectively.The percentage of C_(pos)PI_(neg)PNA_(neg)F_(neg)M_(pos) sperm didnot differ between HF and LF ejaculates; however, thepercentage of F_(neg) cells within the PI_(neg)PNA_(neg) andPI_(neg)M_(pos) sperm populations at 0 h was higher in theHF than in the LF bulls. Applying the random forestensemble learning method, approximately two-thirds ofejaculates could be correctly assigned to their fertilitygroup. The fraction of F_(neg) sperm within the PI_(neg)M_(pos)population at 0 h was the most important fertilitypredictor among the 18 defined sperm populations. Inconclusion, multicolor flow cytometry offered an insightinto the functional heterogeneity of cryopreservedbovine sperm. Indeed, the ability of viable sperm toretain low Ca~(2+) levels differed between bulls of diversefertility. A classifier based on selected sperm populationsassessed through multicolor flow cytometry couldcontribute to the prognosis of bull fertility after AI.