Multicolor flow cytometric analysis of cryopreserved bovine sperm: A tool for the evaluation of bull fertility
Multicolor flow cytometric analysis of cryopreserved bovine sperm: A tool for the evaluation of bull fertility
复制标题
DOI：
10.3168/jds.2019-16572
复制
发表时间：
2019-12-01
影响因子：
3.5
通讯作者：
Bollwein, H.
中科院分区：
文献类型：
--
作者：
Bucher, K.;Malama, E.;Bollwein, H.
本研究旨在利用多色流式细胞术分析冷冻保存的牛精子的功能状态。通过对公牛生育能力的回顾性研究，进一步评价了精子功能性状作为公牛生育能力预测指标的价值。为此，在人工授精（AI）中心，根据人工授精后至少1000次的年56天不回复率（%）选择20头荷斯坦-弗里马公牛作为成熟精子供体，并将其分为高（HF, n(HF) = 10头）和低（LF, n(LF) = 10头）生育公牛。在解冻（0小时）和在38摄氏度孵育3小时（3小时）后立即检测每头公牛冷冻保存的4至5个射精（总共91个射精）。通过3激光流式细胞仪配置5种荧光染料，包括钙绿素紫、碘化丙啶、花生花青素偶联凝集素、Fluo-4和花青素染料DiIC(1)(5)，分别评估精子酯酶活性、质膜完整性、顶体状态、细胞内Ca2+水平和线粒体膜电位。测定了18个精子亚群的相对大小百分比，这些亚群表现出以下2种或更多特征的组合：高酯酶活性（C-pos）、完整的质膜（PIneg）、未染色的顶体（PNA（阴性））、低细胞内Ca2+水平（f -阴性）和高线粒体膜电位（M-pos）。在两个生育组中，M-pos细胞分别在0和3 h时含有超过90%和84%的pingpna（阴性）精子。C(pos)PI（负）PNA（负）F（负）M（pos）精子的百分比在HF和LF之间没有差异；然而，在0 h时，HF公牛的PI（负）、PNA（负）和pegmpos精子群体中f -阴性细胞的百分比高于LF公牛。应用随机森林集合学习方法，大约三分之二的射精可以正确地分配到他们的生育组。在18个确定的精子群体中，0 h时pegmpos群体中f -阴性精子的比例是最重要的生育预测指标。总之，多色流式细胞术提供了对冷冻保存牛精子功能异质性的深入了解。事实上，在不同生育能力的公牛之间，有活力的精子保持低Ca2+水平的能力是不同的。通过多色流式细胞术评估的基于选定精子群体的分类器可以帮助预测人工智能后公牛的生育能力。
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 degrees C (3 h). A panel of 5 fluorochromes including calcein violet, propidium iodide, pycoerythrin-conjugated lectin of Arachis hypogea, Fluo-4, and cyanine dye DiIC(1)(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 (PIneg), 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 PIneg PNA(neg) sperm at 0 and 3 h, respectively. The percentage of C(pos)PI(neg)PNA(neg)F(neg)M(pos) sperm did not differ between HF and LF ejaculates; however, the percentage of F-neg cells within the PI(neg)PNA(neg) and PInegMpos 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 PInegMpos 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.