不同酸碱性稀释液联合一水肌酸对猪X、Y精子分离效果的研究

doi:10.11843/j.issn.0366-6964.2024.10.017

摘要/Abstract

摘要：

旨在探究调整猪精液稀释液酸碱性联合一水肌酸(ceatine monohydrate, CMH)对猪X、Y精子的分离效果。本研究使用8头2~4岁健康的杜洛克公猪采集精液，通过调整精液稀释液Beltsville(BTS)的pH，使用精子计算机辅助分析系统(CASA)检测精子活率和运动轨迹确定X、Y精子分选体系最适pH，利用CASA检测精子运动性能确定一水肌酸最适添加浓度。通过免疫荧光染色和流式细胞术评估X、Y精子分选效果，利用荧光标记法、精子活性氧MitoSOX Red染色等方法检测精子顶体、质膜完整性、活性氧含量等精子质量指标。结果表明，当稀释液pH为6.4和7.1时，孵育60 min对精子活率无显著影响(P＞0.05)，上层精子前向性运动占比极显著高于下层(P＜0.01)。稀释液中添加750 μmol·L^-1一水肌酸孵育60 min时显著提高了精子活力、直线运动速率、曲线运动速率和摆动性(P＜0.05)。使用酸性稀释液联合一水肌酸分选猪X、Y精子，分选后上层精液X精子占比达63.32%；碱性稀释液联合一水肌酸分选猪X、Y精子，分选后上层精液Y精子占比达到67.19%。进一步研究发现，分选后顶体完整性、质膜完整性和ROS水平同对照组相比无显著差异(P＞0.05)，ATP水平、线粒体膜电位和运动性能显著高于对照组(P＜0.05)。综上，本研究通过调整稀释液酸碱性并联合一水肌酸建立了一种简便、价廉和快捷的猪X、Y精子分选体系，该方法不影响分选后精子质量并提高精子运动性能，进一步为猪性控精液新技术研发提供试验依据。

关键词: 猪精子, 酸碱性稀释液, 一水肌酸, 性控精液, X、Y精子分选

Abstract:

The study aimed to explore the separation effect of porcine X, Y sperm by adjusting the acidity and alkalinity of porcine semen diluent combined with creatine monohydrate (CMH). In this study, eight healthy Duroc boars aged 2-4 years were used to collect semen. By adjusting the pH of semen diluent Beltsville (BTS), the optimal pH of the X, Y sperm sorting system was determined by sperm computer-aided analysis system (CASA), and the optimal concentration of creatine monohydrate was determined by CASA. The effect of X, Y sperm sorting was evaluated by immunofluorescence staining and flow cytometry, and sperm quality indexes such as acrosome, plasma membrane integrity, and reactive oxygen species content (ROS) were detected by fluorescence labeling and MitoSOX Red staining of sperm ROS. The results showed that when the pH of the diluent was 6.4 and 7.1, there was no significant effect on sperm viability after incubation for 60 min (P>0.05), and the proportion of sperm in the upper layer moving forward was significantly higher than that in the lower layer (P < 0.01). Adding 750 μmol·L^-1 creatine monohydrate to the diluent and incubating for 60min significantly improved sperm motility, linear movement rate, curve movement rate, and wobble (P < 0.05). Using acid diluent combined with creatine monohydrate to separate pig X, Y sperm, the proportion of X sperm in the upper semen after separation was 63.32%. Alkaline diluent combined with creatine monohydrate was used to separate pig X, Y sperm. After separation, the proportion of Y sperm in the upper semen reached 67.19%. Further study showed that there was no significant difference in acrosome integrity, plasma membrane integrity, and ROS level between the two groups (P>0.05), while ATP level, mitochondrial membrane potential, and exercise performance were significantly higher than those of the control group (P < 0.05) after separation. To sum up, this study established a simple and effective separation system of X and Y sperm in pigs by adjusting the acidity and alkalinity of the diluent in parallel with creatine monohydrate. This method did not affect the quality of sperm and improved the motility of sperm after separation, which further provided a theoretical basis for the research and development of new technologies for controlling semen in pigs.

Key words: pig sperm, acid and alkaline diluent, creatine monohydrate, sexually controlled semen, X, Y sperm sorting

中图分类号:

S828.3

胡冰艳, 刘起昊, 曹超越, 李孟轩, 庞卫军. 不同酸碱性稀释液联合一水肌酸对猪X、Y精子分离效果的研究[J]. 畜牧兽医学报, 2024, 55(10): 4443-4454.

Bingyan HU, Qihao LIU, Chaoyue CAO, Mengxuan LI, Weijun PANG. Study on the Separation Effect of Pig X, Y Sperm of Different Acid-Base Diluents Combined with Creatine Monohydrate[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(10): 4443-4454.

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参数 Parameter	时间/min Time	组别Group
参数 Parameter	时间/min Time	0 μmol·L^-1 CMH	250 μmol·L^-1 CMH	500 μmol·L^-1 CMH	750 μmol·L^-1 CMH	1 000 μmol·L^-1 CMH
活力/% Motility	30	80.71±1.78	80.31±0.45	81.56±2.01	81.34±1.56	81.62±0.79
	45	80.50±0.92	80.52±2.22	80.99±1.33	80.66±0.78	80.09±0.54
	60	80.13±1.45^a	80.82±1.56^a	81.32±0.79^a	82.95±2.02^b	80.55±2.32^a
	90	80.06±2.33^a	80.84±0.78^a	80.95±2.44^a	82.92±0.89^b	78.71±1.39^c
直线运动速率/	30	19.89±0.67	18.91±2.02	18.88±1.12	18.35±0.34	18.87±0.34
(μm·s^-1) VSL	45	16.62±1.23	17.68±1.11	17.03±0.67	17.68±1.54	16.64±0.21
	60	19.40±2.01^a	18.92±0.89^a	19.87±2.21^ab	21.26±0.70^b	21.44±0.66^b
	90	20.89±0.54^a	21.33±0.34^a	21.18±1.55^a	20.98±0.53^ab	17.26±1.20^c
曲线运动速率/	30	45.63±1.89	44.17±1.86	44.05±0.87	44.10±2.52	44.20±1.34
(μm·s^-1) VCL	45	46.42±0.76	45.43±0.99	45.34±2.02	46.75±1.56	45.83±2.01
	60	50.26±2.14^a	48.79±2.11^ab	49.47±1.23^ab	52.44±2.01^c	48.37±2.08^ab
	90	50.87±1.32^a	50.83±1.77^a	50.11±0.54^a	53.98±0.54^b	50.92±1.76^a
摆动性/% WOB	30	31.25±0.88	34.25±0.63	33.10±2.34	35.27±0.90	34.67±0.12
	45	31.42±1.67^a	32.67±2.23^a	32.75±1.01^ab	33.84±2.12^ab	31.79±0.34^a
	60	31.03±2.45^a	31.06±1.45^a	36.30±0.45^b	43.05±2.09^c	37.40±1.90^b
	90	30.26±1.01^a	30.29±0.84^a	34.58±2.22^b	38.58±0.23^c	30.42±1.22^a

精子参数Sperm parameter	pH=6.8	pH=6.4 UP	pH=7.1 UP
ATP水平/(μmol·mL^-1) ATP content	52.45±0.65^a	60.35±1.21^b	62.78±0.97^b
精子活力/% Sperm motility	70.32±0.23^a	73.21±0.97^b	73.67±1.03^b
直线运动速率/(μm·s^-1) Straight-linear velocity	20.63±0.47^a	23.74±1.45^b	25.34±1.87^b
曲线运动速率/(μm·s^-1) Curvi-linear velocity	42.63±1.21^a	45.74±2.53^b	46.34±1.56^b
平均路径速度/(μm·s^-1) Average pathvelocity	27.35±2.23^a	30.87±0.32^b	31.42±1.54^b

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