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

Abstract

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

CLC Number:

S828.3

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.

Figures/Tables 10

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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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Study on the Separation Effect of Pig X, Y Sperm of Different Acid-Base Diluents Combined with Creatine Monohydrate

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