TLR7/8激动剂R848对梅花鹿Y精子分选效率的影响

doi:10.11843/j.issn.0366-6964.2025.07.021

摘要/Abstract

摘要：

旨在利用雷西莫特(resiquimod, R848)和聚乙烯吡咯烷酮(polyvinylpyrrolidone, PVP)联合制备分选梅花鹿精子的分选液，建立一种省时、简便、高效的梅花鹿性Y精子分离方法，在生产中提高繁殖效率有针对性增加雄性后代数量。本研究采集3头健康成年公鹿精液，利用精子活力分析仪和共聚焦显微镜评估5种不同基础液、不同R848、PVP浓度和孵育时间对精子数量、运动参数(活力、前向运动率、平均路径速度、平均曲线速度、平均直线速度、前向性)和精子质量(精子质膜、顶体完整性)的影响。使用单精子核型鉴定技术检测分选后上层单精子的DNA核型，确定Y精子的分选效果，并通过体外受精对分选精液进行受精能力的评价。结果表明，5种基础液中，配方1和2中精子活力显著高于配方3、4、5，且两者之间无显著差异；随着R848和PVP浓度升高，各处理组的上层精子数量均显著降低，在60和90 min时R848组(R组)和R848+PVP组(R+P组)上层精子数量均显著低于对照组(C组)。R组和R+P组的运动参数和精子质量与C组相比，平均路径速度和平均曲线速度显著降低、前向性显著升高，而活力、前向运动率、平均直线速度、质膜和顶体完整率无显著变化；R组上层Y精子检出比率为80%，R+P组为90%，均显著高于C组50%，且R+P组卵裂率与C组无显著差异。本试验中，采用R848+PVP联合孵育的方法，上层Y精子分离率可达90%，初步建立了一种无需CO₂培养箱和精液冷冻，精子即采即分的梅花鹿Y精子分离技术，为梅花鹿性控精液的分离及生产提供了技术支撑。

关键词: 梅花鹿, R848, PVP, Y精子分离

Abstract:

The purpose of this experiment was to use resiquimod (R848) and polyvinylpyrrolidone (PVP) to prepare the sorting solution for sorting sika deer sperm, and to establish a time-saving, simple, and efficient method for separating sika deer Y sperm, to improve the reproductive efficiency and increase the number of male offspring in production. In this study, semen was collected from 3 healthy adult male sika deer, and a sperm viability analyzer and confocal microscope were used to assess the effects of 5 different basal fluids, R848 concentrations, PVP concentrations, and incubation times on sperm counts, motility parameters (viability, progressive motility, average path velocity, curvilinear velocity, straight-line velocity, and straightness), and sperm quality (plasma membrane and acrosome integrity). The DNA karyotype of the sorted upper monosperm was detected using the monosperm karyotyping technique to determine the effect of Y-sperm sorting, and the sorted semen was evaluated for fertilizing ability by in vitro fertilization. The results showed that among the 5 basal fluids, sperm motility was significantly higher in formulation 1 and 2 than that in formulation 3, 4, and 5, and there was no significant difference between formulation 1 and 2; the upper sperm counts were significantly lower in all treatment groups as the concentrations of R848 and PVP increased, and the upper sperm counts in both the R848 group (group R) and the R848+PVP group (group R+P) were significantly lower than those in the control group (group C) at 60 and 90 min. The motility parameters and sperm quality in group R and R+P showed significantly lower average path velocity and curvilinear velocity, significantly higher straightness, and no significant changes in viability, progressive motility, straight-line velocity, plasma membrane and acrosomal integrity, compared to group C. The upper Y spermatozoa detection rate was 80% in group R and 90% in group R+P, which were significantly higher than that in group C (50%), and the spermatozoa cleavage rate of group R+P was not significantly higher than that in group C. In this experiment, the method of R848+PVP combined incubation was used, and the upper Y sperm separation rate was up to 90%. A kind of deer Y sperm separation technology without CO₂ incubator and semen freezing was preliminarily established, which provided technical support for the separation and production of sexed semen of sika deer.

Key words: sika deer, R848, PVP, Y sperm separation

中图分类号:

S814

张旭林, 唐毓, 刘理想, 范冰峰, 张燠诗, 张颖, 邵静, 孙慧敏, 褚萧宇, 彭飞宇, 许保增. TLR7/8激动剂R848对梅花鹿Y精子分选效率的影响[J]. 畜牧兽医学报, 2025, 56(7): 3278-3289.

ZHANG Xulin, TANG Yu, LIU Lixiang, FAN Bingfeng, ZHANG Yushi, ZHANG Ying, SHAO Jing, SUN Huimin, CHU Xiaoyu, PENG Feiyu, XU Baozeng. Effect of TLR7/8 Agonist R848 on the Efficiency of Y Sperm Sorting in Sika Deer[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(7): 3278-3289.

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项目 Item	配方1 Formula 1	配方2 Formula 2	配方3 Formula 3	配方4 Formula 4	配方5 Formula 5
氯化钠 NaCl	114.00	113.10	114.00	138.35	－
氯化钾 KCl	3.21	3.00	3.20	5.30	9.39
磷酸二氢钠 NaH₂PO₄	0.40	－	0.40	1.40	－
碳酸氢钠 NaHCO₃	20.00	－	25.00	37.00	14.28
葡萄糖 D(+)-glucose	－	11.00	－	－	158.10
丙酮酸钠 Sodium pyruvate	－	5.00	0.20	1.25	－
L-半胱氨酸 L-cysteine	－	0.57	－	－	－
乳酸钠 Na-lactate	12.55	－	10.00	－	－
山梨醇 Sorbitol	12.00	－	－	－	－
4-羟乙基哌嗪乙磺酸 HEPES	10.00	－	－	－	－
柠檬酸 Citric acid	－	－	－	－	2.60
番茄红 Lycopene	－	－	－	－	0.37
柠檬酸钠 Sodium citrate	－	－	－	－	6.80
乙二胺四乙酸 EDTA	－	－	－	－	1.34
六水·氯化镁 MgCl₂·6H₂O	0.50	－	0.50	0.70
卡那霉素 Kanamycin	65.23	－	－	－	－
二水氯化钙 CaCl₂·2H₂O	2.00	7.50	2.00	3.90
三(羟甲基)氨基甲烷 Tris base	－	20.00	－	－	47.05
庆大霉素/(μg·mL^-1) L-Gentamicin	－	－	5.00	50.00	－
酚红/% Phenol red	－	－	－	0.50	－
聚乙烯醇/% PVA	0.01	－	－	－	－
牛血清白蛋白/(mg·mL^-1) BSA	－	2.00	6.00	5.00	－

项目 Item	n	活力/% Viability	前向运动率/% PR	平均路径速度/ (μm·s^-1)VAP	平均直线速度/ (μm·s^-1)VSL	平均曲线速度/ (μm·s^-1)VCL	前向性/% STR
对照 Control	3	85.3±3.1	69.3±11.9	80.9±1.4^a	68.6±2.6	138.8±3.2^a	83.3±1.5^b
R848	3	86.0±3.5	70.5±9.6	63.5±6.9^b	62.5±4.0	89.2±7.8^b	90.3±2.1^a
R848+PVP	3	87.0±2.6	76.0±4.0	70.5±1.7^b	67.1±2.0	94.5±2.4^b	93.0±2.0^a

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