Comparison of Six Diluents and Preservation Conditions on Rooster Semen

doi:10.13304/j.nykjdb.2021.0394

Abstract

Abstract:

In order to explore the effects of different diluents on rooster semen cryopreservation， 6 diluents （LR， lake’s， BPSE， modified Sasaki， Beltsville and Nabi） were respectively used for the cryopreservation of Xiayan roosters semen. The semen was briefly stored at 37 and 4 ℃ after thawing. The viability， motility， functional integrity and antioxidant capacity of sperm treated with different diluents and storage conditions were determined. The results showed that lake's diluent got the highest viability （48.20%）， motility （45.70%） and acrosome integrity （45.26%）. The viability in LR diluent treatment after thawing was 48.16%， which the motility rate in LR treatment was significantly lower than those in lake’s diluent treatment. 4 ℃ was more suitable for short-term preservation of rooster sperm after thawing， and the viability of rooster sperm in Lake’s and Nabi diluents could still reach over 30% after 45 min storage. Although the viability and motility of sperm in Nabi diluent were lower than those in LR and lake’s diluents at 0 min， it showed the best viability ability with the extension of preservation time. The mitochondrial activity， plasma membrane integrity and antioxidant capacity of frozen sperm in LR， Lake’s and Nabi diluents decreased with storage time， which the decreased range was more obvious when stored at 37 ℃. Furthermore， the semen in LR diluent showed a worse tolerance on these indicators. In conclusion， the lake’s and Nabi diluents were more suitable for rooster semen freezing. The storage at 4 ℃ after thawing could prolong the survival time of frozen sperm in vitro. The sperm in LR diluent showed a high viability after thawing， but lower motility and preservation resistance.

Key words: chicken, freezing semen, diluent

摘要：

为探究不同稀释液对鸡精液冷冻保存效果的影响，选择LR、Lake’s、BPSE、Modified Sasaki、Beltsville和Nabi共6种稀释液对霞烟鸡精液进行冷冻，解冻后分别在37或4 ℃条件下短时间保存，分析不同冷冻稀释液和保存条件对精子活率、活力、功能完整性和抗氧化指标的影响。结果表明，6种稀释液中Lake’s稀释液的冻后活率、活力和顶体完整性最高，分别为48.20%、45.70%和45.26%；LR稀释液虽然获得了48.16%的冻后活率，但其活力显著低于Lake’s稀释液。解冻后4 ℃保存更适合鸡精子的短时间保存，Lake’s和Nabi稀释液在4 ℃条件下保存45 min后活力仍达30 %以上。虽然Nabi稀释液解冻初始时的精子活率和活力低于LR和Lake’s稀释液，但随着保存时间的延长，其冻后精子存活能力表现最佳。随着冻后保存时间的延长，LR、Lake’s和Nabi稀释液冻后精子的线粒体活性、质膜完整性和抗氧化能力均呈现下降趋势，且37 ℃保存时下降更为明显，同时LR稀释液在这些指标上表现出较差的耐受性。综上所述，Lake’s和Nabi冷冻稀释液对鸡精子冷冻保存的效果最佳；解冻后4 ℃保存可延长冻精的体外存活时间；LR稀释液虽获得了较高的冻后活率，但其活力和冻后耐保存性能较差。

关键词: 鸡, 精液冷冻, 稀释液

CLC Number:

S831.2

Mengqian HE, Junyue WANG, Lingwei SUN, Jiehuan XU, Caifeng WU, Shushan ZHNAG, Janjun DAI, Kaixuan YANG, Defu ZHANG. Comparison of Six Diluents and Preservation Conditions on Rooster Semen[J]. Journal of Agricultural Science and Technology, 2022, 24(10): 53-61.

何孟纤, 汪俊跃, 孙玲伟, 徐皆欢, 吴彩凤, 张树山, 戴建军, 杨凯旋, 张德福. 6种鸡精液冷冻稀释液以及冻后保存条件比较[J]. 中国农业科技导报, 2022, 24(10): 53-61.

Figures/Tables 6

References 35

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配方Formula	LR	Lake’s	BPSE	Modified Sasaki	Nabi	Beltsville
谷氨酸钠 Sodium glutamate/g	1.920	1.920	0.867	1.200	0.867	0.861
乙酸镁-4H₂O Magnesium acetate-4 H₂O/g	0.08			0.08
乙酸钾 Potassium acetate/g	0.500		0.259	0.300
葡萄糖 Glucose/g	0.8			0.2
果糖 Fructose/g		1.0	0.5		0.5	0.5
甘露糖 Mannose/g				3.8
聚乙烯吡啶酮 Povidone/g	0.3
醋酸钠Sodium acetate/g		0.815			0.320	1.430
柠檬酸钾 Potassium citrate/g		0.128	0.606	0.050	0.064	0.064
氯化镁 Magnesium chloride/g		0.068			0.034	0.034
磷酸氢二钾 Dipotassium hydrogen phosphate/g			0.969		0.759	1.270
磷酸二氢钾 Potassium dihydrogen phosphate/g			0.065		0.070	0.006
双（2-羟甲基）氨基-三（羟甲基）甲烷 Bis（2-hydroxyethyl） imino Tris-（hydroxymethyl）-methane/g				0.40
三羟甲基甲胺基乙磺酸 N-［Tris（hydroxymethyl）metyl］-2-aminoethanesulfonic acid/g				0.40	0.32	0.19
水 H₂O/mL	100	100	100	100	100	100
渗透压 Osmotic pressure/（mOsm·kg^-1）	343	333	330	415	310	330
pH	7.0	7.0	7.3	7.2	7.4	7.5

配方Formula	LR	Lake’s	BPSE	Modified Sasaki	Nabi	Beltsville
谷氨酸钠 Sodium glutamate/g	1.920	1.920	0.867	1.200	0.867	0.861
乙酸镁-4H₂O Magnesium acetate-4 H₂O/g	0.08			0.08
乙酸钾 Potassium acetate/g	0.500		0.259	0.300
葡萄糖 Glucose/g	0.8			0.2
果糖 Fructose/g		1.0	0.5		0.5	0.5
甘露糖 Mannose/g				3.8
聚乙烯吡啶酮 Povidone/g	0.3
醋酸钠Sodium acetate/g		0.815			0.320	1.430
柠檬酸钾 Potassium citrate/g		0.128	0.606	0.050	0.064	0.064
氯化镁 Magnesium chloride/g		0.068			0.034	0.034
磷酸氢二钾 Dipotassium hydrogen phosphate/g			0.969		0.759	1.270
磷酸二氢钾 Potassium dihydrogen phosphate/g			0.065		0.070	0.006
双（2-羟甲基）氨基-三（羟甲基）甲烷 Bis（2-hydroxyethyl） imino Tris-（hydroxymethyl）-methane/g				0.40
三羟甲基甲胺基乙磺酸 N-［Tris（hydroxymethyl）metyl］-2-aminoethanesulfonic acid/g				0.40	0.32	0.19
水 H₂O/mL	100	100	100	100	100	100
渗透压 Osmotic pressure/（mOsm·kg^-1）	343	333	330	415	310	330
pH	7.0	7.0	7.3	7.2	7.4	7.5

稀释液 Diluent	活率 Viability/%	活力 Motility/%	顶体完整率 Acrosome integrity/%
LR	48.16±2.65 a	37.80±0.96 b	41.33±0.12 b
Lake’s	48.20±1.91 a	45.70±0.04 a	45.26±0.35 a
BPSE	31.36±1.55 c	24.60±0.24 c	25.01±0.42 c
Modified Sasaki	31.50±1.80 c	27.43±1.17 c	25.03±0.11 c
Beltsville	25.60±2.76 d	19.36±0.70 d	20.96±0.22 d
Nabi	45.73±1.86 b	43.60±0.60 a	40.56±0.42 b

稀释液 Diluent	活率 Viability/%	活力 Motility/%	顶体完整率 Acrosome integrity/%
LR	48.16±2.65 a	37.80±0.96 b	41.33±0.12 b
Lake’s	48.20±1.91 a	45.70±0.04 a	45.26±0.35 a
BPSE	31.36±1.55 c	24.60±0.24 c	25.01±0.42 c
Modified Sasaki	31.50±1.80 c	27.43±1.17 c	25.03±0.11 c
Beltsville	25.60±2.76 d	19.36±0.70 d	20.96±0.22 d
Nabi	45.73±1.86 b	43.60±0.60 a	40.56±0.42 b

稀释液 Diluent	温度 Temperature/℃	时间 Time/min	线粒体活性 Mitochondrial activity/%	质膜完整性 Plasma membrane integrity/%
LR	37	0	43.52±3.10 a	44.50±0.91 a
		15	29.29±0.70 b	29.03±1.46 d
		30	21.02±1.33 f	20.82±0.75 e
	4	0	43.52±3.10 a	44.50±0.91 a
		15	37.19±1.84 b	38.61±1.17 b
		30	28.75±1.56 d	33.40±0.75 c
Lake’s	37	0	43.72±1.72 a	45.36±1.81 a
		15	33.69±1.78 c	40.32±2.73 b
		30	25.42±1.05 de	28.60±0.66 d
	4	0	43.72±1.72 a	45.36±1.81 a
		15	40.11±2.42 b	40.32±2.73 b
		30	32.09±1.63 c	37.56±3.41 b
Nabi	37	0	41.27±1.76 ab	40.63±1.33 b
		15	34.10±1.06 c	36.20±0.81 b
		30	27.17±0.79 d	27.73±0.56 d
	4	0	41.27±1.76 ab	40.63±1.33 b
		15	37.53±0.76 b	39.06±1.55 b
		30	31.00±0.27 cd	34.35±1.26 bc
P值 P value	D×T		<0.001	0.001
	D×C		<0.001	0.001
	C×T		<0.001	0.001
	D×T×C		0.001	0.004