不同冷冻保护剂在鸡精液冷冻中的作用效果分析

本实验采用一定浓度的甘油、乙二醇、二甲基亚砜（ＤＭＳＯ）、二甲基乙酰胺（ＤＭＡ）作为冷冻保护剂，用含冷冻保护剂的稀释液将精液稀释后常温保存，观察精子活率，比较精子生存指数，并进行输精实验，发现在常温下对精子毒害作用最大的是甘油，其次是ＤＭＳＯ，而ＤＭＡ及乙二醇对精子的毒害作用最小。以一定浓度的４种冷冻保护剂将精液冷冻后观察解冻活率，发现以ＤＭＡ作为冷冻保护剂，解冻后精子活率最高。在输精实验中，以ＤＭＡ作为冷冻保护剂采用深阴道输精，取得了５０％的受精率。浅输精取得了４０％的受精率。     

不同冷保保护剂在鸡精液冷冻中的作用效果分析

本实验采用一定浓度的甘油，乙二醇，二甲基亚砜（ＤＭＳＯ），二甲基乙酰胺（ＤＭＡ）作为冷冻保护剂，用含冷冻保护剂的称释法液精稀释后常温保存，观察精子活率，比较精子生存指数，并进行输精实验，发现在常温下对精子毒害作用最大的是甘油，其次是ＤＭＳＯ，而ＤＭＡ及乙二醇对精子的毒害作用最小，以一定浓度的４种冷冻保护剂将精液冷冻后观察冻活率，发现以ＤＭＡ作为冷冻保护剂，解冻后精子活率最高。在输精实验中，以ＤＭＡ     

种鸡精液冷冻保护剂的确定

本实验以农大褐父母代种鸡为材料，以甘油、乙二醇、ＤＭＡ和ＤＭＳＯ为主要冷冻保护剂的试验表明，乙二醇和ＤＭＡ的毒性较小；以Ａ液为稀释液，加抗冷冻剂ＤＭＡ的解冻精子活力为０．５５，且以１０％的ＤＭＡ浓度最佳，精子活力最高为０．６０，显著高于其它组（Ｐ＜０．０５）。以日液为稀释液的解冻结果表明，应用混合冷冻保护剂的保护效果较好，可获得０．４１～０．４５的精子活力，差异显著（Ｐ＜０．０５）。     

种鸡精液冷冻保护剂的确定

以农大褐父母代鸡为材料，以甘油、乙二醇、ＤＭＡ和ＤＭＳＯ为主要冷冻保护剂的试验表明，乙二醇和ＤＭＡ的毒性较小，以Ａ液为稀释液，加抗冷冻剂ＤＭＡ的解冻精子活力为０．５５，且以１０％的ＤＭＡ浓度最佳，精子活力最高为０．６０，显著高于其它组（Ｐ〈０．０５）。以Ｂ液为稀释液的解冻结果表明，应用混合冷冻保护剂的保护效果较好，可获得０．４１￣０．４５的精子活力，差异显著（Ｐ〈０．０５）。     

种鸡精液冷冻保护剂的确定

本实验以农大褐父母代种鸡为材料，以甘油，乙二醇，ＤＭＡ和ＤＭＳＯ为主要冷冻保护剂的表明，乙二醇和ＤＭＡ的毒性较小，以Ａ液为稀释液，加抗冷冻剂ＤＭＡ的解冻精子活力为０．５５， 以１０％的ＤＭＡ浓度最佳，精子活力最高为０．６０，显著高于其它组。以Ｂ液为稀释液的结果表明，应用混合冷冻保护剂的保护效果较好，可获得０．４１－０４５的精子活力，差异显著。     

牛精液保存和质量评定

前言精液可以在-196℃的液氮中保存,解冻后仍具有较高的受精能力。尽管如此,冷冻—解冻后的精液中仍有不少精子死亡和失去活力。因此,为了获得较高的受精率,输精剂量一定要高于新鲜精液。卵黄或牛奶是牛冷冻精液稀释液的基本成分。试验发现与甘油类似的其他冷冻保护剂的     

硫酸鱼精蛋白和谷氨酰胺对鸡精液冷冻的作用

本文以农大褐父母代种鸡为材料，研究硫酸鱼精蛋白和谷氨酰胺对鸡精液冷冻效果的作用表明，Ａ０液＋ＤＭＡ和Ｂ０液＋甘油，解冻后精子活力分别为０．５３，０．４９，均显著高于其它组（Ｐ＜０．０５）；Ａ０液＋硫酸鱼精蛋白、Ａ０液＋谷氨酰胺，解冻后分别获得了０．４６，０．４１的精子活力，均显著高于对照组（Ｐ＜０．０５）；Ｂ０液各组试验效果差异均不显著（Ｐ＞０．０５）。     

硫酸鱼精蛋白和谷氨酰胺对鸡精液冷冻效果的影响

以农大褐父母代种鸡为材料，进行硫酸鱼精蛋白和谷氨酰胺对鸡精液冷冻效果影响表明，Ａ液＋ＤＭＡ和Ｂ液＋甘油，解冻后精子活力分别为０．５３、０．４９，均显著高于其它（Ｐ〈０．０２５），Ａ液＋硫酸鱼精蛋白、Ａ液＋谷氨安、解冻后分别获得了０．４６、０．４１的精子活力、均显著高于对照组（Ｐ〈０．０５）；Ｂ组均不显著（Ｐ〉０．０５）。     

精浆对猪精子冷冻效果影响的研究

为优化猪精子冷冻技术,提高解冻后精子的活力和受精能力,本试验分别以含精浆浓度为10%、20%和30%的冷冻保护剂处理精子,以冷冻前、解冻后精子活力和质膜完整性,解冻后精子进行体外受精(IVF)的卵裂率和囊胚率等作为检测指标,同时以含有卵黄的Tris-柠檬酸－葡萄糖(Tris-citric acid-glucose,TCG)冷冻基础液作为对照研究精浆对猪冷冻精子的保护作用。结果显示,在含有10%精浆浓度的稀释液中,冷冻前质膜完整性,解冻后精子活率、质膜完整性、IVF囊胚率相对于对照组均显著提高(P<0.05);当含有10%精浆的冷冻精液解冻后用于人工授精时,与配母猪妊娠率、窝产仔数、窝产活仔数等仍显著低于鲜精授精组(P<0.05)。上述结果表明,含10%精浆的冷冻保护剂能提高精子的冷冻后活力和IVF胚胎发育率,但用于人工授精配种与鲜精相比还有一定差距。     

巴马香猪精液冷冻保存研究

利用甘油和二甲基亚砜（DMSO）作为冷冻保护剂,以精子存活率为评价指标,研究了冷冻保护剂对香猪精液冷冻保存效果的影响。试验分工、Ⅱ、Ⅲ、Ⅳ4个试验组和1个对照组。以解冻后的精子活力、活率为判断标准,比较了4种冷冻保护液对香猪精液冷冻保存的影响。结果表明,试验Ⅰ、Ⅱ、Ⅳ组精子活率显著高于Ⅲ组（含5％甘油）（P〈0．05）;试验Ⅰ、Ⅱ、Ⅳ组之间差异不显著（P〉0．05）;Ⅰ组精子活率最高,达到53．10％。     

Trehalose in glycerol-free freezing extender enhances post-thaw survival of boar spermatozoa

Cryopreservation of boar semen is still considered suboptimal due to lower fertility as compared with fresh samples when glycerol, a permeating cryoprotectant, is used. Trehalose is a non-permeable cryoprotectant and nonreducing disaccharide known to stabilize proteins and biologic membranes. The aim of this study was to evaluate the cryosurvival and in vitro penetrability of boar spermatozoa when glycerol was replaced with trehalose in a freezing extender. Ejaculated Berkshire semen samples were diluted in egg yolk-based freezing extender containing glycerol (100 mM) or trehalose (0, 50, 100, 150, 200 and 250 mM) and cryopreserved using a straw freezing procedure. Thawed samples were analyzed for motility, viability, mitochondrial membrane potential (MMP), and acrosome integrity. In experiment 2, penetrability of spermatozoa cryopreserved with 100 mM glycerol or trehalose was examined. Replacement of cryoprotectant glycerol (100 mM) with trehalose had no effect on sperm viability, but replacing it with 100 mM trehalose improved motility, MMP and acrosome integrity significantly. Sperm motility and MMP were considerably higher in 100 mM trehalose, whereas the acrosome integrity was substantially higher in 100–250 mM trehalose. The in vitro penetration rate was also significantly higher in spermatozoa cryopreserved with trehalose (61.3%) than in those cryopreserved with glycerol (43.6%). In conclusion, 100 mM non-permeable trehalose can be used to replace glycerol, a permeating cryoprotectant, for maintenance of better post-thaw quality of boar spermatozoa.     

Intrauterine and intravaginal insemination with frozen canine semen using an extender consisting of orvus ES paste-supplemented egg yolk tris-fructose citrate

Our previous report indicated that addition of Orvus ES Paste (OEP) to the extender of frozen canine semen protected acrosomes and maintained sperm motility after thawing. In this study, artificial insemination (AI) using the frozen semen was carried out. The frozen semen was prepared using egg yolk Tris-fructose citrate, and the final concentrations of glycerol and OEP were 7% (v/v) and 0.75% (v/v), respectively. AI was performed during the optimal mating period predicted from the peripheral plasma progesterone level. In intrauterine insemination (IUI), the bitches were laparotomized and 1 x 10(8) spermatozoa were infused into one of the uterine horns. In insemination of non-OEP supplemented semen, 3 x 10(8) spermatozoa were inseminated. In intravaginal insemination (IVI), 10-40 x 10(8) spermatozoa were inseminated. Conception was obtained in nine of 10 bitches (90.0%) that underwent IUI. The number of newborns was from 1 to 7 (mean 3.6 +/- 0.9). The mean ratio of the number of puppies to the number of ovulations in the inseminated uterine horn was 71.8%. The number of puppies did not exceed the number of ovulation in the inseminated uterine horn. Conception using non-OEP supplemented frozen semen was unsuccessful in all four bitches. In IVI, conception was not obtained in any of the six bitches that received insemination of 10 x 10(8) or 40 x 10(8) spermatozoa, but two of three bitches that received insemination of 20 x 10(8) spermatozoa were fertilized. It was shown that a high conception rate can be obtained by IUI using OEP-supplemented frozen canine semen. Developmenmt of a non-surgical method of IUI and a method of freezing canine sperm applicable to IVI is necessary.     

The Effect of Glycerol Concentrations on the Post‐thaw In Vitro Characteristics of Cryopreserved Sex‐sorted Boar Spermatozoa

The objective of this study was to optimize protocols for the cryopreservation of sex‐sorted boar spermatozoa. In the experiment 1, we evaluated the effects of a standard boar sperm cryopreservation procedure (3% final glycerol concentration) on the in vitro characteristics of sex‐sorted sperm frozen at low sperm concentrations (20 × 10⁶ sperm/ml; S20 group). Non‐sorted spermatozoa frozen at 1000 × 10⁶ (C1000 group) and 20 × 10⁶ (C20 group) sperm/ml were used as the freezing control groups. In experiment 2, the effects of different final glycerol concentrations (0.16%, 0.5%, 1.0%, 2.0% and 3.0%) on post‐thaw quality of the S20 and C20 groups were evaluated. In both experiments, the samples were evaluated prior to freezing (5°C) and at 30, 90 and 150 min after thawing. Experiment 1 indicated that freezing sperm at low concentrations decreased (p < 0.05) the total motility (TM) and progressive motility (PM) at 90 and 150 min after thawing regardless of whether the sperm were sorted or not. However, the sperm membrane integrity was not affected at any evaluation step. Inexperiment 2, significant effects on the TM and PM because of increased glycerol concentrations in the S20 and C20 groups were observed only at 90 and 150 min after thawing. The samples frozen in 3% glycerol showed lower (p < 0.05) TM and PM values when compared to those frozen in the presence of 0.5% and 1% glycerol. In both experiments, non‐sorted control samples displayed higher percentages of spermatozoa with damaged DNA than sorted spermatozoa. In conclusion, the optimization of cryopreservation conditions by decreasing the glycerol concentrations can improve post‐thaw motility of sex‐sorted spermatozoa frozen at low concentrations.     

Cryopreserving turkey semen in straws and nitrogen vapour using DMSO or DMA: effects of cryoprotectant concentration,freezing rate and thawing rate on post-thaw semen quality

1. This study was designed to identify a suitable protocol for freezing turkey semen in straws exposed to nitrogen vapour by examining the effects of dimethylacetamide (DMA) or dimethylsulfoxide (DMSO) as cryoprotectant (CPA), CPA concentration, freezing rate and thawing rate on in vitro post-thaw semen quality.

2. Pooled semen samples were diluted 1:1 (v:v) with a freezing extender composed of Tselutin diluent containing DMA or DMSO to give final concentrations of 8% or 18% DMA and 4% or 10% DMSO. The semen was packaged in 0.25 ml plastic straws and frozen at different heights above the liquid nitrogen (LN₂) surface (1, 5 and 10 cm) for 10 min. Semen samples were thawed at 4°C for 5 min or at 50°C for 10 s. After thawing, sperm motility, viability and osmotic tolerance were determined.

3. Cryosurvival of turkey sperm was affected by DMSO concentration. Freezing rate affected the motility of sperm cryopreserved using both CPAs, while thawing rates showed an effect on the motility of sperm cryopreserved using DMA and on the viability of sperm cryopreserved using DMSO. Significant interactions between freezing rate × thawing rate on sperm viability in the DMA protocol were found.

4. The most effective freezing protocol was the use of 18% DMA or 10% DMSO with freezing 10 cm above the LN₂ surface and a thawing temperature of 50°C. An efficient protocol for turkey semen would improve prospects for sperm cryobanks and the commercial use of frozen turkey semen.     

Microencapsulation of bovine spermatozoa

Two experiments were conducted to examine the efficacy of microencapsulation of bovine spermatozoa for use in artificial insemination. In Exp. 1, sperm were encapsulated at three different concentrations (45, 90 and 180 X 10(6) sperm/ml) in either .75- or 1.5-mm (diameter) microcapsules and incubated in vitro for 24 h at 37 C. Unencapsulated samples of each concentration served as controls. Capsule contents were evaluated for percentage of sperm motility and intact acrosomes at 2, 12 and 24 h of incubation. Capsule fragility was evaluated after 24 h incubation. Viability of spermatozoa was not influenced by sperm concentration or capsule size, and compared with controls, cellular injury after encapsulation was not apparent. Fragility of capsules was unaffected by capsule size; however, as the sperm concentration increased, integrity of the capsules decreased (P less than .05). In Exp. 2, using frozen-thawed semen, the effect of egg yolk content, presence of glycerol and viability of spermatozoa on the success of microencapsulation was measured. The extender was 2.9% sodium citrate with glycerol (7% v/v) and either 0, 5, 10 or 15% egg yolk (v/v). Uniformity of capsules in size and shape was evaluated subjectively. Capsule integrity and uniformity were unaffected by glycerol, sperm viability or egg yolk level up to 10% v/v; however, encapsulation of spermatozoa in 15%-yolk buffer increased the heterogeneity in capsule size and shape. Viability of encapsulated spermatozoa was maximal for extenders containing 10 or 15% yolk v/v. Reduced viability for the 5% yolk extender was due to pre-encapsulation injury associated with freezing. Microencapsulation procedures are compatible with sperm viability and can be adapted to an acceptable extender system used in artificial insemination.     

Cryopreservation of Ghagus chicken semen: Effect of cryoprotectants,diluents and thawing temperature

The present study evaluated the effects of cryoprotectants, semen diluents and thawing temperature during Ghagus chicken semen cryopreservation. Four different experiments were conducted; Experiment 1—semen was cryopreserved using 6% dimethylacetamide (DMA) and 2% dimethylsulphoxide (DMSO) in Sasaki diluent (SD) and Lake and Ravie diluent (LR), Experiment 2 and 3—semen was cryopreserved using 8% ethylene glycol (EG) in SD, LRD and Red Fowl Extender (RFE), Experiment 4—semen was cryopreserved using 6% dimethylformamide (DMF) in SD, LR and Beltsville poultry semen extender (BPSE). Semen was cryopreserved in 0.5 ml French straws. Thawing was done at 5°C for 100 s in ice water in Experiments 1, 2 and 4, whereas in Experiment 3 thawing was done at 37°C for 30 s. The post-thaw sperm motility, viable sperm and acrosome-intact sperm were significantly (p < .05) lower in cryopreserved samples in all the experiments. No fertile eggs were obtained from cryopreserved samples in Experiments 1 and 2, except for 8% EG RFE treatment where the fertility was 0.83%. In Experiments 3 and 4, highest fertility was obtained in LR treatment 48.12 and 30.89%, respectively. In conclusion, using cryoprotectant EG (8%) and thawing at 37°C for 30 s, and DMF(6%) resulted in acceptable level of fertility in Ghagus chicken. Though the diluents influenced post-thaw in vitro semen parameters, the fertility was not affected. In addition, results indicated that thawing temperature may be a critical stage in the cryopreservation protocol.     

Deep-freezing of boar semen in plastic film 'cochettes'

The motility and membrane integrity of spermatozoa from nine boars frozen with a programmable freezing machine in plastic bags, 'cochettes', and in 'maxi-straws', in total doses of 5 x 10(9) spermatozoa/5 ml with glycerol (3%) used as cryoprotectant, were assessed after thawing. A computer-based cell motion analyser was used to evaluate sperm motility, while the integrity of the plasmalemma was assessed with fluorescent supravital dyes (C-FDA/PI). The fertilizing capacity of the semen frozen in the two containers was investigated by inseminating (AI) gilts. Pregnancy was monitored by Doppler-ultrasound, and the numbers of corpora lutea and viable embryos counted at slaughter, between days 30 and 38 after AI. The cochettes sustained the overall procedure of freezing/thawing (FT), with 30 min post-thaw (PT) sperm motility being significantly higher than for straws, 46.9 vs. 39.5%. The only significant difference in motility patterns detected when comparing the packages was a higher sperm velocity (VCL) in cochettes at 30 min PT. However, percentages of FT-spermatozoa with intact membranes, detected with the supravital probes, were higher in maxi-straws than in cochettes, 46.8 vs. 43.0% (P < 0.05). There were no significant differences found in fertilizing capacity between spermatozoa frozen in maxi-straws and those frozen in cochettes. The results indicate that although the deep-freezing of AI-doses of boar semen in large plastic bags is feasible, problems such as their inconvenient size for storage and inconsistent thawing must be solved before this type of container can be used for the commercial cryopreservation of boar semen.     

黑丝羽乌骨鸡精液DMA颗粒冷冻技术研究

为研究不同稀释液与解冻装置对黑丝乌骨鸡N,N-二甲基甲酰胺（N,N-dimethylformamide,DMA）颗粒冻精解冻后精子质量的影响,试验首先采用不同稀释液对精液进行稀释并比较其解冻后精子活力与人工输精的受精率;其次,选取不同解冻管及冻精颗粒数进行恒温水浴解冻,比较其活力;最后,依据解冻后的精子活力,筛选恒温水浴、恒温漏斗、恒温板3种解冻装置各自的最佳解冻温度,并利用各自最佳解冻温度解冻后的精液进行人工输精,检测受精率。结果显示：①不同稀释液组的精子活力与受精率高低趋势一致,为LR > F > B > L组,且各组之间差异显著（P < 0.05）。②在60℃恒温水浴中,用薄壁大玻璃管解冻的精子活力最好。③不同解冻装置有各自最佳解冻活力的温度范围,恒温水浴为50~60℃（0.51~0.59）、恒温漏斗为40~45℃（0.42~0.46）、恒温板为50~55℃（0.61~0.63）,每种装置最佳温度段内的精子活力差异不显著（P > 0.05）。④3种解冻装置最佳解冻状态相比：在精子活力上,60℃恒温水浴与55℃恒温板分别显著高于40℃恒温漏斗（P < 0.05）,但60℃恒温水浴与55℃恒温板之间差异不显著（P > 0.05）;在受精率上,55℃恒温板最高（26.91%）,60℃恒温水浴次之（23.08%）、40℃恒温漏斗最低（20.93%）,三者之间差异不显著（P > 0.05）。因此,黑丝羽乌骨鸡精液应采用LR稀释液、DMA冷冻保护剂及颗粒冷冻技术,在54.9℃恒温板解冻可获得较高的受精率。     

Study on Technology of Black Silkies Chicken Semen Freezing by DMA Pellet

The aim was to explore the effects of different kinds of dilution and thawing devices on the N,N-dimethylformamide (DMA) pellet frozen semen of Black Silkies. Firstly,the motility and fertility of the frozen semen thawed by different dilutions were compared;Then,the motility of the frozen semen was compared when the pellets were thawed using different tube and different number.Finally,the motility and fertility of the sperm thawed by three kinds of thawing devices (thermostat water bath,hotfunnel and hotplate ) were tested. The results showed that:①There was similar order of the sperm motility and fertility in the different dilution groups (LR > F > B > L),and there was significant difference among those groups (P < 0.05).②The motility was the best when the frozen semen was thawed with large thin-wall glass tube at 60℃.③The best temperature range of the 3 devices was different. The highest motility for thermostat water bath was 50 to 60℃ (0.51 to 0.59),and thermostat hotfunnel was 40 to 45℃ (0.42 to 0.46),while thermostat hotplate was 50 to 55℃ (0.61 to 0.63).There was no significant difference of the motility in the optimum temperature range for each device (P > 0.05).④The fertility of the different devices in their best thawing temperature was 26.91% (55℃,thermostat hotplate),23.08% (60℃, thermostat water bath), 20.93% (40℃, thermostat funnel),respectively,and there was no significant differences among those groups (P > 0.05).Therefore,the efficiency thawing condition for the Black Silkies frozen semen was the LR diluent,DMA cryoprotectant,pellet freezing,thawed in the thermostat hotplate at 54.9℃.