海藻糖、二甲乙酰胺对公猪精液冷冻保存效果的研究

采用5% 二甲乙酰胺(DMA)(V/V)完全替代甘油,比较乳糖、海藻糖对精液冷冻保存效果的影响。结果表明,海藻糖显著提高了冷冻——解冻后精子成活力(49.32%±1.52%)与顶体完整性 (47.33%±1.16%)(P<0.05)。然后利用海藻糖替代乳糖,评价不同浓度的DMA对公猪精液冷冻保存的影响。结果表明,当DMA添加量为4%时,解冻后精子活率、成活力、顶体完整率分别为(45.17±0.56)%、(50.33±0.67)%、(48.30±1.44)%,均显著高于3% DMA、6% DMA添加组(P<0.05),精子活率显著高于5% DMA添加组(P<0.05),但精子成活力、顶体完整性与其差异不显著(P>0.05)。因此,当利用海藻糖作为冷冻保存基础稀释液,DMA最适添加量为4%。     

Comparison of glycerol, lactamide, acetamide and dimethylsulfoxide as cryoprotectants of Japanese white rabbit spermatozoa

The rabbit is considered to be a valuable laboratory animal. We compared glycerol, lactamide, acetamide, and dimethylsulfoxide (DMSO) as cryoprotectants in egg-yolk diluent of ejaculated Japanese white rabbit spermatozoa for improvement of sperm cryopreservation methods. Rabbit semen was frozen with 1.0 M glycerol, lactamide, acetamide, or DMSO in plastic straws. Forward progressive motility and plasma membrane integrity of the post-thaw spermatozoa were examined. The rate of forward progressive motile spermatozoa in lactamide (37.8 +/- 3.0%) was significantly (P<0.05) higher than in glycerol (17.0 +/- 3.3%). In addition, the rates of sperm plasma membrane integrity in lactamide and acetamide (35.9 +/- 3.3% and 30.2 +/- 3.0%, respectively) were significantly (P<0.05) higher than in glycerol (17.0 +/- 2.6%). The results indicate that 1.0 M lactamide and acetamide have higher cryoprotective effects than 1.0 M glycerol for cryopreservation of Japanese white rabbit spermatozoa.     

二甲基甲酰胺对猪精液冷冻保存效果的影响

用二甲基甲酰胺(DMF)完全替代甘油,比较不同平衡时间和不同DMF添加量对猪精液冷冻保护效果的影响。结果表明,DMF能完全替代甘油,获得较好的冷冻保护效果。最佳平衡时间为90 min,解冻后精子活力为(44.57±0.72)%,显著高于对照组和其他组(P0.05)。当DMF添加量为5%时,冻后精子活力、活率、线粒体活性、顶体完整率和质膜完整率分别为(49.91±0.39)%(、46.51±0.26)%、(47.51±0.52)%(、49.84±0.56)%、(46.30±1.61)%,均显著高于2%、3%、6%DMF添加组(P0.05),但与4%DMF添加组相比,冻后精子活力、活率和质膜完整率差异不显著(P0.05)。本试验结果表明,DMF最适添加量为5%。     

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.     

Effects of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability of boar spermatozoa frozen in straws

Experiments were conducted to study the effect of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability of boar spermatozoa after cryopreservation in straws. Semen (split ejaculate) in maxi-straws (6 mm o.d.) was frozen using a programmable freezing chamber. Three methods for in vitro sperm evaluation were used: motility (MOT), acrosome integrity (NAR) and flow cytometric analysis of sperm treated with carboxyfluorescein diacetate and propidium iodide to assess sperm plasma membrane integrity (PMI). No interactions were found among the three variables evaluated. Length of prefreeze exposure to glycerol, ranging from .5 min to 75 min, had no effect on post-thaw sperm viability. Exposure of sperm to a glycerol-containing extender medium at 5 degrees C gave improved post-thaw viability over that exposed at 0 degree C (P less than .05). Glycerol at a concentration of 3 or 4% resulted in maximum post-thaw MOT. Acrosome integrity values were greatest for 2 and 3% glycerol, whereas PMI was greatest when glycerol concentration was 4 to 6%. The primary cryoprotective effect of glycerol on boar semen may be extracellular. It is concluded that 3 or 4% glycerol gives maximum viability of frozen-thawed spermatozoa when the present methods are employed.     

海藻糖对猪精液冷冻保存效果的影响

在传统的Tris-柠檬酸-葡萄糖稀释液基础上，分别添加25％、50％、75％、100％的海藻糖，研究不同浓度海藻糖对猪精液冷冻后精子质量的影响。结果表明，海藻糖相对于对照TCG稀释液能够显著改善和提高猪精液的冷冻效果，其最佳添加浓度为25％，冷冻-解冻后猪精子活力、活率、线粒体活性、质膜完整性以及顶体完整率均显著提高（P〈0．05），分别达到41．38％、46．34％、44．56％、43．51％和64．09％。海藻糖可以明显抑制精子获能，获能处理前精子获能率仅为3．68％，而获能处理后达到41．82％，有利于促进精子获能。精液稀释液中甘油的适宜添加浓度为2％，海藻糖只有与甘油共同作用，才能在冷冻-解冻过程更加有效地保护精子。猪精子活力、活率、线粒体活性、质膜完整率、顶体完整率等之间存在极显著的正相关关系（P〈0．01），而与获能处理前精子的获能率存在显著的负相关关系（P〈0．05）。     

The Combinatorial Effect of Different Equex STM Paste Concentrations,Cryoprotectants and the Straw‐Freezing Methods on the Post‐Thaw Boar Semen Quality

This study was to evaluate the combinatorial effect (14 treatments, A–N) of different Equex STM paste concentrations, cryoprotectants and the straw‐freezing method on the post‐thaw boar semen quality. Two ejaculates were collected from each of nine boars (three boars from each of three breeds). Semen was diluted in extenders with different concentrations of Equex STM paste and different cryoprotectants [glycerol or dimethylacetamide (DMA)] before cryopreserving via liquid nitrogen or dry ice. Motility, viability, percentage of spermatozoa with intense acrosomal staining and with normal morphology of post‐thaw sperm were evaluated. The qualities of thawed semen were best preserved in treatment H (extender with 0.5% Equex STM paste and 5% glycerol and freezing by dry ice) and were worst in treatment B (extender with 0% Equex STM paste and 5% DMA and freezing by dry ice). Significant difference (p < 0.05) was present in post‐thawed sperm motility (63% vs 27%), sperm viability (70% vs 33%) and sperm acrosomal integrity rate (68% vs 29%) between treatments H and B. However, sperm proportion with normal morphology showed no significant difference among treatments (66% vs 66%; p > 0.05). Moreover, statistical analysis suggests that no significant difference was present in semen quality among breed or individual donors (p > 0.05). These findings suggest that Equex STM paste improved the cryosurvival efficiency of boar sperm, and the favourable straw‐freezing method changes between glycerol and DMA.     

Egg Yolk and Glycerol Requirements for Freezing Boar Spermatozoa Treated with Methyl β-Cyclodextrin or Cholesterol-loaded Cyclodextrin

Egg yolk (EY) and glycerol are common constituents of extenders used for sperm cryopreservation. It has been demonstrated that using cholesterol-loaded cyclodextrins (CLC) improves sperm cryosurvival in several species. However, standard freezing extenders might not be the most appropriate for CLC-treated sperm. This study evaluated the EY and glycerol requirements for freezing CLC-treated boar spermatozoa. Semen samples from 34 ejaculates coming from 4 boars were used. Each ejaculate was split into three aliquots: one was used untreated (control), and the other two were treated with 1 mg of CLC or methyl-β-cyclodextrin/120 × 10⁶ sperm for 15 min at 22 C prior to cryopreservation. Our results indicated that reducing the concentration of EY was detrimental for sperm viability after thawing (31.57 ± 2 vs. 19.89% ± 2 for 20 and 10% EY, respectively; P <0.05), even in semen treated with CLC. On the other hand, it was observed that the traditional concentration of glycerol (3%) was not the appropriate for freezing CLC-treated sperm (61.10 ± 3 vs. 47.87% ± 3 viable sperm for control and CLC-treated sperm, respectively; P <0.05). Thus, CLC-treated sperm showed a higher tolerance to high glycerol concentrations (5%) in terms of sperm viability (59.19% ± 3) than non-treated sperm (45.58% ± 3; P<0.05). Therefore, it could be necessary to modify the freezing extenders for CLC-treated sperm. Nevertheless, additional studies will be needed to evaluate alternative cryoprotectants and to determine the effect of high glycerol concentrations on sperm functionality.     

The Cryoprotective Effect of Trehalose Supplementation on Boar Spermatozoa Quality

In order to improve boar sperm quality during frozen-thawed process, the influence of the presence of trehalose on success of cryopreservation of boar sperm were investigated. We evaluated freeze-thawing tolerance of boar spermatozoa in a base cooling extender with the addition of different trehalose concentrations (0, 25, 50, 100 and 200 m m ), and try to determine the optimum concentration of trehalose. We chose sperm motility, mitochondrial activity, acrosome integrity and membrane integrity as parameters to evaluate cryopreservation capacity of boar spermatozoa. We obtained the best results for 100 m m trehalose-supplemented extenders, with values of 49.89% for motility, 44.69% for mitochondrial activity, 66.52% for acrosome integrity and 44.61% for membrane integrity, while freeze-thawing tolerance diminished significantly for 200 . The synergic effect of trehalose and glycerol resulted in better cryosurvival of boar spermatozoa than that of a single cryoprotectant. In conclusion, when trehalose-supplementation was added up to 100 m m , trehalose confers a greater cryoprotective capacity to the extender, and the sperm motility, mitochondrial activity, membrane integrity and acrosome integrity parameters were significantly improved during frozen-thawed process.     

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。     

Cryopreservation of boar semen by egg yolk-based extenders containing lactose or fructose is better than sorbitol

The present study determined the effect of different types of sugars (lactose, fructose, glucose and sorbitol) used in egg yolk-based extender on the post-thawed boar semen quality. Twenty-two ejaculates from 6 fertility-proven Yorkshire boars were cryopreserved by liquid nitrogen vapor method. Sperm motility, viability, acrosome integrity and intact functional plasma membrane were determined at 0, 2 and 4 hr after thawing. It was found that the lactose-based extender resulted in a higher percentage of post-thawed sperm motility, viability, intact acrosome and functional plasma membrane than sorbitol-based extender (P<0.05) and fructose-based extender yielded a higher post-thawed sperm motility and viability than sorbitol-based extender (P<0.05). It could be concluded that sorbitol was not an effective sugar for the cryopreservation in boar semen.     

Acrosomal ultrastructure of stallion spermatozoa cryopreserved with ethylene glycol using two packaging systems

The present experiments aimed to examine the substitution of glycerol (G) by ethylene glycol (E) as a cryoprotective agent for stallion spermatozoa. Two different ethylene glycol concentrations (5% and 10%) and also the association of glycerol (2%) and ethylene glycol (3%) (E/G) were studied (Experiment 1). In Experiment 2, two packing systems (0.5 x 4.0 ml) were evaluated using both cryoprotectors. In both experiments, the sperm membrane integrity after freezing was evaluated using transmission electron microscopy. The mean post-thaw motility was 34.25, 36.5, 29.25 and 34.75% for G5%, E5%, E10% and E/G, respectively. It was observed that the percentage of motile spermatozoa was significantly smaller (P<0.05) when semen was processed with E10%. A decrease in the acrosome integrity was observed in frozen thawed spermatozoa from all treated groups. It was observed that 28.0, 22.5, 25.5 and 22.5% of the sperm cells had a normal acrosome following freezing with G5%, E5%, E10% and E/G, respectively. Undulation of the outer acrosomal membrane, acrosomal swelling and loss of acrosomal content density and homogeneity were the most evident ultrastructural alterations observed. In Experiment 2, the post-thaw motility was higher (P<0.05) for sperm frozen in 0.5 ml straws than in 4.0 ml straws, regardless of the cryoprotector used. The ultrastructural evaluation showed 26.7 and 16.0% of intact acrosomes for sperm frozen in 0.5 ml and 4.0 ml straws, respectively. We concluded that ethylene glycol has similar cryoprotective properties to glycerol and that utilisation of 0.5 ml straws improved the ability of horse sperm cells to withstand damage after the cryopreservation process.     

L-肉碱对猪精液冷冻保存效果的研究

本试验用5%二甲基甲酰胺(DMF)替代甘油作为冷冻保护剂,研究不同浓度(0、0.02、0.04、0.06、0.08 mg/mL)L-肉碱对猪精液冻后常规指标(精子活率、线粒体活性、顶体完整性、质膜完整性)、过氧化氢酶(CAT)和总抗氧化酶(T-AOC)活性以及丙二醛(MDA)含量的影响。结果表明:添加0.04和0.06 mg/mL的L-肉碱可有效改善猪精液冷冻后效果。0.04 mg/mL组可显著提高精子冻后活率和线粒体活性(P<0.05);0.06 mg/mL组可显著提高顶体完整性和质膜完整性(P<0.05)。添加0.06 mg/mL L-肉碱可显著提高冷冻后精子内T-AOC酶活性并且抑制MDA的产生(P<0.05),但CAT活性与0.04 mg/mL组差异不显著。在冷冻稀释液中添加0.06 mg/mL L-肉碱可以提高猪精液冷冻保存效果。     

Comparison of Different Glycerol and Egg Yolk Concentrations Added to Tris‐based Extender for the Collared Peccaries (Tayassu tajacu) Semen Freezing

This study aimed to evaluate various concentrations of egg yolk (5, 10, or 20%) in combination with different concentrations of glycerol (3% or 6%) added to a Tris‐based extender on the post‐thaw characteristics of sperm obtained from Tayassu tajacu. For this purpose, semen from 10 sexually male mature collared peccaries was collected by electroejaculation and evaluated for sperm motility, vigour, viability, morphology and functional membrane integrity. The ejaculates were initially extended in Tris‐fructose plus egg yolk (5%, 10% or 20%). After cooling, the semen was added to Tris‐egg yolk plus glycerol (6% or 12%), resulting in a final concentration of 3% or 6% glycerol of the extender. Straws were frozen using liquid nitrogen and thawed in a water bath at 37°C for 30 s. The frozen–thawed semen was evaluated as reported for fresh semen. After thawing, a significant decrease was verified for sperm motility and vigour, for all the samples in comparison with fresh semen. However, no differences were evidenced among treatments for any sperm characteristics evaluated (p > 0.05), except for the combination between 10% egg yolk and 6% glycerol, which provided the worst preservation of functional membrane integrity (p < 0.05). The interactions between higher concentrations of egg yolk (20%) and glycerol (6%) and also between lower concentrations of the same substances (5% egg yolk and 3% glycerol) added to the Tris‐based extender negatively affected the preservation of the normal sperm morphology after thawing (p < 0.05). In conclusion, the use of Tris‐based extender added to 10% or 20% egg yolk plus 3% glycerol is recommended for effective sperm cryopreservation in collared peccaries.     

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.     

Aquaglyceroporins but not orthodox aquaporins are involved in the cryotolerance of pig spermatozoa

Background: Aquaporins(AQPs) are a family of transmembrane water channels that includes orthodox AQPs,aquaglyceroporins(GLPs) and super AQPs. AQP3, AQP7, AQP9 and AQP11 have been identified in boar sperm,and they are crucial for sperm maturation and osmoregulation. Water exchange is an important event in cryopreservation, which is the most efficient method for long-term storage of sperm. However, the freezethaw process leads to sperm damage and a loss of fertilizing potential. Assuming that the quality of frozenthawed sperm partially depends on the regulation of osmolality variations during this process, AQPs might play a crucial role in boar semen freezability. In this context, the aim of this study was to unravel the functional relevance of the different groups of AQPs for boar sperm cryotolerance through three different inhibitors.Results: Inhibition of different groups of AQPs was found to have different effects on boar sperm cryotolerance.Whereas the use of 1,3-propanediol(PDO), an inhibitor of orthodox AQPs and GLPs, decreased total motility(P 0.05), it increased post-thaw sperm viability, lowered membrane lipid disorder and increased mitochondrial membrane potential(MMP)(P 0.05). When acetazolamide(AC) was used as an inhibitor of orthodox AQPs, the effects on post-thaw sperm quality were restricted to a mild increase in MMP in the presence of the intermediate concentration at 30 min post-thaw and an increase in superoxide levels(P 0.05). Finally, the addition of phloretin(PHL), a GLP inhibitor, had detrimental effects on post-thaw total and progressive sperm motilities,viability and lipid membrane disorder(P 0.05).Conclusions: The effects of the different inhibitors suggest that GLPs rather than orthodox AQPs are relevant for boar sperm freezability. Moreover, the positive effect of PDO on sperm quality suggests a cryoprotective role for this molecule.     

Effect of the Interaction Between Cryoprotectant Concentration and Cryopreservation Method on Frozen/Thawed Chicken Sperm Variables

This work examines the effect of the interaction between different concentrations of two cryoprotectants – glycerol (GLY) and dimethylacetamide (DMA) – and two methods of cryopreservation – pellets produced by plunging into liquid nitrogen and gradual in‐straw freezing – on frozen/thawed chicken sperm variables. Sperm was cryopreserved using: (i) 6% DMA, following the in‐straw and the pellet methods (ii) 11% GLY, following the in‐straw and the pellet methods; and (iii) 8% GLY in the in‐straw method and 3% DMA in the pellet method (i.e. reduced cryoprotectant concentrations). When 6% DMA was used as the cryoprotectant, no differences were seen between the in‐straw and pellet methods in terms of frozen/thawed sperm variables or fertility (10.8% and 12.8%, respectively). The viability and motility variables of the frozen/thawed sperm produced using the in‐straw method with 11% GLY were higher (p < 0.05) than those recorded for the sperm preserved using the same cryoprotectant and concentration in the pellet method. However, fertility was extremely low in both groups (2.1% and 4.2% for the in‐straw and pellet methods, respectively). Finally, the use of 8% GLY in the in‐straw method returned higher sperm viability, intact acrosome and motility values than the use of 3% DMA in the pellet method (p < 0.01). No differences were seen, however, in the fertility results obtained (28.8% and 25.0%, respectively). These results suggest that cryoprotectant concentrations can be reduced and still provide acceptable fertility rates.     

Extenders containing dimethylformamide associated or not with glycerol are ineffective for ovine sperm cryopreservation

The study aimed at testing the effectiveness of dimethylformamide, alone or combined with glycerol, as cryoprotectant for freezing ram semen. Ejaculates from nine rams were cryopreserved in Tris-based extenders, containing 5% of glycerol, association of dimethylformamide with glycerol, in four proportions achieving 5% of cryoprotectors in the media and pure dimethylformamide (2, 3, 4 and 5%) in replacement to glycerol. The samples were diluted to 100 × 10(6) sptz/ml and stored in 0.25-ml straws in liquid nitrogen. After thawing (37 °C for 30 s), motility was preserved better by the extender containing 5% of glycerol (p < 0.05). The extenders containing pure dimethylformamide, or more than 2% in combination with glycerol, provided sperm motilities close to zero. Plasma and acrosomal membrane integrity were preserved better (p < 0.05) in the extender containing 5% glycerol. It can be concluded that dimethylformamide, alone or combined with glycerol, has no beneficial effects on ovine semen cryopreservation.     

不同稀释液对公猪精液常温保存效果的研究

 为提高猪精液的保存效果,设计了3种常温保存稀释液配方,并对保存后的精子进行了顶体染色,为猪精液常温保存技术及稀释液配方的研究提供参考依据。结果表明：当精子活率为50％与30％时,配方Ⅱ的精子保存时间均显著高于（P<0.05）配方Ⅰ与配方Ⅲ,且配方Ⅱ所保存精子的总存活时间也显著高于（P<0.05）配方Ⅰ与配方Ⅲ;在精液保存24h后,3种配方保存的精子顶体完整率均在95％以上,且差异不显著（P>0.05）,配方Ⅱ对精子的常温保存效果要优于配方Ⅰ与配方Ⅲ。     

不同甘油浓度下稀释液中添加棉子糖对绒山羊冷冻精液品质的影响

在甘油浓度为3%和6%的冷冻精液稀释液中添加不同浓度的棉子糖,旨在优化绒山羊冷冻稀释液配方。结果表明:甘油浓度为3%时,山羊精液稀释液的抗冻保护效果较好,在此基础上添加10 mmol/L棉子糖,精子活率、顶体完整率、生存指数、质膜完整率均高于其他实验组(P<0.05),并且畸形率显著降低(P<0.05);在脂质过氧化反应和抗氧化酶活性方面,该实验组CAT活性高于其他实验组(P<0.05),而MDA浓度低于其他实验组(P<0.05)。