Effect of cryoprotectants and thawing temperatures on chicken sperm quality

There is need for standardization of freezing–thawing protocol for rooster semen to minimize variability among results. Therefore, we aimed to compare effect of four different permeating cryoprotectants and two thawing temperatures (37 vs. 5°C) on sperm post‐thaw motility and to analyse combined effect of the best permeating cryoprotectant (P‐CPA) with one of four non‐permeating cryoprotectants (N‐CPA) on post‐thaw quality of rooster semen evaluated in vitro. Pooled semen from Ross PM3 rooster heavy line was diluted in Kobidil extender and frozen in cryoprotectant solution containing 6% dimethylacetamide, 7.5% dimethylformamide, 9% N‐methylacetamide or 8% ethylene glycol (EG) in liquid nitrogen vapours. To determine the best thawing rate, straws were thawed either at 37 or 5°C. Furthermore, samples were frozen in the presence of the best N‐CPA either with 0.75 mol/L ficoll, 0.2 mol/L sucrose, 0.2 mol/L trehalose or 0.05 mol/L glycine. Sperm motility, membrane destabilization and viability were analysed to compare different freezing–thawing conditions. In addition, morphology and ultrastructure analysis were performed to compare fresh and frozen‐thawed sperm quality. Our results indicate that the combination of EG and the thawing at 5°C improves (p ≤ .05) sperm post‐thaw motility. Moreover, ficoll addition to EG‐based freezing extender provided additional beneficial effect (p ≤ .05) on progressive movement and apoptosis incidence. Further work should evaluate different N‐CPA concentrations to improve freezing protocol. In addition, fertility evaluation and testing on different chicken lines are needed in order to contribute to animal genetic resources bank.     

抗冻剂在水产品冻藏中的应用研究

冷冻保藏可以最大限度地保持水产品的营养价值,但水产品在冻结贮藏中易发生冰晶的长大,对冻结水产品的品质带来很大的影响,使细胞受到机械损伤,蛋白质发生变性,解冻时汁液流失量增加,风味和营养价值下降。添加抗冻剂被认为是防止水产品蛋白质冷冻变性的最有效方法之一。文章探讨了水产品抗冻剂的作用机理,水产品经抗冻剂处理后再冻藏可提高Ca2+-ATPase 活性、增加样品的pH,较好的保持了物料的营养成分,抑制冰晶的生长,降低肌肉组织受到机械损伤;综述了新型抗冻剂如糖类抗冻剂、复合磷酸盐抗冻剂、蛋白水解产物型抗冻剂及抗冻蛋白在水产品中的应用研究进展。今后研究者可利用现代分析技术与方法获得蛋白质分子变化的动态特征,进一步理清水产品蛋白质冷冻变性的机理和作用机制。     

玻璃化冷冻水牛MII期卵母细胞的初步研究

为建立玻璃化冷冻保存水牛MⅡ期卵母细胞的有效程序,试验比较了3种冷冻保护剂组合(Ⅰ:20%乙二醇+20%二甲基亚砜;Ⅱ:25%乙二醇+25%二甲基亚砜;III:25%乙二醇+25%甘油)和3种玻璃化冷冻方法(毛细玻璃管法、铜环法及半麦管法)对水牛MⅡ期卵母细胞的毒性和冷冻效果的影响。结果表明,Ⅲ组的卵母细胞形态正常率显著低于Ⅰ和Ⅱ组(P<0.05),且III组的存活率也显著低于Ⅰ组(P<0.05),而Ⅰ和Ⅱ组的卵母细胞形态正常率和存活率没有显著差异(P>0.05)。进一步孤雌激活发现,Ⅰ组的卵裂率显著高于Ⅲ组(29.17±4.81%,5.56±11.11%,P<0.05),Ⅰ组的囊胚发育率显著高于Ⅱ和Ⅲ组(8.33±2.23%,2.43±2.87%,1.86±3.71%,P<0.05),然而Ⅱ和Ⅲ组间的卵裂率和囊胚发育率没有差别(P>0.05)。采用铜环法的回收率显著高于半麦管法(P<0.05),但和毛细玻璃管法之间差异不显著(P>0.05),冻后卵母细胞孤雌激活后的卵裂率和囊胚发育率差异不显著(P>0.05)。3种冷冻保护剂中Ⅰ组的细胞毒性作用最小,Ⅲ组的细胞毒性作用最大;铜环法和毛细玻璃管法能有效冷冻保存...     

抗冻剂、CB和离心极化对猪GV期卵母细胞冷冻效率的影响

【目的】评价2种冷冻保护剂(EDS和EPS)、细胞松弛素B(CB)和离心极化处理对猪GV期卵母细胞冷冻效果的影响。【方法】分别应用EDS和EPS 2种冷冻保护剂、CB(设5.0,7.5和10.0μg/mL 3个水平)和离心极化处理,对猪卵母细胞进行玻璃化冷冻保存,解冻后用台盼蓝染色法和Hochest33342染色法分析猪卵母细胞存活率及随后的成熟率。【结果】EPS组和EDS组冻融后的猪卵母细胞存活率和成熟率均无显著差异。以EPS作冷冻保护剂时,冻融后裸卵组的存活率显著高于COCs组(P<0.05)。7.5μg/mL CB处理组的裸卵存活率显著高于对照组(P<0.05);7.5μg/mL CB+离心极化处理组裸卵存活率与成熟率均显著高于CB单处理组和对照组(P<0.05)。【结论】同等条件下,猪GV期裸卵的冷冻效果好于COCs;CB或CB+离心极化均能改善GV期猪卵母细胞冷冻效果。     

兔胚胎玻璃化冷冻的研究

探讨用玻璃化冷冻法保存兔胚胎的效果．结果表明,兔早期囊胚在0．5M海藻糖（93．8％）溶液中的存活率显著高于0．5M蔗糖组（70．0％,P〈0．05）．对照组与不同浓度甘油处理间兔胚胎存活率均差异不显著（P〉0．05）．分别用不同浓度乙二醇玻璃化冷冻兔胚胎,解冻回收后正常胚胎数和囊胚孵化率,对照组与10％,20%EG组之间均差异不显著（P〉0．05）,但对照组,10％,20％EG组均显著高于30％EG组（P〈0．05）．解冻回收后正常胚胎数体内胚胎组（79．4％）显著高于体外胚胎（54．1％,P〈0．05）,囊胚孵化率2组之间差异不显著（P〉0．05）．缓慢冷冻法与玻璃化冷冻法之间胚胎解冻回收后正常胚胎数和囊胚孵化率均差异不显著（P〉0．05）．     

小鼠卵母细胞冷冻技术研究

采用PBS作为冷冻基础液,分别用甘油和二甲基亚砜(DMSO)作为冷冻保护液,在程序化冷冻保存和玻璃化冷冻保存条件下,研究小鼠生发泡期(GV期)卵母细胞的抗冻能力。结果表明,2种冷冻方法对小鼠GV期卵母细胞解冻后形态正常率和存活率无显著影响(P>0.05)。冷冻保护剂种类对小鼠GV期卵母细胞解冻后形态正常率无显著影响(P>0.05);但对存活率有显著影响,玻璃化冷冻采用二甲基亚砜作为冷冻保护液效果极显著优于甘油(P<0.01)。以冷冻效果较好的二甲基亚砜作为冷冻保护液,采用玻璃化冷冻不同发育阶段(GV期和MⅡ期)的小鼠卵母细胞,解冻后形态正常率无显著差异(P>0.05),但存活率GV期要显著优于MⅡ期卵母细胞(P<0.05)。     

哺乳动物胚胎冷冻保存研究进展

哺乳动物胚胎冷冻保存效果受冷冻保护剂、冷冻方法、解冻方法等多种因素影响,其中冷冻方法是一个关键性因素.目前胚胎冷冻方法主要有常规慢速冷冻法和玻璃化冷冻法两种.常规慢速冷冻法是指利用甘油、乙二醇等做冷冻保护剂通过缓慢降温的方式进行胚胎冷冻;玻璃化冷冻法是指利用高浓度的冷冻保护剂通过快速降温的方式进行胚胎冷冻.与常规慢速冷冻法相比,玻璃化冷冻法简化了操作过程,大大缩短了操作时间,不需昂贵的程序控制冷冻仪.     

采用主成分分析抗冻剂对冷冻虾肉糜3D可打印性的影响

针对冷冻虾肉糜的蛋白质易发生冷冻变性而失去3D可打印性的问题,该研究以添加不同抗冻剂的虾肉糜为研究对象,对虾肉糜冷冻贮藏过程中3D打印精确性和稳定性、流变和质构特性的变化开展试验研究,采用相关性和主成分分析虾肉糜3D打印精确性和稳定性与流变和质构特性之间的关系。研究结果表明,随着冻藏天数的延长,所有处理虾肉糜的3D打印精确性和稳定性、黏性常数、流变单元之间的连接强度、硬度、弹性、黏附性、持水性等均呈下降趋势;添加抗冻剂的处理可以减缓虾肉糜这些指标的下降;3D打印精确性和稳定性与虾肉糜的黏性常数、流变单元之间的连接强度、弹性、硬度、黏附性、持水性等呈正相关。添加8%海藻糖+0.3%多聚磷酸盐（即TP处理）抑制虾肉糜肌原纤维蛋白冷冻变性的效果最好,冻藏30 d后虾肉糜仍具有较好的3D可打印性,打印精确性和稳定性分别为96.64%和97.61%,该研究结果为保障虾肉糜3D打印的原料供应提供了理论参考。     

Major physiological adjustments in freezing-tolerant grey tiger longi-corn beetle （Xylotrechus rusticus） during overwintering period

1Xylotrechus rusticus(Linnaeus) is one of the most destructive woodborers in northeast China; it damages poplar and is listed as a domestic forestry quarantine pest. Overwintering larvae were collected during October 2012 and March 2013 in Harbin, China, to quantify indicators related to the insect's overwintering strategy and the major cryoprotectants. The supercooling points(SCPs), which ranged from –14.7°C to –2.9°C, were higher than the lethal temperatures of LT50(–33.64°C) and LT99(–40.17°C) after 24 h exposure., also the minimum mean daily temperature(–24.5°C) and mean monthly temperature(–18.0°C) at the sampling site in January during 2008–2012. Thus, X. rusticus is a typical freezing-tolerant insect. Glycerol serves as a major cryoprotectant for overwintering larvae, because it was the only polyol accumulated during the winter and it also had a significant negative correlation with the SCP(p= 0.033, R =0.907). The glycogen and lipid are major sources of energy and their levels decreased substantially in the middle of overwintering, when glycogen had a significant correlation with the SCP(p= 0.006, R =0.971) whereas the lipid contents did not. Moreover, inter-conversions between glycerol and glycogen, as well as mannose and glycogen, were suggested by their negative correlations. The water content did not change obviously during the winter and was not correlated with the SCP. The free amino acids in the hemolymph and the total protein contents of the bodies of larvae changed significantly during winter, although both had no correlations with the SCP.