Low‐density lipoproteins and milk serum proteins improve the quality of stallion sperm after vitrification in straws

Lipids and proteins can be used for sperm vitrification to preserve the integrity of sperm membranes or to increase the viscosity of the medium. This study evaluated the effect of low‐density lipoproteins (LDL) and milk serum proteins (Pronexcell) for stallion sperm vitrification. Hippex extender (Barex Biochemical Products, The Netherlands), plus 1% of bovine serum albumin and 100 mM of trehalose, was used as control for sperm vitrification. In experiment 1, different concentrations of LDL (L1 = 0.25, L2 = 0.5, L3 = 1%) and in experiment 2 of Pronexcell (P1 = 1, P2 = 5, P3 = 10%) were added to control extender. Vitrification was performed in 0.25‐ml straws directly plunged into liquid nitrogen. Total motility (TM, %) and progressive motility (PM, %) were analysed by CASA, and plasma membrane (IMS, %) and acrosome membrane integrity (AIS, %) were assessed under epifluorescence microscopy. Post‐warmed sperm parameters were compared between treatments by ANOVA. Results were expressed as mean ± SEM. In both experiments, the minimum concentration of LDL and Pronexcell obtained significantly higher values (p < 0.01) than the control extender for TM (L1 = 52.95 ± 4.4; P1 = 58.99 ± 4.6; C = 30.88 ± 3.0), PM (L1 = 36.79 ± 5.5; P1 = 47.25 ± 4.3; C = 19.20 ± 2.4), IMS (L1 = 68.88 ± 3.6; P1 = 47.25 ± 4.3; C = 52.81 ± 2.6) and AIS (L1 = 45.88 ± 3.6; P1 = 47.25 ± 4.3; C = 26.00 ± 2.1). No differences in sperm parameters were found among different concentrations of LDL or Pronexcell. In conclusion, the addition of 0.25% LDL and 1% Pronexcell to the vitrification extender is recommended to improve the quality of stallion sperm after vitrification.     

Effect of nicotinic acid on the plasma membrane function and polyunsaturated fatty acids composition during cryopreservation in boar sperm

This study was conducted to evaluate the effect of nicotinic acid on plasma membrane integrity and fatty acid composition in frozen–thawed boar sperm. Boar semen was cryopreserved using freezing extender containing nicotinic acid (NA), then plasma membrane integrity, osmotic equilibration, lipid peroxidation and fatty acid were analysed. The plasma membrane integrity of frozen–thawed sperm was significantly higher in the 10 mM NA than in the 0 and 20 mM NA treatment groups (p < 0.05). Additionally, the osmotic equilibration ability was not different in treatment groups, but lipid peroxidation was significantly decreased in the 10 mM NA treatment group (p < 0.05). The saturated fatty acids were significantly decreased in the 10 mM NA treatment group, and C18:1n‐9, C18:2n‐6, C20:4n‐6, C22:5n‐6 and C22:6n‐3, and total polyunsaturated fatty acids (PUFAs) were significantly increased in the 10 mM NA treatment groups (p < 0.05). In summary, 10 mM NA improved plasma membrane integrity, inhibited lipid peroxidation and increased PUFAs in frozen–thawed boar sperm. These results suggest that NA may be useful to protect the plasma membrane and inhibit the loss of PUFAs for sperm cryopreservation in pigs.     

老年猪耳成纤维细胞建立及克隆胚胎发育

为保存江苏地方猪种的种质资源,通过采集猪耳结合低温运输进行老年猪耳组织块贴壁培养,建立猪耳成纤维细胞,进而将细胞扩繁传代并冷冻保存。通过规范采样流程,结合低温运输方式,使运输时间达到8h以上,扩大了采样范围,同时,使污染风险由前期的30.77%(13头)降低到目前的8.3%(12头)。虽然老年猪耳成纤维细胞在初期游离到铺满培养瓶需要21d,远高于胎儿(怀孕24d的胚胎)成纤维细胞,但其后期的冻存与解冻培养均不受影响。随后,利用手工克隆技术生产克隆胚胎,老年猪耳成纤维细胞作为供体囊胚率(27.04±4.08)%与新生猪耳成纤维细胞作为供体囊胚率(28.8±2.37)%相比,无显著差异(P>0.05)。本试验结果证明老年猪耳成纤维细胞培养及冻存的可行性,为利用体细胞与体细胞核移植对地方猪种种质资源的保存提供支持。     

Initial cooling time before freezing affects post‐thaw quality and reproductive performance of rabbit semen

The aim of this study was to investigate the effect of initial cooling time at 5°C during semen cryopreservation on post‐thaw quality and reproductive performance of rabbit semen. Pooled semen samples (n = 6) were divided into two subsamples and cooled at 5°C for 45 or 90 min. After cooling, the semen samples were diluted to a ratio of 1:1 (v:v) with a freezing extender composed of Tris‐citrate‐glucose (TCG) containing 16% of dimethylsulfoxide and 0.1 mol/L sucrose. The semen was subsequently loaded in 0.25 ml straws, equilibrated at 5°C and frozen in liquid nitrogen vapor. After thawing, sperm motility, viability, osmotic resistance, acrosome and DNA integrity were assessed. Our results indicate that the longer cooling time, that is, 90 min before cryopreservation significantly improves sperm post‐thaw viability, motility and fertility. In fact, reproductive performances obtained with semen frozen after a 90 min cooling time were similar to those produced by fresh semen insemination. Hence, the present research provides an effective freezing protocol for rabbit semen that will allow for the creation of a sperm cryobank for the conservation of Italian rabbit genetic resources, as well as the use of frozen semen doses in commercial farms.     

二倍体和三倍体虹鳟精液低温保存寿命的研究

与二倍体(2n组)精原液做比较,以不同密度为组别,研究了4℃下两组(3n-HD和3n-LD)三倍体虹鳟(Oncorhynchus mykiss)精原液的保存时限。结果发现,随着保存时间的延长,精液中精子的活力和寿命逐渐降低。2n组、3n-HD组和3n-LD组虹鳟精原液中精子分别在保存5d、4.5d和12.5d后失去活力;在7d、15d和14.5 d后死亡,即三倍体虹鳟精原液4℃保存时间约为二倍体的2倍,说明低温短期保存三倍体虹鳟精原液可以取得较好效果。     

Cryopreservation effects on the sperm quality of cachama blanca Piaractus brachypomus (Cuvier 1818)

The effects of straws volume, cryoprotectants and thawing temperatures were evaluated on the sperm quality of cachama blanca Piaractus brachypomus (Cuvier), an important Colombian fish species. Sexually mature fish were induced to ovulation or spermiation with a carp pituitary extract. A pool of suitable sperm samples was diluted in glucose, egg yolk, dimethyl sulphoxide (DMSO‐10%), methanol (MET‐10%) or ethylene glycol (ETG‐5%) and packed in 0.5, 2.5 or 5.0 mL straws and frozen in nitrogen vapour. The thawing process was performed in a 35 or an 80 °C water bath. The fertility was evaluated after 6 h post fertilization. The highest motility percentage (33 ± 3%) was observed with sperm cryopreserved with DMSO, packed in 5 mL straws and thawed at 35 °C. The treatments with DMSO and MET packed in 0.5 and 5.0 mL straws and thawed at 35 °C showed the highest fertility (higher than 71%) and the lowest fertility was obtained with MET‐2.5 mL (9 ± 5%). In all the treatments, a significant decrease in the sperm quality was observed at 80 °C. Sperm cryopreserved with DMSO‐10% or MET‐10%, packed in 2.5 or 5.0 mL straws are suitable to achieve acceptable fertilization and to fertilize high amounts of eggs.     

Cryopreservation of Persian sturgeon (Acipenser persicus) sperm: effects of cryoprotectants,antioxidant, membrane stabilizer,equilibration time and dilution ratio on sperm motility and fertility

Three experiments were performed to develop protocols for cryopreservation of Persian sturgeon Acipenser persicus, sperm. In the first experiment, sperm from six males was individually split in three subsamples and cryopreserved using Modified Tsvetkova's extender (mT) supplemented with dimethyl sulfoxide (DMSO), methanol (MeOH), glycerol (Gly) and ethylene glycol (EG) at concentration of 5%, 10%, 15% and 20%. In the second set of experiments, the effects of six equilibration times (0, 5, 10, 20, 40 and 60 min) and dilution ratios (volume sperm: volume extender 1:0.5, 1:1, 1:2, 1:3, 1:5 and 1:10) and the additive advantage of bovine serum albumin (BSA; 0, 2.5, 5 and 10 mg mL^?1) and ascorbic acid (0, 2.5, 5 and 10 U mL^?1), on the post‐thaw survival of sperm (triplicate set of six fish) were evaluated. Then, sperm was diluted in 1:1 mT extender with 10 mg mL^?1 BSA with selected cryoprotectants (15% MeOH and 10% DMSO) for 5 min. After a month of storage in liquid nitrogen, post‐thawed sperm motility; fertilization and hatching rate and viability of derived larvae were measured (Exp.3). Evaluation of cryoprotectants efficiency showed that MeOH 15% and DMSO 10% were suitable for cryopreservation of Persian sturgeon sperm. Gly and EG resulted in very low post‐thaw motility rates even at lowest concentration. No significant difference was observed among the four different equilibration times (0, 5, 10, 20 min) (P > 0.05) although higher equilibration times than 20 min resulted low post‐thaw motility (P < 0.05). The motility of frozen–thawed sperm did not significantly change when dilution ratio was increased from 1:0.5 to 1:3 (P > 0.05). However, higher dilution ratios (1:5 and 1:10) reduced the percentage of motile sperm. Supplementation of the cryoprotectant solution with 10 mg mL^?1 BSA significantly improved post‐thaw motility (P < 0.05), but ascorbic acid did not improve post‐thaw motility (P > 0.05). The results of experiment 3 showed that the highest fertilization (30.2 ± 5.75) and hatching rates (28.2 ± 5.25) were observed when samples were frozen with 15% MeOH (P > 0.05). Our study indicates that the use of mT extender consisting of 10 mg mL^?1 BSA in 15% MeOH diluted with sperm at 1:1 ratio for 5 min can be recommended cryopreservation method for Persian sturgeon sperm.     

日本蟳精子超低温冷冻保存技术的研究

以精子存活率作为评价指标,采用两步降温法研究了稀释剂、抗冻保护剂和预冷时间对日本蟳精子超低温冷冻保存效果的影响。结果表明:以采用稀释剂II、15%二甲基亚砜(DMSO)作为抗冻保护剂的保存效果最佳。在液氮中保存24 h后,精子存活率可达83.76%,保存一年后可达73.81%。精液的第一次预冷时间以25 min为宜。     

鱼类生殖细胞移植的研究进展及应用前景

生殖细胞移植是指将供体的生殖细胞移植到同种或异种受体体内,供体生殖细胞嵌合到受体性腺,经过增殖、分化并最终发育为功能性配子的过程。作为辅助生殖技术,它不仅为珍稀濒危动物的繁育和保护提供了新途径,同时也为生殖干细胞的功能研究提供了有效手段。鱼类生殖细胞移植研究首先在模式鱼类斑马鱼中开展,经过十多年的发展,取得了一系列突破性的进展:主要包括先后建立了以胚胎、仔鱼和成鱼为受体的生殖细胞移植体系,精原和卵原干细胞的发现拓宽了供体生殖细胞的选择,受体的选择与制备方法的完善。该技术在缩短鱼类性成熟周期、性控育种、珍稀濒危鱼类保护等方面具有巨大的应用前景,已成功在多种淡水和海水鱼类中开展了研究和应用。本文结合作者的研究实践和经验,系统地梳理和总结了鱼类生殖细胞移植的研究进展,指出了该技术实践应用的关键问题,并探讨了其应用前景。     

乌克兰鳞鲤精子超低温冷冻保存方法研究

为研究适用于乌克兰鳞鲤精子的超低温冷冻保存方法,分析比较3种稀释液[Hank′s、Cortland、Freezefish冻精稀释液,精子与每种稀释液均设置3种比例(1∶1、1∶3、1∶5)]及3种体积分数为10%的抗冻剂(二甲基亚砜、1,2-丙二醇和丙三醇)对乌克兰鳞鲤精子低温(4 ℃)保存活力的影响;运用筛选出的冷冻保护液及稀释比例,分析比较3种“3步冷冻法”以及3种解冻温度(20、30、40 ℃)对乌克兰鳞鲤精子活力的影响。试验结果表明,采用Hank′s作为稀释液,10%二甲基亚砜为抗冻剂,精子与稀释液比例为1∶3,平均降温速率为12 ℃/min,解冻温度为30 ℃时,精子活力最高(>68%)。通过对稀释液、抗冻剂、稀释比例、降温速率和解冻温度的层层筛选,建立了适宜乌克兰鳞鲤精子超低温冷冻保存的方法,在其种质保护方面具有重要意义,为开展其他鱼类精子超低温冷冻保存提供参考。