AMPK激活剂在绵羊精液冷冻保存中的作用研究

旨在研究AMPK激活剂二甲双胍（metformin，Met）和阿卡地新（acadesine，AICAR）对绵羊精液冷冻保存效果的影响。本研究首先在冷冻基础稀释液中分别添加不同浓度（0、100、200、300、400、500 μmol·L^-1）的Met和AICAR，冷冻解冻后根据精子活力、运动性能和结构完整性指标筛选出最佳的添加浓度（400 μmol·L^-1 Met、200 μmol·L^-1 AICAR）；然后分别使用不同的冷冻稀释液（对照组：稀释液；Met组：含400 μmol·L^-1 Met的稀释液；AICAR组：含200 μmol·L^-1 AICAR的稀释液）冷冻精液，解冻后检测精子中AMPK蛋白表达、顶体酶活性、代谢指标、线粒体功能以及抗氧化酶活性。结果表明，稀释液中添加400 μmol·L^-1 Met和200 μmol·L^-1AICAR均可显著提高解冻后精子活力、运动性能及精子结构完整性（P<0.05），其中400 μmol·L^-1 Met组精子总活力达43.20%，顶体完整率为91%，质膜完整率为46%。与对照组相比，Met组和AICAR组解冻后精子中AMPK磷酸化水平显著升高（P<0.05）；顶体酶活性显著提高（P<0.05）；丙酮酸水平显著下降（P<0.05），乳酸脱氢酶活性、乳酸以及ATP含量均显著升高（P<0.05）；与对照组相比，Met和AICAR组稀释液更有利于维持线粒体膜电位（P<0.05），提高ATP酶（P<0.05）以及抗氧化酶的活性（P<0.05）。添加适当浓度的AMPK激活剂可以提高绵羊精液冷冻保存的效果。     

马氏珠母贝肉酶解产物的抗酒精性肝损伤作用

为探究马氏珠母贝肉酶解产物(Enzymatic hydrolysate from Pinctada martensii,EP)对酒精性肝损伤(Alcoholic liver damage,ALD)的保护作用,该研究将EP超滤分级为截留分子量>10 kD(EP-Ⅰ)、3~10 kD(EP-Ⅱ)和<3 kD(EP-Ⅲ)3个组分,检测其体外抗肝损伤活性及对ALD小鼠肝保护作用的影响。体外试验结果显示,EP-Ⅲ可显著激活体外乙醇脱氢酶(ADH)活性(P<0.01),3个超滤组分均具有一定的体外抗氧化能力且EP-Ⅲ>EP-Ⅱ>EP-Ⅰ;动物试验结果显示,与模型对照组相比,各超滤组分均能够显著降低小鼠血清中谷丙转氨酶(ALT)和谷草转氨酶(AST)活力、小鼠肝脏指数及肝脏中丙二醛(MDA)和甘油三酯(TG)含量,同时显著增强小鼠肝脏中超氧化物歧化酶(SOD)、乙醇脱氢酶(ADH)和乙醛脱氢酶(ALDH)活力,提高肝脏中的谷胱甘肽(GSH)含量。综上,马氏珠母贝肉酶解超滤组分对急性ALD具有一定的辅助保护作用,其中EP-Ⅲ的保护作用效果最佳,其机制可能与加快机体乙醇代谢和减缓乙醇对机体造成的氧化损伤相关。     

Sperm cryopreservation of the noble Scallop Chlamys nobilis by a programmable freezing method: Effect of cryoprotectant

A study on Chlamys nobilis sperm cryopreservation by a programmable freezing method was conducted under laboratory condition. Four cryoprotectant agents (dimethyl sulfoxide [DMSO], methanol [MET], propanediol[PG] and ethylene glycol [EG]) and four concentrations (5%, 10%, 20% and 30%) were evaluated for their ability to retain sperm motility, movement characteristics and fertility. Results showed that cryopreserved sperm total motility produced by DMSO and MET at 5%, 10% and 20% were higher than other cryoprotectant treatment groups (CPA groups), as well as rapid sperm percentage. The curvilinear (VCL) and straight line (VSL) velocity produced by DMSO at 5% significantly higher than other CPA groups (p < 0.05), while no significant differences were found for average path (VAP) velocity. The lateral head displacement (ALH) in all CPA groups was similar and without significant difference (p > 0.05), as well as the beat‐cross frequency (BCF). A significant higher fertilization rate was produced in DMSO than that in MET at same concentration (p < 0.05), and no significant differences were found for differing concentrations of the same cryoprotectant (p > 0.05). Overall, 5%‐20% DMSO was more suitable for Chlamys nobilis sperm programmable cryopreservation when the calcium‐free Hanks’ balanced salt solution was used as the extender, and 10°C/min from 0°C to ?80°C was used as freezing rate. The findings presented in this study will benefit conservation programs for Chlamys nobilis.     

虎雪兰玻璃化超低温法脱毒技术的研究

以兰属花卉虎雪兰组培原球茎为试材,采用玻璃化超低温法对兰花病毒脱除进行了研究,以期为虎雪兰玻璃化超低温法脱毒体系的建立提供参考依据。结果表明:在蔗糖浓度0.5 mol·L-1预培养4 d,然后在蔗糖0.6 mol·L-1加载液冰上处理50 min,之后转入PVS2溶液冰上玻璃化处理120 min,再液氮冷冻40 min,37℃水浴解冻3 min,最后卸载液(1/2MS+1.2 mol·L-1蔗糖)卸载20 min,待恢复培养后,原球茎成活率可达到65%以上,随机检测不同处理样品的脱毒率可达97%。     

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.     

Retracted: Study of testis histology and sperm morphology in the Bunnei,Barbus sharpeyi (Gunther, 1874) induced by luteinizing hormone‐releasing hormone analogue and carp pituitary extract

This study was conducted to determine the effects of hormone treatment on testis structure in Barbus sharpeyi, as well as the morphology of sperm examined by scanning electronic microscopy (SEM). Male B. sharpeyi were divided into three groups (three fish per group) and injected with luteinizing hormone – releasing hormone analogue (LHRH–A₂) or carp pituitary extract (CPE). The first and second groups were treated with 10 μg kg^?1 LHRH–A₂ and metoclopramide (MET), and their testis were sampled pre‐ and Poststripping respectively. The third group received 2 mg kg^?1 CPE and were killed pre‐stripping. Based on the histological results obtained, the testicular connective tissue of the lumen was thicker, and seminal vesicles were of a lower volume, in fish injected with CPE in comparison to the other groups. After treatment with LHRH–A₂ and MET, not all spermatozoa within the testis were ejaculated, and only a small amount of sperm was obtained by abdominal stripping. The highest and lowest diameters of connective tissue within lobules were observed in fish receiving CPE and LHRH–A₂ treatments respectively. There was a significant difference (P < 0.05) in lobule space between the fish injected with the CPE and the fish injected with the LHRH–A₂ and MET. The SEM results revealed that the spermatozoa of B. sharpeyi were composed of a spherical to elliptical head, a cylindrical midpiece, and a lengthy flagellum. In conclusion, it was found that injection with LHRH–A₂ and MET improved the spermatogenic process in comparison to injection with CPE.     

The Effects of an Oxygen Scavenger and Coconut Water on Equine Sperm Cryopreservation

Alternative sources of lipoproteins in semen extenders could replace animal by-products. We hypothesized that: (1) post-thaw semen parameters and fertility would not be different in coconut water (CW)–treated samples compared with egg yolk (EY)–treated samples and (2) the use of an oxygen scavenger (Oxyrase) would improve post-thaw sperm motility and membrane integrity and decrease lipid peroxidation. Experiment 1: three ejaculates each from five stallions were split into four treatments: EY, CW, egg yolk with Oxyrase, and coconut water with Oxyrase. Computer-assisted sperm analysis measured progressive and total motility, velocity, and linearity. Membrane integrity, apoptosis, and lipid peroxidation were evaluated using propidium iodide, annexin, and BODIPY fluorescent probes, respectively. Samples were cryopreserved, stored in liquid nitrogen, and then thawed to 37°C and analyzed again. Experiment 2: one ejaculate was divided into two aliquots and cryopreserved using either CW or EY. In a crossover design, 12 mares were bred on two consecutive cycles with either EY or CW. Pregnancy evaluations were at 14-day gestation. No differences were detected in sperm parameters between CW and EY (P > .05). Oxyrase did not improve sperm motility parameters in post-thaw samples, nor did it show protective effects for viability or against membrane damage (P > .05). More mares became pregnant using CW than EY (11/12 vs. 6/12, respectively; P = .013). Use of CW is a viable alternative to animal-based products in the cryopreservation of stallion semen.     

Effects of single layer centrifugation (SLC) on bull spermatozoa prior to freezing on post‐thaw semen characteristics

Single layer centrifugation (SLC) has been shown to select the most robust spermatozoa from the ejaculate in several species. Here the effects of SLC prior to freezing on various parameters of frozen‐thawed bovine sperm quality are reported. Semen from 8 bulls was layered on top of a species‐specific colloid, Bovicoll. After centrifugation for 20 min at 300 g, the resulting sperm pellet was resuspended in OPTIXcell^® (IMV Technologies, l′Aigle, France); the SLC‐selected sperm samples and uncentrifuged controls were frozen. On thawing, all sperm samples were analysed for membrane integrity, production of reactive oxygen species, mitochondrial membrane potential (MMP) and chromatin integrity. The SLC‐treated samples had a higher percentage of live, superoxide‐positive spermatozoa than uncentrifuged samples (27.9 ± 5.1% versus 21.7 ± 6.7%; p = .03). They had a higher proportion of spermatozoa with high mitochondrial membrane potential than uncentrifuged samples (55.9 ± 8.2% versus 40.5 ± 15.1%; p = .03) and also a lower proportion of spermatozoa with low mitochondrial membrane potential than non‐treated samples (42.0 ± 8.5% versus 55.9 ± 14.4%; p = .04). No significant effects of treatment were found for membrane integrity or chromatin integrity. The effect of bull was significant on the proportions of dead, superoxide‐positive spermatozoa and live, hydrogen peroxide‐negative spermatozoa, as well as on membrane integrity, but it was not significant for mitochondrial membrane potential or chromatin integrity. These results suggest that SLC selects the most metabolically active bull spermatozoa from the rest of the population in normal ejaculates; the pattern of reactive oxygen species production may be different in SLC‐selected spermatozoa compared to unselected samples.     

Fertilization rate,motility, lipid peroxidation and pH changes after chilled storage of rainbow trout (Oncorhynchus mykiss) eggs and spermatozoa by a RMPI medium

The aim of this study was to evaluate the effects of Roswell Park Memorial Institute (RPMI) 1,640 medium on chilled storage of eggs and spermatozoa of rainbow trout (Oncorhynchus mykiss). After 3 days of storage, eggs in RPMI 1,640 media (pH 8.2, 9 and 10), Cortland medium and coelomic fluid were inseminated with fresh spermatozoa (Experiment 1). Eggs in RPMI 1,640 medium at pH 8.2 shown the lowest thiobarbituric acid‐reactive substances (TBARS, 0.053 ± 0.003 nmol/ml) and pH changes (from 8.20 ± 0.01 to 8.18 ± 0.01), the highest fertilization rate (82 ± 3%). Undiluted and diluted spermatozoa at ratios of 1:2 and 1:9 with RPMI 1,640 media (pH 8.2, 9 and 10) and Cortland medium were inseminated with fresh eggs (Experiment 2). Spermatozoa in RPMI 1,640 medium at pH 9 (1:9) caused the lowest TBARS content (0.037 ± 0.002 nmol/ml) and pH changes (from 9.00 ± 0.01 to 8.98 ± 0.01), the highest fertilization rate (77 ± 2%) and motility parameters. Based on Experiments 1 and 2, eggs and spermatozoa were stored for another 3 days in RPMI 1,640 medium at pH 8.2 and 9 (1:9) respectively (Experiment 3). Fertilization rate of storage eggs and spermatozoa in Experiment 3 was 79 ± 5%, showing successfully storage of rainbow trout gametes with the same medium.     

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。