6种草本药用植物种子超低温保存技术研究

以草本药用植物杜若、过江藤、夏枯草、皱果苋、罗勒和山香的成熟种子为材料,探讨了含水量和冷冻方法对种子超低温保存的影响。结果表明,经液氮超低温冷冻后,6种草本药用植物种子发芽率较对照组均有显著差异(p0.05);适宜的含水量下,种子经过超低温冷冻后其发芽率甚至高于对照组。3种冷冻方法中,玻璃化冷冻法更适合过江藤和山香种子的超低温保存,缓慢冷冻法更适合皱果苋、杜若和夏枯草种子的超低温保存,直接冷冻法适合于杜若和罗勒种子的超低温保存。所以,液氮超低温冷冻法保存杜若等6种草本药用植物种子是可行的;含水量对超低温保存前后夏枯草、山香和罗勒种子的发芽率影响显著。     

The Effect of Trehalose Supplementation of INRA-82 Extender on Quality and Fertility of Cooled and Frozen-Thawed Stallion Spermatozoa

Stallion semen cryopreservation is often associated with poor post-thaw sperm quality. Sugars act as nonpermeating cryoprotectants. The aim of the present study was to evaluate the cryoprotective effect of trehalose on stallion sperm quality and field fertility rates subjected to cooling and freeze–thaw process. Semen samples were collected from six Arabian stallions, divided into five different treatments in a final concentration of 100 × 10⁶ sperm/mL by using INRA-82 extender containing 0, 25, 50, 100, and 200 mM of trehalose then subjected to both cold storage and cryopreservation. Sperm motility, acrosome, plasmatic membrane, and DNA integrity were analyzed, and 57 mares were used to evaluate the field fertility of chilled and frozen-thawed semen. Results showed that the extender containing 100 mM trehalose only increased the functional acrosomal, plasma membrane, and DNA integrities. The inclusion of 50 mM trehalose in semen extender resulted in significantly (P < .05) increased post-thaw total motility compared to the control group, and chilled semen achieved higher pregnancy rates compared to the frozen-thawed one. Pregnancy rate of mares inseminated with frozen-thawed semen (P < .05; 46.15% vs. 36.36%, respectively) was lower than those inseminated with chilled semen (76.47% vs. 68.75%, respectively) but higher than control. In conclusion, addition of 50 mM trehalose yielded the highest quality stallion semen after cooling and post-thawing in terms of motility, integrities of acrosome, membrane, and DNA as well as improved field fertility.     

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.     

Single-Layer Colloid Centrifugation as a Method to Process Urine-Contaminated Stallion Semen After Freezing-Thawing

Urospermia is a major ejaculatory dysfunction affecting stallions. It has been thought that urine-contaminated semen should not be cryopreserved; however, on select cases, urine contamination of semen cannot be avoided. A recent study suggested that urospermic semen can be cryopreserved after cushion centrifugation and extension. Thus, this study aimed to assess the use of single-layer colloid centrifugation (SLC) to process frozen-thawed urine-contaminated stallion semen. Raw ejaculates (n = 55) from eight stallions were split into three groups: no urine, low (20%), or high (50%) urine contamination. Semen was extended 1:1, cushion-centrifuged, and resuspended at 200 million sperm/mL in BotuCrio. Resuspended semen was loaded in 0.5 mL straws and cryopreserved in liquid nitrogen. Samples were thawed (37°C for 30 seconds) and processed by SLC (400 g/30 minutes). Percentages of total motility (TM) and progressive motility (PM) were assessed with computer-assisted semen analyzer. Sperm viability (%VIAB) and yield were assessed with a NucleoCounter before and after gradient centrifugation. Data were analyzed with two-way ANOVA and Tukey’s test. The motility parameters TM before SLC (control: 35 ± 2; low: 33 ± 0.7; high: 22 ± 1.8) after SLC (control: 51 ± 3.6; low: 42 ± 2.2; high: 25 ± 2.8) and PM before SLC (control: 24 ± 1.8; low: 21 ± 1.14; high: 12 ± 1.5) and after SLC (control: 40.3 ± 3.2; low: 31 ± 3.9; high: 14 ± 2) significantly decreased with increasing urine contamination. Urine contamination marginally reduced (P < .05) sperm viability after cryopreservation before SLC (control: 45 ± 0.7; low: 27 ± 0.2; high: 27 ± 0.3) and after SLC (control: 54 ± 0.5; low: 49 ± 0.7; high: 38 ± 0.6). Recovery rates of sperm after centrifugation were not significantly different between groups. In conclusion, urine contamination affects sperm motility parameters in a dose-dependent manner. Post-thaw SLC selected sperm with higher motility and viability in control and low groups but only selected sperm with higher viability in the high group.     

Advances in Holding and Cryopreservation of Equine Oocytes and Embryos

Methods for holding of oocytes and embryos during shipment as well as for their cryopreservation can greatly aid equine reproductive management. Oocytes can be held at room temperature overnight or at cooler temperatures for two nights without affecting maturation or embryo development after intracytoplasmic sperm injection. In contrast, methods for cryopreservation of equine oocytes that support high rates of embryo development have not yet been established. Equine embryos may be held overnight at temperatures from 5°C to 19°C without reduction in viability, but longer holding periods, or higher holding temperatures, may be detrimental. Small equine embryos (<300 μm), either in vivo derived or in vitro produced, can be slow frozen or vitrified successfully. In the last decade, methods have been developed to allow in vivo–derived expanded blastocysts, up to Day 8, to be vitrified successfully after blastocoele collapse. These methods of shipment and preservation allow mare owners in remote locations to have access to sophisticated assisted reproductive technologies.     

High or Low Body Fat Deposition in the Presence of a Normal Oral Sugar Test is Not Associated With Postthaw Semen Parameters in Stallions

This study compared the postthaw semen parameters of stallions with high and low body condition score (BCS) and evaluated associations between body morphometric parameters and postthaw semen parameters. Twenty stallions were split into Low BCS (BCS<7, n = 11) and High BCS (BCS ≥7, n = 9) groups, and underwent a complete morphometric analysis (e.g., neck scores and circumference, crest neck height, body weight, and height), and subcutaneous body fat thickness (SFT) at the tail head, withers, shoulders, and retroperitoneal space. A fasted oral sugar test (OST) was conducted on all stallions. One ejaculate from each stallion was frozen with a commercial egg yolk-based extender. Postthaw sperm motility parameters, plasma membrane integrity, mitochondrial membrane potential, hydrogen peroxide and intracellular superoxide production, and lipid peroxidation were analyzed for all stallions. The circumference at 25% and 50% of the neck’s length were larger for High-BCS stallions (P < .05). There were no differences between groups for the neck crest height (P > .05). Stallions with High BCS had greater SFT at the tail head than stallions with Low BCS (P < .05); however, there were no differences between groups in the SFT at the shoulders and withers (P > .05). All stallions had resting blood glucose below the cutoff for equine metabolic syndrome. There were no differences between groups for resting glucose concentrations or for a peak at 30 or 60 minutes after initiation of the OST (P > .05). There were no differences in sperm parameters between groups (P > .05). Collectively, the findings of the present study suggest that High BCS or Low BCS in the presence of normal OST do not explain post-thaw semen parameters.     

褪黑素在牛体外胚胎生产中的应用研究进展

近年来牛体外胚胎生产取得很大进展,可以稳定地在体外获得囊胚,但体外生产效率偏低,不能满足产业需要。在牛体外胚胎生产过程中很容易产生活性氧和活性氮,导致配子和胚胎受到氧化应激或硝化应激。褪黑素作为一种强有力的抗氧化剂和抗凋亡剂,可以直接清除有毒的氧衍生物,减少活性氧的形成,在牛体外胚胎生产过程中发挥积极作用。本文主要对褪黑素在牛卵母细胞体外成熟、体外受精、早期胚胎发育、配子和胚胎冷冻保存等方面的研究进行综述,为后续体外胚胎生产技术的优化研究和推广应用提供参考。     

银杏愈伤组织超低温保存的研究

本文对银杏子叶愈伤组织进行了超低温保存研究。结果表明 ,冰冻保护剂、冷冻速度对解冻后材料相对存活率影响较大 ,冻前预培养和预冻至某一温度停留一段时间也较为重要。在含高浓度山梨醇或甘露醇的培养基上预培养 2d的愈伤组织 ,用 10 %二甲基亚砜 +0 .5mol·L- 1 山梨醇作为冰冻保护剂 ,以 1℃·min- 1 速度降温 ,在 - 15℃、- 35℃分别停留 10min和 30min后 ,投入液氮中保存 1d ,细胞相对存活率可达 6 0 %以上。再培养时 ,细胞能恢复生长。     

Somatic embryo culture for propagation,artificial seed production,and conservation of sawara cypress (<Emphasis Type="Italic">Chamaecyparis pisifera</Emphasis> Sieb. et Zucc.)

 Somatic embryogenesis in Chamaecyparis pisifera Sieb. et Zucc. was initiated from immature seeds collected from the end of June to early July. Mass propagation through adventitious shoot bud production from somatic embryo culture on Woody Plant (WP) medium and artificial seed production using sodium alginate was achieved. A high bud forming index value (25.8) was obtained on medium supplemented with 1 μM 6-benzylaminopurine. The conversion rates from artificial seeds under aseptic and nonaseptic conditions were 60%–100% and 10%–12%, respectively. For germplasm conservation, somatic embryos and embryogenic cells were successfully stored at 4°C (medium-term storage) and in liquid nitrogen for long-term storage. Received: December 21, 2001 / Accepted: August 1, 2002 Acknowledgments This work was supported in part by the Japan Science and Technology Corporation and in part by a Grant for Research for the Future Program from the Japan Society for the Promotion of Science. Correspondence to:E. Maruyama     

大鼠胚胎冷冻保存技术及研究进展

随着大鼠基因序列测序的完成和基因敲除技术的发展,通过对大鼠进行基因注入和基因敲除后建立的人类疾病模型日渐增多,建立一种完善的大鼠胚胎冷冻保存技术体系保存这些遗传资源至关重要。本综述系统介绍了大鼠胚胎冷冻技术的发展进程及研究现状,并根据其研究现状分别介绍了各类冷冻保护剂的特点及其组合,常用冷冻保存方法的比较及解冻方法;分析了目前大鼠胚胎冷冻保存技术面临的一些难题;介绍了大鼠胚胎冷冻保存技术的应用前景。