卵巢组织玻璃化冷冻研究进展

卵巢组织玻璃化冷冻可替代直接冷冻卵母细胞或胚胎。玻璃化冷冻卵巢组织在辅助生殖上具有优越性。它无需控制供体的生殖周期 ,也无需取出卵泡。同样这一技术可用于保存濒危动物或受意外伤害的人或动物的卵母细胞 ;可为性成熟前失去生殖能力的动物或人提供生殖保险以及增加卵母细胞的来源 ;可用于建立生殖细胞 (卵母细胞 )的冷冻库。而传统的冷冻技术存在很多弊端。文章综述了玻璃化冷冻卵巢组织的研究背景和现状 ,并指出了其广阔的应用前景     

卵巢冷冻保存和移植的历史、现状及前景

随着卵巢组织冷冻保存方法和冷冻技术的发展,生殖系的保存已变成了现实,卵巢组织冷冻和移植将作为一种保存种质资源最长远的选择,如冻存卵巢组织移植为年轻女性患者恢复内分泌及生育能力提供了可能.试验表明,冻存卵巢移植后其功能可恢复,并能保持较长期功能.文章就卵巢冷冻保存和卵巢移植技术的发展进步,卵巢组织冷冻和移植的历史、研究进展、方法、影响因素及临床应用进行综述.     

牛胚胎玻璃化冷冻技术研究进展

胚胎冷冻保存是保存和繁殖遗传优势动物的主要工具,是胚胎移植产业的重要组成部分。目前广泛使用的冷冻方法主要有慢速冷冻和玻璃化冷冻2种。由于玻璃化冷冻具有成本低、效率高、操作简单等优点,玻璃化冷冻法越来越受到人们的重视。经过多年研究,研究人员已经在玻璃化冷冻方法、冷冻液等方面取得了大量进展,玻璃化冷冻法已经开始进入商业化应用。本文综述了牛胚胎冷冻保存技术的研究进展,旨在为相关从业人员提供一定借鉴和参考。     

猪卵母细胞玻璃化冷冻保存技术的研究进展

卵母细胞冷冻保存技术在动物种质资源保存、濒危动物保护等方面具有独特的作用,猪卵母细胞的冷冻保存仍是一项世界性难题,尚处于摸索阶段。现就猪卵母细胞冷冻保存的现状、冷冻原理与方法及冷冻损伤等问题进行综述。     

体细胞冷冻保存技术及其在动物遗传资源保护中的应用研究进展

体细胞冷冻保存技术是指将体细胞置于超低温条件下,新陈代谢几乎停止,从而长期保存体细胞的一种技术,该技术对于体细胞克隆、诱导性多能干细胞等具有重要意义,同时也是建立动物体细胞库的关键技术。本文综述了体细胞冷冻保存原理与冷冻损伤机制、新型冷冻保护剂、冷冻保存方法以及冷冻保存技术在动物遗传资源保护上的应用,以期为体细胞冷冻保存技术的研究与应用提供参考。     

家禽精子冷冻保存技术研究进展

精子冷冻保存技术是一种在体外长期低温保存精子的技术。该技术不仅在人类精子保存方面应用广泛,而且在畜牧业生产和动物保种等领域具有重要意义。然而,目前家禽精子冷冻保存效果还并不理想,大部分精子冷冻复苏后损伤严重,丧失受精能力,限制了该技术的应用和推广。鉴于近年来关于冷冻保护剂、抗氧化剂和药物传递系统在精子冷冻保存技术中的作用,文章从精子冷冻技术的研究历程、冷冻保存程序、冷冻损伤机制和家禽精子冷冻保存液等方面进行综述,为家禽精子冷冻保存技术的研究与应用提供参考。     

去脂对猪卵母细胞和胚胎玻璃化冷冻的影响

卵母细胞和胚胎冷冻保存技术在动物种质资源保存、濒危动物保护等方面具有独特的作用。本文综述了猪卵母细胞和胚胎的玻璃化冷冻及去脂技术,以期为后续研究提供参考,提高冷冻成功率。     

驴精液冷冻保存技术研究进展

精液冷冻技术在驴种质资源的长期保存和引进以及人工辅助繁殖技术应用等方面具有重要意义,精液冷冻保存有利于充分发挥优秀种公驴的繁殖力,降低驴养殖产业的生产成本。近年来,驴精液冷冻保护剂和冷冻方法不断优化,驴精液冷冻保存技术也取得了很大的进步。但由于驴冷冻复苏的精子活力和受胎率低,严重限制了驴冷冻精液的推广与应用。本文对驴精液冷冻保存技术的发展历程、精子冷冻损伤机制、影响驴精液冷冻保存的因素和驴冷冻精液的应用进行综述,为今后驴精液冷冻保存技术的研究提供参考。     

卵巢异种移植在生殖生物学和动物保存中的研究进展

随着关于卵巢组织较有效的冷冻保存方法的发展,生殖系的保存和繁殖已变成了现实的目标。本文就卵巢冷冻保存和卵巢移植上的发展进步,异种移植在保存稀有和濒危物种及一些女性卵巢功能上的潜在价值,以及即将出现的异种移植技术进行综述。     

哺乳动物胚胎冷冻技术的研究进展

胚胎冷冻保存已广泛用于胚胎移植、动物克隆以及动物资源保护。此技术的应用需保证胚胎在冷冻—解冻后具有较高的成活率。自从1972年小鼠胚胎冷冻保存获得成功以来,许多学者在简化冷冻程序、缩短冷冻时间等方面进行了深入的研究。文章就胚胎冷冻的原理、保护剂、冷冻方法以及解冻方法等方面进行了综述。     

Effect of vitrification of feline ovarian cortex on follicular and oocyte quality and competence

Cryopreservation of ovarian cortex has important implications in the preservation of fertility and biodiversity in animal species. Slow freezing of cat ovarian tissue resulted in the preservation of follicular morphology and in the follicular development after xenografting. Vitrification has been recently applied to ovarian tissues of different species, but no information is available on the effect of this method on feline ovarian cortex. Moreover, meiotic competence of fully grown oocytes isolated from cryopreserved tissue has not been reported. The aim of this study was to evaluate the effect of vitrification of feline ovarian cortex on follicular morphology and oocyte integrity, as well as meiotic competence. A total of 352 fragments (1.5-2 mm(3) ) were obtained from ovarian cortical tissues: 176 were vitrified and 176 were used fresh as control. Histological evaluation of fresh and vitrified fragments showed intact follicles after cryopreservation procedures with no statistically significant destructive effect from primordial to antral follicles. After IVM, oocytes collected from vitrified ovarian fragment showed a higher proportion of gametes arrested at germinal vesicle (GV) stage compared to those isolated from fresh control tissue (33.8% vs 2.9%; p < 0.001). However, oocytes isolated from vitrified tissues were able to resume meiosis, albeit at lower rate than those collected from fresh tissues (39.8% vs 85.9%; p < 0.00001). Vitrification induced changes in the organization of cytoskeletal elements (actin microfilaments and microtubules) of oocytes, but significantly only for actin network (p < 0.001). Finally, chromatin configuration within the GV was not affected by the cryopreservation procedure. Our study demonstrated that vitrification preserves the integrity of ovarian follicles and that oocytes retrieved from cryopreserved tissue maintain the capability of resuming meiosis. To our knowledge, this has not previously been reported in the cat.     

哺乳动物的卵巢冷冻保存

经冷冻保存后的卵巢或组织可以保存大量的原始卵泡,并且结构完整,具有活力及发育能力,且已直接用于临床获得妊娠。因此,卵巢冷冻近年来成了生殖生物学、生殖医学的研究热点。本文就卵巢冷冻的历史、基本技术原理、方案选择与评价及其影响因素作一综述。     

新生小鼠卵巢组织玻璃化冷冻保存与异体异位移植研究

开展小鼠新生胎儿卵巢组织冷冻保存与移植研究,为建立家畜卵巢组织冷冻保存与移植技术奠定基础。采用微滴法玻璃化冻存方法处理卵巢,卵巢复苏后移植到昆明系雄性小鼠受体肾囊下,19只雄性受体鼠接受了卵巢移植,每侧肾囊移植2枚,卵巢共移植1日龄小鼠卵巢38个。饲喂28 d有效回收移植卵巢的受体鼠为17只,受体总有效率分别为89.4%;有效回收移植卵巢30个,卵巢总回收率为78.9%。结果表明,小鼠早期卵巢经过冷冻、解冻并异体异位移植后,其原始卵泡能够重新启动生长发育。     

The Use of an Open-freezing System with Self-seeding for Cryopreservation of Mouse Ovarian Tissue

Chemoradiotherapy in young women with cancer has substantially improved life expectancy in these patients, but these treatments often cause infertility. One method of preserving fertility is to cryopreserve ovarian tissue. In this study, an automatic open-vessel freezing system with self-seeding was tested for cryopreservation of murine ovarian tissue; the mouse is a species widely used in human and veterinary medical research. The freezing system concerned, is used for cryopreservation of oocytes and embryos in Europe. Twenty severe combined immunodeficiency (SCID) mice were ovariectomized. The ovarian tissue was either directly transplanted heterotopically into the neck muscle (group 1, n = 6) or cryopreserved after equilibration with 1.5 M dimethylsulphoxide and propanediol. After thawing, the tissue was transplanted in SCID mice (group 2, n = 6). Before and after thawing, a part of the ovarian tissue was examined with the LIVE/DEAD fluorescent viability staining. The count of follicles revealed intact (fresh 24.1%/thawed 21.7%), impaired (fresh 35.1%/thawed 35.4%), and dead follicles (fresh 40.8%/thawed 42.9%). The healthy follicular loss because of the cryopreservation was 10.0%. All recipient mice were killed after 3 weeks. Transplanted ovarian tissue was found macroscopically in all mice. Histological examination showed several growing follicles in all developmental phases in both groups of SCID mice [group 1 (fresh grafts): 315 +/- 76.3 (mean +/- SD); group 2 (cryopreserved grafts): 237 +/- 63.4]. These results demonstrate that the use of an open-freezing system allows the survival of cryopreserved mouse ovarian tissue.     

Cryopreservation of canine ovaries by vitrification

The cryopreservation of ovarian tissues is a technology with significant potential for the preservation of the genetic resource materials of working dogs, including guide dogs for the blind. However, no attempt has been reported on cryopreservation of the canine ovary. Thus, we evaluated a vitrification method for cryopreservation of canine ovaries and determined the potential functionality of vitrified-warmed canine ovaries by means of transplantation into non-obese diabetic-severe combined immunodeficiency (NOD-SCID) mice. All ovarian tissues cryopreserved by vitrification were morphologically normal in terms of histology. Cryopreserved ovaries were transplanted into the ovarian bursa of the NOD-SCID mice, and the xenografts were recovered from 23 of 23 mice (100%) 4 weeks after the operation. The transplanted canine tissue was tightly adhered to the mouse ovary. Although antral follicle formation did not occur after grafting, proliferating cell nuclear antigen immunoreactivity was detectable in many of the granulosa cells in the primary follicles of the grafts. These results indicate that cryopreservation of the canine ovary by vitrification appears to have the potential to restore endocrine function and ovulation potential.     

Morphological and Apoptotic Comparison of Primordial and Primary Follicles in Cryopreserved Human Ovarian Tissue

There are few reports which were designed to compare the survival rate of human primary follicles with primordial follicles after cryopreservation. This study was designed to evaluate whether such a difference occurs. Human ovarian biopsies were cryopreserved using dimethylsulphoxide/sucrose as the cryoprotectants. Fresh and cryopreserved ovarian samples were evaluated for viability differences between the two types of follicles using the endpoints of histology, ultrastructure and DNA fragmentation. In comparison with fresh ovarian tissue (83.9% ± 10.0%), the percentage of morphologically normal primordial follicles was not significantly different in cryopreserved tissue (73.9% ± 17.2%). However, a lower percentage of primary follicles with normal morphology was seen in the cryopreserved group (43.3% ± 25.7% vs 74.8% ± 19.4% for the fresh group). Transmission electron microscopy revealed that the cryopreservation did not appear to affect the structural integrity of primordial follicles; however, varying ultrastructural damage to the cytoplasm was observed in the majority of the cryopreserved primary follicles. Using a DNA fragmentation assay, the percentage of apoptotic primordial and primary follicles in the unfrozen (26.3% and 20%) and frozen (23.3% and 25%) ovarian tissue was similar. A higher proportion of primary follicles, compared to primordial follicles, suffer histological damage after slow freezing.     

哺乳动物配子冷冻保存并应用于珍稀濒危动物保护的技术策略

配子（精子和卵子）冷冻保存是将配子在超低温条件下冷冻,可进行优秀家畜个体和珍稀濒危动物种质资源的长期保存。配子冷冻是珍稀濒危动物种质资源保护、保存和利用的一项重要内容,也是动物种质资源库建设的一项关键技术。本文对国内外精子和卵母细胞冷冻保存利用技术的研究现状进行了详细论述,同时介绍了配子冷冻-解冻后的损伤评估研究,提出了未来我国在濒危珍稀动物配子冷冻保存方面的应用策略和保种建议,以期为我国家畜生物种质资源库建设、家畜种质资源保护和开发利用以及珍稀濒危动物保护提供参考。     

Simple prediction of the survival of follicles in cryopreserved human ovarian tissue

This study examines the possible predictive value of the LIVE/DEAD fluorescence viability assay for evaluation of survival of cryopreserved human ovarian tissue. Ovarian tissue from ten patients was examined by LIVE/DEAD viability staining before and after cryopreservation and after freezing in a -20 C freezer (negative control). After cryopreservation with a slow freezing protocol and cryoprotectant the LIVE/DEAD assay showed 86% viable follicles (an intact oocyte and at least more than 50% of the granulosa cells alive), whereas after freezing at -20 C the survival rate was 67%. The healthy follicular loss after cryopreservation was 4%, whereas with freezing at -20 C, it was 25%. Although this assay overestimates the survival rate of cryopreserved primordial follicles, if the LIVE/DEAD assay yields greater than approximately 85% viable follicles, it can be assumed that the follicles in the cryopreserved tissue have maintained their developmental potential and that the tissue is suitable for retransplantation.