山羊胚胎简易冷冻试验

用RPE冷冻器冷冻20枚山羊桑椹胚和囊胚。冷冻液为1M甘油杜氏磷酸盐缓冲液(PBS)。胚胎装于0.25ml塑料细管内,0℃时置入冷冻器。以1℃/分降温,-6.5～-7℃诱发结晶,再以0.5℃/分(0.3～0.8℃/分)降温至-30℃,然后投入液氮保存5～12天。35℃水浴解冻。20枚胚胎冻后形态正常率为65％(13/20)。将13枝形态基本正常的胚胎移植给5只受体羊,结果4只受体羊妊娠,产羔7只,冻胚存活率为35％(7/20)。除1只冻胚羔产下后90分钟死亡外,另6只冻胚羔生长发育正常。     

不同冷冻保护剂和投入液氮时温度对冷冻兔胚胎存活的影响

将384个 8～16细胞期的兔胚胎分成四个不同的试验组。用PBS+50％兔血清作为保存液,1.5M的DMSO或甘油作冷冻保护剂,以1℃/分的速度从13～17℃降到-40或-78℃投入液氮。用DMSO和甘油作冷冻保护剂,-40℃投入液氮的两组胚胎解冻后,经体外培养发育成胚泡的比率(分别为35.7％和32.8％)较-78℃投入液氮的两组(分别为22.9％和7.0％)为高。将-40℃投入液氮的两组胚胎解冻后,分别移植给5只受体母兔,受体妊娠率在DMSO组为80.0％,甘油组为60.0％;妊娠20天胎儿存活率分别为>44.9％和>34.0％;仔兔出生率分别为2.04％和22.6％。     

影响玻璃化冷冻兔胚胎效果的一些因素

试验对影响玻璃化冷冻兔胚胎效果的一些因素进行探讨，以找出理想的玻璃化冷冻方法。在测试的５种玻璃化溶液中，含３５％乙二醇（ＥＧ）和１．０ｍｏｌ／Ｌ蔗糖的溶液（ＶＳ１）对胚胎的毒性最小。用ＶＳ１冷冻桑椹胚和囊胚的理想程序是：在室温下使胚胎分别在２０％ＥＧ和３５％ＥＧ中平衡２、３分钟后，移入ＶＳ１中，０．５分钟内（囊胚也可在２分钟后）投入液氮中冷冻。桑椹胚的存活率为９１．７％（３３／３６），囊胚的存活率为９７．１％（３３／３４）～９７．３％（３６／３７）。８～１６细胞胚胎的理想冷冻程序为：在室温下使胚胎在２０％ＥＧ、３５％ＥＧ中平衡２、３分钟，移入４℃的３７％ＥＧ＋１．０ｍｏｌ／Ｌ蔗糖溶液中平衡２分或１０分钟后冷冻，胚胎存活率分别为１００％（３７／３７）、８６．１％（３１／３６）。     

影响胚胎冷冻效果的因素分析

多数哺乳动物胚胎保存的基本方法为先将胚胎放入防冻液中平衡一段时间:装管后,以0.3～0.5℃/min的速度降至植冰温度,平衡一段时间,然后以0.3℃/min的降温速率降温至-35℃,平衡10 min后投入液氮中保存.     

马胚冷冻到低温的成活力

对马胚冷冻到0°、-10°、-15°和-20℃或贮存在20°,-5°和-196℃的存活力,通过非手术移植到母马的方法进行了测定。6日胚,7日胚和8日胚(排卵后6,7,8天)用非手术法采集,用 Dulbecco 氏缓冲磷酸盐液冲洗,用1.5M 二甲亚砜(DMSO)或1.0M 甘油在室温平衡,然后以0.5—1.0℃/分的速度冷却到-5℃,放入晶种后保持10分钟,再以0.12—0.27℃/分的速度冷冻到-70℃,然后投入液氮里。在贮存1～214天后,给胚加温,以1.4—10.3℃—/分速度从-70℃加温到-10℃,再以     

猪胚胎的OPS玻璃化冷冻

以广西巴马小型猪为供体,采用超数排卵技术,采集5~6日龄具有完整透明带的胚胎(囊胚/桑葚胚),采用二步法OPS(Open pulled straw)玻璃化冷冻技术进行保存,即胚胎首先在冷冻液1(TCM199 20?S 10%EG 10%DMSO)中平衡3 min,然后立即转入冷冻液2(TCM199 20?S 20%EG 20%DMSO 0.4 mol/LSUC)中并在1 min内装管(每管含2~6枚胚胎),直接投入液氮保存;3个月后解冻移植给8头受体母猪(每头移入25~26枚胚胎),其中1头怀孕产仔(8头活仔),获得猪胚胎超低温(-196℃)冷冻后代。     

不同浓度防冻剂和投液氮温度对牛体外受精胚胎冷冻效果的影响

比较了3种不同浓度的甘油（1.4,1.0,0.7mol/L）和投液氮温度（-25℃、-30℃和-35℃）对牛体外受精胚胎冷冻效果的影响。结果发现,最终投液氮温度为-25℃时,胚胎冻后存活率以1.4mol/L甘油组为最高,同1.0mol/L甘油组相比,差异显著（78.5%vs 64.5%,P〈0.05）;与0.7mol/L甘油组相比,差异极显著（78.5%vs53.5%,P〈0.01）。以1.0mol/L的甘油冷冻,-30℃投液氮的效果优于-25℃的,它们之间冻后胚胎的孵化率差异显著（77.1%vs 64.5%,P〈0.05）。用0.7mol/L甘油冷冻,投液氮温度以-30℃的为最好,冻后胚胎的孵化率（73.3%）极显著（P〈0.01）高于-25℃的（53.5%）。用1.4mol/L甘油冷冻,以-25℃投液氮为最好,冻后胚胎的孵化率（78.5%）显著（P〈0.05）高于-35℃的（65.7%）。结果表明,不同的甘油浓度和投液氮温度对牛体外受精胚胎的冷冻效果有显著的影响。     

影响胚胎冷冻效果因素分析

大多数哺乳动物胚胎保存的基本方法为：先将胚胎放人防冻液中平衡一段时间：装管后,以1 min0．3～0．5℃的速度降至植冰温度,平衡一段时间,植冰后再平衡一段时间,然后以1 min 0．3℃的降温速率降温至-35℃,平衡10min后投入液氮中保存。     

猪冷冻（—20℃）胚胎在我国移植成功

实验Ⅰ将129枚猪胚胎,用于冷冻-解冻-体外培养研究。以含有1.5M甘油的PBS液为冷冻液,以1℃/分的速率从35℃降至-7℃,在-7℃自动诱发给冰,然后以0.3℃/分的速率降至-35℃,再以0.1℃/分的速率降至-36℃,立即投入液氮(-196℃);胚胎解冻在37℃水浴中进行、分步脱出甘油后以改进的mBMOC-Ⅱ为培养液进行体外培养。结果,囊胚、扩张囊胚、刚孵出囊胚和孵化出后期囊胚、解冻后体外培养存活率分别为0％(0 /2)、64％(16/25)、0％(0/9)。在液氮中(-196℃)保存80～103天,扩张囊胚、刚孵出囊胚和孵出后期囊胚解冻后,体外培养存活率分别为41.6％(5/12)、63.7％(7/11)和0％(0/70)。结果表明,在液氮温度(-196℃)保存下,刚孵出囊胚有较高的耐冻性和存活能力。实验Ⅱ进行猪冷冻胚胎解冻后体内发育能力研究。96枚扩张囊胚和刚孵出囊胚,分批冷冻和保存在-20℃、-35℃和-196℃。解冻后移植3头受体,其中2头返情,1头妊娠并于1990年7月31日产仔2头,公母各1头,初生重均为1.35kg,妊娠期为116夭。该结果为我国首例猪冷冻(-20℃)胚胎移植成功,并为今后开展猪胚胎超低温(-196℃)冷冻保存技术研究奠定了基础。     

奶牛胚胎体外保存时间和温度对移植受胎率的影响

试验对胚胎在体外保存时间的长短及温度与移植妊娠率的关系进行统计分析，胚胎收集后分别在0-3h,3-6h和6h后冷冻，解冻后移植受胎率分别为46.07%,43.33%,30.56%,0-3h和3-6h冷冻的胚胎，移植受胎率没有明显差异（P〉0。05），6h后的冻胚与6h以前的冻胚移植受胎率差异显著（P〈0．05）。胚胎分别在室温和37℃保存5h后冷冻，解冻后移植受胎率分别为39．74％、42．24％（P〉0．05）。     

牛体外受精胚胎一步脱防冻剂冷冻方法的研究

为了研究适合于牛体外受精胚胎的一步除防冻剂的冷冻方法，特进行两个试验。试验１分别用１．５Ｍ甘油＋０．２５Ｍ蔗糖、１．５Ｍ乙二醇和１．５Ｍ丙二醇作防冻剂冷冻体外受精后第７天，已发育至囊胚阶段的牛胚胎。使胚胎降温至－７℃后，植冰，然后以０．３℃／分的速率降温至－３０℃，立即将胚胎投入液氮中冷冻保存。在３７℃水中解冻胚胎后，使其直接在含１５％胎犊血清（ＦＣＳ）的磷酸缓冲液（ＰＢＳ）中脱去防冻剂。经体外培养７２ｈ后，３组胚胎的孵化率分别为７７．２７％（１０２／１３２）、７３．２４％（１０４／１４２）、４７．９０％（５７／１１９），第１、２组的孵化率极显著高于第３组（Ｐ＜０．０１），说明用１．５Ｍ丙二醇溶液冷冻的胚胎，在解冻后不宜直接在ＰＢＳ中脱防冻剂。试验２比较用不同浓度（１．０Ｍ０１．５Ｍ，２．０Ｍ）的乙二醇和不同投液氮温度（－２５℃，－３０℃，－３５℃）冷冻胚胎的效果。结果表明乙二醇浓度和投液氮温度对胚胎孵化率均无显著影响（Ｐ＞０．０５），各组胚胎的孵化率变动于７４．４４％－８５．４８％之间。     

Technical note: viability and motility of vitrified/thawed primordial germ cell isolated from common carp (Cyprinus carpio) somite embryos

The feasibility of cryopreserving common carp (Cyprinus carpio) primordial germ cells (PGC) by vitrification of whole embryos at the 22- to 28-somite stage was investigated. Green fluorescent protein (GFP)-labeled PGC were cooled rapidly using liquid nitrogen after exposure to a pretreatment solution containing 1.5 M cryoprotectant (ethylene glycol or dimethyl sulfoxide, 30 or 50 min) and a vitrification solution containing 3 M cryoprotectant and 0.5 M sucrose (5, 10, 20, or 30 min). Embryonic cells that were pretreated for 30 min and vitrified for 20 min with ethylene glycol had the greatest rate of survival of embryonic cells (68.6%; P < 0.01), an optimal highest percentage of viable PGC (73.8 to 74.9%; P < 0.05), and no evidence of ice formation after thawing. The vitrified/thawed PGC were transplanted into blastula-stage embryos from goldfish (Carassius auratus). The PGC maintained their motility and moved to the gonadal ridge of the host embryo. Thus, the combination of vitrification and transplantation to produce germ-line chimeras is a powerful tool for the artificial production of next-generation offspring.     

Use of a rapid method of cryopreservation of bovine embryos for non-surgical transfer in transplantation practice

The embryos were frozen and thawed in Cassou minipaillette by a rapid method. Embryos with cryoprotective agent (glycerol, 1.5 M) were placed directly into the freezing medium at the temperature of -6 to -7 degrees C, frozen after seedling at the temperature decrease by 0.3 to 0.5 degrees C per minute to the temperature of -32 degrees C and then transferred directly into liquid nitrogen. They were thawed in a bath warm 20 to 37 degrees C. After thawed the cryoprotective agent was evacuated in 1.1 M sucrose. The best-quality embryos were selected for freezing. Out of these 366 thawed so far, with average survival of 74.31%. The total of the 268 thawed embryos were transferred ipsilaterally, by a non-surgical method, to 190 synchronised heifers, out of which 105 (55.26%) got in calf. Rapid freezing method based on 1.5 M of glycerol and thawing at the presence of 1.1 M sucrose proved effective and suitable for practice, as not only sufficient reviviscence of embryos and their survival in womb are guaranteed, but also a substantial shortening of the freezing as well as thawing process.     

Effect of slow cooling end point temperature on survival of frozen bovine embryos

One hundred sixty-two embryos were collected from superovulated crossbred beef cattle 7 to 8 d after the onset of estrus. Embryos were frozen in modified Dulbecco's phosphate-buffered saline supplemented with 20% heat-inactivated fetal calf serum (PBS + FCS) and dimethylsulfoxide (DMSO), which was added in three steps to a final concentration of 1.5 M. Embryos were placed in .25 ml of 1.5 M DMSO in PBS + FCS in 1-ml glass ampules and cooled at 1.0 C/min from ambient temperature to -7 C, seeded and then cooled at .3 C/min to -19, -26, -33, -38, -43, -50 or -57 C before immersion (plunging) in liquid nitrogen. Ampules were thawed in 25 C water, and DMSO was removed in six steps at .25 M increments. 10 min/step. After removal of DMSO, embryos were cultured 24 h in PBS + FCS and then fixed and stained. Just after thawing, embryos for which slow cooling was terminated at -50 C were of lower (P less than .05) morphological quality than other groups. After removal of cryoprotectant, embryos from both the -19 and -50 C treatments had deteriorated more (P less than .05) than had embryos from other treatments. After 24-h culture, embryos slow-cooled to -19, -26 and -50 C had a lower rate of survival (P less than .05) than did embryos from -33, -38, -43 and -57 C temperatures. Embryos slow-cooled to -33, -38 and -43 C showed a higher percentage of healthy nuclei than did embryos slow-cooled to -19, -26 and -50 C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions between straw size and thawing rates on the cryopreservation of agouti (Dasyprocta aguti) epididymal sperm

This study verifies the interactions between straw size and thawing rates and their impact on the epididymal sperm from this species. Caudae epididymidum from 10 agoutis were subjected to retrograde washing using a coconut water extender (ACP‐109c^®). Epididymal sperm were evaluated and extended in ACP‐109c^® plus egg yolk (20%) and glycerol (6%). The samples were packaged in 0.25‐ or 0.50‐ml straws, frozen in liquid nitrogen and thawed at 37°C/1 min or 70°C/8 s, followed by a re‐evaluation. The use of 0.25‐ml straws thawed at 37°C/1 min provided a value of 26.6% for sperm motility. No interactions between straw size and thawing rates were verified on agouti sperm (p > 0.05), but when 0.5‐ml straws were thawed at 70°C/8 s, sperm vigour decreased significantly (p < 0.05). It is recommended that the agouti epididymal sperm cryopreserved in ACP‐109c^® extender should be packaged in 0.25‐ or 0.50‐ml straws and thawed at 37°C/60 s.     

Viability of OPS Vitrified Sheep Embryos After Direct Transfer

The aim of this study was to evaluate the viability in the effect of open pulled straw (OPS) vitrification procedure of sheep embryos after direct transference. Embryos were produced in vivo and cryopreserved in slow freezing or OPS vitrification. The survival rates of cryopreserved embryos were compared to non-frozen standard pattern. In a first set of experiments, embryos at morula and blastocyst stages were dived in ethylene glycol (1.5 M) and frozen in an automatic freezer. After being thawed, they were directly or indirectly transferred to ewes recipient. A second group of embryos were drawn into OPS and plunged into liquid nitrogen after being exposed at room temperature for 1 min and 45 s in 10% EG plus 10% dimethyl sulphoxide (DMSO), then again for 30 s in 20% EG + 20% DMSO + 0.5 M sucrose. After being warmed, embryos were also directly transferred using a French mini straw as the catheter for the transplantation process or after in vitro dilution of cryoprotectants (two-step-process). No significant difference was observed among fresh, frozen or vitrified embryos on pregnancy rate (50.0%, 38.6% and 55.8%). However, when we evaluated only the direct transference, the pregnancy rate of OPS vitrified embryos was higher than that of frozen embryos (57.1% vs 34.8%) (p = 0.07). In addition, vitrified morulae had a higher pregnancy rate than the one with frozen embryos (64.0% vs 38.9%) (p = 0.07). Finally, our results indicate that OPS vitrification technique in association with direct transference improves the viability of sheep embryos with potential applications to field conditions.     

The effect of cooling to different subzero temperatures on dog sperm cryosurvival

The objective was to assess the effect of cooling to different subzero temperatures around ice formation (?5°C) on dog sperm cryosurvival and plasma membrane fluidity. Semen was centrifuged, and sperm were resuspended in a Tris‐egg yolk medium (3% glycerol). Diluted sperm were cooled from 22 to 5°C, and then, a Tris‐egg yolk medium containing 7% glycerol was added (final concentration of 5% glycerol and 200 × 10⁶ cells/ml). Sperm were packaged in 0.5‐ml plastic straws, and equilibration was done 16 hr at 5°C before freezing. I. Straws (n = 47) at 5°C were exposed to nitrogen vapours to determine the freezing point. II. Other straws (from different ejaculates) processed as mentioned, were further cooled to ?3, ?5 or ?7°C and immediately rewarmed in a water bath at 37°C. Motility, plasma membrane functionality and acrosome integrity were assessed. III. Other straws (from different ejaculates) processed as mentioned were further cooled to ?3 or ?5°C, frozen over nitrogen vapours and stored in liquid nitrogen for one month. Straws were thawed in a water bath at 38°C for 30 s. Motility, plasma membrane functionality, plasma membrane integrity, acrosome integrity, capacitation status and plasma membrane fluidity were assessed. Ice nucleation temperature was ?14.3 ± 2.05°C (mean ± SD); cooling to +5, ?3, ?5 and ?7°C, without freezing, produces no differences on sperm quality between target temperatures; cooling to +5, ?3, and ?5°C produced no differences on sperm survival and plasma membrane fluidity after freeze–thawing. In conclusion, cooling of dog spermatozoa to different subzero temperatures did not improve sperm cryosurvival and had no effect on plasma membrane fluidity after thawing.     

In vitro Evaluation of in vivo Fertilizing Ability of Frozen Rabbit Semen

The present work studied different spermatozoa parameters and the ability of frozen rabbit spermatozoa to fertilize, in vitro, in vivo‐matured oocytes, as a test to predict their in vivo fertility and prolificacy. Semen from rabbit bucks was frozen using two freezing protocols [in a freezer at ?30°C or in liquid nitrogen vapour (LNV)]. For the in vivo trial, females were inseminated with frozen‐thawed spermatozoa. Oocytes used for in vitro testing were recovered 14 h after ovulation induction from donors and co‐incubated with 2 × 10⁶ frozen‐thawed spermatozoa during 4 h at 37°C in Tyrode's medium under an atmosphere of 5% CO₂ in air with maximal humidity. After co‐incubation period, presumptive zygotes were cultured in TCM199 supplemented with 20% foetal bovine serum (FBS), under the same conditions described above. Although no statistical differences were observed between freezing protocols in seminal parameters [motility rate: 40 and 35%, VCL: 35 and 46 μm/s, amplitude of lateral head displacement (ALH): 1.7 and 2.4 μm, for semen frozen at ?30°C and in LNV, respectively], significant differences were noted in the fertilizing ability in vivo and in vitro. Semen frozen at ?30°C showed the highest fertilizing ability in vitro (26.7% vs 6.2 and 8.7% for semen frozen at ?30°C, in LNV and fresh semen, respectively) and the lowest fertility rate in vivo (21.7% vs 64.2% and 70.6% for semen frozen at ?30°C, in LNV and fresh semen, respectively). Sperm frozen at ?30°C seemed to be more capacitated.     

Production of germ-line chimeras by the transfer of cryopreserved gonadal germ cells collected from 7- and 9-day-old chick embryos

Gonadal germ cells (GGC) were collected from the gonads of 7‐ or 9‐day‐old White Leghorn chick embryos and suspended in freezing medium containing 10% dimethylsulfoxide (DMSO). The cell suspension was frozen at ?1°C/min. until the temperature reached ?80°C. Then, the cells were immersed in liquid nitrogen at ?196°C and stored for 3–4 months. Approximately 50 frozen/thawed GGC were injected into the dorsal aorta of each 2‐day‐old Rhode Island Red (RIR) embryo, from which blood was drawn before germ‐cell injection. The injected embryos were incubated until they hatched and the chicks were raised until sexually mature. On reaching sexual maturity, a progeny test was performed by mating recipient chicks with normal RIR of the opposite sex. Progenies were obtained from male germ cell recipients that were injected with germ cells collected from 7‐ and 9‐day‐old embryos. The results demonstrated that frozen/thawed GGC collected from 7‐ or 9‐day‐old fertilized eggs can be used to produce male germ‐line chimeras.