Equilibrium vitrification of mouse embryos at various developmental stages using low concentrations of cryoprotectants

We previously developed a new vitrification method (equilibrium vitrification) by which two-cell mouse embryos can be vitrified in liquid nitrogen in a highly dehydrated/concentrated state using low concentrations of cryoprotectants. In the present study, we examined whether this method is effective for mouse embryos at multiple developmental stages. Four-cell embryos, eight-cell embryos, morulae, and blastocysts were vitrified with EDFS10/10a, 10% (v/v) ethylene glycol and 10% (v/v) DMSO in FSa solution. The FSa solution was PB1 medium containing 30% (w/v) Ficoll PM-70 plus 0.5 M sucrose. The state of dehydration/concentration was assessed by examining the survival of vitrified embryos after storage at –80°C. When four-cell embryos and eight-cell embryos were vitrified with EDFS10/10a in liquid nitrogen and then stored at –80°C, the survival rate was high, even after 28 days, with relatively high developmental ability. On the other hand, the survival of morulae and blastocysts vitrified in liquid nitrogen and stored at –80°C for four days was low. Therefore, morulae and blastocysts cannot be vitrified in a highly dehydrated/concentrated state using the same method as with two-cell embryos. However, when blastocysts were shrunken artificially before vitrification, survival was high after storage at –80°C for four days with high developmental ability. In conclusion, the equilibrium vitrification method using low concentrations of cryoprotectants, which is effective for two-cell mouse embryos, is also useful for embryos at multiple stages. This method enables the convenient transportation of vitrified embryos using dry ice.     

Transfer of canine embryos at various developmental stages recovered by hysterectomy or surgical uterine flushing

In dogs, embryo transfer (ET) techniques such as induciton of excessive ovulation and synchronization of estrus have not progressed well. Therefore, using embryos at various developmental stages, ET was investigated in dogs from a beagle colony in which the ovulation days were close, as estimated by the progesterone level. Embryos were, recovered 8-11 days after ovulation (4-9 days after mating) by excising the oviducts and uteri (excision method) in 16 animals and by surgical flushing of the uteri at laparotomy (surgical method) in 3 animals. In 24 dogs with -4 to +2 days of difference in the timing of ovulation between donor and recipient dogs, 1-10 embryos at the 8-cell to blastocyst stages were transferred per animal. The mean embryo recovery rate by the excision method (97.1%) was significantly higher than that by the surgical method (42.5%) (p<0.01). Twelve (57.1%) of 21 animals with -1 to +2 days difference in ovulation day became pregnant after the transfer of 8-cell to blastocyst stage embryos. Although 3 dogs with -4 to -2 days of difference of ovulation day underwent ET of morula or compacted morula, none of these dogs became pregnant. The mean ratio of the number of newborns to the number of transferred embryos was only 51.9%. The mean duration of the period between ovulation and delivery in the pregnant recipients was 65.8 days, which tended to be longer than that in natural mating. These results demonstrate that pregnancy can be induced by ET at the 8-cell to blastocyst stage in dogs with -1 to +2 days difference in ovulation day.     

Open-pulled straw (OPS) vitrification of in vitro fertilised mouse embryos at various stages

The present study was designed to investigate the cryotolerance of in vitro fertilised (IVF) mouse embryos at various preimplantation developmental stages. IVF mouse embryos were vitrified by the open-pulled straw (OPS) method. After warming, embryos were morphologically evaluated and assessed by their development to blastocysts, hatched blastocysts or term. The results showed that a high proportion (93.3-100.0%) of vitrified embryos at all developmental stages were morphologically normal after recovery. The developmental rate of vitrified 1-cell embryos to blastocyst (40.0%) or hatched blastocyst (32.7%) or term (9.3%) was significantly lower than that from other stages (P < 0.05). Vitrified embryos from 2-cell to early blastocyst stage showed similar blastocyst (71.8-89.5%) and hatched blastocyst rates (61.1-69.6%) and could develop to term without a significant loss of survival compared with those of fresh embryos (P > 0.05). Vitrified 2-cell embryos showed the highest survival rate in vivo (50.6%, 88/174), compared with that from other stages (9.3-30.5%, P < 0.05). The data demonstrate that the OPS method is suitable for the cryopreservation of IVF mouse embryos from 2-cell stage to early blastocyst stage without a significant loss of survival. Embryos at the 2-cell stage had the best tolerance for cryopreservation in the present study.     

Effects of various cryoprotectants on the survival of mouse embryos cryopreserved by the quick freezing method

The effects of concentrations of glycerol, ethylene glycol or dimethylsulphoxide (DMSO) in the presence of either 0.25 M lactose or sucrose on the post-thaw survival of mouse quickly-frozen compacted morulae were studied. In this method, the embryos were directly frozen in liquid nitrogen (LN2) vapor at approximately -170 degrees C for 2 min before being plunged into LN2. High survival rates of frozen-thawed embryos were obtained when the freezing medium contained 3 M ethylene glycol with either 0.25 M lactose or sucrose (76.5 and 70.2%, respectively). When the embryos were frozen in glycerol, significantly high survival was obtained with 3 M glycerol + 0.25 M sucrose (73.5%, P less than 0.001). However, a freezing medium containing DMSO with either sugar gave lower survival rates. At a higher concentration of 4 M, ethylene glycol with 0.25 M lactose gave significantly higher survival rate than glycerol or DMSO (P less than 0.05). Significantly higher rates were obtained at 2 M with all 3 cryoprotectants when the freezing medium contained lactose rather than sucrose (P less than 0.05). This study showed that glycerol and ethylene glycol were effective cryoprotectants in the quick freezing of mouse embryos, while DMSO was less effective. In addition, the protective effects of these cryoprotectants are affected by their concentrations and the type of sugar used.     

冷冻方法和冷冻保护液对山羊卵母细胞发育的影响

比较了不同冷冻方法和冷冻保护液对山羊卵母细胞冷冻-解冻后体外受精和激活后的发育效果.结果表明,开放式拉长塑料细管(OPS)法的效果好于玻璃化细管法和常规冷冻法;在OPS法中,20%EG 20%DMSO对卵母细胞的冷冻保护效果好于EDFS40,而在玻璃化细管法冷冻中,则是EDFS40好于20%EG 20%DMSO.对于常规冷冻法而言,则是1.5mol/L EG的冷冻效果好于1.5mol/L PROH.     

High developmental rates of mouse oocytes cryopreserved by an optimized vitrification protocol: the effects of cryoprotectants, calcium and cumulus cells

Unfertilized oocytes are one of the most desired germ cell stages for cryopreservation because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, in general, the fertility and developmental ability of cryopreserved oocytes are still low. The aim of the present study was to improve vitrification of mouse oocytes. First, the effects of calcium and cryoprotectants, dimethyl sulfoxide and ethylene glycol (EG), in vitrification medium on survival and developmental ability of vitrified oocytes were evaluated. Oocytes were vitrified by a minimal volume cooling procedure using different cryoprotectants. Most of the vitrified oocytes were morphologically normal after warming, but their fertility and development were low independently of calcium and cryoprotectants. Second, the effect of cumulus cells on ability of oocytes to be fertilized and develop in vitro was examined. The fertility and developmental ability of denuded oocytes (DOs) after IVF were reduced compared with cumulus-oocyte complexes (COCs) both in fresh and cryopreserved groups. Vitrified COCs showed significantly (P<0.05) higher fertility and ability to develop to the 2-cell and blastocyst stages than those of vitrified DOs with cumulus cells and vitrified DOs alone. The vitrified COCs developed to term at a high success rate equivalent to the rate obtained with IVF using fresh COCs. Taken together, the current results clearly demonstrate that, in the presence of surrounding cumulus cells, matured mouse oocytes vitrified using calcium-free media and EG retain their developmental competence. These findings will contribute to improve oocyte vitrification in not only experimental animals but also clinical application for human infertility.     

Survival and developmental competence of bovine embryos at different developmental stages and separated blastomeres after vitrification in different solutions

Generating techniques to enhance the success of blastomere separation is important for bovine economy, because it increases the number of transferable embryos. This study aimed to identify the optimum cryoprotectants for the vitrification of bovine embryos and the separation of blastomeres at different stages. In experiment 1, expanded blastocysts were vitrified in two different vitrification solutions, either (1) ethylene glycol (EG) + propylene glycol (PG) or (2) EG. The survival rate of blastocysts in the EG + PG was higher than that of the EG. In experiment 2, intact two‐cell and eight‐cell stage embryos were vitrified in the same solutions used in experiment 1. The EG + PG produced more dead embryos than the EG (P < 0.05). In the EG, the rate of blastocyst formation was similar for the vitrified two‐ and eight‐cell embryos and the non‐vitrified ywo‐cell embryos. In experiment 3, separated blastomeres of two‐ and eight‐cell embryos were vitrified in EG. There was no difference in the rate of blastocyst formation and total number of cells between the two vitrified groups. In summary, at the blastocyst stage, EG + PG was superior, based on both survival rates and cell numbers; however, at the 2–8 cell stage, the use of EG alone was better than the other groups.     

Wistar大鼠超数排卵及采集不同时期胚胎的初步研究

为了研究获取Wistar大鼠不同发育阶段胚胎所需超排试剂的最佳剂量以及不同发育期胚胎的采集方法,试验采用10 IU/只、20 IU/只和30 IU/只3种剂量的孕马血清促性腺激素-人绒毛膜促性腺激素(PMSG-HCG)对8～9周龄的Wistar大鼠进行超排处理。结果表明:采集2-细胞期胚胎时,分别获得胚胎(12.1±4.6)枚、(23.1±10.2)枚和(24.9±8.2)枚,其中激素剂量为30 IU/只时采集2-细胞期胚胎超排效果最好;在采集桑葚胚期胚胎时,分别可获得胚胎(10.3±5.0)枚、(23.7±17.2)枚和(17.2±5.9)枚,其中激素剂量为20 IU/只的超排效果最好。     

Effect of vitrification at different meiotic stages on epigenetic characteristics of bovine oocytes and subsequently developing embryos

Vitrification by the Cryotop method is frequently used for bovine oocyte cryopreservation. Nevertheless, vitrified oocytes still have reduced developmental competency compared with fresh counterparts. The objective of this study was to compare the effect of vitrification either at the germinal vesicle (GV) stage or at the metaphase II (MII) stage on epigenetic characteristics of bovine oocytes and subsequently developing embryos. Our results demonstrated that vitrification of oocytes at each meiotic stage significantly reduced blastocyst development after in vitro fertilization (IVF). However, vitrification at the GV stage resulted in higher blastocyst development than did vitrification at the MII stage. Irrespective of the meiotic stage, oocyte vitrification did not affect 5-methylcytosine (5mC) immunostaining intensity in oocyte DNA. However, at both stages, it caused a similar reduction of 5mC levels in DNA of subsequently developing blastocysts. Oocyte vitrification had no effect on the intensity of H3K9me3 and acH3K9 immunostaining in oocytes and subsequent blastocysts. The results suggest that irrespective of meiotic stage, oocyte vitrification alters global methylation in resultant embryos although such alteration in the oocytes was not detected. Oocyte vitrification might not influence histone acetylation and methylation in oocytes and resultant embryos. Vitrification at the immature stage was more advantageous for blastocyst development than at the mature stage.     

Cytogenetic analysis and developmental assessment of mouse embryos derived from in vitro fertilization of oocytes reconstructed by meiosis-II chromosome transplantation

An electrofusion methodology for transferring meiosis-II chromosomes (M-II-t) has not been completely established. The present study compared the use of two temperatures (fusion at 37 C for Group A and 25 C for Group B) during an electrofusion procedure for mouse oocyte M-II-t and investigated the cytogenetic normality and developmental competence of embryos derived from in vitro fertilization using oocytes reconstructed by M-II-t. The M-II-t oocytes were fertilized in vitro and cultured to the blastocyst stage; the resultant embryos were analyzed cytogenetically. Subsequently, chromosomal normality of the resultant embryos at the prometaphase stage of first cleavage division and the integrity of the meiosis-II spindles of the reconstructed oocytes were analyzed. The success rate of electrofusion in Group B was 92.1%, which was significantly different from that in Group A (49.2%) (P<0.05). The fertilization rates (A, 80.7%; B, 77.2%) and development rates (A, 70.9%; B, 65.5%) in the M-II-t groups were significantly lower than those in the control group (95.0 and 92.2%, respectively) (P<0.05). The incidence of chromosomal abnormalities in the Group A embryos (20.5%) at the blastocyst stage was significantly higher than that in the control group embryos (8.5%) (P<0.05), but the incidence of chromosomal abnormalities in Group B (12.5%) was not significantly different compared with the other groups. A temperature of 25 C during the electrofusion procedure for M-II-t resulted in a good fusion rate, good development rate, and efficient production of chromosomally normal blastocysts. Furthermore, the incidence of chromosomal abnormalities in the first cleavage embryos at the prometaphase stage in Group B (9.6%) did not differ significantly from that in the control group (6.6%). The spindle morphology of the M-II-t oocytes in Group B was normal.     

小鼠卵母细胞及体细胞核移植重构早期端粒酶活性的测定

采用端粒酶重复序列扩增(telomeric repeat amplification protocol,TRAP)-银染法,对小鼠卵母细胞、体内受精胚胎及重构胚的各阶段端粒酶活性进行测定,对PCR产物的电泳条带进行灰度值分析,计算端粒酶总产品总量。结果表明:(1)体内受精胚胎及核移植重构胚各阶段的端粒酶活性随胚胎分裂呈上升趋势,囊胚期最高(P0.05);(2)体内受精胚胎的端粒酶活性高于体细胞核移植重构胚端粒酶活性。同时进行囊胚计数并测定单卵裂球相对端粒酶活性,结果表明:单卵裂球的端粒酶活性呈下降趋势,囊胚期端粒酶活性最低(P0.05)。以上结果说明小鼠卵母细胞及体细胞核移植重构胚端粒酶活性变化与细胞发育水平及细胞发育全能性有关。     

玻璃化冷冻对猪MII期卵母细胞发育能力的影响

以MII期卵母细胞作为试验材料,应用玻璃化冷冻方法,就目前应用最多的7组冷冻保护液配方进行筛选,并且比较3种不同冷冻载体冷冻卵母细胞的效果。结果发现:7组冷冻保护液对MII期卵母细胞有低毒性作用,各组分裂率、囊胚率、囊胚细胞数均低于对照组;综合各组指标,选取3组对猪MII期卵母细胞的发育能力损伤最小的冷冻保护剂,应用GMP管对其冷冻保护效果进行比较,发现第7组冷冻保护液配方EFS不适于MII期卵母细胞的冷冻,其形态正常率、分裂率分别为1.19%、0,与第3组、第6组形态正常率、分裂率(59.48%、53.55%;24.19%、35.02%)差异显著(P0.05)。综合考虑,选取HM+7.5%(DMSO+EG),HM+17%(DMSO+EG)+0.4 mol/L Su作为冷冻保护剂来比较3种不同冷冻载体冷冻卵母细胞的效果差异,发现半麦管冻融后MII期卵母细胞的分裂率高达53.67%,与OPS法的29.33%及GMP法的35.00%相比差异显著(P0.05),半麦管法更适合MII期猪卵母细胞的玻璃化冷冻。     

Histone deacetylase 1 down-regulation on developmental capability and histone acetylation in bovine oocytes and parthenogenetic embryos

Histone deacetylase 1 (HDAC1) is one of the most conserved enzymes present in the nuclei of cells, including bovine oocytes and pre-implantation embryos. However, the biological functions of HDAC1 in supporting the growth and development of bovine oocytes and embryos are still not fully elucidates. In this study, three siRNAs (si299, si672, and si1272) targeting to HDAC1 mRNA sequence were designed. After transfection into bovine fibroblast cells, si299, the most effective one in HDAC1 knock-down, was selected. The selected siRNA was microinjected into bovine germinal vesicle (GV) stage oocytes to determine the functions of HDAC1 in the maturation of bovine oocytes. Finally, the siRNA was microinjected into mature oocytes, which were then parthenogenetically activated and cultured in vitro until the blastocyst stage. The rates of cleavage, blastocyst development and acetylation of lysine 14 of H3 (H3K14) state were checked. The results suggest that HDAC1 knock-down in oocytes did not influence the rates of maturation or cleavage of parthenogenetic embryos. However, the rates of blastocyst decreased after siRNA microinjection. Furthermore, histone H3K14 acetylation level increased after siRNA microinjection into parthenogenetic embryos.     

In vitro developmental potential of nuclear transfer embryos cloned with enucleation methods using pre-denuded bovine oocytes

Enucleation of a recipient oocyte is an important essential process in the procedure of somatic cell nuclear transfer (SCNT). The present study investigated a method for the improvement of enucleation efficiency. Oocytes were denuded of cumulus cells before the completion of nuclear maturation (pre-denuded) after 12 h of culture at MI stage and subsequently cultured for additional 6 h until the completion of nuclear maturation and extrusion of the first polar body (PB1). The extrusion rate of PB1 was not significantly different in the pre-denuded oocyte group, compared with control oocyte group matured for 18 h. However, the number of oocytes showing the metaphase II (MII) located just underneath the PB1 was significantly higher (p<0.05) in the pre-denuded oocyte group than those in control oocyte group. To test the effect of pre-denuding on the enucleation rate and developmental potential of embryos to blastocyst stage, subsequent somatic cell nuclear transfer comparisons were made with three different methods of enucleation at MII stage using vital dyes (demicoline and Hoescht) or the PB1 (blind enucleation) to localize the chromosome plate. Enucleation rate of the oocytes with demicoline, Hoechst and pre-denuding enucleation groups were significantly higher (p<0.05) than those of blind enucleation groups. However, cleavage rate to two-cell stage and, developmental rate to blastocyst and hatched blastocyst stage, the mean numbers of total and ICM cells in the SCNT embryos with Hoechst enucleation groups were significantly decreased (p<0.05), compared to those of blind, demicoline and pre-denuding enucleation groups. Moreover, the level of telomerase activity was also significantly (p<0.05) decreased in SCNT blastocysts of Hoechst enucleation group, compared to those of blind, demicoline and pre-denuding enucleation groups. Taken together, pre-denuding enucleation group using pre-denuded oocytes was a useful and simple enucleation method for bovine SCNT embryos.     

Uptake of metronidazole in Artemia at different developmental life stages

The purpose of this study was to examine the capacity of live brine shrimp Artemia spp. to accumulate metronidazole at different developmental life stages. Metronidazole is used in fish as an antiparasitic medication. An effective drug delivery method is to enrich the Artemia with metronidazole and offer them as live feed to the infected fish, usually ornamental species and other small fishes. Artemia cysts were hatched and then soaked in a metronidazole solution (0.05%) at instars 1-3 of larval development. Our findings indicated that Artemia were able to accumulate metronidazole at levels considered therapeutic to other animals and humans (25-100 mg/kg). However, the levels varied depending on the stage of larval development. Artemia accumulated the highest levels of metronidazole (137-143 mg/kg) when they started filter feeding (instar 2), whereas newly hatched Artemia (instar 1) contained the lowest level (85 mg/kg). Based on this study and a review of the literature, a new protocol recommended for enriching Artemia with metronidazole consists of soaking the Artemia in a 0.05% metronidazole solution for 3 h at room temperature. Because metronidazole is relatively insoluble in water, it must first be dissolved in warm water with continuous stirring.     

不同发育时期旋毛虫脂肪酸组成的研究

为探讨旋毛虫生长发育过程中脂肪酸组分的变化及外界环境对其影响,本研究采用贝尔曼氏装置分别收集旋毛虫肌幼虫和成虫,同时,将少量骨骼肌和肠黏膜进行酯化处理,通过气相色谱质谱联用技术进行分析。结果显示:肌幼虫和成虫的脂肪酸组分涵盖了C12～C22的22种,主要为16、18和20碳脂肪酸,但旋毛虫肌幼虫与成虫的各脂肪酸相对含量有明显差异(p0.05)。分别对旋毛虫肌幼虫和成虫及其各自寄生部位骨骼肌和肠黏膜中的脂肪酸成份进行比较分析,结果显示:各脂肪酸组成相似,但在相对含量上却有显著差异(p0.05)。表明不同发育时期旋毛虫的脂肪酸组成在种类和相对含量上均存在明显差异,这可能与其所处的不同宿主环境和不同发育时期虫体的特殊生理结构有关。