Transporting bovine oocytes in a medium supplemented with different macromolecules and antioxidants: Effects on nuclear and cytoplasmic maturation and embryonic development in vitro

We investigated whether supplementing the medium used to transport bovine oocytes with different macromolecules [foetal calf serum (FCS) or bovine serum albumin (BSA)] or a mixture of antioxidants (cysteine, cysteamine and catalase) affects their nuclear and cytoplasmic maturation and thereby affects their subsequent embryonic development and cryotolerance. Oocytes were transported for 6 hr in a portable incubator and then subjected to standard in vitro maturation (IVM) for 18 hr. The oocytes in the control groups were cultured (standard IVM) for 24 hr in medium containing 10% FCS (Control FCS) or 10% FCS and the antioxidant mixture (Control FCS+Antiox). The intracellular concentrations of reactive oxygen species (ROS) at the end of IVM period were lower in the oocytes subjected to simulated transport in the presence of a macromolecular supplement or the antioxidant mixture than that of the control group (FCS: 0.62 and BSA: 0.66 vs. Control FCS: 1.00, p < .05; and Transp: 0.58 and Transp Antiox: 0.70 vs. Control FCS: 1.00, p < .05). After IVM, the mitochondrial membrane potentials of the transported oocytes were lower than those of the non‐transported oocytes (FCS: 0.41 and BSA: 0.57 vs. Control FCS: 1.00, p < .05; and Transp: 0.48 and Transp Antiox: 0.51 vs. Control FCS: 1.00 and Control Antiox: 0.84, p < .05). The blastocyst formation rates (36.9% average) and the re‐expansion rates of vitrified‐warmed blastocysts (53%, average) were unaffected (p > .05) by the treatments. In conclusion, supplementing the medium in which bovine oocytes are transported with antioxidants or different macromolecules did not affect their in vitro production of embryos or their cryotolerance.     

The effect of resveratrol on the developmental competence of porcine oocytes vitrified at germinal vesicle stage

We tested the effects of resveratrol both as a pre‐treatment and as a recovery treatment after warming during in vitro maturation (IVM) on the viability and developmental competence of porcine oocytes vitrified at the germinal vesicle stage. Pre‐treatment before vitrification of oocytes for 3 hr with 2 μM resveratrol did not affect survival, oocyte maturation and embryo developmental competence to the blastocyst stage after parthenogenetic activation. However, supplementation of the medium with resveratrol during subsequent IVM after vitrification and warming significantly improved the ability of surviving oocytes to develop to the blastocyst stage, and this effect was observed only on vitrified, but not on non‐vitrified oocytes. The intracellular levels of glutathione and hydrogen peroxide in oocytes were not affected by vitrification and resveratrol treatment. Also, there was no significant difference in the occurrence of apoptosis measured by annexin V binding between vitrified and non‐vitrified oocytes, regardless of the resveratrol treatment. In conclusion, resveratrol did not prevent the cellular damages in immature porcine oocytes during vitrification; however, when added to the IVM medium, it specifically improved the developmental competence of vitrified oocytes. Further research will be necessary to clarify the mechanisms of action of resveratrol on the recovery of vitrified oocytes from vitrification‐related damages.     

Developmental competence of in vitro matured ovine oocytes vitrified in solutions with different concentrations of trehalose

This study aimed to determine the optimum concentration of trehalose in solutions used for vitrification of in vitro matured (IVM) ovine oocytes. IVM oocytes were randomly divided into four experimental (vitrified) and one control (fresh) groups. Experimental groups were treated with different concentrations (0.0, 0.25, 0.5 and 1.0 M) of trehalose. After warming, some viable oocytes were exposed to 0.25% pronase to test zona pellucida hardening, whereas the others were fertilized and cultured in vitro for 8 days to evaluate their developmental competence. Blastocysts quality was assessed by differential staining and TUNEL test. Survival and developmental rates of oocytes vitrified in the presence of 0.5 M trehalose were significantly higher than those of the other vitrified groups. Furthermore, there was a significant difference between fresh and vitrified groups in total blastocyst rate. Analysis of blastocysts quality also revealed a significant difference between the group treated with 0.5 M trehalose and other groups in terms of apoptotic index. Furthermore,zona pellucida digestion time period was longer in trehalose‐free (0.0 M) group compared to other groups. In conclusion, we found that IVM ovine oocytes vitrified in solutions containing 0.5 M trehalose are fertilization‐competent and are able to produce good‐quality blastocysts with an apoptotic index comparable to that of the fresh oocytes. Therefore, 0.5 M may be considered the optimum concentration of trehalose to be used in solutions prepared for vitrification of oocytes.     

Post‐Warming Competence of In Vivo Matured Rabbit Oocytes Treated with Cytoskeletal Stabilization (Taxol) and Cytoskeletal Relaxant (Cytochalasin B) Before Vitrification

The aim of this study was to investigate the effect of Taxol and Cytochalasin B on the spindle, chromosome configuration and development to blastocyst stage after parthenogenesis activation of in vivo matured rabbit oocytes after vitrification. Oocytes were randomized into four groups: oocytes treated with Cytochalasin B or Taxol before vitrification, oocytes without treatment before vitrification and fresh oocytes. Oocytes were vitrified using Cryotop method, and meiotic spindle and chromosomal distribution were assessed with a confocal laser scanning microscopy. To determine oocyte competence, in vitro development of oocytes was assessed with parthenogenesis activation. There were no significant differences in the frequencies of normal spindle (33.0%, 31.0% and 32.6%, for non‐treated, Taxol‐treated and Cytochalasin B‐treated oocytes, respectively) and chromosome (48.3%, 46.6% and 34.8%, for non‐treated, Taxol‐treated oocytes and Cytochalasin B‐treated oocytes respectively) in vitrified groups, but significantly lower than those of fresh group (89.7% and 90.2%, for normal spindle and chromosome organization, respectively). No statistical differences were found in the cleavage and blastocyst development rates between non‐treated and Taxol‐treated oocytes (7.7% and 1.5% and 13.7% and 4.6%, for non‐treated and Taxol‐treated oocytes, respectively), although they were significantly lower than in the fresh group (42.3% and 32.1%, for cleavage and blastocyst development, respectively). Oocytes treated with Cytochalasin B failed to reach blastocyst stage. Normal spindle, chromosome configuration and blastocyst development of in vivo matured rabbit oocytes were damaged in vitrification, which was not improved by Taxol and Cytochalasin B pre‐treatment before vitrification. Moreover, a detrimental effect on blastocyst development of Cytochalasin B pre‐treatment before vitrification was observed.     

Effect of vitrification procedures on the subsequent development of in vitro matured swamp buffalo oocytes following in vitro fertilization

Nowadays, the efficiency of buffalo oocytes cryopreservation is still low. The purpose of this study was to evaluate effects of two combinations of cryoprotectant agents (CPAs) and two vitrification devices for vitrification of swamp buffalo oocytes on their survival after vitrification warming, and subsequent developmental ability after in vitro fertilization. In vitro matured (IVM) oocytes were vitrified by either Cryotop (CT) or solid surface vitrification (SSV) interacting with vitrification solution A (VA) or B (VB). In the VA or VB solution exposed test, the oocytes showed similar survival rates, but decreased blastocyst rates after in vitro fertilization compared with that of untreated oocytes. After vitrification, the CT method combined with VA solution yielded a higher survival rate (91.3 ± 5.84%) of vitrified oocytes than that combined with VB solution (69.8 ± 4.19%–75.8 ± 4.55%); however, all the vitrification treatments showed lower blastocyst rates (1.1 ± 0.07%–5.2 ± 0.24%) compared with that of untreated oocytes (18.0 ± 1.09%). Our results indicated that combined vitrification treatments in this study did not improve the decreased ability of vitrified oocytes developing to the blastocyst stage.     

Effects of polyethylene glycol and a synthetic ice blocker during vitrification of immature porcine oocytes on survival and subsequent embryo development

We evaluated the effects of polyethylene glycol (PEG) and Supercool X‐1000 (SC) as supplements during the vitrification of immature cumulus‐enclosed porcine oocytes in a solution based on 17.5% ethylene glycol + 17.5% propylene glycol. After warming, the oocytes were subjected to in vitro maturation, fertilization and embryo culture. In Experiment 1, equilibration and vitrification solutions were supplemented with or without 2% (w/v) PEG (PEG+ and PEG‐, respectively). The survival rate, cleavage and blastocyst development were similar between PEG+ and PEG‐ groups; however, all values were lower than those in the non‐vitrified control. In Experiment 2, vitrification solution was supplemented with or without 1% (v/v) SC (SC+ and SC‐, respectively). The percentages of survival and blastocyst development were similar between SC+ and SC‐ groups but lower than those in the non‐vitrified control. The percentage of cleavage in the SC‐ group was significantly lower than the control and the SC+ groups, which were in turn similar to one another. In both experiments, the cell numbers in blastocysts were not significantly different among the non‐vitrified and vitrified groups. In conclusion, PEG did not improve oocyte survival and embryo development, whereas SC improved the ability of surviving oocytes to cleave but not to develop into blastocysts.     

Calves Born after Direct Transfer of Vitrified Bovine In Vitro-produced Blastocysts Derived from Vitrified Immature Oocytes

Vitrification has been the method of choice for the cryopreservation of bovine oocytes, as rapid cooling decreases chilling sensitivity. The aim of this study was to determine the in vitro and in vivo survival and the viability of immature oocytes vitrified using super‐cooled liquid nitrogen. Immature oocytes were randomly allocated to three groups: (i) non‐vitrified control group, (ii) vitrified in normal (?196°C) liquid nitrogen (LN₂) and (iii) vitrified in super‐cooled LN₂ (≤?200°C). Open‐pulled glass micropipettes were used as vitrification containers. Immature oocytes were in vitro‐matured, fertilized and cultured to the blastocyst stage. In vitro viability was assessed by cleavage and blastocyst rates on days 2 and 7 of culture respectively. Vitrified blastocysts derived from the immature vitrified oocytes were directly transferred to synchronous recipients. The in vitro embryo development of vitrified immature oocytes was not influenced by the LN₂ state. After direct transfer (one embryo per recipient) of 16 embryos obtained from immature vitrified oocytes (eight from each vitrified group), two healthy calves were born in each group. These results indicated that vitrification of immature bovine oocytes using glass micropipettes under normal or super‐cooled LN₂, resulted in viable blastocysts and live calves following in vitro embryo production.     

Effects of fatty acid-free bovine serum albumin and fetal calf serum supplementing repair cultures on pre- and post-warm viability of biopsied bovine embryos produced in vitro

The objective of this study was to investigate the influence of fatty acid-free bovine serum albumin (BSA) or fetal calf serum (FCS) on the re-expansion of biopsied blastocysts and post-warm viability of subsequently vitrified embryos. Firstly, blastocysts produced in vitro were biopsied at Day 7 and cultured to allow repair in TCM199 with 0.3% BSA or 5% FCS for 24 h. The re-expansion rates and mean total numbers of cells of the re-expanded embryos after the repair culture with BSA were almost the same as that with FCS. Secondly, after biopsied embryos were similarly cultured for repair with BSA or FCS, re-expanded embryos were selected for vitrification. After warming and exposure to 0.5 M sucrose with 20% FCS in mPBS, the embryos were cultured in TCM199 with 5% FCS for 24 h. The re-expansion rate and mean total number of cells in re-expanded blastocysts in the BSA treatment group (97.4 +/- 2.9% and 106 +/- 42) was significantly higher than that in the FCS treatment group (51.6 +/- 9.1% and 61 +/- 38), respectively (P<0.05 and P<0.01). In conclusion, both FCS and BSA supplementation can be useful for repairing cultures of bovine biopsied blastocysts; but, compared with BSA supplementation, FCS supplementation during repair culture reduces the post-warm viability of biopsied and subsequently vitrified embryos.     

Cryopreservation of immature buffalo oocytes: Effects of cytochalasin B pretreatment on the efficiency of cryotop and solid surface vitrification methods

The aim of the present study was to compare the efficiency of the solid surface (SSV), cryotop (CT) vitrification methods and cytochalasin B (CB) pretreatment for cryopreservation of immature buffalo oocytes. Cumulus‐oocyte complexes (COCs) were placed for 1 min in TCM199 containing 10% dimethylsulfoxide (DMSO), 10% ethylene glycol (EG), and 20% fetal bovine serum, and then transferred for 30 s to base medium containing 20% DMSO, 20% EG and 0.5 mol/L sucrose. CB pretreated ((+)CB) or non‐pretreated ((?)CB) COCs were vitrified either by SSV or CT. Surviving vitrified COCs were selected for in vitro maturation (IVM) and in vitro fertilization (IVF). The rate of viable oocytes after vitrification in CT groups (82%) was significantly lower (P < 0.05) than that in a fresh control group (100%), but significantly higher (P < 0.05) than those in SSV groups (71–72%). Among vitrified groups, the highest maturation rate was obtained in the CT (?)CB group (32%). After IVF, the cleavage and blastocyst formation rates were similar among vitrified groups but significantly lower than those of the control group. In conclusion, a higher survival rate of oocytes after vitrification and IVM was obtained in the CT group compared with that in the SSV group, indicating the superiority of the CT method. Pretreatment with CB did not increase the viability, maturation or embryo development of vitrified oocytes.     

Comparison of Ethylene Glycol and Propylene Glycol for the Vitrification of Immature Porcine Oocytes

Our aim was to optimize a cryoprotectant treatment for vitrification of immature porcine cumulus-oocyte complexes (COCs). Immature COCs were vitrified either in 35% ethylene glycol (EG), 35% propylene glycol (PG) or a combination of 17.5% EG and 17.5% PG. After warming, the COCs were in vitro matured (IVM), and surviving oocytes were in vitro fertilized (IVF) and cultured. The mean survival rate of vitrified oocytes in 35% PG (73.9%) was higher (P<0.05) than that in 35% EG (27.8%). Oocyte maturation rates did not differ among vitrified and non-vitrified control groups. Blastocyst formation in the vitrified EG group (10.8%) was higher (P<0.05) than that in the vitrified PG group (2.0%) but was lower than that in the control group (25.0%). Treatment of oocytes with 35% of each cryoprotectant without vitrification revealed a higher toxicity of PG on subsequent blastocyst development compared with EG. The combination of EG and PG resulted in 42.6% survival after vitrification. The maturation and fertilization rates of the surviving oocytes were similar in the vitrified, control and toxicity control (TC; treated with EG+PG combination without cooling) groups. Blastocyst development in the vitrified group was lower (P<0.05) than that in the control and TC groups, which in turn had similar development rates (10.7%, 18.1% and 23.3%, respectively). In conclusion, 35% PG enabled a higher oocyte survival rate after vitrification compared with 35% EG. However, PG was greatly toxic to oocytes. The combination of 17.5% EG and 17.5% PG yielded reasonable survival rates without toxic effects on embryo development.     

Effect of Different Combinations of Cryoprotectants on In Vitro Maturation of Immature Buffalo (Bubalus bubalis) Oocytes Vitrified by Straw and Open‐Pulled Straw Methods

This study was designed to evaluate effects of different combinations of cryoprotectants on the ability of vitrified immature buffalo oocytes to undergo in vitro maturation. Straw and open‐pulled straw (OPS) methods for vitrification of oocytes at the germinal vesicle stage also were compared. The immature oocytes were harvested from ovaries of slaughtered animals and were divided into three groups: (i) untreated (control); (ii) exposed to cryoprotectant agents (CPAs); or (iii) cryopreserved by straw and OPS vitrification methods. The vitrification solution (VS) consisted of 6 m ethylene glycol (EG) as the standard, control vitrification treatment, and this was compared with 3 m EG + 3 m dimethyl sulfoxide (DMSO), 3 m EG + 3 m glycerol, and 3 m DMSO + 3 m glycerol. Cryoprotectants were added in two steps, with the first step concentration half that of the second (and final) step concentration. After warming, oocyte samples were matured by standard methods and then fixed and stained for nuclear evaluation. Rates of MII oocytes exposed to CPAs without vitrification were lower (54.3 ± 1.9% in EG, 47.5 ± 3.4% in EG + DMSO, 36.8 ± 1.2% in EG + glycerol and 29.9 ± 1.0% in DMSO + glycerol; p < 0.05) than for the control group (79.8 ± 1.3%). For all treatments in each vitrification experiment, results were nearly identical for straws and OPS, so all results presented are the average of these two containers. The percentages of oocytes reaching telophase‐I or metaphase‐II stages were lower in oocytes cryopreserved using all treatments when compared with control. However, among the vitrified oocytes, the highest maturation rate was seen in oocytes vitrified in EG + DMSO (41.5 ± 0.6%). Oocytes cryopreserved in all groups with glycerol had an overall low maturation rate 19.0 ± 0.6% for EG + glycerol and 17.0 ± 1.1% for DMSO + glycerol. We conclude that the function of oocytes was severely affected by both vitrification and exposure to cryoprotectants without vitrification; the best combination of cryoprotectants was EG + DMSO for vitrification of immature buffalo oocytes using either straw or OPS methods.     

Vitrification with Glutamine Improves Maturation Rate of Vitrified / Warmed Immature Bovine Oocytes

The current study examined the protective effects of l ‐glutamine and cytochalasin B during vitrification of immature bovine oocytes. Oocyte vitrification solution (PBS supplemented with 10% FCS, 25% EG, 25% DMSO and 0.5 m trehalose) was the vitrification control. Treatments were the addition of 7 μg/ml cytochalasin B, 80 mm glutamine or both cytochalasin and glutaminine for 30 s. After warming, oocytes were matured in vitro for 24 h, fixed and stained with Hoechst (33342) for nuclear maturation evaluation. l ‐glutamine improved the vitrified/warmed immature bovine oocytes viability (32.8%), increasing the nuclear maturation rates compared to other treatments and the no treatment vitrified control (17.4%). There was, however, no effect of cytochalasin B on in vitro maturation (14.4%).     

High survival rate of bovine oocytes matured in vitro following vitrification

Improving pregnancy rates associated with the use of cryopreserved human oocytes would be an important advance in human assisted reproductive technology (ART). Vitrification allows glasslike solidification of a solution without ice crystal formation in the living cells. We have attempted to improve the survival rates of oocytes by a vitrification technique using bovine models. In vitro matured oocytes with or without cumulus cells were vitrified with either 15.0% (v/v) ethylene glycol (EG) + 15% (v/v) dimethylsulfoxide (DMSO) + 0.5 M sucrose or 15% (v/v) EG + 15% (v/v) 1,2-propanediol (PROH) + 0.5 M sucrose, using 'Cryotop' or 'thin plastic sticker', respectively. The oocyte survival rates after vitrifying-warming, and the capacity for fertilization and embryonic development were examined in vitro. The rate of embryonic development to blastocyst was significantly higher (P<0.05) in the oocytes vitrified with 15% (v/v) EG + 15% (v/v) PROH + 0.5 M sucrose than in the oocytes vitrified with 15% (v/v) EG + 15% (v/v) DMSO + 0.5 M sucrose (7.4% +/- 4.1 vs. 1.7% +/- 3.0, respectively). Oocytes vitrified without cumulus cells had a higher survival rate after thawing and a superior embryonic developmental capacity compared with oocytes vitrified with cumulus cells. Prolonged pre-incubation time after thawing adversely affected the rates of embryonic cleavage and development. These results indicate that in vitro matured bovine oocytes can be vitrified successfully with the mixture of the cryoprotectants, EG + PROH, the absence of cumulus cells for vitrification does not affect oocyte survival rate after warming, and vitrified and warmed oocytes do not require pre-incubation before in vitro fertilization.     

玻璃化冷冻对小鼠卵母细胞透明带的影响

本文旨在通过研究玻璃化冷冻小鼠卵母细胞透明带超微结构、透明带厚度(ZPT)、厚度变量(ZPTV)及双折射分值(ZPB)的影响,分析三者间的相关性,探求玻璃化冷冻液的最佳配方。选用4组应用最广泛的冷冻液配方对小鼠卵母细胞进行处理,与未处理的卵母细胞比较存活率、受精率、卵裂率和囊胚率,选出最适冷冻液配方。以新鲜卵母细胞为对照组,进行冷冻程序但并没有进行实际冷冻的卵母细胞为处理组,进行玻璃化冷冻复苏的卵母细胞为冷冻组,扫描电镜观察各组的透明带超微结构变化;检测3组细胞的ZPT和ZPB,并对ZPT和ZPB、ZPTV和ZPB之间相关性进行分析。结果发现HM+7.5%(DMSO+EG),HM+15%(DMSO+EG)+0.5 mol/L Su冷冻液组对卵母细胞发育潜力影响较小。玻璃化冷冻对ZPT(6.05±0.10μm vs 5.77±0.60μm)和ZPB(0.30±0.38 vs 1.22±0.21)有显著影响(P<0.05),且ZPT与ZPB呈负相关(r=-0.299)(P<0.05)。扫描电镜发现玻璃化冷冻造成卵母细胞透明带呈熔融状,表面凹凸不平,窗孔基本不可见,甚至出现透明带部分脱落的情况;冷冻组透明带超微结构的正常率较对照组和处理组下降,其中粗ZP(44.9%对92.9%和84.8%)(P<0.01)和光滑ZP(31.0%对7.4%和9.1%)(P<0.01)。结果表明,玻璃化冷冻影响卵母细胞的发育潜能,低温对透明带的超微结构有较大的负面影响。     

Effects of cryoprotectants and cryoprotectant combinations on viability and maturation rates of Camelus dromedarius oocytes vitrified at germinal vesicle stage

Camel fertility faces many problems, which could be solved by assisted reproductive technologies (ARTs). We designed the experiment to explore the effect of different cryoprotectant concentrations and combinations on viability and maturation rates of vitrified/warmed camel oocytes. We collected ovaries directly after slaughtering from local abattoir and transported them to laboratory in a thermo‐flask containing normal physiological saline. We aspirated the oocytes from follicles, which is 2–8 mm in diameter, washed three times in TCM‐199 and then examined under stereo‐microscope for selection. We selected morphologically normal oocytes with an evenly granulated cytoplasm and a compact cumulus cell layer. We equilibrated morphologically normal oocytes in equilibration solution (ES), which is half concentration of vitrification one. After equilibration, We transported oocytes to vitrification solution using ethylene glycol (EG, 40%), dimethyl sulphoxide (DMSO, 40%) and EG 40% + DMSO 40%. The obtained results revealed that addition of EG 40% + DMSO 40% resulted in the best quality of vitrified/warmed oocytes, which is demonstrated by higher per cent survival rate (90.16%) and maturation rate (58.95%). While DMSO 40% resulted in 62.79% and 29.54%, respectively, EG 40% reported 86.11% and 53.47%, respectively. We could conclude that vitrification of immature camel oocytes by using 40% EG + 40% DMSO is suitable methods to limit drawbacks of vitrification methods, and we need further studies to assess the ability of in vitro‐produced blastocyst to develop in vivo and establish pregnancy after embryo transfer.     

Piezo-actuated zona-drilling improves the fertilisation of OPS vitrified mouse oocytes

The present study was designed to investigate fertilisation of open pulled straw (OPS) vitrified mouse oocytes drilled with piezo-micromanipulation method and their subsequent in vitro and in vivo developmental capacity. Ovulated mouse oocytes were vitrified using the OPS method. After warming, the zona pellucida of a group of vitrified-warmed oocytes was drilled by piezo-micromanipulation. Groups of (a) vitrified, (b) vitrified/drilled and (c) fresh control oocytes were fertilised in vitro. The fertilisation rate of vitrified-warmed oocytes was significantly lower than that of fresh oocytes (45.0 +/- 12.6% vs. 85.2 +/- 6.8%, P < 0.05), and was significantly improved by zona-drilling (85.4 +/- 7.3%). However, blastocyst formation rates of the vitrified and vitrified/drilled groups were significantly lower than those of the fresh controls (65.7 +/- 7.0% and 66.4 +/- 2.5% vs. 86.6 +/- 4.3%, respectively, P < 0.05). The cell number of blastocysts from the vitrified/drilled or the vitrified group was not different from that of the controls. Embryo transfer resulted in pregnancy in all three groups, but the rate of development to term was lower in the vitrified/drilled or vitrified groups than in the controls (16.6 +/- 0.7% or 36.0 +/- 2.4% vs. 51.3 +/- 2.9%, respectively). Our results demonstrated that zona-drilling with piezo-micromanipulation could improve fertilisation in OPS vitrified mouse oocytes but did not increase the overall number of vitrified oocytes developing to term.     

绵羊体外成熟卵母细胞OPS法玻璃化冷冻保存试验

研究以EDFS30为玻璃化冷冻液,以卵母细胞解冻后孤雌激活和体外受精后的卵裂率、囊胚发育率作为评价指标,探讨了以OPS法玻璃化冷冻保存体外成熟绵羊卵母细胞的效果。结果表明:卵母细胞孤雌激活后的卵裂率,冷冻组(64.2%)显著(P<0.05)低于毒性组(76.7%)和对照组(79.1%),而毒性组和对照组无显著(P>0.05)差异;卵母细胞孤雌激活后的囊胚发育率,冷冻组(4.2%)和毒性组(5.8%)均显著(P<0.05)低于对照组(20.2%),毒性组和冷冻组无显著(P>0.05)差异;冷冻组和毒性试验组卵母细胞体外受精后的卵裂率和囊胚发育率(67.6%和7.1%;62.3%和9.1%)均显著低于对照组(78.4%和28.4%)(P<0.05),而毒性组和冷冻组无显著(P>0.05)差异。可见以EDFS30为玻璃化冷冻液,采用OPS法冷冻保存绵羊体外成熟卵母细胞会在一定程度上降低其受精能力和胚胎发育能力。     

Antioxidants and glycine can improve the developmental competence of vitrified/warmed ovine immature oocytes

Despite the numerous potential applications of oocyte cryopreservation, the poor success rate has limited its practical applications. In livestock, particularly in ovine, the oocytes have low developmental competence following vitrification/warming process. Considering the occurrence of osmotic and oxidative stresses during the vitrification/warming process, the application of antioxidants and osmolytes may improve the developmental competence of vitrified/warmed oocytes. In the present study, we aimed to evaluate the effects of the addition of ascorbic acid (AA) and N‐acetyl cysteine (NAC) as antioxidants and glycine as an organic osmolyte either to the vitrification/warming solutions (VWS) or to the IVM medium on the developmental competence of vitrified/warmed ovine germinal vesicle stage oocytes. The survival rate in the vitrified groups was significantly lower than fresh ones. In vitrified/warmed oocytes, there was no significant difference in survival rate between supplemented and non‐supplemented groups. The addition of AA and/or NAC to the VWS or IVM medium and adding glycine to the IVM medium reduced the proportion of apoptotic oocytes and fragmented embryos, which was reflected as an increase in the proportions of metaphase II stage oocytes and blastocyst production. The best result was achieved by supplementing the IVM medium with NAC. In our study condition, antioxidants and glycine could improve the developmental competence of vitrified/warmed ovine immature oocytes, especially when added during IVM.     

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.     

Vitrification of bovine matured oocytes and blastocysts in a paper container

In the present study, we aimed to determine the applicability of a paper container for the vitrification of in vitro matured (IVM) bovine oocytes. In experiment 1, IVM oocytes were exposed to vitrification solution (20% dimethylsulfoxide (DMSO), 20% ethylene glycol (EG), and 5 mol/L sucrose), using a two‐step method, for 30 s; loaded onto either a paper container or Cryotop; and stored in liquid nitrogen. No significant difference (P < 0.05) in the survival and blastocyst formation rates after in vitro vitrification was observed between the paper container and Cryotop. In experiment 2, IVM oocytes were exposed to either a two‐ or three‐step vitrification solution. The three‐step vitrification solution was not significantly different from the two‐step solution in terms of oocyte survival, cleavage and blastocyst rates. In experiment 3, in vitro produced blastocysts were graded according to the manual of the International Embryo Transfer Society (grades 1 and 2) and vitrified using the two‐ and three‐step methods. For grade 2 blastocysts, the three‐step method showed significantly higher (P < 0.05) survival and hatched blastocyst rates than the two‐step method, whereas for grade 1 blastocysts, no significant difference was observed. In conclusion, the paper device and three‐step technique are suitable for oocytes and embryo vitrification.