7日龄小鼠生精上皮单细胞冷冻保存

在DMEM中添加10％小牛血清（NBS），并分别添加不同浓度的抗冻剂二甲基亚砚（DMSO）、丙二醇（PG）、乙二醇（EG）及甘油（G），对7日龄小鼠生精上皮单细胞进行冷冻保存，复苏后台齿蓝染色测定细胞复苏率。结果：当冻存液中DMSO分别为5％、10％、15％、20％时，细胞复苏率分别为77．1％、88．2％、86．5％、73．5％、65．5％，其中10％和15％DMSO组细胞复苏率最高，与其作各组间均差异极显著（P＜0．01）。当冻存液中PG分别为5％、10％、15％、20％、25％时，细胞复苏率分别为66．2％、84．3％、72．1％、69．9％、47．5％，其中10％PG组细胞复苏率最高，与其余各组间均差异极显著（P＜0．01）。当冻存液中EG分别为5％、10％、15％、20％时，细胞复苏率分别为64．9％、81．6％、60．9％、44．7％，其中10％EG组细胞复苏率最高，与其余各组间均差异极显著（P＜0．01）。当冻存液中G分别为5％、10％、20％、25％时，细胞腹苏率均低于10％。10％DMSO组细胞复苏率与10％PG组和10％EG组之间均差异显著（P＜0．05）或极显著（P＜0．01）。结果表明，10％DMSO、10％PG及10％EG均适宜冷冻保存小鼠精原细胞，以及10％DMSO的冻存效果最好，而则不适宜冷冻保存。在含10％DMSO的冻存液中，二步慢速冷冻，液氮储存，37C水浴复苏小鼠精原细胞，是一种具有较高细胞复苏率的冷冻保存方法。     

四种抗冻剂20℃时对小鼠生精上皮单细胞存活率的影响

将小鼠生精上皮单细胞置20℃含不同抗冻剂的冻存液中一定时间后，台盼蓝染色测定细胞存活率，以筛选冷冻保存小鼠生精上皮细胞的侯选抗冻剂及使用浓度。结果，20℃30min，10％浓度的二甲基亚砜（DMSO）、丙二醇（PG）及乙二醇（EG）对7日龄小鼠生精上皮单细胞存活率均无显著影响，而10％甘油（G）则使细胞存活率显著下降；20℃ 30min,10%浓度的DMSO、PG、EG及G对成年小鼠生精上皮单细胞存活率均无显著影响；20℃ 5min,25%浓度的DMSO、PG、EG及G均使7日龄及成年小鼠生精上皮单细胞存活率显著下降。实验结果表明，10％DMSO、PG及EG可作为7日龄小鼠生殖细胞慢速冷冻保存时的侯选抗冻剂，10％DMSO、PG、EG及G可作为成年小鼠生殖细胞慢速冷冻保存时的侯选抗冻剂；在高浓度抗冻剂超速冷冻保存小鼠生殖细胞时，平衡时间应短于5min，或在4℃进行。     

成年小鼠生精小管及睾丸的冷冻保存

采用含不同浓度抗冻剂二甲基亚砜(DMSO)、乙二醇(EG)、丙二醇(PG)及丙三醇(G)的DMEM培养基作为冻存液,对成年小鼠生精小管及完整睾丸进行慢速冷冻,液氮保存,37 ℃水浴复苏,并测定细胞复苏率。结果表明,成年小鼠生精小管在50 mL/L、100 mL/L 及150 mL/L DMSO冻存液中的细胞复苏率分别为92.6%、93.4%及69.4%,在50 mL/L、100 mL/L及150 mL/L PG 冻存液中的细胞复苏率分别为94.2%,93.3%及66. 9%,在50 mL/L、100 mL/L 及150mL/L EG 冻存液中的细胞复苏率分别为87.2%、91. 0%及62. 6%,在50 mL/L、100mL/L及150 mL/L G冻存液中的细胞复苏率分别为90.0%、90.5%及67.1%。各种抗冻剂的最高细胞复苏率之间差异不显著(P> 0. 05 )。成鼠完整睾丸在含100 mL/LDMSO、PG、EG或G的冻存液中的细胞复苏率分别为56.0%、50.0%、50.2%及50.2%。结果显示,DMSO、PG、EG 及G 均适宜用作成年小鼠生精小管的抗冻剂,最佳使用浓度均为50 mL/L~100 mL/L。完整睾丸冷冻后的细胞复苏率低于60%,仍需进一步研究。     

不同冻存保护剂对绵羊前体脂肪细胞冻存效果的影响

<正>【目的】探讨绵羊前体脂肪细胞冷冻保存的最佳冻存保护剂种类和浓度。【方法】在含20%胎牛血清的DMEM/F12培养液中分别添加不同浓度的DMSO、EG、PG、G和PVP作为冻存液,对原代培养的绵羊前体脂肪细胞进行冷冻保存后,检测复苏细胞存活率、细胞增殖能力、细胞中脂肪沉积量,测量GPDH活性,并用实时荧光定量PCR检测PPARγ和LPL mRNA的表达水平,最后对复苏细胞进行染色体核型分析。【结果】各种冻存保护液中,10%DMSO和5%DMSO+5%PVP     

不同冻存液对奶牛乳腺上皮细胞冻存质量的影响

试验旨在探究奶牛乳腺上皮细胞(bovine mammary epithelial cells,BMECs)最佳的冻存液以改善乳腺上皮细胞的冻存质量.BMECs传至第5代后分别加入以下10种不同配方的冻存液.A组:85％ DMEM+10％胎牛血清+5％ DMSO;B组:80％ DMEM+ 10％胎牛血清+10％ DMSO;C组:75％ DMEM+10％胎牛血清+15％ DMSO;D组:70％ DMEM+10％胎牛血清+20％ DMSO;E组:85％ DMEM+5％胎牛血清+10％ DMSO;F组:75％ DMEM+15％胎牛血清+10％ DMSO;G组:70％DMEM+20％胎牛血清+10％DMSO;H组:80％DMEM+10％胎牛血清+10％甘油;I组:70％ DMEM+10％胎牛血清+20％甘油;J组:60％ DMEM+10％胎牛血清+30％甘油,冻存前统一调整细胞密度到1×106个/mL冻存.分别对复苏后的细胞进行台盼蓝染色计算存活率和PI/Hoechst 33258双染计算凋亡率.结果表明,BMECs经不同冻存剂冻存复苏后,细胞活力、形态学及凋亡率表现有所不同,其中B组和G组的活力和24h贴壁率较其他组高,二者的凋亡率较低,二者之间差异无显著性(P＞0.05);传代后B组细胞的生长状况最好.     

鸡胚19期原始生殖细胞慢速冷冻和玻璃化冷冻保存的研究

本研究对发育至第19期的鸡胚性腺原始生殖细胞(primordial germ cells，PGCs)分离提纯后，在DMEM中添加冷冻保护剂DMSO（二甲基亚砜）、EG（乙二醇）、蔗糖、PVP（聚乙烯吡咯烷酮）分别组成6种慢速冷冻保护液和6种玻璃化冷冻保护液，进行超低温冷冻保存。复苏后苔盼蓝染色测定细胞存活率，体外接种培养、传代。结果：①慢速冷冻保存中，PGCs在慢速冷冻液V（10％EG+10% FBS+0.1 mol/L蔗糖）条件下复苏后存活率最高（92.20%），且与慢速冷冻液I（10％ DMSO+10% FBS）存活率之间差异显著（P<0.05）。②玻璃化冷冻保存中，PGCs在玻璃化冷冻液I（10% DMSO+10% EG+20% FBS+10% PVP）条件下复苏后存活率最高（84.15%），且与其余5种玻璃化冷冻液下复苏后存活率之间差异均极显著（P<0.01）。③培养传至第3代的慢速冷冻复苏后PGCs细胞和培养传至第2代的玻璃化冷冻复苏后PGCs细胞，PAS染色、AKP染色呈阳性并保持完整的二倍体核型。     

冷冻保护剂和胎牛血清对牛成纤维细胞冷冻效果的影响

以甘油与DMSO为冷冻保护剂,添加5%-20%胎牛血清的DMEM为基础冻存液,对鲁西黄牛皮肤成纤维细胞进行冷冻保存,结果表明:①同浓度DMSO为冷冻保护剂效果优于同浓度的甘油(P<0.05),但添加50%血清时并不明显(P>0.05),可能与较高血清浓度有关,血清对冻存细胞的影响高于冷冻保护剂的作用。②血清浓度一定时,DMSO和甘油浓度在10%-15%时,冻存效果较好。③当甘油与DMSO浓度一定时,血清浓度越高冷冻保存效果越好。④甘油与DMSO浓度一定时(DMSO浓度5%除外),添加10%和20%血清效果无差异(P>0.05)。因此,在实际应用中,考虑成本等因素,血清浓度达到10%时就已经能达到一般试验要求。     

小鼠4-细胞胚胎OPS法冷冻保存技术

在25℃室温和37℃恒温台条件下，利用玻璃化冷冻溶液EFS30、EFS40、EDFS30或EDFS40，对小鼠4-细胞胚胎进行玻璃化冷冻保存．以解冻后培养72h的囊胚发育率为其体外发育能力的考核指标，同时对解冻后培养1～3h的胚胎进行移植以判定其体内发育潜力。开放式拉长塑料细管（OPS）二步法冷冻保存，即胚胎首先移入预处理液（10％EG或10％EG＋10％DMSO）中平衡30s，再移入玻璃化溶液中洗涤后吸入OPS管中，分别经35、30或25s后直接投入液氮中冷冻保存。一步法冷冻保存则无需预处理液处理。结果表明，小鼠胚胎4-细胞一步法和二步法冷冻后囊胚最高发育率分别为87．7％和88.6％，与对照组（93.0％）差异不显著（P〉0．05）。利用最佳冷冻组获得的143枚胚胎移植于12只假妊娠50～60h的受体母鼠，结果有4只妊娠产仔17只，妊娠产仔率为42．5％（17／40），与对照组59．4％（19／32）差并不显著（P〉0.05）。     

鸡囊胚细胞玻璃化冷冻保存技术的研究

研究不同冷冻液配方对鸡囊胚细胞的玻璃化冷冻保存效果。对新鲜种蛋囊胚细胞分离提纯后,在DMEM中添加不同的冷冻保护剂DMSO（二甲基亚砜）、EG（乙二醇）、蔗糖,分别组成6种玻璃化冷冻保护液,进行超低温冷冻保存。复苏后通过苔盼蓝染色测定活细胞存活率。结果：玻璃化冷冻保存中,囊胚细胞在玻璃化冷冻液配方2（10％EG＋10％DMSO＋0．5mol／L蔗糖＋20％FBS＋DMEM）条件下复苏后存活率最高（71．32％）,与玻璃化冷冻液配方1和3条件下复苏后存活率之间差异显著（P〈0．05）,且与玻璃化冷冻液配方4、5、6条件下复苏后存活率之间差异均极显著（P〈0．01）,可以作为鸡囊胚细胞的玻璃化冷冻液。     

马皮肤成纤维细胞的体外培养与冷冻保存

本研究利用小组织块直接培养法得到了成年马皮肤成纤维细胞的原代培养物,再用酶消化法处理,能够纯化马皮肤成纤维细胞。成纤维细胞的冷冻保存,首先采用手工冷冻法,选用含有不同浓度保护剂如：二甲基亚砜（DMSO）、乙二醇（EG）、甘油（GC））和新生牛血清（NCS）的12种冷冻液对马皮肤成纤维细胞进行冷冻保存;其次用2种冷冻方法对马皮肤成纤维细胞进行冷冻保存,以24 h贴壁率评价冻存效果。结果表明,10% DMSO和20% NCS的DMEM冻存液,对马皮肤成纤维细胞的冻存效果好(24 h贴壁率84.98%)。从冷冻方法来看,程序冷冻法(86.32%）优于手工冷冻法（79.98%)(P<0.05)。     

鸡胚精原干细胞体外保存能力的研究

采用二酶3步法获取孵化第19 d的鸡胚精原干细胞（SSCs）,比较在快速冷冻条件下3种冷冻保护剂（DMSO、乙二醇、甘油）在3个浓度5%、10%、15%条件下对鸡胚精原干细胞的冷冻保存效果进行研究。结果显示：（1）当DMSO的浓度为5%、10%及15%时,复苏后细胞存活率分别为73.1%、88.6%和74.8%,三者差异显著（P〈0.05）;而10%DMSO保存精原干细胞的存活率、复苏后培养细胞生长和集落形成均显著高于其它2个浓度;当乙二醇浓度为5%、10%和15%时,复苏后细胞存活率分别为69.4%、83.1%和65.2%,三者差异显著（P〈0.05）;复苏后无论饲养层存在与否,SSCs均能增殖,但未有AKP阳性集落生成;当甘油浓度为5%、10%、15%时,复苏后细胞存活率均小于15%,三者差异不显著（P〉0.05）;复苏后细胞存活时间极短,仅存活12 h左右;（2）当在10%DMSO浓度条件,复苏后SSCs在有饲养细胞层的条件下,培养5 d形成集落,表明10%DMSO是鸡胚精原干细胞适宜的冷冻保护剂。     

Studies on MⅡ Stage Porcine Oocytes Vitrification

The study was designed to select right cryoprotectants and carrier for porcine matured oocytes to improve oocyte viability after vitrification and lay a good foundation for porcine oocytes continue development. Using 40% EG, 15% EG+15% DMSO+20% FBS, 15% EG+15% DMSO+20% SPS as cryoprotectants and taking GMP and cryotop as carrier to do porcine oocytes vitrification. The results showed that VS3 including SPS made better active of FDA (59.40%±4.93%) than VS1 （42.12%±4.08%） and VS2 (37.57%±1.21%）（P<0.05);VS3 as cryoprotectants and cryotop as carrier made better active of FDA (83.33%±3.33%) than GMP (59.40%±4.93%)(P<0.05). The data demonstrated that use SPS to instead of FBS and cryotop as carrier could significantly improve active of oocyte after vitrification.     

Vitrification of Boer Goat Morulae and Early Blastocysts by Straw and Open-Pulled Straw Method

The aim of this study was to investigate the effects of different vitrification solutions [EFS30 or EFS40 contains 30% (v/v) ethylene glycol (EG), 40% (v/v) EG; EDFS30 or EDFS40 contains 15% (v/v) EG and 15% (v/v) dimethyl sulfoxide (DMSO), 20% (v/v) EG and 20% (v/v) DMSO], equilibrium time during vitrification (0.5-2.5 min) and vitrification protocols [one-step straw, two-step straw and open-pulled straw (OPS)] on in vivo development of vitrified Boer goat morulae and blastocysts after embryo transfer. In the one-step straw method, the lambing rates of vitrified embryos in EFS30 (37.5%), EFS40 (40.5%) or EDFS30 (38.2%) group were similar to that of fresh embryos (57.5%) and conventional freezing method (46.7%) when the equilibrium time was 2 min. In the two-step straw method, the highest lambing rate was obtained when embryos were pretreated with 10% EG for 5 min and then exposed to EFS40 for 2 min (51.4%), showing similar lambing rates compared with fresh embryos (56.1%) or the embryos cryopreserved by conventional freezing method (45.2%). In the OPS method, the lambing rate in EFS40, EDFS30 or EDFS40 groups were similar to that (57.1%) of fresh embryos, or to that (46.0%) of embryos cryopreserved by conventional freezing method. The highest lambing rate (51.4%) of the group of OPS was obtained when the embryos were vitrified with EDFS30. In conclusion, either the two-step straw method in which embryos were pretreated in 10% EG for 5 min and then exposed to EFS40 for 2 min, or the OPS method in which embryos were pretreated in 10% EG + 10% DMSO for 30 s and then exposed to EDFS30 for 25 s was a simple and efficient method for the vitrification of Boer goat morulae and blastocysts.     

Effects of five cryoprotective agents on quality of sheep epididymal spermatozoa during pre-freezing

The objective of this study was to test five cryoprotective agents during ram epidydimal spermatozoa incubation (at 4 °C), of up to 3 h, typical equilibration time before the freezing step begins, in order to establish a starting point for future freezing and thawing protocols. The parameters analyzed were: progressive motility (PM), vitality, and the plasma membrane functional integrity by the hypoosmotic swelling (HOS) test. Testes and epididymides were collected immediately after death. The tail of both epididymides were isolated and spermatozoa were recovered constituting one sample (n=20). A Tes–Tris–Yolk extender was employed. The extender contained five alternative CPAs: Dimethylacetamide (DMA), Dimethyl sulfoxide (DMSO), Ethylene glycol (EG), Glycerol (GLY) and Propylene glycol (PG) at three final concentrations: 2.5%, 5.0% and 10.0%. Control groups consisted of samples mixed only with the extender, without any CPA. All sample groups were exposed to the CPAs for 1 h or 3 h at 4 °C. EG exposure yielded better responses in both PM and HOS test parameters compared to extender only and also the other CPAs. There was no difference among all the treatments regarding vitality. EG (with best results at 2.5%) is thus proposed as a good CPA (followed by DMA as an explorable alternative) for the implementation of forthcoming ram epididymal spermatozoa freezing protocols.     

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.     

Comparison of different intracellular cryoprotectants on the solid surface vitrification of red-rumped agouti (Dasyprocta Leporina Lichtenstein, 1823) ovarian tissue

In contributing to the conservation of wild rodents, the aim of this study was to evaluate the use of distinct cryoprotectants, separately or in combination, for solid surface vitrification (SSV) of red-rumped agouti ovarian tissue. Ovarian cortex from nine females was recovered and fragmented. Fresh fragments (control) were used to analyse the pre-antral follicle (PF) morphology using a histologic procedure, viability using the Trypan blue test, cell proliferation by counting the argyrophilic nucleolar organizing regions (Ag-NORs technique) and DNA integrity using the TUNEL assay. The remaining fragments were vitrified using SSV method with 3 M or 6 M ethylene glycol (EG) or dimethyl sulfoxide (DMSO), or in combination (3 M EG/3 M DMSO), and further evaluated as reported for the fresh samples. All cryoprotectants were effective at preserving PFs morphology compared to the control group (80.7 ± 5.21%), except 6 M EG and 3 M DMSO that provoked a significant (p < .05) decrease on the values of morphologically normal primary (60.0 ± 19.0%) and primordial (44 ± 4.5%) follicles, respectively. Regarding viability, all cryoprotectants provided values similar to that verified for the control group (79.0%), but a significant decrease (p < .05) was observed with EG/DMSO combination (59%). Using Ag-NORs technique, the highest (p < .05) cell proliferative capacity was detected when using EG at each tested concentration. The TUNEL proved the preservation of DNA integrity regardless of the cryoprotectant. In summary, we suggest the use of 3 M EG for the solid surface vitrification of red-rumped agouti ovarian tissue.     

小鼠肾细胞冻存过程中甘油浓度的优化

目的:探讨在冻存小鼠肾细胞过程中,通过优化冷冻保护剂中甘油浓度的加入量,以达到小鼠肾细胞的最佳冻存效果。方法:将小鼠肾细胞制备成单细胞悬液,然后加入不同浓度的冷冻保护剂甘油,冻存细胞,比较细胞冻存效果,计算细胞成活率。四个组的甘油加入量分别为5%、10%、15%、20%。结果:在小鼠肾细胞冻存过程中,当冷冻保护剂中甘油的浓度达到10%时,冻存效果最佳,细胞成活率达到89.57%。     

Optimization of cryoprotectant treatment for the vitrification of immature cumulus-enclosed porcine oocytes: comparison of sugars,combinations of permeating cryoprotectants and equilibration regimens

Our aim was to optimize the cryoprotectant treatment for the preservation of immature porcine cumulus-oocyte complexes (COCs) by solid surface vitrification. In each experiment, the vitrification solution consisted of 50 mg/ml polyvinyl pyrrolidone, 0.3 M of the actual sugar and in total 35% (v/v) of the actual permeating cryoprotectant (pCPA) combination. After warming, the COCs were subjected to in vitro maturation, fertilization and embryo culture. In Experiment 1, trehalose and sucrose were equally effective during vitrification and warming in terms of facilitating oocyte survival and subsequent embryo development. In Experiment 2, when equilibration was performed at 38.5 C in a total of 4% (v/v) pCPA for 15 min, the combination of ethylene glycol and propylene glycol (EG + PG = 1:1) was superior to EG and dimethyl sulfoxide (EG + DMSO = 1:1) in terms of oocyte survival after vitrification and the quality of resultant blastocysts. In Experiment 3, equilibration in 4% (v/v) pCPA for 15 min before vitrification was superior to that in 15% (v/v) CPA for 5 min for achievement of high survival rates irrespective of the pCPA combination used. In Experiment 4, when equilibration was performed in 4% EG + PG for 5 min, 15 min or 25 min, there was no difference in oocyte survival and subsequent embryo development after vitrification and warming; however, the developmental competence of cleaved embryos was tendentiously reduced when equilibration was performed for 25 min. In conclusion, trehalose and sucrose were equally effective in facilitating vitrification, and the optimum pCPA treatment was 5–15 min equilibration in 4% (v/v) of EG + PG followed by vitrification in 35% (v/v) EG + PG.     

Effect of cryoprotectant composition on in vitro viability of in vitro fertilized and cloned bovine embryos following vitrification and in-straw dilution

In this study the efficacy of the combination of glycerol (GLY) and ethylene glycol (EG) as cryoprotectants in a vitrification method developed for direct embryo transfer was evaluated by in vitro development of in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos after vitrification. The IVF and SCNT blastocysts were vitrified in either 40% GLY, 30% GLY + 10% EG, or 20% GLY + 20% EG using French straws. After warming, the straws were held vertically for 1 min without shaking and were then placed horizontally for 5 min to dilute the cryoprotectants. After washing, the vitrified-warmed embryos were cultured in vitro for 72 h. There were no differences among the vitrification solutions with respect to the rates of vitrified-warmed IVF and SCNT embryos surviving and developing to the hatched blastocyst stage. However, the rates of development to the hatched blastocyst stage of the SCNT embryos vitrified with 40% GLY tended to be higher than those vitrified with 30% GLY + 10% EG or 20% GLY + 20% EG (26% vs. 7-8%, respectively). The development rates to the hatched blastocyst stage of the IVF and SCNT embryos vitrified with solution containing EG were significantly lower (P<0.05) than those of non-vitrified embryos. These results suggest that use of the combination of GLY and EG as cryoprotectants had no beneficial effect on the viability of embryos after in-straw dilution. However, this method is so simple that it can be used for practical direct transfer of vitrified embryos in the field.     

Effect of vitrification medium composition and exposure time on post-thaw development of buffalo embryos produced in vitro

This study examined the effects of different vitrification medium compositions and exposure times (2, 4 and 6min) on the post-thaw development of buffalo embryos produced in vitro (IVP). The compositions were (1) 40% ethylene glycol (EG); (2) 25% glycerol (G)+25% EG, and (3) 25% EG+25% dimethylsulfoxide (DMSO). The base medium was 25mM Hepes-buffered TCM-199+10% steer serum +50microg/mL gentamycin. The IVP embryos were cryopreserved by a two-step vitrification method at 24 degrees C. After warming, the embryos were cultured in vitro for 72h. The vitrification of morulae and blastocysts in 25% EG+25% DMSO with an exposure time of 2 and 4min, respectively, resulted in a better hatching rate than other combinations. The hatching rate of morulae vitrified in 25% EG+25% G, 25% EG+25% DMSO, and blastocysts vitrified in 40% EG, 25% EG+25% DMSO were negatively correlated with exposure time. However, the hatching rate of blastocysts vitrified in 25% EG+25% G was positively correlated with exposure time. The study demonstrated that the post-thaw in vitro development of IVP buffalo embryos was affected by the vitrification medium composition and exposure time.