Vitrification and rapid-freezing of cumulus cells from rabbits and pigs

To use adult somatic cloning technology in animal breeding, this technology should be complemented with nuclear donor cell cryopreservation. Two different conventional nonequilibrium methods (vitrification, V: 3.58M EG and 2.82M DMSO in PBS plus 20% FCS and rapid-freezing, RF: 0.25M sucrose, 2.25M EG and 2.25M DMSO in PBS plus 20% FCS) were assayed here on different cumuli types from rabbits and pigs. In rabbits, the cell proliferation capability of fully disaggregated cumuli was not affected by cryopreservation procedures (V: 100% and RF: 82%). Vitrified samples from partially or non-disaggregated cumuli showed the lowest proliferation frequencies (4% and 0%, respectively). In pigs, differences in cell proliferation capability were only observed between vitrified non-disagreggated cumuli and vitrified or rapid-frozen, fully disaggregated cumuli (72% vs 100% or 100%, respectively; P < 0.05). In both species, in vitro cultured sub-confluent samples were able to survive to a second cryopreservation treatment, maintaining the cell proliferation capability in nearly 50% of thawed samples. In conclusion, before cryopreservation, disaggregation of cumulus cells from both species into small clusters of cells improved their viability after thawing. These results allow us to efficiently, easily and rapidly store rabbit and pig cumulus cells, from selected high-merit females.     

Vitrification of in vitro cultured rabbit morulae

In the present work, we attempt to establish an efficient vitrification procedure for 32-cell rabbit embryos obtained in vitro. In experiment 1, both the effect of the composition of the vitrification solutions and the cryoprotectant addition (either in one or two steps) were studied. For one-step addition, straws with embryos in the final vitrification solution (total time 60s) were plunged into liquid nitrogen. For two-step addition, previously embryos were 2 min pre-equilibrated in 0.5 ml of (1:1) PBS plus 20% FCS: vitrification solution without sucrose. Different solutions of cryoprotectants were compared: 25 vol.% ethylene glycol supplemented with 0.25 M sucrose (25EG+S) and 20% ethylene glycol plus 20% dimethyl sulfoxide, alone (20EG+20DMSO-S) or supplemented with 0.25 M sucrose (20EG+20DMSO+S). Six percent (30/487) of the total of 32-cell embryos obtained by in vitro culture in each experimental session was slow-frozen by a classical method as a technical efficiency control. Only 30% slow-frozen embryos reached blastocyst stage. Significant differences in embryo development were detected between the one-step (25EG+S) and two-step (25EG+S) groups and the one-step (20EG-20DMSO+S) and two-step (20EG-20DMSO-S) groups (0-6% versus 36-50%, respectively). Consequently, in the following experiments only these two vitrification procedures were used. In experiment 2, we attempted to substitute the use of PBS by HEPES-buffered Ham's F-10 (h-CM) in all cryoprotective solutions or media. When h-CM was used, a significant reduction in the in vitro embryo development was observed when the HEPES-buffered groups were compared with one-step (20EG-20DMSO+S) group in s-PBS (35-45% versus 73%). In experiment 3, the one-step (20EG+20DMSO+S) and two-step (20EG+20DMSO-S) procedures were assayed using two FCS levels (20 and 40%) in the PBS-based media. Relative to in vitro development, the highest rates were reached with one step (20EG-20DMSO+S), using PBS plus 20% FCS, which was different from two steps (20EG-20DMSO-S), regardless of percentage of FCS in the PBS-based media (81% versus 41-45%; P<0.05). In conclusion, we propose either the one step (20EG-20DMSO+S) or two steps (20EG-20DMSO-S) prepared in PBS plus 20% serum for use in future works.     

Membrane transport properties of equine and macaque ovarian tissues frozen in mixtures of dimethylsulfoxide and ethylene glycol

The rate at which equine and macaque ovarian tissue sections are first cooled from +25 degrees C to +4 degrees C has a significant effect on the measured water transport when the tissues are subsequently frozen in 0.85 M solutions of glycerol, dimethylsulfoxide (DMSO), or ethylene glycol (EG). To determine whether the response of ovarian tissues is altered if they are suspended in mixtures of cryoprotective agents (CPAs), rather than in solutions of a single CPA, we have now measured the subzero water transport from ovarian tissues that were suspended in mixtures of DMSO and EG. Sections of freshly collected equine and macaque ovaries were suspended either in a mixture of 0.9 M EG plus 0.7 M DMSO (equivalent to a mixture of approximately 5% vv of EG and DMSO) or in a 1.6M solution of only DMSO or only EG. The tissue sections were cooled from +25 degrees C to +4 degrees C and then frozen to subzero temperatures at 5 degrees C/min. As the tissues were being frozen, a shape-independent differential scanning calorimeter technique was used to measure water loss from the tissues and, consequently, the best fit membrane permeability parameters (L(pg) and E(Lp)) of ovarian tissues during freezing. In the mixture of DMSO+EG, the respective values of L(pg) and E(Lp) for equine tissue first cooled at 40 degrees C/min between +25 degrees C and +4 degrees C before being frozen were 0.15 microm/min atm and 7.6 kcal/mole. The corresponding L(pg) and E(Lp) values for equine tissue suspended in 1.6M DMSO were 0.12 microm/min atm and 27.2 kcal/mole; in 1.6M EG, the values were 0.06 microm/min atm and 21.9 kcal/mole, respectively. For macaque ovarian tissues suspended in the mixture of DMSO+EG, the respective values of L(pg) and E(Lp) were 0.26 microm/min atm and 26.2 kcal/mole. Similarly, the corresponding L(Lg) and E(Lp) values for macaque tissue suspended in 1.6M DMSO were 0.22 microm/min atm and 31.4 kcal/mole; in 1.6 M EG, the values were 0.20 microm/min atm and 27.9 kcal/mole. The parameters for both equine and macaque tissue samples suspended in the DMSO+EG mixture and first cooled at 0.5 degrees C/min between +25 degrees C and +4 degrees C were very similar to the corresponding values for samples cooled at 40 degrees C/min. In contrast, the membrane parameters of equine and macaque samples first cooled at 0.5 degrees C/min in single-component solutions were significantly different from the corresponding values for samples cooled at 40 degrees C/min. These results show that the membrane properties of ovarian cells from two species are different, and that the membrane properties are significantly affected both by the solution in which the tissue is suspended and by the rate at which the tissue is cooled from +25 degrees C to +4 degrees C before being frozen. These observations suggest that these variables ought to be considered in the derivation of methods to cryopreserve ovarian tissues.     

Calcium concentration in vitrification medium affects the developmental competence of in vitro matured ovine oocytes

The present study was designed to determine whether different calcium concentrations in the vitrification solutions could improve the developmental competence of in vitro matured ovine oocytes after cryopreservation. In vitro matured oocytes were vitrified with 16.5% ethylene glycol (EG) + 16.5% dimethylsulfoxide (DMSO) vitrification media. The base media contain different calcium concentrations, so that five experimental groups were obtained: TCM/FCS (TCM 199 + 20% fetal calf serum (FCS), [Ca²⁺] 9.9 mg/dl); PBS/FCS (Dulbecco Phosphate Buffered Saline (PBS) + 20% FCS, [Ca²⁺] 4.4 mg/dl); PBS^{CaMg free}/FCS (PBS without Ca²⁺ and Mg²⁺ + 20% FCS [Ca²⁺] 2.2 mg/dl); PBS/BSA (PBS + 0.4% bovine serum albumin (BSA), [Ca²⁺] 3.2 mg/dl) and PBS^{CaMg free}/BSA (PBS without Ca²⁺ and Mg²⁺ +0.4% BSA, [Ca²⁺] 0.4 mg/dl). After warming, the oocytes from the five experimental groups were assessed for survival, spontaneous parthenogenetic activation and developmental capacity via in vitro fertilization. Oocyte survival after vitrification procedures was better preserved in group PBS^{CaMg free}/FCS compared to the others (P < 0.05). In addition, a positive correlation was found between calcium concentration in vitrification solutions and spontaneous parthenogenetic activation (correlation index 0,82; P < 0.001). Development of vitrified oocytes was significantly affected by vitrification media composition (P < 0.01). In particular, oocytes from group PBS^{CaMg free}/FCS led to higher cleavage rates and blastocyst rate compared to the others. Our data showed that lowering calcium concentration in the vitrification medium improves the blastocyst rate of vitrified ovine oocytes, probably reducing the effect of EG + DMSO during vitrification. On the contrary, the replacement of FCS with BSA dramatically reduces the developmental potential of these oocytes.     

Vitrification of collared peccary ovarian tissue using open or closed systems and different intracellular cryoprotectants

This study aimed to evaluate different vitrification methods using distinct cryoprotectants (CPAs) for the preservation of collared peccary ovarian preantral follicles (PFs). Ovarian pairs from six females were fragmented and three fragments (fresh control group) were immediately evaluated for morphology, viability, cell proliferation capacity (assessed by quantifying the number of argyrophilic nucleolus organizer regions – NORs), and apoptosis (by the identification of activated caspase-3 expression). The remaining 18 fragments were vitrified using the solid surface vitrification (SSV) method or the ovarian tissue cryosystem (OTC) with 3 M ethylene glycol (EG), 3 M dimethylsulfoxide (DMSO), or a combination of the two (1.5 M EG/1.5 M DMSO). After two weeks, samples were rewarmed and evaluated as described previously. The OTC with any of the CPAs provided a similar conservation of morphologically normal PFs as the fresh control group (75.6 ± 8.6%); however, the SSV was only efficient with DMSO alone (63.9 ± 7.6%). Regarding the viability or cell proliferation, all tested groups provided post rewarming values similar to those observed for the fresh control group, 84.0 ± 2.9% viable cells with 2.0 ± 0.2 NORs. Related to apoptosis analysis, only the OTC with EG (46.7%) and the SSV method with EG (43.4%) or the combination of EG and DMSO (33.4%) provided similar values to those found for the fresh control group (36.7%). Our findings indicate the utilization of a closed system, the OTC, with 3 M EG as the CPA for the vitrification of collared peccary ovarian tissue.     

Combination of intracellular cryoprotectants preserves the structure and the cells proliferative capacity potential of adult collared peccary testicular tissue subjected to solid surface vitrification

The objective was to evaluate different permeating cryoprotectants to vitrify testicular tissue biopsies from adult collared peccaries. Five pairs of testicles were dissected into fragments (9 mm³) that were allocated to non-vitrified (control) and vitrified groups using a solid-surface method following exposure to different cryoprotectants (3.0 M dimethyl sulfoxide (DMSO), 3.0 M ethylene glycol (EG) or 1.5 M DMSO + 1.5 M EG). After warming, samples were evaluated for histomorphology, ultrastructure, viability, and proliferative capacity potential. The appropriate conservation of the ultrastructural organization of the seminiferous tubule in terms of lumen presence and cell junctions was only observed at the use of DMSO/EG combination. Regardless of the cryoprotectant, the vitrification effectively preserved cell nuclear visualization and condensation similarly as observed at the non-vitrified group. Moreover, DMSO/EG combination provided a better preservation of basal membranes of seminiferous tubules than DMSO (P < 0.05). The occurrence of cell swelling was more evident in the use of DMSO than EG (P < 0.05), but both isolate cryoprotectants were similar to the DMSO/EG combination. Only the DMSO/EG combination maintained the proliferative capacity potential for spermatogonia (3.69 NORs/cell) and Sertoli cell (3.19 NORs/cell) similar to controls (3.46 and 3.31 NORS/cell, respectively). Moreover, ~40% cell viability was found after vitrification independent of cryoprotectant. In conclusion, DMSO/EG in combination is better than DMSO or EG alone for SSV of testicular tissue biopsies from adult collared peccaries.     

Developmental competence of frozen-thawed yak (Bos grunniens) oocytes followed by in vitro maturation and fertilization

In the present study, we examined the ability of immature germinal vesicle (GV) and subjected to in vitro matured (MII) yak oocytes to survive after cryopreservation as well as their subsequent development following in vitro maturation and fertilization. Both GV and MII oocytes were cryopreserved by using two different vitrification solutions (VS); VS-I contained 10% ethylene glycol (EG) and 10% dimethylsulfoxide (DMSO) in TCM-199 + 20% (v/v) fetal calf serum (FCS) whereas VS-II contained 40% EG + 18% Ficoll + 0.5 M sucrose in TCM-199 + 20% FCS. The percentage of oocytes found to be morphologically normal was greater (P < 0.01) in VS-I group than in VS-II group. Rates of cleavage (30.6–42.2%) and blastocyst formation (2.9–8.9%) did not differ among groups, but were lower than in unfrozen control (55.7% and 25.4%, P < 0.01). These results show that a combination of EG and DMSO or EG, Ficoll and sucrose can be used to cryopreserve yak oocytes in French straws.     

Effect of macromolecules in solutions for vitrification of mature bovine oocytes

This study was designed to evaluate vitrification procedures for in vitro matured bovine oocytes for efficient blastocyst production after warming, IVF and culture. A second goal was to replace serum as the macromolecular component of the vitrification solution, without compromising efficacy. The first experiment compared two containers, open pulled straws (OPS) versus cryoloops, and two vitrification protocols: short equilibration (H-TCM-199+10% EG+10% DMSO+20% FCS for 30s, followed by H-TCM-199+20% EG+20% DMSO+20% FCS+0.48M galactose for 20s) versus long equilibration (H-TCM-199+3% EG+20% FCS for 10min, followed by H-TCM-199+31% EG+20% FCS+1M galactose for 20s). Subsequent experiments used only cryoloops and the short equilibration protocol to evaluate the effect of replacing FCS with defined macromolecules (BSA, Ficoll, PVP, and PVA) in vitrification solutions. Cryoloops were superior to OPS for vitrification of oocytes as determined by blastocyst production (P<0.05). The short and long vitrification protocols gave similar results. The presence of macromolecules in vitrification solutions for bovine oocytes was necessary for acceptable post-warming developmental capacity; 20% FCS, 1% and 2% BSA, 6% and 18% Ficoll, 6% and 20% PVP, 1% PVA, and the combinations of 18% Ficoll+1% BSA, and 6% PVP+1% BSA provided similar protection during vitrification of oocytes; development ranged from 14.8% to 23.0% blastocysts/oocyte, which was not different (P>0.05) from non-vitrified controls (26.9-34.0% blastocysts/oocyte). Too much (6%) and too little (0.3%) BSA, and 0.3% PVA for vitrification resulted in lower blastocyst production (P<0.05) relative to unvitrified oocytes.     

Rabbit and pig ear skin sample cryobanking: effects of storage time and temperature of the whole ear extirpated immediately after death

The post-mortem temporal and thermal limits within which there will be ample guarantees of rescuing living skin cells from dead specimens of two species, rabbit and pig, were studied. Post-mortem extirpated whole ears were stored (in non-aseptic conditions) either at 4 degrees C or at room temperature (from 22 to 25 degrees C) or at 35 degrees C for different time lapses after animal death. In both species, the post-mortem maximum time lapses where cell viability was not significantly reduced were 240, 72, and 24 h post-mortem (hpm) for 4, 22-25 and 35 degrees C, respectively. Once the post-mortem temporal limits for each tested thermal level at which cells from skin samples are able to grow in culture were defined, the survival ability of skin samples submitted to these temporal limits and cryopreserved were tested. In the pig, skin samples stored at the three tested thermal levels survived after vitrification-warming, reaching confluence in culture. In rabbit, only tissue samples from ears stored at 35 degrees C for 24 hpm did not survive after vitrification-warming. In conclusion, we should remark that cell survival rates obtained according to the assayed post-mortem time lapses and thermal levels are sufficient to collect and to cryopreserve skin samples from the majority of dead specimens.     

Freezing solution containing dimethylsulfoxide and fetal calf serum maintains survival and ultrastructure of goat preantral follicles after cryopreservation and in vitro culture of ovarian tissue

Goat ovarian cortex fragments were subjected to slow freezing in the presence of various solutions containing intracellular cryoprotectants, including 1.0 M ethylene glycol (EG), propanediol (PROH), or dimethyl sulfoxide (DMSO), with or without sucrose and/or fetal calf serum (FCS). Histological examination revealed that only the DMSO-containing solutions were able to maintain a follicular ultrastructure similar to the morphology observed in the fresh control. Therefore, fragments previously cryopreserved in DMSO solutions (with and without sucrose and/or FCS) were cultured in vitro for 48 h and then subjected to viability, histological, and ultrastructural analysis. No significant differences were observed among the percentages of morphologically normal follicles in cryopreserved ovarian tissue before in vitro culture (DMSO: 62.5%; DMSO + sucrose: 68.3%; DMSO + FCS: 60.0%; DMSO + sucrose + FCS: 60.0%) and after culture (DMSO: 60.8%; DMSO + sucrose: 64.2%; DMSO + FCS: 70.8%; DMSO + sucrose + FCS: 55.0%). Following in vitro culture, the viability analysis showed that only the freezing solution containing DMSO and FCS (75.6%) maintained a percentage of viable follicles similar to that observed after culture without cryopreservation (89.3%). As determined by ultrastructural analysis, morphologically normal preantral follicles were detected in the fresh control and in fragments cultured before and after cryopreservation with DMSO and FCS. Thus, a freezing solution containing DMSO and FCS, under the experimental conditions tested here, guaranteed the maintenance of viability and follicular ultrastructure after short-term in vitro culture.     

Cryopreservation of immature bovine oocytes by vitrification in straws

The aim of this study was to cryopreserve by vitrification by ethylene glycol (EG) and dimethyl sulfoxide (DMSO) immature bovine oocytes in straws and to investigate the effects of vitrification on post-thaw oocyte maturation. A total of 575 cumulus oocyte complexes were obtained by follicle aspiration from 238 ovaries of cows slaughtered at a local abattoir. Following selection, oocytes with compacted cumulus cells and evenly granulated ooplasm were vitrified using one of the three different solutions with a non-vitrified group served as control. The first step vitrification solution contained 20% EG while the second step solution contained 40% EG+1M sucrose in a basic media used in group EG. Oocytes were matured in N-2-hidroxyethyl piperazine-N-2-ethanosulfonic acid (HEPES) buffered tissue culture medium (TCM) 199 for 24h at 39 degrees C in a humidified atmosphere of 5% CO2 in air. Oocytes were fixed following evaluation for polar body formation, stained with Giemsa solution and nuclear maturation was examined. The numbers of oocytes which were observed at Metaphase II (MII) stage were 41 (34.1%), 17 (14.9%), 29 (20.7%) and 78 (79.6%) in groups EG, DMSO, Mix and Control, respectively. Maturation rate distribution in group Mix was not statistically different when compared to maturation rate distributions in groups EG and DMSO (p>0.05). Differences between other groups were significant (p<0.001). However, better results were obtained in EG group compared to DMSO and mix groups. Maturation rates were lower in all treatment groups than the control group. The lowest maturation result was obtained in DMSO group. Maturation rate in group Mix was between maturation rates of EG and DMSO groups. Immature bovine oocytes can be vitrified in straws, but maturation success differs with the cryoprotectant and it seems that to obtain better maturation rates, new cryopreservation techniques specific for immature bovine oocytes are needed.     

Osmotic and cryoprotective effects of a mixture of DMSO and ethylene glycol on rabbit morulae

Comparisons were made of the osmotic and cryoprotective effects on rabbit embryos preserved by vitrification with 2 solutions and by conventional freezing. Embryos obtained from rabbits killed 70 to 72 h after mating were used in the study (n = 948). Initially, toxicity of the 3 cryoprotectants was studied in fresh (unfrozen) embryos (n = 135). Subsequently, embryos placed in ethylene glycol (EG, 40% v/v; n = 88) and ethylene glycol with dimethyl sulfoxide (EG+DMSO, 20% v/v each, respectively; n = 344) were loaded into straws and plunged directly into liquid nitrogen. Embryos placed in 1.5 M DMSO and 20% heat inactivated rabbit serum were subjected to conventional freezing in a programmable freezer (control group, n = 363). The osmotic effect was estimated by measuring the changes in the embryonic and interzonal volume (crossectional area) and in the thickness of the mucin coat (n = 18). Cryoprotective effectivity was determined by development to the blastocyst stage in vitro, or birth of normal pups after transfer into synchronized recipients. Osmotic effects of cryoprotective solutions on embryonic and interzonal volume and mucin coat thickness were variable and overall not significant. Survival rate of cryopreserved embryos in vitro and development to blastocysts, was worst in the EG-treated embryos. Survival rate at birth was higher in vitrified vs frozen embryos. We conclude that rabbit morulae can be vitrified successfully in EG+DMSO medium.     

Factors affecting the survival,fertilization, and embryonic development of mouse oocytes after vitrification using glass capillaries

Cryopreservation of mammalian oocytes is an important way to provide a steady source of materials for research and practice of parthenogenetic activation, in vitro fertilization, and nuclear transfer. However, oocytes cryopreservation has not been common used, as there still are some problems waiting to be solved on the repeatability, safety, and validity. Then, it is necessary to investigate the damage occurred from vitrification and find a way to avoid or repair it. In this study, mouse mature oocytes were firstly pretreated in different equilibrium media, such as 5% ethylene glycol (EG) + 5% dimethyl sulfoxide (DMSO), 10% EG + 10% DMSO, and 15% EG + 15% DMSO in TCM199 supplemented with 20% fetal calf serum (FCS), for 1, 3, and 5 min, respectively, and then oocytes were transferred into vitrification solution (20% EG, 20% DMSO, 0.3 M sucrose, and 20% FCS in TCM199, M2, Dulbecco’s phosphate buffered saline, and 0.9% saline medium, respectively) and immediately loaded into glass capillaries to be plunged into liquid nitrogen. After storage from 1 h to 1 wk, they were diluted in stepwise sucrose solutions. The surviving oocytes were stained for cortical granule, meiotic spindles, and chromosomes. Oocytes without treatments were used as controls. The results showed that oocytes pretreated in 5% EG +5% DMSO group for 3–5 min or in 10% EG + 10% DMSO group for 1–3 min were better than other treatments. Oocytes vitrified in TCM199 as basic medium showed higher survival and better subsequent embryonic development than other groups. When the concentration of FCS in vitrification solution reduced below 15%, the rates of survival, fertilization, and developing to blastocyst declined dramatically. The inner diameter (0.6 mm) of glass capillaries and amount of vitrification solution (1–3 μl) achieved more rapid cooling and warming and so reduce the injury to oocytes. Cropreservation led to the exocytosis of cortical granule of oocytes (about 10%) and serious disturbance of microtubules and chromosomes. With 2 h incubation, the microtubules could repolymerize and the rate of fertilization in vitro was much higher than those of 1 and 3 h incubation groups. In conclusion, the protection of basic medium and FCS to oocytes during cryopreservation and sufficient cooling and warming rates using glass capillaries have profound effects on oocytes survival and subsequent embryonic development competence. The appropriate time for fertilization in vitro may be related to the recovery of spindles after incubation and avoiding ageing in the whole process.     

The successful double cryopreservation of rabbit (Oryctolagus cuniculus) semen in large volume using the directional freezing technique with reduced concentration of cryoprotectant

Using directional freezing, our objective was to cryopreserve rabbit semen and achieve fertility that was equal or higher than that achieved with conventional freezing. The working hypothesis was that controlling the ice-front propagation would allow reduction of DMSO concentration to <1M, in addition to the capability to freeze large volumes (2-10 mL). Moreover, single and double freezing of semen were used to demonstrate the abbreviated mechanical stress imparted by directional freezing. Single-cryopreserved semen from 15 males extended with 0% egg yolk/1.75 M DMSO, 15.3% egg yolk/0.88 M DMSO and 20% egg yolk/0M DMSO resulted in lower (P<0.05) mean+/-S.E.M. post-thaw motility (3.6+/-1.1, 28.5+/-2.8 and 36.3+/-1.8%, respectively) compared to fresh semen (73.3+/-1.2%). Semen from seven of these males, subject to double freezing using only egg yolk based extenders, resulted in post-thaw motilities of 18.1+/-2.2 and 16.4+/-3.3%. Despite the reduced functional parameters of cryopreserved semen, fertility and kindling rates of 73.9 and 56.5% for single frozen-thawed, and 28.6 and 35.7% for double frozen-thawed semen were achieved (with insemination of 98 females). There was no significant difference in fertility rate between fresh semen (87.5%) and semen that was single frozen-thawed with the 15.3% egg yolk/0.88 M DMSO extender (73.9%). In conclusion, cryopreservation of rabbit semen in large volumes using directional freezing achieved fertility rates similar to those achieved with fresh semen. Furthermore, acceptable fertility rates with double frozen-thawed semen could facilitate the future use of sex-sorted semen in rabbits.     

Comparison and avoidance of toxicity of penetrating cryoprotectants

The objective of this study was to elucidate the toxicity of widely used penetrating cryoprotective agents (CPAs) to mammalian oocytes. To this end, mouse metaphase II (M II) oocytes were exposed to 1.5 M solutions of dimethylsulfoxide (DMSO), ethylene glycol (EG), or propanediol (PROH) prepared in phosphate buffered saline (PBS) containing 10% fetal bovine serum. To address the time- and temperature-dependence of the CPA toxicity, M II oocytes were exposed to the aforementioned CPAs at room temperature (RT, ～23°C) and 37°C for 15 or 30 minutes. Subsequently, the toxicity of each CPA was evaluated by examining post-exposure survival, fertilization, embryonic development, chromosomal abnormalities, and parthenogenetic activation of treated oocytes. Untreated oocytes served as controls. Exposure of MII oocytes to 1.5 M DMSO or 1.5 M EG at RT for 15 min did not adversely affect any of the evaluated criteria. In contrast, 1.5 M PROH induced a significant increase in oocyte degeneration (54.2%) and parthenogenetic activation (16%) under same conditions. When the CPA exposure was performed at 37°C, the toxic effect of PROH further increased, resulting in lower survival (15%) and no fertilization while the toxicity of DMSO and EG was still insignificant. Nevertheless, it was possible to completely avoid the toxicity of PROH by decreasing its concentration to 0.75 M and combining it with 0.75 M DMSO to bring the total CPA concentration to a cryoprotective level. Moreover, combining lower concentrations (i.e., 0.75 M) of PROH and DMSO significantly improved the cryosurvival of MII oocytes compared to the equivalent concentration of DMSO alone. Taken together, our results suggest that from the perspective of CPA toxicity, DMSO and EG are safer to use in slow cooling protocols while a lower concentration of PROH can be combined with another CPA to avoid its toxicity and to improve the cryosurvival as well.     

Development of vitrified bovine secondary and primordial follicles in xenografts

The objective was to evaluate the effect of various vitrification conditions on the morphology of bovine secondary and primordial follicles, and to use xenografting to confirm their developmental ability. Secondary follicles were placed in vitrification solution containing 15% (v:v) ethylene glycol (EG), 15% (v:v) dimethyl sulfoxide (DMSO), 20% (v:v) fetal calf serum (FCS), and 0, 0.25, or 0.5 M sucrose at room temperature for 1 or 30 min, or at 4 °C for 30 min before being plunged into liquid nitrogen (LN₂). Ovarian tissues with primordial follicles were equilibrated in a solution containing 7.5% EG, 7.5% DMSO, and 20% FCS for 5 or 15 min, and then treated with a vitrification solution (15% EG, 15% DMSO, and 20% FCS) containing 0 or 0.5 M sucrose at room temperature for 1 min, and then plunged into LN₂. One week later, follicles and tissues were warmed, and morphology assessed histologically. Secondary follicles vitrified in sucrose-free solution had more oocytes with shrinkage of the nucleus and abnormal cytoplasm relative to those vitrified in sucrose-containing solution. When primordial follicles were equilibrated for 5 min and vitrified in sucrose-free solution, the percentage of morphologically normal primordial follicles was higher than in the other groups (P < 0.05). After 4 wk and 6 mo of xenografting of vitrified-warmed secondary and primordial follicles, respectively, in SCID mice, follicles developed to the antral stage and oocytes grew. In conclusion, bovine secondary follicles were successfully cryopreserved in sucrose-containing vitrification solutions and maintained their ability to develop to the antral stage and grow oocytes, whereas primordial follicles vitrified in sucrose-free solution maintained their morphology and developed to the antral stage, with oocyte growth.     

Successful vitrification of pronuclear-stage rabbit zygotes by minimum volume cooling procedure

Rabbit zygotes at the pronuclear-stage were cryopreserved by vitrification using a gel-loading tip (GL-tip), Cryoloop or Cryotop. In GL-tip and Cryoloop methods, zygotes were first exposed to 10% ethylene glycol (EG)+10% DMSO in TCM199+20% fetal bovine serum (FBS) for 2 min, and then equilibrated for 30 s in a vitrification solution composed of 20% EG+20% DMSO+0.6 M sucrose in TCM199+20% FBS. In Cryotop method, zygotes were first exposed to 7.5% EG+7.5% DMSO+20% FBS in TCM199 for 3 min, and then equilibrated for 1 min in a vitrification solution composed of 15% EG+15% DMSO+0.5 M sucrose+20% FBS in TCM199. In vitro culture of vitrified-warmed zygotes using GL-tip and Cryoloop resulted in low cleavage rates (2 and 5%, respectively) and no development into blastocysts. In contrast, zygotes vitrified-warmed using Cryotop exhibited higher proportions of cleavage (58%) and development into blastocysts (24%). When compacted morulae or early blastocysts were vitrified by these three procedures, 80-93% of them exhibited blastocoele expansion or zona hatching during the subsequent 48 h of culture. Use of Cryotop instead of GL-tip or Cryoloop for zygote vitrification, without changing conditions of solutions and periods for exposure, equilibration and post-warm dilution, resulted in cleavage and blastocyst development rates of 88 and 45%, respectively. A longer exposure time (10 min) of zygotes to 7.5% EG+7.5% DMSO+20% FBS in TCM199 resulted in higher proportions of zygotes cleaving (94%) and developing into blastocysts (51%) after Cryotop vitrification. Proportions of post-warm zygotes (10-min exposure group) and fresh control zygotes developing into newborn offspring were 36 and 53%, respectively. Pronuclear-stage rabbit zygotes were successfully cryopreserved by vitrification using the Cryotop method.     

不同防冻剂对兔胚胎干细胞慢速冷冻保存的影响

干细胞冷冻保存是干细胞研究和临床应用中的必需技术。为提高兔胚胎干细胞在慢速冻存过程中的保存效果,比较了二甲基亚砜(DMSO)和乙二醇(ethylene glycol,EG)对兔胚胎干细胞冷冻保护效果。对冷冻复苏后的细胞进行台盼蓝染色,并研究其胚胎干细胞分子特性,结果表明DMSO比EG具有更好的冷冻保护效果。再在以10%DMSO为基础的防冻液中添加膜稳定剂海藻糖(trehalose)或谷氨酰胺(glutamine),细胞冷冻复苏后结果显示,谷氨酰胺对兔胚胎干细胞有明显的冷冻保护作用,使细胞存活率从71%提高到83.7%。当谷氨酰胺浓度为0、5、10、20、40mmol/L分别加入防冻液中后,20mmol/L的谷氨酰胺具有最佳的冷冻保护效果。以上结果得出兔胚胎干细胞慢速冷冻的防冻液改进配方为:在胚胎干细胞培养液中添加10%DMSO 20mmol/L谷氨酰胺。     

Ultrastructural features of agouti (Dasyprocta aguti) preantral follicles cryopreserved using dimethyl sulfoxide, ethylene glycol and propanediol

The objective was to develop an efficient protocol for cryopreservation of agouti (Dasyprocta aguti) ovarian tissue. Agouti ovarian fragments were placed, for 10 min, in a solution containing MEM and fetal bovine serum plus 1.5 M dimethyl sulfoxide (DMSO), ethylene glycol (EG) or propanediol (PROH); some of those fragments were subsequently cryopreserved in a programmable freezer. After exposure and/or thawing, all samples were fixed in Carnoy prior to histological analysis. To evaluate ultrastructure, follicles from the control and all cryopreserved treatments were fixed in Karnovsky and processed for transmission electron microscopy. After exposure and freezing, there was a significant decrease in the percentage of morphologically normal preantral follicles in all treatments when compared to the control (92.67 ± 2.79, mean ± SD). However, there were no significant difference when the exposure and freezing procedures were compared using the same cryoprotectant. Moreover, there was no significant difference among cryoprotectants at the time of exposure (DMSO: 64.7 ± 3.8; EG: 70.7 ± 11.2, PROH: 63.3 ± 8.5) or after freezing (DMSO: 60.6 ± 3.6, EG: 64.0 ± 11.9; PROH: 62.0 ± 6.9). However, only follicles frozen with PROH had normal ultrastructure. In conclusion, preantral follicles enclosed in agouti ovarian tissue were successfully cryopreserved using 1.5 M PROH, with satisfactory maintenance of follicle morphology and ultrastructure.     

Survival and viability of vitrified in vitro and in vivo produced ovine blastocysts

Ovine blastocysts were produced by maturation, fertilization and in vitro culture (IVM/IVF/IVC) of oocytes from slaughtered adult and prepubertal ewes and collection from superovulated and inseminated adult animals. Dulbecco's PBS supplemented with 0.3 mM Na Pyruvate and 20% FCS was used as the basic cryopreservation solution. The embryos were exposed to the vitrification solution as follows: 10% glycerol (G) for 5 min, then 10% G +20% ethylene glycol (EG) for 5 min. Embryos were placed into 25% G + 25% EG in the center of 0.25- mL straws and plunged immediately into LN2. Warming was done by placing the straws into a water bath at 37 degrees C for 20 sec, and their contents were expelled into a 0.5 M sucrose solution for 3 min; the embryos were then transferred into 0.25 M and 0.125 M sucrose solution for 3 min each. Warmed blastocysts were transferred to the culture medium for 24 h. Survival was defined as the re-expansion of the blastocoele. All surviving blastocysts were transferred to synchronized recipient ewes, and the pregnancy was allowed to go to term. Of 68 vitrified in vitro produced blastocysts, 46 re-expanded (67.6%) and 10 lambs were born (14.7%). From the 62 in vivo derived and vitrified embryos, 52 re-expanded (83.8%) and 39 lambs were born (62.9%). The lambing rate of in vitro produced fresh transfer embryos was 40% (20 lambs/50 blastocysts transferred), and of the 32 in vivo derived blastocysts and transferred fresh, 26 lambs were born (81.2%). The results indicate that in vitro produced embryos can be successfully cryopreserved by vitrification.