Effect of estrous cow serum during bovine embryo culture on blastocyst development and cryotolerance after slow freezing or vitrification

The present study investigated the effect of estrous cow serum (ECS) during culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. Embryos were derived from in vitro maturation (IVM) and in vitro fertilization (IVF) of abbatoir-derived oocytes. At Day 3, embryos were cultured in three different media: Charles Ronsenkrans medium + amino acids (CR1aa; without bovine serum albumin (BSA)) + 5% estrous cow serum (CR1-ECS), CR1aa + 3 mg/mL BSA (CR1-BSA) or CR1aa + 5% ECS + 3 mg/mL BSA (CR1-ECS-BSA). At 7.5 d post-insemination (PI), blastocyst yield and quality were evaluated; blastocysts and expanded blastocysts from each media were cryopreserved by Open Pulled Straw (OPS) vitrification method or slow freezing (1.5 M ethylene glycol, EM). Total blastocyst yield did not differ among CR1-ECS, CR1-BSA and CR1-ECS-BSA (30.9, 33.1 and 32.9%, respectively, P < 0.05). Embryo survival (hatching rate) was higher in vitrified versus slow-frozen embryos (43% versus 12%, respectively, P < 0.01), and in embryos cultured in CR1-BSA (40.3%) compared with those cultured in serum-containing media (CR1-ECS, 21.5% and CR1-ECS-BSA, 19.8%; P < 0.01). In conclusion: (a) it was possible to produce in vitro bovine embryos in serum-free culture medium without affecting blastocyst yield and quality; (b) serum-free medium produced the best quality embryos (in terms of post-cryopreservation survival); and (c) vitrification yielded the highest post-cryopreservation survival rates, regardless of the presence of serum in the culture medium.     

The effect of equilibration time on survival and development rates of mouse pronuclear-stage embryos vitrified in solid surface (SSV) and convential straws: in vitro and in vivo evaluations

The objective of this study was to improve the efficiency of cryopreservation of pronuclear-stage (PN) mouse embryos. A novel vitrification technique (solid surface vitrification, SSV) was compared with a convential one in straws both for cryosurvival and obtaining progeny from cryopreserved PN mouse embryos. In the SSV method, 15-20 PN embryos were exposed to vitrification solutions for approximately 20 sec after equilibration, and then they were dropped in 2 microl drops onto a pre-cooled (-150 to -180 degrees C) metal surface. In the straws method, groups of 5-10 PN embryos were loaded in a single straw after equilibration. In experiment I, it was compared the effect of the vitrification solutions alone, without vitrification. No reduction was detected in survival, cleavage and blastocysts rates and the lowest development rate was obtained from hatched blastocyst for 20 min equilibration (24.5%). In experiment II, SSV method resulted in significantly higher survival and cleavage rates than that of in-straw vitrified 15-20 min group (87% vs. 60%, 83% vs. 67%, respectively; P < 0.05). There were no statistical differences among any of the blastocyts groups. However, there was a statistical difference in hatched blastocysts between 15 to 5, 10, and 20 min (P < 0.05). In experiment III, it was found no major effect among equilibration time periods in toxicity groups according to the mean cell number of blastocysts developed from PN embryos. But, there was a significant differences between 15 min SSV and 10 min in straw vitrified according to the mean cell number of blastocysts developed from PN embryos following vitrification (P < 0.05). The good results were obtained from 15 min equilibration group for SSV and 10 min equilibration group for straw vitrification. In the last experiment, embryo transfer after vitrification and toxicity was investigated. There were significant differences between SSV and straw just on the rate of pups born (30% and 20.5% respectively; P < 0.05). In conclusion, vitrification of PN mouse embryos by SSV can result in high rates of in vitro development to expanded and hatched blastocyst stage and in vivo development to live pups.     

Effects of bovine serum proteins in culture medium on post-warming survival of bovine blastocysts developed in vitro

Experiments were conducted to investigate the factors affecting the survival of bovine blastocysts produced in vitro after cryopreservation by vitrification. Zygotes were obtained by in vitro maturation and fertilization of oocytes. Embryos used in this study were developed in vitro at Day 7 and 8 (Day 0 = insemination day) in modified synthetic oviduct fluid medium supplemented with calf serum or BSA. Embryos were cryopreserved in a two-step protocol consisting of exposure to 10% ethylene glycol for 5 min, followed by the original vitrification solution (designated as VS) consisting of 40% (v/v) ethylene glycol, 6% (w/v) polyethylene glycol and 0.5 M sucrose in phosphate-buffered saline for 1 min. After warming, embryos were cultured in modified TCM-199 for an in vitro survival assay. The highest survival rate was obtained from the warmed embryos developed at Day 7 in medium supplemented with BSA (82.6%), and there were significant differences between results with calf scrum and BSA treatment (42.4 and 70.7%, respectively; P < 0.01). However, there were no significant differences in the cell numbers of embryos among the treatments. These results suggest that the survival of embryos developed in medium with BSA is superior to that of embryos developed in medium containing calf serum, although the cell numbers of the embryos developed under both media were similar.     

The survival of whole and bisected rabbit morulae after cryopreservation by the vitrification method

The survival of whole and bisected rabbit morulae cryopreserved by the vitrification method was investigated. The embryos were loaded in a column of vitrification solution (VS, a mixture of 25% glycerol and 25% 1, 2-propanediol in PBS+16% calf serum), which was located between two columns of 1 M sucrose solution in a plastic straw. The embryos were frozen by being plunged into liquid nitrogen and thawed in a water bath at 20 degrees C. Two methods of loading embryos into straws were used: the single and double column vitrification solution methods. The embryonic survival rates between these two methods were compared. Seventy-one (86.6%) out of 82 morulae vitrified in double column straws developed into the blastocyst stage in vitro. Eleven (18.3%) live fetuses were obtained after the transfer of 60 frozen-thawed morulae to four recipients. By contrast, the survival rate (36.5%, 27 74 ) of embryos vitrified in the single column straws was significantly lower (P<0.05). The vitrification solution of the single column straws became opaque, indicating ice-crystal formation, upon thawing in 5 of 11 straws, which was assumed to have damaged the embryos. More than 80% (29 36 ) of the bisected morulae frozen and thawed in the double column straws developed to the blastocyst stage in vitro when cryoprotectant was diluted stepwise with 1 M and 0.25 M sucrose solution. When the cryoprotectant was removed by one-step dilution with 1 M sucrose solution, swelling in blastomeres was observed and the development rate of the recovered embryos decreased (45.8%, 11 24 ). These results indicate that the vitrification method employed in our experiment is not only efficient for the cryopreservation of rabbit morulae, but it can also be used for the preservation of bisected rabbit morulae, which had not been successful using previous methods.     

牛血清白蛋白对小鼠原核期胚胎玻璃化冷冻的影响

以小鼠原核期胚胎为对象,以胚胎的存活率、卵裂率、囊胚率以及囊胚细胞数作为检测指标,在M2液的基础上添加8种浓度(0,2,4,8,16,32,64,96mg/mL)牛血清白蛋白(BSA)配置防冻液,探讨防冻液和玻璃化冷冻后对胚胎发育的影响。BSA防冻液对胚胎发育影响的实验结果表明,8个浓度组间以及与对照组间胚胎的卵裂率、囊胚率以及囊胚细胞数无显著差异(P>0.05),说明在防冻液中加入一定浓度的BSA对小鼠胚胎无毒性作用。防冻液经玻璃化冷冻后对胚胎发育影响的实验表明,8个浓度组间冷冻胚胎复苏后的存活率、卵裂率、囊胚率及囊胚细胞数无显著差异(P>0.05)。表明BSA在这种防冻液中没有明显的保护作用。从经济、实用、生物安全角度考虑,不支持在玻璃化防冻液中添加BSA。     

Production of transgenic mice from vitrified pronuclear-stage embryos.

Cryopreservation of pronuclear-stage embryos would be useful for transgenic technology and genome preservation purposes. We compared a novel vitrification technique (solid surface vitrification, SSV) with another vitrification method in straws for cryosurvival and to generate transgenic progeny from cryopreserved mouse zygotes following microinjection. The SSV solution consisted of 35% ethylene glycol (EG), 5% polyvinyl-pyrrolidone (PVP), and 0.4 M trehalose in M2 supplemented with 4 mg/ml BSA; the in straw vitrification solution was 7 M EG in M2 plus BSA. In experiment I, we compared the effect of the vitrification solutions alone, without cooling. No reduction was detected in survival and cleavage rates. In experiment II, SSV yielded a significantly higher percentage of morphologically normal zygotes (96%) that also cleaved at significantly higher rates (80%) when compared to that following "in straw" vitrification (68 and 66%, respectively). Cleavage rate in the non-vitrified control group (93%) was significantly higher than that of both vitrified groups. Following embryo transfer, there was no difference in the rate of pups obtained from the SSV, "in straw" vitrified, and control groups (97/457, 21%; 15/75, 20% and 56/209, 27%, respectively). In experiment III, SSV vitrified and fresh embryos were used for pronuclear DNA injection. Survival rate of vitrified embryos after microinjection was reduced compared to nonvitrified ones (64 vs. 72%, respectively; P < 0.05); however, development to two-cell stage was not different (76 vs. 72%, respectively). Following embryo transfer of vitrified vs. fresh microinjected embryos, in both cases 10% live pups were generated, including transgenic pups. The results demonstrated that the efficiency of generating transgenic pups from SSV vitrified pronuclear zygotes is comparable to that from fresh embryos.     

The protective action of polyvinyl alcohol during rapid-freezing of mouse embryos

Biological products like serum and BSA are routinely used in embryo freezing solutions. These products are undefined and can potentially expose the embryos to infectious agents. Therefore, this experiment was designed to evaluate in vitro and in vivo survival of mouse embryos frozen in solutions supplemented with a chemically defined macromolecule, polyvinyl alcohol (PVA). Morula-stage embryos from superovulated mice were collected, frozen by a rapid freezing procedure, and cryoprotectant diluted out (after thawing) in media supplemented with either 10% fetal calf serum (FCS), 0.1 mg/mL PVA, or a combination of 10% FCS and 0.1 mg/mL PVA. Frozen-thawed (good to excellent quality) and nonfrozen (control, collected in FCS supplemented medium) embryos were cultured in medium M16 (32) supplemented with either 4 mg/mL BSA or 0.1 mg/mL PVA for 72 h. Embryos frozen in solutions supplemented with FCS or PVA and nonfrozen embryos were transferred to pseudopregnant recipients. Recipients were humanly killed on Day 15 after transfer, and the rate of implantation and percentage of live fetuses were recorded. The supplementation of collection, freezing and cryoprotectant dilution solutions with FCS, PVA or FCS plus PVA did not influence (P > 0.05) the rate of survival and in vitro development of embryos to hatched/hatching blastocyst-stage. However, a higher (P < 0.01) in vitro development rate to hatching/hatched-stage was recorded when frozen-thawed embryos were cultured in medium supplemented with BSA than with PVA. There was no difference (P > 0.05) in the rate of implantation (68 vs 72%) or percentage of live fetuses (62 vs 60%) between pregnant recipients with embryos frozen in medium with FCS or PVA. The rate of implantation and development of embryos frozen in medium supplemented with PVA or FCS was comparable (P > 0.05) to that of nonfrozen embryos. It may be concluded that PVA can be substituted for FCS in medium for freezing mouse embryos; however, it can not be completely substituted for BSA in the in vitro culture of embryos to the hatched blastocyst stage.     

In vitro and in vivo viability of vitrified and non-vitrified embryos derived from eCG and FSH treatment in rabbit does

This study aimed to evaluate the in vitro and in vivo viability of vitrified and non-vitrified embryos derived from eCG and FSH treatments in rabbit does. Ninety-six nulliparous does were randomly subjected to consecutive superovulation treatments with eCG (20 IU/kg body weight intramuscularly (i.m.), eCG group), FSH (3 x 0.6 mg/doe at 24 h intervals i.m., FSH group), or without superovulation treatment (control group). Does were artificially inseminated 3 days later and ovulation was induced immediately by hCG (75 IU/doe intravenous). Seven experimental groups were differentiated: first FSH and eCG treatment, second FSH and eCG treatment, eCG-interchanged group (does with previous FSH treatment), FSH-interchanged group (does with previous eCG treatments) and control group. Embryos were collected in vivo by laparoscopy 76-80 h post-insemination in the first and second recovery cycles and post mortem in the third recovery cycles. The ovulation rate was significantly higher in does treated with the first-FSH than in those treated with eCG or in control does (25.2+/-2.0 versus 19.2+/-1.4 to 11.0+/-1.5, and 12.2+/-1.2, first-FSH, first-eCG to second-eCG and control groups, respectively, P < 0.05). Significant differences were observed in the total recovery influenced by ovulation rate in each group (20.3+/-2.2 to 9.4+/-1.2, first-FSH to control groups). Embryo donor rate (donor with at least one normal embryo) was similar among groups with an overall of 75.1%. The number of normal embryos recovered per doe with at least one normal embryo increased significantly in relation to ovulation rate (17.7+/-2.2 to 8.41+/-3, first-FSH and control groups). The vitrification of embryos negatively affected their in vitro development to hatched blastocyst in all groups (88.1% versus 48%, P > 0.05). However, after embryo transfer, this negative effect was only observed in superovulated vitrified embryos (16.8 and 12.8% versus 39.4% total born rate from eCG, FSH and control vitrified groups, P < 0.05). Results indicated that the primary treatments with eCG or FSH increased the number of normal embryos recovered per donor doe, but these embryos are more sensitive to vitrification protocols.     

Banking of embryos of mutated, paralytic tremor rabbit by means of vitrification

Cryopreservation enables banking of embryos for future use in medicine and in animal breeding. It also enables protection of germ plasm of endangered species and unique strains or lanes of laboratory animals. This paper describes an example of employing a vitrification method for banking of embryos of a unique lane of rabbit. The paralytic tremor (pt) rabbit is an X-linked recessive mutant lane of the Chinchilla breed characterized by hypomyelination of the central nervous system. In order to obtain a sufficient number of embryos, pt females were subjected to superovulation and surgical embryo collection. All suitable embryos were vitrified in 0.25 mL insemination straws in a modified EFS vitrification solution comprised of ethylene glycol (40%), Ficoll 70 (18%) and sucrose (0.3 M) in Hepes buffered TCM medium containing 20% fetal calf serum. In order to assess the efficiency of the vitrification procedure, a representative portion of vitrified embryos was warmed after a period of storage. Warmed embryos were subjected to in vitro culture for 72 h or were transferred to the uterus of synchronized recipients. The majority of the 141 warmed embryos survived vitrification and 100/141 (71%) developed to the blastocyst stage. Moreover, out of an additional 34 warmed embryos transferred to four recipients, eight (23.5%) developed to term and seven live pups were born. Six of the rabbit pups exhibited paralytic tremor symptoms typical for the pt lane. Although the overall efficiency of the vitrification method was lower compared with the effects usually achieved for 'healthy' embryos, results presented confirm the real possibility of the future restoration of the colony of pt rabbit, if sufficient number of embryos are cryopreserved.     

Conventional slow freezing, vitrification and open pulled straw (OPS) vitrification of rabbit embryos

Three different methods of cryopreservation viz., conventional slow freezing, vitrification and open pulled straw vitrification were compared for their ability to support post thaw in vitro and in vivo development of rabbit embryos. Morula stage rabbit embryos were collected from super-ovulated donor does. They were randomly allocated to different freezing methods and stored up to 3 months in liquid nitrogen. After thawing and removal of cryoprotectants, embryos exhibiting intact zona pellucida and uniform blastomeres were considered suitable for in vitro culture and/or transfer. Three to five cryopreserved embryos placed in approximately 1 ml of culture medium (TCM 199 supplemented with foetal calf serum and antibiotics) were incubated for up to 72 h under humidified atmosphere of 5% CO2 in air at 39 degrees C. Development to hatched blastocyst stage was considered the initial indicator of success of cryopreservation of embryos. Of the embryos cryopreserved by programmed freezing, open pulled straw vitrification, vitrification-55 h pc and vitrification-72 h pc 55, 71, 17 and 48%, respectively, developed into hatched blastocysts. Similarly 19, 29, and 4% of embryos cryopreserved by programmed freezing, open pulled straw vitrification and vitrification -72 h pc developed into live offspring on transfer to recipient does. This is the first report on open pulled straw vitrification of rabbit embryos. Present results, suggest that (a) open pulled straw vitrification supports better in vitro survival of frozen thawed rabbit morulae; (b) both programmed freezing and OPS are similar but superior to vitirification in supporting in vivo survival of frozen thawed rabbit embryos.     

The cell agglutination agent, phytohemagglutinin-L, improves the efficiency of somatic nuclear transfer cloning in cattle (Bos taurus)

One of the several factors that contribute to the low efficiency of mammalian somatic cloning is poor fusion between the small somatic donor cell and the large recipient oocyte. This study was designed to test phytohemagglutinin (PHA) agglutination activity on fusion rate, and subsequent developmental potential of cloned bovine embryos. The toxicity of PHA was established by examining its effects on the development of parthenogenetic bovine oocytes treated with different doses (Experiment 1), and for different durations (Experiment 2). The effective dose and duration of PHA treatment (150 microg/mL, 20 min incubation) was selected and used to compare membrane fusion efficiency and embryo development following somatic cell nuclear transfer (Experiment 3). Cloning with somatic donor fibroblasts versus cumulus cells was also compared, both with and without PHA treatment (150 microg/mL, 20 min). Fusion rate of nuclear donor fibroblasts, after phytohemagglutinin treatment, was increased from 33 to 61% (P < 0.05), and from 59 to 88% (P < 0.05) with cumulus cell nuclear donors. The nuclear transfer (NT) efficiency per oocyte used was improved following PHA treatment, for both fibroblast (13% versus 22%) as well as cumulus cells (17% versus 34%; P < 0.05). The cloned embryos, both with and without PHA treatment, were subjected to vitrification and embryo transfer testing, and resulted in similar survival (approximately 90% hatching) and pregnancy rates (17-25%). Three calves were born following vitrification and embryo transfer of these embryos; two from the PHA-treated group, and one from non-PHA control group. We concluded that PHA treatment significantly improved the fusion efficiency of somatic NT in cattle, and therefore, increased the development of cloned blastocysts. Furthermore, within a determined range of dose and duration, PHA had no detrimental effect on embryo survival post-vitrification, nor on pregnancy or calving rates following embryo transfer.     

Comparison on in vitro fertilized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrification

The objectives of this study were to identify an improved in vitro cell-free embryo culture system and to compare post-warming development of in vitro produced (IVP) bovine embryos following vitrification versus slow freezing. In Experiment 1, non-selected presumptive zygotes were randomly allocated to four medium treatments without co-culture: (1) SOF + 5% FCS for 9 days; (2) KSOM + 0.1% BSA for 4 days and then KSOM + 1% BSA to Day 9; (3) SOF + 5% FCS for 4 days and then KSOM + 1% BSA to Day 9; and (4) KSOM + 0.1% BSA for 4 days and then SOF + 5% FCS to Day 9. Treatment 4 (sequential KSOM-SOF culture system) improved (P > 0.05) morulae (47%), early blastocysts (26%), Day-7 blastocysts (36%), cell numbers, as well as total hatching rate (79%) compared to KSOM alone (Treatment 2). Embryos cultured in KSOM + BSA alone developed slowly and most of them hatched late on Day 9, compared to other treatments. In Experiment 2, the sequential KSOM-SOF culture system was used and Day-7 blastocysts were subjected to following cryopreservation comparison: (1) vitrification (VS3a, 6.5 M glycerol); or (2) slow freezing (1.36 M glycerol). Warmed embryos were cultured in SOF with 7.5% FCS. Higher embryo development and hatching rates (P < 0.05) were obtained by vitrification at 6h (71%), 24h (64%), and 48h (60%) post-warming compared to slow freezing (48, 40, and 31%, respectively). Following transfer of vitrified embryos to synchronized recipients, a 30% pregnancy rate was obtained. In conclusion, replacing KSOM with SOF after 4 days of culture produced better quality blastocysts. Vitrification using VS3a may be used more effectively to cryopreserve in vitro produced embryos than the conventional slow freezing method.     

Effects of fetal calf serum, phenazine ethosulfate and either glucose or fructose during in vitro culture of bovine embryos on embryonic development after cryopreservation

This study investigated effects of hexoses, fetal calf serum (FCS), and phenazine ethosulfate (PES) during the culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. The basal, control medium was chemically defined (CDM) plus 0.5% fatty acid-free BSA. In vitro-produced bovine zygotes were cultured in CDM-1 with 0.5 mM glucose; after 60 hr, 8-cell embryos were cultured 4.5 days in CDM-2. The 8-cell embryos were randomly allocated to a 2 x 3 x 2 x 3 factorial experimental design with two energy substrates (2 mM glucose or fructose); three additives (0.3 microM PES, 10% FCS, and control); two cryopreservation methods using no animal products (conventional slow freezing or vitrification); and semen from three bulls with two replicates for each bull. A total of 1,107 blastocysts were produced. Fructose resulted in 13% more blastocysts per oocyte than glucose (37.2% vs. 32.9%), and per 8-cell embryo (51.3% vs. 45.3%; P < 0.01). No differences were found for additives (P > 0.1) control, FCS, or PES for blastocysts per oocyte or per 8-cell embryo. There was a significant interaction (P < 0.05) between additives and hexoses for blastocyst production; although trends were similar, the benefit of fructose compared to glucose was greater for controls than for FCS or PES. Culture of embryos with PES, which reduces cytoplasmic lipid content, improved cryotolerance of bovine embryos; post-cryopreservation survival of blastocysts averaged over vitrification and slow freezing (between which there was no difference) was 91.9%, 84.9%, and 60.2% of unfrozen controls (P < 0.01) for PES, control, and FCS groups, respectively.     

Improved vitrification method allowing direct transfer of goat embryos

The aim of this study was to design a vitrification method suited to field embryo transfer experiments in goat. In a first experiment, a standard vitrification protocol, previously designed for sheep embryos was compared to slow freezing of goat embryos. No significant difference was observed on kidding rate (48% versus 69%, respectively), nor on embryo survival rate (35% versus 45%). Second experiment: all embryos were vitrified. After warming, embryos were either transferred directly (direct transfer), or after in vitro dilution of the cryoprotectants (conventional transfer). The kidding rate was not affected by the transfer method (38% versus 23%, respectively). However, embryo survival rate tended to be higher after direct transfer (26% versus 14%). Third experiment: OPS vitrification was compared to standard vitrification. The kidding rate was not affected (22% versus 39%, respectively), but the embryo survival rate was lower after OPS (14% versus 28%). Fourth experiment: 0.4M sucrose was added with cryoprotectants in vitrification. The kidding rate after direct transfer was significantly enhanced after addition of sucrose (56% versus 27%, respectively), whereas embryo survival rate was not significantly affected (32% versus 18%). Fifth experiment: vitrification with sucrose supplementation was compared to slow freezing. No significant difference was observed after direct transfer on kidding rate (52% versus 31%, respectively), but embryo survival rate tended to be higher after vitrification (34% versus 21%). In conclusion, our results indicate that addition of 0.4M sucrose in association with direct transfer improves significantly the viability of goat vitrified embryos.     

Stage-dependent viability of vitrified rabbit embryos

The aim of the work was to determine the susceptibility of rabbit embryos to vitrification at different developmental stages. The experiment was carried out on 676 embryos at 1-, 2- and 8-to 16-cell stages as well as the morula and blastocyst stages. As a vitrification medium, a mixture of 30% 1,2-propanediol + 30% glycerol (Solution I), or 35% 1,2-propanediol + 35% glycerol (Solution II), was used. The embryos were frozen in glass ampules placed in nitrogen vapour for 5 min before being plunged into liquid nitrogen. Dilution after rapid thawing was done in one step in a 1-M sucrose solution. After vitrification in Solution I, none of the 1- or 2-cell embryos survived, whereas the survival rate of 8-to 16-cell embryos, morula and blastocysts, was 23.0, 82.7 and 78.5%, respectively. After vitrification in Solution II, the survival rate of 1-, 2- and 8-to 16-cell embryos was 20.0, 43.8 and 92.9%, respectively. The proportion of live offspring on the Day 28 after transfer of 68 vitrified morula was 26.5% compared with 24.0% in the control group. Thus, the proposed vitrification procedures can be useful in the cryopreservation of rabbit embryos.     

Cryopreservation of porcine embryos derived from in vitro-matured oocytes

This study describes a cryopreservation method for porcine in vitro-produced (IVP) embryos using as a model parthenogenetic embryos derived from in vitro-matured (IVM) oocytes. IVP embryos at the expanded blastocyst stage were cryopreserved by vitrification using the minimum volume cooling (MVC) method and exhibited an embryo survival rate of 41.2%. Survival was then significantly improved (83.3%, P < 0.05) by decreasing the amount of cytoplasmic lipid droplets (delipation) prior to vitrification. IVP embryos at the 4-cell stage also survived cryopreservation when vitrified after delipation (survival rate, 36.0%), whereas post-thaw survival of nondelipated embryos was quite low (9.7%). Furthermore, it was demonstrated that porcine IVP morulae can be cryopreserved by vitrification following delipation by a noninvasive method (survival rate, 82.5%). These results clearly confirm that porcine embryos derived from IVM oocytes can be effectively cryopreserved with high embryo survival using the MVC method in conjunction with delipation.     

Development of Cheaper Embryo Vitrification Device Using the Minimum Volume Method

This study was designed to compare the efficiency of the Cryotop and Calibrated plastic inoculation loop (CPIL) devices for vitrification of rabbit embryos on in vitro development and implantation rate, offspring rate at birth and embryonic and fetal losses. CPIL is a simple tool used mainly by microbiologists to retrieve an inoculum from a culture of microorganisms. In experiment 1, embryos were vitrified using a Cryotop device and a CPIL device. There were no significant differences in hatched/hatching blastocyst stage rates after 48 h of culture among the vitrified groups (62±4.7% and 62±4.9%, respectively); however, the rates were significantly lower (P<0.05) than those of the fresh group (95±3.4%). In experiment 2, vitrified embryos were transferred using laparoscopic technique. The number of implanted embryos was estimated by laparoscopy as number of implantation sites at day 14 of gestation. At birth, total offspring were recorded. Embryonic and fetal losses were calculated as the difference between implanted embryos and embryos transferred and total born at birth and implanted embryos, respectively. The rate of implantation and development to term was similar between both vitrification devices (56±7.2% and 50±6.8% for implantation rate and 40±7.1% and 35±6.5% for offspring rate at birth); but significantly lower than in the fresh group (78±6.6% for implantation rate and 70±7.2% for offspring rate at birth, P<0.05). Likewise, embryonic losses were similar between both vitrification devices (44±7.2% and 50±6.8%), but significantly higher than in the fresh group (23±6.6%, P < 0.05). However, fetal losses were similar between groups (10±4.4%, 15±4.8% and 8±4.2%, for vitrified, Cryotop or CPIL and fresh, respectively). These results indicate that the CPIL device is as effective as the Cryotop device for vitrification of rabbit embryos, but at a cost of €0.05 per device.     

Improved cryopreservation of bovine preimplantation embryos cultured in chemically defined medium

The aim of this study was to examine the effects of modifications to a standard slow freezing protocol on the viability of in vitro produced bovine embryos. Bovine oocytes were matured, fertilized with frozen-thawed semen, and presumptive zygotes cultured in defined two-step culture media. The standard freezing medium was 1.5M ethylene glycol (EG), 0.1M sucrose, 10% fetal bovine serum (FBS) in Dulbecco's phosphate buffered saline (D-PBS). A preliminary trial showed that in vitro produced embryos cryopreserved in this medium had a survival rate of 74.6% at 24h and 53.5% at 48 h post-thaw. Experiment 1 studied the effects of omitting the sucrose supplement or replacing it with 0.1M xylose. In Experiment 2, the effects of partial (0%, 25% or 50%) or total (100%) replacement of sodium chloride with choline chloride in the cryopreservation medium were examined (the medium with 100% replacement was designated CJ1). The effects of replacing the 10% FBS with 0.4% BSA or 0.4% lipid-rich BSA (Albumax I) in CJ1 was studied in Experiment 3. In Experiment 4, pregnancy/calving rates following the post-thaw transfer of in vitro produced embryos cryopreserved in the standard freezing medium were compared with those of in vitro and in vivo produced embryos cryopreserved in the improved medium (Albumax I in CJ1). Supplementation of the cryopreservation medium with 0.1M sucrose resulted in higher post-thaw survival rates at 24 h (71.3% versus 53.5 and 51.7%; P<0.05), 48 h (51.1% versus 45.3 and 40.2%), and 72 h (34.0% versus 24.4 and 23.0%) than 0.1M xylose or no supplement, respectively, in Experiment 1. Experiment 2 showed that embryos cryopreserved in the standard medium had poorer survival rates at 24 h (72.8% versus 86.5%; P<0.05), 48 h (53.1% versus 66.3%) or 72 h (28.4% versus 44.9%) than those frozen in CJ1. The post-thaw survival rate of embryos frozen in medium supplemented with Albumax I was better than that for the FBS or BSA supplements at 24h (92.0% versus 90.7 and 87.3%), 48 h (87.3% versus 76.9 and 70.9%; P<0.05), and 72 h (70.4% versus 49.1 and 46 4%; P<0.05; Experiment 3). In Experiment 4, in vitro produced embryos cryopreserved in CJ1 medium supplemented with Albumax I resulted in higher pregnancy rates at Day 35 (31.9% versus 22.9%) and Day 60 (24.1% versus 14.3%) of gestation, and calving rates (22.6% versus 10.0%; P<0.05) than similar embryos frozen in the standard medium. However, in vivo produced embryos cryopreserved in Albumax I in CJ1 resulted in higher pregnancy rates at Day 35 (50.7%; P<0.05) and Day 60 (45.1%; P<0.05) of gestation, and calving rate (43.7%; P<0.05). It was concluded that modification of the freezing medium by addition of lipid-rich BSA and replacing sodium chloride with choline chloride improves the post-thaw survival of in vitro produced embryos, and their viability post-transfer.     

The effect of media, serum and temperature on in vitro survival of bovine blastocysts after Open Pulled Straw (OPS) vitrification

The recently introduced Open Pulled Straw (OPS) vitrification technique has successfully been used for cryopreserving porcine embryos as well as for bovine embryos and oocytes. The aim of this work is to investigate several factors on the in vitro survival of bovine blastocysts. In 5 experiments, a total of 862 in vitro produced blastocysts and expanded blastocysts was vitrified and warmed using the OPS technology, then cultured in vitro for an additional 3 days. The culture medium in Experiments 1 to 4 was SOFaa with supplements and 5% calf serum (CS). In Experiment 1, the replacement of TCM-199 + 20% CS with PBS + 20% CS in the holding medium during vitrification and warming did not result in significant differences in the re-expansion (92 vs 95%) and hatching rates (79 vs 72%). In Experiment 2, the PBS holding medium was supplemented with either 20% CS, 5 mg/mL bovine serum albumin (BSA) or 3 mg/mL polyvinylalcohol (PVA). Although the re-expansion rates did not differ (98, 95 and 93%, respectively), there was a decrease in the hatching rate after vitrification with PVA (77 and 78 vs 51%, respectively). In Experiment 3, the influence of temperature of equilibration media prior to and rehydration media after the vitrification was investigated. When the temperature of these media was adjusted to 20 degrees C instead of the standard 35 degrees C, both the re-expansion and the hatching rates decreased markedly. However, increasing the time of equilibration with the diluted cryoprotectant solution at 20 degrees C eliminated these differences. In Experiment 4, the ethylene-glycol and dimethyl sulfoxide cryoprotectant mixture was replaced with ethylene glycol-ficoll-trehalose solution. No difference in the re-expansion (89 vs 96%, respectively) or hatching rate (79 vs 84%, respectively) was detected. In Experiment 5, the vitrified-warmed blastocysts were cultured in SOFaa medium supplemented with 5% CS or 5 mg/mL BSA. Although the re-expansion rates were identical in the 2 groups (95%), the hatching rates were lower when embryos were cultured in BSA (71 and 47%, respectively). These findings indicated the possible broader application for OPS, as they demonstrated that the physical advantages of rapid cooling and warming may be accompanied by different chemical composition (holding media, cryoprotective additives) according to the requirements of the biological structure. Our study also shows the need for serum supplementation of the medium for hatching to occur after OPS vitrification.     

Effect of biopsy and vitrification on in vitro survival of ovine embryos at different stages of development

The objective of the present study was to assess the in vitro viability of ovine embryos at different stages of development after combining cell sampling and vitrification. Precompacted morulae, compacted morulae and blastocysts were obtained from superovulated Sarda ewes at 4, 5 or 6 d following insemination. Embryo cell biopsy was carried out in a 100-microl drop of PBS + 10% fetal calf serum (FCS) with 10 micromol nocodazole and 7.5 microg/ml cytochalasin-b by aspiration (3-5 cells). Embryos were cryopreserved at room temperature after exposure of 2 solutions for 5 min, transferred into a vitrification solution, loaded into the center of 0.25-ml straws separated by air bubbles from 2 columns of sucrose 0.5 M and plunged immediately into liquid nitrogen. In Experiment 1, the in vitro viability of manipulated or vitrified embryos after in vitro co-culture in TCM 199 medium with 10% FCS and sheep oviductal epithelial cells (SOEC) in 5% CO2 humidified atmosphere in air at 39 degrees C was significantly lower (P < 0.05 and P < 0.01, respectively) at precompacted morula (60 and 30%) and compacted morula (62 and 39%) stages than intact embryos at the same stages (87 and 88%). No differences were found at the blastocyst stage. In Experiment 2, the in vitro survival rate of precompacted morulae which were manipulated and immediately vitrified was lower (P < 0.05) than in those manipulated and, after a temporary period of culture, vitrified at blastocyst stage (21 vs 48%); while no differences were found at compacted morula and blastocyst stages. The results show that 1) the stage of development influences the subsequent in vitro viability of manipulated and vitrified ovine embryos, 2) temporary culture after manipulation and before vitrification improves the in vitro viability of embryos, and 3) the hole in the zona pellucida resulting from biopsy does not affect blastocyst survival after subsequent vitrification.