scholarly journals 143 IN VITRO DEVELOPMENT OF OVINE OOCYTES FROM EWES OF CONTRASTING VITAMIN B12 STATUS

2005 ◽  
Vol 17 (2) ◽  
pp. 222
Author(s):  
L.M. Mitchell ◽  
G. McCallum ◽  
K. Mackie ◽  
M. Ewen ◽  
A. Ainslie ◽  
...  
Keyword(s):  
Vitamin B12 ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Culture Period ◽  
Deficient Diet ◽  
In Vitro Development ◽  
Cell Numbers ◽  
Vitro Development

Suboptimal circulating concentrations of vitamin B12 are commonly found in cattle and sheep grazing cobalt-deficient pastures. Vitamin B12 is a co-factor for enzymes involved in energy metabolism (methylmalonyl CoA mutase) and DNA synthesis/methylation (methionine synthase), and vitamin B12 status may therefore impact on cell division and gene expression in early embryos. The aim of this study was to determine the effect of vitamin B12 status on the in vitro development of ovine oocytes to the blastocyst stage. Mature Scottish Blackface ewes from cobalt-deficient farms were housed for ∼ four months and fed a cobalt-deficient diet (0.06 mg cobalt kg DM−1). At housing, 55 of the ewes were given an intra-ruminal slow-release cobalt bolus to compensate for the dietary deficit, and 52 remained untreated. The ovaries of all ewes were recovered at slaughter within the natural breeding season. Oocytes were aspirated and those with evenly granulated cytoplasm and >3 layers of cumulus cells were pooled according to ewe cobalt treatment, matured, fertilized, and cultured in vitro (∼20 oocytes per 50-μL drop under mineral oil). Oocytes were matured for 24 h in M199 + 10% fetal calf serum at 38.5°C in a humidified atmosphere of 5% CO2 in air prior to co-incubation for 18 h with frozen-thawed semen from a single ejaculate (1 × 106 live sperm mL−1). Presumptive zygotes were cultured for 7 Days in synthetic oviduct fluid + 0.4% fatty acid-free BSA (5% CO2, 5% O2, 90% N). Blastocysts formed at the end of the culture period were fixed and stained (Hoechst 33258) to count cell numbers. Data were analyzed by ANOVA and chi-square. For cobalt-supplemented and non-supplemented ewes, circulating concentrations of vitamin B12 at the time of slaughter were 1244 ± 52.5 and 372 ± 27.9 pmol L−1 (P < 0.001), respectively. Numbers of small (<5 mm) follicles per ewe were 17.6 ± 1.22 and 17.1 ± 1.31, and large (>5 mm) follicles per ewe were 1.8 ± 0.16 and 1.6 ± 0.18, respectively (NS). Cobalt-supplemented ewes yielded a lower proportion of matured oocytes that cleaved but an increased proportion of cleaved oocytes that formed blastocysts by Day 6 of the culture period (Table 1). The proportion of grade 1 and 2 blastocysts was also increased but cobalt treatment did not affect blastocyst cell numbers. In conclusion, results suggest that cleaved eggs derived from ewes of adequate, compared to suboptimal vitamin B12 status have improved developmental competence in vitro. Table 1. Effect of cobalt/vitamin B12 status on the in vitro development of ovine oocytes This work was funded by the Scottish Executive Environment and Rural Affairs Department.

362 DIFFERENT IN VITRO DEVELOPMENT AFTER AGGREGATION OF BOVINE AND FELINE PARTHENOGENETIC EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 269
Author(s):  
A. De Stefano ◽  
A. Gambini ◽  
D. Salamone
Keyword(s):  
Cumulus Cells ◽  
Cell Number ◽  
Total Cell ◽  
In Vitro Development ◽  
Cell Numbers ◽  
Blastocyst Quality ◽  
Vitro Development ◽  
Parthenogenetic Embryos

Embryo aggregation has been shown to improve embryo development in several species. However, the effects seem to be different among species. Thus, the aim of this study was to compare the effect of embryo aggregation over in vitro development and blastocyst quality of bovine and feline parthenogenetic (PA) embryos. To this aim, bovine cumulus-oocyte complexes (COC) were collected from slaughterhouse ovaries, whereas cat ovaries were obtained from ovariectomized animals. The COC were in vitro matured in TCM199 supplemented following standard protocols for each species. After 24 h, cumulus cells and zona pellucidae were removed. Matured oocytes were selected and activated by 5 µM ionomycin treatment for 4 min followed by incubation in 1.9 mM 6-DMAP. Bovine and feline PA embryos were cultured in SOF medium in the well of well system in two different groups: only one PA embryo per microwell (1X); and three PA embryos per microwell (3X, aggregated embryos). Cleavage and blastocyst rates from all groups were assessed at Days 2 and 7, respectively. Size of blastocysts was measured at Day 7 using a millimetre eyepiece, and total cell number was determined by Hoechst 33342 staining. Blastocyst rates and embryo size were analysed by Fisher's test (P < 0.05) and total cell numbers by Kruskal–Wallis test with Dunn's correction (P < 0.05). Statistical differences were found in PA blastocyst rates between experimental groups (1X: 15/104, 24.6% v. 3X: 27/37, 62.2% for feline; and 1X: 21/113, 19.4% v. 3X: 20/32, 62.5% for bovine), but no differences were found between species. In addition, there was no statistical difference in the number of blastocysts obtained per oocyte used in any of the experimental groups. Bovine aggregated PA blastocysts were significantly larger than non-aggregated embryos (>200 microns, 1X: 2/20, 10% v. 3X: 9/19, 47.4%), but no differences were found in cell number. On the other hand, cat aggregated PA blastocysts had significantly higher cell numbers (1X: 122.4 ± 79.66 cells v. 3X: 259.8 ± 137.1 cells), but no differences were found in blastocyst size. This observation can contribute in the understanding of embryo physiology, suggesting that benefits of embryo aggregation in parthenogenic embryos vary among these species.

scholarly journals Effect of granulosa and cumulus cells on in vitro development of the bovine follicular oocytes

1995 ◽  
Vol 8 (4) ◽  
pp. 317-320
Author(s):  
K. S. Im ◽  
H. J. Kim ◽  
K. M. Chung ◽  
H. S. Kim ◽  
K. W. Park ◽  
...  
Keyword(s):  
Cumulus Cells ◽  
In Vitro Development ◽  
Vitro Development

Effects of cycloheximide treatment and interval between fusion and activation on in vitro development of pig nuclear transfer embryos

2002 ◽  
Vol 14 (4) ◽  
pp. 191 ◽  
Author(s):  
M. A. Martinez-Diaz ◽  
K. Ikeda ◽  
Y. Takahashi
Keyword(s):  
Nuclear Transfer ◽  
Kinase Activity ◽  
Short Interval ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
In Vitro Development ◽  
M Phase ◽  
Cytostatic Factor ◽  
Vitro Development

The effects of cycloheximide (CHX) treatment and the interval between fusion and activation on the development of pig nuclear transfer (NT) embryos constructed with enucleated oocytes and serum-starved granulosa/cumulus cells were examined. One group of couplets was fused and activated simultaneously (FAS) by a single electrical pulse (activation pulse). Another three groups of couplets were fused electricaly 1.5, 2.5 or 4.5 h before being subjected to the activation pulse (FBA). Each group was divided into two subgroups and incubated with or without CHX. The NT embryos treated with CHX showed a high and stable cleavage rate, regardless of the interval between fusion and activation; however, development to blastocysts was improved only when the NT embryos were subjected to FAS with CHX. These results indicate that CHX-sensitive events occurring shortly after FAS may be responsible for the development to blastocysts. Fusion pulse rarely activated M II oocytes, but rapidly dropped the p34cdc2 kinase activity in NT embryos. A pronucleus-like structure was observed 2-2.5 h after the activation pulse with CHX in NT embryos of both the FAS and FBA groups. Therefore, successive inactivation of M-phase promoting factor and cytostatic factor at a certain short interval may also play an important role in the development of NT embryos.

65 CLONED EMBRYOS FROM HORSE SOMATIC CELLS AND ENUCLEATED BOVINE AND OVINE OOCYTES AND THEIR DEVELOPMENTAL COMPETENCE

2008 ◽  
Vol 20 (1) ◽  
pp. 113
Author(s):  
H. M. Zhou ◽  
B. S. Li ◽  
L. J. Zhang
Keyword(s):  
Somatic Cell ◽  
Somatic Cells ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
Electrical Pulses ◽  
Mongolian Horse

The objective of this study was to investigate the reprogramming potential of equine somatic cell donor nuclei in either bovine or ovine recipient oocyte cytoplasmic environments. Heterogeneous embryos were reconstructed by somatic cell nuclear transfer (NT). The percentage of fusion and developmental competence, assessed by rates of cleavage and morula and blastocyst formation, were determined. Skin fibroblast cells, obtained from the ear of an adult female Mongolian horse, were dissociated using 0.25% trypsin and cultured in vitro in a humidified atmosphere of 5% CO2 in air at 37°C. Donor somatic cells were serum-starved before NT and used between passages 4 and 6. Bovine and ovine oocytes derived from slaughterhouse ovaries were matured in vitro for 17–19 and 22–24 h, respectively, in a humidified atmosphere of 5% CO2 in air at 38.5°C, before they were enucleated and used as recipient cytoplasts. The fibroblasts were injected under the zona pellucida of the cytoplasts and electrically fused by 2 DC electrical pulses of 1.58 kV cm–1 for 10 μs, with an interval of 0.13 s. The reconstructed embryos were then activated with 5 μm ionomycin in H-M199 for 5 min and then in 2 mm 6-DMAP for 4 h. The equine-bovine and equine-ovine reconstructed embryos were co-cultured, respectively, with bovine and ovine cumulus cells in synthetic oviduct fluid supplemented with amino acids (SOFaa) and 10% fetal calf serum (FCS) for 168 h. The data were analyzed with ANOVA and differences among the groups were evaluated with t-test. The results of the percentages of fusion, cleavage, and development to morula (8 to 64 cells) and blastocyst stages of equine-bovine and equine-ovine heterogeneous embryos are shown in Table 1. This study demonstrates that heterogeneous embryos can undergo early embryonic divisions and that reprogramming of equine fibroblast nuclei can be initiated in foreign cytoplasts. It appears that embryos reconstructed with equine somatic nuclei and ovine cytoplasts have a higher developmental potential than those using bovine cytoplasts. Table 1. Developmental competence of equine-bovine and equine-ovine reconstructed embryos

294 IN VITRO DEVELOPMENT OF RAT OOCYTES FOLLOWING MICROINJECTION OF A CUMULUS CELL INTO THE OOPLASM AND CHEMICAL ACTIVATION

2007 ◽  
Vol 19 (1) ◽  
pp. 262
Author(s):  
W. Fujii ◽  
H. Funahashi
Keyword(s):  
Chemical Activation ◽  
Formation Rate ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Female Rats ◽  
Blastocyst Formation ◽  
In Vitro Development ◽  
Pronuclear Formation ◽  
Vitro Development

If diploid zygotes constituted with a somatic and a maternal genome could successfully develop to term, a new reproductive method would be developed to produce animals. However, there appears to be little information on this subject. In the present study, in vitro early development of the constituted zygotes was examined. A cumulus cell was microinjected into a rat non-enucleated oocyte, the reconstructed oocyte was chemically activated, and the pronuclear formation and in vitro development of the embryo was observed. Prepubertal Wistar female rats (21–27 days old) were induced to superovulate with an IP injection of 15 IU of eCG, followed by 15 IU of hCG 48 h later. Cumulus cells were removed from oocytes by pipetting with 0.1% hyaluronidase. Experiment 1: The DNA content of cumulus cells for microinjection was evaluated by flow cytometry. Experiment 2: The optimal concentration of SrCl2 for activation of rat oocytes was examined. Experiment 3: Cumulus cells were injected into mature oocytes in BSA-free HEPES-buffered mKRB containing 0.1% polyvinyl alcohol (PVA) and cytochalasin B (5 �g mL-1), and were then chemically activated by treatment in Ca2+-free mKRB containing 5 mM SrCl2 for 20 min at 0 to 0.5 (A), 1 to 1.5 (B), or 3 to 3.5 h (C) after injection. Activated embryos were cultured in droplets of mKRB in an atmosphere of 5% CO2 in air at 37�C for 9 to 12 h. After being observed for pronuclear formation, the embryos were transferred into mR1ECM-PVA, and the cleavage and blastocyst formation rates were examined 24 and 120 h later, respectively. Results from 3 to 7 replicates were analyzed by ANOVA and Duncan's multiple range test. A total of 90.0 and 9.5% of cumulus cells derived from ovulated oocyte–cumulus complexes contained 2C and 4C DNA contents, respectively. Survival rates did not differ among oocytes stimulated with 0 to 5 mM SrCl2 (96.7–100%) but did differ between those stimulated with 1.25 and 10 mM SrCl2 (100 and 72.9%, respectively). Activation rates of oocytes increased at higher SrCl2 concentrations and were higher at 5 and 10 mM (92.6 and 98.5%, respectively) than at other concentrations. When cumulus-injected oocytes were activated after various periods after the injection, the incidences of pronuclear formation and cleavage did not differ among the periods (A: 95.0 and 81.3%; B: 85.6 and 85.0%; and C: 82.7 and 84.6%, respectively). Although a majority of the embryos developed to the 2- to 4-cell stages (78.7%; 152/208), the blastocyst formation rate was very low (0.8%; 2/208). In conclusion, rat non-enucleated oocytes injected with a cumulus cell can form pronuclei and cleave following chemical activation, but blastocyst formation of the embryos is very limited.

64 VALPROIC ACID ENHANCES IN VITRO DEVELOPMENT OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS IN PIGS

2010 ◽  
Vol 22 (1) ◽  
pp. 190
Author(s):  
Y. J. Kim ◽  
K. S. Ahn ◽  
M. J. Kim ◽  
H. Shim
Keyword(s):  
Stem Cells ◽  
Valproic Acid ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Developmental Competence ◽  
Control Group ◽  
In Vitro Development ◽  
Vitro Development ◽  
And Control

Epigenetic modification influences reprogramming and subsequent development of somatic cell nuclear transfer embryos. Such modification includes an increase of histone acetylation and a decrease of DNA methylation in transferred donor nuclei. Histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) and valproic acid (VPA) have been known to maintain high cellular levels of histone acetylation. Hence, the treatment of HDACi to NT embryos may increase efficiency of cloning. Indeed, TSA treatment has significantly enhanced the developmental competence of nuclear transfer embryos in several species including pigs (Zhang et al. 2007 Cloning Stem Cells 9, 357-363; Li et al. 2008 Theriogenology 70, 800-808). Valproic acid, another type of HDACi, has often been used to assist reprogramming of somatic cells into induced pluripotent stem cells in mice. In the present study, we tested the potency of VPA compared with TSA on the enhancement of in vitro development in porcine nuclear transfer embryos. Reconstructed embryos were produced by transferring nuclei of adult ear skin fibroblasts into enucleated oocytes. After electrical activation, these embryos were cultured in PZM-3 containing no HDACi (control), 5 mM VPA, or 50 nM TSA for 24 h, and another 5 days thereafter without HDACi. At least 3 replicates were conducted for the following experiments. The rates of cleavage were not different among the VPA, TSA, and control groups. However, the rate of blastocyst development was significantly higher (P < 0.05) in embryos treated with VPA than in those treated with TSA and without HDACi (125/306, 40.8% v. 94/313, 30.0% v. 80/329, 24.3%). Differential staining of inner cell mass (ICM) and trophectoderm (TE) also supported the beneficial effect of VPA treatment in NT embryos. Compared with the control group, the number of TE cells was significantly increased (P < 0.05) in the VPA and TSA treatment groups (79.3 ± 7.4 v. 74.6 ± 9.2 v. 40.0 ± 6.7). Moreover, VPA treatment significantly increased (P < 0.05) the number of ICM cells compared with the control (15.6 ± 1.7 v. 10.8 ± 2.6), whereas no differences were observed between the TSA treatment and control group (12.9 ± 3.0 v. 10.8 ± 2.6). The present study demonstrates that VPA enhances in vitro development of nuclear transfer embryos, in particular by an increase of blastocyst formation and the number of ICM cells, suggesting that VPA may be more potent than TSA in supporting developmental competence of cloned embryos. However, long-term effects of different HDACi in the development of nuclear transfer embryos, including any adverse outcome from destabilizing epigenetic condition, remain to be determined by further in vivo embryo transfer studies.

Oesophagostomum radiatum: inhibition of in vitro development by newborn calf serum

1990 ◽  
Vol 64 (1) ◽  
pp. 9-14
Author(s):  
I. J. East ◽  
C. J. Fitzgerald
Keyword(s):  
Inhibitory Action ◽  
Natural Infection ◽  
Calf Serum ◽  
Foetal Calf Serum ◽  
Newborn Calf Serum ◽  
In Vitro Development ◽  
Specific Antibodies ◽  
Serum Inhibition ◽  
Vitro Development

ABSTRACTOesophagostomum radiatum developed to fourth stage larvae after 14 days in in vitro culture. However, development was totally inhibited if the standard 50% foetal calf serum in the medium was replaced by newborn calf serum. Inhibition did not occur with serum from cattle immune to O. radiatum through natural infection or experimental vaccination irrespective of the titre of specific antibodies to O. radiatum in each serum. The inhibitory action of NCS could be abolished by heat treatment at 56°C for 1 h but not by dialysis or repeated freeze-thawing. The inhibition was not consistent with observed differences in the activity of 19 enzymes in the various sera or the absence of various thiol-containing stimulants of worm development.

scholarly journals 205.The effect of FSH concentration during IVM and gamete co-incubation length during IVF on the development of unstimulated prepubertal ewe oocytes

2004 ◽  
Vol 16 (9) ◽  
pp. 205 ◽  
Author(s):  
K. M. Morton ◽  
W. M. C. Maxwell ◽  
G. Evans
Keyword(s):  
Blastocyst Stage ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
In Vitro Development ◽  
Oocyte Collection ◽  
Chi Squared ◽  
Development In Vitro ◽  
Stage 1 ◽  
Vitro Development

The developmental competence of prepubertal oocytes can be increased by the administration of gonadotrophins prior to oocyte collection (1); but this is not possible with abattoir-sourced oocytes, and modifications to the IVP system may increase in vitro development. Experiments were conducted to determine the effects of FSH concentration (10, 20 or 60 μg mL-1) during IVM (5 replicates) and gamete co-incubation length (short: 2-3 h, long: 18-20 h) during IVF (6 replicates) on subsequent embryonic development. For both experiments ovaries were collected from prepubertal lambs (16-24 weeks) slaughtered at an abattoir and embryos produced in vitro (1). Data were analysed by chi-squared test. Oocyte cleavage at 48 hours post-insemination (hpi) was higher for oocytes matured in medium containing 20 (60/77; 77.9%) and 60 (56/73; 76.7%) than 10 μg mL-1 (40/67; 59.7%) FSH. Blastocyst formation (% cultured oocytes) on Day 7 (Day 0 = IVF) was higher for oocytes matured with 20 (31/77; 40.3%) than 10 (16/67; 23.9%) or 60 μg mL-1 (20/73; 27.4%). Oocyte cleavage at 48 hpi was reduced for short (36/57; 63.2%) compared with long (49/55; 89.1%) co-incubation, although blastocyst formation (% cultured oocytes; Day 7) did not differ between groups (22/57; 38.6% and 23/55; 41.8%, respectively). These results demonstrate that increasing the FSH concentration above normal levels during IVM of prepubertal lamb oocytes improves development in vitro. Gamete co-incubation length did not influence the proportion of oocytes progressing to the blastocyst stage. (1) Morton et al. (2003) Proc. Soc. Reprod. Fert. P18.

scholarly journals Antioxidant Nobiletin Enhances Oocyte Maturation and Subsequent Embryo Development and Quality

2020 ◽  
Vol 21 (15) ◽  
pp. 5340
Author(s):  
Yulia N. Cajas ◽  
Karina Cañón-Beltrán ◽  
Magdalena Ladrón de Guevara ◽  
María G. Millán de la Blanca ◽  
Priscila Ramos-Ibeas ◽  
...  
Keyword(s):  
Embryo Development ◽  
Biological Effects ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Cortical Granules ◽  
Cleavage Rate ◽  
Cytoplasmic Maturation

Nobiletin is a polymethoxylated flavonoid isolated from citrus fruits with wide biological effects, including inhibition of reactive oxygen species (ROS) production and cell cycle regulation, important factors for oocyte in vitro maturation (IVM). Therefore, the objective of the present study was to evaluate the antioxidant activity of nobiletin during IVM on matured bovine oocyte quality (nuclear and cytoplasmic maturation; oocyte mitochondrial activity; intracellular ROS and glutathione (GSH) levels) and their developmental competence, steroidogenesis of granulosa cells after maturation, as well as quantitative changes of gene expression in matured oocytes, their cumulus cells, and resulting blastocysts. Bovine cumulus-oocyte complexes were in vitro matured in TCM-199 +10% fetal calf serum (FCS) and 10 ng/mL epidermal growth factor (EGF) (Control) supplemented with 10, 25, 50, or 100 μM of nobiletin (Nob10, Nob25, Nob50, and Nob100, respectively) or 0.1% dimethyl sulfoxide (CDMSO: vehicle for nobiletin dilution). A significantly higher percentage of matured oocytes in metaphase II was observed in Nob25 and Nob50 compared to other groups. Similarly, cleavage rate and cumulative blastocyst yield on Days 7 and 8 were significantly higher for Nob25 and Nob50 groups. Oocytes matured with 25 and 50 μM nobiletin showed a higher rate of migration of cortical granules and mitochondrial activity and a reduction in the ROS and GSH content in comparison with all other groups. This was linked to a modulation in the expression of genes related to metabolism (CYP51A1), communication (GJA1), apoptosis (BCL2), maturation (BMP15 and MAPK1), and oxidative stress (SOD2 and CLIC1). In conclusion, nobiletin offers a novel alternative for counteracting the effects of the increase in the production of ROS during IVM, improves oocyte nuclear and cytoplasmic maturation, and subsequent embryo development and quality in cattle.

89 IMPROVED POST-WARMING DEVELOPMENTAL COMPETENCE OF OPEN PULLED STRAW-VITRIFIED IN VITRO-PRODUCED BOVINE BLASTOCYSTS BY SUBLETHAL HYDROSTATIC PRESSURE PRETREATMENT

2008 ◽  
Vol 20 (1) ◽  
pp. 125 ◽  
Author(s):  
C. Pribenszky ◽  
F. E. Siqueira ◽  
M. Molnár ◽  
A. Harnos ◽  
R. Rumpf
Keyword(s):  
Hydrostatic Pressure ◽  
Developmental Competence ◽  
Equilibration Time ◽  
In Vitro Development ◽  
Custom Made ◽  
Boar Semen ◽  
Pre Treatment ◽  
Vitro Development ◽  
Hatching Rates

Vitrification of in vitro (IVP)-produced bovine blastocysts is well established, reaching post-warming hatching rates close to 70–80% in vitro. Nevertheless, improvements still are needed regarding realizable pregnancy rates. High hydrostatic pressure (HHP) treatment of fresh boar semen before freezing increased the litter size achieved by insemination of frozen–thawed boar semen (Kuo et al. 2007 6th Int. Conf. Boar Semen Pres, Alliston, ON, Canada, poster #22); HHP treatment-related improvements were also observed in the in vitro cryosurvival of mouse blastocysts (Pribenszky et al. 2004 Reprod. Fertil. Dev. 17, 199–200), bull and boar semen, and pig oocytes, theoretically by the sublethal stress-induced production/stabilization of shock proteins (Pribenszky et al. 2006 Reprod. Fertil. Dev. 18, 162–163; 2007 Reprod. Fertil. Dev. 19, 181–182; and b; Du et al. 2007). The aim of the present study was to improve the post-warming in vitro developmental competence of vitrified bovine IVP blastocysts through the application of HHP as pre-treatment, in order to apply the protocol in later in vivo experiments. Day 7 IVP blastocysts were aspirated in TQC holding medium (AB Technology, Sao Paulo, Brazil) into 0.25-mL straws. Straws were pressure-treated in a custom-made hydrostatic pressure chamber (Cryo-Innovation Ltd., Budapest, Hungary), using water as pressure medium. Six hundred bar pressure was applied for 60 min at 32�C. Immediately after pressure treatment, or following 60- or 120-min incubation, embryos were vitrified and warmed using open pulled straws (OPS) according to the method of Vajta et al. (1998 Mol. Reprod. Dev. 51, 53–58). Untreated blastocysts were vitrified as controls. After warming, embryos were cultured in vitro in SOF (Holm et al. 1999 Theriogenology 52, 683–700) for 72 h. Embryos were checked for re-expansion and hatching at 4, 24, 48, and 72 h post-warming. For the experiment, 404 blastocysts were used in 5 replicates. Logistic regression was used for statistical evaluation. All vitrified groups were inferior compared to the non-vitrified control (97%, 97%, 98, and 100% expansion; 0%, 23%, 72 and 91% hatching at 4, 24, 48, and 72 h, respectively). HHP treatment had a significant effect (P > 0.05) on the post-warming developmental competence of vitrified blastocysts. HHP treatment followed by 60 min of equilibration proved to be superior among all treatment groups regarding both re-expansion and hatching rates and the speed of resumption of normal in vitro development (HHP treatment followed by a 60-min equilibration time before vitrification/warming: re-expansion rates: 88%, 89%, 85, and 90%; hatching rates: 0%, 22%, 51, and 73% v. non-treated vitrified/warmed controls: re-expansion rates: 63%, 69%, 71, and 81%; hatching rates: 0%, 6%, 43, and 63%; at 4, 24, 48, and 72 h post-warm, respectively). In conclusion, hydrostatic pressure pre-treatment significantly improved in vitro survival and hatching rates as well as the speed of resumption of normal in vitro development. Further studies are needed to reveal the molecular-biological implications of the HHP treatments, as well as field trials to test if the in vitro improvements can be confirmed by pregnancy and birth rates. This work was supported by EMBRAPA and a Kozma grant, Hungary.

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