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.

Vitrification of bovine blastocysts pretreated with sublethal hydrostatic pressure stress: evaluation of post-thaw in vitro development and gene expression

2011 ◽  
Vol 23 (4) ◽  
pp. 585 ◽  
Author(s):  
E. Siqueira Filho ◽  
E. S. Caixeta ◽  
C. Pribenszky ◽  
M. Molnar ◽  
A. Horvath ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hydrostatic Pressure ◽  
Transcript Abundance ◽  
Developmental Competence ◽  
Hatching Rate ◽  
In Vitro Development ◽  
Stress Treatment ◽  
Pressure Stress ◽  
Vitro Development

Sublethal stress treatment has been reported to enhance gametes’ performance in subsequent procedures, such as cryopreservation. The aim of the present study was to evaluate the effect of different equilibration times between the termination of a sublethal hydrostatic pressure (HP) stress treatment and the initiation of vitrification on the post-thaw survival, continued in vitro development, hatching rate and gene expression of selected candidate genes of in vitro-produced (IVP) expanded bovine blastocysts. Day 7 IVP blastocysts were subjected to 600 bar pressure for 60 min at 32°C. Immediately after pressure treatment (HP0h) or after 1 or 2 h incubation (HP1h and HP2h groups, respectively), embryos were either vitrified and warmed using the open pulled straw method, followed by 72 h in vitro culture or were stored at –80°C until gene expression analysis. Re-expansion and hatching rates after vitrification–warming were significantly (P < 0.05) higher in the HP0h (88 and 76%, respectively) and HP1h (90 and 75%, respectively) groups than in the untreated (82 and 63%, respectively) and HP2h groups (79 and 70%, respectively). Moreover, the HP1h group showed further improvement in the speed of re-expansion and resumption of normal in vitro development. Cumulative analysis of all genes (SC4MOL, HSP1A1A, SOD2 and GPX4) revealed a similar pattern of expression, with a tendency for peak transcript abundance 1 h after HP treatment. Application of HP stress treatment was found to be efficient in increasing the in vitro developmental competence of vitrified bovine embryos.

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.

Cells under pressure: how sublethal hydrostatic pressure stress treatment increases gametes' and embryos' performance

2011 ◽  
Vol 23 (1) ◽  
pp. 48 ◽  
Author(s):  
Csaba Pribenszky ◽  
Gabor Vajta
Keyword(s):  
Hydrostatic Pressure ◽  
Cell Survival ◽  
New Approach ◽  
In Vitro Development ◽  
Stress Treatment ◽  
Sublethal Stress ◽  
Minimal Damage ◽  
Pressure Stress ◽  
Vitro Development

The principal approach in in vitro embryo culture and manipulation has been a defensive one: procedures aim to satisfy passively the supposed or real physiological needs of gametes and embryos. Similarly, during cryopreservation the aim is to cause minimal damage to cells whilst attempting to obtain the highest achievable cell survival. However, carefully chosen and precisely controlled sublethal stress treatment of cells has been described to improve embryos’ and gametes’ performance, and, as a consequence, subsequent morphological survival, fertilisation, in vitro development, pregnancy and farrowing rates improved considerably compared with untreated controls. This review summarises studies that open up a new approach: instead of – and besides – trying to passively reduce the harm to cells during in vitro manipulations and culture, procedures may also prepare the cells themselves to ward off or reduce the damage by turning up the cells’ own, inner capacities.

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.

17 TREATMENT WITH MGCD 0103 IMPROVES THE IN VITRO DEVELOPMENT OF PORCINE EMBRYOS DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER

2016 ◽  
Vol 28 (2) ◽  
pp. 138
Author(s):  
H.-Y. Zhu ◽  
L. Jin ◽  
Q. Guo ◽  
Y.-C. Zhang ◽  
X.-C. Li ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
In Vitro Development ◽  
Single Donor ◽  
Or Groups ◽  
Humidified Air ◽  
Vitro Development

We use MGCD 0103 to test whether the treatment with this novel histone deacetylase inhibitor improves the in vitro development of porcine somatic cell NT (SCNT) embryos. Matured eggs were cultured in medium supplemented with 0.05 M sucrose and 0.4 μg mL–1 demecolcine for 1 h. Treated eggs with a protruding membrane were transferred to medium supplemented with 5 μg mL–1 cytochalasin B and 0.4 μg mL–1 demecolcine. Protrusions were then removed by aspirating with a 15-μm inner diameter glass pipette. A single donor cell was inserted into the perivitelline space of each egg and electrically fused using 2 direct pulses of 150 V mm–1 for 50 μs in 0.28 M mannitol. Fused eggs cultured for 1 h were activated by 2 direct pulses of 100 V mm–1 for 20 μs and incubated with 2 mM 6-DMAP for 4 h. Subsequently, the cloned embryos were cultured in medium for 7 days at 38.5°C in 5% CO2 humidified air. In Experiment 1, after activation and treatment with 6-DMAP for 4 h, the SCNT embryos were cultured in medium supplemented with 0, 0.2, 2, or 20 μM MGCD 0103 for 24 h and then transferred to medium without MGCD 0103. In Experiment 2, SCNT embryos were cultured in medium supplemented with 0.2 μM MGCD 0103 for 0, 6, 24, or 48 h and then transferred to medium without MGCD 0103. As shown in Table 1, development to the blastocyst stage increased in SCNT embryos treated with 0.2 μM MGCD 0103 compared with the control or groups treated with 2 or 20 μM MGCD 0103 (25.51 v. 10.74, 3.53, 3.20%, respectively; P < 0.05). As shown in Table 1, treatment for 6 h with 0.2 μM MGCD 0103 significantly improved the rate of blastocyst formation compared with the control or groups treated for 24 or 48 h (21.17 v. 10.48, 19.23, 10.20%, respectively; P < 0.05). Our results suggested that 0.2 μM MGCD 0103 treatment for 6 h can improve in vitro developmental competence of porcine SCNT embryos. Table 1.In vitro development of pig SCNT embryos with different concentrations of MGCD 0103 for 24 h, and with 0.2 μM MGCD 0103 for different durations

scholarly journals Developmental Competence of CMV-Fut Transgenic and Non-Transgenic Pig Embryos Cultured in Vitro / Potencjał Rozwojowy CMV-Fut Transgenicznych I Nietransgenicznych Zarodków Świni Hodowanych In Vitro

2013 ◽  
Vol 13 (4) ◽  
pp. 765-769
Author(s):  
Jacek Jura ◽  
Zdzisław Smorąg ◽  
Barbara Gajda ◽  
Daniel Lipiński ◽  
Ryszard Słomski
Keyword(s):  
Blastocyst Stage ◽  
Developmental Competence ◽  
Transgenic Pig ◽  
Transgenic Pigs ◽  
In Vitro Development ◽  
Gene Construct ◽  
Significant Difference ◽  
Developmental Capacity ◽  
Vitro Development

Abstract Possible influence of a transgene on life functions of embryos makes it reasonable to confirm or deny it for a particular gene construct. In vitro development of an embryo is a widely used criterion of its competence. The aim of the study was to compare in vitro developmental capacity of transgenic and non-transgenic pig embryos. The results showed a statistically significant difference in in vitro developmental capacity of embryos obtained from transgenic and non-transgenic pigs. Developmental competence of embryos (morula and blastocyst stage) produced from zygotes obtained from transgenic sows decreased compared to that obtained from non-transgenic sows.

scholarly journals 321 DEVELOPMENT IN VIVO AND IN VITRO OF PORCINE OOCYTES FERTILIZED BY INTRACYTOPLASMIC INJECTION OF A FREEZE - DRIED SPERM HEAD

2005 ◽  
Vol 17 (2) ◽  
pp. 311
Author(s):  
M. Nakai ◽  
K. Kikuchi ◽  
A. Takizawa ◽  
M. Ozawa ◽  
J. Noguchi ◽  
...  
Keyword(s):  
Sperm Head ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Vitro Fertilization ◽  
In Vitro Development ◽  
Sperm Suspension ◽  
Freeze Dried ◽  
Vitro Development

The present study investigated the development in vivo and in vitro of in vitro matured porcine oocytes injected with a freeze-dried (FD) boar sperm head. In mice, DNA damage was induced during the holding period after rehydration and before sperm injection (Wakayama, T. and Yanagimachi, R. 1998, Nat. Biotechnol., 16, 639–641). Here, we examined the relationship between duration of rehydration of FD sperm and in vitro development of FD sperm-injected porcine oocytes. We also assessed the in vivo developmental competence of the injected oocytes after embryo transfer. Ejaculated boar spermatozoa were suspended in Pig-FM (Suzuki, K. et al. 2002, Int. J. Androl. 25, 84–93) and sonicated for 1 min to separate sperm heads from the tails. An aliquot (100 μL) of the sperm suspension was put into a glass tube and then pre-cooled at −40°C for 6 h. Each tube was attached to a freeze-dry system (DuraDry μP, FTS Systems, Stone Ridge, NY, USA) for 12 h. The ampules were closed and stored at 4°C for more than 7 days before use. For rehydration, 100 μL of distilled water was added into the ampules. In Experiment I, we injected FD sperm heads which were kept for 0–60, 60–120, or 120–180 min after rehydration. At 1 h after the injection, the injected oocytes were stimulated with a DC pulse and cultured for 6 days. The rate of blastocyst formation and the number of cells in the blastocysts were examined. Embryos after in vitro fertilization (IVF) were evaluated as a control. As shown in Table 1, the rates of blastocyst formation were not different (by χ2 test) for duration of rehydration and the control. However, the cell numbers of FD groups were lower (P < 0.05; by Student's t-test) than that in the control. In Experiment II, oocytes injected with a single FD sperm head and stimulated were transferred to both oviducts of a total of ten recipient gilts. Two recipients were diagnosed as pregnant at Day 30 of gestation. At Day 39, one of the pregnant recipients had an abortion, and two fetuses were recovered. The other pregnancy was not maintained. The results suggest that oocytes fertilized with a single FD sperm head have competence to be implanted and to develop to the early fetal stage, and also that the duration for rehydration does not influence in vitro developmental ability in pigs. Table 1. Effects of the duration from rehydration of freeze-dried sperm heads to the injection of the heads into in vitro matured oocytes on in vitro development of the oocytes in pigs

345 INFLUENCE OF DIFFERENT MATURATION MEDIA ON IN VITRO SWINE EMBRYO DEVELOPMENT

2006 ◽  
Vol 18 (2) ◽  
pp. 280
Author(s):  
J. A. Visintin ◽  
A. S. Lima ◽  
A. B. Nascimento ◽  
A. C. Nicacio ◽  
M. G. Marques ◽  
...  
Keyword(s):  
Embryo Development ◽  
In Vitro Development ◽  
Significant Difference ◽  
Vitro Development ◽  
Blastocyst Hatching ◽  
Hatching Rates

The aim of this work was to study the influence of maturation media NCSU23, Whitten, and TCM199 on in vitro development of swine embryos. Oocytes from slaughtered gilt ovaries were in vitro-matured (IVM) with NCSU23 (n = 148), Whitten (n = 151), and TCM199 (n = 170) media for 44 h. After IVM, all oocytes were fertilized in mTBM medium for 6 h and cultured in NCSU23 with 0.4% BSA for 5 days. After Day 5, all embryos were cultured in NCSU23 supplemented with 20% FCS for 4 days. Cleavage, blastocyst, and hatching rates were evaluated. Cleavage rates were 29% for NCSU23, 35.8% for Whitten, and 42.3% for TCM199 maturation media, with no significant difference among media (P > 0.05). Blastocyst rates were similar (P > 0.05), 18.9%, 21.8%, and 20%, respectively, for NCSU23, Whitten and TCM199 maturation media. Hatching rates were 16.1%, 34.3%, and 32.3%, respectively, for NCSU23, Whitten, and TCM199 maturation media, with no significant differences (P > 0.05). In conclusion, all three maturation media supported IVF, allowing embryo development from cleavage until blastocyst hatching.

24 EFFECT OF TREATMENT WITH TRICHOSTATIN A ON IN VITRO DEVELOPMENT OF BOVINE NUCLEAR-TRANSFERRED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 130 ◽  
Author(s):  
S. Akagi ◽  
K. Fukunari ◽  
K. Matsukawa ◽  
S. Watanabe ◽  
S. Takahashi
Keyword(s):  
Trichostatin A ◽  
Chemical Activation ◽  
Calcium Ionophore ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Fibroblast Cells ◽  
In Vitro Development ◽  
Vitro Development

It has been reported that 5 or 50 nM trichostatin A (TSA) treatment after somatic cell nuclear transfer (NT) improves the success rate of mouse cloning (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189). In this study, we examined the effect of TSA treatment on the in vitro development of bovine NT embryos. As donor cells for NT, bovine fibroblast cells of passages 3 to 5 were used following culture in serum-starved medium for 5 to 7 days. Oocytes were enucleated after in vitro maturation in TCM-199 supplemented with 10% fetal bovine serum. Enucleated MII oocytes were fused with fibroblast cells by a DC pulse of 25 V/150 µm for 10 µs in Zimmerman mammalian cell fusion medium. Fused oocytes were activated by 10 µM calcium ionophore for 5 min, followed by incubation with 2.5 µg mL−1 cytochalasin D, 10 µg mL−1 cycloheximide, and 5 or 50 nM TSA for 1 h, and then cycloheximide and 5 or 50 nM TSA for 4 h. After chemical activation, NT embryos were cultured in IVD-101 (Research Institute of Functional Peptide Co., Ltd., Yamagata, Japan) with 5 or 50 nM TSA for 10 h and subsequently cultured in IVD-101 without TSA. Control NT embryos were cultured in the same medium without TSA after fusion. After in vitro culture for 8 days, blastocyst formation and cell numbers of blastocysts were examined. The fusion rate of enucleated oocytes with fibroblast cells was 81% (199/247). In vitro development of NT embryos is summarized in Table 1. There were no differences in the cleavage rate and development rate to the blastocyst stage of NT embryos among control, and 5 and 50 nM TSA treatments. The cell number of 50 nM TSA-treated NT embryos at the blastocyst stage was higher than that of control NT embryos without TSA treatment. In conclusion, 50 nM TSA treatment for 15 h after activation did not affect the in vitro developmental competence, but increased total cell number in bovine NT embryos. These results suggest that TSA treatment may improve the quality of blastocysts in bovine NT. Table 1. Effects of TSA treatment on in vitro development of NT embryos derived from fibroblast cells

125 SWINE EMBRYOS WITH HIGH POTENTIAL TO DEVELOP IN VITRO HAVE LESS γH2A.X AND MORE NBS1 PROTEINS

2010 ◽  
Vol 22 (1) ◽  
pp. 221
Author(s):  
A. R. S. Coutinho ◽  
V. Bordignon
Keyword(s):  
Dna Damage ◽  
In Vitro Culture ◽  
Western Blotting ◽  
Protein Detection ◽  
Developmental Competence ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Artificial Environment ◽  
Vitro Development

The developmental potential of embryos produced in vitro is lower than those produced in vivo. The artificial environment and the stressful conditions of culture may affect embryo development through various mechanisms including DNA damage and, consequently, cell death. We hypothesized that the developmental competence of in vitro-cultured embryos is influenced by mechanisms signalling DNA damage and repair processes. Therefore, the aim of the study was to assess these processes by systematic quantification of phosphorylated histone H2A.X (γH2A.X) and p95 or nibrin protein (NSB1) in early- and late-cleaved swine embryos cultured in vitro. Studies from several groups including ours have demonstrated superior in vitro development for early-cleaved (within 24 h of culture) compared with late-cleaved (between 24 and 48 h) embryos. The presence of γH2A.X is associated with the DNA double-strand breaks, and NBS1 is involved in the process of DNA damage repair. These proteins were detected by both immunofluorescence and western blotting. Swine embryos were produced by parthenogenetic activation using in vitro-matured oocytes. Oocyte maturation, activation, and embryo culture were conducted as previously described (Che L et al. 2007 Theriogenology 67 1297-1304). At 24 and 48 h after activation, embryos were categorized as early- and late-cleaved, and were collected for protein detection on D2-3, D4-5, or D6-7 of culture. A minimum of 3 replicates were performed per treatment. The amount of protein in relation to the β-actin at D2-3, D4-5, and D6-7 as revealed by western blotting was 76.4% ± 1.3, 63.3% ± 10.5, and 43.2% ± 11.2 for γH2A.X and 60.2% ± 4.2, 67.3% ± 13.2, and 61.3% ± 6.2 for NBS1, respectively. Comparisons between early and late-cleaved groups were then performed by immunoflorescence detection of both proteins. Differences between groups were verified using Student’s t-test. The average proportion of cells that were positively stained for γH2AX at D2-3, D4-5, and D6-7 of culture was 64.4% ± 2.6 (n = 178) v. 65.92% ± 3.7 (n = 114; P = 0.7), 55.7% ± 2.4 (n = 121) v. 59.8% ± 4.7 (n = 62; P = 0.4) and 29.1% ± 2.1 (n = 137) v. 43.5% ± 3.4 (n = 41; P = 0.001), for early v. late-cleaved embryos. The values for NSB1 staining were 13.9% ± 3.8 (75) v. 3.9% ± 3.0 (34; P = 0.09), 50.5% ± 4.2 (66) v. 35.8% ± 6.0 (33; P = 0.05), and 51.0% ± 4.5 (n = 54) v. 38.2% ± 5.5 (n = 24; P = 0.1). These findings confirm the presence of γH2A.X and NBS1 proteins in swine embryos during all stages of in vitro culture. We further show that early cleaved embryos have a lower proportion of γH2A.X and a higher proportion of NSB1-positive cells compared with late-cleaved embryos. Together, these findings suggest that early cleaved embryos that have a superior capacity for in vitro development are better prepared to repair DNA damage during in vitro culture. Supported by NSERC.

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