Birth of cloned calves from vitrified–warmed zona-free buffalo (Bubalus bubalis) embryos produced by hand-made cloning

2013 ◽  
Vol 25 (6) ◽  
pp. 860 ◽  
Author(s):  
Ambikaprasanna Saha ◽  
Sudeepta K. Panda ◽  
Manmohan S. Chauhan ◽  
Radhey S. Manik ◽  
Prabhat Palta ◽  
...  
Keyword(s):  
Live Birth ◽  
Bubalus Bubalis ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Total Cell Number ◽  
Fetal Fibroblast ◽  
Before And After ◽  
Blastocyst Rate ◽  
Effective Use

The availability of techniques for the vitrification of cloned blastocysts can improve their effective use. The present study compared the developmental competence of buffalo cloned embryos derived from adult (BAF), newborn (BNF) and fetal fibroblast (BFF) before and after vitrification. Despite similar cleavage rates among the three groups, the blastocyst rate was lower for BAF- than BNF- and BFF-derived embryos (30.2 ± 2.2% vs 41.7 ± 1.7% and 39.1 ± 2.1%, respectively; P < 0.01). The total cell number of BNF-derived blastocysts was significantly higher (P < 0.01) than that of BFF-derived blastocysts, which, in turn, was higher (P < 0.01) than that of BAF-derived blastocysts. Following transfer of vitrified–warmed blastocysts to recipients, no pregnancy was obtained with fresh (n = 8) or vitrified–warmed (n = 18) BAF-derived blastocysts, whereas transfer of fresh BNF- (n = 53) and BFF-derived (n = 32) blastocysts resulted in four and three pregnancies, respectively, which aborted within 90 days of gestation. The transfer of vitrified–warmed BNF-derived blastocysts (n = 39) resulted in the live birth of a calf weighing 41 kg, which is now 23 months old and has no apparent abnormality, whereas the transfer of vitrified–warmed BFF-derived blastocysts (n = 18) resulted in one live birth of a calf that died within 6 h. These results demonstrate that cloned buffalo embryos cryopreserved by vitrification can be used to obtain live offspring.

48 DEVELOPMENTAL COMPETENCE OF CLONED OR PARTHENOGENETICALLY ACTIVATED PORCINE EMBRYOS: EFFECT OF DIAMETER OF PREPUBERTAL GILT OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 130
Author(s):  
J. Li ◽  
J. Adamsen ◽  
R. Li ◽  
H. Pedersen ◽  
Y. Liu ◽  
...  
Keyword(s):  
Chemical Activation ◽  
Cell Number ◽  
Visual Observation ◽  
Total Cell ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Total Cell Number ◽  
Mean Diameter ◽  
Blastocyst Rate

One of the primary factors influencing the developmental ability of cloned embryos is the oocyte′s diameter (Hirao et al. 1994 J. Reprod. Fertil. 100, 333–339). However, the oocyte donor's age (i.e. its sexual maturity) is also important to consider, because a high proportion of immature oocytes can be expected (Ikeda and Takahashi 2003 Reprod. Fertil. Dev. 15, 215–221). The present study was to investigate the effect of diameter of oocytes collected from prepubertal gilts weighing 100 to 120 kg on the developmental ability of cloned and parthenogenetically activated (PA) embryos. Cumulus–oocyte complexes collected from ovaries of prepubertal gilts were in vitro matured for 42 to 44 h as described for sow oocytes (Li et al. 2008 Theriog 70, 800–808). After removal of the cumulus cells, the matured oocytes were sorted into 2 groups based on visual inspection: large (L) and small (S) oocytes, whereas non-sorted oocytes were used as control (C). In addition, 1 batch from each of the 3 groups of oocytes had their mean size measured. Subsequently, all 3 groups were used for handmade cloning (HMC; Li et al. 2009 Reprod. Domest. Anim. 44, 122–127) or parthenogenetic activation (PA; Kragh et al. 2005 Theriogenology 64, 1536–1545). Then a chemical activation with 5 μg mL–1 cytochalasin B and 10 μg mL–1 cycloheximide in PZM-3 medium was applied for 4 h on both HMC and PA embryos. Finally, the activated embryos were washed and cultured in PZM-3 medium using the WOW system. The embryo development was evaluated by cleavage rate (Day 2), blastocyst rate (Day 6), and total cell number in blastocysts. Data were analysed by ANOVA with single factor in Excel (Microsoft Excel 2007, Redmond, WA, USA). The results showed (Table 1) that by simple visual observation, oocytes could be easily sorted into the following groups: L group (mean diameter 110 μm, from 105 to 116 μm), S group (mean diameter 101 μm, from 93 to 106 μm) and C group (mean diameter 107 μm, from 93 to 116 μm). Cleavage rates and total cell number were similar in the 3 groups. However, the blastocyst rate in L group either for HMC or PA was higher than S group. The data confirm that prepubertal gilt oocytes are useful for cloning and PA, but developmental rates can be increased by selection of large oocytes by simple visual observation. Table 1.Data analysis results

253 DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES MATURED IN SERUM-FREE MEDIUM UNDER LOW OXYGEN TENSION

2009 ◽  
Vol 21 (1) ◽  
pp. 224
Author(s):  
M. M. Pereira ◽  
F. Q. Costa ◽  
P. H. A. Campos ◽  
R. V. Serapiao ◽  
J. Polisseni ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Embryo Quality ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Total Cell Number ◽  
Blastocyst Rate ◽  
Bovine Oocytes

In vitro maturation (IVM) is a critical step in in vitro bovine embryo production. A number of factors can influence the IVM environment, such as media composition and protein supplementation. Serum and higher O2 tension have been shown to reduce embryo quality; however, little is known about the effects of serum and O2 tension during IVM on embryo quality and development. This study aimed to evaluate the effect of serum and O2 tension on IVM of bovine oocytes. Immature oocytes obtained from slaughterhouse ovaries were randomly distributed in 4 groups of IVM: G1 (n = 253), 0.1% polyvinyl alcohol (PVA) in air; G2 (n = 248), 10% inactivated estrous cow serum (ECS) in air; G3 (n = 270), 0.1% PVA under 5% O2; and G4 (n = 236), 10% ECS under 5% O2. In vitro maturation was performed with TCM-199 culture medium supplemented with 20 μg mL–1 FSH, under 5% CO2 at 38.5°C for 24 h. After maturation, oocytes were in vitro fertilized with 2.0 × 106 sperm mL–1 in Fert TALP medium, supplemented with heparin, for 20 h. Presumptive zygotes were denuded by vortexing and cultured in CR2aa medium with 2.5% fetal calf serum under 5% CO2 and 5% O2 at 38.5°C. Cleavage rate was evaluated 72 h postfertilization, and blastocyst rate and total cell number were evaluated 8 days postfertilization. Morphological classification of embryos was performed at Day 8 according to the International Embryo Transfer Society manual (1998). Cleavage, blastocyst, and grade 1 embryo rates were analyzed by chi-square, and total cell number was analyzed by ANOVA, with means compared by LSD. Results are presented as mean ± SEM. There was no difference (P > 0.05) in cleavage rates among G1, G2, and G4 (61.6, 65.3, and 57.6%, respectively), but cleavage rate was lower (P < 0.05) in G3 (52.5%). Blastocyst rates among G1, G3, and G4 (15.8, 17.7, and 20.3%, respectively) were similar (P > 0.05). However, blastocyst rate in G2 (25.0%) was higher (P < 0.05) than in G1 and G3, but was similar to G4 (P > 0.05). Total cell number was similar (P > 0.05) among G2 (194.1 ± 13.1), G3 (173.3 ± 9.0), and G4 (163.8 ± 8.7), but was lower (P < 0.05) in G1 (124.5 ± 11.4). The grade 1 embryo rate was lower (P < 0.05) in G1 (70.3%) than in G2 (89.5%), but was similar (P > 0.05) to G3 (77.0%) and G4 (83.9%). The results suggest that IVM with PVA in TCM-199 medium under 5% O2 can be performed without reducing embryo development and quality, when compared with ECS. On the other hand, oocyte developmental competence seems to be affected when IVM is performed with PVA under air conditions. Financial support: CNPq, FAPEMIG.

scholarly journals Effect of insulin–transferrin–selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation on in vitro bovine embryo development

Zygote ◽  
2016 ◽  
Vol 24 (6) ◽  
pp. 890-899 ◽  
Author(s):  
A.L.S. Guimarães ◽  
S.A. Pereira ◽  
M. N. Diógenes ◽  
M.A.N. Dode
Keyword(s):  
Ascorbic Acid ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Bovine Embryo ◽  
Total Cell Number ◽  
Embryo Production ◽  
Embryo Size ◽  
Blastocyst Rate

SummaryThe aim of this study was to evaluate the effect of adding a combination of insulin, transferrin and selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation (IVM) and in vitro culture (IVC) on in vitro embryo production. To verify the effect of the supplements, cleavage and blastocyst rates, embryo size and total cell number were performed. Embryonic development data, embryo size categorization and kinetics of maturation were analyzed by chi-squared test, while the total cell number was analyzed by a Kruskal–Wallis test (P < 0.05). When ITS was present during IVM, IVC or the entire culture, all treatments had a cleavage and blastocyst rates and embryo quality, similar to those of the control group (P < 0.05). Supplementation of IVM medium with ITS and AA for 12 h or 24 h showed that the last 12 h increased embryo production (51.6%; n = 220) on D7 compared with the control (39.5%; n = 213). However, no improvement was observed in blastocyst rate when less competent oocytes, obtained from 1–3 mm follicles, were exposed to ITS + AA for the last 12 h of IVM, with a blastocyst rate of 14.9% (n = 47) compared with 61.0% (n = 141) in the control group. The results suggest that the addition of ITS alone did not affect embryo production; however, when combined with AA in the last 12 h of maturation, there was improvement in the quantity and quality of embryos produced. Furthermore, the use of ITS and AA during IVM did not improve the competence of oocytes obtained from small follicles.

225 DEVELOPMENTAL COMPETENCE AND QUALITY OF PIG EMBRYOS OBTAINED FROM STANDARD IN VITRO FERTILIZATION OR INTRACYTOPLASMIC SPERM INJECTION

2013 ◽  
Vol 25 (1) ◽  
pp. 260 ◽  
Author(s):  
I. Grad-Mandryk ◽  
J. Kosenyuk ◽  
B. Gajda
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Quality Criteria ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Tunel Staining ◽  
In Vitro Production ◽  
Total Cell Number ◽  
Vitro Fertilization

In vitro production of porcine embryos is still relatively inefficient. The main reasons for this limited performance are polyspermy after IVF and the poor developmental ability of obtained zygotes. Intracytoplasmic sperm injection (ICSI) is one possible solution to eliminate polyspermy. The aim of this study was to compare the developmental competence of pig zygotes, total cell number, and DNA fragmentation of pig blastocysts derived from IVF or ICSI. Cumulus–oocyte complexes were obtained by aspiration from antral follicles of ovaries collected from slaughtered gilts. The oocytes were then cultured in modified TC-199 medium to metaphase II for 42 h. Semen for IVF was incubated in modified capacitation medium (M199) for 1 h. The sperm fraction (1 × 106 cells mL–1) was introduced into droplets containing oocytes, and then gametes were co-incubated for 4 h in modified TC-199 medium. Intracytoplasmic sperm injection was performed using a mechanical micromanipulator (Research Instruments Limited, Cornwall, UK). Micromanipulation was carried out in modified NCSU-37 medium. The tails of spermatozoa were broken, and then single spermatozoa were aspirated into the injection pipette. The oocyte was fixed by a holding pipette, and the sperm head was then introduced into the oocyte cytoplasm. Presumptive zygotes were cultured in vitro for 144 h in NCSU-23 medium. The embryo quality criteria were developmental competence (morula and blastocyst rates), total cell number per blastocyst, and degree of apoptosis assessed by TUNEL staining. Data were analysed by chi-squared test. The experiment was performed on 136 zygotes (6 replicates) obtained after IVF and 83 zygotes (4 replicates) obtained after ICSI. Percentages of embryos developed to the morula and blastocyst stages were 42.3 ± 6.1 and 28.8 ± 4.7 after IVF, respectively, and 51.7 ± 15.4 and 34.5 ± 18.9 after ICSI, respectively (no differences were observed). Significant differences were noticed in total number of cells per blastocyst between embryos after IVF and ICSI (33.7 ± 5.39 v. 22.8 ± 3.22; P < 0.01). However, there was no difference in the degree of apoptosis between IVF and ICSI embryos (5.14 ± 3.49 and 6.14 ± 4.88, respectively). Our preliminary studies demonstrated a higher proportion of cell numbers in IVF-derived embryos compared with those produced by ICSI, but the developmental competence and degree of apoptosis, as evaluated by the TUNEL method, in both groups were comparable. This study was funded by project N N311 516140 by the NCN, Poland.

171 Effect of progesterone and prolactin on the developmental competence of bovine oocytes during the terminal phase of in vitro maturation

2019 ◽  
Vol 31 (1) ◽  
pp. 210
Author(s):  
G. Singina ◽  
E. Shedova ◽  
T. Taradajnic ◽  
V. Konnova ◽  
E. Tsyndrina
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Terminal Phase ◽  
Cleavage Rate ◽  
Total Cell Number ◽  
One Step ◽  
Group 2 ◽  
The One

To date, considerable progress has been achieved in in vitro production (IVP) technologies in cattle; however, developmental potentials of oocytes matured in vitro remain low compared with in vivo-matured oocytes. Thus, a better understanding of different aspects of oocyte maturation may allow us to increase the embryo development rate. Our study was aimed to assess the effects of progesterone (P4) and prolactin (PRL) on the bovine oocyte developmental competence. Bovine cumulus-enclosed oocytes (CEO) were matured using either one-step or two-step maturation conditions. For the one-step protocol, CEO were cultured for 24h in TCM-199 supplemented with 10% fetal calf serum (FCS), 10μg mL−1 porcine FSH, and 10μg mL−1 ovine LH (standard medium). For the two-step procedure, CEO were first cultured for 16h in the standard medium (n=1263) and then transferred to 1 of 3 experimental media and cultured for additional 8h in either absence or presence of either P4 (50 ng mL−1) or bovine PRL (50ng mL−1). The 3 media tested in the two-step maturation were (1) TCM-199 containing 10% FCS (group 1), (2) TCM-199 containing 3mg mL−1 BSA (group 2), or (3) Fert-TALP medium supplemented with 6mg mL−1 BSA (group 3). Fert-TALP was selected because it can potentially be used throughout maturation and fertilization. Following in vitro maturation, all oocytes underwent an IVF/in vitro culture procedure as described previously (Singina et al. 2014 Reprod. Fertil. Devel. 26, 154). The embryo development was evaluated at Days 2 and 7 for cleavage and blastocyst rates. In addition, obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using 4′,6-diamidino-2-phenylindole (DAPI) and TUNEL staining. The data from 4 to 5 replicates (113-159 oocytes per treatment) were analysed by ANOVA. For oocytes matured for 24h in the one-step culture, the cleavage rate, blastocyst rate, total cell number, and apoptotic nuclei per blastocyst were 66.1±1.1, 23.7±2.0, 71.4±9.1, and 4.8±1.2%, respectively. For the two-step culture, the cleavage rate did not differ from that of the one-step culture system, ranging from 64.8 to 76.5%. Also, no effects of the two-step systems were observed on total cell number (63.0-78.8) or the proportion of apoptotic nuclei (3.3-5.3%) at the blastocyst stage. The culture of CEO in group 1 (without the supplements) had a reduced blastocyst rate (17.4±0.4%; P&lt;0.05) compared with the standard one-step maturation group, and the addition of P4 (but not PRL) improved the blastocyst yield (P&lt;0.05). Furthermore, when P4 (but not PRL) was added to group 2 and group 3 media, blastocyst rates increased significantly (32.9±3.1 and 32.8±2.7%, respectively) compared with those of the one-step group (P&lt;0.05), but did not differ from those of untreated groups 2 and 3 (26.2±2.7 and 30.0±3.0%, respectively). Our data indicate that P4 supplementation during the terminal phase of two-step IVM can enhance the developmental competence of bovine oocytes and that the nature of this effect depends on the composition of IVM medium, whereas no effect of PRL supplementation was observed. The study was supported by RFBR (No. 17-29-08035).

34 Effect of polysaccharide from Flammulina velutipes on the vitrification of bovine oocytes

2020 ◽  
Vol 32 (2) ◽  
pp. 143
Author(s):  
Y. Ihara ◽  
K. Tatakura ◽  
Y. Wada ◽  
H. Kawahara ◽  
K. Yamanaka
Keyword(s):  
Tissue Culture ◽  
Culture Medium ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Flammulina Velutipes ◽  
Control Group ◽  
Cleavage Pattern ◽  
Total Cell Number ◽  
Bovine Oocytes

The developmental competence of oocytes after cryopreservation is compromised by the physical injury due to the ice crystallisation. Recent studies have reported that polysaccharide (xylomannan) derived from the mycelium and fruit body of the basidiomycete Flammulina velutipes inhibits the ice recrystallisation in the cryopreserved Chinese hamster ovary cells. In this study, we aimed to clarify the effect of xylomannan from Flammulina velutipes on the developmental competence of bovine vitrified oocytes. Bovine ovaries were obtained from a local abattoir, and cumulus-oocyte complexes (COCs) were aspirated from follicles (2-6mm in diameter) using a 19-gauge needle attached to a syringe. The COCs were matured for 22h in tissue culture medium-199 supplemented with 5% fetal bovine serum (FBS), 0.02IUmL−1 FSH, and 10μgmL−1 gentamycin. After maturation, COCs were incubated in base solution (BS: 10% FBS-tissue culture medium-199, control group; n=149) or BS supplemented with 100μgmL−1 xylomannan (xylomannan group; n=175) for 1h before vitrification. All vitrification procedures were performed at room temperature. The COCs were equilibrated in BS with 3% ethylene glycol for 12min and then in vitrification solution (BS with 30% ethylene glycol, 1.0M sucrose) for 1min. The COCs were loaded on a Cryotop (Kitazato) and transferred into liquid nitrogen. The warming procedure was performed on a warm plate (42°C). The COCs were placed into BS supplemented with 0.5, 0.25, 0.125, and 0M sucrose for 5min each. After washing with IVF100 solution (Research Institute for the Functional Peptide), COCs were applied for IVF. The viability of putative zygotes was morphologically evaluated following IVF, and ones that survived were cultured in CR1aa supplemented with 5% FBS. The cleavage pattern was evaluated at 28h after IVF as follows: embryos with blastomeres of the same size without fragmentation were classified as normal cleavage; embryos with 2 blastomeres and several small fragments, direct cleavage from the 1-cell stage to 3 or 4 blastomeres, or 2 blastomeres of different size were classified as abnormal cleavage. The rates of cleavage and blastocyst formation were calculated on 2 and 8 days after culture, respectively. Total cell number and apoptosis of blastocysts were measured by terminal deoxynucleotidyl transferase dUTP nick end labelling assay. All data were obtained from more than four replicates. Viability and invitro development data were analysed using the chi-squared test. Total cell number and apoptosis data were analysed by a Student's t-test. Although no significant differences in viability, cleavage pattern, and cleavage rate (85.8 vs. 80.3%, 17.2 vs. 14.8%, and 35.4 vs. 36.7%, respectively) were observed, the developmental rate to blastocysts in the xylomannan group was significantly higher than that in the control group (68.6 vs. 42.2%; P&lt;0.01). The present results suggest that co-incubation with xylomannan before vitrification is an effective method to improve the vitrification outcome in bovine oocytes.

scholarly journals High revivability of vitrified–warmed bovine mature oocytes after recovery culture with α-tocopherol

Reproduction ◽  
2015 ◽  
Vol 149 (4) ◽  
pp. 347-355 ◽  
Author(s):  
Ikuko Yashiro ◽  
Miho Tagiri ◽  
Hayato Ogawa ◽  
Kazuya Tashima ◽  
Seiji Takashima ◽  
...  
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Cortical Granules ◽  
Total Cell Number ◽  
Recovery Culture ◽  
Bovine Oocytes

The objective of this study was to investigate whether developmental competence of vitrified–warmed bovine oocytes can be improved by antioxidant treatment during recovery culture. In experiment 1, one of the two antioxidants (either l-ascorbic acid or α-tocopherol) was added as a supplement to the recovery culture medium to which postwarming oocytes were exposed for 2 h before IVF. The exposure to α-tocopherol had a positive effect on rescuing the oocytes as assessed by the blastocyst yield 8 days after the IVF (35.1–36.3% vs 19.2–25.8% in untreated postwarming oocytes). Quality of expanding blastocysts harvested on Day 8 was comparable between α-tocopherol-treated vitrification group and fresh control group in terms of total cell number and chromosomal ploidy. In experiment 2, level of reactive oxygen species, mitochondrial activity, and distribution of cortical granules in α-tocopherol-treated postwarming oocytes were assessed. No obvious differences from the control data were found in these parameters. However, the treatment with α-tocopherol increased the percentage of zygotes exhibiting normal single aster formation (90.3% vs 48.0% in untreated postwarming oocytes; 10 h post-IVF). It was concluded that α-tocopherol treatment of vitrified–warmed bovine mature oocytes during recovery culture can improve their revivability, as shown by the high blastocyst yield and the higher mean total cell number in the blastocysts.

Effect of oxygen tension and serum during IVM on developmental competence of bovine oocytes

2010 ◽  
Vol 22 (7) ◽  
pp. 1074 ◽  
Author(s):  
Michele M. Pereira ◽  
Marco A. Machado ◽  
Fernanda Q. Costa ◽  
Raquel V. Serapiao ◽  
Joao H. M. Viana ◽  
...  
Keyword(s):  
Oxygen Tension ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Total Cell Number ◽  
Effect Of Oxygen ◽  
Vitro Production ◽  
Bovine Oocytes

With an aim to improve the in vitro production of bovine embryos, the present study investigated the effect of serum and oxygen tension during IVM on oocyte developmental competence. Four experimental groups were evaluated: G1, 10% oestrus cow serum (OCS) with 20% O2; G2, 0.1% polyvinyl alcohol (PVA) with 20% O2; G3, 10% OCS with 5% O2; and G4, 0.1% PVA with 5% O2. The proportion of MII oocytes, blastocyst rates and total cell number were not affected (P > 0.05) when the OCS was replaced with PVA under 5% O2, whereas a higher (P < 0.05) blastocyst rate and total cell number were found with OCS compared with PVA under 20% O2. The apoptosis index was lower in blastocysts from oocytes matured with PVA under 5% O2 (G4) compared with other groups (G1, G2 and G3), but no differences (P > 0.05) were found in maturation and blastocyst rates. Significant differences were found in the amount of specific transcripts in oocytes matured under different conditions. In conclusion maturation with PVA and 5% O2 provides an efficient in vitro culture condition for the maturation of bovine oocytes.

Sperm-borne small RNAs improve the developmental competence of pre-implantation cloned embryos in rabbit

Zygote ◽  
2021 ◽  
pp. 1-6
Author(s):  
Hongyu Qin ◽  
Pengxiang Qu ◽  
Huizhong Hu ◽  
Wenbin Cao ◽  
Hengchao Liu ◽  
...  
Keyword(s):  
Small Rnas ◽  
Apoptotic Cell ◽  
Epigenetic Modification ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Total Cell Number ◽  
Cell Stage ◽  
Micro Rnas

Summary The low efficiency of somatic cell nuclear transfer (SCNT) greatly limits its application. Compared with the fertilized embryo, cloned embryos display abnormal epigenetic modification and other inferior developmental properties. In this study, small RNAs were isolated, and miR-34c and miR-125b were quantified by real-time PCR; results showed that these micro-RNAs were highly expressed in sperm. The test sample was divided into three groups: one was the fertilized group, one was the SCNT control group (NT-C group), and the third group consisted of SCNT embryos injected with sperm-borne small RNA (NT-T group). The level of tri-methylation of lysine 9 on histone H3 (H3K9me3) at the 8-cell stage was determined by immunofluorescence staining, and the cleavage ratio, blastocyst ratio, apoptotic cell index of the blastocyst and total cell number of blastocysts in each group were analyzed. Results showed that the H3K9me3 level was significantly higher in the NT-C group than in the fertilized group and the NT-T group. The apoptosis index of blastocysts in the NT-C group was significantly higher than that in the fertilized group and the NT-T group. The total cell number of SCNT embryos was significantly lower than that of fertilized embryos, and injecting sperm-borne small RNAs could significantly increase the total cell number of SCNT blastocysts. Our study not only demonstrates that sperm-borne small RNAs have an important role in embryo development, but also provides a new strategy for improving the efficiency of SCNT in rabbit.

190 EFFECT OF TRANS-ε-VINIFERIN ON IN VITRO PORCINE OOCYTE MATURATION AND SUBSEQUENT DEVELOPMENTAL COMPETENCE IN PRE-IMPLANTATION EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 207
Author(s):  
Y. Jeon ◽  
S.-S. Kwak ◽  
S.-A. Jeong ◽  
R. Salehi ◽  
Y. H. Seong ◽  
...  
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Vitis Amurensis ◽  
Total Cell Number ◽  
Treatment Groups ◽  
Nuclear Maturation ◽  
Porcine Oocyte

Trans-ε-viniferin is a naturally occurring polyphenol belonging to the stilbenoids family. Trans-ε-viniferin is isolated from Vitis amurensis, 1 of the most common wild grapes in Korea, Japan and China. We investigated the effects of trans-ε-viniferin on in vitro maturation (IVM) and developmental competence after IVF or parthenogenesis (PA). At the laboratory of Veterinary Pharmacology, College of Veterinary Medicine, Chungbuk National University, trans-ε-viniferin was purified from the leaves and stems of Vitis amurensis. Data were analyzed with SPSS 17.0 using Duncan's multiple range test. First, in total, 594 cumulus–oocyte complexes (COC) were used for the evaluation of nuclear maturation. The COC were matured in TCM-199 medium supplemented with various concentrations of trans-ε-viniferin (0, 0.1, 0.5, 1.0 and 5.0 μM) with 10% porcine follicular fluid, 10 IU mL–1 of eCG and 10 IU mL–1 of hCG. After 22 h in maturation culture, the COC were cultured in hormone-free medium supplemented with various concentrations of trans-ε-viniferin for an additional 22 h and then nuclear maturation was evaluated. Second, in total, 300 matured oocytes were used to examine the effects of different trans-ε-viniferin concentrations (0, 0.5 and 5.0 μM) on porcine oocyte intracellular glutathione (GSH) and reactive oxygen species (ROS) levels. Lastly, the developmental competence of oocytes matured with different concentrations of trans-ε-viniferin (0, 0.5 and 5.0 μM) was evaluated after IVF or PA. In total, 711 embryos were evaluated. As results, we observed that trans-ε-viniferin treatment during IVM did not improve the nuclear maturation of oocytes in any group (84.2, 86.6, 85.5, 83.3 and 79.2%, respectively), but significantly increased (P < 0.05) intracellular GSH levels in the 0.5 μM group (0 μM vs 0.5 μM; 14.6 vs 16.8 pmol oocyte–1) and reduced ROS levels (0 μM vs 0.5 μM and 50 μM; 174.6 vs 25.7 and 23.8 pixel oocyte–1). Oocytes treated with trans-ε-viniferin during IVM did not have significantly different cleavage rates or blastocyst formation rates after IVF, but total cell numbers were significantly higher (P < 0.05) in the 0.5 and 5.0 μM treatment groups (53.6 ± 4.0 and 47.9 ± 3.1) compared to the control group (36.4 ± 2.2). The PA embryos showed similar results; there were no significant differences in cleavage rates and blastocyst formation rates, but the total cell number significantly increased in the 0.5 and 5.0 μM treatment groups (59.6 ± 4.2 and 60.8 ± 4.6) compared to the control group (43.1 ± 2.1). In conclusion, these results indicate that trans-ε-viniferin treatment during porcine IVM increased total cell number of blastocysts, possibly through increasing intracellular GSH synthesis and reducing ROS levels. This work was supported by a grant from the Korea institute of Planning & Evaluation for Technology in Food, Agriculture, Forestry & Fisheries, Republic of Korea.

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