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.

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<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.

PSX-B-11 Comparing effects of FGF2 and IGF1 on the development competence of parthenogenetic activated bovine oocytes maturing in vitro

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 327-328
Author(s):  
Galina Singina
Keyword(s):  
Growth Factor ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Control Group ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Maturation Medium ◽  
Bovine Oocytes

Abstract The oocyte quality acquired during in vitro maturation (IVM) are the main limitative factors affecting the embryo production. The aim of the present research was to study effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 1 (IGF1) during IVM of bovine oocytes on their developmental potential after parthenogenetic activation. Bovine cumulus-oocyte complexes (COC; n = 1176) were cultured for 22h in either standard maturation medium (TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2 mM sodium pyruvate, 10 μg/ml FSH and 10 μg/ml LH; Control) or maturation medium supplemented with different concentrations (5–160 ng/ml) of FGF2 and IGF1. After IVM, matured oocytes activated by sequential treatment with ionomycin followed by DMAP and cyclohexamide and then cultured up to the blastocyst stage. The obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using DAPI and TUNEL staining. The data from 4 replicates (77–91 oocytes per treatment) were analyzed by ANOVA. Cleavage rates of activated oocytes did not differ between groups and ranged from 63.7 to 68.1%. The addition of 10, 20 and 40 ng/ml of FGF2 to the IVM medium led to an increase in the yield of blastocysts [from 19.6±1.8% (Control) to 35.2±3.4, 29.8±1.9 and 31.1±2.1%, respectively (P<0.05)] and in the total cell number in embryos that developed to the blastocyst stage (P<0.05). Meanwhile, the blastocyst yield and the total cell number in blastocysts in the IGF1-treated groups were similar to that in the control group. No effects of both growth factors on the proportion of apoptotic nuclei in blastocysts (5.3–7.1%) were observed. Thus, FGF2 (but not IGF1) are able to maintain competence for parthenogenetic development of bovine COC during their maturation invitro. Supported by RFBR (18-29-07089) and the Ministry of Science and Higher Education of Russia.

152 Activation of Bovine Oocytes Using the Zinc Chelator TPEN

2018 ◽  
Vol 30 (1) ◽  
pp. 216
Author(s):  
C. L. Timlin ◽  
K. Uh ◽  
V. R. G. Mercadante ◽  
K. Lee
Keyword(s):  
Polar Body ◽  
Subsequent Development ◽  
Cell Number ◽  
Total Cell ◽  
Cell Counting ◽  
Control Group ◽  
Oocyte Activation ◽  
Total Cell Number ◽  
Zinc Chelator ◽  
Bovine Oocytes

Traditionally, artificial oocyte activation has been induced by stimulating intracellular calcium increase in the oocyte. Recently, the use of zinc chelators has also shown to be effective in inducing activation by decreasing intracellular concentration of zinc, mimicking events during fertilization; however, this has not been demonstrated in bovine oocytes. The use of artificial activation in bovine has potential for overcoming subfertility-related production loss and aid in livestock cloning. In this study, we determined whether bovine oocytes could be artificially activated in the presence of the zinc chelator TPEN [N,N,N’,N’-tetrakis(2-pyridylmethyl) ethane-1,2-diamine]. Bovine cumulus–oocyte complexes (COC) were collected from abattoir-derived ovaries and incubated for 24 h in TCM-199 maturation medium supplemented with fetal bovine serum, sodium pyruvate, Glutamax, oestradiol, and FSH. The COC were denuded by vortexing in denuding medium containing 0.1% hyaluronidase, and individual oocytes were selected based on presence of a visible polar body. Matured oocytes were then incubated in TL-HEPES medium supplemented with 1 of 5 treatments for parthenogenetic activation: (1) DMSO for 2 h (control, n = 116), (2) 100 µM TPEN for 45 min (100-45, n = 103), (3) 100 µM TPEN for 120 min (100-120, n = 102), (4) 200 µM TPEN for 45 min (200-45, n = 63), or (5) 200 µM TPEN for 120 min (200-120, n = 142). After treatment, oocytes were washed with culture media and incubated in droplets of SOF-Be1 medium under oil to monitor subsequent development. The number of blastocysts was recorded on Day 10 of culture. Blastocysts were stained with Hoechst for 15 min to evaluate total cell number. The frequencies of blastocyst formation were compared using the Chi-squared test, and differences in total cell number were compared using the Student’s t-test. All TPEN treatments significantly increased the number of oocytes developed to the blastocyst stage relative to the control group, which was unable to form blastocysts (P < 0.01). The 100-45 treatment had a greater % blastocysts compared with the 200-120 treatment (16.5% vs 7.77%; P < 0.05), tended to be greater than 100-120 treatment (16.5% vs 7.8%, P = 0.058), and was numerically greater than the 200-45 treatment (16.5% vs 7.94%; P = 0.114). Three treatments that resulted in blastocysts were analyzed for cell counting: 200-120 (n = 5), 100-120 (n = 4), and 100-45 (n = 7). Average total cell number was 119.20 ± 52.28 for the 200-120 group, 83.75 ± 51.06 for the 100-120 group, and 111.71 ± 59.06 for the 100-45 group. There was no difference in total cell number among groups (P ≥ 0.341). Here, we demonstrated that mature bovine oocytes can successfully be parthenogenetically activated by incubating with the zinc chelator TPEN. Oocytes incubated with 100 µM TPEN for 45 min provided the greatest blastocyst yield. Total cell number did not differ between treatments, but all groups analyzed showed blastocysts containing over 100 cells, demonstrating the effectiveness of the oocyte activation approach. Further studies will focus on optimizing the use of TPEN to activate bovine oocytes.

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.

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.

The combined treatment of calcium ionophore with strontium improves the quality of ovine SCNT embryo development

Zygote ◽  
2012 ◽  
Vol 21 (2) ◽  
pp. 139-150 ◽  
Author(s):  
Inchul Choi ◽  
Jie Zhu ◽  
Keith H. S. Campbell
Keyword(s):  
Treatment Group ◽  
Combined Treatment ◽  
Calcium Ionophore ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Oocyte Activation ◽  
Total Cell Number

SummaryPoor embryo quality is a major problem that contributes to the failure of pregnancy in somatic cell nuclear transfer (SCNT). The aims of this study were to improve the quality of ovine SCNT embryos by modifying the conventional activation protocol with the addition of SrCl2. In order to achieve this objective we conducted a series of experiments with in vitro-matured oocytes to optimize conditions for oocyte activation with strontium, and subsequently applied the protocol to SCNT embryos. The results showed that in vitro-matured oocytes could be activated effectively by 10 mM SrCl2 + 5 mg/ml cytochalasin B (CB) for 5 h in the absence of Ca2+ and that the blastocyst rate on day 7 (33.2%) was similar to that in the control group (31.0%) (5 M calcium ionophore [IP] A23187 for 5 min and cultured in CB/cycloheximide [CHX] for 5 h; P > 0.05). In SCNT experiments, the total cell number/blastocyst (104.12 ± 6.86) in the IP + SrCl2/CB-treatment group was, however, significantly higher than that in the control group (81.07 ± 3.39; P < 0.05). Apoptotic index (12.29 ± 1.22%) was significantly lower than the control (17.60 ± 1.39%; P < 0.05) when a combination of IP and SrCl2/CB was applied to SCNT embryos. In addition, karyotyping of the SCNT embryos showed that the percentage of diploid blastocysts in the IP + SrCl2/CB-treatment group was slightly higher than that in the control (P > 0.05). We conclude that the modified activation protocol with IP + SrCl2/CB can improve significantly the quality of ovine SCNT embryos in terms of total cell number, apoptosis and ploidy.

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.

Effect of vitrification technique and assisted hatching on rabbit embryo developmental rate

Zygote ◽  
2009 ◽  
Vol 17 (1) ◽  
pp. 57-61 ◽  
Author(s):  
M. Popelková ◽  
Z. Turanová ◽  
L. Koprdová ◽  
A. Ostró ◽  
S. Toporcerová ◽  
...  
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Assisted Hatching ◽  
Hatching Rate ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Significant Difference ◽  
Rabbit Embryos

SummaryThe aim of the study was to determine the efficiency of two vitrification techniques followed by two assisted hatching (AH) techniques based on post-thaw developmental capacity of precompacted rabbit embryos and their ability to leave the zona pellucida (hatching) during in vitro culture. The total cell number and embryo diameter as additional markers of embryo quality after warming were evaluated. In vivo fertilized, in vitro cultured 8–12-cell rabbit embryos obtained from superovulated rabbit does were cryopreserved by two-step vitrification method using ethylene glycol (EG) as cryoprotectant or by one-step vitrification method with EG and Ficoll (EG+Ficoll). Thawed embryos were subjected to enzymatic or mechanical AH. Vitrified EG group showed significantly lower (P < 0.05) blastocyst rate (22.5%) and hatching rate (15%) than those vitrified with EG + Ficoll (63 and 63% resp.) and that of control (97 and 97% respectively). Significantly lower values of total cell number (P < 0.05) as well as embryo diameter (P < 0.01) in EG group compared with EG + Ficoll and control group were recorded. No significant difference was found in developmental potential of warmed embryos treated by either mechanical or enzymatic AH. The present study demonstrates that the EG + Ficoll vitrification protocol provides superior embryo survival rates over the EG vitrification protocol for 8–12-cell stage precompacted rabbit embryos. No positive effect of either mechanical or enzymatic AH on the post-thaw viability and quality of rabbit embryos in vitro was observed.

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