133 EFFECT OF PROGESTERONE SUPPLEMENTATION OF MATURATION MEDIUM ON THE DEVELOPMENT OF IN VITRO-MATURED-IN VITRO-FERTILIZED-IN VITRO-CULTURED BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 180 ◽  
Author(s):  
K. Syoji ◽  
K. Imai ◽  
H. Koyama ◽  
O. Dochi
Keyword(s):  
Cell Mass ◽  
Calf Serum ◽  
Total Cell ◽  
Differential Staining ◽  
Control Group ◽  
Blastocyst Formation ◽  
Cell Numbers ◽  
Maturation Medium ◽  
Significant Difference

The objective of this study was to investigate whether progesterone (P4) supplementation to in vitro maturation (IVM) medium could affect the competence of bovine oocyte to develop into blastocysts in vitro. Cumulus–oocyte complexes (COC) were collected by aspiration of ovarian follicles (2 to 6 mm in diameter) obtained from a local abattoir. The COC were matured for 20 h in TCM-199 supplemented with 5% calf serum and 0.02 AU mL–1 FSH at 38.5° in an atmosphere of 5% CO2 in air. After 18 h of gamete co-culture (5 × 106 sperms mL–1), presumptive zygotes were cultured in CRlaa containing 5% calf serum at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 for 9 days (fertilization = Day 0). Progesterone was added to the IVM medium 10 h after the start of the culture (1 μg group = 1 μg mL–1 of P4; 5 μg group = 5 μg mL–1 of P4; control group = no P4). The maturation (MII) rates were investigated after 20 h of starting the IVM culture. After maturation, the COC were denuded mechanically, and a part of the oocytes were mounted on slides, fixed with aceto-alcohol (1 : 3) solution for 48 h, stained with aceto-orcein, and observed under a phase-contrast microscope to determine their nuclear status (1 μg group: n = 32; 5 μg group: n = 28; control group: n = 31). The remaining COC were used for IVF. The cleavage rates were investigated on day 2, and the blastocyst formation rates were investigated on Days 7 to 9, respectively (1 μg group: n = 264; 5 μg group: n = 274; control group: n = 277). The blastocysts from Day 7 were used for differential staining of the inner cell mass (ICM) and trophectoderm cells (TE). The total cell numbers, ICM, and TE in the blastocysts were counted (1 μg group: n = 28; 5 μg group: n = 24; control group: n = 24). The rates of MII, cleavage, and blastocyst formation were expressed and analysed by the chi-squared test. Each set of cell numbers (mean ± standard error) was analysed by the unpaired t-test. The MII rate in the control group (76.7%) was significantly lower (P < 0.05) than that in the 1 μg group (93.8%). The cleavage rate in the 1 μg group (85.6%) was significantly higher (P < 0.05) than those in the control group (74.7%) and 5 μg group (77.4%). Further, the blastocyst formation rate in the 1 μg group (47.7%) and 5 μg group (43.4%) were significantly higher (P < 0.05) than that in the control group (35.0%). The ICM numbers (mean ± s.e.) were 39.5 ± 13.8 to 36.2 ± 8.9, the TE numbers were 74.4 ± 22.4 to 66.2 ± 12.9, the total cell numbers of blastocysts were 110.6 ± 28.2 to 103.0 ± 13.8. There was no significant difference in cell numbers among the groups. These results indicate that the cleavage and blastocyst formation rates can be improved by the addition of 1 μg mL–1 of P4 to the maturation medium.

Development of bovine embryos cultured in CR1aa and IVD101 media using different oxygen tensions and culture systems

2010 ◽  
Vol 58 (4) ◽  
pp. 465-474 ◽  
Author(s):  
Tamás Somfai ◽  
Yasushi Inaba ◽  
Yoshio Aikawa ◽  
Masaki Ohtake ◽  
Shuji Kobayashi ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Calf Serum ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Cell Numbers ◽  
Culture Systems ◽  
Significant Difference

The aim of the present study was to optimise the culture conditions for the in vitro production of bovine embryos. The development of in vitro fertilised bovine oocytes in CR1aa supplemented with 5% calf serum and IVD101 culture media were compared using traditional microdrops and Well of the Well (WOW) culture systems either under 5% or 20% oxygen tension. After 7 days of culture, a significantly higher blastocyst formation rate was obtained for embryos cultured in CR1aa medium compared to those cultured in IVD101, irrespective of O 2 tensions and culture systems. The blastocyst formation in IVD101 was suppressed under 20% O 2 compared to 5% O 2 . Despite their similar total cell numbers, higher rates of inner cell mass (ICM) cells were observed in blastocysts developed in IVD101 medium than in those developed in CR1aa, irrespective of O 2 tensions. There was no significant difference in blastocyst formation, total, ICM and trophectoderm (TE) cell numbers between embryos obtained by microdrop and WOW culture systems irrespective of the culture media and O 2 tensions used. In conclusion, CR1aa resulted in higher blastocyst formation rates irrespective of O 2 tension, whereas IVD101 supported blastocyst formation only under low O 2 levels but enhanced the proliferation of ICM cells.

41 EFFICIENT STRATEGY FOR INTERSPECIFIC CLONING IN FELIDS

2014 ◽  
Vol 26 (1) ◽  
pp. 134
Author(s):  
L. N. Moro ◽  
M. I. Hiriart ◽  
J. Jarazo ◽  
C. Buemo ◽  
A. Sestelo ◽  
...  
Keyword(s):  
Species Conservation ◽  
Total Cell ◽  
Domestic Cat ◽  
Control Group ◽  
Felis Silvestris ◽  
Blastocyst Formation ◽  
Acinonyx Jubatus ◽  
Cell Numbers ◽  
Number Of Cells

Most of the 36 species of wild felids are at a level of threat, and interspecific SCNT (iSCNT) comes as a strategy to contribute to these species conservation. The aim of this study was to evaluate the effect of embryo aggregation in cheetah (Ch, Acinonyx jubatus), bengal (Ben, a hybrid between Felis silvestris and Prionailurus bengalensis), and domestic cat (DC, Felis silvestris) embryos generated by cloning. DC oocytes were in vitro matured and zona-free SCNT (with DC fibroblasts) or iSCNT (with Ch or Ben fibroblasts) was performed. The reconstructed embryos were activated with 5 μM ionomycin and 1.9 mM 6-DMAP, and cultured in SOF using microwells. Cloned embryos were cultured individually or as 2-embryo aggregates. The experimental groups were Ch1X, Ch2X, Ben1X, Ben2X, and the control groups were DC1X and DC2X. Embryo development was compared by Fisher's exact test (P ≤ 0.05). Embryo aggregation improved cleavage (Day 2) and blastocyst (Day 7) rates per well in all the groups (87.2% v. 96.7%, 83.8% v. 93.3% and 87.6% v. 98.2% for cleavage; and 13.7% v. 28.6%, 33.3% v. 43.8% and 27.4% v. 47.7% for blastocyst, for Ch1X (n = 102), Ch2X (n = 91), Ben1X (n = 154), Ben2X (n = 105), DC1X (n = 113), and DC2X (n = 109), respectively. Moreover, the Ch2X blastocyst rate was statistically similar as the control group DC1X. The mean total cell numbers of the blastocysts obtained were 264 ± 211 and 400.8 ± 97 for Ch1X and Ch2X, 278 ± 62 and 517 ± 104 for Ben1X and Ben2X, 385 ± 127 and 625 ± 183 for DC1X and DC2X, respectively. Although no statistical differences were obtained between the 1X and 2X groups, the 2X groups nearly doubled the average number of cells compared with the 1X groups. Blastocysts were also classified as grade 1 (expanded blastocysts with a well-defined ICM), grade 2 (expanded blastocysts without a well-defined ICM), and grade 3 (not expanded blastocysts). This classification showed an increase in grade 1 DC2X blastocyst compared with DC1X blastocysts (36.7% v. 16.1%), but no differences were observed in the other species. Expression of OCT-4 was assessed by inmunocytochemistry. The cheetah blastocysts markedly over-expressed this protein: the percentage of cells that expressed OCT-4 in Ch1X, Ch2X, Ben1X, Ben2X, DC1X, and DC2X was 88.2, 80.2, 46.3, 45.4, 51, and 47.4%, respectively, with statistical differences among all the groups except Ben1X and Ben2X. The proportion of OCT-4 expressing cells over total cell numbers was analysed by the difference of proportions test (P ≤ 0.05). In conclusion, iSCNT resulted in high rates of blastocyst formation, especially when embryo aggregation was applied. This strategy has not been previously evaluated in felids or iSCNT procedures, and has been demonstrated to improve blastocyst formation, the number of cells in the 3 groups, and the blastocyst quality in the DC. Other pluripotent genes besides OCT-4 should be studied to determine whether the overexpression of this gene in cheetah embryos is the consequence of an inefficient nuclear reprogramming that prevents a correct regulation. Finally, the iSCNT and embryo aggregation could contribute to species conservation in felids.

158 ASTAXANTHIN EFFECTS ON MATURATION, FERTILIZATION, AND DEVELOPMENT OF PORCINE OOCYTES CULTURED UNDER HEAT STRESS

2014 ◽  
Vol 26 (1) ◽  
pp. 193 ◽  
Author(s):  
L. T. K. Do ◽  
V. V. Luu ◽  
Y. Sato ◽  
M. Taniguchi ◽  
T. Otoi
Keyword(s):  
Heat Stress ◽  
Dna Fragmentation ◽  
Cell Number ◽  
Total Cell ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Control Group ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Maturation Medium ◽  
Significant Difference

Heat stress can engender various disorders in reproductive functions such as impairment of oocyte maturation, fertilization, and embryonic development. Astaxanthin, an extremely common carotenoid, is a typical fat-soluble antioxidant that scavenges ROS and blocks lipid peroxidation. Moreover, astaxanthin has been shown to improve the development of embryos exposed to heat stress by a reduction in stress-inducible genes. This study was conducted to investigate the effects of astaxanthin supplementation on the meiotic competence, fertilization, and development of porcine oocytes exposed to high temperature (41°C) during maturation culture. Cumulus–oocyte complexes (COC) collected from ovaries were transferred into maturation medium supplemented with astaxanthin (0, 0.25, 0.5, or 1.0 ppm) and were then cultured for 46 h at 41°C or 38.5°C. After maturation culture, the COC were subjected to IVF and embryo culture to evaluate the fertility and development of oocytes. The total cell number and DNA fragmentation in the blastocysts were assessed using terminal deoxynucleotidyl transferase dUTP nick end labelling and Hoechst 33342 staining. The total numbers of oocytes matured at 41°C and 38.5°C in each treatment group were 432 to 470 and 426 to 444, respectively. Data were analysed using ANOVA, followed by Fisher's protected least significant difference test. Exposure to elevated temperatures during maturation culture significantly reduced the proportions of oocytes that reached metaphase II. When the COC were cultured in the maturation medium supplemented with 0.5 and 1.0 ppm of astaxanthin under heat stress conditions (41°C), the supplementation of astaxanthin significantly improved the proportions of maturation, fertilization, and blastocyst formation compared with the control group (0 ppm) (50–52%, 45–55%, and 11–12% v. 17, 25, and 6%, respectively). The supplementation of the maturation medium with 0.25 ppm of astaxanthin improved only blastocyst formation (9.6%). Similarly, the supplementation of astaxanthin at 1.0 ppm improved the proportions of maturation, fertilization, and blastocyst formation of oocytes matured at 38.5°C s compared with the control group (67, 57, and 18% v. 48, 33, and 12%, respectively). However, no beneficial effect of astaxanthin supplementation was found in the total cell number or DNA fragmentation in the blastocysts, irrespective of culture temperature. Our findings show that the supplementation of astaxanthin to maturation medium improves maturation, fertilization, and embryo development of porcine oocytes exposed to heat stress during maturation culture.

133 EFFECTS OF FROZEN STORED CULTURE MEDIA ON PRE-IMPLANTATION DEVELOPMENT OF PARTHENOTES AND CLONED EMBRYOS IN PIGS

2009 ◽  
Vol 21 (1) ◽  
pp. 166
Author(s):  
Y. H. Zhang ◽  
H. T. Xi ◽  
Y. Liu ◽  
J. Li ◽  
A. Pederson ◽  
...  
Keyword(s):  
Culture Media ◽  
Polar Body ◽  
Frozen Storage ◽  
Test Group ◽  
Total Cell ◽  
Control Group ◽  
Cell Numbers ◽  
Significant Difference ◽  
And Control

The present study was designed to examine if frozen storage of porcine zygote medium (PZM3) plus 3 mg mL–1 BSA (Yoshioka et al. 2002 Bio. Reprod. 66, 112–119) is feasible to culture pig embryos produced by parthenogenetic activation and somatic nuclear transfer. Slaughterhouse-derived sow cumulus–oocyte complexes (COCs) were matured in TCM199 supplemented with 10% porcine follicle fluid, 5% cattle serum, 10 IU mL–1 eCG, 5 IU mL–1 hCG, 0.8 mm L-glutamine and 0.05 mg mL–1 gentamicin at 38.5°C, 100% humidity and 5% CO2 in air. For activation, cumulus cells were removed after 42 to 44 h of maturation, and the denuded oocytes with 1st polar body were activated with a double 160 V mm–1, 100 μs direct pulse followed by culture in PZM3. Each experiment was replicated at least three times. Data were expressed as mean ± SEM and analyzed by using chi-square module in SPSS 11.0, with P < 0.05 denoting significant difference. In Experiment 1, after preparation, liquid PZM3 was aliquoted to 50 mL falcon centrifuge tubes. Randomly, half of the tubes with PZM3 were put into –80°C freezers, and the rest were placed into 4°C refrigerator. Within one week after storage, a tube of frozen PZM3, while that stored at 4°C served as control, was warmed at 38.5°C in CO2 incubator, and more than three 4-well culture dishes were then made with 400 μL PZM3 in each well and balanced for at least 4 h in the incubator before experiment. The results showed that both cleavage (78/93, 83.9 ± 1.2% v. 87/103, 84.5 ± 1.8%, P > 0.05) and blastocyst (60/93, 65.2 ± 2.1% v. 65/103, 63.1 ± 3.8%, P > 0.05) rates were similar between frozen-warmed PZM3 and control, as was total cell numbers per blastocyst (50 ± 7 v. 47 ± 5, P > 0.05) between groups. In Experiment 2, we used somatic cloned embryos to investigate the effect of frozen-warmed PZM3 on pre-implantation development of such embryos. Our results indicated that no significant difference in rates of cleavage (68/95, 71.5 ± 5.1% v. 78/100, 78.1 ± 1.9%, P > 0.05), blastocyst formation (33/95, 34.6 ± 7.6% v. 78/100, 38.2 ± 3.5%, P > 0.05) and total cell numbers per blastocyst (40 ± 11 v. 48 ± 9, P > 0.05) was found between the test and control groups, designed the same as in Experiment 1. In Experiment 3, we tested whether PZM3 in frozen storage for 5 months was able to support in vitro development of parthenotes comparable to freshly-made ones. PZM3 after frozen storage for 5 months was warmed using the same method as Experiment 1, and the newly made PZM3 within 1 week of storage at 4°C acted as control. The results showed that although the cleavage (135/138, 97.8 ± 2.7% v. 117/129, 90.7 ± 3.1%, P > 0.05) and blastocyst (104/138, 75.4 ± 1.6% v. 84/129, 65.1 ± 2.3, P > 0.05) rates in control group were both slightly higher than that in the test group, no statistical differences was observed. We also found no significant difference in total cell numbers per blastocyst (48 ± 7 v. 46 ± 6, P > 0.05) between groups. Taken together, our results imply that frozen storage of PZM3 is feasible, and of practical value for culture pig embryos.

350 EFFECT OF PROGESTERONE SUPPLEMENTATION OF MATURATION MEDIUM ON DEVELOPMENT OF IVM-IVF-IVC BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 331
Author(s):  
K. Miyata ◽  
H. Koyama ◽  
C. Lessard ◽  
J. Singh ◽  
O. Dochi
Keyword(s):  
In Vitro Maturation ◽  
Formation Rate ◽  
Calf Serum ◽  
Control Group ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Oocyte Competence ◽  
Maturation Medium ◽  
Progesterone Supplementation

Follicular fluid from small and large bovine follicles contains large amounts of progesterone, and during preovulatory period progesterone concen- tration increase markedly by 18 h after LH surge. Furthermore, cumulus cells express membrane progestin receptor beta (Liu et al. 2008 Steroids 73, 1416-1423). For these reasons, we hypothesized that progesterone supports maturation of preovulatory bovine oocytes to MII stage. The object of this study was to investigate the effect of progesterone supplementation of in vitro maturation medium on competence of bovine oocyte to develop into blas- tocysts in vitro. COCs were collected by the aspiration of 2-6 mm follicles from ovaries within 6 h of slaughter. The COCs were divided into 5 groups: (1) a control group, TCM-199 supplemented with 5% calf serum (CS) as IVM medium, and (2 to 5) progesterone (P4) supplementation groups, TCM- 199 supplemented with 5% CS and 1, 3, 5, and 10 μg mL-1 of P4. Groups of 10 COCs were incubated in 50-μL drops of IVM media at 38.5°C under an atmosphere of 5% CO2 in air for 20 h. The matured COCs were inseminated with 3 × 106 sperm mL-1. After 18 h of gamete co-culture, the pre- sumptive zygotes were cultured in CR1aa media supplemented with 5% CS for 9 days at 38.5°C under an atmosphere of 5% CO2, 5%O2, and 90% N2. Embryonic development was evaluated at 48 h after IVF (total cleavage rates) and on Days 7 to 9 (blastocyst rate). Data was analyzedby chi-square test. The results are presented in Table 1. There were no significant differences in the cleavage rates between treatments. However, the blastocyst formation rate of 5 μg mL-1 P4 supplementation group was significantly higher than that of 10 μg mL-1 P4 supplementation group (P < 0.05). In addition, the blastocyst formation rates of 10 μg mL-1 P4 supplementation group was lower than the other groups. These results suggest that progesterone supple- mentation of in vitro, maturation medium affects the competence of the oocytes to develop into blastocysts in vitro, and 5 μg mL-1 P4 supplementation may be effective in increasing embryo production. Furthermore, 10 μg mL-1 P4 supplementation has negative effect on the oocyte competence. Table 1.Effect of progesterone supplementation on development of IVF bovine embryos

Blastocyst production by in vitro maturation and development of porcine oocytes in defined media following intracytoplasmic sperm injection

Zygote ◽  
2007 ◽  
Vol 15 (2) ◽  
pp. 93-102 ◽  
Author(s):  
M. Kobayashi ◽  
S. Asakuma ◽  
Y. Fukui
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Subsequent Development ◽  
Control Group ◽  
Control Medium ◽  
Blastocyst Formation ◽  
Cell Numbers ◽  
Defined Media ◽  
Significant Difference ◽  
The Mean

SummaryThe present study was carried out to establish porcine defined IVP. In Experiments 1 and 2, we investigated the efficacy of additional 0.6 mM cystine and/or 100 µM cysteamine (Cys) to a defined TCM199 maturation medium with regard to the intracellular glutathione (GSH) concentration and the developmental competence of in vitro matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The control medium was a modified TCM199 containing 0.05% (w/v) polyvinyl alcohol (PVA). Cys and/or cystine were added to the control medium. The control group and immature oocytes (presumptive germinal vesicle oocytes; GV) were prepared for GSH assay. In Experiment 3, the efficacy of epidermal growth factor (EGF) addition to a modified porcine zygote medium (mPZM) for in vitro culture (IVC) medium was investigated on embryonic development and the mean cell number of blastocysts following ICSI. As a positive or negative control, 0.3% BSA (mPZM-3) or 0.3% PVA (mPZM-4), respectively, was added to the base medium. The defined IVC medium was supplemented with 5 or 10 ng/ml EGF. In Experiment 1, no significant difference was found in the rates of cleavage (31.4–64.3%) and blastocyst formation (6.5–22.9%) among the treatment and control groups. The mean cell numbers per blastocyst ranged from 30 to 48 among the groups without significant differences. However, in Experiment 2, the intracellular GSH concentrations in the oocytes cultured in the medium supplemented with 100 µM Cys (9.6 pmol/oocyte) or Cys + cystine (9.9 pmol/oocyte) were significantly (p < 0.05) higher than the control (2.5 pmol/oocyte) and 0.6 mM cystine (6.5 pmol/oocyte) groups, but not different from the GV group (9.0 pmol/oocyte). The GSH concentration in the cystine group was also significantly (p < 0.05) higher than that in the control group, but not different from the GV group. In Experiment 3, the rates of cleavage and blastocyst formation and the mean cell numbers of blastocysts were not significantly different among the groups. However, the addition of 5 ng/ml EGF into the mPZM-4 resulted in a significantly (p < 0.05) higher blastocyst rate per cleaved embryo than the other two defined groups (mPZM-4 + 5 ng/ml: 48.6%, mPZM-4 and mPZM-4 +10 ng/ml: 23.4% and 23.1%, respectively).The present results indicate that the addition of Cys to a defined medium for in vitro maturation (IVM) of porcine oocytes increases intracellular GSH concentration. Further addition of cystine into the IVM medium containing 100 µM Cys is not necessary and TCM199 plus Cys (100 µM) could be used as a defined IVM medium for porcine oocytes. The addition of 5 ng/ml EGF to a defined IVC medium has enhanced subsequent development after ICSI. This study shows that porcine blastocysts can be produced by defined media throughout the steps of IVP (IVM, ICSI and IVC).

The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro

Development ◽  
1988 ◽  
Vol 102 (4) ◽  
pp. 793-803 ◽  
Author(s):  
V.E. Papaioannou ◽  
K.M. Ebert
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Staining Technique ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Morphological Development ◽  
Total Cell Number

Total cell number as well as differential cell numbers representing the inner cell mass (ICM) and trophectoderm were determined by a differential staining technique for preimplantation pig embryos recovered between 5 and 8 days after the onset of oestrus. Total cell number increased rapidly over this time span and significant effects were found between embryos of the same chronological age from different females. Inner cells could be detected in some but not all embryos of 12–16 cells. The proportion of inner cells was low in morulae but increased during differentiation of ICM and trophectoderm in early blastocysts. The proportion of ICM cells then decreased as blastocysts expanded and hatched. Some embryos were cultured in vitro and others were transferred to the oviducts of immature mice as a surrogate in vivo environment and assessed for morphology and cell number after several days. Although total cell number did not reach in vivo levels, morphological development and cell number increase was sustained better in the immature mice than in vitro. The proportion of ICM cells in blastocysts formed in vitro was in the normal range.

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.

Use of chemically defined system for the direct comparison of inner cell mass and trophectoderm distribution in murine, porcine and bovine embryos

Zygote ◽  
1997 ◽  
Vol 5 (4) ◽  
pp. 309-320 ◽  
Author(s):  
Rabindranath de la Fuente ◽  
W. Allan King
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Number ◽  
Total Cell ◽  
Differential Staining ◽  
Similar Proportion ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Cell Numbers

SummaryThe mammalian blastocyst comprises an inner cell mass (ICM) and a trophectoderm cell layer. In this study the allocation of blastomeres to either cell lineage was compared between murine, porcine and bovine blastocysts. Chemical permeation of trophectoderm cells by the Ca2+ ionophore A23187 in combination with DNA-specific fluorochromes resulted in the differential staining of trophectoderm and ICM. Confocal microscopy confirmed the exclusive permeation of trophectoderm and the internal localisation of intact ICM cells in bovine blastocysts. Overall, differential cell counts were obtained in approximately 85% of the embryos assessed. Mean (±SEM) total cell numbers were 72.2 ± 3.1 and 93.1±5 for in vivo derived murine (n = 41) and porcine (n = 21) expanded blastocysts, respectively. Corresponding ICM cell number counts revealed ICM/total cell number ratios of 0.27 and 0.21, respectively. Comparison of in vivo (n = 20) and in vitro derived bovine embryos on day 8 (n = 29) or day 9 (n = 29) revealed a total cell number of 195.25±9.9, 166.14±9.9 and 105±6.7 at the expanded blastocyst stage with corresponding ICM/total cell ratios of 0.27, 0.23 and 0.23, respectively. While total cell numbers differed significantly among the three groups of bovine embryos (p<0.05), the ICM/total cell ratio did not. These results indicate that a similar proportion of cells is allocated to the ICM among blastocysts of genetically divergent species.

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&lt;0.05)] and in the total cell number in embryos that developed to the blastocyst stage (P&lt;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.

Sign in / Sign up

Export Citation Format

Share Document