Supplementation of insulin–transferrin–selenium to embryo culture medium improves the in vitro development of pig embryos

Zygote ◽  
2013 ◽  
Vol 22 (3) ◽  
pp. 411-418 ◽  
Author(s):  
Ziban Chandra Das ◽  
Mukesh Kumar Gupta ◽  
Sang Jun Uhm ◽  
Hoon Taek Lee
Keyword(s):  
Nuclear Transfer ◽  
Culture Medium ◽  
Mammalian Species ◽  
Oxygen Species ◽  
Embryo Culture Medium ◽  
Maturation Medium ◽  
Blastocyst Quality ◽  
Vitro Development

SummaryInsulin, transferrin and selenium (ITS) supplementation to oocyte maturation medium improves the post-fertilization embryonic development in pigs. ITS is also commonly used as a supplement for the in vitro culture (IVC) of embryos and stem cells in several mammalian species. However, its use during IVC of pig embryos has not been explored. This study investigated the effect of ITS supplementation to IVC medium on the in vitro development ability of pig embryos produced by parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT). We observed that ITS had no significant effect on the rate of first cleavage (P > 0.05). However, the rate of blastocyst formation in ITS-treated PA (45.3 ± 1.9 versus 27.1 ± 2.3%), IVF (31.6 ± 0.6 versus 23.5 ± 0.6%) and SCNT (17.6 ± 2.3 versus 10.7 ± 1.4%) embryos was significantly higher (P < 0.05) than those of non-treated controls. Culture of PA embryos in the presence of ITS also enhanced the expansion and hatching ability (29.1 ± 3.0 versus 18.2 ± 3.8%; P < 0.05) of blastocysts and increased the total number of cells per blastocyst (53 ± 2.5 versus 40.9 ± 2.6; P < 0.05). Furthermore, the beneficial effect of ITS on PA embryos was associated with significantly reduced level of intracellular reactive oxygen species (ROS) (20.0 ± 2.6 versus 46.9 ± 3.0). However, in contrast to PA embryos, ITS had no significant effect on the blastocyst quality of IVF and SCNT embryos (P > 0.05). Taken together, these data suggest that supplementation of ITS to the IVC medium exerts a beneficial but differential effect on pig embryos that varies with the method of embryo production in vitro.

235 DEVELOPMENT OF CAMEL (CAMELUS DROMEDARIUS) OOCYTES AFTER CHEMICAL ACTIVATION

2008 ◽  
Vol 20 (1) ◽  
pp. 197
Author(s):  
N. A. Wani
Keyword(s):  
Sodium Bicarbonate ◽  
Embryo Culture ◽  
Culture Medium ◽  
Chemical Activation ◽  
Blastocyst Stage ◽  
Camelus Dromedarius ◽  
Dromedary Camel ◽  
Embryo Culture Medium ◽  
Maturation Medium

Identification of an optimal protocol for activation of the MII oocytes in a species like camel not only allows us to evaluate the quality of oocytes after their in vitro maturation, but also is required for the success of advanced technologies like cloning. The present study was aimed to determine activation of in vitro-matured dromedary (Camelus dromedarius) oocytes using ionomycin or ethanol followed by sequential culture in phosphorylation inhibitor (6-dimethylaminopurine) or the specific maturation promoting factor inhibitor (roscovitine). Cumulus–oocyte complexes (COCs), collected from slaughterhouse ovaries, were randomly distributed to 4-well culture plates (20–25 COCs/well) containing 500 µL of the maturation medium. The maturation medium consisted of TCM-199 supplemented with 0.15 mg mL–1 L-glutamine, 2.1 mg mL–1 sodium bicarbonate, 0.22 mg mL–1 pyruvate, 20 ng mL–1 epidermal growth factor, 50 µg mL–1 gentamycin, 10 µg mL–1 bFSH, 10 µg mL–1 bLH, 1 µg mL–1 estradiol, and 10% estrous dromedary serum (EDS). The COCs were cultured at 38.5�C in an atmosphere of 5% CO2 in air for 36–40 h. The COCs were either fertilized in vitro (positive control) using epididymal spermatozoa collected from slaughtered males or activated with 5 µm ionomycin for 5 min or 7% ethanol for 7 min, both followed by exposure to 2 mm 6-DMAP or 50 µm roscovitine for 4 h. After being washed thoroughly in embryo culture medium, they were cultured for a period of 7 days at 38.5�C in an atmosphere of 5% CO2, 5% O2, and 90% N2 in air. The embryo culture medium consisted of TCM-199 supplemented with 0.15 mg mL–1 L-glutamine, 2.1 mg mL–1 sodium bicarbonate, 0.22 mg mL–1 pyruvate, 50 µg mL–1 gentamicin, 1% insulin-transferrin-selenium (ITS) media supplement, and 10% EDS. First cleavage was recorded on Day 2 and the number of embryos developing to morulas and blastocysts was recorded on Day 7 of culture. The proportions of oocytes cleaved were 58.6 � 4.4, 55.9 � 4.5, 49.1 � 5.3, 43.2 � 6.05, and 54.1 � 3.3%, while the proportions of cleaved oocytes reaching blastocyst stage were 22.5 � 0.9, 19.1 � 2.8, 9.04 � 3.3, 8.2 � 3.8, and 15.2 � 2.3%, and those at morula stage were 61.1 � 4.9, 54.6 � 6.2, 67.1 � 7.2, 57.8 � 4.6, and 53.6 � 5.6% in the ionomycin/ 6-diethylaminopurine, ionomycin/roscovitine, ethanol/6-diethylaminopurine, ethanol/roscovitine, and IVF groups, respectively. The proportions of blastocysts obtained in the ionomycin/6-diethylaminopurine and ionomycin/roscovitine groups were higher (P < 0.05) when compared with the ethanol/6-diethylaminopurine and ethanol/roscovitine groups. Also, the proportion of blastocysts obtained in the ionomycin/6-diethylaminopurine group was higher than that in the in vitro-fertilized group. In summary, methods for oocyte or cytoplast activation in dromedary camel incorporating ionomycin/6-diethylaminopurine and ionomycin/roscovitine giving better results than those incorporating ethanol/6-diethylaminopurine and ethanol/roscovitine.

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

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

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

Cysteamine supplementation of in vitro maturation medium, in vitro culture medium or both media promotes in vitro development of buffalo (Bubalus bubalis) embryos

2008 ◽  
Vol 20 (2) ◽  
pp. 253 ◽  
Author(s):  
T. Anand ◽  
D. Kumar ◽  
M. S. Chauhan ◽  
R. S. Manik ◽  
P. Palta
Keyword(s):  
In Vitro Culture ◽  
In Vitro Maturation ◽  
Culture Medium ◽  
Bubalus Bubalis ◽  
Cell Number ◽  
Hatching Rate ◽  
Cleavage Rate ◽  
Maturation Medium ◽  
Vitro Development

The effects of supplementation of in vitro maturation (IVM) or in vitro culture (IVC) or both IVM and IVC media with cysteamine on the yield, hatching rate (HR) and total cell number (TCN) of buffalo blastocysts were examined. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to IVM and IVF. The IVM or IVC media were supplemented with 0, 50, 100 or 200 µm cysteamine. Supplementation of IVM medium with 50 µm cysteamine increased (P < 0.01) the cleavage rate and blastocyst yield without affecting the HR and TCN whereas a higher concentration of 200 µm significantly (P < 0.05) reduced the blastocyst yield but not TCN. Similar increases in blastocyst yield, without any effect on HR and TCN were observed after supplementation of the IVC medium with 100 (P < 0.01) or 50 µm (P < 0.05) cysteamine, whereas 200 µm cysteamine was ineffective. Supplementation of both IVM medium with 50 µm cysteamine and of IVC medium with 100 µm cysteamine increased the yield of blastocysts and hatched blastocyst by over 100% (P < 0.01) compared with the controls without any adverse effects on HR or TCN. The results of the present study suggest that supplementation of both IVM and IVC media improves the yield of blastocysts without compromising their health.

scholarly journals The effects of supplemented sericin on in vitro maturation and preimplantation development of mouse embryos: An experimental study

2021 ◽  
pp. 921-928
Author(s):  
Omid Banafshi ◽  
Sherko Nasseri ◽  
Leila Farhadi ◽  
Masoud Alasvand ◽  
Mohammad Bagher Khadem-Erfan ◽  
...  
Keyword(s):  
Embryo Culture ◽  
In Vitro Maturation ◽  
Culture Medium ◽  
Control Group ◽  
Vitro Fertilization ◽  
In Vitro Development ◽  
Embryo Culture Medium ◽  
Vitro Development

Background: Mouse embryo culture condition is an essential part of transgenic, reproductive and developmental biology laboratories. Mouse embryonic culture media may have a high risk of serum contamination with pathogens.  Objective: To investigate the effect of sericin as an embryo culture medium supplement on in vitro maturation (IVM), in vitro fertilization (IVF), and development of the preimplantation embryo in mice. Materials and Methods: The effects of sericin at three concentrations (subgroups) of 0.1%, 0.5%, and 1% as a medium supplement on IVM, IVF, and in vitro development of mouse embryos were separately investigated and compared with a sericin-free (control) group. The cumulative effect of the three concentrations was evaluated for IVM + in vitro development and IVF + in vitro development as follow-up groups. Results: In the IVM group, compared to the control group, the number of oocysts reaching the MII stage was significantly higher when 1% sericin was used (161/208 = 77.4%). No significant results were observed in the IVF and in vitro development groups with different concentrations of sericin compared to the control group. Among the follow-up groups, in the IVM + in vitro development group, the number of oocytes was higher after passing the IVM and IVF and reaching the blastocysts stage when 1% sericin was used, compared with other sericin subgroups. A significant difference was also noted when compared with the control group (p = 0.048). The IVF + in vitro development study group, on the other hand, did not show any significant relationship. Conclusion: It can be concluded that 1% sericin can be used as a supplement in mouse embryo cultures to improve the IVM rate. Also, based on the findings, sericin appears to be an effective supplement which can have a positive effect on the development of embryos derived from IVM. Key words: Sericin, In vitro maturation, In vitro fertilization, Preimplantation embryo, Culture medium, Mice.

In vitro development of equine nuclear transfer embryos: effects of oocyte maturation media and amino acid composition during embryo culture

Zygote ◽  
2003 ◽  
Vol 11 (1) ◽  
pp. 77-86 ◽  
Author(s):  
Y.H. Choi ◽  
Y.G. Chung ◽  
S.C. Walker ◽  
Westhusin M.E. ◽  
K. Hinrichs
Keyword(s):  
Amino Acids ◽  
Embryo Culture ◽  
Nuclear Transfer ◽  
Culture Medium ◽  
Essential Amino Acids ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Embryo Culture Medium ◽  
Igf I

This study was conducted to evaluate the effects of insulin-like growth factor I (IGF-I) and other media factors during oocyte maturation, and the presence of different compositions of amino acids in embryo culture medium, on the development of equine embryos. Oocytes recovered from slaughterhouse-derived ovaries were matured in vitro for 24 h and those with a polar body were subjected to intracytoplasmic sperm injection (ICSI) or nuclear transfer with adult fibroblasts (NT). For ICSI embryos, there were no significant differences in rates of morphological cleavage, cleavage with normal nuclei or average nucleus number at 96 h post-ICSI between the absence and presence of IGF-I in maturation medium, or between embryos cultured in G1.2 or a modified CZB medium (CZB-C). Embryos produced by interspecies NT (equine donor cells into bovine cytoplasts) also showed no difference in cleavage rate or average nucleus number whether cultured in G1.2 or in CZB-C. The rates of cleavage, cleavage with normal nuclei and average nucleus number of equine NT embryos were not significantly different among oocytes matured in M199 with FSH in the presence or absence of IGF-I, or in EMMI medium, which contains IGF-I, epidermal growth factor, steroid hormones, FSH and LH. There were no differences in development of equine NT embryos cultured in any of three amino acid treatments (with or without non-essential amino acids, or containing taurine, hypotaurine and cysteine only). The cleavage rate and average nucleus number of parthenogenetically activated oocytes (treated similarly to NT oocytes but not enucleated or subjected to donor cell injection) were significantly (p < 0.05) higher than those for NT embryos. These results indicate that the presence of IGF-I or of EMMI medium during in vitro maturation of equine oocytes does not have a beneficial effect on their developmental competence as assessed at 96 h. Presence or absence of non-essential amino acids in embryo culture medium does not affect development of NT embryos within the first 96 h of culture. Factors associated with enucleation or nuclear transfer decrease the developmental competence of equine NT embryos. CZB-C medium may be used for culture of equine embryos with results similar to those obtained with G1.2 medium, thus providing a base medium that may be modified for further study of culture requirements of equine embryos.

Anethole improves blastocysts rates together with antioxidant capacity when added during bovine embryo culture rather than in the in vitro maturation medium

Zygote ◽  
2019 ◽  
Vol 27 (6) ◽  
pp. 382-385 ◽  
Author(s):  
J.C. Anjos ◽  
F.L.N. Aguiar ◽  
N.A.R. Sá ◽  
J.F. Souza ◽  
F.W.S. Cibin ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Culture Medium ◽  
Cumulus Cells ◽  
Bovine Embryo ◽  
Oxygen Species ◽  
Embryo Production ◽  
Maturation Medium ◽  
Bovine Oocytes ◽  
Medium Supplementation

SummaryWe performed the exposure of bovine oocytes to anethole during in vitro maturation (0 or 300 µg/ml), during in vitro embryo production (0, 30, 300 or 2000 µg/ml), or during both periods to determine the rates of 2−4 cells embryos, blastocysts rates and cells numbers, as well as the production of reactive oxygen species (ROS). Bovine ovaries (n = 240) were collected from a local abattoir after slaughter and cumulus–oocyte complexes (COCs) with homogeneous and non-dark cytoplasm, surrounded by two or more compact layers of cumulus cells, and an intact zona pellucida were selected for in vitro maturatuion (IVM). Mature oocytes were then submitted to in vitro fertilization (IVF) and in vitro embryo production (IVP) in culture medium supplemented or not with different concentrations of anethole, as described above. Although IVM medium supplementation with 300 µg/ml anethole improved the rates of bovine blastocysts formation, we demonstrated that IVP medium supplementation with 30 µg/ml anethole, regardless of IVM medium enrichment, considerably enhanced blastocysts rates. Furthermore, ROS levels were decreased only when anethole was added to the IVP medium without previous IVM medium supplementation.

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