75 Improvement of Developmental Competence of Bovine In Vitro-Produced Embryos by Using Charcoal:Dextran-Stripped Fetal Bovine Serum on In Vitro Culture Media

2018 ◽  
Vol 30 (1) ◽  
pp. 176
Author(s):  
A. Mesalam ◽  
R. Kong ◽  
B.-H. Choi ◽  
K.-L. Lee ◽  
B.-Y. Park ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Bovine Serum ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Mrna Levels ◽  
Fetal Bovine

Serum has widely been used as a main supplement to embryo in vitro culture media as it contains embryotrophic factors. Charcoal:dextran treatment of fetal bovine serum (FBS) removes lipophilic chemicals and certain steroid hormones and growth factors. The objective of this study was to investigate the effects of charcoal:dextran-stripped fetal bovine serum (CDS FBS) and heat-inactivated FBS (HI FBS) in embryo culture medium (SOF-BE1 medium supplemented with 10% of serum) on their ability to support in vitro development of bovine embryos. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, and cryo-tolerance. The experiment was conducted in 6 replicates (350 oocytes per group). The differences in embryo development, integrated optical intensity, and expression levels of the various genes between experimental groups were analysed by one-way ANOVA. Duncan’s multiple range tests were used to test the differences between the treatments. The level of statistical significance was set at P < 0.05. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly (P < 0.05) higher in medium containing CDS FBS than in medium containing HI FBS (42.84 ± 0.78% v. 36.85 ± 0.89%, respectively). The total number of cells per Day 8 blastocyst was not significantly different (P > 0.05) between the CDS FBS group (208.40 ± 14.77) and the HI FBS group (195.11 ± 19.15). Furthermore, the beneficial effects of CDS FBS on embryos were associated with a significantly increased mitochondrial activity, as identified by MitoTracker Green, and reduced intracellular lipid content, as identified by Nile red staining, which increased their cryo-tolerance. The post-thaw survival rate of blastocysts was significantly (P < 0.05) higher after 24 h in the CDS FBS than in the HI FBS group (85.33 ± 4.84% v. 68.67 ± 1.20%). Quantitative reverse transcription PCR showed that the mRNA levels of lipid metabolism-related genes, acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain, and the cholesterol metabolism related gene hydroxymethylglutaryl-CoA reductase were significantly increased upon culture with CDS FBS. Moreover, the mRNA levels of survival gene sirtuin 1, antioxidant gene superoxide dismutase 2, and anti-apoptotic associated gene B-cell lymphoma 2 in frozen–thawed blastocysts were significantly (P < 0.05) higher in the CDS FBS group than in the HI FBS group; however, the mRNA level of the pro-apoptotic gene BCL2-associated X protein was significantly reduced. In conclusion, these data suggest that supplementation of in vitro culture medium with CDS FBS improves in vitro bovine embryo developmental competence and the quality of blastocysts in terms of their crytolerance and gene expression. This research was supported by grant from the Next-Generation BiogGeen21 (No. PJ01107703), IPET (No. 315017-5 and 117029-3), Allergy free cat (Co.. Felix Pets), BK21plus, and KGSP.

76 Tetrahydrofuran does not have Embryo Toxic Effects on In Vitro-Produced Bovine Embryos

2018 ◽  
Vol 30 (1) ◽  
pp. 176
Author(s):  
M. M. R. Chowdhury ◽  
I. Khan ◽  
A. Mesalam ◽  
K.-L. Lee ◽  
J.-Y. Hwang ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Bovine Serum ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Control Group ◽  
Bovine Embryos ◽  
Sodium Pyruvate ◽  
Fetal Bovine ◽  
The Impact

In vitro embryo developmental potentials are still suboptimal compared with in vivo potential due to the challenge of various unknown stressors that must be overcome by in vitro-cultured oocytes. To improve existing embryo developmental potentials, many chemicals have been treated in maturation media by dissolving in toxic substances such as dimethyl sulfoxide (DMSO) or other carrier molecule. The foremost effort of this study was to investigate the impact of the solvent tetrahydrofuran (THF) on the cytotoxicity of in vitro embryo production (IVP). The experiment was completed within 8 replicates. Statistical analyses were performed using SPSS version 22.0 (IBM/SPSS, Armonk, NY, USA), a one-way ANOVA followed by multiple pairwise comparisons (Tukey’s test), and Duncan’s multiple range post hoc test. The level of statistical significance was considered P < 0.05. Oocytes were cultured in vitro maturation media (IVM) followed by in vitro fertilization (IVF), in vitro culture media 1 (IVC1), and in vitro culture media 2 (IVC2). Composition of the media was as follows: IVM medium was TCM-199 supplemented with 10% (v/v) fetal bovine serum, 1 µg mL−1 oestradiol-17β, 10 µg mL−1 FSH, 0.6 mM cysteine, and 0.2 mM sodium pyruvate. The IVC1 medium consisted of CR1-aa supplemented with 44 µg mL−1 sodium pyruvate, 14.6 µg mL−1 glutamine, 10 IU mL−1 penicillin, 0.1 mg mL−1 streptomycin, 3 mg mL−1 BSA, and 310 µg mL−1 glutathione. The IVC2 medium was the same composition as IVC1 except that BSA was replaced with 10% (v/v) fetal bovine serum. The final concentration of the optimized (0.5 µM) THF in culture medium was 0.4%. When coculturing with 0.5 µM THF in the IVM stage, the cleavage rate (58.65 ± 1.90% v. 56.87 ± 1.68%) was not significantly different, but the blastocyst rate (35.21 ± 1.44% v. 28.34 ± 2.11%) was significantly higher compared with the control group. The TUNEL assay confirmed that apoptotic nuclei in THF group were significantly reduced compared with the control group (2.32 ± 0.14 v. 5.65 ± 0.12). The total cell number of trophectoderm (TE) in control and THF groups was 115.34 ± 0.98 and 132.13 ± 1.55, and that of the inner cell mass (ICM) was 29.67 ± 0.40 and 39.94 ± 0.44, respectively. However, the ICM:TE ratio in control and treated blastocysts was 1:3.34 and 1:3.9, which was not statistically significant. Immunocytochemistry analysis (using antibodies to IKBKB, NFkB, COX2, CASP9, and CASP3) demonstrated that THF supplementation significantly attenuated expression of these proteins. The quantitative recerse transcription PCR data established that relative mRNA expression level of the anti-apoptotic gene BCL2 was up-regulated, whereas that of COX2, iNOS, BAX, IKBKB, NFkB, CASP9, and CASP3 were significantly down-regulated in the THF treated group compared with the control. In conclusion, 0.5 µM THF supplement in the IVM media did not have injurious effects on in vitro-cultured bovine embryos. This work was supported by grant from the Next-Generation BiogGeen21 (No. PJ01107703), IPET (No. 315017-5 and 117029-3), Allergy free cat (Co.. Felix Pets) and BK21plus.

scholarly journals Fetal Bovine Serum-Derived Extracellular Vesicles Persist within Vesicle-Depleted Culture Media

2018 ◽  
Vol 19 (11) ◽  
pp. 3538 ◽  
Author(s):  
Brandon Lehrich ◽  
Yaxuan Liang ◽  
Pooya Khosravi ◽  
Howard Federoff ◽  
Massimo Fiandaca
Keyword(s):  
Cell Growth ◽  
Extracellular Vesicles ◽  
Bovine Serum ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Cellular Growth ◽  
Primary Astrocyte ◽  
Fetal Bovine ◽  
Cell Growth And Viability

It is known that culture media (CM) promotes cellular growth, adhesion, and protects explanted primary brain cells from in vitro stresses. The fetal bovine serum (FBS) supplement used in most CM, however, contains significant quantities of extracellular vesicles (EVs) that confound quantitative and qualitative analyses from the EVs produced by the cultured cells. We quantitatively tested the ability of common FBS EV-depletion protocols to remove exogenous EVs from FBS-supplemented CM and evaluated the influence such methods have on primary astrocyte culture growth and viability. We assessed two methodologies utilized for FBS EV removal prior to adding to CM: (1) an 18-h ultracentrifugation (UC); and (2) a commercial EV-depleted FBS (Exo-FBS™). Our analysis demonstrated that Exo-FBS™ CM provided the largest depletion (75%) of total FBS EVs, while still providing 6.92 × 109 ± 1.39 × 108 EVs/mL. In addition, both UC and Exo-FBS™ CM resulted in poor primary astrocyte cell growth and viability in culture. The two common FBS EV-depletion methods investigated, therefore, not only contaminate in vitro primary cell-derived EV analyses, but also provide a suboptimal environment for primary astrocyte cell growth and viability. It appears likely that future CM optimization, using a serum-free alternative, might be required to advance analyses of cell-specific EVs isolated in vitro.

scholarly journals Influence of recombinant hematopoietins and of fetal bovine serum on the globin synthetic pattern of human BFUe

Blood ◽  
1990 ◽  
Vol 76 (6) ◽  
pp. 1150-1157 ◽  
Author(s):  
AR Migliaccio ◽  
G Migliaccio ◽  
M Brice ◽  
P Constantoulakis ◽  
G Stamatoyannopoulos ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Mrna Level ◽  
Fetal Hemoglobin ◽  
Mrna Levels ◽  
Gm Csf ◽  
Gamma Globin ◽  
Fetal Bovine ◽  
Globin Synthesis

Abstract We have studied the effects of recombinant hematopoietic growth factors, granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or interleukin-3 (IL-3) on the globin program of adult human erythroid progenitors (BFUe) stimulated to terminal differentiation by erythropoietin under fetal bovine serum (FBS)-supplemented or FBS- deprived culture conditions. Fetal globin production by BFUe-derived erythroblasts was assessed at the protein and mRNA level and its cellular distribution was evaluated by immunofluorescence. Although hemoglobinization and maturation of BFUe-derived erythroblasts was by and large comparable in FBS-replete versus FBS-deprived cultures, the latter had significantly less (up to 20-fold) gamma-globin and gamma- globin mRNA levels. Reduced gamma-globin in serum-deprived cultures was also reflected by a smaller proportion of erythroblasts with detectable gamma-globin by immunofluorescence. Erythroid bursts induced by either GM-CSF or IL-3 produced similar levels of gamma-globin both in FBS- supplemented and in FBS-deprived cultures. These results, obtained even in cultures of highly enriched BFUe, suggest that GM-CSF and IL-3, although they significantly increase the number and size of erythroid bursts, do not by themselves exert a direct influence on the level of fetal globin synthesis. By contrast, factor(s) present in FBS appear to exert a dominant influence on fetal globin synthesis in vitro. Although FBS-deprived conditions appear to largely abrogate the in vitro activation of fetal hemoglobin (Hb F) in normal samples, they do support increased Hb F production in samples from patients with hereditary persistence of fetal hemoglobin or from cord blood.

302 REPLACEMENT OF FETAL BOVINE SERUM WITH KNOCKOUT SERUM REPLACER AND STEM CELLS CONDITIONED MEDIUM DURING IN VITRO CULTURE OF BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 307
Author(s):  
M. M. Souza ◽  
N. Z. Saraiva ◽  
C. S. Oliveira ◽  
T. A. D. Tetzner-Nanzeri ◽  
R. Vantini ◽  
...  
Keyword(s):  
Stem Cells ◽  
In Vitro Culture ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Protein Supplementation ◽  
Control Group ◽  
Bovine Embryos ◽  
Fetal Bovine

The use of fetal bovine serum (FBS) as protein supplementation in IVP of bovine embryos has presented difficulties because it can introduce a number of pathogenic components in culture systems, can be related to the birth of calf with abnormal growth and development, and precludes the establishment of the actual nutritional needs of the embryo, because it contains an unlimited variety of substances. This study evaluated the replacement of the FBS in the medium of in vitro culture (IVC) of bovine embryos, using the knockout serum replacer (KSR) as protein supplementation and culture medium conditioned with stem cells. Therefore, bovine oocytes from ovaries of slaughterhouse were selected and matured in vitro in TCM-199 medium supplemented with 10% FBS (Crypion), 1.0 μg mL-1 FSH (Pluset®, Calier, Barcelona, Spain), 50 μg mL-1 hCG (Profasi®, Serono, Geneva, Switzerland), 1.0 μg mL-1 estradiol (Sigma E-2758, Sigma Chemical, St. Louis, MO, USA), 0.2 mM sodium pyruvate, and 83.4 μg mL-1 amikacin for 24 h. After that, 1144 oocytes were fertilized in IVF-TALP medium containing 6 mg mL-1 of BSA. After 18 to 22 h, the zygotes were cultured in SOF + 5% FBS (group 2); SOF + 5% KSR (group 3); SOF (5% FBS) + 10% SOF (5% FBS) conditioned by stem cells (group 4); or SOF (5% KSR) + 10% SOF (5% KSR) conditioned by stem cells (group 5), in an atmosphere of 5% O2 at 38.5°C for 8 days. A control group outside the controlled atmosphere was added, supplemented with 5% FBS (group 1). The SOF medium supplemented with 5% FBS or KSR was conditioned by stem cells and added to SOF medium for the culture of embryo at a concentration of 10%. The rates of cleavage and production of blastocysts were assessed 48 hours and 7 days after IVF, respectively, and analyzed by chi-square test, with a significance level of 5% in the statistical program Minitab® (release 14.1, Minitab, State College, PA, USA). On the eighth day, the TUNEL test for determination of the percentage of apoptosis and the differential staining technique for determination of inner cell mass (ICM) and trophoblast (TF) were performed. The results were submitted to ANOVA, followed by comparing the means by Tukey’s test using the program GraphPad Prism (GraphPad, San Diego, CA, USA). The treatments did not differ in the production of embryos, being similar to the control group: G1 = 31.75% (74/233), G2 = 35.26% (79/224), G3 = 32.70% (74/226), G4 = 28.76% (63/219), and G5 = 26.85% (65/242). With regard to the assessment of embryonic quality, the treatments showed similar results to the control groups. No differences were observed among groups both in color and ICM/TF ratio (G1 = 0.60, G2 = 0.62, G3 =0.65, G4 = 0.60, and G5 = 0.60). Furthermore, the TUNEL showed no significant difference in the percentage of apoptosis among groups (G1 = 7.10%, G2 = 3.76%, G3 = 5.58%, G4 = 4.50%, and G5 = 4.11%). The data obtained so far indicate that it is possible to produce embryos in vitro by replacing the FBS in the culture, achieving results similar to those obtained with serum. Financial support: FAPESP 2007/58506-6.

scholarly journals Influence of recombinant hematopoietins and of fetal bovine serum on the globin synthetic pattern of human BFUe

Blood ◽  
1990 ◽  
Vol 76 (6) ◽  
pp. 1150-1157 ◽  
Author(s):  
AR Migliaccio ◽  
G Migliaccio ◽  
M Brice ◽  
P Constantoulakis ◽  
G Stamatoyannopoulos ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Mrna Level ◽  
Fetal Hemoglobin ◽  
Mrna Levels ◽  
Gm Csf ◽  
Gamma Globin ◽  
Fetal Bovine ◽  
Globin Synthesis

We have studied the effects of recombinant hematopoietic growth factors, granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or interleukin-3 (IL-3) on the globin program of adult human erythroid progenitors (BFUe) stimulated to terminal differentiation by erythropoietin under fetal bovine serum (FBS)-supplemented or FBS- deprived culture conditions. Fetal globin production by BFUe-derived erythroblasts was assessed at the protein and mRNA level and its cellular distribution was evaluated by immunofluorescence. Although hemoglobinization and maturation of BFUe-derived erythroblasts was by and large comparable in FBS-replete versus FBS-deprived cultures, the latter had significantly less (up to 20-fold) gamma-globin and gamma- globin mRNA levels. Reduced gamma-globin in serum-deprived cultures was also reflected by a smaller proportion of erythroblasts with detectable gamma-globin by immunofluorescence. Erythroid bursts induced by either GM-CSF or IL-3 produced similar levels of gamma-globin both in FBS- supplemented and in FBS-deprived cultures. These results, obtained even in cultures of highly enriched BFUe, suggest that GM-CSF and IL-3, although they significantly increase the number and size of erythroid bursts, do not by themselves exert a direct influence on the level of fetal globin synthesis. By contrast, factor(s) present in FBS appear to exert a dominant influence on fetal globin synthesis in vitro. Although FBS-deprived conditions appear to largely abrogate the in vitro activation of fetal hemoglobin (Hb F) in normal samples, they do support increased Hb F production in samples from patients with hereditary persistence of fetal hemoglobin or from cord blood.

90 OVIDUCTAL EPITHELIAL CELLS Co-CULTURE PROMOTES GOAT (CAPRA HIRCUS) IN VITRO PARTHENOGENETIC EMBRYO DEVELOPMENT

2017 ◽  
Vol 29 (1) ◽  
pp. 152
Author(s):  
M. Mahajan ◽  
D. Nagoorvali ◽  
N. Rawat ◽  
M. S. Chauhan ◽  
R. S. Manik ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Epithelial Cells ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Calcium Ionophore ◽  
Mrna Abundance ◽  
Developmental Competence ◽  
Capra Hircus ◽  
Fetal Bovine

Co-culture of pre-implantation embryos with oviducal epithelial cells mimics the in vivo conditions, thus, playing a crucial role in embryo metabolism and gene expression and finally supporting embryonic developmental competence in several ways. Hence, the objective of the present study was to evaluate the effect of goat oviducal epithelial cells (GOEC) co-culture on goat parthenogenetic embryonic development, quality, and relative mRNA abundance of genes related to developmental competence and oxidative stress. The GOEC were obtained from goat oviducts by squeezing and thorough washing with TCM-199 + 10% fetal bovine serum. Goat cumulus–oocyte complexes were collected from slaughterhouse ovaries and matured in TCM-199 + 10% fetal bovine serum supplemented with 5 μg mL−1 of FSH, 10 μg mL−1 of LH, and 1 μg mL−1 of β-oestradiol for 27 h in CO2 incubator with 5% CO2 and at 38.5°C with >95% RH. In vitro matured cumulus–oocyte complexes were denuded and activated with 5 μM calcium ionophore and 2 mM 6-DMAP. Following activation, embryos were co-cultured with and without GOEC (control) in mCR2aa media. The blastocyst development rate was significantly (P < 0.05) higher (23.00 ± 1.15% v. 17.33 ± 1.45%) in the media cultured with GOEC than in control. The total cell number of blastocysts (n = 4) was also found to be significantly more (167.25 ± 17.51 v. 110.25 ± 12.02) than that of control (P < 0.05). However, the apoptotic index (3.76 ± 0.23% v. 7.97 ± 1.99%) was not significantly different in both groups. Further, RNA was isolated from both groups (20 each) of blastocysts on Day 8, and cDNA was prepared. Analysis by qPCR revealed that the relative mRNA abundance of development related genes, i.e. VEGF, BMP4, and CCNB1, showed significantly high (P < 0.05) expression, whereas the expression of CRABP1 was significantly low (P < 0.05) in GOEC co-culture than control. Oxidative stress related genes GPX-1 and SOD2 had comparable expression in both the culture systems, whereas a nonsignificant (P < 0.05) increase in expression of PRDX1 was observed in GOEC co-culture group. In conclusion, co-culture of embryos with GOEC in the simple culture media like mCR2aa helps in improving developmental competence and quality of parthenogenetic embryos. This work was supported by the NFBSFARA Project on Parthenogenetic Goat (CA-4002), New Delhi, India.

scholarly journals 2POSTHATCHING DEVELOPMENT SYSTEM: A NOVEL IN VITRO CULTURE OF BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 123 ◽  
Author(s):  
D.O. Brandão ◽  
G. Vajta ◽  
P. Maddox-Hyttel ◽  
D. Stringfellow ◽  
P. Lövendahl ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Bovine Serum ◽  
Culture System ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Fetal Bovine Serum ◽  
Cell Mass ◽  
Embryo Morphology ◽  
Fetal Bovine

Although high blastocyst rates can be achieved in somatic cell nuclear transfer, abortions and developmental abnormalities still hamper advancement. Reliable and practical methods to evaluate early embryonic development and differentiation are required to understand and overcome the problem. Our aim was to establish an in vitro culture system for monitoring posthatching development (PHD). Slaughterhouse-derived bovine oocytes were matured in vitro, fertilized (Day 0) and cultured (Holm et al., 1999, Theriogenology, 52, 683–700). On Day 8, degenerated embryos were removed from each well and 400L of modified culture medium (SOFaaci plus 0.5% glucose and 10% fetal bovine serum) were added. At Day 11, hatched blastocysts were selected by scoring them as Quality 1 (Q1: &gt;1.0mm, clear trophoblast, compact inner cell mass), Quality 2 (Q2: 0.5mm, dark spots in the trophoblast, less compact inner cell mass), or Quality 3 (Q3: &lt;0.5mm, many dark spots in the trophoblast, spread inner cell mass). The resulting 304 blastocysts in 12 replicates were then loaded into 15mm×1.2 gel tunnels of 2.4% agarose in PBS, supplemented with either 5% (Agar5) or 10% (Agar10) fetal bovine serum, covered with the modified culture medium, and then incubated at 38.5°C in 5% CO2, 5% O2, 90% N2. Embryo morphology and length were evaluated using a stereomicroscope on Days 12, 13, 14 and 15. On Day 14, 75 embryos were removed, biopsed (1mm) for sex determination of each embryo, and processed for light and transmission electron microscopy. Qualitative and quantitative data were analyzed by χ2 test and GLM procedure of SAS, respectively, with P level of 0.05. A total of 170 embryos (56% of total) initiated elongation. This percentage was higher (LSmeansSD, n=12; P&lt;0.05) in Agar10 v. Agar5 in both Q1 (889 v. 637), Q2 (667 v. 485) and Q3 embryos (529 v. 278). Mean embryo length (mm; LSmeansSEM) on Day 13 was higher (P&lt;0.05) in Q1 (2.10.2, n=49) and Q2 (1.71.4, n=98) than Q3 (1.20.3, n=23). On Day 14, Q1 embryos (3.50.2) were longer (P&lt;0.01) than Q2 and Q3 embryos (2.70.1 and 2.00.3). On Day 15, Q1, Q2 and Q3 embryos (4.40.5, n=24, 4.00.3, n=45 and 2.90.6, n=14, respectively) had similar length, probably influenced by the low number of Q3 embryos. The percentage of males was higher (P&lt;0.001) in Q1 (95%; n=40), but similar in Q2 (39%; n=26) and Q3 (71%; n=7). Light microscopy confirmed hypoblast and epiblast formation. Ultrastructural analysis revealed that the latter had penetrated the trophoblast (Rauber’s layer), forming an embryonic disc including many degenerative cells. In conclusion, this culture system represents the first model for rapid growth, elongation, and initial differentiation of bovine posthatching embryos.

185 PARTHENOGENETIC ACTIVATION OF SIKA DEER (CERVUS NIPPON TEMMINCK) OOCYTES WITH CHEMICALS

2012 ◽  
Vol 24 (1) ◽  
pp. 204
Author(s):  
Y. P. Yin ◽  
L. N. Tang ◽  
A. R. Fan ◽  
S. Zhang ◽  
X. Ma ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Sika Deer ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Cumulus Cells ◽  
Control Group ◽  
Oocyte Activation ◽  
Parthenogenetic Activation ◽  
Fetal Bovine

Parthenogenetic activation of the oocyte represents an important step in the somatic cell nuclear transfer. The aim of the present study was to establish optimizing conditions for parthenogenetic activation of Sika deer oocytes necessary for cloning Sika deer. Sika deer ovaries were collected from a slaughter house during oestrus season (October and November), placed into saline (25°C) supplemented with 1% (v/v) penicillin and streptomycin and transported into the laboratory within 4 h. The small vesicular follicles (diameter, 2–5 mm) on the ovarian surface were incised with a scalpel in a Petri dish containing PBS to release the cumulus–oocyte complexes (COC). Only COC with uniform cytoplasm and at least 3 layers of compact cumulus cells were cultured in vitro for 24 h. The media of in vitro maturation (IVM) was TCM-199 supplemented with 10% fetal bovine serum, 10 μg mL–1 FSH, 1 μg mL–1 LH, 0.2 mM cysteamine and 50 ng mL–1 epidermal growth factor. After IVM, the cumulus cells were denuded with 0.2% hyaluronidase in TCM-199 at 38.5°C by pipetting. The cumulus-free Sika deer oocytes were stimulated by 1 of the following treatments: 1) ethanol + 6-DMAP, treated with 7% ethanol for 7 min and 2 mM 6-dimethylaminopurine (6-DMAP) in DSOF for 4 h; or 2) ionomycin + 6-DMAP, treated with 5 μM ionomycin for 5 min and 2 mM 6-DMAP in DSOF for 4 h. Then, oocytes were transferred into culture media for 7 days [Day 0 (D0) = activation]. On D3, embryos were transferred into fresh DSOF drops supplemented with 10% (v/v) fetal bovine serum. All cultures were overlaid with mineral oil and kept in a humidified modular incubation chamber gassed with 5% CO2. Effects of these chemicals on oocyte activation were then examined and compared with the controls, in which oocytes were cultured in TCM-199 for 4 h without chemical supplement. Our results showed that rates of cleavage, morula and blastocyst were 72.7, 43.9 and 32.4% (n = 139), respectively, by treatment with ionomycin + 6-DMAP. And rates of cleavage, morula and blastocyst were 61.1, 29.7 and 17.8% (n = 134), respectively, by treatment with ethanol + 6-DMAP. However, the rates of cleavage, morula and blastocyst were 5, 0 and 0% (n = 101) in the control group. Meanwhile, the rates of oocyte cleavage (72.7% vs 61.1%), morula (43.9% vs 29.7%) and blastocyst (32.4% vs 17.8%) between 2 treatments of ionomycin + 6-DMAP and ethanol + 6-DMAP were significantly different (P < 0.05). In conclusion, parthenogenetic activation of Sika deer oocytes with ionomycin + 6-DMAP is more effective than that with ethanol + 6-DMAP. These results have begun to elucidate parameters important for animal modeling and cloning with the Sika deer and should facilitate the development of genetically defined animal models in this species. This work was supported by the grant from the China Postdoctoral Science Foundation (No. 20090451135).

scholarly journals Study on the growth promoting capacity of calf and fetal bovine serum for animal cells "in vitro" II: electrophoretic study and survey on the antiproteolytic activity of pools of calf and fetal bovine serum

1984 ◽  
Vol 26 (2) ◽  
pp. 97-104 ◽  
Author(s):  
Edda de Rizzo ◽  
Celidéia Aparecida Coppi Vaz ◽  
Inácio França Mendes ◽  
Anatércia Ferreira Bonfim Yano
Keyword(s):  
Bovine Serum ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Calf Serum ◽  
Animal Origin ◽  
Electrophoretic Study ◽  
Fetal Bovine ◽  
Growth Promoting ◽  
Antiproteolytic Activity

Calf serum and fetal bovine serum present great variability as to its growth promoting efficiency (GPE). As supplement of culture media to cultivate cells of animal origin they stimulate the "in vitro" multiplication and maintain cell viability. When fourteen lots of calf sera of variable GPE had the total protein contents as well as the percentages of serum fractions determined, no significant differences that could possibly explain the variability of the GPE were observed. Evaluation of the antiproteolytic activity of nineteen lots of calf serum and eighteen serum lots of younger calves showed that the former exhibited lower antiproteolytic titers (1:40 to 1:80) than the latter (1:80 to 1:160). Twelve lots of fetal bovine serum studied in parallel, showed the highest concentration of antiproteolytic factors, with titers equal to 1:320. Sera of bovine origin, but not fetal sera, are usually heat-inactivated, what was demonstrated to be responsible for the decrease of the antiproteolytic activity of 75% of the lots tested. This could explain the inability of certain heat-inactivated sera in promoting multiplication of some cells "in vitro", as verified with primary monkey kidney cells. The results obtained in this study indicated the convenience of submiting each lot of serum to be introduced in cell culture to previous determination of its characteristics, such as growth promoting efficiency, antiproteolytic activity and also toxicity, absence of extraneous agents, etc., in order to minimize the possibility of using serum lots of questionable quality, thus preventing not only the loss of cell lines, but also undesirable and sometimes expensive delays.

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