134 EFFECT OF REPLACING FETAL BOVINE SERUM BY DIFFERENT GROWTH FACTORS IN THE PRE-IMPLANTATION DEVELOPMENT OF BOVINE EMBRYOS GENERATED BY IN VITRO FERTILIZATION

2014 ◽  
Vol 26 (1) ◽  
pp. 180
Author(s):  
R. Felmer ◽  
T. Vargas ◽  
R. Sanchez ◽  
M. E. Arias
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Culture Medium ◽  
Fetal Bovine Serum ◽  
Fibroblast Growth Factor 2 ◽  
Bovine Embryos ◽  
Epidermal Growth ◽  
Fetal Bovine ◽  
Defined Culture

Different culture systems have been studied that support pre-implantation development of bovine embryos up to the blastocyst stage. However, the use of chemically defined culture systems has been less studied. The objective of the present study was to evaluate the effect, in the developmental potential of in vitro-produced bovine embryos, of replacing fetal bovine serum (FBS) by different growth factors in the maturation and embryo culture media. In experiment 1, oocytes collected by aspiration of ovaries from a local slaughterhouse were matured in standard TCM-199 culture medium at 38.5°C, 5% CO2, and saturation humidity. The effect of insulin-like growth factor 1 (100 ng mL–1), epidermal growth factor (10 ng mL–1), and fibroblast growth factor 2 (500 ng mL–1) was evaluated at 24 h by the presence of a polar body after removal of cumulus-oocyte complexes. In experiment 2, oocytes matured in vitro in the presence of FBS were fertilized by co-incubation with commercial sperm (mL) for 18 h in standard fertilization medium (Fert-TALP). The presumptive zygotes were denuded and randomly allocated in a chemically defined culture medium based on KSOM supplemented with polyvinyl alcohol (PVA), fructose, and each of the growth factors listed previously. Undefined cultured medium was based on KSOM supplemented with 5% FBS. Embryos were cultured at 38.5°C in a mixture of gases and saturation humidity. Cleavage and blastocyst rates were recorded on Days 3 and 7, respectively. Analysis of variance was used to test for statistically significant differences between groups (P < 0.05) using Stat Graphics Plus 2 Software. In cases where statistically significant differences were observed, a multiple comparison test was run using Tukey's test. In experiment 1, a similar maturation rate was observed in all treatments relative to the undefined maturation medium (range = 88–91%). In experiment 2, no differences were observed in the cleavage (79, 87, 85, and 85%) and the blastocyst rates (24, 25, 26, and 30%) for the epidermal growth factor, insulin-like growth factor 1, fibroblast growth factor 2, and FBS treatments, respectively. In conclusion, we demonstrated that maturation of bovine oocytes can be achieved in chemically defined conditions by replacing FBS by each of the growth factors evaluated herein. Furthermore, chemically defined KSOM medium supplemented by any of these growth factors can generate a similar rate of blastocyst than the undefined medium containing FBS. Analyses are under way to evaluate the effect of completely defined culture conditions (maturation and embryo culture) on the pre-implantation development of embryos produced in the presence of these growth factors.

Keratinocytes can differentiate into eccrine sweat ducts in vitro: involvement of epidermal growth factor and fetal bovine serum

2003 ◽  
Vol 33 (3) ◽  
pp. 141-150 ◽  
Author(s):  
Takanori Shikiji ◽  
Mitsuyoshi Minami ◽  
Toshiyuki Inoue ◽  
Kenji Hirose ◽  
Hajimu Oura ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Epidermal Growth ◽  
Fetal Bovine ◽  
Eccrine Sweat

189 EFFECT OF PROGESTERONE AND EPIDERMAL GROWTH FACTOR ON IN VITRO-PRODUCED EIGHT-CELL BOVINE EMBRYOS IN A SERUM-FREE CULTURE MEDIUM

2007 ◽  
Vol 19 (1) ◽  
pp. 211 ◽  
Author(s):  
B. Merlo ◽  
E. Iacono ◽  
G. Mari
Keyword(s):  
Growth Factor ◽  
Embryo Development ◽  
Culture Medium ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Serum Free ◽  
Epidermal Growth ◽  
Blastocyst Rate

The role of progesterone (P4) and epidermal growth factor (EGF) in early bovine embryo development is still not clear. P4 has been administered at different times of embryo development, and a direct effect on IVF-derived bovine 8-cell embryos has been noted even if there was an interference due to the P4 vehicle (Ferguson et al. 2005 Reprod. Fertil. Dev. 17, 219 abst). EGF has been added to the culture medium from the presumptive zygote stage at different concentrations, resulting in improved blastocyst rates compared to that in control medium (Mtango et al. 2003 Theriogenology 59, 1393–1402; Sirisathien et al. 2003 Anim. Reprod. Sci. 77, 21–32), and gave results similar to those with 5% or 10% FCS (Palasz et al. 2000 Anim. Reprod. Sci. 58, 229–240). The objective if this experiment was to determine the effect of P4 and EGF on development of in vitro-produced bovine embryos when administered alone or in combination at the 8-cell stage in the absence of serum. In vitro-produced bovine 8-cell embryos were randomly allotted to treatments: (1) control, SOFaaBSA medium (BSA, 16 mg mL−1; n = 198); (2) P4, SOFaaBSA + P4 (15 ng mL−1 in ethanol; n = 198); (3) EGF, SOFaaBSA + EGF (25 ng mL−1; n = 200); (4) P4 + EGF, SOFaaBSA + P4 (15 ng mL−1 in ethanol) + EGF (25 ng mL−1; n = 201); and (5) FBS, SOFaaBSA + FBS (5%; n = 197). In order to minimize the toxic effect of ethanol, it was allowed to evaporate from the culture dish and then medium was added. All in vitro procedures were carried out at 38.5°C in a humidified atmosphere of 5% CO2 in air; presumptive zygotes were cultured in SOFaaBSA until 8-cell stage. Embryo development was evaluated on Day 6 and on Day 8 after IVF (Day 0), and rates calculated from 8-cell embryos. The study was done in 4 replicates and chi-square test was used for statistical analysis (Statistica for Windows; Stat Soft Inc., Tulsa, OK, USA); significance was assessed at P &lt; 0.05. Results are reported in Table 1. No differences were found in the number of morulae between P4 and control, between P4 + EGF and FBS, and between P4 + EGF and EGF (P &gt; 0.05), whereas the combination P4 + EGF was better than P4 alone (P &lt; 0.05). Blastocyst rate was not different (P &gt; 0.05) among EGF, P4 + EGF, and FBS groups. P4 achieved an higher (P &lt; 0.05) blastocyst rate than control but it was lower (P &lt; 0.05) than that of P4 + EGF or FBS. In conclusion, P4 alone improves embryo development from the 8-cell embryo to the blastocyst stage in a serum-free culture system, and EGF alone achieves a blastocyst rate not significantly different from that of FBS; furthermore, the combination of P4 and EGF can be considered the most suitable as an alternative to FBS because similar results were obtained in terms of both morulae and blastocysts. Table 1.Eight-cell bovine embryo development in SOFaaBSA medium in presence of P4, EGF, P4+EGF, or FBS

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.

Effects of amino acids on development in vitro of cleavage-stage bovine embryos into blastocysts

1996 ◽  
Vol 8 (5) ◽  
pp. 835 ◽  
Author(s):  
T Pinyopummintr ◽  
BD Bavister
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Embryo Development ◽  
Culture Medium ◽  
Essential Amino Acids ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Development In Vitro ◽  
Defined Culture

Effects of amino acids on early bovine embryo development in vitro were examined using a chemically-defined, protein-free culture medium. Bovine embryos produced in vitro were cultured from 18 h to 72 h post insemination in a simple medium containing lactate as the only energy source except for the amino acid treatments. Subsequently, embryos were transferred to TCM-199 supplemented with serum for blastocyst development to substantiate their developmental competence. Treatments were: (1) non-essential amino acids from TCM-199 (NEA); (2) essential amino acids from TCM-199 (EA); (3) NEA+EA; (4) Eagle's minimum essential medium amino acids (MEM AA); (5) 11 amino acids present in HECM-6 (11 AA); and (6) 0.2 mM glutamine (GLN). A higher proportion of embryos (percentage of inseminated ova) cleaved to the > or = 8-cell stage by 72 h post insemination in NEA (56.7%), EA (41.2%), 11 AA (40.3%) and GLN (51.1%) than in either NEA+EA (30.0%) or MEM AA (33.1%). However, after transfer to complex medium, embryos that had developed in EA, as well as those in MEM AA or NEA+EA, produced significantly fewer blastocysts (37.1%, 34.4% and 25.6% respectively) than those in NEA (56.7%), GLN (48.9%) or 11 AA (37.7%). The ability of blastocysts to hatch from their zonae pellucidae was also affected by amino acid treatment during cleavage stages. The present study indicated that the addition of NEA or GLN or 11 AA to a chemically-defined culture medium during the cleavage phase of bovine embryo development increases their subsequent ability to reach the blastocyst stage. These data have implications for understanding the nutritional needs of bovine embryos produced in vitro and for optimizing the composition of culture media to support their development.

133 EFFECTS OF ACTIVINA ON mRNA EXPRESSION IN IN VITRO FERTILIZED BOVINE EMBRYOS CULTURED FROM CHEMICALLY DEFINED TWO-STEP CULTURE MEDIUM

2008 ◽  
Vol 20 (1) ◽  
pp. 147
Author(s):  
J. E. Park ◽  
G. Jang ◽  
H. J. Oh ◽  
S. G. Hong ◽  
I. S. Yang ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Early Stage ◽  
Activin A ◽  
Developmental Competence ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Defined Culture

During the preimplantation stage, embryo development occurs in a maternal environment within the oviducts and uterine horns. It has been speculated that both the embryo itself and the maternal reproductive tract provide paracrine factors that influence embryo development (Jones et al. 2006 Reproduction 132(5), 799–810). Activins are known for FSH releasers, and several previous studies have reported that activin subunits and activin receptors mRNA were expressed in oocytes, zygotes, and oviduct (Yoshioka et al. 1998 Reprod. Fertil. Dev. 10(3), 293–298; Gandolfi et al. 1995 Mol. Reprod. Dev. 40(3), 286–291). The purposes of the present study were Experiment 1) to evaluate the effects of activin A on developmental competence of bovine embryos derived from two-step defined culture medium (Lim et al. 2007 Theriogenology 67(2), 293–302) and Experiment 2) to analyze the effects of activin A on transcriptional level of the genes in IVF embryos. Cumulus–oocyte complexs were harvested from ovaries obtained from a local slaughter house, matured, and fertilized in vitro. In vitro fertilized zygotes cultured in media supplemented with activin A in the early stage at the concentrations of 0, 10, or 100 ng mL–1 or in the later stage medium at the concentrations of 0, 10, or 100 ng mL–1. Data were analyzed using the Statistical Analysis System (SAS) program. In Exp. 1, although the development competence of embryos that cultured with activin A in the early stage medium was not significantly different, development to blastocysts on day 8 in the later stage medium with 100 ng mL–1 activin A was significantly higher than the control group [22.4% (54/264) v. 34.7% (76/233); P < 0.05]. Hatching rate of blastocyst on day 8 was significantly higher in the presence of 100 ng mL–1 activin A in the later stage culture medium compared with the control group [9.3% (5/54) v. 22.4% (17/76); P < 0.05]. In Exp. 2, the relative expression of 3 genes (Na/KATPase, E-cad, Glut-1) related to blastocyst hatching and implantation was analyzed. The relative abundance (ratio to GAPDH mRNA) of gene transcripts in blastocysts was measured by conventional semi-quantitative reverse transcription-polymerase chain reaction. The expression level of the Na/K ATPase, E-cad, and Glut-1 gene were higher in the presence of activin A in the culture medium compared with the control group. In conclusion, this study suggests that activin A during the later stage of in vitro bovine embryo development can enhance the developmental competence of preimplantation embryos, increase the hatching rate, and affect expression level of genes related to hatching and implantation in defined culture medium. This study was financially supported by KOSEF (grant ? M10625030005-07N250300510) and the Korean MOE, through the BK21 program for Veterinary Science.

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