Culture of early somite mouse embryos during organogenesis

Development ◽  
1979 ◽  
Vol 49 (1) ◽  
pp. 17-25
Author(s):  
T. W. Sadler
Keyword(s):  
Total Protein ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Normal Growth ◽  
Protein Analysis ◽  
Mouse Embryos ◽  
Culture Period ◽  
Similar Time ◽  
Rat Serum

Early somite (2—4) mouse embryos were explanted and then maintained in culture for 24 or 48 h intervals. Various types of media were tested and it was determined that rat serum supported normal growth over a period of 48 h, based on total protein analysis and histological comparisons with in vivo specimens. Other media including fetal calf serum and fetal calf serum + Waymouth's (1:1) supported some growth, but did not equal the success of using rat serum alone. During the 48 h culture period in rat serum, embryos developed to stages indistinguishable from embryos maintained for a similar time in vivo.

Culture of mouse embryos during neurulation

Development ◽  
1981 ◽  
Vol 66 (1) ◽  
pp. 109-116
Author(s):  
T. W. Sadler ◽  
D. A. T. New
Keyword(s):  
Growth And Development ◽  
Calf Serum ◽  
Mouse Embryos ◽  
Rat Serum ◽  
Developmental Abnormalities ◽  
Time Period ◽  
Lower Protein ◽  
Made In

A comparison between static versus rotator culture systems and a variety of media (rat serum, new born calf serum, DMEM and Waymouth's) was made in an attempt to promote in vitro growth of mouse embryos from the beginning of neurulation (headfold stage) to the closure of the neural tube and formation of the limb buds (48 h). The results demonstrate that good development can be achieved for 48 h using a rotator system and that 80% of embryos cultured on rotators show growth and differentiation similar to that obtained for the same time period in vivo. Static cultures are less successful and embryos grown in this system show lower protein content and somite numbers than those maintained on rotators. Undiluted rat serum is superior to all other media tested and supports better growth and development as monitored by total protein and developmental abnormalities.

Effects of storage methods of fetal calf serum on the development of mouse embryos

1990 ◽  
Vol 33 (1) ◽  
pp. 265
Author(s):  
K. Kaneyama ◽  
T. Takeda ◽  
T. Onihara
Keyword(s):  
Fetal Calf Serum ◽  
Calf Serum ◽  
Mouse Embryos ◽  
Storage Methods

Effects of bovine oviduct epithelial cells, fetal calf serum and bovine serum albumin on gene expression in single bovine embryos produced in the synthetic oviduct fluid culture system

2005 ◽  
Vol 17 (8) ◽  
pp. 751 ◽  
Author(s):  
Mona E. Pedersen ◽  
Øzen Banu Øzdas ◽  
Wenche Farstad ◽  
Aage Tverdal ◽  
Ingrid Olsaker
Keyword(s):  
Gene Expression ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Glut 1 ◽  
Significant Difference ◽  
Oviduct Fluid

In this study the synthetic oviduct fluid (SOF) system with bovine oviduct epithelial cell (BOEC) co-culture is compared with an SOF system with common protein supplements. One thousand six hundred bovine embryos were cultured in SOF media supplemented with BOEC, fetal calf serum (FCS) and bovine serum albumin (BSA). Eight different culture groups were assigned according to the different supplementation factors. Developmental competence and the expression levels of five genes, namely glucose transporter-1 (Glut-1), heat shock protein 70 (HSP), connexin43 (Cx43), β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), analysed as mRNA by using reverse transcription–polymerase chain reaction, were measured on bovine embryos cultured for 9 days. Gene expression of these in vitro-produced embryos was compared with the gene expression of in vivo-produced embryos. There was no significant difference found in embryo developmental competence between the Day 9 embryos in BOEC co-culture, FCS and BSA supplements in SOF media. However, differences in gene expression were observed. With respect to gene expression in in vivo and in vitro embryos, BOEC co-culture affected the same genes as did supplementation with FCS and BSA. HSP was the only gene that differed significantly between in vitro and in vivo embryos. When the different in vitro groups were compared, a significant difference between the BOEC co-culture and the FCS supplementation groups due to Glut-1 expression was observed.

Effect of culture conditions on artificial activation of porcine oocytes matured in vitro

1996 ◽  
Vol 8 (8) ◽  
pp. 1153 ◽  
Author(s):  
N Yamauchi ◽  
H Sasada ◽  
S Sugawara ◽  
T Nagai
Keyword(s):  
In Vitro Maturation ◽  
Fetal Calf Serum ◽  
Culture Media ◽  
Calf Serum ◽  
Treated Group ◽  
Culture Conditions ◽  
Culture Period ◽  
Subsequent Response ◽  
Artificial Activation

The effects of culture media used and culture period for in vitro maturation of porcine oocytes on their subsequent response to chemical and electrical activation, were investigated. Activated oocytes were identified by the presence of a pronucleus(ei) or cleavage. Porcine oocytes were cultured for 24, 30, 36, 42 and 48 h in TCM199 with Earle's salts (199) supplemented with 10% fetal calf serum (199-FCS) before electrical stimulation. Although few oocytes were activated after 24 h and 30 h of culture (5.4% and 6.1% respectively), the percentage of activated oocytes increased significantly to 93.2% after 42 h in culture (P < 0.05); however, when the culture period was extended to 48 h, there was a significant decrease to 56.7% (P < 0.05). Oocytes were also cultured in four types of media: (1) 199-FCS; (2) 199 supplemented with 5 mg mL-1 bovine serum albumin (199-BSA); (3) Kreb's-Ringer bicarbonate solution supplemented with 10% FCS (KRB-FCS); and (4) KRB supplemented with BSA (KRB-BSA). After 42 h of culture in each medium, the oocytes were electrically activated. Although rates of maturation of oocytes cultured in the four media were similar (74.0-80.8%), all oocytes except those cultured in 199-FCS failed to be activated. In addition, oocytes were cultured for 36, 42 and 48 h in 199-FCS and then stimulated by treatment with ethanol. Significantly fewer oocytes were activated in the chemically-treated group than in the electrically-treated group. These results indicate that culture conditions used for the culture of porcine oocytes in vitro are important with respect to their subsequent response to artificial activation.

scholarly journals Fetal Calf Serum-Free Generation of Functionally Active Murine Dendritic Cells Suitable for In Vivo Therapeutic Approaches

2000 ◽  
Vol 114 (1) ◽  
pp. 142-148 ◽  
Author(s):  
Gabriele Müller ◽  
Anke Müller ◽  
Helmut Jonuleit ◽  
Kerstin Steinbrink ◽  
Claudia Szalma ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Therapeutic Approaches ◽  
Serum Free ◽  
Free Generation

Forskolin effect on the cryosurvival of in vitro-produced bovine embryos in the presence or absence of fetal calf serum

Zygote ◽  
2012 ◽  
Vol 22 (2) ◽  
pp. 146-157 ◽  
Author(s):  
Daniela Martins Paschoal ◽  
Mateus José Sudano ◽  
Midyan Daroz Guastali ◽  
Rosiára Rosária Dias Maziero ◽  
Letícia Ferrari Crocomo ◽  
...  
Keyword(s):  
Culture Medium ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Similar Rate ◽  
Bovine Embryos ◽  
Embryo Survival ◽  
Vitrification Solution ◽  
Oviductal Fluid

SummaryThe objective of this study was to assess the viability and cryotolerance of zebu embryos produced in vitro with or without the addition of fetal calf serum (FCS) and forskolin (F). Embryos produced in vivo were used as a control. Presumptive zygotes were cultured in modified synthetic oviductal fluid supplemented with amino acids (SOFaa), bovine serum albumin (BSA) and with (2.5%) or without (0%) FCS. On day 6 of growth, the embryos from each group were divided into treatments with or without 10 μM F to induce embryonic lipolysis, comprising a total of four experimental groups: 2.5% FCS, 0% FCS, 2.5% + F and 0% + F. For vitrification, embryos were exposed to vitrification solution 1 (5 M EG (ethylene glycol)) for 3 min and then transferred to vitrification solution 2 (7 M EG, 0.5 M galactose solution and 18% (w/v) Ficoll 70) before being introduced to liquid nitrogen. The presence of FCS in the culture medium resulted in the production of embryos with a similar rate of damaged cells compared with in vivo-produced embryos. After vitrification, the 2.5% FCS group had a significantly higher rate of damaged cells when compared with the other groups (P < 0.05). The results of this experiment indicated that the omission of FCS and the addition of forskolin do not have deleterious effect on embryo production rates. In addition, embryos produced in the presence of FCS had greater sensitivity to cryopreservation, but this effect was reversed when forskolin was added to the medium, which improved embryo survival without affecting embryo development and quality after vitrification.

Developmental competence in vitro and in vivo of cryopreserved, hatched blastocysts from the dromedary camel (Camelus dromedarius)

2004 ◽  
Vol 16 (6) ◽  
pp. 605 ◽  
Author(s):  
J. A. Skidmore ◽  
M. Billah ◽  
N. M. Loskutoff
Keyword(s):  
Survival Rate ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Sodium Lactate ◽  
Developmental Competence ◽  
Penicillin G ◽  
Dromedary Camel ◽  
Dromedary Camels

The present paper describes experiments designed to investigate methods for cryopreserving embryos from dromedary camels. Because preliminary studies had shown ethanediol to be the best cryoprotectant to use for camel embryos, the current experiments were performed to determine the minimum exposure time to 1.5 m ethanediol required to achieve cryoprotection. The uteri of 30 donor camels were flushed non-surgically 8 days after mating. Embryos were recovered and 158 were assigned to one of three groups, which were exposed to 1.5 m ethanediol for either 10 min (n = 67), 5 min (n = 51) or 1 min (n = 40). Embryos were subsequently thawed and rehydrated by expelling either directly into holding medium (HM; HEPES-buffered Tyrode's medium containing sodium lactate and 3 mg mL−1 bovine serum albumin, 10% fetal calf serum, 100 IU mL−1 penicillin G, 100 μg mL−1 streptomycin and 25 μg mL−1 amphotercin B) or initially into HM containing 0.2 m sucrose for 5 or 10 min. The survival rate of all embryos immediately post-thawing, as judged by the morphological appearance of the embryos, was high (91%), but was greatly reduced after 2 h culture (59%). Ninety-two embryos were transferred to recipient camels resulting in 18 viable fetuses (1 min ethanediol exposure, n = 1/15; 5 min ethanediol exposure, n = 3/34; 10 min ethanediol exposure, n = 14/43). Of the embryos rehydrated directly in HM, six of 65 resulted in viable fetuses and those rehydrated initially in 0.2 m sucrose for 5 or 10 min resulted in nine of 47 and three of 46 fetuses respectively. From these experiments, we conclude that camel embryos can be cryopreserved using ethanediol as a cryoprotectant when the embryos are cooled slowly (to 33°C) before being plunged into liquid nitrogen for storage.

74 EFFECT OF SIZE AND CULTURE PERIOD OF FROZEN–THAWED BOVINE TROPHOBLASTIC VESICLES ON INTERFERON-τ SECRETION

2014 ◽  
Vol 26 (1) ◽  
pp. 151
Author(s):  
Y. Hashiyada ◽  
H. Takahashi ◽  
D. Yamaguchi ◽  
K. Imai ◽  
M. Geshi
Keyword(s):  
Significant Positive Correlation ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Calf Serum ◽  
Culture Period ◽  
Fetal Bovine ◽  
Student’S T ◽  
Student’S T Test

Frozen–thawed bovine trophoblastic vesicles (bTV) derived in vivo could secrete interferon-τ (IFN-τ) at the same level as fresh bTV on Days 4 to 6 after thawing. However, amounts of IFN-τ decreased following continuous in vitro culture (Hashiyada et al. 2012 38th IETS). Co-transfer of frozen–thawed bTV improved pregnancy rate of embryos due to the effects of IFN-τ secreted by bTV (Hashiyada et al. 2008 41th SSR). However, the relation between bTV size and IFN-τ secretion level during culture has not been well documented. The objective of present study was to characterise the concentration of IFN-τ related bTV volume and culture period after thawing of cryopreserved bTV. The bTV were prepared from Day 16 elongating blastocysts recovered nonsurgically. The dissected trophoblastic fragment, 1 to 1.5 mm in width, was cultured using TCM-199 supplemented with 20% (vol/vol) fetal bovine serum and 0.1 mM β-mercaptoethanol. Formed vesicles after 24 h of culture were cryopreserved using D-PBS supplemented with 20% calf serum and 1.8 M ethylene glycol. After thawing, bTV were cultured individually with 100 μL/well/day until Day 2 (i.e. the day of thawing was defined as Day 0), and thereafter changed to 200 μL/well/day to termination at Day 10. Collection of culture media and measurement of bTV diameter were performed before cryopreservation and after thawing for every day. Interferon-τ in collected media was measured by radioimmunoassay. The estimated bTV volume was calculated based on the diameter. Data were analysed by Student's t-test. Nine fresh bTV before cryopreservation were used to assess the IFN-τ secretion for 24 h in relation to bTV volume. A significant positive correlation was observed between secreted IFN-τ (mean ± s.e.M, 19.9 ± 3.1 ng mL–1) and bTV volume (1.49 ± 0.6 mm3, r = 0.91; P < 0.01). Initial IFN-τ secretion from bTV cultured for 24 h after thawing was significantly decreased compared with that before cryopreservation (29.1 ± 2.1 ng mL–1 and 58.4 ± 4.8 ng mL–1; P < 0.01, n = 27). In continuous culture of bTV (n = 8), IFN-τ secretion increased gradually from Day 2 (23.1 ± 9.0 ng mL–1) to Day 4 (32.2 ± 8.4 ng mL–1), and then maintained this level until Day 7 (33.4 ± 14.9 ng mL–1). However, this amount of IFN-τ tended to decrease on Day 8 (24.8 ± 5.0 ng mL–1), 9 (16.5 ± 4.4 ng mL–1), and 10 (12.0 ± 1.7 ng mL–1). Interferon-τ secretion from bTV on Day 9 and 10 was lower than that on Day 3, 4, 5, 6, and 8, respectively (P < 0.05). Volume of bTV increased also from Day 2 (0.2 ± 0.1 mm3) to Day 5, 6 (0.8 ± 0.3 mm3) and 7 (0.7 ± 0.2 mm3). However, bTV volumes shrank drastically on Day 8 (0.3 ± 0.1 mm3), 9, and 10 (0.2 ± 0.1 mm3). In comparison with bTV during culture, volumes on Day 4, 5, and 7 were greater than those on Day 2 and 3, and volumes on Day 6 and 7 were greater than on Day 8, 9, and 10 (P < 0.05). These results indicate that the dynamics of IFN-τ secretion reflected the expansion or reduction of bTV in continued culture after thawing. Interferon-τ secretion might be related to bTV volume. Moreover, we reconfirmed that cryopreserved bTV highly express IFN-τ during 4 to 7 days after thawing.

Response of preimplantation rat blastocysts in vitro to extracellular uterine luminal components, serum and hormones

1977 ◽  
Vol 25 (1) ◽  
pp. 265-277
Author(s):  
M.A. Surani
Keyword(s):  
Calf Serum ◽  
Giant Cells ◽  
Biologically Active ◽  
Foetal Calf Serum ◽  
Active Components ◽  
Rat Serum ◽  
Pregnant Females ◽  
Trophoblast Giant Cells

The influence of extracellular environmental factors on preimplantation rat blastocysts was tested by determining the number of embryos which escaped from their zonae pellucidae, followed by attachment and outgrowth of trophoblast giant cells, after 72 h in culture Uterine luminal ocmponents from individual females, or hormones, were included in Dulbecco's medium which contained 4 mg/ml bovine serum albumin. In about 20% of cases, uterine fluids were embryotonic. However, uterine fluids from day-5 pregnant females, the day of implantation in the rat, were more potent in these tests than uterine fluids obtained from ovariectomized females treated with progesterone alone. The potency of a mixture of the 2 fluids was also high. Uterine fluids obtained at 14 h after an injection of oestradiol and progesterone to the ovariectomized females, were also effective in these tests. Rat serum and foetal calf serum were effective too, but steroids or insulin alone in the medium had no detectable influence on embryos. Serum or uterine luminal proteins appear to be essential for maintaining the viability of the blastocysts and for inducing the responses observed here. In the uterine fluids, some proteins released into the lumen after treatment of females with oestradiol and progesterone appear to be the biologically active components. Differences in the responses of blastocysts in vitro are compared with those in vivo.

A factor present in fetal calf serum enhances oncogene-induced transformation of rodent fibroblasts

1987 ◽  
Vol 7 (10) ◽  
pp. 3380-3385
Author(s):  
W L Hsiao ◽  
C A Lopez ◽  
T Wu ◽  
I B Weinstein
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Time Course ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Fold Increase ◽  
Fibroblast Cell ◽  
Focus Formation ◽  
Heat Stable

Our previous studies indicated that addition of the tumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) or teleocidin to Dulbecco modified Eagle medium supplemented with calf serum enhanced T24-induced focus formation in both the murine C3H 10T1/2 and rat 6 embryo fibroblast cell lines. In the present studies we have found that fetal calf serum (FCS) is more potent than 12-O-tetradecanoylphorbol-13-acetate in enhancing T24-induced focus formation, in terms of the number and size of the foci, in both C3H 10T1/2 and rat 6 cells. Time course studies indicate that FCS can exert this enhancing effect when it is added several days after the transfection with T24 DNA. In rat 6 cells, an 11-fold increase in T24-induced focus formation occurred when the transfected cultures were maintained for only 1 day in 5% FCS, starting 4 days after the transfection. Several known growth factors, including epidermal growth factor, transforming growth factors alpha and beta, insulin, and platelet-derived growth factor, did not enhance T24-induced transformation in these cell systems. Fractionation studies indicate that the factor present in FCS has a molecular weight of about 1,300, is not lipid soluble, and is acid, base, and heat stable. These findings suggest that a factor(s) normally present in serum may enhance the emergence of tumor cells in vivo, by acting in concert with an activated oncogene, during the multistage carcinogenic process.

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