67 The growth and development of invivo-derived dromedary embryos during short-term incubation: Use of embryo holding medium and the effect of embryonic morphology

2021 ◽  
Vol 33 (2) ◽  
pp. 140
Author(s):  
B. Asadi ◽  
F. Seyedasgari
Keyword(s):  
Growth And Development ◽  
Culture Medium ◽  
Calf Serum ◽  
Daily Basis ◽  
Developmental Competence ◽  
Dromedary Camel ◽  
Short Term ◽  
Experimental Media ◽  
Further Development ◽  
Short Term Culture

Production of invivo embryos for transfer in dromedary camel is a well-established practice, whereas freezing of these embryos is still an ongoing challenge. A common approach in evaluation of freeze–thawing method is achieved by studying invitro development of frozen–thawed embryos. However, not much is known about the development pattern of fresh dromedary embryos during incubation. The objectives of this study were to evaluate the usefulness of commercial holding media for easy short-term culture of these embryos and to provide preliminary insights on the growth and development of hatched blastocysts with different shapes. Recovered hatched blastocysts from superovulated donors were graded as transferable and non-transferable. Embryos with significant folding or crinkliness were further categorized as collapsed, whereas those with a round or oval appearance were categorized as spherical. Culture was performed in 500-μL drops at 38.5°C, 5% O2, 0–6% CO2, and maximum humidity in groups of 2 to 4. The 4 experimental media included culture medium (CM; TCM-199, 10% fetal calf serum (FCS), 0.3mM sodium pyruvate, 2.2mg mL−1 sodium bicarbonate), serum-supplemented holding medium (SSH; Syngro+10% FCS), serum-free holding medium (Syngro) and V-Onestep (Vitromed). In experiment 1, a total of 36 embryos were assigned to 4 groups and further development of the embryos was monitored up to 96h by morphological evaluations, identifying static and degenerating embryos on daily basis. In experiment 2, a total of 16 spherical and 16 collapsed embryos were cultured in SSH and CM and two-thirds of the culture drop was replaced with fresh medium at 72h. The proportion of developing embryos and their size expansion was compared between treatments by Fisher’s test and Mann–Whitney U test, respectively. Statistically similar proportions of embryos continued to develop in all media within the first 48h despite a numeric advantage in CM group; at 72h, the proportion of growing embryos was significantly higher in CM (77.8%) and SSH (66.6%) compared with SFH (11.1%) and OneStep (22.2%) (P<0.05). None of the embryos in SFH and only 1 embryo in the V-Onestep group survived beyond 72h, whereas 3/9 embryos in SSH and 7/9 embryos in CM continued to expand. In experiment 2, the proportion of spherical embryos that developed was higher compared with their collapsed counterparts (8/8 in both groups vs. 5/8 and 4/8 in CM and SSH, respectively) at 24h. However, remaining collapsed embryos grew and expanded at similar rates to spherical ones in each group (P>0.05). Replacing the medium did not favour continuation of embryonic growth in SSH beyond 72 h; only 5/16 embryos survived to 96h compared with 12/16 in CM. In conclusion, serum-supplemented commercial holding preparations provide comparable results to culture medium for short-term incubation of invivo dromedary embryos. Natural collapsing of hatched blastocysts might be associated with lower developmental competence.

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.

scholarly journals Tissue culture of sockeye salmon intestine: functional response of Na+-K+-ATPase to cortisol

2005 ◽  
Vol 288 (6) ◽  
pp. R1598-R1605 ◽  
Author(s):  
Philip A. Veillette ◽  
Graham Young
Keyword(s):  
Atpase Activity ◽  
Sockeye Salmon ◽  
Culture Medium ◽  
Slow Release ◽  
Short Term ◽  
Direct Target ◽  
Dose Dependent ◽  
Short Term Culture

A method to culture tissue explants of the intestine from freshwater-adapted sockeye salmon ( Oncorhynchus nerka) was developed to assess possible direct effects of cortisol on Na+-K+-ATPase activity. As judged by several criteria, explants from pyloric ceca and the posterior region of the intestine remained viable during short-term (6-day) culture, although Na+-K+-ATPase activity declined and basolateral components of the enterocytes were observed to be partially degraded. Addition of cortisol to the culture medium maintained Na+-K+-ATPase activity (over 2–12 days) above that of control explants and, in some cases, was similar to levels before culture. The response to cortisol was dose dependent (0.001–10 μg/ml). Within the physiological range, the response was specific for cortisol and showed the following hierarchy: dexamethasone ≥ cortisol > 11-deoxycortisol > cortisone. Insulin maintained Na+-K+-ATPase activity over controls in explants of ceca but not posterior intestine. To compare in vivo and in vitro responses, slow-release implants of cortisol (50 μg/g) were administered to salmon for 7 days. This treatment elevated plasma cortisol levels and stimulated Na+-K+-ATPase activity in both intestinal regions. The results demonstrate that the teleost intestine is a direct target of cortisol, this corticosteroid protects in vitro functionality of Na+-K+-ATPase, and explants retain cortisol responsiveness during short-term culture.

165 THE EFFECTS OF L-CARNITINE AND LINOLEIC ACID ALBUMIN SUPPLEMENTATION ON THE DEVELOPMENT AND CRYOSURVIVAL OF BOVINE IN VITRO-MATURED/IN VITRO-FERTILIZED EMBRYOS IN IN VITRO CULTURE MEDIUM

2012 ◽  
Vol 24 (1) ◽  
pp. 194
Author(s):  
S. Miyashita ◽  
Y. Inaba ◽  
T. Somfai ◽  
M. Geshi ◽  
T. Nagai ◽  
...  
Keyword(s):  
Linoleic Acid ◽  
In Vitro Culture ◽  
Culture Medium ◽  
Formation Rate ◽  
Calf Serum ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Formation ◽  
Cleavage Rate

The objective of this study was to investigate the effects of the supplementation of a lipid metabolism inducer, L-carnitine (LC) and a membrane stabilizer, linoleic acid albumin (LAA), on the developmental competence and cryosurvival of bovine in vitro-matured/in vitro-fertilized embryos in in vitro culture medium. Cumulus–oocyte complexes collected from the ovaries of slaughtered cattle were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 AU mL–1 of FSH at 38.5°C in an atmosphere of 5% CO2 in air. After IVF (Day 0), presumptive zygotes were cultured in CRlaa containing 5% CS at 38.5°C in an atmosphere of 5% CO2, 5% O2 and 90% N2 for 9 days. The culture medium was supplemented with 0.6 mg mL–1 of LC (LC group; n = 180) or with 0.25 mg mL–1 of LAA (LAA group; n = 180) or with both LC and LAA (LC + LAA group; n = 180) or without LC and LAA (control; n = 178). The cleavage rates were recorded on Day 2 and the blastocyst formation rates were recorded on Day 7 to 9. Expanded blastocysts harvested on Day 7 and 8 (LAA group: n = 31; LC group: n = 29; LC + LAA group: n = 25; control group: n = 33) were used for freezing in modified PBS supplemented with 1.5 M ethylene glycol, 0.1 M sucrose and 20% CS. After thawing, they were cultured in TCM-199 supplemented with 20% FBS and 0.1 mM β-mercaptoethanol at 38.5°C under 5% CO2 in air for 72 h. The rates of re-expansion, hatching and formation of hatched blastocysts were determined at 24, 48 and 72 h after thawing, respectively. The rates of cleavage and blastocyst formation were expressed as mean ± s.e.m. and analysed by ANOVA. The post-thaw survival rates of frozen embryos were analysed by chi-square test. The cleavage rate in the control group (69.1 ± 2.5%) was significantly lower than that in the LAA (81.8 ± 3.8%) and LC + LAA groups (77.9 ± 1.4%) but did not differ from that in the LC group (73.8 ± 2.4%). The blastocyst formation rate in the control group (21.7 ± 2.8%) was significantly lower (P < 0.05) than those in the LAA and LC + LAA groups (33.5 ± 2.8% and 31.4 ± 2.4%, respectively), but it did not differ significantly from that of the LC group (32.1 ± 3.3%) despite a strong tendency (P = 0.06). There were no significant differences among the control, LC, LAA and the LC + LAA groups in post-thaw re-expansion rates (66.7, 75.9, 67.7 and 76.0%, respectively), hatching rates (48.5, 69.0, 58.1 and 64.0%, respectively) and rates of formation of hatched blastocysts (51.5, 62.1, 61.3 and 64.0%, respectively). These results indicate that the addition of LC and LAA to the medium for in vitro culture of in vitro-matured/in vitro-fertilized bovine embryos improved their ability to develop to the blastocyst stage; however, the effects on the freezing tolerance were not verified.

98 EIGHT-CELL STAGE DELINEATES CRYOTOLERANCE OF VITRIFIED RABBIT EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 208
Author(s):  
T.-A. Lin ◽  
C.-H. Chen ◽  
L.-Y. Sung ◽  
J.-C. Ju ◽  
E. Chen ◽  
...  
Keyword(s):  
Culture Medium ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Developmental Competence ◽  
Cell Stage ◽  
General Linear Model Analysis ◽  
Developmental Rates ◽  
Humidified Air ◽  
Rabbit Embryos ◽  
Hatching Rates

A number of groups have successfully vitrified rabbit embryos at morula or blastocyst (BL) stage. For earlier stages (1- to 8-celled), however, there are very limited studies and the results are generally unsatisfactory. In this study, we examined the survival and developmental competence of rabbit embryos vitrified at different preimplantational stages. Sexually matured female New Zealand White (NZW) rabbits were superovulated with our standard protocols, followed by mating with NZW males. At 18h post-hCG treatment, viable fertilized embryos were collected and cultured in 2.5% FBS B2 medium (Laboratories CCD, Paris, France) in 5% CO2 with humidified air at 38.5°C. A total of 691 rabbit embryos at pronuclear, 2-celled, 4-celled, 8-celled, and morula/early blastocyst (BL; Day 3) stages were vitrified by the open pulled straw (OPS) method in HEPES buffered TCM-199 medium containing 20% fetal calf serum, ethylene glycol, and dimethyl sulfoxide. After stored in liquid nitrogen for at least 1 month, embryos were sequentially warmed, rehydrated, and washed before culture in B2 culture medium. Survival and developmental rates were analyzed by general linear model analysis (SPSS 11.0, SPSS Inc., Chicago, IL, USA). Embryos vitrified at 8-celled stage or beyond showed greater survival, and expanded BL and hatching rates than those at pronuclear, 2-celled and 4-celled embryos (Table 1). In particular, the expanded and hatched BL rates were significantly higher in the 8-celled group than those in 4-celled group, suggesting that the 8-celled is the threshold stage to tolerate the vitrification procedure in rabbit embryos. In addition, the rates of expanded and hatched blastocysts in morula/BL group were still significantly higher than those in the 8-celled group. Our results may provide the proper timing of cryopreserving fertilized, transgenic, and/or cloned rabbit embryos at early stages for biomedical research. Table 1.Survival and developmental competence of rabbit embryos vitrified at different stages after thawing This study was supported by NIH1R43 RR023774-01A1 and 5R44HL091605-03.

Marrow transplantation measured by uptake of Fe59 by spleen

1964 ◽  
Vol 206 (6) ◽  
pp. 1244-1250 ◽  
Author(s):  
L. H. Smith
Keyword(s):  
Bone Marrow ◽  
Plasma Clearance ◽  
Marrow Cell ◽  
Calf Serum ◽  
X Rays ◽  
Cell Integrity ◽  
Short Term ◽  
X Ray ◽  
Short Term Culture

Transplantation of isologous (syngeneic) bone marrow into lethally X-irradiated mice has been quantified in terms of the degree to which the spleen incorporates Fe59. Spleen uptake of Fe59 is linear with bone marrow cell dose and reflects the erythropoietic integrity of the transplanted cells. Parameters of the assay were investigated including X-ray exposure to the recipient, time of assay, Fe59 uptake time and plasma clearance of the isotope. The following protocol was adopted: (C3H x C57BL) F1 male mice are a) exposed to 850 r of X rays, b) injected with bone marrow 24 hr later, c) given Fe59 8 days later, and d) killed 6 hr after isotope injection to obtain spleens for Fe59 analysis. The assay was used to determine radiosensitivity of the erythropoietic compartment of marrow and to determine integrity of marrow cells after short-term culture as a means of studying in vitro recovery from radiation damage. Results showed that the in vitro radiation exposure required to reduce the surviving cells to 37% (D37) was 75 r. Studies of normal cells in short-term culture showed that cell integrity could be maintained for at least 6 hr in Tyrode's solution with calf serum at 22 C but that integrity was reduced after 6 hr at 37 C in all media tested.

scholarly journals Alpha lipoic acid (ALA) effects on developmental competence of equine preantral follicles in short-term culture

2018 ◽  
Vol 105 ◽  
pp. 169-173 ◽  
Author(s):  
R.G. Gomes ◽  
C.B. Silva ◽  
S.M. González ◽  
R.L. Oliveira ◽  
M.C. Max ◽  
...  
Keyword(s):  
Lipoic Acid ◽  
Developmental Competence ◽  
Alpha Lipoic Acid ◽  
Short Term ◽  
Preantral Follicles ◽  
Short Term Culture

scholarly journals Evaluating Smartphone Pressure Observations for Mesoscale Analyses and Forecasts

2017 ◽  
Vol 32 (2) ◽  
pp. 511-531 ◽  
Author(s):  
Luke E. Madaus ◽  
Clifford F. Mass
Keyword(s):  
United States ◽  
Rural Areas ◽  
Mesoscale Convective System ◽  
Convective System ◽  
Active Period ◽  
Eastern United States ◽  
Short Term ◽  
Mesoscale Convective ◽  
Dynamic Structures ◽  
Further Development

Abstract Smartphone pressure observations have the potential to greatly increase surface observation density on convection-resolving scales. Currently available smartphone pressure observations are tested through assimilation in a mesoscale ensemble for a 3-day, convectively active period in the eastern United States. Both raw pressure (altimeter) observations and 1-h pressure (altimeter) tendency observations are considered. The available observation density closely follows population density, but observations are also available in rural areas. The smartphone observations are found to contain significant noise, which can limit their effectiveness. The assimilated smartphone observations contribute to small improvements in 1-h forecasts of surface pressure and 10-m wind, but produce larger errors in 2-m temperature forecasts. Short-term (0–4 h) precipitation forecasts are improved when smartphone pressure and pressure tendency observations are assimilated as compared with an ensemble that assimilates no observations. However, these improvements are limited to broad, mesoscale features with minimal skill provided at convective scales using the current smartphone observation density. A specific mesoscale convective system (MCS) is examined in detail, and smartphone pressure observations captured the expected dynamic structures associated with this feature. Possibilities for further development of smartphone observations are discussed.

Proliferation and agglutinability of primary and transformed human epithelial cells in culture

1976 ◽  
Vol 21 (3) ◽  
pp. 553-561
Author(s):  
M.A. Ricard ◽  
R.J. Hay
Keyword(s):  
Culture Medium ◽  
Monolayer Culture ◽  
Primary Cultures ◽  
Short Term ◽  
Chloromethyl Ketone ◽  
Normal Populations ◽  
Short Term Exposure ◽  
Cell Strains ◽  
Human Placentae

Primary epithelial populations (HAM) were obtained by dissociation of the amniotic membrane stripped from human placentae. Agglutinability of cells from such normal populations and of cells from the transformed epithelial line WISH was then compared using concavanalin A as mediator. Extensive similar studies have previously been reported with cell strains isolated from other species. Freshly dissociated HAM cells from primary cultures agglutinated much less readily than did cells from WISH populations. Furthermore, the former exhibited a drastic decline in agglutinability as a function of time in suspension culture after trypsinization. Short-term exposure (60 h) of HAM cells in monolayer culture to 5-bromodeoxyuridine (BrdU) elicited heightened agglutinability detectable through 22 days in vitro. Addition of the protease inhibitors n-tosyl-L-lysyl-chloromethyl ketone (TLCK) or p-tosyl-L-arginine-methyl ester (TAME) to the culture medium inhibited proliferation of the WISH line by 40–50% while effecting only a 10–15% inhibition of HAM cells. These results also confirm data with other cell species indicating that high proteolytic activity at the surface of transformed cells may be related to the rapid proliferation rate.

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