scholarly journals 32 Bovine embryo cryopreservation in a chemically defined medium

2020 ◽  
Vol 32 (2) ◽  
pp. 142
Author(s):  
A. Østergaard ◽  
L. Gavin-Plagne ◽  
M. Guedes Teixeira ◽  
S. Buff ◽  
T. Joly
Keyword(s):  
Ethylene Glycol ◽  
Calf Serum ◽  
Defined Medium ◽  
Good Manufacturing Practice ◽  
Pathogen Transmission ◽  
Embryo Cryopreservation ◽  
Bovine Embryo ◽  
Chemically Defined Medium ◽  
Significant Difference ◽  
Chi Squared

Embryo cryopreservation media often contain an animal-derived component, such as bovine serum albumin (BSA) or fetal calf serum. However, the industry is faced with the issue of composition variability between batches and, most importantly, the risk of pathogen transmission. The aim of this study was to compare the effectiveness of two embryo cryopreservation ethylene glycol-based media: IMV's freezing medium with BSA (IMV Technologies) and a chemically defined medium containing STEMALPHA.CRYO3, called CRYO3 (Ref 5617, Stem Alpha). CRYO3 is produced according to EU good manufacturing practice, ensuring the composition and quality of the product. Bovine morulae were collected invivo and split into two groups to be slow frozen in both media. All results were analysed with chi-squared tests with Yates's correction. For the invitro approach of this study, frozen morulae (n=86) were thawed, cultured in medium (1:1:1 mixture of RPMI, Dulbecco's modified Eagle's medium, and Ham's F10) at 38.5°C with 5% CO2, and monitored every 24h for 72h to evaluate survival and expansion rates. Survival rate was higher in the CRYO3 group than in the BSA group (74 and 50%, respectively; P<0.05). Expansion and hatching rates followed the same trend: 38 and 14%, respectively, at 48h (P<0.05) and 40 and 18% at 72h (P<0.05). For the invivo approach of this study, frozen morulae (n=123) were thawed and transferred on the field to evaluate pregnancy and calving rates. Field results showed no significant difference between CRYO3 and the BSA-based medium for pregnancy rates (73 and 63%; P=0.3006) nor for the delivery rates (64 and 55%; P=0.3614).The trial was conducted with different AI teams and in 10 commercial herds, thus adding robustness to the data. This study has shown that CRYO3 can replace BSA in an ethylene glycol cryopreservation medium for invivo-produced bovine embryos. Therefore, this product eliminates some sanitary risk in embryo commerce, which is a concern in an international context.

scholarly journals 312 BLASTOCYST DEVELOPMENT OF MALE AND FEMALE BOVINE EMBRYOS PRODUCED BY IVF WITH FLOW CYTOMETRICALLY-SORTED SPERM

2005 ◽  
Vol 17 (2) ◽  
pp. 306
Author(s):  
M. Zhang ◽  
K.H. Lu ◽  
G.E. Seidel Jr
Keyword(s):  
Y Chromosome ◽  
Calf Serum ◽  
Defined Medium ◽  
Chemically Defined Medium ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Male And Female ◽  
Significant Difference

Several studies have demonstrated that male bovine embryos produced in vitro develop faster than female embryos produced in vitro, which results in more male than female blastocysts. The objective of this study was to compare the rate of blastocyst development of male and female bovine embryos derived from sexed sperm and cultured in a chemically defined medium + fatty acid-free (FAF)-BSA. Bovine oocytes (n = 1364) were fertilized with two types of frozen-thawed sperm (X- or Y-chromosome-bearing sperm sorted at 90% accuracy). Oocytes, aspirated from slaughterhouse ovaries, were matured in TCM199 supplemented with 10% fetal calf serum plus hormone additives (15 ng FSH, 1 mg LH, 1 mg 17 β-estradiol mL−1) for 22–24 h at 39°C, 5% CO2 in air with maximum humidity. Semen from one bull was sorted by flow cytometry into X- and Y-chromosome bearing sperm and frozen for later use with IVF. The procedures for IVF and IVC have been previously described (Lu KH, et al. 1999 Theriogenology 52, 1393–1405). Presumptive zygotes were removed from culture and placed in chemically defined medium (CDM-1 [Zhang M et al. 2003 Theriogenology 60, 1657–1663]) 6–7 h after insemination and cultured for 65–66 h. Embryos which had cleaved by 72 h post insemination were further cultured 96 h in CDM-2 (Zhang M et al. 2003 Theriogenology 60, 1657–1663) containing 0.12 IU insulin mL−1. Cleavage and blastocyst rates per oocyte inseminated were recorded on Day 3 and Days 7–8 after insemination, respectively. Data were analyzed by ANOVA procedures with replicates and treatments in the model. There was no significant difference in cleavage rate or blastocyst rate between X and Y sperm treatments. These results indicate that embryos produced with Y sperm do not reach the blastocyst stage in significantly higher proportions than embryos produced with X sperm in this chemically defined medium + FAF-BSA. Apparently, this IVC system leads to a more synchronous development of male and female embryos than other methods of producing bovine embryos in vitro. Table 1. Cleavage and blastocyst rates with X and Y sperm This research was supported by XY, Inc., Fort Collins, CO, USA.

scholarly journals A Chemically Defined Medium for Rabbit Embryo Cryopreservation

PLoS ONE ◽  
2013 ◽  
Vol 8 (8) ◽  
pp. e71547 ◽  
Author(s):  
Pierre Bruyère ◽  
Anne Baudot ◽  
Thierry Joly ◽  
Loris Commin ◽  
Elodie Pillet ◽  
...  
Keyword(s):  
Defined Medium ◽  
Embryo Cryopreservation ◽  
Chemically Defined Medium ◽  
Rabbit Embryo

scholarly journals Expression of Virulence Factors by Staphylococcus aureus Grown in Serum

2011 ◽  
Vol 77 (22) ◽  
pp. 8097-8105 ◽  
Author(s):  
Yuichi Oogai ◽  
Miki Matsuo ◽  
Masahito Hashimoto ◽  
Fuminori Kato ◽  
Motoyuki Sugai ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Factors ◽  
Iron Acquisition ◽  
Calf Serum ◽  
Growth Conditions ◽  
Defined Medium ◽  
Chemically Defined Medium ◽  
Regulatory Factor ◽  
Content Type

ABSTRACTStaphylococcus aureusproduces many virulence factors, including toxins, immune-modulatory factors, and exoenzymes. Previous studies involving the analysis of virulence expression were mainly performed byin vitroexperiments using bacterial medium. However, whenS. aureusinfects a host, the bacterial growth conditions are quite different from those in a medium, which may be related to the different expression of virulence factors in the host. In this study, we investigated the expression of virulence factors inS. aureusgrown in calf serum. The expression of many virulence factors, including hemolysins, enterotoxins, proteases, and iron acquisition factors, was significantly increased compared with that in bacterial medium. In addition, the expression of RNA III, a global regulon for virulence expression, was significantly increased. This effect was partially restored by the addition of 300 μM FeCl3into serum, suggesting that iron depletion is associated with the increased expression of virulence factors in serum. In chemically defined medium without iron, a similar effect was observed. In a mutant withagrinactivated grown in serum, the expression of RNA III,psm, andsec4was not increased, while other factors were still induced in the mutant, suggesting that another regulatory factor(s) is involved. In addition, we found that serum albumin is a major factor for the capture of free iron to prevent the supply of iron to bacteria grown in serum. These results indicate thatS. aureusexpresses virulence factors in adaptation to the host environment.

Development in vitro of mouse embryos from the two-cell egg stage to the early somite stage

Development ◽  
1974 ◽  
Vol 31 (1) ◽  
pp. 235-245
Author(s):  
Yu-Chih Hsu ◽  
John Baskar ◽  
Leroy C. Stevens ◽  
John E. Rash
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Defined Medium ◽  
Chemically Defined Medium ◽  
Cell Stage ◽  
Cord Serum ◽  
Somite Stage ◽  
Development In Vitro

About 1–3% of mouse blastocysts, which had initially been cultured from the two-cell stage in chemically defined medium or about 3–5% of blastocysts which were explanted from the uterus, developed to the early somite stage when cultured in vitro on collagen. Two-cell eggs were initially cultivated in chemically defined medium to the blastocyst stage. Blastocysts were then transferred to Eagle's minimal essential medium (MEM) plus 10% heat-inactivated calf serum. Two barriers to further development were overcome. First, the formation of endoderm and ectoderm from the inner cell mass immediately after attachment to collagen. Second, formation of the embryo proper from the embryonic region. Both barriers were overcome by using heat-inactivated human cord serum after the blastocysts hatched from the zona pellucida and attached to collagen. After attachment, embryos were cultured in MEM plus 20% heat-inactivated human cord serum which was changed daily until early somite stages. Apparently normal healthy development in vitro occurred, as judged by light and electron microscopic examination.

scholarly journals Expired Platelet Concentrate as a Source of Human Platelet Lysate for Xenogeneic-Free Culture of Human Dermal Fibroblasts

2021 ◽  
Vol 50 (8) ◽  
pp. 2355-2365
Author(s):  
Muhammad Najib Fathi Bin Hassan ◽  
Zheng Yie Yap ◽  
Yee Loong Tang ◽  
Min Hwei Ng ◽  
Jia Xian Law
Keyword(s):  
Gene Expression ◽  
Wound Healing ◽  
Human Platelet ◽  
Defined Medium ◽  
Pathogen Transmission ◽  
Dermal Fibroblasts ◽  
Platelet Lysate ◽  
Human Dermal Fibroblasts ◽  
Chemically Defined Medium ◽  
Human Platelet Lysate

Dermal fibroblasts have been used clinically to promote wound healing and to reduce wrinkles. Most of the time, fetal bovine serum (FBS) is used for the expansion of fibroblasts. In addition, chemically defined medium can also be used for fibroblast expansion. Nonetheless, both FBS and chemically defined medium are not ideal to culture cells that will be used clinically as FBS has the risk of pathogen transmission and induction of xenogeneic immune response whilst chemically defined medium is extremely expensive. In this study, we examine the potential of using human platelet lysate (hPL) prepared from expired platelet concentrates to culture human dermal fibroblasts. For the experiments, fibroblasts were cultured with 5 and 10% hPL, with 10% FBS as the control group to compare the cell morphology, viability, growth rate, extracellular matrix gene expression and wound healing. Results showed that fibroblasts cultured with hPL were more elongated and smaller in size. The cell viability was higher than 90% for all groups. Expansion with 10% hPL significantly shorten the population doubling time compared to the 5% hPL and 10% FBS groups. However, fibroblasts cultured with hPL have lower expression of type I collagen, type III collagen and fibronection as well as slower wound closure. In summary, hPL has the potential to be used as a serum substitute for FBS to expand fibroblasts as it significantly increases the cell proliferation. However, further studies are required to determine if the changes in the ECM gene expression and migration of the hPL-expanded fibroblasts will affect the efficacy of the cells in promoting in vivo wound healing.

scholarly journals 318EFFECT OF PIG FOLLICLE FLUID AND FETAL CALF SERUM ON PORCINE OOCYTE MATURATION AND SUBSEQUENT DEVELOPMENT AFTER ACTIVATION AND SOMATIC CELL NUCLEAR TRANSFER

2004 ◽  
Vol 16 (2) ◽  
pp. 278
Author(s):  
Z. Liu ◽  
L. Lai ◽  
G. Im ◽  
M. Samuel ◽  
D. Wax ◽  
...  
Keyword(s):  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
In Vitro Maturation ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Defined Medium ◽  
Chemically Defined Medium ◽  
Maturation Rate ◽  
Blastocyst Rate

In vitro maturation of porcine oocytes is very important for understanding porcine somatic cell nuclear transfer (SCNT). In order to develop an in vitro maturation system that can provide more high quality oocytes, the effect of porcine follicle fluid (pFF) (gathered from 3–5-mm porcine follicles) and fetal calf serum (FCS: Sigma, St. Louis, MO, USA), as an important additional component of a chemically-defined medium was studied. Cumulus-oocyte complexes (COC) derived from follicles 3–5mm in diameter were cultured in three different media: a chemically-defined medium (CDM: TCM-199 with 0.1mgmL−1 cysteine, 10ngmL−1 EGF, 0.5μgmL−1 LH and 0.5μgmL−1 FSH); CDM with 10% pFF (CDM+p); and CDM with 10% FCS (CDM+F). After 42–44h of maturation, oocytes with a clear polar body were classified as matured oocytes. Matured oocytes stimulated by electric pulse (120v, 30μs, 2 pulse), or enucleated and fused with fibroblasts to construct SCNT embryos by using the same electrical parameters. All of these parthenogenetic and SCNT embryos were cultured in Porcine Zygote Medium-3. The blastocyst rate was assessed under a stereomicroscope on Day 6, and the number of nuclei in the blastocysts was counted under a fluorescent microscope after staining with 5μgmL−1 of Hoechst 33342. All data were subjected to a Generalized Linear Model Procedure (PROC-GLM) of Statistical Analysis System (SAS). The maturation rates of porcine oocytes in CDM and CDM+p were 53.2±3.8% (539/1050) and 69.7±3.8% (587/847), respectively;; in CDM and CDM+F, 61.1±3.1% (471/776) and 70.2±3.7% (577/844), respectively. Oocytes matured in CDM+p and CDM+F showed a higher (P<0.05) maturation rate than those in CDM. The percentages of parthenogenetic blastocysts of oocytes matured in CDM and CDM+p were 13.9±2.1% (35/250) and 20.2±5.3% (64/300), and the numbers of nuclei in these blastocysts were 25.8±2.3 and 25.8±1.4, respectively. The blastocyst rate from CDM- and CDM+F-matured oocytes were 20.1±2.0% (53/272) and 22.2±4.7%(71/298), and the numbers of nuclei in these blastocysts were 24.7±1.5 and 25.3±1.5, respectively. There were no significant (P>0.05) differences in the percentages of parthenogenetic blastocysts and nuclei numbers between CDM and CDM+p, or CDM and CDM+F. The percentages of blastocysts in SCNT embryos derived from CDM and CDM+p were 8.1±1.5% (14/192) and 12.3±1.9% (24/192), while the nuclei numbers in these blastocysts were 26.6±1.2 and 34.5±2.2, respectively. The percentages of blastocysts after SCNT from oocytes matured in CDM and CDM+F were 24.3±4.9% (35/139) and 27.1±5.5% (45/176), while the numbers of nuclei were 29.8±2.5 and 32.2±1.9, respectively. There were no significant (P>0.05) differences between CDM and CDM+p, or CDM and CDM+F in SCNT embryo blastocyst rate, but the SCNT embryos derived from CDM+p showed a higher (P<0.05) nuclear number. In conclusion, these results indicate that 10% pFF or FCS in CDM can promote a higher maturation rate of porcine oocytes. As recipient cytoplasm for SCNT, oocytes matured in CDM+p can support development of blastocysts that contain more nuclei than those matured in CDM alone. Supported in part by Food for the 21st Century and RR13438.

90 OPEN PULLED STRAW VITRIFICATION OF IN VITRO PORCINE BLASTOCYTS IN A CHEMICALLY DEFINED MEDIUM

2011 ◽  
Vol 23 (1) ◽  
pp. 150
Author(s):  
J. Sanchez-Osorio ◽  
C. Cuello ◽  
J. Gomis ◽  
C. Maside ◽  
M. A. Gil ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Formation Rate ◽  
Calf Serum ◽  
Defined Medium ◽  
Basic Medium ◽  
Chemically Defined Medium ◽  
Cleavage Rate ◽  
Number Of Cells ◽  
Serum Components

The aim of this study was to design a chemically defined medium for the vitrification of in vitro produced porcine blastocysts avoiding the use of serum or serum components. Cumulus–oocyte complexes were matured in vitro in NCSU-23 for 44 h and were inseminated with frozen–thawed spermatozoa. Presumptive zygotes were cultured for 16 h to assess in vitro fertilization (IVF) parameters (N = 200) or for 6 days (N = 600) in order to obtain blastocysts. For chemically delipidation, 10 μM forskolin was added to the culture medium on Day 5 of in vitro culture. On Day 2, embryos were evaluated for cleavage rate. On Day 6, embryos were assessed for blastocyst formation; only those blastocysts showing excellent morphological appearance were selected for vitrification. Blastocysts were vitrified using as basic medium TCM-199 HEPES supplemented with 20% of newborn calf serum (NBCS; n = 65), with 0.1% of polyvinyl alcohol (PVA; n = 64) or without additives (WA; n = 65). The OPS-vitrification and warming were performed as described by (Sanchez-Osorio et al. 2010 Theriogenology 73, 300–308) using 16% of Etylenglycol and 16% of dimetyl sulfoxide as final concentrations of cryoprotectants. Vitrified blastocysts were warmed and cultured in vitro for 24 h to assess their viability. Blastocysts that totally reformed their blastocoel cavity showing a normal or excellent morphology were considered viable. In addition, after in vitro culture vitrified-warmed viable embryos were fixed in 4% paraformaldehyde in PBS medium and stained with Hoechst 33342 in order to assess the total number of cells. Data were analysed by using the MIXED procedure of SPSS. The threshold for significance was set at P < 0.05. Results are expressed as least squares means ± SEM. The maturation, penetration, and monospermy rates were 98.5 ± 1.2%, 85.3 ± 3.6%, and 48.8 ± 5%, respectively. The efficiency of IVF (defined as the ratio of monospermic oocytes to the total number of inseminated oocytes) was 41.0 ± 4.9%. The values of cleavage rate at Day 2 and blastocysts formation rate were 67.8 ± 1.4% and 37.3 ± 1.6%, respectively. After vitrification and warming, similar survival rates were observed for NBCS (33.8 ± 5.9) PVA (40.6 ± 6.0), and WA (30.8 ± 5.9) groups. No significant differences were found for the total number of cells (ranged from 35.4 ± 6.8 to 50.8 ± 8.3) among vitrification groups. In conclusion, in vitro derived porcine blastocysts can be vitrified in the absence of serum and serum components. Furthermore, PVA is a suitable substitute for serum in vitrification solutions with no detrimental effect on the viability of in vitro produced pig blastocysts. This study was supported by the Seneca foundation of Murcia (GERM 04543/07).

THE METABOLISM OF YEAST SPORULATION: I. EFFECT OF CERTAIN METABOLITES AND INHIBITORS

1956 ◽  
Vol 2 (6) ◽  
pp. 519-537 ◽  
Author(s):  
J. J. Miller ◽  
Clara Halpern
Keyword(s):  
Carbon Source ◽  
Ethylene Glycol ◽  
Marked Effect ◽  
Defined Medium ◽  
Yeast Nitrogen Base ◽  
Chemically Defined Medium ◽  
Optimum Growth ◽  
Nitrogen Base ◽  
Acetate Concentration ◽  
Growth Experiments

Cells of bakers' yeast harvested from a chemically-defined medium were placed in buffer for sporulation experiments and in Wickerham's yeast nitrogen base for growth experiments. The metabolites were included in these two solutions in concentrations up to 1% usually, and growth and sporulation were compared under conditions as nearly equivalent as possible. Optimum sporulation was observed in 0.033–0.1% glucose, 1% pyruvate, 0.033% ethanol, 0.033% acetaldehyde, and 0.33% acetate. Optimum growth in one day occurred in 1% glucose, 1% pyruvate, 0.1–0.33% ethanol, 0.0033% acetaldehyde, and 0.033% acetate. Very few asci were found in 0.33% glucose or in 1% ethanol, but cells transferred to buffer after one day in such solutions sporulated well. The addition of 0.33% glucose or 1% ethanol to 0.3% acetate suppressed sporulation. In 0.1% acetaldehyde no sporulation or growth was evident, with or without the addition of 0.3% acetate. Acetoin, 2,3-butylene glycol, and ethylene glycol had no marked effect on sporulation and growth. Sporulation and growth were inhibited by 7 × 10−4M fluoroacetate with 0.3% acetate or 0.3% ethanol as the carbon source. Inhibition was much greater when the acetate concentration was decreased to 0.03%. Sporulation in glucose was inhibited by fluoroacetate, while growth in glucose was little affected. With acetate as the carbon source, sporulation was more sensitive than growth to urethane, while the reverse was found with azide, malachite green, cyanide, and dinitrophenol. The two processes seemed about equally sensitive to arsenite, p-nitrophenol, and malonic acid. Most of the asci that formed in glucose solutions were two-spored. In acetate, ethanol, and pyruvate three- and four-spored asci usually predominated, except in the weaker concentrations.

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