scholarly journals In vitro production of cattle blastocysts in chemically defined medium with or without insulin supplementation

1996 ◽  
Vol 5 (5) ◽  
pp. 509-514 ◽  
Author(s):  
Kristiina Bredbacka ◽  
Peter Bredbacka
Keyword(s):  
Bovine Serum Albumin ◽  
Culture Medium ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Chemically Defined Medium ◽  
In Vitro Production ◽  
Cell Numbers ◽  
Vitro Production ◽  
Bovine Oocytes

In this study we evaluated the use of a chemically defined medium in the production of blastocysts from bovine oocytes fertilized in vitro. As culture medium we used CRI-PVP, a modification of CRlaa medium with bovine serum albumin replaced by polyvinylpyrrolidone. After 168 h of culture (192 h after insemination) 8.7%, 10.5 and 12.8% of the cleaved embryos developed to the blastocyst stage in the presence of 0, 2 or 200 nM insulin, respectively. The supplementation of 200 nM insulin tended to increase cell numbers in morulae and blastocysts (P=0.10). It is concluded that CRI-PVP can be used as a chemically defined medium in the production of blastocysts from bovine 1-cell embryos. However, further modifications are needed, and the insulin concentrations used may be below the optimum for blastocyst production.

255. In vitro maturation of bovine oocytes in serum-free media

2005 ◽  
Vol 17 (9) ◽  
pp. 102
Author(s):  
S. Zhang ◽  
A. J. French ◽  
R. T. Tecirlioglu
Keyword(s):  
Embryo Development ◽  
Bovine Serum ◽  
Culture Medium ◽  
Polar Body ◽  
In Vitro Production ◽  
Serum Free ◽  
Cell Numbers ◽  
Defined Culture ◽  
Vitro Production

Culture medium supplemented with sera is commonly used for the in vitro production (IVP) of livestock embryos. However, serum induced complications including batch variation, the potential risk of virus and mycoplasma contamination and the implication in the large offspring syndrome in domestic animals impels the development of a serum-free culture system. In this study, we investigated whether replacement of fetal bovine serum (FBS) with bovine serum albumin (BSA) in three maturation media, tissue culture medium-199 (TCM-199), a modified synthetic oviduct fluid (mSOF) routinely used in our laboratories and a commercially available SOF-VC (Vitro Cleave, Cook Australia). Harvested oocytes were matured, parthenogenetically activated and in vitro cultured (Day 7) to measure maturation efficiency, embryo development and quality with the aim of developing a simplified and defined culture medium for the in vitro production of bovine embryos. Abattoir derived cumulus oocyte complexes were matured in TCM-199, mSOF and SOF-VC media supplemented with LH and beta-estradiol in the presence of 15% FBS or 0.08% BSA at 39ºC in 5% CO2 in air. Polar body extrusion was assessed twenty-two hour post maturation and selected MII occytes were activated using calcium ionophore/6-dimethylaminopurine and cultured for seven days in SOF medium supplemented with 0.8% BSA. On day seven, blastocyst development was assessed and randomly selected blastocysts were stained to determine inner cell mass (ICM), trophectoderm (TE) and total cell numbers (TCN). Supplementation with either BSA or FCS did not significantly affect the maturation efficiency, blastocyst rates or differential cell numbers within each maturation media tested. However, maturation efficiency and blastocyst rates were significantly lower (P < 0.01) when oocytes were matured in either mSOF or SOF-VC regardless of FBS or BSA supplementation. From this study, we conclude that BSA effectively replaces FCS and TCM-199 is superior to SOF (mSOF or SOF-VC) in terms of oocyte maturation regardless of protein source. Once matured SOF and TCM-199 parthenogenetically blastocysts were equivalent in terms of embryo development and quality.

scholarly journals In vitro production of Sudanese camel (Camelus dromedarius) embryos from epididymal spermatozoa and follicular oocytes of slaughtered animals

2017 ◽  
Vol 20 (1) ◽  
pp. 95-101 ◽  
Author(s):  
A.E. Abdelkhalek ◽  
Sh.A. Gabr ◽  
W.A. Khalil ◽  
Sh.M. Shamiah ◽  
L. Pan ◽  
...  
Keyword(s):  
Culture Medium ◽  
Fertilization Rate ◽  
Blastocyst Stage ◽  
Dromedary Camel ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Maturation Rate ◽  
Epididymal Spermatozoa ◽  
Vitro Production

Abstract Application of assisted reproductive technology in camelidea, such as artificial insemination (AI) and embryo transfer, has been slow in comparison to that for other livestock species. In Egypt, there are few attempts to establish in vitro maturation (IVM) and fertilization (IVF) techniques in dromedary camel. The present study was carried out to produce Sudanese camel embryos using in vitro matured oocytes and epididymal spermatozoa. Dromedary camel ovaries were collected from abattoirs and then, the oocytes were aspirated from all the visible follicles on the ovarian surface (~2-8 mm in a diameter). Meanwhile, Fetal Dromedary Camel Serum (FDCS) was obtained from camel fetuses after slaughtering. Thereafter, only Cumulus Oocyte Complexes (COCs) were matured in vitro in the Tissue Culture Medium (TCM-199) complemented with 10% FDCS. Spermatozoa required for in vitro fertilization were collected from testes (epididymal cauda) of the slaughtered camel bulls. The results clearly showed that the maturation rate of oocytes at metaphase II was about 59.5% while the fertilization rate was around 70.4%. Intriguingly, the embryo rates determined were 13.1%, in 2-cell; 0.0%, in 4-cell; 34.7%, in 8-16% cell; 39.1%, in morula and 13.1% in a blastocyst stage. This study represented a successful in vitro production of Sudanese dromedary camel embryos from epididymal sperm cells and in vitro matured oocytes recovered from slaughtered camels.

45 EFFECT OF OSMOLARITY IN CULTURE MEDIUM ON THE PRE-IMPLANTATION DEVELOPMENT OF PORCINE NT AND IVF EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 141
Author(s):  
I. S. Hwang ◽  
H. J. Moon ◽  
J. H. Shim ◽  
M. R. Park ◽  
D. H. Kim ◽  
...  
Keyword(s):  
Culture Medium ◽  
Developmental Rate ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Final Concentration ◽  
Control Group ◽  
In Vitro Production ◽  
Fetal Fibroblasts ◽  
Vitro Production

In vitro production of the pig embryo is very important as an initial step to improve its application in biotechnology. The in vitro production system for pig embryos, however, has been plagued by the high incidence of polyspermy and poor embryo quality. The present study was conducted to examine the relationship between apoptosis and osmolarity of culture medium in pre-implantation development of porcine NT and IVF embryos. Oocytes were aspirated from ovaries collected from a local abattoir, and then matured in TCM-199 for 40–44 h. Fresh semen was diluted and equilibrated at 16�C. The final concentration of motile spermatozoa was adjusted to 5 � 105 cells/mL in fertilization medium. Fetal fibroblasts were prepared from a 35-day-old porcine fetus for use as donor cells. The NT and IVF embryos were cultured in PZM-3 supplemented with 0.05 M sucrose or a final concentration of 138 mM NaCl (280–320 mOsmol) for the first 2 days, and then cultured in PZM-3 (250–270 mOsmol) for the remaining days. For the control, NT and IVF embryos were cultured in PZM-3 for whole culture period. After 6 days of culture, the developmental ability of embryos, total cell numbers, ratio of ICM/TE, and apoptosis of cells in blastocysts were examined. The developmental rate to the blastocyst stage of NT embryos was significantly higher (P &lt; 0.05) in the sucrose and NaCl groups than in the control [14.7% (21/153) and 21.7% (34/154) vs. 11.5% (18/152), respectively]. Also, the developmental rate to the blastocyst stage after IVF was slightly higher in embryos cultured in the medium supplemented with NaCl than in the control group [21.8% (49/235) and 26.4% (61/237) vs. 18.9% (44/247)]. For apoptosis, both NT and IVF blastocysts produced in the sucrose and NaCl groups showed slightly lower frequency of apoptosis compared to that of the control (2.2% and 2.8% vs. 3.1% for NT; 0.9% and 0.7% vs. 1.1% for IVF). These studies suggest that the high osmolarity in the early embryo culture stage could enhance the in vitro development of both porcine NT and IVF embryos to the blastocyst stage and could reduce the apoptosis of cells.

scholarly journals 290EFFECTS OF HYALURONAN ON IN VITRO DEVELOPMENT OF PORCINE EMBRYOS CULTURED IN A CHEMICALLY DEFINED MEDIUM

2004 ◽  
Vol 16 (2) ◽  
pp. 264
Author(s):  
K. Yoshioka ◽  
H. Ekwall ◽  
H. Rodriguez-Martinez
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Cell Counting ◽  
Transmission Electron Microscopy Data ◽  
Chemically Defined Medium ◽  
In Vitro Production ◽  
Microscopy Data

Hyaluronan (HA), a glycosaminoglycan present in follicular and oviductal fluids, has been related to sperm capacitation, fertilization and embryo development. We have previously developed an in vitro-production (IVP) system of porcine embryos, where porcine blastocysts can be produced by IVF and IVC in chemically defined media and can develop to full-term by transfer to recipients. The application of a chemically defined medium to IVP in pigs allows the analysis of the physical action of substances on the development of pre-implantation embryos. In the present study, the effects of HA on the development of porcine embryos in a chemically defined medium were investigated. Porcine presumptive zygotes were produced by IVM and IVF of COC from pre-pubertal gilts and frozen-thawed ejaculated boar semen. The zygotes were cultured in Porcine Zygote Medium (PZM)-5 containing different concentrations of HA (0 [control], 1, 2, 5, 10, 20 and 50μgmL−1) until 6 days after IVF, and representative specimens were fixed for cell counting and transmission electron microscopy. Data of percentages and cell numbers were statistically analyzed by one-way ANOVA and Fisher’s PLSD test. The percentage of embryos that developed to the blastocyst stage (15.8% [23/144] to 19.5% [27/139]) did not differ among treatments. However, addition of 5 or 10μgmL−1 HA increased (P&lt;0.05) the total number of cells in blastocysts (56.1 and 58.3 cells [n=22 and 23], respectively) compared to control (no HA, 42.0 cells [n=23]). To evaluate proliferation rates of inner cell mass (ICM) and trophectoderm (TE), embryos were cultured in PZM-5 for various periods of exposure to 10μgmL−1 HA. The numbers of ICM and TE cells in Day-6 blastocysts cultured in the presence of exogenous HA from Day 0 to Day 3 (18.3 and 34.4 cells, respectively [n=38]) or Day 6 (17.9 and 35.9 cells, respectively [n=36]) were significantly (P&lt;0.05) higher than those cultured without HA through the culture period (13.5 and 24.2 cells, respectively [n=26]). In the presence of HA from Day 3 to 6, only the number of TE cells (37.1 cells [n=33]) increased (P&lt;0.05), compared to PZM-5 alone. Differences in ultrastructure were noticed among blastocysts cultured with or without 10mgmL−1 HA. Blastocysts cultured with HA had mainly mature mitochondria while many mitochondria appeared morphologically immature in the blastocysts cultured without HA. Lipid droplets in the blastocysts cultured with HA seemed to be more homogeneous in comparison with those in the blastocysts cultured in PZM-5 alone. Further differences were seen in the numbers of lysosome-like structures, which were greater in blastocysts cultured with HA. This study demonstrates that exogenous HA improves cell proliferation and normality of ICM and TE in porcine embryos cultured in a chemically defined medium, depending on the exposure periods to HA. (Supported by MAFF, Japan and STINT, Sweden.)

scholarly journals Cytokinin Mediated Increased In Vitro Production of Secondary Metabolites with Special Reference to Solasodine in Solanum erianthum

2021 ◽  
Vol 8 (02) ◽  
pp. e62-e68
Author(s):  
Jeeta Sarkar ◽  
Nirmalya Banerjee
Keyword(s):  
Secondary Metabolites ◽  
High Performance ◽  
Culture Medium ◽  
In Vitro Production ◽  
Leaf Extracts ◽  
Plant Parts ◽  
Regenerated Plants ◽  
Vitro Production ◽  
Regenerated Plantlets

AbstractSteroid alkaloid solasodine is a nitrogen analogue of diosgenin and has great importance in the production of steroidal medicines. Solanum erianthum D. Don (Solanaceae) is a good source of solasodine. The aim of this study was to evaluate the effect of different cytokinins on the production of secondary metabolites, especially solasodine in the in vitro culture of S. erianthum. For solasodine estimation, field-grown plant parts and in vitro tissues were extracted thrice and subjected to high-performance liquid Chromatography. Quantitative analysis of different secondary metabolites showed that the amount was higher in the in vitro regenerated plantlets compared to callus and field-grown plants. The present study critically evaluates the effect of the type of cytokinin used in the culture medium on solasodine accumulation in regenerated plants. The highest solasodine content (46.78±3.23 mg g-1) was recorded in leaf extracts of the in vitro grown plantlets in the presence of 6-γ,γ-dimethylallylamino purine in the culture medium and the content was 3.8-fold higher compared to the mother plant.

scholarly journals In Vitro Production of Erythropoietin by Kidneys Perfused With a Serum-free Solution

Blood ◽  
1974 ◽  
Vol 44 (1) ◽  
pp. 77-85 ◽  
Author(s):  
Allan J. Erslev
Keyword(s):  
Tissue Culture ◽  
Atmospheric Pressure ◽  
Culture Medium ◽  
Kidney Tissue ◽  
In Vitro Production ◽  
In Vitro Perfusion ◽  
Serum Free ◽  
Enzymatic Activation ◽  
Vitro Production

Abstract Normal rabbits exposed to 0.4 atmospheric pressure for 3 hr will generate about 40-60 U of erythropoietin during a subsequent 3-hr period. If the kidneys were removed from 3-hr hypoxic animals, washed carefully, and perfused for 3 hr by recirculation with a serum-tissue culture mixture, each kidney generated about 14 U of erythropoietin in vitro. Perfusion of normal kidneys did not result in the production of erythropoietin, and only small amounts were generated if the perfusate contained Puromycin. Three-hour hypoxic kidneys perfused for 3 hr with a serum-free tissue culture medium were found to generate about 8 U of erythropoietin per kidney and similar kidneys perfused with saline about 1 U. These results indicate that erythropoietin is synthesized by kidney tissue and not produced by enzymatic activation of a plasma substrate.

197 EFFECTS OF HYALURONAN ON OXYGEN CONSUMPTION AND ATP CONTENT IN PIG BLASTOCYSTS PRODUCED IN VITRO

2007 ◽  
Vol 19 (1) ◽  
pp. 215
Author(s):  
K. Yoshioka ◽  
M. Yokoo ◽  
T. Ozawa ◽  
C. Suzuki ◽  
H. Abe ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Metabolic Activity ◽  
Formation Rate ◽  
Defined Medium ◽  
Cell Number ◽  
Total Cell ◽  
Chemically Defined Medium ◽  
Atp Content ◽  
Cell Numbers

Hyaluronan (HA), a glycosaminoglycan present in follicular and oviductal fluids, has been related to sperm capacitation, fertilization, and embryo development. We have found that exogenous HA improves cell proliferation of porcine embryos cultured in a chemically defined medium (Yoshioka et al. 2004 Reprod. Fertil. Dev. 16, 264–265). Moreover, mitochondrial maturation was clearly more advanced in blastocysts cultured with HA compared to those cultured without HA, as seen by transmission electron microscopy. In the present study, the effects of HA on oxygen consumption and ATP content of blastocysts, produced in a defined system which reflects metabolic activity, were investigated. Porcine immature oocytes were matured for 44 h in porcine oocyte medium (POM) and subsequently fertilized with frozen–thawed ejaculated semen in porcine gamete medium supplemented with theophylline, adenosine, and cysteine (PGMtac4). Both POM and PGMtac4 were chemically defined media modified from porcine zygote medium (PZM)-5. After IVF, presumptive zygotes were cultured in PZM-5 containing HA (from the microorganism, Nacalai tesque, Kyoto, Japan) at concentrations of 0 [control], 10 [HA10], or 100 [HA100] �g mL-1 until 5 days after IVF. Blastocyst formation rate and total cell numbers/blastocyst at Day 5 were assessed. In addition, oxygen consumption and ATP content of single Day 5 blastocysts were measured. Blastocyst oxygen consumption was quantified using scanning electrochemical microscopy (HV-403; Research Institute for the Functional Peptides, Yamagata, Japan), and embryonic ATP content was determined using a commercial assay based on the luciferin-luciferase reaction (ATPlite; PerkinElmer, Groningen, The Netherlands). Data were statistically analyzed by ANOVA and Fisher&apos;s PLSD test. While the percentage of embryos that developed to the blastocyst stage [30.5% (63/206) to 31.7% (65/206)] did not differ among treatments, blastocyst cell number in the HA100 group [57.9 cells (n = 64)] was greater (P &lt; 0.05) compared to those in the control [48.6 cells (n = 63)] or HA10 [50.0 cells (n = 65)] groups. Blastocyst oxygen consumption rate in the HA100 group [0.629 � 10-14 mol s-1 (n = 15)] was significantly higher than in the control [0.500 � 1-14 mol s-1 (n = 16)] or HA10 [0.464 � 10-14 mol s-1 (n = 14)] groups. ATP content/blastocyst did not differ among treatments [control: 0.645 pmol (n = 38), HA10: 0.727 pmol (n = 42), and HA100: 0.704 pmol (n = 43)]. It is concluded that HA affects the metabolic activity of pig blastocysts developed in a chemically defined medium, enhancing oxygen consumption and their total cell numbers, thus improving the quality of IVP blastocysts.

232 ADDITION OF VERY LOW AMOUNTS OF SERUM (ESTRUS COW SERUM) IMPROVES IN VITRO EMBRYO PRODUCTION IN DAIRY CATTLE

2015 ◽  
Vol 27 (1) ◽  
pp. 205 ◽  
Author(s):  
E. Mullaart ◽  
F. Dotinga ◽  
C. Ponsart ◽  
H. Knijn ◽  
J. Schouten
Keyword(s):  
Culture Medium ◽  
Culture Media ◽  
Calf Serum ◽  
High Serum ◽  
In Vitro Production ◽  
Embryo Production ◽  
Chi Square ◽  
Embryo Yield ◽  
Vitro Production

Improving the efficiency of the in vitro production (IVP) process is very important because it results in more embryos to be used in breeding programs or as commercial service. At CRV, a culture medium consisting of SOF with amino acids and BSA is used. In the past, richer culture media were used with 10% fetal calf serum combined with BRL cell co-culture. Although the efficiency of the IVP process of these media was good, these rather high serum concentrations were quite often related to large offspring syndrome (LOS). The switch to a culture system without serum resulted in a significant reduction in LOS but also in a reduction of embryo yield. The aim of the present study was to investigate the effect of adding low amounts of serum to the culture medium on efficiency of embryo production. Immature cumulus-oocyte complexes (COC) were recovered from ovaries 6 to 8 h upon slaughter. The COC were matured in vitro in TCM199/FCS/LH/FSH supplemented with cysteamine (0.1 mM). Subsequently, matured oocytes were fertilised with frozen-thawed gradient-separated semen and further cultured for 7 days in SOFaaBSA. The SOF medium contained either 0 (control), 0.1, 0.5, or 1.0% oestrus cow serum (ECS). Embryos development was scored at Day 7. Three replicates were performed and results were analysed by chi-square analyses. The results clearly show that adding ECS significantly improved embryo production (Table 1). Interestingly, already very low amounts (0.1%) of serum gave a significant increase in embryo percentage. In conclusion, addition of very low amounts of ECS (0.1%) is beneficial for embryo production, resulting in significantly higher embryo production (from 19 to 27%). In a subsequent field trial with OPU-derived embryos, the effect of addition of 0.1% ECS on birth weight (LOS) of the calves has to be investigated. Table 1.Percentage of blastocysts at Day 7 after culture in SOF medium with different amounts of serum

Developments in in vitro technologies for swine embryo production

2004 ◽  
Vol 16 (2) ◽  
pp. 15 ◽  
Author(s):  
Matthew B. Wheeler ◽  
Sherrie G. Clark ◽  
David J. Beebe
Keyword(s):  
Culture Medium ◽  
Culture Media ◽  
Physical Culture ◽  
Efficient Production ◽  
Media Composition ◽  
In Vitro Production ◽  
Considerable Research ◽  
Blastocyst Rate ◽  
Vitro Production

Several modifications have been made to in vitro production (IVP) systems to allow more efficient production of viable porcine embryos. Although in vitro production of pig embryos has been studied for over 30 years, the overall blastocyst production rate remains low. The low blastocyst rate is due to several factors, including polyspermic oocyte penetration, low rate of male pronucleus formation and less than optimal in vitro culture systems. These conditions are all inherent problems in porcine IVP and many of the mechanisms involved remain unknown. Considerable research has examined culture medium and the techniques used during the various stages of in vitro production. However, changes to the physical culture system used during IVF have remained unchanged until recently. The present paper will summarise selected developments in fertilisation and embryo culture media composition and focus on the development of modified equipment to improve the conditions used during the IVP of porcine oocytes and embryos.

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