scholarly journals 302 PROTEIN SUPPLEMENTATION TO IVM MEDIUM IN RELATION TO THE INCIDENCE OF APOPTOSIS IN BOVINE OOCYTES MATURED IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 302
Author(s):  
E. Warzych ◽  
K. Matulewicz ◽  
A. Nogowska ◽  
D. Lechniak
Keyword(s):  
Culture Media ◽  
State Committee ◽  
Protein Supplementation ◽  
Developmental Competence ◽  
Vitro Fertilization ◽  
Maturation In Vitro ◽  
Wide Range ◽  
Defined Culture

Mammalian embryos derived from in vitro fertilization display lower developmental competence and quality when compared to their in vivo counterparts. The composition of culture media significantly contributes to this phenomenon. Media supplemented with FBS or the serum derivate BSA are described as biochemically undefined. Those macromolecules were shown to exert a wide range of effects on cultured embryos, dependent on batch-to-batch variability. Therefore, replacement of these protein sources with a synthetic macromolecule such as polyvinyl pyrrolidone (PVP) or polyvinyl alcohol (PVA) provides a possibility to use a chemically defined culture medium (Ali et al. 2002 Biol. Reprod. 66, 901–905). Apoptosis as programmed cell death naturally occurs in mammalian oocytes and embryos; however, its incidence is significantly higher in vitro. The aim of this study was to investigate whether protein supplementation (FBS, fatty acid-free (faf)-BSA, PVP40) of IVM medium affects the incidence of apoptotic oocytes. In the present study, the IVM system previously described by Makarevich et al. (2002 Biol. Reprod. 66, 386–392) was used. Briefly, follicular oocytes aspirated from slaughterhouse ovaries were matured in vitro in one of three maturation media supplemented with FBS (10%), faf-BSA (6 mg mL−1) or PVP40 (4 mg mL−1). The terminal TUNEL assay kit was used to detect the DNA fragmentation in apoptotic cells (DeadEndTM Fluorometric TUNEL system, Promega, Madison, WI, USA). The data were analyzed by chi-square test of independence. Altogether, 630 oocytes collected during 12 IVM experiments were subjected to the Tunel test, and 563 (89.4%) of them were successfully investigated: 426 after maturation in vitro and 137 follicular, non-matured. The remaining 67 cells were lost during manipulation. The rate of Tunel-positive cells differed (P < 0.001) between matured (11.8%) and follicular oocytes (1.5%). Protein supplementation of IVM media did not significantly affect the rate of apoptotic oocyte occurrence, which was 9% in the faf-BSA group, 11.5% in the FBS group, and 15% in the PVP group. No differences were observed in the rate of Tunel-positive cells between oocytes at MII and MI stages. In conclusion, protein supplementation of IVM medium used in the present study did not affect the incidence of apoptotic oocytes after maturation in vitro. This research was supported by the State Committee for Scientific Research as a Solicited Project PBZ-KBN-084 from 2003 to 2005 year.

scholarly journals Maturation of human oocytes in vitro and their developmental competence

Reproduction ◽  
2001 ◽  
pp. 51-75 ◽  
Author(s):  
A Trounson ◽  
C Anderiesz ◽  
G Jones
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Growth Phase ◽  
Developmental Competence ◽  
Minimum Size ◽  
Success Rates ◽  
Human Oocytes ◽  
Maturation In Vitro

Complete maturation of oocytes is essential for the developmental competence of embryos. Any interventions in the growth phase of the oocyte and the follicle in the ovary will affect oocyte maturation, fertilization and subsequent embryo development. Oocyte size is associated with maturation and embryo development in most species examined and this may indicate that a certain size is necessary to initiate the molecular cascade of normal nuclear and cytoplasmic maturation. The minimum size of follicle required for developmental competence in humans is 5-7 mm in diameter. Maturation in vitro can be accomplished in humans, but is associated with a loss of developmental competence unless the oocyte is near completion of its preovulatory growth phase. This loss of developmental competence is associated with the absence of specific proteins in oocytes cultured to metaphase II in vitro. The composition of culture medium used successfully for maturation of human oocytes is surprisingly similar to that originally developed for maturation of oocytes in follicle culture in vitro. The presence of follicle support cells in culture is necessary for the gonadotrophin-mediated response required to mature oocytes in vitro. Gonadotrophin concentration and the sequence of FSH and FSH-LH exposure may be important for human oocytes, particularly those not exposed to the gonadotrophin surge in vivo. More research is needed to describe the molecular and cellular events, the presence of checkpoints and the role of gene expression, translation and protein uptake on completing oocyte maturation in vitro and in vivo. In the meantime, there are very clear applications for maturing oocytes in human reproductive medicine and the success rates achieved in some of these special applications are clinically valuable.

scholarly journals IN VITRO CALLUS INDUCTION AND COMPARATIVE GC-MS ANALYSIS OF METHANOLIC EXTRACTS OF CALLUS AND LEAF SAMPLES OF AMPELOCISSUS LATIFOLIA (ROXB.) PLANCH

2018 ◽  
Vol 10 (9) ◽  
pp. 68
Author(s):  
Deep Chhavi Anand ◽  
Rishikesh Meena ◽  
Vidya Patni
Keyword(s):  
Callus Induction ◽  
Culture Media ◽  
Biological Activities ◽  
Gas Chromatography Mass Spectrometry ◽  
Dodecanoic Acid ◽  
Stem Explants ◽  
Ms Analysis ◽  
Wide Range

Objective: The aim of the present study was to develop a callus induction protocol and comparative study of therapeutic phytochemicals present in in vivo leaf and in vitro callus extracts through Gas Chromatography-Mass Spectrometry analysis.Methods: Murashige and Skoog media was used as culture media for callus induction. In vitro callus induction protocol was developed by studying the effects of various plant growth regulators like auxin, 2, 4-D (2,4-dichlorophenoxyacetic acid), NAA (naphthalic acetic acid), alone and in combination with cytokinin BAP (benzyl aminopurine), on leaf and stem explants. The GC-MS analysis of Ampelocissus latifolia was carried out on Shimadzu QP-2010 plus with thermal desorption system TD 20 to study the phytochemical profile.Results: In vitro callus induction protocol was developed for the plant and callusing was done from leaf and stem explants of Ampelocissus latifolia. The best result for callus induction was obtained using leaf explant, and callus production were maximum in Murashige and Skoog medium fortified with BAP (0.5 mg/l) and NAA (1.0 mg/l). Major compounds identified in the GC-MS analysis were Campesterol, Stigmasterol, Beta-Sitosterol, Docosanol, Dodecanoic acid, etc., in in vitro extract and Beta Sitosterol, Tocopherol, Squalene, Bergamot oil, Margarinic acid, Hexadecanoic acid, etc., in in vivo extract. The different active phytochemicals identified have been found to possess a wide range of biological activities, thus this analysis forms a basis for the biological characterization and importance of the compounds identified for human benefits.Conclusion: This is the first report on callus induction in Ampelocissus latifolia. From the results obtained through the in vitro callus induction and its comparative GCMS analysis with in vivo extract, it is revealed that Ampelocissus latifolia contains various bioactive compounds that are of importance for phytopharmaceutical uses. The GCMS analysis revealed that the amount of Beta-sitosterol and 5-Hydroxymethylfurfural (HMF) was very high in in vitro extract as compared to in vivo extract.

scholarly journals 146COMPARISON OF EMBRYO CULTURE MEDIA FOR BLASTOCYST DEVELOPMENT AFTER INTRACYTOPLASMIC SPERM INJECTION OF IN VITRO-MATURED EQUINE OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 195
Author(s):  
Y.H. Choi ◽  
D.D. Varner ◽  
K. Hinrichs
Keyword(s):  
In Vitro Culture ◽  
Embryo Culture ◽  
Intracytoplasmic Sperm Injection ◽  
Culture Media ◽  
Polar Body ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Vitro Fertilization ◽  
Significant Difference

Research on in vitro culture of equine embryos has been scant, due to failure of equine in vitro fertilization to be repeatably successful. We have recently obtained high fertilization rates of equine oocytes via intracytoplasmic sperm injection (ICSI) using a piezo drill (Choi et al., 2002 Reproduction 123, 455–465). Culture of presumptive zygotes in G1.2/2.2 medium resulted in 63% cleavage and an average of 15 cells at 4d, but only 2 to 9% blastocyst development at 7 days (Choi et al., 2003 Theriogenology 59, 1219–1229). In the present study, we evaluated the effect of two different culture media, G1.3/G2.3 v. DMEM/F-12, with or without FBS, on blastocyst development after ICSI. Oocytes were collected from slaughterhouse-derived ovaries by follicular scraping and were matured in vitro for 24h in M199 with 10% FBS and 5μUmL−1 FSH. After culture, oocytes having a polar body (198/305; 65%) were fertilized by ICSI with frozen-thawed equine sperm using a piezo drill. Presumptive zygotes were cultured in 1 of 4 media: G1.3/G2.3 (which includes 0.8% BSA) with or without 10% FBS, or in DMEM/F-12 with 0.5% BSA, with or without 10% FBS. Culture was performed in microdroplets at 5μL/zygote under oil at 38.2°C in an atmosphere of 5% CO2, 5% O2 and 90% N2 for 7.5 days. In G1.3/2.3 treatments, G1.3 media were completely refreshed at 48h, zygotes were transferred to G2.3 (with or without FBS as per the first stage) at 96h, and were completely refreshed with the same media at 144h. In DMEM/F-12 treatments, media were completely refreshed every other day. Three to 5 replicates were performed in each treatment, and data were analyzed by chi-square test. There were no significant differences in cleavage rates (59–64%) among treatments. The rate of development to blastocyst, per oocyte injected, in G1.3/G2.3/BSA (1/49, 2%) was significantly lower (P&lt;0.05) than that for the other three treatments: G1.3/2.3/BSA/FBS (9/49, 18%), DMEM/F-12/BSA (9/50, 18%), or DMEM/F-12/BSA/FBS (10/50, 20%). There was no significant difference in blastocyst development among the latter three treatments. These findings indicate that G1.3/2.3 media with BSA only do not adequately support growth of equine embryos. Development of up to 20% of injected oocytes to the blastocyst stage in G media supplemented with FBS, in DMEM/F-12/BSA or in DMEM/F-12/BSA/FBS represents the highest in vitro equine blastocyst rate in medium alone (i.e. without co-culture) yet reported. The success of DMEM/F-12 as an embryo culture medium may provide a relatively simple basis for equine in vitro culture programs. To determine whether this medium was able to support further developmental competence, we cultured equine embryos resulting from nuclear transfer of in vitro-matured oocytes in DMEM/F-12+10% FBS (without BSA). We transferred 4 resulting blastocysts to recipient mares by transcervical transfer; one pregnancy is ongoing at 230d gestation at the time of this writing. This work was supported by the Link Equine Research Endowment Fund, Texas A&amp;M University.

scholarly journals Developmental Competence of Domestic Cat Vitrified Oocytes in 3D Enriched Culture Conditions

Animals ◽  
2019 ◽  
Vol 9 (6) ◽  
pp. 329 ◽  
Author(s):  
Martina Colombo ◽  
Maria Giorgia Morselli ◽  
Mariana Riboli Tavares ◽  
Maricy Apparicio ◽  
Gaia Cecilia Luvoni
Keyword(s):  
Developmental Stages ◽  
Culture Conditions ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Control Group ◽  
Vitro Fertilization ◽  
In Vitro Embryo Production ◽  
Physical And Chemical

Cryoinjuries severely affect the competence of vitrified oocytes (VOs) to develop into embryos after warming. The use of culture conditions that provide physical and chemical support and resemble the in vivo microenvironment in which oocytes develop, such as 3D scaffolds and coculture systems, might be useful to improve VOs outcomes. In this study, an enriched culture system of 3D barium alginate microcapsules was employed for the in vitro embryo production of domestic cat VOs. Cryotop vitrified-warmed oocytes were in vitro matured for 24 h in the 3D system with or without fresh cumulus-oocyte complexes (COCs) in coculture, whereas a control group of VOs was cultured in traditional 2D microdrops of medium. After in vitro fertilization, presumptive embryos were cultured in 3D or 2D systems according to the maturation conditions. Vitrified oocytes were able to mature and develop into embryos in 3D microcapsules (17.42 ± 11.83%) as well as in 2D microdrops (14.96 ± 8.80%), but the coculture with companion COCs in 3D resulted in similar proportions of VOs embryo development (18.39 ± 16.67%; p = 1.00), although COCs presence allowed for blastocyst formation (0.95 ± 2.52%). In conclusion, embryos until late developmental stages were obtained from cat VOs, and 3D microcapsules were comparable to 2D microdrops, but improvements in post-warming conditions are still needed.

Alteration of extracellular cation concentrations and ratios in culture medium does not affect first cleavage division of hamster zygotes in vitro nor overcome the 'two-cell block'

1989 ◽  
Vol 1 (3) ◽  
pp. 231 ◽  
Author(s):  
BD Bavister ◽  
M Golden
Keyword(s):  
Culture Medium ◽  
Culture Media ◽  
Cell Block ◽  
Cleavage Division ◽  
Cell Stage ◽  
Wide Range ◽  
Standard Culture ◽  
Simple Medium

In vivo fertilized hamster one-cell eggs (embryos) were cultured in a simple medium that was modified to provide a wide range of concentrations and ratios of the four major cation components (sodium, potassium, calcium and magnesium) while maintaining total osmotic pressure at 290 +/- 5 mosm. Embryos were cultured in these media to find the optimum cation concentrations for supporting the first cleavage division in vitro and to determine if physiologically abnormal cation concentrations and/or ratios in standard culture media could account for the 'two-cell block' to development in vitro in this species. Despite using a broad range of ratios for sodium:potassium (from 45:1 to 5:1) and for calcium:magnesium (from 17:1 to 1:1), there were no significant differences in the proportions of fertilized eggs that underwent the first cleavage division (approx. 60-80% across all treatments), and none of the two-cell embryos underwent further cleavage during extended culture. These data demonstrate that the first cleavage division of hamster embryos in vitro is insensitive to extracellular concentrations and ratios of the major cations, and that the non-physiological concentrations and/or ratios of these cations in the culture medium are not the primary reason for the failure of hamster zygotes to develop past the two-cell stage in vitro.

scholarly journals Effects of Progesterone on in Vitro Developmental Competence of Bovine Embryos

2020 ◽  
Vol 8 (1) ◽  
pp. 42
Author(s):  
Orhan Örnek ◽  
Yusuf Ziya Güzey
Keyword(s):  
Embryo Development ◽  
Culture Media ◽  
Early Embryo ◽  
Developmental Competence ◽  
Direct Role ◽  
Vitro Fertilization ◽  
Morula Stage ◽  
Vitro Development

Progesterone plays a key role in the establishment and maintenance of pregnancy in mammalian. Increasing levels of circulating progesterone in the post-conception period are associated with conceptus elongation and high pregnancy rates in cattle. Contradictory results are available on the direct role of progesterone in early embryo development. The objective of this study was to evaluate direct effects of progesterone on in vitro development of cattle embryos. Immature oocytes collected from slaughtered animals and cultured in the presence of different concentrations of progesterone (25, 50, 100 ng/mL) following in vitro fertilization. Cleavage rates in 25 and 50 ng/mL concentrations of progesterone were significantly higher than those in controls and 100 ng/mL. Rate of embryos that reached to the morula stage was similar in all groups. Supplementation of 25 and 50 ng/mL progesterone to the culture media significantly increased blastocyst yield while 100 ng/mL progesterone resulted in a decrease. As a conclusion, we can suggest that progesterone supplementation in in vitro culture may support embryo development at low levels.

86 IN VITRO-MATURED GILT OOCYTES CAN HAVE EQUAL OR BETTER DEVELOPMENTAL COMPETENCE THAN SOW OOCYTES WITH NEW MATURATION MEDIA

2017 ◽  
Vol 29 (1) ◽  
pp. 150 ◽  
Author(s):  
L. D. Spate ◽  
S. L. Murphy ◽  
J. A. Benne ◽  
A. Giraldo ◽  
D. Hylan ◽  
...  
Keyword(s):  
Growth Factor ◽  
Somatic Cell ◽  
Culture Media ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Development In Vitro ◽  
Humidified Air

It has long been thought that oocytes obtained from sows yielded a higher level of developmental competence compared with oocytes obtained from prepubertal gilts. Because gilt-derived oocytes are more readily available to our laboratory and they are less developmentally competent, we hypothesised that by making alterations to our maturation system we could improve the developmental competence of the gilt-derived oocytes to that of their sow-derived counterparts. We performed 2 experiments that evaluated the ability of each source of oocyte to develop to the blastocyst stage, using altered maturation media. The first experiment focused on the developmental ability of each source of oocytes, through IVF and culture. The second experiment again focused on the developmental competence of each oocyte source but through somatic cell NT. For both experiments, the sow-derived oocytes were obtained from Desoto Biosciences and the gilt ovaries were collected from Smithfield Inc. in Milan, Missouri. Both sets of oocytes were in vitro matured in M199 supplemented with 0.57 mM cysteine, 5 μg mL−1 LH and FSH, and 10 ng mL−1 epidermal growth factor; however, the gilt derived media was altered to contain 40 ng mL−1 fibroblast growth factor 2 and 20 ng mL−1 insulin-like growth factor and leukemia inhibitory factor. Additionally, the maturation media for the sow-derived oocytes contained the addition of 5 μg mL−1 insulin and 10% follicular fluid. In the first experiment we performed IVF on oocytes from the 2 sources as per our laboratory standard IVF procedure, co-incubating the oocytes with 0.25 × 106 porcine semen for 4 h, followed by washing and moving the oocytes to MU2 culture media at 38.50°C in 5% CO2, humidified air overnight. After overnight culture the presumptive zygotes were transferred to the same conditions with 5% CO2, 5% O2, and 90% N2. After an additional 5 days, blastocyst development was assessed. The gilt oocytes yielded 39.3a ± 7.2% blastocyst, and the sow oocytes had a blastocyst rate of 24.9b ± 6.9%, with an n of 389 and 313, respectfully. Statistical analysis was performed by using Genmod in SAS 9.4. In the second experiment, using standard laboratory protocol for somatic cell NT, we activated both sets of oocytes with 200 μM thimerosal for 10 min followed by 30-min incubation with 4 mM dithiothreitol. The embryos were co-incubated for 15 h with 500 nM Scriptaid in the MU2 culture media in 5% CO2, humidified air; then these embryos were also moved to 5% CO2, 5% O2, and 90% N2 and cultured to Day 6. The sow oocytes produced a blastocyst percentage of 38.6%, and the gilt oocyte group had a blastocyst percentage of 43.5%, with an n of 290 and 285, respectfully. There was no difference statistically between these treatments. Both gilt and the sow oocyte sources have yielded live piglets at this time. We concluded that the maturation system used for our gilt-derived oocytes resulted in equal or better development in vitro compared with the sow-derived oocytes. Follow-up experiments evaluating in vivo development are needed for a complete comparison. This work was funded by Food for the 21st Century University of MO, and the NIH U42OD011140.

scholarly journals Metabolomics analysis of follicular fluid coupled with oocyte aspiration reveals importance of glucocorticoids in primate periovulatory follicle competency

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sweta Ravisankar ◽  
Kelsey E. Brooks ◽  
Melinda J. Murphy ◽  
Nash Redmayne ◽  
Junghyun Ryu ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Paracrine Signaling ◽  
Vitro Fertilization ◽  
Maturation In Vitro ◽  
Variable Capacity ◽  
Notable Increase ◽  
Oocyte Aspiration ◽  
Metabolomics Analysis

AbstractGonadotropin administration during infertility treatment stimulates the growth and development of multiple ovarian follicles, yielding heterogeneous oocytes with variable capacity for fertilization, cleavage, and blastocyst formation. To determine how the intrafollicular environment affects oocyte competency, 74 individual rhesus macaque follicles were aspirated and the corresponding oocytes classified as failed to cleave, cleaved but arrested prior to blastulation, or those that formed blastocysts following in vitro fertilization. Metabolomics analysis of the follicular fluid (FF) identified 60 unique metabolites that were significantly different between embryo classifications, of which a notable increase in the intrafollicular ratio of cortisol to cortisone was observed in the blastocyst group. Immunolocalization of the glucocorticoid receptor (GR, NR3C1) revealed translocation from the cytoplasm to nucleus with oocyte maturation in vitro and, correlation to intrafollicular expression of the 11-hydroxy steroid dehydrogenases that interconvert these glucocorticoids was detected upon an ovulatory stimulus in vivo. While NR3C1 knockdown in oocytes had no effect on their maturation or fertilization, expansion of the associated cumulus granulosa cells was inhibited. Our findings indicate an important role for NR3C1 in the regulation of follicular processes via paracrine signaling. Further studies are required to define the means through which the FF cortisol:cortisone ratio determines oocyte competency.

scholarly journals 321 DEVELOPMENT IN VIVO AND IN VITRO OF PORCINE OOCYTES FERTILIZED BY INTRACYTOPLASMIC INJECTION OF A FREEZE - DRIED SPERM HEAD

2005 ◽  
Vol 17 (2) ◽  
pp. 311
Author(s):  
M. Nakai ◽  
K. Kikuchi ◽  
A. Takizawa ◽  
M. Ozawa ◽  
J. Noguchi ◽  
...  
Keyword(s):  
Sperm Head ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Vitro Fertilization ◽  
In Vitro Development ◽  
Sperm Suspension ◽  
Freeze Dried ◽  
Vitro Development

The present study investigated the development in vivo and in vitro of in vitro matured porcine oocytes injected with a freeze-dried (FD) boar sperm head. In mice, DNA damage was induced during the holding period after rehydration and before sperm injection (Wakayama, T. and Yanagimachi, R. 1998, Nat. Biotechnol., 16, 639–641). Here, we examined the relationship between duration of rehydration of FD sperm and in vitro development of FD sperm-injected porcine oocytes. We also assessed the in vivo developmental competence of the injected oocytes after embryo transfer. Ejaculated boar spermatozoa were suspended in Pig-FM (Suzuki, K. et al. 2002, Int. J. Androl. 25, 84–93) and sonicated for 1 min to separate sperm heads from the tails. An aliquot (100 μL) of the sperm suspension was put into a glass tube and then pre-cooled at −40°C for 6 h. Each tube was attached to a freeze-dry system (DuraDry μP, FTS Systems, Stone Ridge, NY, USA) for 12 h. The ampules were closed and stored at 4°C for more than 7 days before use. For rehydration, 100 μL of distilled water was added into the ampules. In Experiment I, we injected FD sperm heads which were kept for 0–60, 60–120, or 120–180 min after rehydration. At 1 h after the injection, the injected oocytes were stimulated with a DC pulse and cultured for 6 days. The rate of blastocyst formation and the number of cells in the blastocysts were examined. Embryos after in vitro fertilization (IVF) were evaluated as a control. As shown in Table 1, the rates of blastocyst formation were not different (by χ2 test) for duration of rehydration and the control. However, the cell numbers of FD groups were lower (P < 0.05; by Student's t-test) than that in the control. In Experiment II, oocytes injected with a single FD sperm head and stimulated were transferred to both oviducts of a total of ten recipient gilts. Two recipients were diagnosed as pregnant at Day 30 of gestation. At Day 39, one of the pregnant recipients had an abortion, and two fetuses were recovered. The other pregnancy was not maintained. The results suggest that oocytes fertilized with a single FD sperm head have competence to be implanted and to develop to the early fetal stage, and also that the duration for rehydration does not influence in vitro developmental ability in pigs. Table 1. Effects of the duration from rehydration of freeze-dried sperm heads to the injection of the heads into in vitro matured oocytes on in vitro development of the oocytes in pigs

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