125 INTRACYTOPLASMIC SPERM INJECTION (ICSI)-BASED MOUSE EMBRYO ASSAY: CHOICE OF EMBRYO CULTURE SYSTEM OUTWEIGHS THE EFFECT OF FERTILIZATION PROCEDURE ON EMBRYO DEVELOPMENT

2013 ◽  
Vol 25 (1) ◽  
pp. 209
Author(s):  
C. Schwarzer ◽  
T. C. Esteves ◽  
S. Le Gac ◽  
V. Nordhoff ◽  
S. Schlatt ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Mouse Embryo ◽  
Embryo Culture ◽  
Intracytoplasmic Sperm Injection ◽  
Culture Medium ◽  
Culture Media ◽  
Blastocyst Formation ◽  
The Absolute ◽  
Mouse Embryo Assay

Human embryo culture media, intended for assisted reproductive technologies (ARTs), are released for clinical use if they pass the mouse embryo assay (MEA). This assay prescribes that at least 70% of in vivo fertilized mouse 1-cell embryos form blastocysts, in order to grant the culture medium approval. In the fertility clinic, however, human embryos undergo more manipulation than their MEA counterparts through, for example, fertilization by intracytoplasmic sperm injection (ICSI); further, only a minority of the embryos transferred to the uterus goes on to establish gestations. In this context, we asked if the results of the MEA only depend on the type of in vitro culture, or are also affected by the method of fertilization. Superovulated B6C3F1 mouse oocytes were fertilized by ICSI using C57Bl/6 sperm. Pronuclear-stage eggs were allocated to four developmental environments: two ART culture protocols (HTF/MultiBlast, Irvine Scientific; ISM1/ISM2, Origio), standard mouse culture medium (KSOM(aa), made in-house) and the oviduct of pseudopregnant CD1 mice. As control for the invasive manipulation, pronuclear-stage eggs were generated by mating (B6C3F1 × C57Bl/6) and cultured in KSOM(aa) medium. Embryos were recovered from culture or from the CD1 uterus and scored for blastocyst formation at 96 h of development (Table 1). For these blastocysts, we determined the number of total, inner cell mass (ICM), and trophectoderm (TE) cells (Table 1) by confocal immunofluorescence microscopy (Schwarzer et al. 2012 doi:10.1093/humrep/des223). Our results show that ART culture protocols applied to mouse ICSI embryos are not equivalent in supporting blastocyst formation. Based on blastocyst rates, the ranking observed here after ICSI, reflects the ranking reported by us for IVF embryos (Schwarzer et al. 2012); that is, KSOM(aa) > HTF/MultiBlast > oviduct > ISM1/2. This similarity suggests that the effect of in vitro culture on mouse development exceeds the effect of ICSI, provided gametes are of good quality. From the analysis of cell numbers, we note that while the ICM/TE ratios are not of easy interpretation, the absolute numbers of cells in the ICM draw a clear line between the environment of the oviduct and those of culture media. Irrespective of the ICM/TE ratio, only the oviduct environment secures 8 cells in the ICM (Table 1). Soriano and Jaenisch (1986 Cell 46, 19–29) reported that 8 cells of the ICM are set aside to give rise to the body of a mouse. In summary, the current MEA is a valuable assay to assess the quality of culture medium, however, its refinement is necessary to better model the adaptive properties of embryo culture when different methods of fertilization are applied. Until the MEA is extended into postimplantation development, as we advocate (Schwarzer et al. 2012), the absolute numbers of cells in the ICM may be a better gauge of embryo quality than the blastocyst rates. Table 1.Mouse embryo assay outcomes after ICSI

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<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&M University.

scholarly journals Rheological and Microstructural Features of Plant Culture Media Doped with Biopolymers: Influence on the Growth and Physiological Responses of In Vitro-Grown Shoots of Thymus lotocephalus

2021 ◽  
Vol 2 (2) ◽  
pp. 538-553
Author(s):  
Natacha Coelho ◽  
Alexandra Filipe ◽  
Bruno Medronho ◽  
Solange Magalhães ◽  
Carla Vitorino ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Average Pore Size ◽  
Physiological Responses ◽  
Gum Arabic ◽  
Shoot Elongation ◽  
Hydroxyethyl Cellulose ◽  
Plant Culture

In vitro culture is an important biotechnological tool in plant research and an appropriate culture media is a key for a successful plant development under in vitro conditions. The use of natural compounds to improve culture media has been growing and biopolymers are interesting alternatives to synthetic compounds due to their low toxicity, biodegradability, renewability, and availability. In the present study, different culture media containing one biopolymer (chitosan, gum arabic) or a biopolymer derivative [hydroxyethyl cellulose (HEC), carboxymethyl cellulose (CMC)], at 100 or 1000 mg L−1, were tested regarding their influence on the growth and physiological responses of Thymus lotocephalus in vitro culture. Cellulose-based biopolymers (HEC and CMC) and gum arabic were used for the first time in plant culture media. The results showed that CMC at 100 mg L−1 significantly improved shoot elongation while chitosan, at the highest concentration, was detrimental to T. lotocephalus. Concerning only the evaluated physiological parameters, all tested biopolymers and biopolymer derivatives are safe to plants as there was no evidence of stress-induced changes on T. lotocephalus. The rheological and microstructural features of the culture media were assessed to understand how the biopolymers and biopolymer derivatives added to the culture medium could influence shoot growth. As expected, all media presented a gel-like behaviour with minor differences in the complex viscosity at the beginning of the culture period. Most media showed increased viscosity overtime. The surface area increased with the addition of biopolymers and biopolymer derivatives to the culture media and the average pore size was considerably lower for CMC at 100 mg L−1. The smaller pores of this medium might be related to a more efficient nutrients and water uptake by T. lotocephalus shoots, leading to a significant improvement in shoot elongation. In short, this study demonstrated that the different types of biopolymers and biopolymer derivatives added to culture medium can modify their microstructure and at the right concentrations, are harmless to T. lotocephalus shoots growing in vitro, and that CMC improves shoot length.

scholarly journals Factors affecting developmental competence of equine oocytes after intracytoplasmic sperm injection

Reproduction ◽  
2004 ◽  
Vol 127 (2) ◽  
pp. 187-194 ◽  
Author(s):  
Y H Choi ◽  
L B Love ◽  
D D Varner ◽  
K Hinrichs
Keyword(s):  
Embryo Culture ◽  
Intracytoplasmic Sperm Injection ◽  
Room Temperature ◽  
Culture Media ◽  
Developmental Competence ◽  
Glucose Medium ◽  
Factors Affecting ◽  
Nuclear Maturation ◽  
High Glucose Medium

This study was conducted to evaluate the effect of initial cumulus morphology (expanded or compact) and duration of in vitro maturation (24, 30 or 42 h) on the developmental competence of equine oocytes after intracytoplasmic sperm injection (ICSI). The effect of manipulation temperature (room temperature vs 37 °C) at the time of ICSI and concentration of glucose (0.55 vs 5.5 mM) during embryo culture was also investigated. The nuclear maturation rates of expanded (Ex) oocytes were significantly (P < 0.001) higher than those of compact (Cp) oocytes at all maturation times (61–72 vs 23–25% respectively). Forty-eight hours after ICSI of mature Ex oocytes, the rate of cleavage with normal nuclei was significantly (P < 0.05) higher for oocytes matured for 24 h than for those matured for 30 or 42 h (73 vs 57–59% respectively). For Cp oocytes, the morphologic cleavage rates for oocytes matured for 30 h were significantly higher (P < 0.05) than for those matured for 24 or 42 h (86 vs 55–61% respectively). The overall proportion of embryos having more than four normal nuclei at 48 h culture was significantly higher (P < 0.05) for Cp than for Ex oocytes. Manipulation temperature did not affect development of embryos from Ex or Cp oocytes at 96 h after ICSI. Culture in high-glucose medium significantly increased morphologic cleavage of Cp, but not Ex, oocytes (P < 0.05). Embryos from Cp oocytes had a significantly higher average nucleus number after 96-h culture than did embryos from Ex oocytes. These data indicate that developmental competence differs between Ex and Cp equine oocytes, and is differentially affected by the duration of maturation and by composition of embryo culture media.

80 DEVELOPMENT OF A SYNTHETIC MEDIUM FOR THE IN VITRO CULTURE OF BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 154 ◽  
Author(s):  
D. Moreno ◽  
A. Neira ◽  
L. Dubreil ◽  
L. Liegeois ◽  
S. Destrumelle ◽  
...  
Keyword(s):  
Growth Factors ◽  
Embryo Culture ◽  
Disease Transmission ◽  
Culture Medium ◽  
Culture Media ◽  
Synthetic Medium ◽  
Wilcoxon Test ◽  
Blastocyst Development ◽  
Negative Effect

In the majority of media for embryo culture, 2 of typical components used are FCS or BSA; however, the presence of FCS in the culture medium has been shown to have a negative effect on embryo quality and the use of animal-derived proteins in culture media increases the risks of disease transmission through in vitro embryo production. The aim of this study was to develop an in vitro embryo culture medium free from FCS and BSA, but with the addition of various growth factors and cytokines (GF-CYK: IGF-I, IGF-II, bFGF, LIF, GM-CSF) 50 ng mL–1 and (TGF-β1) 100 ng mL–1 supplemented with hyaluronan (HA) and recombinant albumin (RA). Bovine oocytes (n = 1043, 6 replicates) from abattoir ovaries were matured in TCM-199 medium with 60 μg mL–1 penicillin, 60 μg mL–1 streptomycin, and 10 ng mL–1 EGF for 24 h at 39°C and 5% CO2 in humidified air. Afterward, the oocytes were fertilized in IVF-TALP medium with 6 mg mL–1 fatty acid-free BSA and 1.7 IU mL–1 heparin for 18 h under the same conditions. After fertilization, presumptive zygotes were divided into two groups and cultured in 30 μL droplets of SOF supplemented with (1) 0.4% BSA + 5 μg mL–1 insulin, 5 μg mL–1 transferrin, and 5 ng mL–1 selenium (ITS) as a control; or (2) GF-CYK + 0.5 mg mL–1 HA + 0.15% RA (M1). Droplets were preserved under mineral oil in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 at 39°C. Blastocyst development and blastocyst diameter was observed at 7 and 8 days post-fertilization (dpf). Developmental and diameter data were analysed using the Wilcoxon test by using R software. The blastocyst rates were not significantly different between the control and M1 medium: at 7 dpf (22.9% ± 4.8 and 30.2% ± 3.0), and at 8 dpf (29.6% ± 5.1 and 37.4% ± 2.0 respectively; P > 0.05). The blastocyst diameter obtained with the M1 medium was significantly greater (P < 0.05) than that of the control at 7 dpf (173.3 μm ± 4.9 and 157.2 μm ± 4.1, respectively); however, no significant differences were observed at 8 dpf (190.3 μm ± 5.2 and 179.7 μm ± 5.3, respectively). In conclusion, the FCS- and BSA-free medium with GF-CYK, HA, and RA (M1) showed a comparable development rate to the control medium at 7 and 8 dpf. These growth factors and cytokines in association with hyaluronan and recombinant albumin have a synergistic action by promoting an increase in the blastocyst diameter at 7 dpf. This is fully synthetic method of embryo culture; it presents a valuable tool to reduce the risks of disease transmission via embryo transfer.

160 COMPARATIVE STUDY OF IN VITRO CULTURE MEDIA AND ASSISTED HATCHING TECHNIQUES ON MOUSE EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 188
Author(s):  
N. C. Negota ◽  
L. P. Nethenzheni ◽  
M. L. Mphaphathi ◽  
D. M. Barry ◽  
T. L. Nedambale
Keyword(s):  
In Vitro Culture ◽  
Chorionic Gonadotropin ◽  
Culture Medium ◽  
Culture Media ◽  
Assisted Hatching ◽  
Hatching Rate ◽  
Significant Difference ◽  
Equine Chorionic Gonadotropin ◽  
F1 Generation

The in vitro culture media and assisted hatching techniques remain challenging obstacles to be utilised widely. Mechanical, chemical, enzymatic thinning, and laser-assisted techniques have been used previously but information is still lacking on its application in livestock. The aim of this study was to compare the effect of 2 in vitro culture media (Hamster F10 and TMC-199) and 4 (mechanical, chemical, enzymatic, and laser) assisted hatching techniques on blastocyst formation and hatching rate using murine embryos as a model. The C57/b and Balb/c breeds were raised until they reached maturity and bred naturally to produce F1 generation. The light in the breeding house was controlled at 14 h light and 10 h dark. Feed and water were provided ad libitum for the mice. Superovulation of females were stimulated using equine chorionic gonadotropin and human chorionic gonadotropin. The F1 generation was used for the collection of the 400 blastocysts and randomly allocated into 4 assisted hatching techniques. Blastocysts were paired into a group of 10 and replicated 4 times for each assisted hatching technique. The general linear model of SAS version 9.4 (SAS Institute Inc., Cary, NC, USA) was used to analyse the data. Assisted hatching techniques of laser, mechanical, enzymatic, and chemical yielded 46.9 ± 37.1, 51.1 ± 40.2, 39.1 ± 35.8, and 33.3 ± 4.5%, respectively, under in vitro culture of Hamster F10. The TCM-199, laser, mechanical, enzymatic, and chemical assisted hatching techniques yielded 56.3 ± 43.3, 52.6 ± 35.5, 49.2 ± 37.5, and 33.9 ± 35.5%, respectively, with a significant difference. There was no significant difference observed in assisted hatching techniques and Hamster F10 culture medium. However, the hatching rate of embryos for all techniques was higher when in vitro cultured in TCM than cultured in Hamster F10. Hatching rate of blastocysts increased from chemical, enzymatic, mechanical, and laser with response to Hamster F10 and TCM; thus, laser is a suitable assisted hatching technique with TCM-199.

scholarly journals ISOPEROXIDASES PATTERN OF ROBINIA PSEUDOACACIA VAR. OLTENICA IN VITRO CULTURE ON DIFFERENT MEDIA

1970 ◽  
Vol 62 ◽  
Author(s):  
Cristina Babeanu ◽  
Georgeta Ciobanu ◽  
Mihaela Corneanu ◽  
Gabriela Marinescu ◽  
C. G. Corneanu
Keyword(s):  
Thermal Stability ◽  
In Vitro Culture ◽  
Humic Acids ◽  
Culture Medium ◽  
Culture Media ◽  
Robinia Pseudoacacia ◽  
Magnetic Fluids ◽  
Electrophoretic Separation

In this work is studied the isoperoxidase pattern of the in vitro subculture of Robinia pseudoacacia var. oltenica, on various culture media, with various content of myo-inositol, humic acids and magnetic fluids. The electrophoretic separation of the isoperoxidases was performed from extracts obtained from 30 days’old vegetal material. The isoperoxidase fractions were characterized in terms of quantity and quality and a study of their thermal stability was performed. The obtained data show that the isoperoxidase activity is modulated in various ways by the presence of the studied factors into the culture medium.

scholarly journals 316 SUPPLEMENTAL CYSTEINE PRESENCE DURING THE DECONDENSATION OF SPERM CHROMATIN IMPROVES FERTILIZATION AND BLASTOCYST FORMATION AFTER INTRACYTOPLASMIC SPERM INJECTION IN PIGS

2005 ◽  
Vol 17 (2) ◽  
pp. 308
Author(s):  
M. Katayama ◽  
T. Cantley ◽  
A. Rieke ◽  
B. Day
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Culture Media ◽  
Morphological Changes ◽  
Formation Rate ◽  
Sperm Chromatin ◽  
Blastocyst Formation ◽  
Fertilization Rates ◽  
Duration Time

The effect of a cysteine supplement in culture media for oocytes matured in vitro after intracytoplasmic sperm injection (ICSI) on fertilization and embryo development were examined. In the first experiment, sperm injected oocytes were cultured in NCSU23 (control) or NCSU23 supplemented with 0.57–3.71 mM cysteine (0.57–3.71 Cys) for 12 h after ICSI, and then fixed to observe pronuclear formation. In the second experiment, to examine the appropriate duration time of cysteine supplement to support fertilization, sperm-injected oocytes were transferred into NCSU23 following culture in NCSU23 supplemented with 1.71 mM cysteine for 1, 2, 3, 4, 5, 6, or 9 h after ICSI, and then fixed at 12 h. At the same time, morphological changes of sperm heads in oocytes cultured in NCSU23 (1.71 Cys) were observed. In the third experiment, to examine the developmental ability of ICSI embryos fertilized in NCSU23 (1.71 Cys), sperm injected oocytes were cultured under the following conditions for a total of 168 h; NCSU23 (control), NCSU23 (1.71 Cys) for 3 h followed by transfer into NCSU23 (1.71 Cys-3 h), NCSU23 (1.71 Cys) for 12 h followed by transfer in NCSU23 (1.71 Cys-12 h), or NCSU23 (1.71 Cys) (1.71 Cys). Data were pooled from at least five replicates. Values in each replicate were analyzed using one-way ANOVA. Significance of differences was assessed by Student's t-test. Culture with several concentrations of cysteine for 12 h showed that 1.71–3.71 Cys significantly (P < 0.05) increased fertilization rates above controls or 0.57 Cys (56–60%, 35%, or 48%, respectively). Culture for several duration times with 1.71 Cys showed that fertilization rates increased as the duration time increased to 3 h which was significantly (P < 0.05) higher than controls (68% and 34%, respectively), and culture times of greater than 3 h did not increase fertilization rates (58–68%). At 3 h, 59% of oocytes cultured in NCSU23 (1.71 Cys) had decondensed sperm heads and 16% of those had enlarged sperm heads. At 6 h, 50% of oocytes cultured in NCSU23 (1.71 Cys) had male pronuclei. Blastocyst formation rate in 1.71 Cys-3 h was 29% which was higher than for controls (20%). On the other hand, 1.71 Cys-12 h cultures showed low blastocyst formation rates, and continuous culture in NCSU23 (1.71 Cys) for 168 h (1.71 Cys) significantly (P < 0.05) decreased blastocyst rates (16% and 7%, respectively). We found that the supplement of 1.71 mM cysteine to NCSU23 for culture of oocytes after ICSI improved fertilization rates. However, the presence of 1.71 mM cysteine for 12 h or longer after ICSI had adverse effects on embryo development. Since 1.71 mM cysteine supplement for 3 h after ICSI improved blastocyst formation with the same fertilization rates as when supplemented for 12 h, the presence of cysteine only during the decondensation of sperm chromatin was found to be associated with the improvement of fertilization and also the promotion of blastocyst formation.

168 CORRELATION OF DEVELOPMENT KINETICS AND SEX OF IN VITRO-PRODUCED BOVINE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 196
Author(s):  
A. R. Buzzo ◽  
A. R. Pupulim ◽  
J. Mazucheli ◽  
F. V. Meirelles ◽  
I. P. Emanuelli
Keyword(s):  
In Vitro Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Proteinase K ◽  
Bovine Embryos ◽  
Male And Female ◽  
Stage Of Development ◽  
Males And Females ◽  
Kinetics Of

Approaches to improve the culture medium for in vitro production (IVP) of bovine embryos have been continuous because of the high commercial demand and a portion of this attempts the production of female cattle (dairy cows and stud cattle). However, in some embryonic in vitro culture systems, the development kinetics is faster in male than in female embryos (Avery 1992 Mol. Reprod. Dev. 32, 265–70; Xu 1992 Mol. Reprod. Dev. 31, 249–50). The aim of this work was to relate the kinetics of blastocyst expansion with the production rates of male and female embryos. Cumulus–oocyte complexes (n = 917; classes I and II) of cows from a slaughterhouse were matured with TCM-199 bicarbonate and 10% FCS (38.5°C, 5% CO2) for 24 h and fertilized with frozen-thawed semen in TALP-IVF medium for 18 h. Presumptive zygotes were culture in SOF medium supplemented with 10% FSB (5% O2, 38.5°C). Seven days after IVF, embryos were divided in 2 groups according to their kinetic stage of development: nonexpanded blastocysts (n = 175), or hatched and expanded blastocysts (n = 146). Hence, embryos were individually frozen in LN and stored in cryotubes. After thawing, Proteinase K (16 mg mL–1) was added to each tube and the tubes were incubated for 60 min at 37°C. Proteinase was denatured at 98°C for 10 min and the contents of each tube were divided into 2 samples (A and B) and subjected to the PCR technique. Two pairs of primers for the specific sequence of the Y chromosome were used to amplify the sequence of 210 and 250 bp for the male bovine and 1 pair of primers was used for the autosomal bovine sequence with a 280-bp fragment. Female embryos with a 280-bp product were observed in sample A and none were observed in sample B. The presence of 2 amplicons (280 and 210 bp) in sample A and 1 amplicon of 250 bp in sample B indicated that the embryo was male. A chi-square test was used to evaluate homogeneity. An analysis of the percentage of males and females between the experimental groups was performed by logistic regression and significance was considered when P < 0.05. There was no difference in the proportions of males and females in the nonexpanded blastocyst group (49.71 and 50.29%; P > 0.05). In the hatched and expanded blastocyst group, the proportion of males (65.75%) was statistically different from the proportion of females (34.25%); that is, the chance of the embryo being male was twice as high (P < 0.0038). These results suggest that there is a difference in the kinetics of embryo development between male and female embryos and that blastocyst expansion can point that out. In vitro culture media with FCS support the development of expanded male blastocysts. Further research in culture medium modifications (FCS, the energy source, amino acids and others) are needed to respond to the trend in the production of sex-defined embryos.

291 EFFECTS OF ANTIOXIDANT AND CULTURE MEDIUM ON THE DEVELOPMENT OF PORCINE IN VITRO-MATURED - IN VITRO-FERTILIZED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 261
Author(s):  
C. Choe ◽  
D.-S. Son ◽  
S.-H. Choi ◽  
S.-R. Cho ◽  
H.-J. Kim ◽  
...  
Keyword(s):  
Culture Medium ◽  
Culture Media ◽  
Culture Conditions ◽  
Free Oxygen Radicals ◽  
Blastocyst Formation ◽  
Free Oxygen ◽  
Statistical Analysis System ◽  
Developmental Rates ◽  
Analysis System

Most cells cultured in vitro are exposed to the risk of injury by free oxygen radicals (FOR). However, some of FOR-induced injury could be reduced by the antioxidants and culture medium used for in vitro embryos. This study was undertaken to examine the effects of the antioxidant and culture medium on the development of porcine in vitro-matured–in vitro-fertilized embryos. In Experiment 1, we treated the porcine oocytes in NCSU23 medium with various concentrations of β-mercaptoethanol (β-ME) to determine the effective concentration of antioxidants during IVM of porcine oocytes. In Experiment 2, we tested different culture media to find the proper culture conditions for in vitro porcine embryos. The porcine oocytes that were matured in NCSU23 medium and then fertilized in mTBM medium were cultured in NCSU23 or porcine zygote medium-5. All steps (maturation, fertilization, and development) were carried out in vitro. Differences were analyzed among treatments using the general linear model (GLM) procedure in the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). The results were summarized as follows. Various concentrations of β-ME showed different developmental rates in porcine embryos. The rates of blastocyst formation at Day 7 after IVF were 9.2 � 1.8 (n = 65), 10.0 � 4.2 (n = 80), 17.5 � 1.1 (n = 63), 20.7 � 1.7 (n = 82), and 14.6 � 1.4 (n = 82) in oocytes treated with β-ME at 0, 10, 25, 50, and 100 �M during IVM, respectively. Of the concentrations of β-ME tested, 50 �M β-ME markedly increased the rates of blastocyst formation at Day 7 (P &lt; 0.05). The rates of blastocyst formation at Day 7 in the NCSU23 and PZM-5 culture media of porcine IVF-derived embryos were 18.8 � 2.6 (n = 96) and 15.6 � 7.1 (n = 77), respectively. The developmental rates were slightly increased in NCSU23, compared with those in PZM-5, but there were no significant differences (P &lt; 0.05) between the NCSU23 and PZM-5 media. In conclusion, these results suggest that the addition of 50 �M β-ME in the IVM medium can improve developmental the rates of porcine embryos in vitro.

scholarly journals In vitro culture of Neoechinorhynchus buttnerae (Acanthocephala: Neoechinorhynchidae): Influence of temperature and culture media

2018 ◽  
Vol 27 (4) ◽  
pp. 562-569 ◽  
Author(s):  
Carinne Moreira de Souza Costa ◽  
Talissa Beatriz Costa Lima ◽  
Matheus Gomes da Cruz ◽  
Daniela Volcan Almeida ◽  
Maurício Laterça Martins ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Treatment Strategies ◽  
Infected Fish ◽  
In Vivo Studies ◽  
Colossoma Macropomum ◽  
Tank Water

Abstract Infection by the acantocephalan Neoechinorhynchus buttnerae is considered one of most important concerns for tambaqui fish (Colossoma macropomum ) production. Treatment strategies have been the focus of several in vivo studies; however, few studies have been undertaken on in vitro protocols for parasite maintenance. The aim of the present study was to develop the best in vitro culture condition for N. buttnerae to ensure its survival and adaptation out of the host to allow for the testing of substances to be used to control the parasite. To achieve this, parasites were collected from naturally infected fish and distributed in 6-well culture plates under the following treatments in triplicate: 0.9% NaCl, sterile tank water, L-15 Leibovitz culture medium, L-15 Leibovitz + agar 2% culture medium, RPMI 1640 culture medium, and RPMI 1640 + agar 2% culture medium. The plates containing the parasites were maintained at 24 °C, 28 °C, and 32 °C. The RPMI 1640 + agar 2% culture medium showed the best survival of 24 days at 24 °C. No body alterations such as swollen parasites, body deformation, dehydration and hardening were observed in the RPMI 1640 + 2% culture medium.

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