237 CHROMOSOMAL ABNORMALITIES IN IN VITRO-PRODUCED EARLY BOVINE EMBRYOS: USE OF HOMOLOGOUS FOLLICULAR FLUID SUPPLEMENTATION IN THE OOCYTE MATURATION MEDIA

2013 ◽  
Vol 25 (1) ◽  
pp. 266
Author(s):  
S. Demyda-Peyrás ◽  
M. Hidalgo ◽  
J. Dorado ◽  
L. De Luca ◽  
E. Genero ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Follicular Fluid ◽  
Chromosomal Abnormalities ◽  
Culture Media ◽  
Control Group ◽  
Bovine Embryos ◽  
Humid Atmosphere ◽  
Serum Replacement ◽  
Laboratory Techniques

Chromosomal abnormalities were described as a possible cause of embryo failures in cattle, even more so when they are in vitro produced. It has been widely demonstrated that the post-fertilization culture environment affects the frequency of blastomeric aneuploidies. However, the literature concerning the effect of the oocyte maturation techniques on in vitro-produced embryos is scarce. The aim of this study was to determine the effect of homologous bovine follicular fluid (BFF) as a possible replacement for commercial sera in the appearance of chromosomal abnormalities on early IVF-produced embryos. Cumulus–oocyte complexes obtained from ovarian puncture were maturated in modified bicarbonate-buffered TCM-199 media, supplemented with glutamine, sodium pyruvate, FSH, LH, oestradiol, and gentamicin in three different groups. Two treatments were performed: 1) base media supplemented with BFF, obtained aseptically from follicles between 4 and 10 mm in diameter (10 and 20%), and 2) a control group, with base media supplemented with 10% FCS without BFF. After 20 h of culture at 38.5°C in a 5% CO2 humid atmosphere, cumulus–oocyte complexes from both treatments were fertilized in IVF media and then cultured for 72 h in SOF media, according to our laboratory techniques. A total of 152 early embryos were cytogenetically evaluated following our standard laboratory techniques. Developed embryos were individually fixed onto a slide, disaggregated into blastomeres with acetic acid, and stained with Giemsa solution. Chromosomal numerical abnormalities were evaluated in each embryo by direct observation at 1250× magnification using a bright field microscope. Results were statistically compared among treatments by the expected proportion test. No significant differences (P > 0.05) were found between different culture media on the percentages of normal diploid embryos or each kind of numerical abnormality. According to our results (Table 1), the use of homologous follicular fluid as a supplement on the oocyte maturation media did not influence the appearance of abnormal complements in the embryos produced compared with the use of FCS. In conclusion, homologous follicular fluid may be considered a valid serum replacement in the maturation media on IVF-produced bovine embryos. Table 1.Analysis of chromosomal complements of Day 3 in vitro-produced bovine embryos derived from oocytes maturated in culture media with different serum supplementation1

87 EVALUATION OF THE NUMERICAL CHROMOSOMAL ABNORMALITIES ON IN VITRO EARLY BOVINE EMBRYOS: EFFECT OF THE CELL CO-CULTURE WITH GRANULOSA CELLS

2014 ◽  
Vol 26 (1) ◽  
pp. 157
Author(s):  
S. Demyda-Peyrás ◽  
M. Hidalgo ◽  
J. Dorado ◽  
M. Moreno-Millan
Keyword(s):  
Embryo Development ◽  
Granulosa Cells ◽  
Chromosomal Abnormalities ◽  
Culture Media ◽  
Gradient Centrifugation ◽  
Early Embryo ◽  
Bovine Embryos ◽  
Humid Atmosphere ◽  
Early Embryos

Chromosomal numerical abnormalities (CNA) were described as a major cause of developmental failures in in vitro-produced (IVP) embryos. It has been described that CNA are influenced by the post-fertilization culture environment of the embryo. Furthermore, it was demonstrated that the use of different culture media affects the CNA rates. The addition of granulosa cells during early embryo development is a well-known procedure to simplify the culture of bovine IVP and cloned embryos. This technique avoids the use of culture environments saturated with N2 (tri-gas chambers). The aim of this study was to determine the effect of the addition of granulosa cells in the chromosomal abnormalities of IVP cattle embryos. Cumulus–oocyte complexes (COC) were matured in TCM-199 medium, supplemented with glutamine, sodium pyruvate, FSH, LH, oestradiol, and gentamicin during 20 h at 38.5°C in a 5% CO2 humid atmosphere. Subsequently, matured oocytes were fertilized in IVF-TALP medium using 1 × 106 spermatozoa mL–1, selected through a Percoll gradient centrifugation. After fertilization, zygotes were divided in 2 groups and cultured in TCM-199 medium for 48 h, with (TCM-GC) or without (TCM) the addition of 1 × 106 granulosa cells. These cells were obtained by centrifuging and washing the follicular fluid remaining from searching dishes and adjusted to the working concentration. After culture, a total of 106 early embryos (72 hpi) were cytogenetically evaluated following our standard laboratory techniques. Embryos showing normal development were individually fixed onto a slide, disaggregated into blastomeres with acetic acid, and stained with Giemsa solution. Chromosomal numerical abnormalities were evaluated by direct observation at 1250× magnification in a brightfield microscope. Percentage of normal diploid embryos (D) and abnormal haploid (H), polyploid (P), or aneuploid (A) embryos were determined. Results were statistically compared between treatments using a Z test for proportions. Results were: D = 81.4%, H = 7.2%, P = 7.2%. and A = 3.6% in TCM and D = 84.3%, H = 3.9%, P = 9.8%, and A = 1.9% in TCM-GC. No significant differences (P > 0.05) were found between culture media in the chromosomal abnormality rates. According to our results, the use of somatic cells in co-culture during embryo development did not influence the appearance of abnormal complements in the produced embryos. This would allow the use of GC as a potential complement to simplify the techniques used in the culture of bovine embryos until Day 3.

185 OVIDUCTAL CO-CULTURE CELL DID NOT REDUCE THE RATE OF CHROMOSOMAL ABNORMALITIES IN IN VITRO-PRODUCED BOVINE EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 201
Author(s):  
S. Demyda-Peyrás ◽  
P. Peral-García ◽  
M. Moreno-Millán
Keyword(s):  
Embryo Quality ◽  
Chromosomal Abnormalities ◽  
Culture Media ◽  
Score Test ◽  
Culture Cell ◽  
Bovine Embryos ◽  
Bright Field ◽  
Laboratory Techniques ◽  
Standard Procedures

One of the major causes of embryo failure on in vitro-produced cattle embryos is their increased rate of chromosomal abnormalities. It was demonstrated that they can be increased by the pre- and post-environmental conditions of culture, such as the culture media and supplementation and culture atmosphere among others. Furthermore, it was suggested that the percentage of chromosomal abnormalities detected could be used as an indirect methodology to evaluate the embryo stress during culture. The use of oviduct cells in co-culture was employed as a technique that improves the embryo quality in different culture systems in cattle, probably by modulating the variations in the embryo environment or detoxifying the culture media. For that reason, we aimed to determine if the use of an oviducal co-culture system could reduce the percentage of chromosomal abnormalities in in vitro-produced cattle embryos. Oviducal cells were obtained from abattoir oviducts, and plated and cultured in TCM-199 media following standard procedures. Cumulus-oocyte complexes were also obtained from the abattoir and matured in a standard media (TCM199, with glutamine, pyruvate, FSH, LH, and antibiotics) for 20 h (38.5°C, 5% CO2). Matured oocytes were fertilized in IVF-TALP with 1 × 106 spermatozoa/mL (previously selected through a 45–90% Percoll™ gradient) for 18 h using the same conditions. Presumptive zygotes (n = 653) were divided into 2 groups and cultured in the mSof medium for 72 h, with (SOV) or without (SOF) the addition of oviducal cells. In total, 108 embryos were successfully karyotyped following our standard laboratory techniques. Chromosomal numerical abnormalities were evaluated by direct observation at 1250× magnification in a bright-field microscope using a simple Giemsa staining. The percentage of diploid embryos (54/58 in SOV and 47/53 in SOF) and abnormal embryos (4/58 in SOV and 6/53 in SOF) were nonsignificant (P > 0.05; Z-score test for 2 population proportions). Our results suggest that the use of oviducal cells as a co-culture supplementation in in vitro-produced cattle embryos do not improve the percentage of chromosomal abnormalities detected.

scholarly journals 311EFFECT OF OOCYTE MATURATION MEDIA ON THE SPEED OF MEIOTIC PROGRESSION AND BLASTOCYST DEVELOPMENT OF BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 275
Author(s):  
D. Fischer ◽  
J. Bordignon ◽  
C. Robert ◽  
D. Betts
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Immunofluorescence Microscopy ◽  
Chromosomal Abnormalities ◽  
Culture Media ◽  
Blastocyst Formation ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
Meiotic Progression

Environment is crucial for in vitro development of gametes and embryos. The recent progression of culture media towards defined conditions brought to surface the impact of different medium supplements on oocyte and embryo development. In this work we evaluate the effect of various oocyte culture media on bovine oocyte maturation and subsequent embryo development. Bovine cumulus-oocyte complexes were recovered from slaughterhouse ovaries and matured in vitro in either TCM-199 (Gibco) or SOF (Synthetic Oviduct Fluid) media supplemented with BSA (fatty acid-free) or serum (fetal bovine serum). Oocytes from each treatment group were denuded and fixed at 18, 20, 22, 24, 26 and 28h post-maturation (p.m.). Oocyte meiotic progression was monitored in each of the groups (n=28–40 oocytes/group) by immunofluorescence microscopy of chromatin. Oocytes matured in SOF showed a slower rate of meiotic progression when compared to the other groups, with the highest percentage of oocytes reaching the MII stage by 28h p.m. (60.71% SOF-BSA, 71.43% SOF-Serum). The fastest developmental rate was observed in oocytes matured in TCM-serum (77.15% at 24h p.m.) followed by oocytes matured in TCM-BSA (74.29% at 26h p.m.). In order to evaluate the effect of nuclear maturation on chromosome segregation, chromosomal organization of MII oocytes was evaluated by immunofluorescence microscopy within each media group (n=26–31 oocytes/group) at 18, 22 and 26h p.m.. No chromosomal abnormalities were found at 18h p.m.. Both media supplemented with BSA induced lower frequencies of chromosomal abnormalities (0 to 3.23%) and (3.57 to 7.69%) for SOF and TCM, respectively, when compared to their serum-supplemented counterparts (7.14 to 11.54%) and (10 to 10.71%) for SOF and TCM, respectively at 22 and 26h p.m.. Remarkably, the maturation medium and its supplements influenced the speed of blastocyst development. For this experiment, oocytes were matured in TCM-BSA, TCM-Serum, SOF-BSA or SOF-serum, fertilized in vitro in a TALP-base media supplemented with BSA and cultured in SOF-BSA. Blastocyst development was assessed at 7, 8 and 9 days of culture. Cleavage rates were similar between the groups (84–90%), whereas development rates to blastocyst stage varied among treatment groups. Maturation in SOF-BSA induced a delay in blastocyst formation that reached its highest percentage only on day 9 of culture (30.8%); moreover, blastocyst development was carried over until Day 12. When oocytes were matured in the presence of serum, the number of blastocysts did not increase after Day 8 of culture (26.6%, TCM-serum). These results provide evidence of a severe impact of oocyte culture media on the nuclear maturation of oocytes and their subsequent embryonic development after IVF. Moreover, the difference in the rate of oocyte maturation and blastocyst formation emphasizes the necessity for reviewing and adapting current protocols to new systems such as SOF-BSA. [Research funded by NSERC and OMAF of Canada.]

scholarly journals Use of Melatonin in the In Vitro Production of Bovine Embryos

2020 ◽  
Vol 21 ◽  
Author(s):  
Alan da Silva LIRA ◽  
Ricardo de Macedo CHAVES ◽  
Felipe de Jesus MORAES JUNIOR ◽  
Sergio Henrique COSTA JUNIOR ◽  
Brenda Karine Lima do AMARAL ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Control Group ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Maturation Medium ◽  
Significant Difference ◽  
Maturation Rate ◽  
Vitro Production

ABSTRACT We aimed to assess the effects of melatonin in the in vitro production of bovine embryos. Our experiment was conducted at the Laboratório de Reprodução Animal of the Universidade Estadual do Maranhão. The cumulus-oocyte complexes (COCs) were distributed among treatments at concentrations of 0, 10-1, 10-3 and 10-5 µMol/L melatonin. Our experiment was further divided into two: the first was to assess the effect of different concentrations of melatonin (treatments) on the maturation rate of COCs, and the second was to assess the effects of melatonin treatments on the in vitro production of bovine embryos. The results from the first experiment demonstrated no significant difference between the in vitro maturation rate of the cultivated COCs in treatments with melatonin. In the second experiment, however, melatonin treatments yielded statistically higher cleavage, morula and blastocyst rates in the 10-5 µM group (52.9%, 52.9%, and 35.3%, respectively), and lower rates in the 10-1 µM group (19.5%, 19.5% and 7.8%, respectively), compared to the others. The control group (no melatonin) and the 10-3 µM group showed similar results. We concluded that supplementation of melatonin in the in vitro maturation medium resulted in no improvement in the oocyte maturation rate, but in the in vitro production of embryos at different concentrations, the 10-5 µM group displayed better results, but with no improvement in the variables (P < 0.05).

191 IN VITRO CULTURE DURING OOCYTE MATURATION AND FERTILIZATION INFLUENCES THE TRANSCRIPTOME PROFILES OF BOVINE BLASTOCYSTS

2015 ◽  
Vol 27 (1) ◽  
pp. 186
Author(s):  
A. Gad ◽  
U. Besenfelder ◽  
V. Havlicek ◽  
M. Hölker ◽  
F. Rings ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Oocyte Maturation ◽  
Microarray Data ◽  
In Vitro Maturation ◽  
Culture Conditions ◽  
Control Group ◽  
Bovine Embryos ◽  
Vitro Fertilization

Early embryonic development, the period from oocyte maturation until blastocyst formation, is the most critical period of mammalian development. It is well known that in vitro maturation, fertilization, and culture of bovine embryos is highly affected by culture conditions. However, the stage-specific effect of culture environment is poorly understood. Therefore, we aimed to examine the effect of in vitro culture conditions during oocyte maturation and fertilization on the transcriptome profile of the resulting blastocysts. Bovine oocytes were matured in vitro and then either directly transferred to synchronized recipients, fertilized, and cultured in vivo (Vitro_M), or transferred after in vitro fertilization (Vitro_F), or at zygote stage (Vitro_Z) and blastocysts were collected at Day 7 by uterine flushing. For in vivo or in vitro fertilization, the same frozen-thawed commercial bull semen has been used. Complete in vitro (IVP) and in vivo produced blastocysts were used as controls. Gene expression patterns between each blastocyst group and in vivo produced blastocyst group were compared using EmbryoGENE's bovine microarray (EmbryoGENE, Québec, QC, Canada) over six replicates of each group (10 blastocyst/replicate). Microarray data were statistically analysed using the Linear Models for Microarray Data Analysis (LIMMA) package under the R program (The R Project for Statistical Computing, Vienna, Austria). Results showed that, the longer the embryos spent under in vitro conditions, the higher was the number of differentially expressed genes (DEG, fold-change = 2 with adjusted P-value = 0.05) compared with in vivo control group. The Vitro_M group showed the lowest number of DEG (149); in contrast IVP group represented 841, DEG, respectively compared to in vivo control group. Ontological classification of DEG showed that lipid metabolism was the most significant function influenced by in vitro maturation conditions. More than 55% of DEG in the Vitro_M group were involved in the lipid metabolism process and most of them showed down-regulation compared to in vivo control group. On the other hand, Vitro_F and Vitro_Z groups showed nearly similar numbers of DEG (584 and 532, respectively) and the majority of these genes in both groups were involved in cell-death- and cell-cycle-related functions. Pathway analysis revealed that retinoic acid receptor activation pathways were the common ones in the Vitro_M and Vitro_F groups. However, different signalling pathways were commonly dominant in the Vitro_F and Vitro_Z groups. This study provides the transcriptome elasticity of bovine embryos exposed to different environments during maturation, fertilization, and culture periods of development.

292 EVALUATION OF THREE PROTEIN SOURCES FOR THE IN VITRO PRODUCTION OF BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 302
Author(s):  
E. A. Ordoñez-Leon ◽  
G. Cancino ◽  
J. Hernandez-Ceron ◽  
J. A. Medrano ◽  
Y. C. Ducolomb ◽  
...  
Keyword(s):  
Embryo Development ◽  
Culture Media ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Embryo Production ◽  
Serum Free ◽  
Serum Replacement ◽  
Knockout Serum Replacement

Bovine embryo development in vitro can be affected by many factors, including protein source, which can cause embryo development failure. The use of in vitro culture media supplemented with serum-free compounds could allow a better understanding of embryo requirements during the preimplantation stages by eliminating a highly variable and undefined compound such as serum. The objective of this study was to evaluate the effect of 3 different protein supplements used during IVM, IVF, and IVC on embryo production. Ovaries were collected from slaughtered cows and then aspirated to obtain oocytes for in vitro embryo production procedures. A total of 2056 oocytes were used, from which 685 were processed with maturation medium supplemented with 10% serum replacement (SR) (Gibco Knockout Serum Replacement, Invitrogen, Carlsbad, CA, USA), a defined serum-free formulation (TCM-199 + SR), fertilization medium with SR (TALP + SR), and culture medium with SR (SOF + SR). These were compared with 675 and 696 oocytes processed with the same IVM, IVF, and IVC media, but supplemented with 10% FCS or 10% heat-inactivated estrous cow serum (ECS), respectively. Data obtained from the variables studied were processed by analysis of variance and means were compared by Tukey’s test. The percentages of embryos produced with FCS (52.4%) and ECS (52.7%) were significantly higher compared with the percentage obtained with SR (41.5%) (P < 0.05). The percentages of morulae were similar in the groups supplemented with FCS (36.5%) and SR (36.7%), but significantly higher than the percentage in the ECS group (26.9%) (P < 0.05). For blastocysts, the percentages of embryos developed with FCS (35.2%) and ECS (35.6%) were significantly higher than that obtained with SR (29.2%) (P < 0.05). When evaluating expanded blastocysts, the percentage obtained in the FCS (45.9%) group was significantly higher than that in the ECS group (33.2%), and this was significantly higher than that obtained in SR (21%), with all these differences being significant (P < 0.05). It is concluded that it is possible to produce bovine embryos in vitro using FCS, ECS, or SR as supplements in IVM, IVF, and IVC media. Significant differences were found in different embryo stages, with the highest proportion of embryos developing with the addition of FCS, whereas supplementation with SR only improved the production of morulae. We thank Consejo Nacional de Ciencia y Tecnologia (CONACYT-Mexico) for the graduate student’s scholarship.

Effect of culture media on porcine embryos produced by in vitro fertilization or parthenogenetic activation after oocyte maturation with cycloheximide

Zygote ◽  
2011 ◽  
Vol 19 (4) ◽  
pp. 331-337 ◽  
Author(s):  
Mariana Groke Marques ◽  
Anibal Ballarotti Nascimento ◽  
Renato Pereira da Costa Gerger ◽  
José Sergio de Arruda Gonçalves ◽  
Ana Rita de Sousa Coutinho ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Culture Media ◽  
Cell Number ◽  
Mature Stage ◽  
Control Group ◽  
High Quality ◽  
Vitro Fertilization ◽  
Parthenogenetic Activation ◽  
Effect Of Treatment

SummaryThis study evaluated the effects of reversible meiotic inhibition and different culture media (PZM3 or NCSU23) on production of porcine embryos by either in vitro fertilization (IVF) or parthenogenetic activation (PA). Oocytes from abattoir-derived ovaries were allocated into two groups for maturation: CHX (5 μg/ml cycloheximide for 10 h) or Control (no CHX). The percentage of metaphase II (MII) oocytes was determined at 36, 40 or 44 h of in vitro maturation. For IVF and PA, denuded oocytes were fertilized with purified sperm for 6 h or activated by electric stimuli. Zygotes were then subdivided into two culture groups: NCSU23 or PZM3. No effect of treatment with CHX and culture media was observed on cleavage (D3) and blastocyst (D7) rates in IVF and PA groups. There are no differences of quality or development rates between IVF-derived embryos cultured in NCSU23 or PZM3. However, we observed high quality PA embryos in PZM3 compared with NCSU23. Maturation arrest with CHX decreased the average blastocyst cell number in IVF while it was increased in PA embryos. As older oocytes are more effectively activated, CHX– blocked oocytes reached the mature stage faster than the control group. In conclusion, the CHX treatment for 10 h, followed by oocyte maturation for 40 h, is an efficient protocol to produce high quality parthenote embryos, especially when they are cultured in PZM3. However, this protocol is not satisfactory for IVF embryos production. In this case, a shorter maturation period could provide better embryo quality.

Corrigendum to: 131 CHROMOSOMAL ABNORMALITIES IN IN VITRO-PRODUCED 4-DAY-OLD CATTLE EMBRYOS: INFLUENCE OF THE OOCYTE MATURATION MEDIA

2015 ◽  
Vol 27 (1) ◽  
pp. 157
Author(s):  
S. Demyda Peyrás ◽  
M. Moreno Millán
Keyword(s):  
Oocyte Maturation ◽  
Chromosomal Abnormalities ◽  
Culture Media ◽  
Cleavage Rate ◽  
Physiological Factors ◽  
Follicular Aspiration ◽  
Multiple Ovulation ◽  
Early Embryos ◽  
Established Technique

Chromosomal abnormalities were related to embryo developmental failures in cattle, being highly increased in IVF-produced compared with multiple-ovulation embryo transfer-produced embryos. The origin of this difference remains unclear, but they were related to the suboptimal environmental culture laboratory conditions. We studied the influence of 3 different maturation media in the appearance of chromosomal abnormalities in early in vitro-produced embryos. A total of 562 oocytes classified as Class A were collected by follicular aspiration in slaughterhouse ovaries and matured by 20 h in 3 different culture media supplemented with 10% FCS and antibiotics as follows: TCM-199 (T, 193 oocytes); DME (D, 178 oocytes); and RPMI-1640 (R, 191 oocytes). Matured oocytes were fertilized by 16 h with 1 × 106 spz mL–1 in IVF-TALP media and cultured by 72 h in SOF media. Later, 48 h after fertilization, 562 presumptive embryos were evaluated showing a normal cleavage rate of 64.7, 55.2, and 54.9% in T, D, and R groups, respectively. After culture, only 181 early embryos (90 hpi) showing normal development were correctly karyotyped following our standard laboratory techniques. Individual blastomeres were stained with Giemsa and assessed by direct observation at ×1250 magnification in a brightfield microscope. The percentage of normal diploid embryos (D) and abnormal haploid (H), polyploid (P), or aneuploid (A) embryos were determined. Only embryos showing almost 2 different blastomeres correctly karyotyped were included in this study. Cleavage rates were statistically higher (P < 0.05) in the oocytes matured in T media in comparison with the oocytes matured in D or R media. The percentage of diploid embryos were almost equals in the 3 groups evaluated (Table 1). There was some variation when each kind of chromosomal abnormality was assessed individually, but no statistical differences were observed. These results are in consonance with our previous studies in which we demonstrated the maturation time and morphological quality are the 2 main oocyte-derived factors influencing the ploidy of early embryos. It was also demonstrated that chromosomal complements were affected, in a much lesser extent, by the maturation media supplementation. However, in the present study, the maturation media did not statistically affected embryo ploidy. However, the higher percentage of cleaved embryos using TCM-199 observed agree with fact that this maturation media is one of the most widely used in IVF procedures in cattle. Based on our data, we suggest that oocyte maturation, a well established technique in cattle, could be performed using different maturation media without expecting major differences in the embryo ploidy, and therefore, the differences observed in cleavage rate must be originated in other physiological factors. Table 1.Results

76 Tetrahydrofuran does not have Embryo Toxic Effects on In Vitro-Produced Bovine Embryos

2018 ◽  
Vol 30 (1) ◽  
pp. 176
Author(s):  
M. M. R. Chowdhury ◽  
I. Khan ◽  
A. Mesalam ◽  
K.-L. Lee ◽  
J.-Y. Hwang ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Bovine Serum ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Control Group ◽  
Bovine Embryos ◽  
Sodium Pyruvate ◽  
Fetal Bovine ◽  
The Impact

In vitro embryo developmental potentials are still suboptimal compared with in vivo potential due to the challenge of various unknown stressors that must be overcome by in vitro-cultured oocytes. To improve existing embryo developmental potentials, many chemicals have been treated in maturation media by dissolving in toxic substances such as dimethyl sulfoxide (DMSO) or other carrier molecule. The foremost effort of this study was to investigate the impact of the solvent tetrahydrofuran (THF) on the cytotoxicity of in vitro embryo production (IVP). The experiment was completed within 8 replicates. Statistical analyses were performed using SPSS version 22.0 (IBM/SPSS, Armonk, NY, USA), a one-way ANOVA followed by multiple pairwise comparisons (Tukey’s test), and Duncan’s multiple range post hoc test. The level of statistical significance was considered P < 0.05. Oocytes were cultured in vitro maturation media (IVM) followed by in vitro fertilization (IVF), in vitro culture media 1 (IVC1), and in vitro culture media 2 (IVC2). Composition of the media was as follows: IVM medium was TCM-199 supplemented with 10% (v/v) fetal bovine serum, 1 µg mL−1 oestradiol-17β, 10 µg mL−1 FSH, 0.6 mM cysteine, and 0.2 mM sodium pyruvate. The IVC1 medium consisted of CR1-aa supplemented with 44 µg mL−1 sodium pyruvate, 14.6 µg mL−1 glutamine, 10 IU mL−1 penicillin, 0.1 mg mL−1 streptomycin, 3 mg mL−1 BSA, and 310 µg mL−1 glutathione. The IVC2 medium was the same composition as IVC1 except that BSA was replaced with 10% (v/v) fetal bovine serum. The final concentration of the optimized (0.5 µM) THF in culture medium was 0.4%. When coculturing with 0.5 µM THF in the IVM stage, the cleavage rate (58.65 ± 1.90% v. 56.87 ± 1.68%) was not significantly different, but the blastocyst rate (35.21 ± 1.44% v. 28.34 ± 2.11%) was significantly higher compared with the control group. The TUNEL assay confirmed that apoptotic nuclei in THF group were significantly reduced compared with the control group (2.32 ± 0.14 v. 5.65 ± 0.12). The total cell number of trophectoderm (TE) in control and THF groups was 115.34 ± 0.98 and 132.13 ± 1.55, and that of the inner cell mass (ICM) was 29.67 ± 0.40 and 39.94 ± 0.44, respectively. However, the ICM:TE ratio in control and treated blastocysts was 1:3.34 and 1:3.9, which was not statistically significant. Immunocytochemistry analysis (using antibodies to IKBKB, NFkB, COX2, CASP9, and CASP3) demonstrated that THF supplementation significantly attenuated expression of these proteins. The quantitative recerse transcription PCR data established that relative mRNA expression level of the anti-apoptotic gene BCL2 was up-regulated, whereas that of COX2, iNOS, BAX, IKBKB, NFkB, CASP9, and CASP3 were significantly down-regulated in the THF treated group compared with the control. In conclusion, 0.5 µM THF supplement in the IVM media did not have injurious effects on in vitro-cultured bovine embryos. This work was supported by grant from the Next-Generation BiogGeen21 (No. PJ01107703), IPET (No. 315017-5 and 117029-3), Allergy free cat (Co.. Felix Pets) and BK21plus.

Influences of zinc on fertilisation and development of bovine oocytes in vitro

Zygote ◽  
1999 ◽  
Vol 7 (3) ◽  
pp. 195-201 ◽  
Author(s):  
J.L. Stephenson ◽  
B.G. Brackett
Keyword(s):  
Sperm Motility ◽  
Oocyte Maturation ◽  
Follicular Fluid ◽  
Bovine Embryos ◽  
Sperm Preparation ◽  
Morula Stage ◽  
Zinc Concentrations ◽  
Bovine Oocytes

The effects of zinc (as ZnCl2) on in vitro production of bovine embryos (IVMFC) and components of the procedure, that is in vitro oocyte maturation (IVM), fertilisation (IVF) and embryo development in culture (IVC), and the effect of added zinc on sperm motility were studied. Immature cumulus oocyte complexes (COCs) were aspirated from ovarian follicles (2-5 mm diameter) at slaughter, and matured, fertilised and cultured in chemically defined conditions. The presence of zinc (10, 100 or 1000 μg added per millilitre) throughout IVMFC inhibited fertilisation. After addition of 10 μg zinc per millilitre separately to media for IVM and IVF, fertilisation was inhibited only when zinc was present for IVM. When present for IVF, 80% of oocytes selected for IVM reached 2- to 4-cell stages by 46 h after insemination whereas 67% of control oocytes (inseminated without added zinc) cleaved. Higher zinc concentrations (100 and 1000 μg added per millilitre) for IVF inhibited fertilisation. Sperm motility was reduced with addition of 10 μg per millilitre of zinc for sperm preparation (i.e. capacitation interval). Addition of 1.0 μg zinc per millilitre to media used through IVMFC, or to the IVC medium alone, resulted in inhibition of development after 2- to 4-cell stages. When added to IVM or to both IVM and IVF media 1.0 μg/ml of zinc compromised development to the morula stage and beyond. Maturing bovine oocytes may be more sensitive to 1.0 μg ml of zinc in vitro than in vivo because a concentration of 3.0 μg/ml has been reported for bovine follicular fluid. Fertilisation was not adversely affected by 10 μg/ml of zinc; however, higher concentrations were inhibitory.

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