4 DEVELOPMENTAL OUTCOMES AND EFFICIENCY OF TWO CRISPR/Cas9 MICROINJECTION METHODS IN BOVINE ZYGOTES

2015 ◽  
Vol 27 (1) ◽  
pp. 94
Author(s):  
Y. S. Bogliotti ◽  
M. Vilariño ◽  
J. L. Chitwood ◽  
J. Wu ◽  
A. Mutto ◽  
...  
Keyword(s):  
Gene Disruption ◽  
Laser System ◽  
Cumulus Cells ◽  
Easy Method ◽  
Intracytoplasmic Injection ◽  
Developmental Rates ◽  
Glass Needle ◽  
Biallelic Mutations ◽  
Knockout Animals

The CRISPR/Cas9 system is a fast, effective, and easy method for gene disruption, allowing generation of knockout animals by direct zygote injection. To date there is no report on the efficiency of this microinjection system in bovine zygotes and its effects on early development. The aim of this study was to compare 2 microinjection methods on developmental rates and efficiency to induce gene disruption. Microinjection effects on embryo development were evaluated by blastocyst (BL) formation rates at Day 8 of culture and by the proportion of lysed embryos (damaged during injection); while the efficiency of CRISPR/Cas9 RNA to create targeted mutations was studied by sequencing resulting blastocysts. Three groups were evaluated: (1) noninjected (control), (2) direct intracytoplasmic injection (direct-ICI), and (3) laser-assisted ICI (laser-ICI). Direct-ICI was performed with a beveled spiked glass needle (5 μm ID; Origio, Måløv, Denmark) to pass the zona pellucida (ZP) and deliver CRISPR/Cas9 RNA as earlier described (Ross et al. 2008 BMC Dev. Biol. 8, 16). For laser-ICI, a Research Instruments (RI) Saturn 5 Active™ laser system (Research Instruments Ltd., Falmouth, United Kingdom) was used to perforate the ZP, and a blunt-end glass needle (5–6 μm ID) used to deliver CRISPR/Cas9 RNA. In both cases, cytoplasm was aspirated into the pipette to disrupt the plasma membrane and the aspirated cytoplasm and CRISPR/Cas9 injected back into the embryo. Embryos were obtained by IVF of in vitro-matured oocytes aspirated from abattoir ovaries. At 18 h post-IVF (hpf), zygotes were denuded from cumulus cells and cultured in groups of 25 in 50-μL drops of KSOM (Evolve, Zenith Biotech, Guilford, CT, USA) with 4 mg mL–1 of BSA. Zygotes were injected at 20 to 22 hpf. Four biological replicates were assessed for BL rates (258 embryos total). A 2-tailed t-test was used to evaluate statistically significant differences (P ≤ 0.05) between groups. Direct-ICI had a greater proportion of lysed embryos (29.5 ± 10.6%) compared with laser-ICI (15.6 ± 5%; P = 0.056). BL development was significantly lower on direct-ICI (15.5 ± 8%) compared with laser-ICI (31.4 ± 5.9%; P = 0.02) and control groups (32.8 ± 6.6%; P = 0.01). These results indicate that laser-ICI causes less damage and results in normal BL rates after microinjection (P = 0.24). Because laser-ICI had normal BL rates and less embryo lysis, we used this method to evaluate efficiency of the CRISPR/Cas9 system to induce genomic mutations. Twelve BL were sequenced. Analysis of the CRISPR targeted region showed that 50% of the embryos had biallelic mutations, 33% monoallelic mutations, and 17% were wild type. These results show that laser-ICI method was very efficient for injecting CRISPR/Cas9 RNA into zygotes, resulting in normal developmental rates and a high amount of mutations in BL. The authors thank Research Instruments Ltd. for providing the laser used in this study.

47 ACTIVATION WITH IONOMYCIN FOLLOWED BY DEHYDROLEUCODINE AND CYTOCHALASIN B OF CLONED BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 181
Author(s):  
N. Canel ◽  
R. Bevacqua ◽  
D. Salamone
Keyword(s):  
Cytochalasin B ◽  
Combined Treatment ◽  
Cumulus Cells ◽  
Normal Embryo ◽  
Intracytoplasmic Injection ◽  
Glass Slides ◽  
Cloned Bovine ◽  
Pronuclear Formation ◽  
Vitro Development

A combined treatment of dehydroleucodine (DhL) and cytochalasin B (CB) was previously demonstrated to induce pronuclear formation of bovine oocytes (Canel and Salamone 2008 Reprod. Fertil. Dev. 21, 214-215). The aim of this study was to evaluate the potential of DhL combined with CB to induce diploid activation of parthenogenetic embryos and to employ this treatment to assist cloning by intracytoplasmic injection of whole cumulus cells. To do that, COCs were collected from cow ovaries obtained from a slaughterhouse and in vitro-matured in TCM-199, at 39°C under 6% CO2 in air for 24 h. After removal of cumulus cells, metaphase II (MII) oocytes were treated with 5 μM ionomycin (Io) for 4 min and randomly assigned to the following activation groups: a) DhL/CB (incubation with 1 μM DhL and 5 μg mL-1 CB, for 3 h); b) DhL/long CB (treatment DhL/CB for 3 h, followed by exposure to 5 μg mL-1 CB alone, for 3 additional hours); and c) DMAP (incubation with 2 mM 6-DMAP for 3 h). In experiment 1, activated oocytes underwent IVC for 48 h and cleaved embryos were treated with 1 μg mL-1 colchicine for 6 h, fixed on glass slides, and stained with 5% vol/vol Giemsa solution to assess chromosomal complements. In experiment 2, MII oocytes were mechanically enucleated and injected with whole cumulus cells obtained from IVM COCs. After 2 h, reconstructed eggs were treated with 5 μM Io for 4 min and randomly exposed to the activation treatments a, b, or c. Parthenogenetic control groups were also included. All embryos were cultured in SOF medium and rates of cleavage, morulae, and blastocysts were evaluated on Days 2, 5, and 8 (Table 1). Results showed that DhL/long CB diploidy rates were significantly higher than those of DhL/CB and DMAP (63.8, 40. and 31.6%, respectively; Fisher’s test, P < 0.05). Both DhL treatments induced polyploidy rates lower than DMAP (5.2, 10.6, and 31.6%, respectively; P < 0.05). Finally, Io followed by DhL/CB or DhL/long CB was able to induce cloned blastocyst rates not statistically different from Io plus DMAP (P > 0.05), but presumably with a higher degree of normal embryo ploidy. Table 1.In vitro development of bovine cloned embryos activated with DhL and CB

33 Improved Dog Cloning Efficiency Using Post-Activation with Ro-3306, a Cdk1 Inhibitor

2018 ◽  
Vol 30 (1) ◽  
pp. 156
Author(s):  
M. J. Kim ◽  
H. J. Oh ◽  
E. M. N. Setyawan ◽  
S. H. Lee ◽  
B. C. Lee
Keyword(s):  
Calcium Ionophore ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
Serum Progesterone ◽  
Significance Level ◽  
Chi Squared ◽  
Pregnancy And Delivery ◽  
Developmental Rates

Inactivation of maturation promoting factor requires proteolytic destruction of cyclin B that results in the loss of cyclin-dependent kinase 1 (Cdk1) activity and exit from metaphase. The aim of this study was to investigate that treatment of Ro-3306, a Cdk1 inhibitor, during post-activation could increase the development of somatic cell nuclear transfer (SCNT) embryos in dogs. Mixed breed female dogs aged at 1 to 5 years and weighing 20 to 35 kg were used in this study (approval number: SNU-160602-14-1). Canine cumulus–oocyte complexes were collected surgically by flushing oviducts with HEPES-buffered TCM-199 medium ~72 h after ovulation, which was determined by serum progesterone concentration. After removal of cumulus cells from oocytes by repeated pipetting in hyaluronidase, matured oocytes were selected for the following experiment. In experiment I, oocytes were activated with (1) 10 μM calcium ionophore and then post-activated with 1.9 mM DMAP (control); (2) DMAP along with 10 μM Ro-3306 (10 μM group); or (3) DMAP along with 50 μM Ro-3306 (50 μM group). Parthenotes were cultured in the synthetic oviducal fluid (SOF) medium after post-activation, and in vitro development was evaluated at 48 h (2-4 cell) and 72 h (6-8 cell). In experiment II, SCNT embryos were produced after oocyte enucleation, donor cell injection, fusion, and activation. Only fused cytoplasts were activated with (1) 1.9 mM DMAP (control) or (2) DMAP along with 50 μM Ro-3306 (50 μM group) and transferred to the oviducts of recipients. The day of embryo transfer was regarded as Day 0. Pregnancy diagnosis was performed by ultrasonography after Day 28 and cloned puppies were delivered Day 58 to 60. Embryo developmental rates in experiment I and II were analysed by one-way ANOVA and t-test, respectively, and pregnancy and delivery rate were analysed by chi-squared test using Graph Prism software (GraphPad, San Diego, CA, USA). The significance level was P < 0.05. Results in experiment I showed that cleavage rate of parthenogenetic embryos in the 50 μM group (89.3 ± 6.8%) was significantly higher than that of 10 μM group or control (50.8 ± 9.9% and 55.4 ± 18.8%, respectively). However, embryonic development to 4 cells and 6-8 cells was not different between treatments. In experiment II, pregnancy rates of recipients receiving embryos in 50 μM group (3/5, 60.0%) were significantly higher than that of control (2/6, 33.3%), but the number of healthy cloned puppies delivered in the 50 µM group (n = 6) versus the control (n = 2) was not different. In conclusion, post-activation with 50 μM Ro-3306 may enhance nuclear reprogramming of dog cloned embryos. This study was supported by RDA (#PJ010928032017), Korea IPET (#316002-05-2-SB010), Research Institute for Veterinary Science, Natural Balance Korea and the BK21 plus program.

303 EFFECT OF SEMEN BATCHES AFTER SEXING ON PRODUCTION EFFICIENCY OF BOVINE EMBRYOS IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 307
Author(s):  
T. L. G. Torregrossa ◽  
M. B. Fernandes ◽  
R. B. Prado ◽  
R. A. Vila ◽  
F. P. Elias ◽  
...  
Keyword(s):  
Production System ◽  
Production Efficiency ◽  
Animal Health ◽  
Personal Communication ◽  
Cumulus Cells ◽  
In Vitro Production ◽  
Significance Level ◽  
Sexed Semen ◽  
Developmental Rates

Within an in vitro production system, bulls differ with respect to their semen potential in generating embryos when the variables of maternal effects are minimized (Marquant-le-Guienne and Humblot 1992 Ann. Zootech. 41, 361-370). We have tested the hypothesis that even with this variation among bulls, there is also an intra-bull variation among frozen sexed semen batches (Sexing Technologies, Navasota, TX, USA; personal communication) when used with IVF. In an embryo commercial production system, 5058 viable oocytes obtained by ovum pick-up with ultrasound from 193 Nelore cows (Bos indicus) over a 12-month period were matured in vitro for 24 h in TCM-199 (Gibco, Life Technologies, Carlsbad, CA, USA) containing 0.5 μg mL-1 FSH (Bioniche Animal Health, Belleville, Ontario, Canada), 50 μg mL-1 LH (Intervet, Boxmeer, the Netherlands) and 10% fetal bovine serum (Gibco). Mature oocytes were inseminated in vitro for 18 h in IVF-Talp medium (BSA-FAF 6 mg mL-1; 10 ng of heparin, Sigma, France), using 3 different batches (I, II, III) of frozen-thawed sexed semen from 4 bulls (A, B, C, D), separated with a Percoll gradient (45:90; Sigma, St-Quentin Fallavier, France). Putative zygotes surrounded in cumulus cells were transferred in CR2aa medium droplets (Rosenkrans and First 1994 J. Anim. Sci. 72, 434-437) with 3 mg mL-1 BSA-FV (Sigma) for 163 h in a humidified incubator at 39°C, with an atmosphere of 5% CO2 in air. Total number of oocytes, total number of blastocysts, and embryonic developmental rates for each bull and respective batch are reported in Table 1. The chi-square test was measured with a significance level of P < 0.05 and showed that there is difference between the batches used with respect to developmental rates of blastocysts. Therefore, there is intra-bull variation in the ability to produce in vitro embryos according to the batch of frozen sexed semen. Table 1.Viable oocytes (VO), total blastocysts (TB), and embryo development rate (%E) by bull and batch used in IVF

scholarly journals 142 IN VITRO DEVELOPMENT OF BOVINE EMBRYOS CULTURED IN KSOM, CR1AA, OR KSOM/CR1aa

2005 ◽  
Vol 17 (2) ◽  
pp. 221 ◽  
Author(s):  
M.R.B. Mello ◽  
C.E. Ferguson ◽  
A.S. Lima ◽  
M.B. Wheeler
Keyword(s):  
Embryo Culture ◽  
Cumulus Cells ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Cell Stage ◽  
In Vitro Development ◽  
Significant Difference ◽  
Developmental Rates ◽  
Vitro Development

In vitro embryo culture is an important step of in vitro production of bovine embryos. It has been shown that IVF-derived bovine embryos cultured in KSOM or CR1aa have high development rates. In our laboratory, we have observed that 8-cell embryos are morphologically superior when embryos are cultured in KSOM whereas blastocysts are morphologically superior when embryos are cultured in CR1aa. Based on these observations, we hypothesized that development of IVF-derived bovine embryos can be improved by sequential use of these media (KSOM and CR1aa). The aim of this experiment was to compare the in vitro development of bovine embryos cultured in KSOM, CR1aa or KSOM/CR1aa supplemented with BSA at Day 0 and BSA and FBS at Day 3. In order to accomplish the sequential culture, fertilized oocytes where cultured in KSOM to the 8-cell stage and then transferred to CR1aa for further development. Oocytes were purchased from Bomed (Madison, WI, USA), and after 22 hours of maturation were fertilized with frozen-thawed semen for 5 hours at 39°C in 5% CO2. After fertilization, the presumptive zygotes were denuded from cumulus cells by votexing and were randomly allotted to one of 3 treatments: (1) cultured only in KSOM (n = 110), (2) cultured only in CR1aa (n = 102), and (3) cultured in KSOM in the first 3 days and then in CR1aa from Day 3 to Day 9 (n = 110). The embryo culture was carried out in 50-μL droplets of medium that were placed in an airtight modular incubator filled with 5% CO2, 5% O2 and 90% N2. The embryos were evaluated on Days 6 to 9 post insemination. All embryo developmental rates were calculated from presumptive zygotes. The Day 6 morula rates were 52%, 40%, and 47% for KSOM, CR1aa, and KSOM/CR1aa, respectively. The Day 7 blastocyst rates for KSOM (40%), CR1aa (25%), and KSOM/CR1aa (30%) were not significantly different; however, Day 9 hatched blastocyst rates were significantly higher (P < 0.05) for KSOM (22%) compared to CR1aa (9%) but not different from KSOM/CR1aa (14%). Regarding embryo quality, Day 7 transferable embryos rates (Grade 1 and Grade 2) were 35%, 25%, and 30%, respectively for KSOM, CR1aa, and KSOM/CR1aa; however, no significant difference was observed. These results indicate that IVF-derived bovine embryos can develop in KSOM, CR1aa, or KSOM/CR1aa with no significant difference among morula, blastocyst and hatched blastocyst rates. However, the combination of KSOM and CR1aa during in vitro culture did not decrease the morula and blastocyst rates.

121 DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS CULTURED IN SEQUENTIAL OR CONTINUOUS MEDIA

2008 ◽  
Vol 20 (1) ◽  
pp. 141
Author(s):  
L. S. Amorim ◽  
D. J. Walker ◽  
G. E. Seidel Jr
Keyword(s):  
Culture Media ◽  
Cumulus Cells ◽  
Similar Efficacy ◽  
Defined Medium ◽  
Bovine Embryos ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Developmental Rates ◽  
Effects Of Media

Slaughtered bovine females have different characteristics including age, nutritional status, breed, and management system, all of which may affect the results obtained in in vitro embryo production. Another key consideration is that early embryos move from the oviduct to a slightly different environment in the uterus, which has led to development of sequential embryo culture media (e.g. Lane M et al. 2003 Theriogenology 60, 407–419). However, the benefits and importance of using sequential media are not fully known. Therefore, the aim of the present study was to compare developmental rates of oocytes obtained from slaughterhouse-derived ovaries from cows or heifers after culture in sequential media (CDM-1, CDM-2) or in a continuous medium (C-CDM). The experiment was a 3 × 2 × 2 factorial design [bulls (A, B, or C), source (cows or heifers), and medium (sequential or continuous)]. Cumulus–oocyte complexes were aspirated, within 5 h of slaughter, from 3- to 8-mm ovarian follicles of cows (1482 oocytes) and fattened heifers usually fed melengesterol acetate (2818 oocytes). Embryos were produced in vitro as described by De La Torre-Sanchez et al. 2006 Reprod. Fertil. Devel. 18, 585–596, with slight modifications. Presumptive zygotes were vortexed to remove cumulus cells and cultured for 2.5 d in C-CDM (CDM supplemented with 5.0 mm L-lactate, essential and nonessential amino acids, and 0.5% FAF-BSA, or in CDM-1 (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Devel. 18, 585–596) at 39°C in a humidified incubator under 5% CO2, 5% O2, and 90% N2. Cleavage was assessed after 2.5 d; 2- to 6-cell embryos were considered as cleaved, but were not cultured further. Embryos at the 7- to 8-cell stage were cultured for an additional 4.5 d in fresh C-CDM or CDM-2. The percentage blastocysts per oocyte was assessed after 7 and 8 days of culture. Data were arcsin-transformed and evaluated by ANOVA. There was a significant interaction between bull and ovary source for both 8-cell embryos and cleavage rate (P < 0.05); however, this interaction was no longer significant for blastocysts. No other interactions were significant nor a source of ovaries. Culturing embryos in CDM-C refreshed after cleavage evaluation (continuous) or culturing embryos in CDM-1 early and CDM-2 after cleavage evaluation (sequential) resulted in similar cleavage and blastocyst rates (Table 1). We conclude that bovine embryos can be produced using a single chemically defined medium (+BSA) with similar efficacy as a system using 2 sequential media. Table 1. Effects of media on embryonic development (mean ± SE)

39 BOVINE SOMATIC CELL NUCLEAR TRANSFER USING CUMULUS - OOCYTE COMPLEXES COLLECTED FROM THE IDENTICAL INDIVIDUAL BY OVUM PICKUP

2007 ◽  
Vol 19 (1) ◽  
pp. 138 ◽  
Author(s):  
K. Hasegawa ◽  
S. Takahashi ◽  
S. Akagi ◽  
K. Takeda ◽  
K. Imai ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Cumulus Cells ◽  
Single Strand Conformation Polymorphism ◽  
Blastocyst Stage ◽  
Polymorphism Analysis ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Developmental Rates ◽  
Vitro Development

We previously produced a cloned calf by nuclear transfer (NT) using cumulus cells removed from cumulus–oocyte complexes (COCs) after IVM. If both cumulus cells and oocytes are obtained identically and individually, and can be used simultaneously for NT, the production of cloned cows will be more expedient. And the cloned offspring produced from them will not exhibit the heteroplasmic mixed mtDNAs of donor cells and recipient oocytes. In this study, we examined the developmental potential of NT embryos using cumulus–oocyte complexes (COCs) collected from cows individually by ovum pickup (OPU). The cumulus cells were removed from COCs after IVM. The cumulus cells and cumulus-free MII oocytes derived from the same cow were used as donor nuclei and recipient oocytes, respectively. NT was performed as previously described (Akagi et al. 2003 Clon Stem Cells 5, 101–108). In Experiment 1, we examined the in vitro development of NT embryos using COCs collected by OPU. The aspiration of the follicles was performed once a week consecutively for 6 weeks in 6 cows (Cows A, B, C, D, E, and F) without hormone stimulation. In Experiment 2, we examined fetal development after the transfer of NT embryos. A Japanese black cow (Cow G) was used for OPU. On Day 7, 13 NT blastocysts were transferred to 7 recipient cows. The mtDNA genotypes of the donor cow and the cloned calf were analyzed by PCR-mediated single-strand conformation polymorphism analysis as previously described (Takeda et al. 2003 Mol. Reprod. Dev. 64, 429–437). The results of Experiment 1 are summarized in Table 1. Fusion rates did not differ among individual cows. However, the developmental rates of NT embryos at the blastocyst stage varied widely among individual cows, with a range of 19 to 64%. In Experiment 2, 2 of 7 recipient cows became pregnant on Day 30. One pregnant cow aborted on Day 60, and another cow calved a healthy calf. The mtDNA genotype of the cloned calf was confirmed to be identical with that of the donor cow. These results indicate that COCs from an identical individual can be used as donor nuclei and recipient oocytes for NT in order to produce female clones with the same mtDNA as that of the donor cow. Table 1.In vitro development of NT embryos using COCs collected by OPU

Effect of ornidazole on fertility of male rats: inhibition of a glycolysis-related motility pattern and zona binding required for fertilization in vitro

Reproduction ◽  
2000 ◽  
pp. 127-135 ◽  
Author(s):  
W Bone ◽  
NG Jones ◽  
G Kamp ◽  
CH Yeung ◽  
TG Cooper
Keyword(s):  
Zona Pellucida ◽  
Glucose Utilization ◽  
Cumulus Cells ◽  
Glycolytic Enzyme ◽  
Male Rats ◽  
Fertilization In Vitro ◽  
Swimming Pattern ◽  
Principal Piece ◽  
Free Media

The effects of the male antifertility agent ornidazole on glycolysis as a prerequisite for fertilization were investigated in rats. Antifertility doses of ornidazole inhibited glycolysis within mature spermatozoa as determined from the lack of glucose utilization, reduced acidosis under anaerobic conditions and reduced glycolytic enzyme activity. As a consequence, cauda epididymidal spermatozoa from ornidazole-fed rats were unable to fertilize rat oocytes in vitro, with or without cumulus cells, which was not due to transfer of an inhibitor in epididymal fluid with the spermatozoa. Under IVF conditions, binding to the zona pellucida was reduced in spermatozoa from ornidazole-fed males and the spermatozoa did not undergo a change in swimming pattern, which was observed in controls. The block to fertilization could be explained by the disruption of glycolysis-dependent events, since reduced binding to the zona pellucida and a lack of kinematic changes were demonstrated by control spermatozoa in glucose-free media in the presence of respiratory substrates. The importance of glycolysis for binding to, and penetration of, the zona pellucida, and hyperactivation in rats is discussed in relation to the glycolytic production of ATP in the principal piece in which local deprivation of energy may explain the reduced force of spermatozoa from ornidazole-fed males.

scholarly journals The Differential Metabolomes in Cumulus and Mural Granulosa Cells from Human Preovulatory Follicles

2021 ◽  
Author(s):  
Er-Meng Gao ◽  
Bongkoch Turathum ◽  
Ling Wang ◽  
Di Zhang ◽  
Yu-Bing Liu ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Granulosa Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Cumulus Cells ◽  
Cholesterol Transport ◽  
Vitro Fertilization ◽  
Expression Of Genes ◽  
Preovulatory Follicles ◽  
Morphological Differences

AbstractThis study evaluated the differences in metabolites between cumulus cells (CCs) and mural granulosa cells (MGCs) from human preovulatory follicles to understand the mechanism of oocyte maturation involving CCs and MGCs. CCs and MGCs were collected from women who were undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment. The differences in morphology were determined by immunofluorescence. The metabolomics of CCs and MGCs was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) followed by quantitative polymerase chain reaction (qPCR) and western blot analysis to further confirm the genes and proteins involved in oocyte maturation. CCs and MGCs were cultured for 48 h in vitro, and the medium was collected for detection of hormone levels. There were minor morphological differences between CCs and MGCs. LC-MS/MS analysis showed that there were differences in 101 metabolites between CCs and MGCs: 7 metabolites were upregulated in CCs, and 94 metabolites were upregulated in MGCs. The metabolites related to cholesterol transport and estradiol production were enriched in CCs, while metabolites related to antiapoptosis were enriched in MGCs. The expression of genes and proteins involved in cholesterol transport (ABCA1, LDLR, and SCARB1) and estradiol production (SULT2B1 and CYP19A1) was significantly higher in CCs, and the expression of genes and proteins involved in antiapoptosis (CRLS1, LPCAT3, and PLA2G4A) was significantly higher in MGCs. The level of estrogen in CCs was significantly higher than that in MGCs, while the progesterone level showed no significant differences. There are differences between the metabolomes of CCs and MGCs. These differences may be involved in the regulation of oocyte maturation.

scholarly journals 46 Effect of the addition of α-tocopherol to in vitro maturation media of bovine oocytes

2020 ◽  
Vol 98 (Supplement_2) ◽  
pp. 2-3
Author(s):  
Theisy P Acosta Pérez
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Control Group ◽  
Chi Square ◽  
Cumulus Expansion ◽  
Minimum Concentration ◽  
Maturation Rate ◽  
Chi Square Test ◽  
System Data

Abstract α-tocopherol is known to be a powerful antioxidant, in this regard, it was added to bovine oocyte in vitro maturation media to evaluate its effect on oocyte maturation. Oocytes (n = 624) aspirated from ovaries of slaughtered cows were classified by quality and divided in four categories according to cytoplasm appearance and cumulus cells layers. Oocytes were washed in TCM-199 supplemented with fetal bovine serum (FBS) and FSH, then distributed in maturation media (TCM-199 supplemented with FBS, FSH and gentamicin). Three experimental groups of α-tocopherol (50, 100 and 200 mM) and a control group without α-tocopherol were used. Maturation was carried 22 h at 38.5°C in a 5% CO2 atmosphere. Oocytes were examined to determine cumulus expansion as categorical data (expansion or no expansion), as well as cumulus expansion Index (CEI). For CEI determination oocytes were graded 0 to 4, being 0 those with null expansion and 4 those with a noticeable cell expansion, then the number of oocytes were multiplied by the grade given and a sum of the totals was obtained, the new total was divided by the total of oocytes in the group and the result obtained corresponded to the CEI of the group. Results were analyzed with Chi Square test (for maturation rates) and an ANOVA (for the CEI) using the SAS system, data are presented as mean ± standard error. There was no statistical difference between control and α-tocopherol groups (P &gt;0.05). Numerically, the control group showed a higher maturation rate (100%) and obtained a higher CEI (2.44±0.20), followed by the 50 mM group (98.16%; 2.39±0.13), the groups 200 mM (97.40%; 2.00±0.14) and 100 mM (96.25%; 2.06±0.24) were the lowest. The addition of the minimum concentration (50 mM) of α-tocopherol to the maturation media could improve maturation rates without exposing oocytes to toxic effects.

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