73 Cytokine addition does not increase developmental competence of invitro-produced bovine embryos

2020 ◽  
Vol 32 (2) ◽  
pp. 162
Author(s):  
C. M. Helland ◽  
M. Barcelo-Fimbres ◽  
L. F. Campos-Chillon
Keyword(s):  
Lipid Content ◽  
Culture Media ◽  
Nile Red ◽  
Cell Number ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Lipid Levels ◽  
Fluorescent Intensity ◽  
Mitotracker Red ◽  
Main Effects

Recently in porcine, the addition of 3 cytokines (FGF2, LIF, and IGF1) improved oocyte maturation, quadrupling the number of piglets born per oocyte collected (Yuan et al. 2017 Proc. Natl. Acad. Sci. USA 114, E5796-E5804). We hypothesised that in the bovine, addition of these cytokines to maturation (MCyt) and culture media (CCyt) would lower lipid content and increase mitochondrial activity, representing an improved developmental competence when compared with standard maturation (MCon) and culture (CCon) conditions. The experimental design was a 2 (MCon and MCyt)×2 (CCon and CCyt) factorial in 8 replicates, testing the interactions of each maturation medium with each culture medium. Invitro-produced embryos produced aspirating cumulus-oocyte complexes (COCs) from 2 to 8mm follicles of abattoir ovaries. The COCs (n=2156) were matured for 23h in MCon or MCyt media at 6% CO2 in air, fertilised with semen from one of two bulls, and cultured in CCon or CCyt media at 38.5°C, 6% CO2, 5% O2, and 89% N2. On Day 7.5 post-fertilisation, blastocyst rates were evaluated and embryos (n=4/replicate/group) were stained with 1µgmL−1 Nile Red for lipid quantification or 300 nM MitoTracker Red CMX-Rosamine for mitochondrial polarity. Images were obtained with confocal microscopy and fluorescent intensity (AFU) was measured by Image J software (National Institutes for Health) adjusted per cell number. Data were analysed by GLM using ANOVA and l.s.d. with SAS (SAS Institute Inc.). Results (Table 1) indicated similar cleavage and blastocyst rates between all groups (P>0.05). The combination of MCon×CCon resulted in higher mitochondrial activity than any other combination (P<0.05). The MCon×CCyt showed the highest lipid levels, whereas MCyt×CCyt showed the lowest lipid levels (P<0.05). Results suggest that the combination of MCyt×CCyt media produces the lowest lipid levels, whereas the MCon×CCon media lead to the highest mitochondrial activity. The addition of cytokines to both maturation and culture media maintains competence and lowers lipid content; however, it also seems to lower mitochondrial activity. Cryopreservation studies and evaluation of pregnancy rates are ongoing. Table 1.Oocyte and embryo developmental competence matured and cultured in control and cytokine-added media Treatment Oocytes (n) Cleavage (%) Blastocysts per oocyte (%) Nile Red per cell (AFU) MitoTracker per cell (AFU) Maturation main effects MCon 1000 96.8±0.4 29.9±2.7 36.1±2.1a 385.1±65.8a MCyt 1156 96.0±0.4 26.2±2.7 30.0±2.1b 209.1±65.8b Culture main effects CCon 1036 96.2±0.4 28.0±2.7 33.1±2.1 392.0±65.8a CCyt 1120 97.7±0.4 28.1±2.7 33.0±2.1 202.2±65.8b Interactions MCon×CCon 461 96.6±0.8 27.1±3.7 33.6±3.0ab 559.0±91.1a MCon×CCyt 539 95.3±0.9 29.5±2.7 38.6±3.0a 211.2±91.1b MCyt×CCon 575 95.4±0.6 27.4±2.1 32.7±3.0bc 224.9±91.1b MCyt×CCyt 581 95.6±0.9 23.2±2.9 27.4±3.0c 193.1±91.1b a-cValues with different superscript in the same column differ (P<0.05).

45 SURVIVAL OF HOLSTEIN IN VITRO-PRODUCED EMBRYOS CULTURED IN NOVEL SYNTHETIC OVIDUCTAL FLUID MEDIA (SCF1) AND DEHYDRATED PRIOR TO CRYOPRESERVATION

2017 ◽  
Vol 29 (1) ◽  
pp. 129 ◽  
Author(s):  
C. M. Owen ◽  
M. Barceló-Fimbres ◽  
J. L. Altermatt ◽  
L. F. Campos-Chillon
Keyword(s):  
Lipid Content ◽  
Nile Red ◽  
Equilibration Time ◽  
Step Size ◽  
A Cell ◽  
Blastocyst Rate ◽  
Mitotracker Red ◽  
Main Effects ◽  
Conventional Freezing

In vitro-produced (IVP) embryos experience poor cryotolerance due to metabolic changes during in vitro culture causing increased lipid accumulation and apoptosis post-thaw. We hypothesised that embryos cultured in a novel SOF for conventional freezing media (SCF1), dehydrated, and allowed longer equilibration before conventional slow freezing would increase post-thaw survival and decrease apoptosis. IVP embryos were produced in 9 replicates by oocytes (n = 3172) aspirated from abattoir ovaries, matured for 23 h, fertilized with semen from 1 of 4 bulls, and cultured in conventional SOF media or SCF1 in 38.5°C in 5% O2, 5% CO2, and 90% N2. Stage 7 blastocysts were stained with 1 µg mL−1 Nile Red for lipid content and 300 nM Mitotracker Red CMX-Rosamine for mitochondrial polarity. Remaining blastocysts were slow-frozen by 1 of 4 protocols: 2-min dehydration in 0 or 0.6 M sucrose in holding media before equilibration (10 or 20 min) in conventional freezing media (1.5 M ethylene glycol and 0.5 M sucrose in holding media). Embryos were thawed and assessed for re-expansion at 48 h and surviving embryos were stained with 4′6-diamidino-2-phenylindole (DAPI) and a TUNEL assay to determine apoptosis. Ten images per embryo were acquired by confocal microscopy using a 5-µM step size at 40× magnification. Fluorescence of Nile Red and Mitotracker was measured by IMAGE PRO software, and cells stained for TUNEL were analysed by a cell counter plug-in. Blastocyst rate, Nile Red, and Mitotracker data (Table 1) were analysed by one-way ANOVA and means separated by Tukey’s HSD. Post-thaw survival and apoptotic levels (Table 1) were analysed as a factorial 2 (SOF and SCF1) × 2 (0 and 0.6 M sucrose) × 2 (10 and 20 min), and means separated by Tukey’s HSD. No interactions occurred between factors so they were dropped from the model and only main effects are shown. Results indicate that SCF1 increased blastocyst rate, mitochondrial polarity, and post-thaw survival and decreased lipid content and post-thaw apoptosis (P < 0.01). A 20-min equilibration time decreased apoptosis (P < 0.01) and tended to increase post-thaw survival (P < 0.1), suggesting that cryotolerance is improved in embryos cultured in SCF1 and equilibrated for 20 min. Table 1.Effect of media on development, lipid content and mitochondrial polarity (top) and of media, equilibration and dehydration on post-thaw survival and apoptosis (bottom)

81 Effects of Day 5 or 6 HEPES-Buffered Transportation Culture Medium on Developmental Competence of Bovine In Vitro-Produced Embryos

2018 ◽  
Vol 30 (1) ◽  
pp. 179
Author(s):  
W. Choi ◽  
C. M. Owen ◽  
M. Barcelo-Fimbres ◽  
J. L. Altermatt ◽  
L. F. Campos-Chillon
Keyword(s):  
Lipid Content ◽  
Embryo Culture ◽  
Culture Medium ◽  
Nile Red ◽  
Developmental Competence ◽  
Fluorescent Intensity ◽  
Embryo Culture Medium ◽  
Thermo Fisher Scientific ◽  
And Control

Most in vitro-produced (IVP) bovine embryos are transferred fresh. Use of a HEPES/bicarbonate embryo culture medium for transportation would offer flexibility for embryo shipment and transfer. We hypothesized that embryos cultured for 36 (Day 6 embryos) or 60 h (Day 5 embryos) in a novel SCF1T medium (SOF for Conventional Freezing 1 supplemented with HEPES) would maintain developmental competence compared with bicarbonate-buffered medium SCF1 (control). In 5 replicates, IVP embryos were produced by aspirating cumulus–oocyte complexes (COC) from 2-to 8-mm follicles of abattoir ovaries. The COC (n = 1036) were matured for 23 h, fertilized with semen from 1 of 3 bulls, and cultured in SCF1 at 38.5°C, 5% CO2, 5% O2, and 90% N2 (Owen et al. 2017 Reprod. Fertil. Dev. 29, 129-130). Randomly, on Day 5 and 6 after fertilization, a subset of presumptive embryos were moved into 500-µL polystyrene vials containing 100 µL of SCF1T medium, covered with 300 µL of sterile mineral oil and cultured in a portable incubator (MicroQ iQ2, Scottsdale, AZ, USA) at 38.5°C for 60 and 36 h, respectively. On Day 7.5 post-fertilization, blastocyst rates were evaluated and embryos (n = 8) from each group were stained with 1 µg mL−1 Nile Red for lipid quantification, and 300 nM Mitotracker Red CMX-Rosamine (Thermo Fisher Scientific, Waltham, MA, USA) for mitochondrial polarity. Images were obtained with confocal microscopy and fluorescent intensity (AFU) was measured by Image J software (National Institutes of Health, Bethesda, MD, USA). Data were analysed by one-way ANOVA and means separated by Tukey’s HSD. Results (Table 1) indicate similar blastocyst rates and lipid content between embryos cultured for 36 to 60 h in SCF1T and control media (P > 0.05). However, mitochondrial polarity was lower in the Day 5 group (P < 0.05) compared with Day 6 and control groups. Results suggest that culturing embryos in SCF1T medium for 36 h maintains developmental competence compared with bicarbonate-buffered media and offers an alternative for shipment and transfer of IVP embryos. Studies involving evaluation of pregnancy rates of the present study are ongoing. Table 1.Effects of Day 5 or 6 SCF1T embryo culture medium on development, lipid content, and mitochondrial polarity

79 Effect of Polydatin on Development and Cryotolerance of In Vitro-Produced Bovine Embryos

2018 ◽  
Vol 30 (1) ◽  
pp. 178
Author(s):  
C. M. Owen ◽  
M. Barceló-Fimbres ◽  
J. L. Altermatt ◽  
L. F. Campos-Chillon
Keyword(s):  
Lipid Content ◽  
In Vitro Maturation ◽  
Nile Red ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Treatment Groups ◽  
Significant Difference ◽  
Blastocyst Rate ◽  
Conventional Freezing

In vitro-produced (IVP) cattle embryos have high reactive oxygen species levels resulting in poor development and cryotolerance. Polydatin, a naturally occurring antioxidant, improves embryonic metabolism when added to maturation media; however, it has not been evaluated at other stages of embryo production. We hypothesised that embryos cultured with polydatin during maturation and fertilization would have increased development and cryotolerance. Therefore, IVP embryos were produced in 8 treatment groups supplemented with 1 µM polydatin during in vitro maturation, fertilization, and culture, or a combination of the different production stages, and each assigned a letter (Table 1). Embryos were produced in 7 replicates by oocytes (n = 3320) aspirated from abattoir ovaries, matured for 23 h in TCM-199 plus 10% fetal bovine serum and gonadotropins, fertilized with semen from 1 of 3 bulls, and cultured in SCF1 (SOF for Conventional Freezing 1; Owen et al. 2017 Reprod. Fertil. Dev. 29, 129-130) in 38.5°C in 5% O2, 5% CO2, and 90% N2. Stage 7 blastocysts were stained with 1 µg mL−1 Nile Red for lipid content or 300 nM Mitotracker Red CMX-Rosamine (Thermo Fisher Scientific, Waltham, MA, USA) for mitochondrial activity. Ten images per embryo were acquired using confocal microscopy at 5 µM step size at 40× magnification, and fluorescence was measured by Image Pro software (Media Cybernetics, Rockville, MD, USA). Remaining blastocysts were slow frozen following a 20-min equilibration in conventional freezing medium (1.5 M ethylene glycol and 0.5 M sucrose in holding medium) with 1 mm l-ascorbic acid. Embryos were thawed and assessed for re-expansion at 48 h. Blastocyst rate, Nile Red, Mitotracker, and re-expansion data were analysed by one-way ANOVA and means separated by least significant difference. Results indicate that treatment B had a higher blastocyst rate than treatment H (P < 0.01), lower lipid content than all other treatment groups (P < 0.01 or 0.05), and higher level of mitochondrial polarity than treatments A, D, E, and G (P < 0.01 or 0.05), suggesting enhanced metabolic activity. Additionally, this treatment enhanced cryotolerance compared with treatment H (P < 0.01). These results suggest that adding polydatin to maturation media has the most effect on embryo developmental competence and cryotolerance. Table 1.Effect of polydatin addition during in vitro maturation (IVM), fertilization (IVF), and culture (IVC) on blastocyst rate, lipid content, Mitotracker, and cryotolerance (± SEM)

113 EFFECT OF PLANT PROTEIN ON DEVELOPMENT AND QUALITY OF CULTURED IN VITRO PORCINE ZYGOTES

2009 ◽  
Vol 21 (1) ◽  
pp. 157 ◽  
Author(s):  
B. Gajda ◽  
I. Grad ◽  
Z. Smorag
Keyword(s):  
Serum Albumin ◽  
Culture Media ◽  
Quality Criteria ◽  
Cell Number ◽  
Developmental Competence ◽  
Plant Protein ◽  
Control Group ◽  
Group 1

Basic culture media are usually supplemented with serum albumin or serum, which contain amino acids that play an important role as energy sources, osmoregulators, and pH stabilizers. However, the presence of undefined serum in culture media introduces a variation from batch to batch and increases viral or prion contamination risk. The aim of the present study was to investigate the possibility of using plant protein substitute (PP) in place of bovine serum albumin (BSA) during in vitro culture of porcine zygotes. The PP is a mixture of several plant proteins and soya lecithin prepared using a high pressure homogenization process. The experiment was done on pig zygotes obtained surgically from superovulated gilts at 24–26 h after insemination. Morphologically normal zygotes were cultured in vitro in 5% CO2 in air at 39° in NCSU-23 medium supplemented with: 0.002 g mL–1 (group 1), 0.004 g mL–1 (group 2), 0.008 g mL–1 (group 3) PP or 0.004 g mL–1 BSA (control group). Embryo quality criteria were developmental competence (cleavage, morula and blastocyst rates), total cell number per blastocyst and degree of apoptosis as assessed by TUNEL method. Results were analyzed by Chi-square test. There were no differences in cleavage rates on Day 2 between zygotes cultured in NCSU-23 medium supplemented with PP (86.0, 88.0, 84.8; group 1 to 3, respectively) and BSA (91.0%, control group). Culture with 0.008 g mL–1 PP increased morula (85.7%) and blastocyst (69.2%) production as compared with control (75.0% and 56.3%, respectively; P < 0.05) and 0.002 g mL–1 PP (79.5% and 51.8%, respectively; P < 0.05). The mean number of cells in Day 7 blastocysts cultured in NCSU-23 medium + 0.004 g mL–1 BSA was lower (P < 0.05) than in NCSU-23 + 0.004 g mL–1 PP (39.1 v. 43.7, respectively). The blastocysts cultured in NCSU-23 medium + 0.002 g mL–1 PP had higher average number of apoptotic nuclei (13.0) as compared with the control (6.5) and 0.004 g mL–1 PP (6.9). In conclusion, this study suggest the positive effect of PP on development in vitro of porcine zygotes to the morula/blastocyst stage. However, further studies are required to determine the quality of the embryos cultured with PP. This study was supported by Scientific Net of Animal Reproduction Biotechnology.

76 Effects of serum type in maturation medium on in vitro development of bovine embryos

2019 ◽  
Vol 31 (1) ◽  
pp. 163
Author(s):  
A. Mesalam ◽  
S. Zhang ◽  
K.-L. Lee ◽  
S.-H. Song ◽  
L. Xu ◽  
...  
Keyword(s):  
Lipid Content ◽  
Cumulus Cell ◽  
Cell Number ◽  
Mitochondrial Activity ◽  
Bovine Embryos ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Maturation Medium ◽  
Gap Junctional

This study investigated the effect of bovine serum albumin (BSA), charcoal:dextran stripped fetal bovine serum (CDS FBS), and heat-inactivated FBS (HI FBS) in maturation medium on their ability to support in vitro oocyte maturation, cumulus cell-oocyte gap junctional communication, and development of bovine embryos. Charcoal:dextran treatment of FBS removes lipophilic chemicals, certain steroid hormones, and certain growth factors; however, HI FBS have a lot-to-lot variation in steroid hormones level that can affect the reproducibility of experimental findings. Oocytes were cultured in TCM-199 supplemented with either 8% (w/v) BSA, 10% (v/v) CDS FBS, or 10% (v/v) HI FBS and 1µg mL−1 oestradiol-17β, 10µg mL−1 FSH, 10ng mL−1 epidermal growth factor, 0.6mM cysteine, 0.2mM sodium pyruvate, and followed by IVF, and the zygotes were cultured in SOF-BE1 medium. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, immunocytochemistry, and cryo-tolerance. The differences in embryo development between experimental groups were analysed by 1-way ANOVA. The Duncan’s multiple range tests were used to test the differences between the treatments. The level of statistical significance was set at P&lt;0.05. We have shown that CDS FBS significantly improved (P&lt;0.05) the percentage of MII oocytes compared with that in the BSA supplemented group (77.2±1.0% v. 69.3%±2.3%, respectively). Moreover, CDS FBS had a higher significant (P&lt;0.05) effect on the rate of blastocyst formation compared with HI FBS and BSA (45.2±0.7% v. 37.4±1.5% and 31.1±3.9%, respectively; 6 replicates were performed). Culture of oocytes with CDS FBS increased (P&lt;0.05) the expression of gap junction proteins, CX37 and CX43, at both transcriptional and translation levels as determined by quantitative RT-PCR and immunofluorescence analysis, respectively. We also found that CDS FBS significantly increased total cell number and decreased the apoptotic index in Day-8 blastocysts compared with the BSA group. Furthermore, the beneficial effects of CDS FBS on embryos were associated with significantly reduced intracellular lipid content and increased mitochondrial activity in both oocytes and blastocysts as identified by Nile red and MitoTracker Green staining, respectively. Taken together, these data suggest that supplementation of maturation medium with CDS FBS, as an alternative to HI FBS, affected cumulus cell-oocyte gap junctional communication, and subsequently improved in vitro developmental competence of bovine oocytes and embryos. Research was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agri-Bio industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant numbers: 117029-3 and 315017-5).

scholarly journals High revivability of vitrified–warmed bovine mature oocytes after recovery culture with α-tocopherol

Reproduction ◽  
2015 ◽  
Vol 149 (4) ◽  
pp. 347-355 ◽  
Author(s):  
Ikuko Yashiro ◽  
Miho Tagiri ◽  
Hayato Ogawa ◽  
Kazuya Tashima ◽  
Seiji Takashima ◽  
...  
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Cortical Granules ◽  
Total Cell Number ◽  
Recovery Culture ◽  
Bovine Oocytes

The objective of this study was to investigate whether developmental competence of vitrified–warmed bovine oocytes can be improved by antioxidant treatment during recovery culture. In experiment 1, one of the two antioxidants (either l-ascorbic acid or α-tocopherol) was added as a supplement to the recovery culture medium to which postwarming oocytes were exposed for 2 h before IVF. The exposure to α-tocopherol had a positive effect on rescuing the oocytes as assessed by the blastocyst yield 8 days after the IVF (35.1–36.3% vs 19.2–25.8% in untreated postwarming oocytes). Quality of expanding blastocysts harvested on Day 8 was comparable between α-tocopherol-treated vitrification group and fresh control group in terms of total cell number and chromosomal ploidy. In experiment 2, level of reactive oxygen species, mitochondrial activity, and distribution of cortical granules in α-tocopherol-treated postwarming oocytes were assessed. No obvious differences from the control data were found in these parameters. However, the treatment with α-tocopherol increased the percentage of zygotes exhibiting normal single aster formation (90.3% vs 48.0% in untreated postwarming oocytes; 10 h post-IVF). It was concluded that α-tocopherol treatment of vitrified–warmed bovine mature oocytes during recovery culture can improve their revivability, as shown by the high blastocyst yield and the higher mean total cell number in the blastocysts.

75 Improvement of Developmental Competence of Bovine In Vitro-Produced Embryos by Using Charcoal:Dextran-Stripped Fetal Bovine Serum on In Vitro Culture Media

2018 ◽  
Vol 30 (1) ◽  
pp. 176
Author(s):  
A. Mesalam ◽  
R. Kong ◽  
B.-H. Choi ◽  
K.-L. Lee ◽  
B.-Y. Park ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Bovine Serum ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Mrna Levels ◽  
Fetal Bovine

Serum has widely been used as a main supplement to embryo in vitro culture media as it contains embryotrophic factors. Charcoal:dextran treatment of fetal bovine serum (FBS) removes lipophilic chemicals and certain steroid hormones and growth factors. The objective of this study was to investigate the effects of charcoal:dextran-stripped fetal bovine serum (CDS FBS) and heat-inactivated FBS (HI FBS) in embryo culture medium (SOF-BE1 medium supplemented with 10% of serum) on their ability to support in vitro development of bovine embryos. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, mitochondrial activity, gene expression, and cryo-tolerance. The experiment was conducted in 6 replicates (350 oocytes per group). The differences in embryo development, integrated optical intensity, and expression levels of the various genes between experimental groups were analysed by one-way ANOVA. Duncan’s multiple range tests were used to test the differences between the treatments. The level of statistical significance was set at P < 0.05. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly (P < 0.05) higher in medium containing CDS FBS than in medium containing HI FBS (42.84 ± 0.78% v. 36.85 ± 0.89%, respectively). The total number of cells per Day 8 blastocyst was not significantly different (P > 0.05) between the CDS FBS group (208.40 ± 14.77) and the HI FBS group (195.11 ± 19.15). Furthermore, the beneficial effects of CDS FBS on embryos were associated with a significantly increased mitochondrial activity, as identified by MitoTracker Green, and reduced intracellular lipid content, as identified by Nile red staining, which increased their cryo-tolerance. The post-thaw survival rate of blastocysts was significantly (P < 0.05) higher after 24 h in the CDS FBS than in the HI FBS group (85.33 ± 4.84% v. 68.67 ± 1.20%). Quantitative reverse transcription PCR showed that the mRNA levels of lipid metabolism-related genes, acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain, and the cholesterol metabolism related gene hydroxymethylglutaryl-CoA reductase were significantly increased upon culture with CDS FBS. Moreover, the mRNA levels of survival gene sirtuin 1, antioxidant gene superoxide dismutase 2, and anti-apoptotic associated gene B-cell lymphoma 2 in frozen–thawed blastocysts were significantly (P < 0.05) higher in the CDS FBS group than in the HI FBS group; however, the mRNA level of the pro-apoptotic gene BCL2-associated X protein was significantly reduced. In conclusion, these data suggest that supplementation of in vitro culture medium with CDS FBS improves in vitro bovine embryo developmental competence and the quality of blastocysts in terms of their crytolerance and gene expression. This research was supported by grant from the Next-Generation BiogGeen21 (No. PJ01107703), IPET (No. 315017-5 and 117029-3), Allergy free cat (Co.. Felix Pets), BK21plus, and KGSP.

89 EFFECT OF MEDIA, METABOLIC REGULATORS, AND STAGE OF DEVELOPMENT ON LIPID CONTENT AND MITOCHONDRIAL POLARITY OF IN VITRO-PRODUCED HOLSTEIN EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 174
Author(s):  
M. A. Roberts ◽  
L. F. Campos-Chillon ◽  
M. Barceló-Fimbres ◽  
J. L. Altermatt
Keyword(s):  
Fluorescence Intensity ◽  
Lipid Content ◽  
Lipid Accumulation ◽  
Metabolic Regulation ◽  
Nile Red ◽  
Bovine Embryo ◽  
Stage Of Development ◽  
Mitotracker Red ◽  
Metabolic Regulators

Current bovine embryo culture methods result in accumulation of lipids and reactive oxygen species, possibly due to sub-optimal metabolic regulation. These effects decrease the cryopreservation survival and implantation potential of in vitro-produced (IVP) embryos. Forskolin has been shown to decrease lipid accumulation, and vitamin K2 (Vit K2) is thought to decrease oxidative stress from in vitro conditions. The aims of this study were (1) to assess lipid content of embryos cultured with or without forskolin and Vit K2 in both continuous and sequential SOF-based medium, and (2) to examine individual and combined effects of forskolin and Vit K2 on mitochondrial polarity. For Experiment 1, a 2 × 2 × 2 factorial design was used to compare culture systems (continuous v. 3-step sequential), additives (no additive v. Vit K2 (0.5 mM at Day 3) plus forskolin (10 µM at Day 5), and blastocyst stage [6 (early) v. 7 (late)] on overall lipid content. For Experiment 2, mitochondrial polarity of stage 7 blastocysts was analysed from the following groups: no additive, Vit K2 (0.5 mM at Day 3), forskolin (10 µM at Day 5), and Vit K2 plus forskolin. IVP embryos (n = 199, Experiment 1; n = 45, Experiment 2) were produced by standard procedures and cultured at 38.5°C in 5% O2, 5% CO2, and 90% N2. For Experiment 1, embryos were stained with 1 μg mL–1 Nile Red, and two images per embryo were taken along the equatorial plane at 40× magnification. For Experiment 2, embryos were stained with 300 nM MitoTracker Red CMX-Rosamine, and 10 images per embryo were acquired by confocal microscopy with a 5-μm step size at 40× magnification. For both experiments, fluorescence intensity (FI) of each image was measured by Image PRO software with embryo controlled for and background fluorescence corrected. Data (Table 1) were analysed by ANOVA and means were compared by Tukey’s HSD. In Experiment 1, embryos cultured with forskolin and Vit K2 showed decreased lipid content in both the early and late stage (P < 0.05), with no effect from culture system (P > 0.05). In Experiment 2, forskolin and Vit K2 individually increased mitochondrial polarity (P < 0.05), but had no combined effect (P > 0.05). In conclusion, these data suggest that while a combination of forskolin and Vit K2 as media additives reduces lipid accumulation, the interaction between these metabolic regulators may negate their individual effects on mitochondrial polarity. Table 1.Fluorescence intensity of Nile Red and MitoTracker Red dyes between treatment groups

scholarly journals Lipid content, mitochondrial activity and early embryo development in oocyte collected from crossbred cows (bos taurus indicus)

2018 ◽  
Vol 25 (1) ◽  
pp. 120-131
Author(s):  
Yoeli Mendez ◽  
Nohely Parra ◽  
Francisco Baez ◽  
Robert Valeris ◽  
Patricia Villamediana
Keyword(s):  
Lipid Content ◽  
Lipid Droplets ◽  
Oocyte Quality ◽  
Early Embryo ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Vitro Fertilization ◽  
Immature Oocytes ◽  
Crossbred Cows

The objective of this research was to evaluate the effect of phenotypic predominance on lipid content, mitochondrial activity and early developmental competence as indicators of oocyte quality. Cumulus-oocyte complexes (COCs) were recovered through follicular aspiration, and underwent in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC) of presumptive zygotes. Lipid content and mitochondrial activity in immature and IVM oocytes were determined. A maturation rate of 80.6% and 69.3% was found for oocytes predominantly B. indicus and predominantly B. taurus, respectively. Total fertilization rate was 27.6%; 26.1% for predominantly B. indicus oocytes and 29% for predominantly B. taurus oocytes. A total of 55.5% and 57.5% of cleaved embryos after 48 and 72 h post-insemination (hpi) in predominantly B. indicus group were observed, respectively. As for the predominantly B. taurus group, 48.6% and 60.4% of cleaved embryos were found after 48 and 72 hpi, respectively. In both groups, immature oocytes showed a greater amount of small lipidic droplets (p <0.0001); IVM decreased the number of small lipid droplets (p < 0.0001) and increased the number of medium and large lipid droplets (p < 0.0001). Predominantly B. indicus oocytes had a greater number of small and medium-sized lipid droplets, while there were no significant differences in large lipid droplets. IVM oocytes had higher mitochondrial activity than immature oocytes group (p < 0.05) without any effect of phenotypic predominance on this parameter. Assessment of lipid content was not a predictive factor of oocyte quality in crossbred cows.

scholarly journals Brilliant Cresyl Blue stain selects largest oocytes with highest mitochondrial activity, maturation-promoting factor activity and embryo developmental competence in prepubertal sheep

Reproduction ◽  
2011 ◽  
Vol 142 (4) ◽  
pp. 517-527 ◽  
Author(s):  
Maria Gracia Catalá ◽  
Dolors Izquierdo ◽  
Svetlana Uzbekova ◽  
Roser Morató ◽  
Montserrat Roura ◽  
...  
Keyword(s):  
Cell Number ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Oocyte Diameter ◽  
Constitutive Function ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue ◽  
Maturation Promoting Factor

The aim of this study was to test the Brilliant Cresyl Blue (BCB) stain to select prepubertal sheep oocytes for in vitro blastocyst production. Oocyte diameter, mitochondrial activity, maturation-promoting factor (MPF) activity and mRNA relative expression (RE) of genes related to metabolism (ATPase Na+/K+ transporting α 1 (ATP1A1) and cytochrome c oxidase subunit 1 (COX1)) and constitutive function of the cell (cytoplasmic polyadenylation-element-binding protein (CPEB) and S100A10) were assessed. Immature oocytes were exposed to different BCB concentrations (13, 26, 39 and 52 μM) and classified according to their cytoplasm colouration as grown BCB+ (blue cytoplasm) and growing BCB− (colourless cytoplasm). Staining oocytes with 13 μM BCB during 60 min allows selection of (BCB+) the largest (123.66 μm) and most competent oocytes to develop to the blastocyst stage (21%) with a higher number of cells (69.71±6.19 s.e.m.) compared with non-stained BCB− oocytes (106.82 μm, 9% and 45.91±3.35 s.e.m. respectively). Mitochondrial activity, assessed by MitoTracker Orange CMTMRos probe, was significantly higher in BCB+ than in BCB− oocytes after in vitro maturation (3369 and 1565 AU respectively). MPF activity was assessed by CDC2 kinase activity assay showing significantly higher activity at metaphase II stage in BCB+ than in BCB− oocytes (1.479±0.09 and 1.184±0.05 optical density respectively). The genes analysed in this work, ATP1A1, COX1, CPEB and S100A10, did not show significant effect in mRNA RE between BCB selected oocytes. In conclusion, BCB stains larger and more competent oocytes to develop to the blastocyst stage with more active mitochondria and MPF activity and higher blastocyst cell number.

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