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)

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)

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&gt;0.05). The combination of MCon×CCon resulted in higher mitochondrial activity than any other combination (P&lt;0.05). The MCon×CCyt showed the highest lipid levels, whereas MCyt×CCyt showed the lowest lipid levels (P&lt;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&lt;0.05).

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

213 EFFECT OF NOVEL SOF MEDIUM AND L-ASCORBIC ACID DURING CRYOPRESERVATION OF IN VITRO-PRODUCED JERSEY CATTLE EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 215 ◽  
Author(s):  
A. R. Higginbotham ◽  
C. M. Owen ◽  
M. Barceló-Fimbres ◽  
L. F. Campos-Chillon
Keyword(s):  
Reactive Oxygen Species ◽  
Ascorbic Acid ◽  
Lipid Content ◽  
Nile Red ◽  
Reactive Oxygen ◽  
Molecular Probes ◽  
Oxygen Species ◽  
High Lipid ◽  
Blastocyst Rate

Jersey embryos have high lipid content and poor cryotolerance. High lipid and reactive oxygen species concentrations are associated with poor post-thaw survival and increased post-thaw apoptosis. It was hypothesized that culturing embryos in SOF-based medium (SCF1; SOF for conventional freezing will decrease lipid content, and adding l-ascorbic acid (l-AA) to freezing media will increase cryotolerance and decrease post-thaw apoptosis. A 2 × 2 factorial design was used to compare SOF v. SCF1 and additives in freezing media (control v. L-AA). In vitro-produced blastocysts were produced in 5 replicates by aspirating oocytes (n = 975) from 2 to 8 mm follicles of abattoir ovaries, maturing for 23 h, fertilizing with semen from 1 of 2 bulls, and culturing in SOF medium or SCF1 in 38.5°C in 5% O2, 5% CO2, and 90% N2. Randomly selected Day 7 blastocysts were stained with 1 µg mL−1 Nile Red for lipid content and 300 nM Mitotracker Red CMX-Rosamine (Molecular Probes Inc., Eugene, OR, USA) for mitochondrial polarity. Remaining blastocysts were placed in 0.6 M sucrose in holding media for 2 min followed by equilibration in 1.5 M ethylene glycol and 0.5 M sucrose in holding media for 10 min with 0 or 0.1 mM l-AA. Blastocysts were thawed and assessed for re-expansion at 24 and 48 h, then stained with 4′,6-diamidino-2-phenylindole and a TUNEL assay to measure apoptosis. Ten images per stained blastocyst were acquired by confocal microscopy using a 5 µM step size at 40× magnification. Image Pro software was used to measure fluorescence of Nile Red and Mitotracker, 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 1-way ANOVA and means were separated by Tukey’s HSD. Post-thaw survival and apoptotic levels (Table 1) were analysed as a factorial 2 (SOF and SCF1) by 2 (0 and 0.1 mM l-AA) and means were separated by Tukey’s HSD. Results (Table 1) indicate SCF1 increased blastocyst rate and post-thaw survival and decreased lipid content (P < 0.01) with no effect on mitochondrial polarity. Post-thaw, l-AA (Table 1) increased survival (P < 0.05) but had no effect on apoptosis. The SCF1 medium increases development and lowers lipid content, whereas l-AA may lower reactive oxygen species to increase cryotolerance. Table 1. Effect of media on development, lipid content, and mitochondrial polarity (top part), and of media and l-AA on post-thaw survival and apoptosis (lower part)

211 EFFECT OF AMNIOTIC FLUID DERIVED STEM CELL-CONDITIONED MEDIUM ON DEVELOPMENT AND POST-THAW SURVIVAL OF IN VITRO-PRODUCED HOLSTEIN EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 214
Author(s):  
K. Krautkramer ◽  
C. M. Owen ◽  
M. Barceló-Fimbres ◽  
L. F. Campos-Chillon
Keyword(s):  
Stem Cell ◽  
Amniotic Fluid ◽  
Lipid Content ◽  
Culture Conditions ◽  
Molecular Probes ◽  
Developmental Competence ◽  
Step Size ◽  
Conventional Freezing

In vitro-produced (IVP) embryos have altered metabolism with lower blastocyst rates and higher lipid accumulation compared with IVP embryos. Culturing embryos with amniotic fluid derived stem cell-conditioned media (AFS) may help mimic in vivo conditions and improve embryo development. We hypothesised that culturing embryos with AFS will improve in vitro culture conditions and developmental competence. The IVP embryos were produced in 5 replicates by aspirating oocytes (n = 1469) of 2 to 8 mm from abattoir ovaries. Oocytes were matured for 23 h, fertilized with semen from 1 of 2 bulls, and cultured in a novel SOF medium (SOF for conventional freezing media) in 38.5°C in 5% O2, 5% CO2, and 90% N2. Media was supplemented with either 0 (control), 5, or 10% AFS. Stage 7 blastocysts were stained with 1 µg mL−1 of Nile Red and 300 nM Mitotracker Red CMX-Rosamine (Molecular Probes Inc., Eugene, OR, USA) to measure lipid content and mitochondrial polarity, respectively. Ten images per stained embryo were acquired by confocal microscopy using a 5 µM step size at 40× magnification. Remaining blastocysts were slow frozen using 2 min of dehydration in 0.6 M sucrose before equilibration for 10 min in a conventional freezing medium (1.5 M ethylene glycol and 0.5 M sucrose). Embryos were thawed and re-expansion was assessed at 24 and 48 h. Data were analysed by general linear model using a one-way ANOVA with means separated by Tukey’s HSD. Results (Table 1) indicated no difference in development, lipid content, mitochondrial polarity, or post-thaw survival (P > 0.01), suggesting that growth factors present in AFS did not improve culture conditions. Table 1. Main effect of AFS on embryo developmental competence and post-thaw survival rates

161 LIPID CONTENT, RESISTANCE TO CRYOPRESERVATION, AND SEX RATIO OF IN VITRO BOVINE BLASTOCYSTS PRODUCED IN A SERUM-FREE SYSTEM

2006 ◽  
Vol 18 (2) ◽  
pp. 188
Author(s):  
F. George ◽  
C. Daniaux ◽  
G. Genicot ◽  
F. Focant ◽  
B. Verhaeghe ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Lipid Content ◽  
Culture Medium ◽  
Nile Red ◽  
Embryo Cryopreservation ◽  
Hatching Rate ◽  
Free System ◽  
Serum Free

In vitro-produced (IVP) bovine blastocysts are known to be more sensitive to cryopreservation than their in vivo counterparts. Removing serum from the culture medium decreases sanitary risk and could improve embryo resistance to cryopreservation by preventing the accumulation of intracellular lipids. Our objectives were to evaluate the lipid content, resistance to cryopreservation, and sex ratio of IVP embryos cultured in a serum-free system. Oocytes from slaughterhouse ovaries were matured in a serum-free enriched medium (Donnay et al. 2004 Reprod. Fertil. Dev. 16, 274) and cultured in 5% O2 in modified SOF supplemented with 5% FCS (FCS) or with insulin-transferrin-selenium (ITS) and 0.1 mg/mL polyvinylpyrrolidone (PVP) (ITS-PVP) or 4 mg/mL BSA (ITS-BSA) (Daniaux et al. 2005 Reprod. Fertil. Dev. 17, 217). Day 5 morulae were stained with the fluorescent dye Nile Red in order to evaluate their lipid content (Genicot et al. 2005 Theriogenology 63, 1181). Day 7 blastocysts (diameter ≥160 µm) were selected, classified according to their size, and frozen in HEPES-SOF containing 1.5 M ethylene glycol, 0.1 M sucrose, and 1.8 mg/mL wheat peptones (George et al. 2002 Reproduction 29, 51). The lipid content was significantly lower in morulae cultured in ITS-BSA compared with the two other media (320 ± 10 arbitrary fluorescence units vs. 383 ± 12 in FCS and 406 ± 10 in ITS-PVP; n = 271; ANOVA2: P < 0.01). After cryopreservation, a higher total hatching rate was found 24 h post-thawing in blastocysts cultured in ITS-BSA and for both serum-free conditions at 48 h (Table 1). In particular, embryos ≤180 µm cultured in FCS were less resistant to cryopreservation than embryos of the same size produced without serum. Expanded blastocysts cultured in ITS-BSA were sexed by PCR (Grisart et al. 1995 Theriogenology 43, 1097) and a higher proportion of male embryos was found (62.7%; n = 51). In conclusion, a complete serum-free system was set up from oocyte maturation to embryo cryopreservation that gave high quality embryos resistant to cryo-preservation. Embryos produced in ITS-BSA presented a lower lipid content, but a shift of the expanded blastocyst sex ratio toward males was observed. Table 1. Hatching rates post-thawing as a function of the blastocyst size and the culture medium

83 VITAMIN K2 SUPPLEMENTATION IMPROVES BLASTOCYST RATE BY RECOVERY OF MITOCHONDRIA IN IN VITRO-CULTURED BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 155
Author(s):  
L. Baldoceda ◽  
C. Vigneault ◽  
P. Blondin ◽  
C. Robert
Keyword(s):  
In Vitro Culture ◽  
Fluorescent Microscopy ◽  
Vitamin K2 ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Rate ◽  
Mitotracker Red

Mitochondria play an important role during early mammalian embryo development through their diverse cellular functions, in particular creating balance between production of ATP by electron transport chain and oxidative stress. Embryonic mitochondria are inherited maternally and independently of the nuclear genome. They show limited activity during the early developmental stages before embryonic genome activation. It has been shown that in vitro culture (IVC) has an adverse effect on mitochondrial function in embryos. So far several attempts have been performed to improve and rescue the impaired mitochondria. It has been shown that vitamin K2 (a membrane-bound electron carrier, similar to ubiquinone) was used to rescue mitochondrial dysfunction and resulted in more efficient ATP production in eukaryotic cells (Vos et al. 2012 Science 336, 1306–1310). Therefore, the aim of the present study was to investigate the effects of supplementation of vitamin K2 on mitochondrial activity and blastocyst rate. Cumulus–oocytes complexes (n = 687) recovered from slaughtered animals, were matured and fertilized in vitro according to our standard procedures. After fertilization, zygotes were cultured in SOF media supplemented with 10 mg mL–1 BSA. At 96 h post-fertilization, vitamin K2 was added to the culture media (n = 448 oocytes). On Day 7, treatment embryos were compared with untreated controls (n = 239 oocytes). In vitro culture was carried out at 38.5°C under 5% CO2, 7% O2, and 88% N2. Differences among groups in blastocyst yield were analysed by ANOVA. Mitochondrial activity data was analysed by unpaired 2-tailed t-tests. Results show that the vitamin K2-treated group had a significantly (P < 0.05) higher blastocyst rate (+8.6%), expanded blastocyst rate (+7.8%), as well as better morphological quality compared with the control group. Furthermore, to evaluate mitochondria activity, pools of embryos of each treatment were labelled with a specific dye for active mitochondria (Mitotracker Red). A significantly higher intensity of Mitotracker Red (P < 0.05) was observed in the vitamin K2 treatment versus control group, as measured by fluorescent microscopy. In conclusion, for the first time, our data prove that supplementation of vitamin K2 during IVC of bovine embryos increases blastocyst rates and embryo quality. Future studies will focus on gene expression to identify targets implicated in impaired mitochondrial activity in in vitro bovine embryo production.

109 EFFECTS OF LIPOLYTIC AGENTS FORSKOLIN, EPINEPHRINE AND CAFFEINE ON EMBRYONIC DEVELOPMENT AND LIPID CONTENT OF BOVINE EMBRYOS PRODUCED IN VITRO

2009 ◽  
Vol 21 (1) ◽  
pp. 154 ◽  
Author(s):  
M. Barcelo-Fimbres ◽  
G. E. Seidel
Keyword(s):  
Amino Acids ◽  
Fatty Acid ◽  
Embryonic Development ◽  
Lipid Content ◽  
Lipid Accumulation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Nile Red ◽  
Essential Amino Acids

The objective of this experiment was to evaluate lipid accumulation and embryonic development of bovine morulae treated with different chemicals. A total of 2619 slaughterhouse oocytes from heifers and mature cows were matured in CDM medium (similar to SOF) plus 0.5% fatty acid-free BSA and hormones (M-CDM) for 23 h at 38.5°C in 5% CO2 in air. Frozen–thawed sperm were centrifuged through a Percoll gradient and co-cultured with matured oocytes for 18 h in F-CDM (CDM+heparin). Zygotes were cultured at 38.5°C in 5% CO2/5% O2/90% N2 in CDM-1 with nonessential amino acids, 10 μm EDTA, 0.5% fatty acid free BSA, and 0.5 mm fructose. After 60 h, resulting 8-cell embryos were cultured 120 h in CDM-2 (CDM-1+essential amino acids and 2 mm fructose). A factorial design was used with 7 treatments, 2 ovary sources (cows v. heifers), and 3 bulls (A, B and C) replicated twice for each bull (6 replicates). At Day 2.5 embryo cleavage and 8-cell rates were evaluated, and on Day 6 a total of 755 morulae were randomly assigned to the 7 treatments (control, 2 and 8 mm caffeine, 1 and 4 μm epinephrine, and 10 and 40 μm forskolin). To quantify lipid accumulation, Day 7 blastocysts were fixed and stained with 1 μg mL–1 Nile red dye, after which a digital photograph of the equatorial part of the embryo (including the inner cell mass) was taken at 200×, and fluorescence intensity was measured with Image Pro software from 0 to 255 shades for each pixel (0 = no lipids; 255 = greatest lipid accumulation), as previously reported (Biol. Reprod. 2007 (Suppl. 1), 87–88). Data were analyzed by ANOVA. No differences in cleavage rates (75 v. 68 ± 3.6%) or eight cell rates (61 ± v. 57 ± 2.8%) were found for heifer v. cow oocytes (P > 0.1); however, blastocyst rates per oocyte and per 8-cell embryo were greater for cows than heifers (20 v. 10 ± 2.1%, and 68 v. 35 ± 3.8%, respectively; P < 0.05). Treatments: 2 and 8 mm caffeine produced fewer blastocysts per morula than 1 and 4 μm epinephrine, 10 and 40 μm forskolin and the control (39, 5 v. 54, 49, 48, 54 and 52 ± 5.8%; respectively) (P < 0.01). More lipid content was found in whole embryos and trophoblast of heifer-derived than cow blastocysts (P < 0.05), and forskolin resulted in less lipid content than control, caffeine- and epinephrine-treated morulae in whole embryos, embryonic mass and trophoblasts (P < 0.05; Table 1). In conclusion, mature cows were a better source of oocytes than feedlot heifers for embryonic development. High doses of caffeine were detrimental to embryos, and the addition of the lypolitic agent forskolin reduced lipid content relative to control, caffeine and epinephrine-treated embryos. Table 1.Main effect treatment means of lipid content (arbitrary fluorescence units)

175 EFFECT OF ACETYL-CoA CARBOXYLASE (ACC) INHIBITOR ON THE LIPID CONTENT AND NUCLEAR MATURATION OF CANINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 202 ◽  
Author(s):  
J. McGill ◽  
G. Reddy ◽  
L. Simon ◽  
G. Wirtu
Keyword(s):  
Lipid Content ◽  
In Vitro Maturation ◽  
Animal Health ◽  
Fluorescent Microscopy ◽  
Cumulus Cell ◽  
Nile Red ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Nuclear Maturation

Compared with other domestic species, embryo technologies are least developed for the dog. This is mainly due to difficulties in producing mature oocytes in vitro. Canine oocytes contain exceptionally high amounts of lipid. High lipid content increases the chilling sensitivity of oocytes and embryos. Mechanical and chemical reductions of the lipid content have been used to improve the cryotolerance of oocytes. Additionally, chemical stimulation of lipid catabolism improved oocyte in vitro maturation (IVM) rates in other species (You et al. 2012 Theriogenology 78, 235–543). Acetyl-CoA carboxylase (ACC) is the rate-limiting enzyme in de novo lipogenesis and its expression has been reported in oocytes and embryos. In somatic cells, inhibition of ACC reduces lipogenesis and enhances β-oxidation. Our hypothesis is that treatment of oocytes with an inhibitor of ACC (CP640186, Pfizer Animal Health, New York, NY, USA) reduces lipid content and improves IVM rate of oocytes. Ovaries were collected from a spay clinic and sliced in HEPES-buffered TCM-199 to recover oocytes. In vitro maturation was conducted at 38.5°C, 5% CO2, and high humidity in TCM-199 supplemented with 1% fetal bovine serum, glutamine, sodium pyruvate, β-mercaptoethanol, oestradiol, epidermal growth factor, and antimicrobial agents (Songsasen et al. Mol. Reprod. Dev. 79, 186–196). During the first 19 to 21 h, the IVM media contained 4 concentrations of the inhibitor (0+DMSO, 0.02, 0.1, and 0.5 μM, designated as treatments 1, 2, 3, and 4, respectively) and then oocytes were transferred to a medium without the inhibitor and cultured for an additional 27 to 29 h. At the end of culture (total of 48 h), oocytes were denuded of cumulus layers, washed, fixed, and stained with Nile red (lipid) and Hoechst-33342 (chromatin), and then mounted on a microscope slide. Lipid content and chromatin status were evaluated using fluorescent microscopy (TRITC and DAPI filters, respectively). The relative lipid content was measured by the corrected total cell fluorescence (CTCF) using ImageJ software (http://rsbweb.nih.gov/ij/). Data on CTCF and proportions of chromatin status of oocytes were analysed using one-way ANOVA (SigmaPlot 11.0). The mean CTCF for each treatment was 5.5 × 109 (n = 51, 5.2 × 109 (n = 44), 4.5 × 109 (n = 31), and 4.8 × 109 (n = 34), respectively (P = 0.3; 4 replicates). At the highest dose, the agent induced relatively more cumulus cell layer expansion but inhibited their attachment to the dish; the latter effect was reversible because cumulus cells attached and proliferated after washing the oocytes of the agent. Metaphase II was rare (≤3.1%); however, the proportion of oocytes developing to ≥GVBD stage (Trt 1 14%, n = 37; Trt 2 41%, n = 56; Trt 3 5%, n = 22; Trt 4 11%, n = 43) was affected by treatments. Our preliminary data indicate that a low concentration of ACC inhibitor has a positive effect on the nuclear maturation of canine oocytes but the effect on lipid content as estimated by using Nile red fluorescence intensity appears to be minimal.

131 Fatty acid binding protein inhibition as a strategy to reduce the lipid content of in vitro-produced bovine embryos

2019 ◽  
Vol 31 (1) ◽  
pp. 191
Author(s):  
L. H. Aguiar ◽  
A. C. Denicol
Keyword(s):  
Fatty Acid ◽  
Fluorescence Intensity ◽  
Lipid Content ◽  
Fatty Acid Binding Protein ◽  
Nile Red ◽  
Staining Intensity ◽  
Blastocyst Development ◽  
Fatty Acid Binding ◽  
Acid Binding Protein

Lipid accumulation decreases cryopreservation survival of in vitro-produced embryos, reducing pregnancy rate after embryo transfer. Fatty acid binding protein 3 (FABP3) plays a role in lipid transport from cumulus cells to the oocyte during maturation. Blocking this transport could reduce lipid content in the oocyte and embryo and increase cryopreservation survival. This preliminary study aimed to test the effect of α-truxillic acid (FABP-I), a chemical molecule that inhibits FABP3/5 action by receptor competition, on lipid content of matured oocytes and blastocysts after culture. Slaughterhouse-derived cumulus-oocyte complexes were matured with 0 (control), 10, 50, 100 and 500nM FABP-I for 22h. In Experiment 1, 346 oocytes in 3 replicates were fixed following maturation and stained with 1μg mL−1 Nile Red to evaluate total lipid content; maturation was assessed by nuclear staining with 10μg mL−1 Hoechst 33342[ACD1]. In Experiment 2, 876 cumulus-oocyte complexes in 5 replicates were matured for 22h under the same concentrations of FABP-I, then fertilized for 18h and cultured for 7 days. Cleavage and blastocyst development were evaluated on Day 2 and 7, respectively. Blastocysts were fixed at Day 7 and stained with Nile Red. Fluorescence intensity was measured in arbitrary units using ImageJ (NIH), and data was analysed using GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA). In Experiment 1, maturation rate did not differ among treatments (70.2±8.7; P=0.7). There were significant effects of treatment, replicate and interaction of treatment by replicate on fluorescence intensity. Compared with control (23.6±0.6), intensity was lowest in oocytes matured with 500nM FABP-I (21.2±0.6; P&lt;0.01) and highest in the 10nM group (26.5±0.6; P&lt;0.01). Staining intensity tended to decrease in the 100nM group (22.1±0.6; P=0.09) and was not different in the 50nM group (24.0±0.7; P=0.6). In Experiment 2, cleavage rate (75.8±2.9; P=0.3) did not differ and blastocyst development tended to be different among treatments (P=0.06). Compared with the control (33.3±4.8), the 500nM group had lower development (17.0±4.8; P&lt;0.03); 10 and 50nM groups had numerically lower (24.7 and 24.0±4.8) and the 100nM group had the highest development rate (37.3±4.8), although either was significant. Treatment tended to affect fluorescence intensity of blastocysts (P=0.07; n=209), and there were significant effects of replicate and interaction between replicate and treatment. Compared with the control (11.7±1.3), fluorescence intensity was lower in the 50nM group (6.8±1.3; P&lt;0.01), whereas 10nM had a tendency for lower intensity (8.3±1.2; P=0.06). Groups 100 and 500nM were not significantly different from controls (9.4±0.9 and 10.7±1.5, respectively). In conclusion, addition of FABP-I up to 500nM did not affect maturation or embryo cleavage but altered blastocyst development. Exposure to 50nM reduced staining intensity in blastocysts without significant decrease in development, whereas 100nM resulted in numerically lower oocyte staining intensity and higher blastocyst development. Future experiments will evaluate cryopreservation survival of embryos treated with FABP-I, and embryo transfer.

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