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.

43 WARMING TEMPERATURE AFFECTS THE VIABILITY AND MEIOTIC COMPETENCE OF IMMATURE PORCINE OOCYTES VITRIFIED IN A CHEMICALLY DEFINED SOLUTION

2014 ◽  
Vol 26 (1) ◽  
pp. 135
Author(s):  
D. Takahashi ◽  
H. Funahashi
Keyword(s):  
In Vitro Maturation ◽  
Fluorescent Microscopy ◽  
Dibutyryl Cyclic Amp ◽  
Nuclear Maturation ◽  
Porcine Oocyte ◽  
Different Temperatures ◽  
Maturation Rate ◽  
Post Hoc ◽  
Meiotic Competence

The aim of this study was to examine the viability and meiotic competence of porcine oocytes when immature porcine cumulus-oocyte complexes (COC) were pretreated for vitrification at different temperatures (25 and 39°C), vitrified in a chemically defined solution, and warmed at different temperatures (39 and 60°C). Cumulus-oocyte complexes were aspirated from middle-size follicles (3–6 mm in diameter) of abattoir-derived porcine ovaries. After collection, the COC were pretreated with cryoprotectants at different temperatures (25 and 39°C) and vitrified in a serum-free chemically defined solution containing 0.6 mg mL–1 of hydroxypropyl cellulose, basically according to a commercial protocol (Cryotop, Kitazato BioPharma Co. Ltd., Fuji, Japan). The vitrified COC were warmed in 1 M trehalose solution at 39 for 60 s or at 60°C for 30 s. The COC were cultured for in vitro maturation (IVM) in modified porcine oocyte medium (POM) supplemented with 50 μM β-mercaptoethanol, 10 IU mL–1 of eCG, 10 IU mL–1 of hCG, and 1 mM dibutyryl cyclic AMP (dbcAMP) for 20 h and then in the fresh medium without hormonal supplements and dbcAMP for another 24 h. Viability of COC was evaluated under fluorescent microscopy after stain with fluorescein diacetate and propidium iodide. Nuclear maturation of the oocytes was evaluated after 44 h of IVM. Statistical analyses of results from 5 replicated trials were performed by ANOVA with a Bonferroni/Dunn post-hoc test (significance, P < 0.05). Although viabilities of vitrified oocytes after 44 h of IVM [6.0% (9/149) to 37.8% (59/155)] were significantly lower than fresh controls [98.8% (158/160)], the viabilities of vitrified oocytes warmed at 60°C [32.0% (49/160) to 37.8% (59/155)] were significantly higher than those warmed at 39°C [6.0% (9/149) to 10.0% (16/160)]. Maturation rates in vitrified oocytes [2.7% (4/149) to 19.8% (31/155)] were also significantly lower than fresh controls [74.8% (120/160)]. Regardless of temperature during pretreatment for vitrification (25 and 39°C), maturation rate of the oocytes warmed at 60°C after vitrification [16.4% (25/154) to 19.8% (31/155)] was significantly higher than that warmed at 39°C [3.1% (5/160) to 2.7% (4/149)]. In conclusion, these results demonstrate that warming at 60°C for 30 s maintains the viability and meiotic competence of immature porcine COC.

353 EFFECTS OF CULTURE DURATION AND CUMULUS CELL REMOVAL ON CANINE OOCYTE IN VITRO MATURATION AND FERTILIZATION USING VASAL SPERM

2007 ◽  
Vol 19 (1) ◽  
pp. 292
Author(s):  
M. Ridha-Albarzanchi ◽  
J. Liu ◽  
M. Kjelland ◽  
D. Kraemer
Keyword(s):  
In Vitro Maturation ◽  
Vas Deferens ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Culture Period ◽  
Cell Contact ◽  
Nuclear Maturation ◽  
Sperm Penetration ◽  
Culture Duration

The objective of this study was to test the hypothesis that in vitro maturation (IVM) and fertilization (IVF) rates of canine oocytes could be improved by increasing culture duration or decreasing/increasing cumulus cell contact with the oocytes when using sperm retrieved from the vas deferens. The canine oocyte is ovulated at the germinal vesicle stage, and maturation of the oocyte occurs in the oviduct and requires up to five days. Therefore, an increase in the culture duration may cause an increase in oocyte nuclear maturation. Canine ovaries and testes were collected from a local clinic, placed in warm saline solution, and transported to the laboratory. Two distinct experiments were carried out, one involving IVM (M-II) after cumulus cell removal at 72 h and 96 h for nuclear maturation evaluation, and the second experiment the same but continued up to IVF. The oocytes were recovered from the ovaries by mincing them in warm Medium-199 with Hanks salts, L-glutamine, and HEPES (GIBCO, Grand Island, NY, USA; Invitrogen Co., Carlsbad, CA, USA). Canine oocytes with a dark cytoplasm and at least 2 layers of cumulus cells were cultured in Medium-199 supplemented with Earle&apos;s salts, 2200 mg mL&minus;1 sodium bicarbonate, 25 mM HEPES, 2 mM sodium pyruvate, 5 &micro;g mL&minus;1 progesterone, 100 ng mL&minus;1 epidermal growth factor, 10 IU mL&minus;1 human chorionic gonadotropin (HCG), 5 &micro;g mL&minus;1 insulin, 0.50 mM epinephrine, 10&percnt; estrus bitch serum, 0.01 mM nonessential amino acids, and 20 &micro;g mL&minus;1 gentamicin. The oocytes were cultured for 72, 96, 120, or 144 h at 38.5&deg;C in 5&percnt; CO2 in humidified air. The cumulus cells were removed after either a 72- or 96-h culture period. For the first 48 h, the cumulus&ndash;oocyte complexes were cultured in the modified Medium-199 containing 10 IU mL&minus;1 HCG and then cultured in the same medium free of HCG. The oocytes were denuded by pipetting, stained with Hoechst 33342, and examined for nuclear maturation. ANOVA was used for statistical analysis of the data. The IVM rate (MII) was significantly higher (P &lt; 0.05) at 72 and 96 h compared to 48, 120, and 144 h (15.1&percnt; and 16.9&percnt; vs. 6&percnt;, 12.4&percnt;, and 9.1&percnt;, respectively). The removal of cumulus cells at 72 h and 96 h resulted in 17.9&percnt; (43/240) and 14.8&percnt; (35/236) IVM rate (MII), respectively (P &gt; 0.05). The sperm motility index (SMI &equals; motility percentage &times; sperm activity grade) was significantly higher in sperm retrieved from the vas deferens (vasal sperm) compared to epididymal and testicular sperm (259 vs. 95 and 19.2, respectively, P &lt; 0.05). The mature oocytes were inseminated by vasal sperm following in vitro hyperactivation with HEPES solution supplemented with 3 mg mL&minus;1 bovine serum albumin. The IVF rates of the oocytes following 72 and 96 h of maturation in vitro were 48.2&percnt; and 40&percnt;, respectively (P &gt; 0.05). Sperm penetration was significantly higher at 96 h compared to 72 h, and the number of sperm heads inside the ooplasm was 3.2 for the 72 h group vs. 4.8 for the 96 h group (P &lt; 0.05). In conclusion, increasing the IVM culture period beyond 72 h did not increase the oocyte maturation rates, and increasing the culture time to 96 h without cumulus cells present increased the rate of sperm penetration.

scholarly journals The influence of ascorbic acid on in vitro maturation of canine oocytes

2016 ◽  
Vol 73 (2) ◽  
pp. 230
Author(s):  
Anamaria Jeni Pernes ◽  
Ileana Miclea ◽  
Marius Zahan ◽  
Vasile Miclea ◽  
Delia Orlovschi ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Meiotic Maturation ◽  
Control Group ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
Oocyte Meiotic Maturation ◽  
Metaphase I

Abstract It is known that L-ascorbic acid (vitamin C) can modulate many biochemical processes intracellularly or extracellularly as antioxidant. The aim of the present study was to examine the effects of media supplementation with ascorbic acid on canine oocyte meiotic maturation, viability and the cumulus cell expansion. Various concentrations of ascorbic acid supplemented in in vitro maturation (IVM) media were tested. Canine oocyte was exposed to different levels of ascorbic acid (0, 50, 150, 250, 500, 750µM). Cumulus expansion, meiotic maturation and degeneration of oocytes were assessed 72 h after in vitro culture. As results, on the group treated with 250µM ascorbic acid was a significant difference compared to the control group on nuclear maturation in stages metaphase I (MI) and metaphase II (MII) (26.98% vs. 6.00%). The groups treated with 50, 150, 250, 500µM had an increase in stage (GVBD), and a significant decrease of degenerate-undefined oocytes compared with the control (23.31%, 18.85%, 13.41% vs 40.80). Concentration 750µM had similar effect to that in the control group. The groups treated with 50, 150, 250, 500µM had an increase in meiosis resumption(GVBD), metaphase I (MI) and metaphase II (MII) with the best result in the group treated with 250 µM ascorbic acid.

271 NUCLEAR MATURATION OF WOOD BISON (BISON BISON ATHABASCAE) CUMULUS - OOCYTE COMPLEXES

2013 ◽  
Vol 25 (1) ◽  
pp. 283
Author(s):  
M. P. Cervantes ◽  
M. Anzar ◽  
R. J. Mapletoft ◽  
J. M. Palomino ◽  
G. P. Adams
Keyword(s):  
In Vitro Maturation ◽  
Transvaginal Ultrasound ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Bison Bison ◽  
Nuclear Maturation ◽  
Wood Bison

Methods of producing wood bison embryos in vivo and in vitro are being developed in an effort to preserve the genetic diversity of this threatened species. Previous data from our laboratory suggest that oocytes collected 24 h after LH treatment had not yet achieved nuclear maturation. The objectives of this study were (1) to determine the optimal interval of time after hCG treatment required for in vivo maturation of cumulus–oocyte complexes (COC) in wood bison, and (2) to compare the maturational characteristics of COC after in vitro v. in vivo maturation. Follicular wave emergence was synchronized among bison cows (n = 25) by follicular ablation (Day –1) from May to June. Ovarian superstimulation was induced with FSH IM diluted in 5 mg mL–1 of hyaluronan (MAP-5, Bioniche, Belleville, Ontario, Canada) given on Day 0 (300 mg) and Day 2 (100 mg). Superstimulated cows were assigned randomly to 5 groups (n = 5/group): COC collected on Day 4 with no maturation (control), or matured in vitro for 24 or 30 h, or collected 24 or 30 h after treatment with 2000 IU of hCG IM on Day 4. The COC were collected by transvaginal ultrasound-guided follicle aspiration. In vitro maturation was done in TCM-199 with 5% calf serum, 5 µg mL–1 of LH, 0.5 µg mL–1 of FSH, and 0.05 µg mL–1 of gentamicin, at 38.5°C and in 5% CO2. To assess nuclear maturation, oocytes were stained with anti-lamin AC/DAPI (4′,6-diamidino-2-phenylindole). Nuclear stages were classified as germinal vesicle (GV), GV breakdown (GVBD), metaphase I (MI), or metaphase II (MII). Comparisons among groups were made by ANOVA and Fisher’s exact test (Table 1). A mean (± SEM) of 7.6 ± 0.6 COC was collected per bison; no differences were observed among groups (P = 0.37). Cumulus cell expansion was more extensive after in vivo than in vitro maturation, and the percentage of fully expanded COC was highest in the in vivo 30-h group (97%; P < 0.05). No COC were expanded in the control (0 h) group, and none reached MI. Maximal nuclear maturation was achieved in vitro by 24 h; that is, there was no difference in the proportion of MII-stage COC at 24 versus 30 h. However, between 24 and 30 h of in vivo maturation, the percentage of nuclear stages GV + GVBD decreased from 54 to 24% (P < 0.05), whereas nuclear stages MI + MII increased from 39 to 74% (P < 0.05). In conclusion, nuclear maturation occurred earlier in vitro versus in vivo, but the consequences of this difference are unknown. Although more than one-third of oocytes matured in vivo for 30 h were mature enough to permit immediate IVF, whether additional in vivo maturation time would be beneficial to fertilization rates remains to be tested. Table 1.Nuclear status of wood bison oocytes after in vitro or in vivo maturation Thanks to Bioniche Canada.

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)

scholarly journals Effect of D-Glucuronic Acid and N-acetyl-D-Glucosamine Treatment during In Vitro Maturation on Embryonic Development after Parthenogenesis and Somatic Cell Nuclear Transfer in Pigs

Animals ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 1034
Author(s):  
Joohyeong Lee ◽  
Eunhye Kim ◽  
Seon-Ung Hwang ◽  
Lian Cai ◽  
Mirae Kim ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
In Vitro Maturation ◽  
Glucuronic Acid ◽  
Cumulus Cell ◽  
Cortical Granule ◽  
Oocyte Diameter

This study aimed to examine the effects of treatment with glucuronic acid (GA) and N-acetyl-D-glucosamine (AG), which are components of hyaluronic acid (HA), during porcine oocyte in vitro maturation (IVM). We measured the diameter of the oocyte, the thickness of the perivitelline space (PVS), the reactive oxygen species (ROS) level, and the expression of cumulus cell expansion and ROS-related genes and examined the cortical granule (CG) reaction of oocytes. The addition of 0.05 mM GA and 0.05 mM AG during the first 22 h of oocyte IVM significantly increased oocyte diameter and PVS size compared with the control (non-treatment). The addition of GA and AG reduced the intra-oocyte ROS content and improved the CG of the oocyte. GA and AG treatment increased the expression of CD44 and CX43 in cumulus cells and PRDX1 and TXN2 in oocytes. In both the chemically defined and the complex medium (Medium-199 + porcine follicular fluid), oocytes derived from the GA and AG treatments presented significantly higher blastocyst rates than the control after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). In conclusion, the addition of GA and AG during IVM in pig oocytes has beneficial effects on oocyte IVM and early embryonic development after PA and SCNT.

Relative Activities of Acetyl-CoA Carboxylase and Fatty Acid Synthetase in Chick Liver: Effects of Dietary Fat

1973 ◽  
Vol 51 (7) ◽  
pp. 1029-1033 ◽  
Author(s):  
Gregory I. Liou ◽  
W. E. Donaldson
Keyword(s):  
Fatty Acid ◽  
Corn Oil ◽  
Fatty Acid Synthetase ◽  
Basal Diet ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Carboxylase Activity ◽  
Cytosol Fraction ◽  
Activity Concentrations

The specific activities of acetyl-CoA carboxylase and fatty acid synthetase were measured in the cytosol fraction of livers from chicks fed various levels of corn oil, cottonseed oil, corn-oil free fatty acids, or crude (79%) oleic acid. Activities of both enzymes were depressed by the addition of fat to a fat-free basal diet. The ratios of synthetase to carboxylase activity were greater than unity when up to 4% fat was fed, but less than unity when 8% or higher levels of fat were fed. The depressions of the activities of these enzymes appeared to be unrelated to the dietary level of linoleate. In in vitro experiments, 2 μM concentrations of palmityl-CoA or oleoyl-CoA depressed acetyl-CoA carboxylase activity. Concentrations of 20 μM of these acyl-CoA esters did not affect the activity of fatty acid synthetase.

scholarly journals P5375A default in acetyl-CoA carboxylase phosphorylation increases thrombus growth in vitro and in vivo, and in a collagen-dependent manner

2017 ◽  
Vol 38 (suppl_1) ◽  
Author(s):  
S. Lepropre ◽  
S. Kautbally ◽  
L. Bertrand ◽  
G.R. Steinberg ◽  
B.E. Kemp ◽  
...  
Keyword(s):  
Dependent Manner ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa

scholarly journals Effects of BMP15 and GDF9 on Ovine Oocytes in an In Vitro Maturation System

2021 ◽  
Author(s):  
◽  
Aanchal Singh
Keyword(s):  
Cell Death ◽  
Litter Size ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Oocyte Quality ◽  
Mature Oocyte ◽  
High Ratio ◽  
High Fecundity ◽  
Species Specific

<p>Oocyte developmental competency is the intrinsic measure of oocyte quality and the capacity for a mature oocyte to support the early stages of embryo development and implantation. Oocyte-secreted factors (OSFs), such as growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), play a pivotal role in regulating the synchrony of various complex maturation events within the cumulus-oocyte complex (COC) through the induction of paracrine and endocrine signalling. These proteins act synergistically to influence the proliferation and differentiation of granulosa cells (GCs), cumulus cell (CC) expansion, promote survival, ovulation, the attainment of developmental competency and fertility. Species-specific ratios suggest that poly-ovulatory mammals have increased fecundity due to high ratios of GDF9:BMP15, which is directly reflected in their large litter size. Interestingly, it has also been found that higher ratios of GDF9:BMP15 also increased blastocyst rate in sheep implying that these embryos develop from oocytes that are more developmentally competent.  In this study, I investigated the hypothesis that supplementing a commercial in vitro maturation (IVM) system with a high ratio of GDF9:BMP15 would increase the developmental competency sheep oocytes; a species with low-moderate litter size. To test this hypothesis, ovine oocytes were matured in a biphasic IVM system containing GDF9 and BMP15 at three divergent ratios (1:6, 1:1, 6:1). The results herein show that the 6:1 ratio resulted in higher levels of reagent transfer to the ovine oocyte through gap junctions (GJs) after 24 hours of incubation. Similarly, it was also observed that at the higher ratio, glutathione (GSH) levels were higher at 7.5 hours of incubation. The high GDF9:BMP15 ratio also facilitated the increased consumption of pyruvate by the COC consistently throughout the culture period. Importantly, the high GDF9:BMP15 ratio showed higher expression of the gene that encodes GJ (CX43) at 24 hours relative to the control. It was also demonstrated through decreased apoptotic factor (BAX:BCL2) ratios, that the addition of OSFs, regardless of ratio, protected against cell death. In summary, this study provides novel results that support the notion that a high GDF9:BMP15 ratio improves oocyte quality by delaying the timing of meiotic resumption. This subsequently improves the transport of key metabolites and antioxidants to protect against oxidative stress and cell death and aid in the completion of maturation, ultimately resulting in the increased developmental competency observed in high fecundity poly-ovulatory species.</p>

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