Developmental Competence Of Bovine Oocytes Research Articles

Physiological high temperatures alter the amino acid metabolism of bovine early antral follicles.

Heat stress reduces the developmental competence of bovine oocytes during the growth phase; however, the detailed mechanisms remain unclear. Amino acids play various critical roles in follicular development, including protein synthesis and as energy sources. We performed in vitro growth (IVG) culture of oocyte-cumulus-granulosa complexes (OCGCs) to assess the amino acid metabolism of small follicles at high temperatures. We isolated OCGCs from early antral follicles (0.5-1.0 mm) and subjected them to IVG culture for 12 days. OCGCs in the heat shock group were cultured under a temperature cycle of (38.5°C: 5 h, 39.5°C: 5 h, 40.5°C: 5 h, and 39.5°C: 9 h) to reproduce the body temperature of lactating cows under a hot environment. OCGCs in the control group were cultured at a constant temperature of 38.5°C for 24 h. Of the surviving OCGCs, those showing similar morphology and size between the groups were selected for amino acid analysis. We analyzed the free amino acids and their metabolites in the culture medium and calculated the depletion or appearance of molecular species. The depletion of three essential amino acids (isoleucine, leucine, and valine), two non-essential amino acids (aspartic acid and glycine), and ornithine was higher in the heat shock group (P < 0.05). Alanine depletion was lower in the heat shock group (P < 0.05). We concluded that heat exposure alters the amino acid metabolism of OCGCs isolated from early antral follicles, which might be involved with the diminished developmental potential of oocytes during summer.

Factors defining developmental competence of bovine oocytes collected for in vitro embryo production.

Despite the currently relatively low effectiveness of producing bovine embryos in vitro, there is a growing interest in applying this laboratory method in the field of reproduction. Many aspects of the procedure need to be improved. One of the main problems is the inferior developmental competence of in vitro matured oocytes that are collected using the ovum pick-up (OPU) method. The mechanisms of oocyte capacitation and maturation, as well as the in vivo conditions in which they grow and mature, should be carefully analyzed. A deliberate application of the identified mechanisms and beneficial factors affecting the in vitro procedures seems to be essential for achieving higher developmental competence of the oocytes that are subjected to fertilization. The results may be improved by developing and employing a laboratory maturation protocol that corresponds with appropriate preparation of donors before the OPU, an optimized hormonal treatment program, the appropriate size of ovarian follicles at the time of aspiration, and a fine-tuned coasting period.

198 Seasonal effect of in vitro prematuration with follicular fluid and follicular fluid small extracellular vesicles on bovine oocyte developmental competence

198 Seasonal effect of in vitro prematuration with follicular fluid and follicular fluid small extracellular vesicles on bovine oocyte developmental competence

Effect of the aflatoxins B1 and M1 on bovine oocyte developmental competence and embryo morphokinetics

Aflatoxins are considered as reproductive toxins for mammalian species. Here, we studied the effect of aflatoxin B1 (AFB1) and its metabolite aflatoxin M1 (AFM1) on the development and morphokinetics of bovine embryos. Cumulus oocyte complexes (COCs) were matured with AFB1 (0.032, 0.32, 3.2, 32 µM) or AFM1 (0.015, 0.15, 1.5, 15, 60 nM), then fertilized and the putative zygotes were cultured in an incubator equipped with a time-lapse system. Exposing COCs to 32 µM AFB1 or 60 nM AFM1 reduced the cleavage rate, whereas exposing them to 3.2 or 32 µM AFB1 further reduced the blastocyst formation. A delay was recorded for the first and second cleavages in a dose-dependent manner for both AFB1- and AFM1-treated oocytes. A delay was recorded in the third cleavage in the AFM1-treated group. To explore potential mechanisms, subgroups of COCs were examined for nuclear and cytoplasmic maturation (n = 225; DAPI and FITC-PNA, respectively), and mitochondrial function was examined in a stage-dependent manner. COCs were examined for their oxygen consumption rates (n = 875; Seahorse XFp analyzer) at the end of maturation, MII-stage oocytes were examined for their mitochondrial membrane potential (n = 407; JC1), and putative zygotes were examined using a fluorescent time-lapse system (n = 279; IncuCyte). Exposing COCs to AFB1 (3.2 or 32 µM) impaired oocyte nuclear and cytoplasmic maturation and increased mitochondrial membrane potential in the putative zygotes. These alterations were associated with changes in the expression of mt-ND2 (32 µM AFB1) and STAT3 (all AFM1 concentrations) genes in the blastocyst stage, suggesting a carryover effect from the oocyte to the developing embryos.

028 In-vitro study to examine the effect of the foodborne mycotoxin aflatoxin B1 on bovine oocyte developmental competence and embryo kinetics

028 In-vitro study to examine the effect of the foodborne mycotoxin aflatoxin B1 on bovine oocyte developmental competence and embryo kinetics

Downregulation of PI3K/AKT/mTOR Pathway in Juglone-Treated Bovine Oocytes.

We have previously reported that juglone, a natural compound found in Juglandaceae with a wide range of biological activities, can reduces the developmental competence of bovine oocytes. In the current study, we investigated the possible mechanisms behind the toxicity of juglone and the relationship with PI3K/AKT/mTOR signaling during the in vitro maturation (IVM) of oocytes. Results show that oocyte exposure to juglone was associated with a significant decrease in filamentous actin (F-actin) accumulation. The RT-qPCR showed downregulation of the meiosis progression indicator GSK-3A, oocyte development marker BMP15, mitochondria fusion controlling MFN1, oxidative stress-related OGG1, and histone methylation-related EZH1, EZH2, SUZ12, G9a, and SUV39H2 genes in juglone-treated oocytes. In addition, glycolysis- (PFK1 and GLUT1), ATP synthesis- (ATPase8 and ATP5F1B), and OXPHOS-specific markers (SDHA and SDHD), as well as the oocyte survival regulators (SOD2, VEGF, and MAPK1) significantly decreased upon juglone treatment. Moreover, lower expression of PI3K, AKT, and mTOR was observed at the transcriptional and/or translational level(s). The autophagy markers LC3B and beclin-1 as well as the DNA damage-specific marker 8-OxoG displayed overexpression in juglone-exposed oocytes. Taken together, our results show that administration of juglone during the IVM can reduce the quality and developmental health of bovine oocytes through downregulation of the PI3K/AKT/mTOR pathway and its downstream signaling cascades.

Developmental competence of bovine oocytes in maturation media supplemented with follicular fluid exosomes

The present study evaluated the role offollicular fluid exosomes on the developmental competence of bovine oocytes. Ovaries from slaughtered crossbred cattle were collected, all visible surface follicles aspirated, and culture-grade oocytes were subjected to further study. Exosomes were isolated from bovine follicular fluid by differential ultracentrifugation. A total of 358 oocytes selected for the study were randomly divided into two groups. Group I constituted 111 oocytes, in which normal maturation was carried. Group II constituted 247 oocytes, in which in vitro maturation (IVM) medium was supplemented with follicular fluid exosomes at 1 μL/100 μL IVM medium. Maturation was assessed after 24h of culture in a CO2 incubator maintained at 38.5°C in 95 per cent humidified atmosphere of 5 per cent CO2. Following IVM of oocytes for 24 h, in vitro fertilisation (IVF)was carried out by co-incubating with capacitated spermatozoa for 18 h and embryo culture was carried out subsequently. In group II oocytes supplemented with exosomes, a significantly higher maturation rate (p ≤0.01) (95.80 ±1.67 vs76.10 ± 0.95), fertilisation rate (53.68 ± 3.02vs37.85 ± 7.01) and cleavage rate (p ≤0.01) (43.66 ± 2.13vs(32.47 ± 5.23) was noticed compared to oocytes in group I without any supplementation.The present study established that supplementation of follicular fluid exosomescould improve the developmental competence of bovine oocytes.

Pre-maturational culture promotes the developmental competence of bovine oocytes derived from an 8-day in vitro growth culture system.

In this study, we evaluated the effects of pre-maturational culture (pre-IVM) on the developmental competence of bovine oocytes derived from an 8-day in vitro growth (IVG) culture system. IVG oocytes were subjected to 5 h pre-IVM before in vitro maturation, followed by in vitro fertilization (IVF). The proportion of oocytes that progressed to the germinal vesicle breakdown stage was similar between groups with and without pre-IVM. Although metaphase II oocytes and cleavage rates after IVF were similar regardless of pre-IVM culture, the blastocyst rate was significantly higher in the group with pre-IVM (22.5%) than without pre-IVM (11.0%, P < 0.05). In conclusion, pre-IVM culture improved the developmental competence of bovine oocytes derived from an 8-day IVG system.

97 Effect of season and oxygen tension on developmental competence of bovine oocytes

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Inhibition of Hsp90 during in vitro maturation under thermoneutral or heat shock conditions compromises the developmental competence of bovine oocytes.

Heat shock protein 90 (Hsp90) is critical for cell homeostasis but its role on bovine oocyte maturation is not well known. We investigated the importance of Hsp90 for competence of bovine oocyte using 17-(allylamino)-17-demethoxygeldanamycin (17AAG), an inhibitor of Hsp90, during in vitro maturation (IVM). Three experiments evaluated the effect of 17AAG on developmental competence of oocytes matured in vitro under thermoneutral (38.5ºC) or heat shock (HS; 41.5ºC) temperatures. The first experiment found that the blastocyst rates were lower (P < 0.05) with 2 µM 17AAG compared with the untreated control (0 µM). The abundance of HSF1 transcripts was higher in oocytes matured with 2 µM than with 0 and 1 µM 17AAG, whereas the abundance of HSP90AA1 and HSPA1A transcripts was lower (P < 0.05) with 1 and 2 µM than with 0 µM. The second experiment found that 2 µM 17AAG for 12 or 24 h during IVM decreased (P < 0.05) the blastocysts rates. In the third experiment, the association of 2 μM 17AAG with HS for 12 h during IVM resulted in lower (P < 0.05) blastocysts rates than 17AAG, HS or untreated control. In conclusion, inhibition of Hsp90 during in vitro maturation compromises further embryo development; the association of Hsp90 inhibition with HS aggravates the deleterious effect of both on oocyte developmental competence.

The effect of quercetin in the maturation media on cumulus-granulosa cells and the developmental competence of bovine oocytes

The present study was designed to investigate the effects of Quercetin on the developmental competence of bovine oocytes and cumulus-granulosa cells (CGs). Two groups of immature cumulus-oocyte complexes (COCs) were subjected to IVM with or without Quercetin. The viability, nuclear status, early and late apoptosis of oocytes and CGs were evaluated using gene expression analysis and staining methods. Embryonic development was assessed morphologically by recording Post-IVF survival, cleavage, and blastocyst rates. The proportion of oocytes reaching the MII stage was greater and the number of early-apoptotic oocytes was lower in the group matured with Quercetin compared to the Control (p < 0.05). Relative upregulation of OCT-4, IGF2R and Bcl-2, and downregulation of CHOP was seen in treated oocytes. Also, downregulation of Bax and upregulation of Glut-4 was detected in treated CGs. The treated oocytes experienced higher post-IVF survival and cleavage rates compared to the untreated group (p < 0.05); more cleaved embryos reached ≥16-cell stage and blastocyst at days 4th and 7th respectively. In addition, total blastocyst rate was significantly improved. It is concluded that supplementing maturation media with Quercetin can enhance the quality of bovine oocytes and endow them with protective potential against early apoptotic damage possibly through CHOP regulation of BCL2 gene family, triggering expression of a gene in CGs to maintain the intactness of oocyte against apoptotic signals and providing oocytes with more energy substrates. It also boosts the subsequent development of oocyte to blastocyst and improves the efficacy of bovine embryo production in vitro.

P-200 An addition of nicotinamide adenine dinucleotide (NAD+) precursor to oocyte maturation medium improves the developmental competence of bovine oocytes after IVF

Abstract Study question Does an increase of nicotinamide adenine dinucleotide (NAD+) level in oocytes lead to an improvement of blastulation after IVF? Summary answer The addition of NAD+ precursor to the oocyte maturation medium increased NAD+ levels in the oocytes, improving the developmental competence to the blastocyst after IVF. What is known already With maternal ageing, the enzyme that degrades NAD+ (CD38) increases in cells and the amount of NAD+ in cells, including oocytes, decreases. The degradation of NAD+ is also increased when cells are isolated from tissues. NAD+, which is an electron carrier used by all eukaryotes, is reduced to NADH via beta-oxidation of fatty acids and the TCA cycle, enhancing ATP synthesis in the mitochondrial electron transfer system. In addition, an enzyme in the inner mitochondrial membrane donates protons from NADH to NADP+ to synthesize NADPH, which maintains the intracellular redox state. Study design, size, duration This study is a prospective study using bovine oocytes retrieved from ovaries obtained from a local slaughterhouse in Osaka city. A total of 2,501 bovine oocytes were used in this study. Participants/materials, setting, methods Bovine cumulus-oocyte-complexes (COCs) were matured in vitro in the culture (IVM) medium supplemented with 0-100 μM nicotinamide mononucleotide (NMN). Matured oocytes were inseminated with the washed spermatozoa and cultured to assess the blastulation. Matured oocytes were also used to examine their NAD+, reactive oxygen species (ROS) and ATP levels. Main results and the role of chance The addition of NMN to the IVM medium increased intracellular NAD+ levels of bovine mature oocytes at 1 µM and above. As a result, it increased intracellular ATP levels and decreased ROS levels at 10 µM and above. It also improved blastocyst formation rates after IVF at 100 µM (P &amp;lt; 0.05; 35%) compared with control (22.2%). Moreover, the supplementation of NMN to the IVM medium decreased the percentage of apoptotic cells and intracellular ROS levels in cumulus cells surrounding oocytes. Limitations, reasons for caution This study was conducted in vitro using bovine oocytes. Thus, studies using human samples should be performed before clinical application. Wider implications of the findings By replenishing NAD+, we have found a clue to improving the IVM and IVF outcome, which has been shown to have a lower blastocyst formation rate than conventional IVF methods using in vivo matured oocytes. It may also improve the developmental potential of oocytes retrieved from older women. Trial registration number not applicable

Effect of heat exposure on the growth and developmental competence of bovine oocytes derived from early antral follicles

In dairy cows, low fertility caused by summer heat stress continues into the cooler autumn season. This can be caused by impaired oocyte quality in small growing follicles during summer. Here, we subjected oocyte–cumulus–granulosa complexes (OCGCs) derived from early antral follicles (0.5–1 mm) to in vitro growth (IVG) culture under two different temperature settings (the control and heat shock groups), and evaluated effects of heat exposure on growth and developmental competence of oocytes, factors affecting the developmental competence of oocytes (steroidogenesis of granulosa cells, oxidative stress in oocytes, and cell-to-cell communication between oocytes and somatic cells). Oocyte diameters after culture were smaller in the heat shock group. Although nuclear maturation and cleavage rates were similar between the groups, blastocyst rates were lower in the heat shock group (0.0%) than in the control group (27.7%), and reduced glutathione (GSH) levels in oocytes were lower in the heat shock group. Supplementation of cysteine, which stimulates GSH synthesis, increased GSH level and improved blastocyst rate of heat shocked oocytes (27.9%). These results suggest that heat exposure impairs the growth and developmental competence of oocytes in early antral follicles through GSH depletion, which can induce low fertility during summer and the following autumn.

L-Cysteine improves bovine oocyte developmental competence in vitro via activation of oocyte-derived growth factors BMP-15 and GDF-9.

This study was designed to investigate the effect of different concentrations of L-cysteine supplementation into the maturation medium on the oocyte nuclear maturation, cumulus cell expansion, ultrastructure of the oocytes and the expression of oocyte-derived growth factors BMP-15, GDF-9 and CB-1 genes. Cumulus oocyte complexes (COCs) were collected from cow's ovaries obtained from abattoir and incubated at 38.5°C in maturation media supplemented with 0, 0.6, 0.8 or 1mM L-cysteine in 5% CO2 under humidified air for 24hr. We found that a significantly higher percentage of oocytes progressed to metaphase II stage in the in vitro maturation (IVM) medium supplemented with L-cysteine, particularly 0.8mM group, compared with untreated control oocytes. Additionally, L-cysteine treatment significantly increased the number of expanded COCs and the degree of expansion of individual COCs. Results of RT-qPCR showed significant increase in expression levels of BMP-15 and GDF-9 in L-cysteine-treated groups compared with control one. Electron microgram showed improvement of cytoplasmic maturation regarding ultrastructure of the oocytes and oocyte-cumulus cell gap junction communication in all L-cysteine-treated groups especially 0.8mM L-cysteine-treated one. In conclusion, supplementation of IVM medium with a potential anti-oxidant, L-cysteine can effectively improve in vitro oocytes cytoplasmic and nuclear maturation via activation of oocyte maturation related BMP-15 and GDF-9 genes in bovine oocytes, benefiting the extended researches about the potential applications of L-cysteine in mammalian breeding technologies.

Effects of lipopolysaccharide on follicular estrogen production and developmental competence in bovine oocytes

Reproductive efficiency and female fertility is essential for productive and sustainable beef cattle operations. Gram-negative bacterial infections cause release of the endotoxin lipopolysaccharide (LPS) which initiates immune responses shown to alter ovarian steroidogenesis and impair oocyte development. The current study was designed to investigate the impact of varying levels of naturally occurring infection and follicular LPS on estradiol (E2) production and oocyte maturation. Bovine ovary pairs were harvested from a slaughterhouse, and oocytes were aspirated from small follicles and matured in vitro. Meiotic events were evaluated on nuclear maturation and spindle morphology to classify oocytes as normal or abnormal. Follicular fluid LPS concentrations were measured and subsequently separated into Low or High LPS groups. A marked difference was detected between the percent of abnormal oocytes matured from Low LPS follicles, compared to the percent of abnormal oocytes matured from High LPS follicles (P = 0.1). Follicular E2 concentrations tended to be greater for high LPS follicles (P = 0.1), however, relative abundance of mRNA transcripts for aromatase (P = 0.93) and beta-catenin (P = 0.63) were similar between groups. No changes were detected in Toll-like Receptor 4 (P = 0.15), Myeloid Differentiation Factor-2 (P = 0.61), or cluster of differentiation 14 (P = 0.46) mRNA transcript abundance in follicles with high LPS, compared to low. Therefore, even Low levels of follicular LPS indicating a subacute infection is capable of impacting the ovarian milieu and may represent an unappreciated factor leading to reduced female fertility and decreased cow retention.

5-Aminolevulinic acid combined with sodium ferrous citrate mitigates effects of heat stress on bovine oocyte developmental competence.

High summer temperatures have deleterious effects on oocyte developmental competence. The antioxidant and autophagy-related properties of 5-aminolevulinic acid (5-ALA) gives the compound a broad range of biological activities. This study aimed to evaluate the effects of: 1) a high temperature-humidity index (THI) on the developmental competence of bovine oocytes, and 2) 5-ALA administration in combination with sodium ferrous citrate (SFC) during in vitro maturation (IVM) on bovine oocyte developmental competence evaluated at high THI. Bovine ovaries were collected from a local slaughterhouse at moderate environmental temperature (MT; THI of 56.2) and high environmental temperature (HT; THI of 76.7) periods; cumulus-oocyte complexes (COCs) were aspirated from medium-sized follicles, matured in vitro for 22 h, fertilized, and cultured for 10 days. For COCs collected during the HT period, 0 (control), 0.01, 0.1, 0.5, or 1 µM 5-ALA was added to the maturation medium in combination with SFC at a molar ratio of 1:0.125. The results showed that HT adversely affected blastocyst and hatching rates compared with MT. Adding 5-ALA/SFC (1 µM/0.125 µM) to the maturation medium of oocytes collected during the HT period improved cumulus cell expansion and blastocyst rates compared with the no-addition control. In conclusion, this study showed that high THI can disrupt bovine oocyte developmental competence. Adding 5-ALA to SFC ameliorates this negative effect of heat stress and improves subsequent embryo development.

Effects of melatonin in the maturation medium on developmental competence of bovine oocytes exposed to heat shock and on embryo quality

Context Heat stress has been related to reduced fertility in cattle, which may reflect damage to developing oocytes. The deleterious effects of heat stress on oocytes may involve an increased production of reactive oxygen species (ROS). The addition of antioxidants, such as melatonin, to the in vitro maturation medium of heat-stressed bovine oocytes may be advantageous for reducing ROS concentrations, thereby enhancing the subsequent fertilisation and development to the blastocyst stage. Aims The present study evaluated the in vitro production and quality of bovine embryos derived from oocytes matured under heat-shock conditions with increasing melatonin concentrations. Methods Cumulus–oocyte complexes from Bos taurus × Bos indicus cows were matured under heat shock (12 h at 41°C, followed by 12 h at 38.5°C) in medium without melatonin (control) or with 10−12, 10−9, 10−6 and 10−3 mol/L melatonin. Logistic regression equations were adjusted when the main effect of melatonin was observed. Key results The cleavage rate on Day 3 of culture, the production of blastocysts on Days 7 and 8, the number and proportion of apoptotic cells in expanded blastocysts, and the proportion of apoptotic cells in the inner cell mass (ICM) were not affected by melatonin. The proportion of 8- to 16-cell-stage embryos on Day 3 and the proportion of ICM cells in expanded blastocysts were influenced by the melatonin concentration. Through logistic regression, it was estimated that the proportion of 8- to 16-cell-stage embryos increased linearly as the melatonin increased until 10−3 mol/L, and that the proportion of ICM cells in blastocysts was maximised between 10−6 and 10−4 mol/L melatonin, and decreased thereafter. Conclusions Melatonin at 10−3 mol/L in the in vitro maturation (IVM) of heat-shocked oocytes stimulated the first embryo cleavages but reduced the ratio of ICM cells in blastocysts. The embryo quality may be improved by melatonin between 10−6 and 10−4 mol/L. Implications The addition of melatonin to IVM medium has the potential to improve the quality of bovine embryos from heat-stressed oocytes.

Influence of Follicular Fluid on &amp;lt;i&amp;gt;in Vitro&amp;lt;/i&amp;gt; Maturation and Fertilization of Bovine Oocytes

The aim of this study was to investigate the effect of time on in vitro maturation of bovine oocytes and of the addition of follicular fluid on meiotic progression. The cumulus-oocyte complexes (COCs) collected from 3 to 6 mm follicles were obtained from ovaries of slaughtered female animals. The medium of maturation was supplemented or not with 20 μL follicular fluid (FF); 661 oocytes were matured in vitro (extrusion of the first polar corpuscle) for 22 hours with added follicular fluid (AFF) (72.01%) or without follicular fluid (WFF) (67.53%) and 679 oocytes were matured in vitro for 26 hours (extrusion of the first polar corpuscle) with AFF (92.1%) and WFF (77.15%). The results of extrusion of the second polar corpuscle as an event related to the fertilization percentages showed that the increase in the fertilization rate is maintained at 26 hours with AFF (79.45%), but the percentage decreases WFF (65.08%). After 22 hours, the fertilization rate was 62.38% AFF and 53.40% WFF. The developmental competence of bovine oocytes is affected by the duration of maturation in vitro and the inclusion in the FF culture medium. The use of follicular fluid in the in vitro maturation medium may be a biological strategy to increase the cumulus expansion, the nuclear maturation and the in vitro fertilization.

Cyclic adenosine monophosphate modulator supplementation on in vitro maturation of bovine oocytes

Study evaluated the role of cAMP modulator (Forskolin and 3-isobutyl-1- methyl xanthine) supplementation on developmental competence of bovine oocytes. Cumulus oocyte complexes recovered from bovine ovaries of unknown reproductive status were used for the study. Oocytes retrieved by aspiration method were graded based on cumulus cell distribution and culture grade oocytes were selected for the study. A total of 414 culture grade oocytes were taken and divided into two groups. Group I constituted of 201 oocytes in which pre-maturation was carried out for a period of 2 h. In group II, 213 oocytes were selected in which normal maturation was carried out. Maturation was assessed after 24h of culture in CO2 incubator maintained at 38.5o C in 95 per cent humidified atmosphere of 5 per cent CO2 . Fertilisation was carried out using frozen thawed semen and the presumptive zygotes were then transferred to culture media and cleavage was assessed 48 h after insemination. A significantly higher maturation rate (p ?0.05) was observed in group I compared to group II (86.85 ± 1.19 vs 79.88 ± 2.67). There was a highly significant increase (p ?0.01) in cleavage rate in group I (65.92 ± 1.23) compared to group II (59.29 ± 1.50). A higher fertilisation rate was observed in group I (75.35 ± 1.19) than group II (71.88 ± 2.56). It could be concluded that pre-maturation with cAMP modulators improved the developmental competence of bovine oocytes.

Low oxygen environment and astaxanthin supplementation promote the developmental competence of bovine oocytes derived from early antral follicles during 8 days of in vitro growth in a gas-permeable culture device

We evaluated the effects of a constant low (5-5%) and modulated (5–20%) oxygen environments on the in vitro development of bovine oocyte-cumulus-granulosa cell complexes (OCGCs) cultured in the presence or absence of an antioxidant (astaxanthin: Ax). OCGCs were cultured in a gas permeable culture device for 8 days in 5-5% O2 (±Ax) and 5–20% O2 (±Ax) culture conditions. In the oxygen modulated culture conditions, the oxygen concentration was switched from 5% to 20% on day 4 of culture. Ax promoted the viability of OCGCs (P < 0.05), but both oxygen and Ax had a significant effect on ROS production levels by OCGCs (P < 0.05). Specifically, ROS levels were significantly lower and higher under 5-5% O2 (+Ax) and 5–20% O2 (-Ax) conditions, respectively (P < 0.05), with intermediate levels observed in the 5-5% O2 (-Ax) and the 5–20% O2 (+Ax) culture conditions. The steroidogenic pattern was characterized by increasing estradiol-17β but with constant progesterone production levels regardless of culture conditions, suggesting the inhibition of luteinization-like changes in granulosa cells. OCGCs cultured in the 5–20% O2 (+Ax) had higher nuclear maturation rates (P < 0.05) that were similar to the oocytes grown in vivo. However, there was no clear difference in the subsequent cleavage rates among the 5-5% O2 (±Ax) and the 5–20% O2 (+Ax) culture conditions (P > 0.05). A constant low oxygen environment significantly promoted the blastocyst rates (P < 0.05); however, the presence of Ax in the 5–20% O2 (+Ax) condition also promoted development similar to the OCGCs cultured in the 5-5% O2 (-Ax) condition (P > 0.05). In conclusion, exposure of OCGCs to constant low oxygen or oxygen modulation in the presence of Ax promotes the healthy development of OCGCs during the 8-day IVG culture using the gas permeable culture device.