Nuclear Maturation Rates Research Articles

Beneficial effects of fibroblast growth factor 10 supplementation during in vitro maturation of buffalo cumulus-oocyte complexes

Fibroblast growth factor 10 (FGF10) is an important regulator of the mammalian cumulus-oocyte complex that plays a crucial role in oocyte maturation. In this study, we investigated the effects of FGF10 supplementation on the in vitro maturation (IVM) of buffalo oocytes and its related mechanisms. During IVM, the maturation medium was supplemented with a range of concentrations of FGF10 (0, 0.5, 5, and 50 ng/mL) and the resulting effects were corroborated using aceto-orcein staining, TUNEL apoptosis assay, detection of Cdc2/Cdk1 kinase in oocytes, and real-time quantitative PCR. In matured oocytes, the 5 ng/mL-FGF10 treatment resulted in a significantly increased nuclear maturation rate, which increased the activity of maturation-promoting factor (MPF) and enhanced buffalo oocyte maturation. Furthermore, it treatment significantly inhibited the apoptosis of cumulus cells, while simultaneously promoting its proliferation and expansion. This treatment also increased the absorption of glucose in cumulus cells. Thus, our results indicate that adding an appropriate concentration of FGF10 to a maturation medium during IVM can be beneficial to the maturation of buffalo oocytes and improve the potential of embryo development.

Succinate dehydrogenase participation in porcine gamete function

Context Porcine gametes require energy for the physiological processes that allow fertilisation. Succinate dehydrogenase (SDH) plays a pivotal role in both, the tricarboxylic acid (TCA) cycle and the respiratory chain. Aims The aim of this work was to study the participation of SDH in the in vitro oocyte maturation, sperm capacitation and acrosome reaction in porcine species. Methods Cumulus–oocyte complexes (COCs) from abattoir-derived porcine ovaries were collected by aspiration and were incubated in maturation media, with the addition of increasing concentrations (0, 1, 5 and 10 mM) of malonate (a specific inhibitor of SDH). Nuclear maturation and cytoplasmatic maturation were analysed. Semen samples were incubated for 2 h in capacitating medium with 40 mM sodium bicarbonate, as sperm capacitation inducer, and the addition of increasing concentrations of malonate (0, 1, 5 and 10 mM). Sperm capacitation state and true acrosomal reaction were evaluated. SDH activity was determined in sperm and oocyte extracts by the spectrophotometric method. Key results The addition of 10 mM of malonate decreased both nuclear and cytoplasmic maturation rates (P < 0.05) without affecting COC viability (assessed using fluorescein diacetate). A lower level of capacitation (induced by bicarbonate) and acrosome reaction (induced by follicular fluid) was observed with the addition of 5 mM of malonate (P < 0.05) without affecting motility and viability of sperm at this concentration. The activity of SDH was 0.35 ± 0.1 × 10−5 and 2.37 ± 0.9 × 10−5 U/COC for immature and in vitro matured COC extracts (P < 0.05) respectively, and 0.44 ± 0.16 U/1010 sperm for boar sperm extracts. Conclusions In conclusion, because it has been proposed that aerobic and anaerobic metabolic pathways of cells are changed depending on the oxygen availability and the composition of metabolic substrates in their environment, our results suggest that energy obtained through the mitochondrial respiration (TCA cycle and oxidative phosphorylation) is necessary to support oocyte maturation, sperm capacitation and acrosome reaction in the porcine species. Implications The study of enzymatic activity in gametes is essential for understanding the mechanisms that control the energy production required to achieve successful fertilisation. This knowledge has significant implications for the development of assisted reproductive technologies.

Oocytes Population and Development Competence of Bali Cattle Embryo In Vitro with Different Ovarian Reproductive Statuses

The present study aims to determine the potential of Bali cattle ovaries as sources of oocytes for in vitro embryo production based on different ovarian reproductive statuses. The ovaries were grouped into 4 categories: ovaries with no corpus luteum and dominant follicles (CL-DF-), those with corpus luteum and no dominant follicles (CL+DF-), those without corpus luteum but with dominant follicles (CL-DF+), and those with corpus luteum and dominant follicle (CL+DF+). The oocytes were collected via the slicing technique and grouped into 4 grades (a, b, c, and d). The oocyte’s maturation was performed using tissue culture medium 199 basic media. A drop sample (10–15 oocytes/drop) covered with mineral oil was then placed in a 5% CO2 incubator at a temperature of 38.5 ℃ for 24 h. Then, the samples were fertilized in 80 μL of fertilization medium with a final spermatozoa concentration of 1.5×106 spermatozoa/mL. After 5–6 h of in vitro fertilization, the oocytes were washed four times using the Charles Rosenkrans 1aa (CR1aa) medium. Then, the samples were cultured using the CR1aa as a base medium. The results showed no significant difference (p>0.05) for the 4 groups based on the oocyte population collected from one pair ovary as well as the number of oocytes that were suitable for maturation. However, group CL-DF+ showed a significant difference (p<0.05) in the rate of nuclear maturation (80.00±12.84), fertilization rate (80.00±4.72), and the ability of embryo development (60.19±22.45) when compared to group CL-DF-, CL+DF-, and CL+DF+. This study determines that the oocyte population of Bali cattle ovary pairs and oocytes quantity that are fit for maturation is not influenced by the reproductive status of the ovaries. However, the level of nuclear maturation, fertilization, and the ability of embryo development is higher in the ovaries without corpus luteum but with dominant follicles.

Time-lapse imaging and developmental competence of donkey eggs after ICSI: Effect of preovulatory follicular fluid during oocyte in vitro maturation

Equus members exhibit very divergent karyotype, genetic plasticity, and significant differences in their reproductive physiology. Despite the fact that somatic cell nuclear transfer and intracytoplasmic sperm injection (ICSI) has gained relevance in the last few years in horses, few reports have been published exploring ovum pick up (OPU) and in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) in donkeys. Yet, some donkey species and breeds are considered endangered, and these assisted-reproductive technologies could help to preserve the genetic of valuable individuals. In this study, we tested the hypothesis that supplementation with jenny preovulatory follicular fluid (PFF) during IVM could improve oocyte developmental competence in the donkey. For this, in vitro nuclear maturation rates, cumulus cell expansion, and embryo development after ICSI of donkey COCs matured in culture media supplemented with fetal bovine serum (FBS) or donkey PFF, with a known metabolomic profile, were assessed. Time-lapse imagining was performed after ICSI of horse and donkey oocytes. Eight OPU sessions were done in five jennies with an average recovery rate of 69.2% (n = 45 COCs). Although lower cumulus cells expansion was observed in oocytes of PFF group (P = 0.0010), no significant differences were described in nuclear maturation rates and preimplantation embryo development between groups. Donkey ICSI embryos showed similar morphokinetics to horse ICSI embryos. Our study shows that supplementing IVM media with FBS or donkey PFF supports nuclear maturation and early preimplantation embryo development after ICSI in donkeys. To our knowledge, the present study is the first report of ICSI, time-lapse imaging and in vitro blastocyst production in donkey.

Ramelteon Reduces Oxidative Stress by Maintenance of Lipid Homeostasis in Porcine Oocytes.

This study aimed to determine the underlying mechanism of ramelteon on the competence of oocyte and subsequent embryo development in pigs during in vitro maturation (IVM). Our results showed that the cumulus expansion index was significantly lower in the control group compared to the ramelteon groups (p < 0.05). Moreover, supplementation of 10−11 and 10−9 M ramelteon significantly increased the cumulus expansion and development-related genes expression, and reduced apoptosis in cumulus cells (p < 0.05). In oocytes, the nuclear maturation rate was significantly improved in 10−11, 10−9, and 10−7 M ramelteon groups compared to the control (p < 0.05). Additionally, the level of intracellular GSH was significantly increased and ROS was significantly decreased in ramelteon-supplemented groups, and the gene expression of oocyte development and apoptosis were significantly up- and down-regulated by 10−11 and 10−9 M ramelteon (p < 0.05), respectively. The immunofluorescence results showed that the protein levels of GDF9, BMP15, SOD1, CDK1, and PGC1α were significantly increased by 10−11 M ramelteon compared to the control (p < 0.05). Although there was no significant difference in cleavage rate, the blastocyst formation rate, total cell numbers, and hatching/-ed rate were significantly improved in 10−11 M ramelteon group compared to the control (p < 0.05). Furthermore, embryo development, hatching, and mitochondrial biogenesis-related genes were dramatically up-regulated by 10−11 M ramelteon (p < 0.05). In addition, the activities of lipogenesis and lipolysis in oocytes were dramatically increased by 10−11 M ramelteon compared to the control (p < 0.05). In conclusion, supplementation of 10−11 M ramelteon during IVM improved the oocyte maturation and subsequent embryo development by reducing oxidative stress and maintenance of lipid homeostasis.

L-Carnitine Supports the In Vitro Growth of Buffalo Oocytes.

Simple SummaryThe objective of this study was to examine the effect of L-carnitine on the growth and subsequent nuclear maturation of buffalo growing oocytes in vitro. Oocyte-granulosa cell complexes (OGCs) were collected from early antral follicles of slaughtered buffaloes and cultured in in vitro growth (IVG) medium with the supplementation of different concentrations (0, 1.25, 1.875 or 2.5 mM) of L-carnitine for 6 days. The results showed that L-carnitine increased the diameter of buffalo oocytes in vitro. L-carnitine also enhanced the antrum-like structure formation and prevented the degeneration of buffalo oocytes in culture. Furthermore, L-carnitine-treated oocytes showed a higher rate of nuclear maturation up to the metaphase II (MII) stage and a lower rate of degeneration. In conclusion, L-carnitine enhances the growth, prevents degeneration, promotes the formation of antrum-like structures and supports nuclear maturation of buffalo oocytes in vitro.This study aimed to determine the effect of L-carnitine on the growth and subsequent nuclear maturation of buffalo small growing oocytes (92–108 µm in diameter) in vitro. Oocyte-granulosa cell complexes (OGCs) were dissected from early antral follicles of slaughtered buffaloes and cultured in in vitro growth (IVG) medium with the supplementation of different concentrations (0, 1.25, 1.875 or 2.5 mM) of L-carnitine for 6 days. The results revealed that L-carnitine increased the diameter of buffalo oocytes in vitro. The degeneration rate was significantly (p < 0.05) lower in 2.5 mM of L-carnitine-treated oocytes (10%) than others (55%, 45% and 32.5% in 0, 1.25 and 1.875 mM of L-carnitine-supplemented groups, respectively). The OGCs showed antrum-like structures significantly (p < 0.05) higher in the 2.5 mM of L-carnitine group (74.0%) than the 0- and 1.25-mM groups (34.6% and 38.1%, respectively). Furthermore, in vitro grown oocytes were placed in in vitro maturation (IVM) medium for 24 h to examine meiotic competence of in vitro grown oocytes with L-carnitine. The L-carnitine (1.875 and 2.5 mM) treated oocytes showed a higher rate of nuclear maturation up to the metaphase II (MII) stage and a lower rate of degeneration. In conclusion, L-carnitine enhances the growth, prevents degeneration, promotes the formation of antrum-like structures and supports nuclear maturation of buffalo oocytes in vitro.

Effect of three schemes of ovum pick-up on the follicular dynamics, gene expression, and in-vitro developmental competence of oocytes in Sahiwal cattle.

This study aimed to compare the effect of three schemes of ovum pick-up (OPU) on follicular dynamics, oocytes recovery, oocytes quality, gene expression, nuclear maturation and in-vitro developmental competence of oocytes in Sahiwal cattle. Considering the follicle population, all the cows were divided equally in a 3× 3 cross over design, and each cow received one of the three treatments: (a) twice weekly (TW; n=6), (b) once weekly (OW; n=6) and (c) bi-weekly OPU (BW; n=6) in three periods, with the first OPU conducted on 4, 7 and 14 days after second dominant follicle puncture (DFP) in the TW, OW and BW OPU interval groups, respectively. The collected cumulus oocytes complexes (COCs) were graded into A, B, C and D grades depending on the number of layers of cumulus cells and homogeneous nature of cytoplasm. Nuclear maturation was assessed by staining the oocytes with Hoechst 33342. The growth rate (mm/day) of dominant follicle (DF) (F1) (0.49 ± 0.21 vs. 0.71 ± 0.26 vs. 1.30 ± 0.27) and first subordinate follicle (F2) (0.85 ± 0.27 vs. 0.71 ± 0.25 vs. 1.06 ± 0.29) did not differ (p > .05) among all the three groups. The proportion of animals bearing a corpus luteum (CL) in the BW OPU interval group (53.3%) was significantly higher (p < .05) as compared to TW (13.3%) and OW (18.3%) OPU interval groups. The number of medium-sized follicles and oocyte with grade A and B were significantly higher (p < .05) in the TW (1.16 ± 0.21 and 33.88 ± 0.03) OPU interval group as compared to the OW (0.88 ± 0.22 and 21.54 ± 0.03) and BW (0.55 ± 0.21 and 21.89 ± 0.02) OPU interval groups. However, the number of degenerated oocytes in BW (0.85 ± 0.16) OPU interval group was significantly higher (p < .05) as compared to the TW (0.16 ± 0.15) and OW (0.44 ± 0.16) OPU interval groups. Expression level of growth differentiation factor 9 in TW OPU interval group was significantly higher (p < .05) as compared to the OW and BW OPU interval groups. Likewise, expression level of bone morphogenetic protein 15 (BMP15) in the TW and BW OPU interval groups was significantly higher (p < .05) as compared to the OW OPU interval group. The nuclear maturation rate was significantly higher in the TW (63.64 ± 0.07) and BW (59.26 ± 0.08) OPU groups as compared to OW (51.43 ± 0.06) OPU interval group. However, the cleavage rate (59.30 ± 0.06 vs. 44.29 ± 0.06 vs. 56.67 ± 0.06) did not differ (p > .05) among the three groups. Whereas, the blastocyst rate tended to be higher (p=.06) in the TW (29.07 ± 0.05) and BW (28.33 ± 0.04) OPU interval groups as compared to OW (18.57 ± 0.05) OPU interval group. Taken together, it can be concluded that TW OPU interval scheme enhances the medium-sized follicles resulting in good quality oocytes, regulates the oocyte-derived paracrine factors, leading to higher nuclear maturation rates and improved embryonic development in-vitro.

Effect of enzymatic pro-oxidant and antioxidant systems on bovine oocyte in vitro maturation

Abstract The role of reactive oxygen species (ROS) during oocyte in vitro maturation (IVM) is still controversial. Although an increase in ROS production may cause deleterious effects in cells, these reactive species may also act as signaling molecules influencing different cell functions. The aim of this study was to examine the effect of varying endogenous ROS levels during IVM on the process of bovine oocyte maturation. To do so, different enzymatic antioxidant (catalase, or superoxide dismutase + catalase, or diphenyl iodonium) or pro-oxidant systems (xanthine + xanthine oxidase, or xanthine + xanthine oxidase + catalase) were added to the culture medium. ROS levels were determined by 2′,7′-dichlorodihydrofluorescein diacetate stain, nuclear maturation was evaluated by the presence of the metaphase II chromosome configuration at 22h of IVM and cleavage rate was recorded 48h post- in vitro fertilization. ROS levels were only significantly increased (P&lt;0.05) by the O2 .- generating system (xanthine + xanthine oxidase + catalase), but meiotic maturation rates were significantly lower (P&lt;0.05) in all the evaluated systems compared with the control, except for the diphenyl iodonium group. However, this last group presented a significantly lower (P&lt;0.05) cleavage rate in comparison to the control group. These results indicate that ROS would play an essential role during oocyte maturation, since its increase or decrease beyond a physiological level significantly reduced nuclear or cytoplasmic maturation rates in bovine oocytes.

Extracellular vesicles from follicular fluid may improve the nuclear maturation rate of in vitro matured mare oocytes

The in vitro maturation (IVM) of equine oocytes is still not efficient and does not yield consistent results. The specific requirements of equine oocytes during this process are still largely unknown, which hinders the development of assisted reproductive techniques (ART) in this species. Because the ovarian follicle microenvironment supports oocytes in their acquisition of developmental competence, follicular fluid seems to be a substantial source of bioactive factors that could support the IVM process. Extracellular vesicles (EVs) are cell-secreted molecules in body fluids that are able to deliver molecular signals and transfer genetic information (mRNA, miRNA) between donor and recipient cells. Hence, our hypothesis is that follicular fluid EVs (ffEVs) from small (<20 mm) ovarian follicles can improve the in vitro maturation rate of mare oocytes. To test our hypothesis, equine ovarian follicular fluid was aspirated and ffEVs were isolated by ultracentrifugation, then characterized using nanoparticle tracking analysis and flow cytometry. Additionally, ffEVs were labeled using the ExoGlow-protein EV labeling kit (System Biosciences, Palo Alto, CA). Cumulus-oocyte complexes (COCs) were matured using a one-step method (Method I, continuous culture for 24–38 h) or a two-step method (Method II, initial denudation after 24 h), in the presence (200 μg protein/ml) or absence of ffEVs. The results show the internalization of ffEVs by equine cumulus cells and, for the first time, also by oocytes. The ffEV treatment during two-step culture had a positive effect on the maturation rate of compacted COCs compared to the control group (45.7% and 20.5%, respectively; p < 0.05). No effect of supplementation was observed on the maturation rate during one-step culture. Our results indicate that the supplementation of culture media with EVs isolated from the follicular fluid of small follicles can improve the IVM rate of mare oocytes, suggesting that ffEVs play an important role during this process and may enhance the development of equine ART.

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.

Bovine In Vitro Oocyte Maturation and Embryo Production Used as a Model for Testing Endocrine Disrupting Chemicals Eliciting Female Reproductive Toxicity With Diethylstilbestrol as a Showcase Compound.

Endocrine disrupting chemicals (EDCs) can interfere with normal hormonal action and regulation. Exposure of women to EDCs has been associated with adverse reproductive health outcomes. The assays currently used to identify EDCs that elicit female reproductive toxicity lack screening tests that address effects on the maturation of oocytes, a process that enables them to be fertilized and develop into embryos. Here, a screening method employing the bovine model of in vitro oocyte maturation and embryo production is described. Endpoints explored address important events in oocyte maturation and developmental competence acquisition. To test the method, the effects of the known human EDC diethylstilbestrol (DES; an estrogen receptor agonist) were evaluated in a range of concentrations (10–9 M, 10–7 M, 10–5 M). Bovine oocytes were exposed to DES during in vitro maturation (IVM) or embryos were exposed during in vitro embryo culture (IVC). The endpoints evaluated included nuclear maturation, mitochondrial redistribution, cumulus cell expansion, apoptosis, and steroidogenesis. DES-exposed oocytes were fertilized to record embryo cleavage and blastocyst rates to uncover effects on developmental competence. Similarly, the development of embryos exposed to DES during IVC was monitored to assess the impact on early embryo development. Exposure to 10–9 M or 10–7 M DES did not affect the endpoints addressing oocyte maturation or embryo development. However, there were considerable detrimental effects observed in oocytes exposed to 10–5 M DES. Specifically, compared to vehicle-treated oocytes, there was a statistically significant reduction in nuclear maturation (3% vs 84%), cumulus expansion (2.8-fold vs 3.6-fold) and blastocyst rate (3% vs 32%). Additionally, progesterone and pregnenolone concentrations measured in IVM culture media were increased. The screening method described here shows that bovine oocytes were sensitive to the action of this particular chemical (i.e., DES), albeit at high concentrations. In principle, this method provides a valuable tool to assess the oocyte maturation process and early embryo development that can be used for reproductive toxicity screening and possibly EDC identification. Further studies should include EDCs with different mechanisms of action and additional endpoints to further demonstrate the applicability of the bovine oocyte model for chemical risk assessment purposes and EDC identification.

Effect of dimethyl alpha-ketoglutarate supplementation on the in vitro developmental competences of ovine oocytes

The supplementation of dimethyl alpha-ketoglutarate (DMKG) during the in vitro maturation (IVM) process has been shown to improve the in vitro developmental competences of porcine oocytes. Here, the effects of DMKG supplementation in IVM medium on the development competencies of ovine oocytes were investigated by analyzing the nuclear maturation rate to metaphase II (MII) stage, ATP synthesis, cortical granules (CGs) dynamic, F-actin polymerization, mitochondrial activity, mitochondrial damage, reactive oxygen species (ROS) production, intracellular glutathione (GSH) production, DNA damage, cellular apoptosis, fertilization capacity and blastocyst development potential of ovine oocytes. In addition, the oxidative stress damage model induced by H2O2 treatment was applied to confirm the antioxidative effect of DMKG supplementation on the development of ovine oocytes. The results showed that compared with MII oocytes without DMKG supplementation (Control group), 3 mM DMKG supplementation during IVM significantly (P < 0.05) increased nuclear maturation rate, ATP synthesis, CGs dynamic, F-actin polymerization, mitochondrial activity, GSH production and embryonic developmental competence and decreased ROS production, mitochondrial damage, DNA damage and cellular apoptosis level of ovine MII oocytes. Moreover, the reductions in the developmental competences of ovine MII oocytes caused by H2O2 induced oxidative stress damages were effectively ameliorated by the co-supplementation in IVM of 3 mM DMKG (P < 0.05).Our results demonstrate the promising effect of DMKG supplementation on the in vitro developmental competence of ovine oocytes via the reduction of oxidative stress damages and indicates further research into the clinical applications of DMKG and the development of ovine breeding technologies is warranted.

Docosahexaenoic acid and phenazine ethosulfate are not efficient lipid modulators for porcine in vitro maturation systems.

The high lipid content in porcine oocytes impairs in vitro embryo production (IVP). Here, we evaluated the influence of two different lipid modulators during in vitro maturation (IVM) on the embryo development and the lipid content of oocytes and embryos. In Experiment I, oocytes were exposed to 50μM docosahexaenoic acid (DHA) with (+) or without (-) the presence of porcine follicular fluid (pFF). In Experiment II, phenazine ethosulfate (PES) was added during IVM at two concentrations (0.5 and 0.05μM). The pFF- with 50μM DHA treatment impaired nuclear maturation, cleavage and blastocyst rates (p < 0.05). Oocytes in pFF- media accumulated less lipids (p < 0.05). The addition of 0.5μM PES reduced all development rates (p < 0.05) and resulted in higher lipid content for oocytes and embryos. Only 0.05μM PES oocytes matured similarly to the control (p > 0.05), although embryo development and embryo lipid content was similar to 0.5μM PES oocytes (p > 0.05). Thus, 50μM DHA supplementation in the IVM medium without pFF impaired oocyte maturation and embryo development rates without interfering in oocyte lipid content even in the presence of pFF. Maturation with PES neither favored porcine embryo development nor reduced their lipid content.

Idebenone relieves the damage of heat stress on the maturation and developmental competence of porcine oocytes.

The reproductive function of animals is often affected by climatic conditions. High-temperature conditions can cause damage to oocyte maturation and embryonic development in a variety of ways. The purpose of this study was to prove that supplementation idebenone (IDB) to the maturation medium can improve the maturation and development of porcine oocytes after heat stress (HS). Porcine cumulus-oocyte complexes (COCs) were cultured in the maturation medium with different concentrations of IDB (0, 0.1, 1 and 10μM) for 44hr at either 38.5°C or under the HS conditions. The cumulus oophorus expansion, nuclear maturation and blastocyst rate after parthenogenetic activation (PA) were measured. We found that HS (in vitro maturation 20-24hr, 42°C) exposure significantly reduced cumulus expansion index and maturation rate of oocytes and the blastocyst rate of PA embryos, while IDB supplementation significantly improved oocyte maturation and development to the blastocysts stage after PA. Moreover, the addition of IDB decreased the intracellular level of ROS and increased GSH content, hence enhancing the antioxidant capacity of oocytes under HS. Meanwhile, IDB treatment also obviously improved the mitochondrial membrane potential and ATP synthesis of oocytes under HS conditions. Furthermore, IDB treatment increased the expression of GDF9 and BMP15 in IVM oocytes which attribute to improve the quality and outcome of IVM oocytes and the development competence of PA embryos in pigs. In summary, we demonstrated that IDB supplementation into the maturation medium exerted protective effects and improved the ability of maturation and developmental competence of porcine oocytes exposed to HS.

Comparison of Two In vitro Maturation Media in Their Efficiency for Production of Competent Alpaca Oocytes

Oocyte maturation is the most critical process in in vitro culture, since the oocyte acquires competence for future processes, which involve the resumption of meiosis, epigenetic regulation, polyspermy block, activation of the zygote, exchange of protamines to histones, cleavage, cell polarization, embryonic stem cell proliferation, and implantation. Therefore, the objective of this study was to evaluate the efficiency of two different in vitro maturation media in the production of competent alpaca oocytes matured in vitro. The competency evaluated by morphometry and cytoplasmic and nuclear staining techniques. Alpaca ovaries (n=44) were sourced from slaughterhouse, transported to laboratory in thermoses with saline solution at 33-36ºC and subjected to follicular aspiration and/or dissection. A total of 212 oocytes were obtained. The oocytes were selected, randomized and distributed in one of the two experimental groups: Maturation medium 1 (MM1) and Maturation medium 2 (MM2) with three replicates per experiment. In vitro maturation was carried out in a portable incubator for 40h under 5% CO2 conditions (produced by effervescent granules) and 38.5ºC (by thermostatic bath). Nuclear maturation was assessed using acetate-orcein staining, cytoplasmic maturation using Brilliant Cresyl Blue (BCB) staining and morphometry using ImageJ Software. Nuclear maturation was classified according to the meiotic stage. Cytoplasmic maturation was classified by the presence (BCB+) or absence (BCB-) of BCB staining. Morphometry evaluated the measurements of the diameter and thickness of the zona pellucida and perivitelline space of the oocyte. Nuclear maturation rate was evaluated in Metaphase, observing no significant differences (P=0.088) between MM1 (46.08 ± 5.90 %) and MM2 (54.65 ± 3.00%) in this parameter. The cytoplasmic maturation rate of MM1 (67.78 ± 4.50%) was significantly lower compared to MM2 (77.61 ± 3.58) % (P=0.014). For the diameter of the oocyte, MM1 (133.42 ± 14.56 μm) and MM2 (136.14 ± 15.2 μm) were no different (P=0.397), same for the thickness of the zona pellucida, MM1 (16.84 ± 3.96 μm) and MM2 (16.73 ± 4.98 μm) were no different (P=0.919). For the perivitelline space area, MM1 (11018.91 ± 2465.40 μm2 ) and MM2 (8686.18 ± 3016.88 μm2 ) were significantly different (P=0.0005). In conclusion, the present study shows that the MM2 medium results are better in cytoplasmic maturation and the area of the perivitelline space. The other parameters (nuclear maturation, diameter of the oocyte and thickness of the zona pellucida) were no different between the two maturation media evaluated.

Impact of Presence or Absence of Trehalose during Vitrification on Viability and Development of Vitrified/Warmed Immature Dromedary Camel Oocytes

Vitrification of immature oocytes at the germinal vesicle (GV) stage is important to preserve female gametes. The standard formula for vitrification solutions has long been a debate. Herein, we investigated the effect of the presence or absence of trehalose in vitrification solution on viability, in vitro maturation (IVM) rates, and development of vitrified/warmed immature dromedary camel oocytes. Cumulus oocyte complexes (COCs) obtained at slaughter from the ovaries of mature she-camels were randomly allocated into three groups; namely, control group, oocytes were directly subjected to IVM without vitrification, vitrification solution 1 (VS1) group, oocytes were vitrified in a solution composed of 25% ethylene glycol (EG) plus 25% dimethyl sulfoxide (DMSO) + 0.5 M trehalose; and vitrification solution 2 (VS2) group, oocytes were vitrified in a solution composed of 25% EG plus 25% DMSO. Vitrification of COCs was conducted by open pulled straws (OPS) method. Following vitrification and warming, morphologically viable oocytes were matured in vitro for 36 h. COCs were then fertilized and cultured in vitro for 7 days. The percentage of viable oocytes was significantly higher (P 0.05) in VS2 than VS1 group (80.0% vs. 63.3%, respectively). Nuclear maturation, cleavage (48 h post-insemination; pi), and blastocyst rates (7 days pi) were significantly higher (P camel oocytes in trehalose-free solution (VS2) was more advantageous than that in trehalose supplemented media since it did not reduce viability and development.

Beneficial Effects of Neurotrophin-4 Supplementation During in vitro Maturation of Porcine Cumulus-Oocyte Complexes and Subsequent Embryonic Development After Parthenogenetic Activation.

Neurotrophin-4 (NT-4) is a neurotrophic factor that plays an important role in follicular development and oocyte maturation. However, it is not yet known whether NT-4 is related to oocyte maturation and follicular development in pigs. This study aims to investigate the effects of NT-4 supplementation during in vitro maturation (IVM) of porcine oocytes and subsequent embryonic development after parthenogenetic activation (PA). First, NT-4 and its receptors (TrkB and p75NTR) were identified through fluorescent immunohistochemistry in porcine ovaries. NT-4 was mainly expressed in theca and granulosa cells; phospho-TrkB and total TrkB were expressed in theca cells, granulosa cells, and oocytes; p75NTR was expressed in all follicular cells. During IVM, the defined maturation medium was supplemented with various concentrations of NT-4 (0, 1, 10, and 100 ng/mL). After IVM, the nuclear maturation rate was significantly higher in the 10 and 100 ng/mL NT-4 treated groups than in the control. There was no significant difference in the intracellular reactive oxygen species levels in any group after IVM, but the 1 and 10 ng/mL NT-4 treatment groups showed a significant increase in the intracellular glutathione levels compared to the control. In matured cumulus cells, the 10 ng/mL NT-4 treatment group showed significantly increased cumulus expansion-related genes and epidermal growth factor (EGF) signaling pathway-related genes. In matured oocytes, the 10 ng/mL treatment group showed significantly increased expression of cell proliferation-related genes, antioxidant-related genes, and EGF signaling pathway-related genes. We also investigated the subsequent embryonic developmental competence of PA embryos. After PA, the cleavage rates significantly increased in the 10 and 100 ng/mL NT-4 treatment groups. Although there was no significant difference in the total cell number of blastocysts, only the 10 ng/mL NT-4 treatment group showed a higher blastocyst formation rate than the control group. Our findings suggest that supplementation with the 10 ng/mL NT-4 can enhance porcine oocyte maturation by interacting with the EGF receptor signaling pathway. In addition, we demonstrated for the first time that NT-4 is not only required for porcine follicular development, but also has beneficial effects on oocyte maturation and developmental competence of PA embryos.

Exogenous progesterone-dependent modulation in the follicular dynamics of Bos indicus cattle undergoing repeated ovum pick-up sessions.

The objective of the present study was to evaluate the role of serum progesterone (P4) in follicular dynamics, oocytes' recovery and quality and their in vitro developmental competence during consecutive ovum pick-up (OPU) sessions in Bos indicus dairy cows. Wave-synchronized Sahiwal cattle (n=20) were randomly divided into treatment (n=10) and control (n=10) groups. CIDR was used as a source of external progesterone in the treatment group. Four consecutive OPU sessions at 96-hr intervals were conducted and repeated ultrasonography at 12-hr intervals was done to monitor follicular dynamics. The viable oocytes were processed for IVC following IVM and IVF until day 7. The serum P4 concentrations in the P4 and control groups were recorded as 2.31±0.059 versus.0.32±0.065ng/ml, respectively (p<.05). In the treatment group, the total number of recorded follicles was higher (p<.05; 12.05±0.37 versus. 10.87±0.40), whilst the growth rate (mm/day) of follicles was lower (p>.05). Per session recovered oocytes (5.31±0.19 versus. 3.58±0.21; p<.0001) and recovery rate (54.23 versus. 42.53%; p<.05) were also higher in the treatment group compared to control. Similarly, the viable oocytes (4.54±0.187 versus. 3.06±0.199) and the number of grade I and II oocytes per session (3.37±0.196 versus. 2.06±0.21) were higher (p<.05) in the treatment group compared with the control group. However, the nuclear maturation, cleavage, and blastocyst rate did not differ (p>.05) between the groups. Taken together, during OPU sessions, serum P4 improves oocytes' recovery and quality, whilst does not affect the in vitro developmental competence of recovered 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.

Effects of Antioxidants on Developmental Competence of Matured Dromedary Camel Oocytes

The purpose of this work was to look into the impact of antioxidants on the viability and mitochondrial distribution of dromedary camel oocytes, through 1) Study the effect of vitamin C (ascorbic acid) and melatonin on the maturation rate and fertilization rate of dromedary camel oocytes, 2) Study the impact of vitamin C and melatonin on the viability, mitochondrial distribution, and intensity of in-vitro matured dromedary camel oocytes. Camel oocytes were aspirated from ovaries. Oocytes of excellent and good quality were in-vitro matured using in vitro maturation media (TCM-199 + 10 ug/ml FSH + 10%fetal calf serum + 100 IU/ml Pregnant mare serum + 50 mg/ml gentamycin) without (control group) or with antioxidant groups (50 µg/ml vitamin C or 100 µM/ml melatonin) and incubated in CO2 incubator (38.5 C, 5% CO2, 20% O2 and 95% humidity) for 40 hours. In vitro matured oocytes with 1st polar body were either in vitro fertilized using epididymal spermatozoa for detection the fertilization rate or stained with Hoechst 33342 (bis-Benzimide H33258) dye and MitoTracker red dye for detection the viability and mitochondrial distribution and intensity using confocal microscope. Results showed that, the nuclear maturation rate of dromedary camel oocytes in vitro matured with antioxidants were significantly (P˂0.001) higher than the control group. The fertilization rate of in vitro matured oocytes in the control group (56.26 ± 0.63) was lower than vitamin C and melatonin group (70.88 ± 0.87 and 76.63 ± 0.82, respectively). The mitochondrial intensity was significantly increased in the vitamin C (118.3 ± 1.13) and melatonin (231.1 ± 4.06) groups when compared with control group (102.5 ± 2.16). The melatonin group was significantly higher in peripheral mitochondrial distribution when compared with control and vitamin C groups, while the diffused distribution of mitochondria was markedly greater in the vitamin C and control group when compared with melatonin group. In-conclusion, Supplementation of vitamin C or melatonin on in vitro maturation media were significantly improved the maturation rate and fertilization rate through enhancing the mitochondrial intensity and distribution in Dromedary camel oocytes.