Contrasting effects of heat shock during in vitro maturation on development of in vitro‐fertilized and parthenogenetic bovine embryos

This study investigated the influence of heat shock during in vitro maturation on embryo development following in vitro fertilization (IVF) or parthenogenesis (Part). Immature bovine cumulus–oocyte complexes were exposed to heat shock (41.0°C) during the first 12 hr of in vitro maturation (IVM), followed by 12 hr at 38.5°C. Control group consisted of in vitro maturation for 24 hr at 38.5°C. Oocytes were in vitro‐fertilized or activated with ionomycin and cultured in vitro for 192 hr post‐in vitro insemination or parthenogenetic activation (hpia). There was an interaction (p < .01) between temperature of IVM and method of oocyte activation (IVF or Part) for cleavage at 48 hpia. Heat shock had a negative impact (p < .01) on cleavage of IVF embryos, whereas no (p > .05) effect was found in the Part embryos. Embryo development towards blastocyst stage at 168 and 192 hpia decreased in both IVF and Part embryos derived from heat‐shocked oocytes. Heat shock increased (p < .05) the apoptotic index in Part blastocysts, but no effect (p > .05) was found in IVF counterparts. Heat shock also down‐regulated the expression of AQP3 (p < .01) and up‐regulated the expression of HSP70.1 (p < .01) in Part blastocysts, whereas it down‐regulated the expression of ATP1A1 (p < .05) in IVF blastocysts. In conclusion, the effects of heat shock during IVM on early embryo cleavage and blastocyst apoptosis are influenced by the method of oocyte activation and expression of some genes can be disturbed in embryos derived from heat‐shocked oocytes.     

Presence of chlorogenic acid during in vitro maturation protects porcine oocytes from the negative effects of heat stress

Chlorogenic acid (CGA) is known to protect oocytes from oxidative stress. Here we investigated the effects of CGA on porcine oocyte maturation under heat stress and subsequent embryonic development after parthenogenetic activation. For in vitro maturation (IVM) at 41.0°C (hyperthermic condition), supplementation of the maturation medium with 50 μM CGA significantly improved the percentage of matured oocytes and reduced the rate of apoptosis relative to oocytes matured without CGA (p < .05). CGA treatment of oocytes during IVM under hyperthermia tended to increase (p < .1) percentage of blastocyst formation after parthenogenesis and significantly increased (p < .05) the total cell number per blastocyst relative to oocytes matured without CGA. For IVM at 38.5°C (isothermic condition), CGA significantly improved the rate of blastocyst development compared with oocytes matured without CGA (p < .05), but did not affect oocyte maturation, apoptosis rate or the number of cells per embryo. Omission of all antioxidants from the IVM medium significantly reduced the rate of oocyte maturation, but the rate was restored upon addition of CGA. These results demonstrate that CGA is a potent antioxidant that protects porcine oocytes from the negative effects of heat stress, thus reducing the frequency of apoptosis and improving the quality of embryos.     

Transporting bovine oocytes in a medium supplemented with different macromolecules and antioxidants: Effects on nuclear and cytoplasmic maturation and embryonic development in vitro

We investigated whether supplementing the medium used to transport bovine oocytes with different macromolecules [foetal calf serum (FCS) or bovine serum albumin (BSA)] or a mixture of antioxidants (cysteine, cysteamine and catalase) affects their nuclear and cytoplasmic maturation and thereby affects their subsequent embryonic development and cryotolerance. Oocytes were transported for 6 hr in a portable incubator and then subjected to standard in vitro maturation (IVM) for 18 hr. The oocytes in the control groups were cultured (standard IVM) for 24 hr in medium containing 10% FCS (Control FCS) or 10% FCS and the antioxidant mixture (Control FCS+Antiox). The intracellular concentrations of reactive oxygen species (ROS) at the end of IVM period were lower in the oocytes subjected to simulated transport in the presence of a macromolecular supplement or the antioxidant mixture than that of the control group (FCS: 0.62 and BSA: 0.66 vs. Control FCS: 1.00, p < .05; and Transp: 0.58 and Transp Antiox: 0.70 vs. Control FCS: 1.00, p < .05). After IVM, the mitochondrial membrane potentials of the transported oocytes were lower than those of the non‐transported oocytes (FCS: 0.41 and BSA: 0.57 vs. Control FCS: 1.00, p < .05; and Transp: 0.48 and Transp Antiox: 0.51 vs. Control FCS: 1.00 and Control Antiox: 0.84, p < .05). The blastocyst formation rates (36.9% average) and the re‐expansion rates of vitrified‐warmed blastocysts (53%, average) were unaffected (p > .05) by the treatments. In conclusion, supplementing the medium in which bovine oocytes are transported with antioxidants or different macromolecules did not affect their in vitro production of embryos or their cryotolerance.     

Sucrose assists selection of high‐quality oocytes in pigs

We investigated whether high‐quality in vitro matured (IVM) oocytes can be distinguished from poor ones based on the morphological changes after treatment with hyperosmotic medium containing 0.2 mol/L sucrose in pigs. We hypothesize that IVM oocytes maintaining round shape have higher quality than mis‐shapened oocytes following dehydration. Oocyte quality was verified by determining embryonic developmental competence using in vitro fertilization, nuclear transfer and parthenogenetic activation. In all cases, the round oocytes had greater (p < .05) developmental competence than that of mis‐shapened oocytes in terms of blastocyst rate and total cell number in blastocysts obtained after 6 days of in vitro culture. We also confirm that round aged oocytes are higher in quality than mis‐shapened aged oocytes. In an attempt to find out why high‐quality oocytes maintain a round shape whereas poorer oocytes become mis‐shapened following sucrose treatment, we examined the arrangement of actin microfilaments and microtubules. Abnormal organization of these cytoskeletal components was higher (p < .05) in mis‐shapened oocytes compared to round oocytes after 52 hr of IVM. In conclusion, sucrose treatment helps selection of high‐quality oocytes, including aged oocytes, in pigs. Abnormal cytoskeleton arrangements partly explain for low developmental competence of mis‐shapened oocytes.     

Effects of the E1 activating enzyme UBA2 on porcine oocyte maturation,apoptosis, and embryonic development in vitro

The purpose of this study was to investigate the effect of the E1 activating enzyme UBA2 on the expression of the SUMO-1 protein during in vitro maturation (IVM) of pig oocytes and embryonic development. In the 5 μg/ml UBA2 treatment group, the expression of the anti-apoptotic gene Bcl-2 and the embryo cleavage rate was significantly increased, while the proapoptotic gene Bax was significantly reduced. When 10 μg/ml UBA2 was added, the in vitro maturation rate, blastocyst rate, and SUMO-1 protein content of oocytes increased significantly (p < .05), and the expression of proapoptotic gene Caspase3 was significantly decreased (p < .05), while the viability of cumulus cells was extremely significantly reduced (p < .01). In summary, UBA2 can regulate the content of the SUMO-1 protein in mature pig oocytes in vitro, which in turn affects the maturation rate of oocytes, expression of apoptosis genes, cumulus cell viability, and the development of embryos after fertilization.     

Kaempferol alleviates the reduction of developmental competence during aging of porcine oocytes

Kaempferol (KAE) is a natural flavonoid present in different plant species and exhibits anti‐inflammatory, antioxidant, and anticancer therapeutic properties. In the present study, we investigated the influence and underlying mechanisms of KAE supplementation on porcine oocytes during in vitro aging. The results show that KAE treatment can alleviate the aging‐related reduction of developmental competence. We observed that the blastocyst production rate in aged oocytes treated with 0.1 μM KAE was significantly higher than in untreated aging oocytes (36.78 ± 0.86% vs. 27.55 ± 2.60%, respectively, p < .05). The KAE‐treated aging oocytes had significantly reduced levels of reactive oxygen species (p < .05). Furthermore, the mRNA levels of the embryonic pluripotency‐related genes Oct4, NANOG, and ITGA5 were significantly increased in blastocysts derived from KAE‐treated oocytes (p < .05). During excessive oocyte culture, KAE treatment maintained the mitochondrial membrane potential and reduced apoptosis; however, this was not observed in untreated aging oocytes. In conclusion, our results suggest that KAE treatment can alleviate the aging of porcine oocytes by reducing oxidative stress and improving mitochondrial function.     

Effects of E2 binding enzyme UBC9 on porcine oocyte maturation,apoptosis and embryo development

SUMOylation is a dynamic post-translational modification process. However, the function of small ubiquitin-like modifiers (SUMOs) in the maturation of porcine oocytes and embryo growth is not well known. Therefore, the aim of this study was to investigate the effect of E2 binding enzyme UBC9 on the expression of SUMO-1 protein during the in vitro maturation of porcine oocytes and embryo development after in vitro fertilization. Four groups were used: 0 (Control), 5, 10 and 15 µg/ml UBC9. Western blotting, flow cytometry and RT-qPCR were used to detect the in vitro maturation of porcine oocytes, SUMO-1 content, viability and the expression of apoptotic genes. Compared to those in the control treatment, the maturation rate (p < .05) and viability (p < .01) of oocytes in the 5 μg/ml treatment group decreased significantly. SUMO-1 protein markers appeared at 59 and 71 kDa and the content of SUMO-1 protein in the 10 µg/ml treatment group decreased significantly (p < .05). In the expression of apoptosis-related genes, Bcl-2 gene expression was significantly downregulated in the 10 μg/ml treatment group (p < .05). However, Bax and Caspase-3 were significantly upregulated in the 5 μg/ml treatment group (p < .05). During embryonic development, the cleavage rate of oocytes in the 10 µg/ml treatment group was significantly reduced (p < .05), whereas blastocyst formation rate in the 5 µg/ml treatment group was significantly reduced. UBC9 regulates SUMO-1 content in mature pig oocytes in vitro, which affects oocyte maturation rate, viability, apoptotic genes expression and embryo development after fertilization.     

The effects of canthaxanthin on porcine oocyte maturation and embryo development in vitro after parthenogenetic activation and somatic cell nuclear transfer

The objective of this study was to examine the effects of canthaxanthin (Cx) treatment during in vitro maturation (IVM) of porcine oocytes on embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), on intracellular glutathione (GSH) and reactive oxygen species (ROS) levels in mature oocytes, and on gene expression in both PA‐ and SCNT‐derived blastocysts. To determine the optimal effective concentration of Cx, porcine oocytes were cultured in IVM medium supplemented with various concentrations (0, 20, 40 and 80 μM) of Cx for 22 hr. Compared to other groups, supplementation with 40 μM Cx significantly improved blastocyst formation rates after PA (p < .05), but no significant differences were observed among groups in total blastocyst cell numbers. Subsequently, oocytes were cultured in IVM medium supplemented with or without 40 μM Cx. Oocytes treated with 40 μM Cx showed significantly increased cleavage and blastocyst formation rates after SCNT compared to the control group (p < .05). Moreover, significantly increased intracellular GSH and reduced ROS levels were observed in the Cx‐treated group (p < .05). In addition, both PA‐ and SCNT‐derived blastocysts from the 40 μM Cx‐treated group showed significantly increased mRNA expression of Bcl2 and Oct4 and decreased Caspase3 expression level (p < .05), when compared with the control group. PA‐derived blastocysts from the 40 μM Cx‐treated group also exhibited significantly decreased expression of Bax (p < .05). Our results demonstrated that treatment with 40 μM Cx during IVM improves the developmental competence of PA and SCNT embryos. Improvement of embryo development by Cx is most likely due to increased intracellular GSH synthesis, which reduces ROS levels in oocytes, and it may also positively regulate apoptosis‐ and development‐related genes.     

Effects of meiotic inhibitors and gonadotrophins on porcine oocytes in vitro maturation,fertilization and development

This study evaluated the effect of three reversible meiotic inhibitors (MINs) and their interaction with gonadotrophins (Gns) on the meiotic maturation and developmental competence of porcine oocytes. In experiment 1, the oocytes were matured for 22 hr in the presence or absence of dbcAMP (1 mM), cycloheximide (7 μM) or cilostamide (20 μM) with or without Gns, and for an additional 22 hr in the absence of MINs and Gns. At 22 hr of maturation, regardless of the presence of Gns, a higher proportion (p < .001) of oocytes cultured in the presence of MINs were effectively arrested at the germinal vesicle stage compared with the oocytes cultured without MINs. At 44 hr of maturation, the proportion of oocytes that reached MII was higher (p < .05) in groups with Gns compared with groups without Gns. In experiment 2, oocytes that were matured as in experiment 1 were inseminated and cultured for 7 days to evaluate fertilization parameters and blastocyst formation. Only oocytes from the dbcAMP + Gns group had higher (p < .05) efficiency of fertilization compared with the other treatment groups. The presence of dbcAMP during maturation also increased (p < .05) blastocyst formation and efficiency of blastocyst formation in both the presence and absence of Gns. These results indicate that the interaction of Gns with the tested MINs improved meiotic progression. In addition, regardless of supplementation with Gns, the presence of dbcAMP during the first maturation period increased and even doubled the capacity of oocytes to develop to the blastocyst stage.     

Oocyte holding in the Iberian red deer (Cervus elaphus hispanicus): Effect of initial oocyte quality and epidermal growth factor addition on in vitro maturation

Current in vitro embryo production protocols in the Iberian red deer (Cervus elaphus hispanicus) need to be optimized; oocyte harvesting in situ followed by overnight holding could reduce the human effort and shipping costs. In our work, post‐mortem ovaries were retrieved, and the oocytes were harvested and allocated to G1 group (good quality) or G2 + G3 group (low quality). The oocytes were separately subjected to immediate in vitro maturation (IVM) or held overnight in a holding medium composed of 40% of TCM 199 with Earle's salts, 40% TCM 199 with Hanks' salts and 20% fetal bovine serum (FBS), at room temperature (16 hr). In vitro maturation was carried out in a basal medium supplemented or not with 50 ng/ml of epidermal growth factor (EGF). Our data showed that addition of EGF to the maturation medium increases the percentage of G1 oocytes reaching metaphase II (3.9% vs. 50%, basal vs. EGF; p < .001) and decreased their degeneration rate (69.9% vs. 22.2%, basal vs. EGF; p < .01) when oocytes were immediately matured. Overnight holding increased the meiotic competence of G1 oocytes (37.5% matured in basal medium) and EGF increased prophase arrest in G2 + G3 oocytes (16.1% vs. 38.8% in germinal vesicle [GV] stage in basal medium vs. EGF added medium; p < .05). Our data demonstrate that oocyte holding can be used in Iberian red deer oocytes. Interestingly, EGF addition increases the oocytes' meiotic competence in immediately matured oocytes but not after oocyte holding depending upon initial oocyte quality.     

Use of protocatechuic acid as the sole antioxidant in the base medium for in vitro culture of ovine isolated secondary follicles

This study evaluated the effect of the protocatechuic acid (PCA) as the sole antioxidant in the base medium for in vitro culture of ovine secondary follicles. Secondary follicles (200‐230 μm) were isolated and cultured in α‐minimal essential medium supplemented with BSA, insulin, glutamine and hypoxanthine (α‐MEM: antioxidant‐free medium) or α‐MEM also added by transferrin, selenium and ascorbic acid (α‐MEM+: with antioxidant) or α‐MEM added by PCA (56.25; 112.5; 225; 450; or 900 μg/ml). Moreover, after culture, oocytes were matured and the chromatin configuration and DNA fragmentation were evaluated. After 12 days, the treatment containing 56.25 μg/ml PCA showed higher percentage of normal follicles than control medium or the other treatments (p < .05), except for 900 μg/ml PCA (p > .05). The antrum formation was significantly higher in treatments containing 56.25, 112.5 or 900 μg/ml PCA, compared to the α‐MEM and similar (p > .05) to the other treatments. The rates of fully grown oocytes (≥110 μm) were similar (p > .05) among all treatments containing PCA and α‐MEM+, and those were superior (p < .05) than α‐MEM, except for 450 μg/ml PCA (p > .05). GSH levels and mitochondrial activity were higher (p < .05) in α‐MEM+ than in α‐MEM and similar (p > .05) to all PCA treatments. The rates of meiotic resumption and DNA fragmentation were similar (p > .05) among α‐MEM+ and 56.25 μg/ml PCA. In conclusion, PCA at 56.25 μg/ml as the sole antioxidant added to the medium for ovine isolated secondary follicle culture maintains follicular survival, GSH and active mitochondria levels, meiotic developmental competence and DNA integrity of cultured oocytes.     

m‐carboxycinnamic acid bishydroxamide improves developmental competence,reduces apoptosis and alters epigenetic status and gene expression pattern in cloned buffalo (Bubalus bubalis) embryos

Incomplete or aberrant reprogramming of nuclear genome is one of the major problems in somatic cell nuclear transfer. In this study, we studied the effect of histone deacetylase inhibitor m‐carboxycinnamic acid bishydroxamide (CBHA) on in vitro development of buffalo embryos produced by Hand‐made cloning. Cloned embryos were treated with CBHA (0, 5, 10, 20 or 50 μM) for 10 hr from the start of reconstruction till activation. At 10 μM, but not at other concentrations examined, CBHA increased (p < .05) the blastocyst rate (63.77 ± 3.97% vs 48.63 ± 3.55%) and reduced (p < .05) the apoptotic index of the cloned blastocysts (8.91 ± 1.94 vs 4.36 ± 1.08) compared to untreated controls, to levels similar to those in IVF blastocysts (4.78 ± 0.74). CBHA treatment, at all the concentrations examined, increased (p < .05) the global level of H3K9ac in cloned blastocysts than in untreated controls to that observed in IVF blastocysts. Treatment with CBHA (10 μM) decreased (p < .05) the global level of H3K27me3 in cloned blastocysts than in untreated controls but it was still higher (p < .05) than in IVF blastocysts. CBHA (10 μM) treatment increased (p < .05) the relative expression level of pluripotency‐related genes OCT‐4 and NANOG, and anti‐apoptotic gene BCL‐XL, and decreased (p < .05) that of pro‐apoptotic gene BAX than in untreated controls but did not affect the relative expression level of apoptosis‐related genes p53 and CASPASE3 and epigenetics‐related genes DNMT1, DNMT3a and HDAC1. These results suggest that treatment of cloned embryos with 10 μM CBHA improves the blastocyst rate, reduces the level of apoptosis and alters the epigenetic status and gene expression pattern.     

A new three-dimensional glass scaffold increases the in vitro maturation efficiency of buffalo (Bubalus bubalis) oocyte via remodelling the extracellular matrix and cell connection of cumulus cells

At present, many three-dimensional (3D) culture systems have been reported, improving the oocyte quality of in vitro maturation (IVM), yet the mechanism still needs to be further explored. Here we examined the effects of a new self-made 3D glass scaffold on buffalo oocyte maturation; meanwhile, the underlying mechanism on buffalo oocyte maturation was also detected. Compared to the two-dimensional (2D) glass dish culture, results revealed that the 3D culture can improve the first polar body rate of oocytes, subsequent cleavage and blastocysts rate of parthenogenetic activation embryos (p < .05). The extracellular matrix-related proteins COL1A1, COL2A1, COL3A1, FN and cell connection-related proteins N-cadherin, E-cadherin, GJA1 were found higher in cumulus cells of 3D culture. Moreover, in cumulus cells, proteins of the PI3K/AKT pathway reported being regulated by FN and E-cadherin including PI3K P85 and p-AKT were also higher in 3D culture. Furthermore, proapoptosis proteins P53, BAX, caspase-3 were lower in both cumulus cells and oocytes in 3D culture, while proteins PCNA and BCL2 showed the opposite result. Results also showed that the apoptosis was inhibited, and the proliferation was enhanced in cumulus cells of 3D culture. Finally, the cumulus expansion-related genes HAS2, CD44, HMMR, PTX3, PTGS2 were found higher in cumulus cells of 3D culture. Taken together, the 3D culture could promote oocyte maturation by regulating proteins correlated with the ECM, cell connection and PI3K/AKT pathway, inhibiting the apoptosis of cumulus cells and oocytes, enhancing the proliferation of cumulus cells and the cumulus expansion.     

Developmental regulation and modulation of apoptotic genes expression in sheep oocytes and embryos cultured in vitro with L‐carnitine

The objective of this study was to find out the impact of L‐carnitine (10 mM) on developmental regulation of preimplantation sheep embryos cultured in vitro when supplemented in maturation medium and post‐fertilization medium separately. Subsequent objective was to observe the L‐carnitine‐mediated alteration in expression of apoptotic genes (Bcl2, Bax, Casp3 and PCNA) in sheep oocytes and developing embryos produced in vitro. Oocytes matured with L‐carnitine showed significantly (p < .05) higher cleavage (67.23% vs 43.12%), morula (47.65% vs 28.58%) and blastocysts (32.12% vs 13.24%) percentage as compared to presumptive zygotes cultured with L‐carnitine during post‐fertilization period. So it is suggested to use L‐carnitine during maturation than post‐fertilization period. Antiapoptotic and proliferative effects of L‐carnitine were confirmed by inducing culture medium with actinomycin D (apoptotic agent) and TNFα (antiproliferative agent), respectively, with and without L‐carnitine. Oocytes and embryos cultured with actinomycin D and TNFα showed developmental arrest with significant (p < .05) decrease in morula and blastocysts percentage but s upplementation of L‐carnitine to actinomycin D and TNFα induced culture medium showed similar result as that of control . L‐carnitine supplementation during IVM significantly (p < .05) upregulated the expression of Bcl2 and PCNA genes in majority of the developmental stages. Although L‐carnitine upregulated the expression of Bax in initial developmental stages but downregulated at latter part, whereas the expression of Casp3 was upregulated upto 16‐cell stage but after that there was no difference in expression. Expression of GAPDH gene was not affected by L‐carnitine supplementation. In conclusion, L‐carnitine acted as an antiapoptotic and proliferative compound during embryo development and supplementation of L‐carnitine during IVM altered the expression of apoptotic genes in the developmental stages of embryos.     

Effect of pterostilbene on development,equatorial lipid accumulation and reactive oxygen species production of in vitro-produced bovine embryos

The pterostilbene (PT) molecule is a phytoalexin with a reducing effect on reactive oxygen species (ROS) and with a capacity to block lipogenesis. However, the potential reducing effects of PT on equatorial lipid accumulation and ROS have not yet been elucidated for in vitro-derived bovine embryos. The present study evaluated the effects of concentrations of 3, 1, 0.33, 0.11 μM PT, and a vehicle group on the percentage of cleaved embryos, embryos with more than 6 cells, percentage of blastocyst on Day 7 and 8, percentage of transferable embryos on Day 7, the cell count and relative concentration of lipids. In the second experiment, the effects of 0.33 μM PT and a vehicle group within two different O₂ environments (5% and 20%) were evaluated for ROS generation and the percentage of Day 8 blastocysts. In the first experiment, no significant differences were found between the treatments with PT and the vehicle group (p > .05) concerning the percentage of cleaved embryos and embryos with more than 6 cells. Lipid reduction was observed in the groups treated with PT versus the vehicle group (p < .05). The vehicle group showed a higher rate of blastocyst production on Days 7 and 8 (p < .05) and an increase in the percentage of transferable embryos on Day 7 compared to the PT treatment groups (p < .05). Cell counts were not significantly different between treatments with PT and the vehicle group (p > .05). In the second experiment, the O₂ concentration did not significantly affect ROS generation (p > .05); however, the groups treated with PT (0.33 μM) had a reduction in ROS (p < .05). The O₂ concentration also did not significantly affect the rate of blastocyst production on Day 8 (p = .7696). Future research should be conducted to ascertain whether the reduction of lipids could enhance the cryopreservation and post-thaw viability of PT-treated embryos.     

Inhibition of PSMD4 alters ZP1 ubiquitination state and sperm–oocyte‐binding ability in pigs

The aim of this study was to determine how the duration of culture affects the ubiquitination of zona pellucida (ZP) proteins (ZP1, ZP2 and ZP3) during porcine oocyte maturation in vitro. We analysed the changes in ZP protein ubiquitination under three conditions: (i) during oocyte maturation from stage GV to MII; (ii) in oocytes cultured for different periods of time; and (iii) in oocytes treated with an antibody against PSMD4. Our results show that ZP1 and ZP2 are ubiquitinated at the GV stage, while ZP1, ZP2 and ZP3 are ubiquitinated at the MII stage, and band intensities for these proteins were significantly different between the GV and MII stages (p < .05). We also found that ubiquitination occurs in ZP1, ZP2 and ZP3 after cultured for 46, 52, 58 and 64 hr, and that the level of ubiquitinated ZP1 was significantly different in oocytes that were cultured for different time periods. Finally, treatment with an antibody against PSMD4 resulted in a significant decrease in ZP1 ubiquitination (p < .05), without affecting ZP2 or ZP3. The number of attached sperms per oocyte was also significantly different between control and anti‐PSMD4‐treated groups. Thus, we concluded that ZP1 and ZP2 are ubiquitinated at the GV stage, and ZP1, ZP2 and ZP3 are ubiquitinated at the MII stage. As the duration of culture increases, the ubiquitination levels of ZP proteins decrease. We also found that PSMD4 improves ZP1 ubiquitination during in vitro culture of porcine oocytes and effectively inhibits sperm–oocyte binding.     

Effects of supplementation of medium with different antioxidants during in vitro maturation of bovine oocytes on subsequent embryo production

The aim of this study was to assess the effects of different antioxidants on the levels of reactive oxygen species (ROS) and glutathione (GSH) in oocytes during in vitro maturation (IVM), as well as on the production of embryos. Oocyte of slaughterhouse‐derived cattle ovaries were placed in IVM with different antioxidants: quercetin (2 μM), cysteamine (100 μM), carnitine (0.5 mg/ml), vitamin C (50 μg/ml) or resveratrol (2 μM). Oocytes matured without any antioxidant supplementation were used as control. The oocytes were assessed for maturation rates and for ROS and GSH levels by fluorescence staining in 2′,7′‐dichlorodihydrofluorescein diacetate and Cell Tracker Blue, respectively. Embryo production was assessed in terms of cleavage, blastocysts and hatching rates and embryo cell numbers. The results expressed in arbitrary fluorescence units showed ROS reduction (p < .05) in the groups with quercetin (27.5 ± 3.4), vitamin C (27.1 ± 3.0) or resveratrol (28.1 ± 4.7), in comparison with those with cysteamine (34.9 ± 4.5), carnitine (34.6 ± 3.8) or to the control group (36.5 ± 5.2). GSH levels increased (p < .05) in cysteamine (63.5 ± 5.5) or carnitine (60.8 ± 4.4) groups in comparison with quercetin (52.7 ± 5.1), vitamin C (53.0 ± 3.8), resveratrol (53.1 ± 4.4) or to the control (49.6 ± 4.5). Nuclear maturation cleavage and hatched blastocysts rates did not differ (p > .05) between groups. However, blastocyst rates after in vitro fertilization in quercetin (53.5 ± 3.9%), vitamin C (52.1 ± 3.1%) resveratrol (54.2 ± 4.0%), cysteamine (52.4 ± 2.7%) or carnitine (54.2 ± 3.1%) groups were higher (p < .05) than in the control (47.2 ± 2.7%). Total cell numbers in embryos from the vitamin C, resveratrol, cysteamine or carnitine groups were higher than in quercetin and control groups, which were similar to each other. The results suggest that using antioxidants during IVM may reduce oxidative stress either by decreasing ROS levels directly or by increasing GSH levels in oocytes, depending on the type of antioxidant used. Overall, oxidative stress control during IVM with the antioxidants examined here improved blastocyst development with similar efficacy.     

Survivability and developmental competences of domestic cat (Felis catus) oocytes after Cryotech method vitrification

The aim of this study was to evaluate the applicability of the Cryotech technique for the vitrification of domestic cat (Felis catus) oocytes, as a model for other feline species threatened with extinction. This technique, in which oocytes are stored in a minimal volume of medium, is already widely used in human assisted reproductive technology. In the first part of this study, a viability test (EtBr/FDA) was used to evaluate the toxicity of the vitrification media (solutions). After IVM, oocytes were placed in vitrification and warming solutions according to the manufacturer's procedure, with or without exposure to liquid nitrogen. The solutions and the vitrification procedure each caused a reduction in oocyte viability, with survival rates of 71.4% in oocytes exposed to the Cryotech media (without cooling in liquid nitrogen), and 62% in oocytes that were vitrified. In the second part of the experiment, parthenogenetic activation was used to evaluate the developmental potential of oocytes previously vitrified using the Cryotech method. After warming, the oocytes were activated using a combination of 0.7 µM ionomycin in TCM 199 medium (5 min) followed by 2 mM 6-DMAP in TCM 199 supplemented with 10% FBS (3 hr), then cultured and evaluated every 24 hr for parthenogenetic cleavage. In the experimental group, 23/50 (46%) cleaved embryos were obtained. Domestic cat oocytes, vitrified by the Cryotech method, are characterized by high survival rates. However, it is necessary to improve the technique to increase the developmental competence of embryos obtained from vitrified oocytes.