Optimizing the Conditions for In Vitro Maturation and Artificial Activation of Sika Deer (Cervus nippon hortulorum) Oocytes

With the goal of establishing experimental protocols for cloning sika deer, various conditions for in vitro maturation (IVM) and artificial activation of sika deer oocytes were examined. In vitro maturation was evaluated in seven different culture media. The highest rate of oocyte maturation was 75.4% in 10 μg/ml follicle‐stimulating hormone (FSH), 1 μg/ml LH, 0.2 mm cysteamine and 50 ng/ml epidermal growth factor (EGF) after 24 h of IVM. The efficiency after 24 h of IVM did not differ significantly (p > 0.05) from that observed after 20 h. Cysteamine (0.2 mm ) significantly increased the maturation rates after 20 h (from 59.1% to 67.2%, p < 0.05) and after 24 h (from 63.2% to 71.6%, p < 0.05) of IVM. The IVM rates of oocytes collected during the oestrous season (75.4%) and the anoestrous season (23.3%) were significantly different at 24 h. The 20 μg/ml FSH, 2 μg/ml LH, 0.4 mm cysteamine and 100 ng/ml EGF significantly increased the maturation rates (from 23.3% to 54.2%, p < 0.01) at 24 h during the anoestrous season. For the activation experiments, the most effective method was chemical activation [ionomycin + 6‐dimethylaminopurine (6‐DMAP)], which promoted the development of sika deer oocytes to the blastocyst stage (32.4%). Our results indicate that in vitro matured sika deer oocytes are good candidates for parthenogenetic activation and that chemical treatment is needed for relatively efficient activation of the oocytes. These optimized conditions for IVM and parthenogenetic activation may be useful for efforts to restore populations of the endangered sika deer using the somatic cell nuclear transfer technique.     

Comparative study of the developmental competence of cloned pig embryos derived from spermatogonial stem cells and fetal fibroblasts

Spermatogonial stem cells (SSC) are promising resources for genetic preservation and restoration of male germ cells in humans and animals. However, no studies have used SSC as donor nuclei in pig somatic cell nuclear transfer (SCNT). This study investigated the potential for use of porcine SSC as a nuclei donor for SCNT and developmental competence of SSC‐derived cloned embryos. In addition, demecolcine was investigated to determine whether it could prevent rupture of SSC during SCNT. When the potential of SSC to support embryonic development after SCNT was compared with that of foetal fibroblasts (FF), SSC‐derived SCNT embryos showed a higher (p < .05) developmental competence to the blastocyst stage (47.8%) than FF‐derived embryos (25.6%). However, when SSC were used as donor nuclei in the SCNT process, cell fusion rates were lower (p < .05) than when FF were used (61.9% vs. 75.8%). Treatment of SSC with demecolcine significantly (p < .05) decreased rupture of SSC during the SCNT procedure (7.5% vs. 18.8%) and increased fusion of cell‐oocyte couplets compared with no treatment (74.6% vs. 61.6%). In addition, SSC‐derived SCNT embryos showed higher blastocyst formation (48.4%) than FF‐derived embryos without (28.4%) and with demecolcine treatment (17.4%), even after demecolcine treatment. Our results demonstrate that porcine SSC are a desirable donor cell type for production of SCNT pig embryos and that demecolcine increases production efficiency of cloned embryos by inhibiting rupture of nuclei donor SSC during SCNT.     

Optimizing injection time of GFP plasmid into sheep zygote

Microinjection of exogenous DNA into the cytoplasm of matured oocytes or zygotes is a promising technique to generate transgenic animals. However, the data about the microinjection time and procedure in sheep are limited and have not treated in detail. To obtain more in-depth information, the Sarda sheep oocytes from abattoir-derived ovaries were subjected to IVM and IVF. Then, the GFP plasmid as a reporter gene was injected into the cytoplasm of MII oocytes (n: 95) and zygotes at different post-insemination intervals (6–8 hpi, n: 120; 8–10 hpi, n: 122; 10–12 hpi, n: 110 and 12–14 hpi, n: 96). There were no significant differences in the cleavage rates between the groups. However, blastocyst rate of injected zygotes at all-time intervals was significantly lower than injected MII oocytes and control group (p < 0.05). Interestingly, the proportion of GFP-positive embryos was higher at 8–10 hpi compared with other injected groups (4 % versus 0 %, p  < 0.01). Among these, the proportion of mosaic embryos was high and two of those embryos developed to the blastocyst stage. In conclusion, we settled on the cytoplasmic microinjection of GFP plasmid at 8–10 hpi as an optimized time point for the production of transgenic sheep and subsequent experiments.     

Melatonin Supplementation During In Vitro Maturation and Development Supports the Development of Porcine Embryos

Melatonin has been reported to improve the in vitro development of embryos in some species. This study was conducted to investigate the effect of melatonin supplementation during in vitro maturation (IVM) and development culture on the development and quality of porcine embryos. In the first experiment, when the in vitro fertilized embryos were cultured with different concentrations of melatonin (0, 10, 25 and 50 ng/ml) for 8 days, the blastocyst formation rate of embryos cultured with 25 ng/ml melatonin (10.7%) was significantly increased (p < 0.05) compared to the control embryos cultured without melatonin (4.2%). The proportion of DNA‐fragmented nuclei in blastocysts derived from embryos cultured with 50 ng/ml melatonin was significantly lower (p < 0.05) than that of embryos cultured without melatonin (2.1% vs 7.2%). In the second experiment, when oocytes were cultured in the maturation medium supplemented with different concentrations of melatonin (0, 10, 25 and 50 ng/ml), fertilized and then cultured with 25 ng/ml melatonin for 8 days, there were no significant differences in the rates of cleavage and blastocyst formation among the groups. However, the proportions (2.7–5.4%) of DNA‐fragmented nuclei in blastocysts derived from oocytes matured with melatonin were significantly decreased (p < 0.05) compared to those (8.9%) from oocytes matured without melatonin, irrespective of the concentration of melatonin. Our results suggest that supplementation of the culture media with melatonin (25 ng/ml) during IVM and development has beneficial effects on the developmental competence and quality of porcine embryos.     

Effects of demecolcine and sucrose on the incidence of cytoplasmic protrusions containing chromosomes in pig oocytes matured in vitro

The present study was carried out to examine whether demecolcine and sucrose affect the formation of a cytoplasmic protrusion containing chromosomes in pig oocytes independently or in combination. In the presence of 20 mM sucrose, the rates of oocytes with a cytoplasmic protrusion after culture for 60 min with 0.2-1.0 microg/ml demecolcine were significantly higher than those with 0.01-0.05 microg/ml demecolcine. When oocytes were cultured for 15 min in the presence of 0.2 microg/ml demecolcine and 20 mM sucrose, 35.1% of them extruded a cytoplasmic protrusion; this rate was significantly lower than those of oocytes cultured for 30-90 min. In the presence of 0.2 microg/ml demecolcine, significantly fewer oocytes extruded a cytoplasmic protrusion after culture for 30 min with 160 mM sucrose than with 0-80 mM sucrose. Significantly more oocytes extruded a cytoplasmic protrusion after culture for 30 min with 0.2 microg/ml demecolcine than without it, regardless of the presence or absence of 20 mM sucrose. In 88.9-100% of the oocytes, the cytoplasmic protrusions contained chromosomes with no significant differences among the different concentrations of demecolcine and sucrose and among the different treatment times. The results of the present study show that the cytoplasmic protrusion containing chromosomes in the pig oocyte is attributable to demecolcine, but sucrose does not affect its formation.     

Comparison of In Vitro Developmental Competence of Cloned Caprine Embryos Using Donor Karyoplasts from Adult Bone Marrow Mesenchymal Stem Cells vs Ear Fibroblast Cells

The aim of this study was to produce cloned caprine embryos using either caprine bone marrow‐derived mesenchymal stem cells (MSCs) or ear fibroblast cells (EFCs) as donor karyoplasts. Caprine MSCs were isolated from male Boer goats of an average age of 1.5 years. To determine the pluripotency of MSCs, the cells were induced to differentiate into osteocytes, chondrocytes and adipocytes. Subsequently, MSCs were characterized through cell surface antigen profiles using specific markers, prior to their use as donor karyoplasts for nuclear transfer. No significant difference (p > 0.05) in fusion rates was observed between MSCs (87.7%) and EFCs (91.3%) used as donor karyoplasts. The cleavage rate of cloned embryos derived with MSCs (87.0%) was similar (p > 0.05) to those cloned using EFCs (84.4%). However, the in vitro development of MSCs‐derived cloned embryos (25.3%) to the blastocyst stage was significantly higher (p < 0.05) than those derived with EFCs (20.6%). In conclusion, MSCs could be reprogrammed by caprine oocytes, and production of cloned caprine embryos with MSCs improved their in vitro developmental competence, but not in their fusion and cleavage rate as compared to cloning using somatic cells such as EFCs.     

Effect of Sericin Supplementation During In Vitro Maturation on the Maturation,Fertilization and Development of Porcine Oocytes

This study aimed to examine the effects of sericin supplementation during in vitro oocyte maturation on the nuclear maturation, fertilization and development of porcine oocytes. Cumulus‐oocyte complexes (COCs) were cultured in maturation medium supplemented with 0 (control), 0.1, 0.5, 1.0, 2.5 or 5.0% sericin and were then subjected to in vitro fertilization and embryo culture. More COCs matured with 1.0% sericin underwent germinal vesicle breakdown and reached metaphase II compared with the control COCs matured without sericin (p < 0.01). The proportions of oocytes with DNA‐fragmented nuclei did not differ between the groups, regardless of the sericin level. The total fertilization rate of oocytes matured with 1.0% sericin was higher (p < 0.05) than that of oocytes matured with 0.1%, 2.5% and 5.0% sericin. Supplementation with more than 1.0% sericin decreased the DNA fragmentation index of the blastocysts compared with the control group (p < 0.05). However, the supplementation of the maturation medium with sericin had no beneficial effects on the cleavage, development to the blastocyst stage and the total cell number of the embryos. Our findings indicate that supplementation with 1.0% sericin during maturation culture may improve the nuclear maturation and the quality of the embryos but does not affect blastocyst formation.     

Effect of Follicle Size on In Vitro Maturation of Pre‐Pubertal Porcine Cumulus Oocyte Complexes

Very small follicles (<3.0 mm diameter) are over‐represented on the surface of ovaries of non‐cycling pigs, and the oocytes collected from these follicles generally have reduced developmental competence in vitro. This study examined the effect of follicle size on the nuclear maturation (n = 608), the potential of parthenogenetic activation (n = 243) and the cyclic AMP (cAMP) content of pre‐pubertal porcine oocytes (n = 480). In addition, the influence of follicle size on steroid hormone synthesis was analysed. Cumulus oocyte complexes (COCs) flushed from small (2.5–4.0 mm) or large (4.5–6.0 mm) ovarian follicles were cultured for 0, 28 and 46 h. After 46 h of IVM, a greater proportion of oocytes from 4.5‐ to 6.0‐mm follicles reach metaphase II (MII) compared with those from follicles with 2.5–4.0 mm of diameter (96.1 vs 77.0%, respectively; p < 0.001). Parthenogenetic activation of oocytes from large follicles produced higher developmental rates than oocytes from large follicles (p < 0.05). At 28 h, the IVM medium with oocytes from large follicles contained significantly more 17ß‐oestradiol (E₂) than the medium with oocytes from small follicles (5.55 vs 3.45 ng/ml, respectively; p < 0.05) and at 46 h, the medium with oocytes from small follicles contained significantly more progesterone (P₄) than the medium with oocytes from large follicles (276.7 vs 108.2 ng/ml, respectively, p < 0.05). Porcine oocytes from large follicles have higher nuclear and cytoplasmic maturation capacities, but the differences did not appear to be cAMP‐mediated. Our findings also suggest that COCs from small follicles undergo more intensive luteinization than COCs from large follicles. The results show that oocytes from follicles with a diameter greater than 4.0 mm are more suitable for in vitro studies.     

Production of Wild Buffalo (Bubalus arnee) Embryos by Interspecies Somatic Cell Nuclear Transfer Using Domestic Buffalo (Bubalus bubalis) Oocytes

The objective of this study was to explore the possibility of producing wild buffalo embryos by interspecies somatic cell nuclear transfer (iSCNT) through handmade cloning using wild buffalo somatic cells and domestic buffalo (Bubalus bubalis) oocytes. Somatic cells derived from the ear skin of wild buffalo were found to express vimentin but not keratin and cytokeratin‐18, indicating that they were of fibroblast origin. The population doubling time of skin fibroblasts from wild buffalo was significantly (p < 0.05) higher, and the cell proliferation rate was significantly (p < 0.05) lower compared with that of skin fibroblasts from domestic buffalo. Neither the cleavage (92.6 ± 2.0% vs 92.8 ± 2.0%) nor the blastocyst rate (42.4 ± 2.4% vs 38.7 ± 2.8%) was significantly different between the intraspecies cloned embryos produced using skin fibroblasts from domestic buffalo and interspecies cloned embryos produced using skin fibroblasts from wild buffalo. However, the total cell number (TCN) was significantly (p < 0.05) lower (192.0 ± 25.6 vs 345.7 ± 42.2), and the apoptotic index was significantly (p < 0.05) higher (15.1 ± 3.1 vs 8.0 ± 1.4) for interspecies than that for intraspecies cloned embryos. Following vitrification in open‐pulled straws (OPS) and warming, although the cryosurvival rate of both types of cloned embryos, as indicated by their re‐expansion rate, was not significantly different (34.8 ± 1.5% vs 47.8 ± 7.8), the apoptotic index was significantly (p < 0.05) higher for vitrified–warmed interspecies than that for corresponding intraspecies cloned embryos (48.9 ± 7.2 vs 23.9 ± 2.8). The global level of H3K18ac was significantly (p < 0.05) lower in interspecies cloned embryos than that in intraspecies cloned embryos. The expression level of HDAC1, DNMT3a and CASPASE3 was significantly (p < 0.05) higher, that of P53 was significantly (p < 0.05) lower in interspecies than in intraspecies embryos, whereas that of DNMT1 was similar between the two types of embryos. In conclusion, these results demonstrate that wild buffalo embryos can be produced by iSCNT.     

Effect of Temporary Meiotic Attenuation of Oocytes with Butyrolactone I and Roscovitine in Resistance to Bovine Embryos on Vitrification

This study aimed to produce in vitro bovine embryos by the addition of two drugs, which is responsible for oocyte meiosis inhibition: roscovitine (ROS) and butyrolactone I (BL‐I). Oocytes were recovered from slaughtered cows and matured in a commercial medium and maintained in a 5% CO₂ atmosphere. Oocytes were maintained for 6 h in an in vitro maturation (IVM) medium containing ROS (12.5 μm ), BL‐I (50 μm ) and association of drugs (ROS 6.25 μm and BL‐I 25 μm ). Oocytes were cultured for 18 h in an agent‐free medium for the resumption of meiosis. After 24 h of maturation, oocytes were inseminated in the commercial in vitro fertilization (IVF) medium. Presumptive zygotes were cultured in SOFaa medium in a 5% CO₂ atmosphere. On day 3, rate of cleavage was evaluated and on days 6 and 7, rate of blastocyst formation. BL‐I and its association with the ROS increased the rates of cleavage and blastocyst formation (p < 0.05). The ROS alone was inefficient, impairing embryonic development, with low rates of blastocyst formation when compared to the control group and other treatments (p < 0.05). The embryos from BL‐I and ROS+BL‐I groups presented higher number of cells and lower rates of cellular apoptosis compared to other groups, either for the fresh or for post‐thawing embryos. Embryos from ROS+BL‐I group showed to be more resistant to the vitrification process, presenting a higher rate of embryonic re‐expansion (p < 0.05). In conclusion, block of meiosis using BL‐I or its association with ROS increased the rate of blastocyst formation, and the association of ROS+BL‐I resulted in a better resistance to the embryo cryopreservation process.     

Efficiency of Two Enucleation Methods Connected to Handmade Cloning to Produce Transgenic Porcine Embryos

The purpose of our work was to establish an efficient-oriented enucleation method to produce transgenic embryos with handmade cloning (HMC). After 41–42 h oocytes maturation, the oocytes were further cultured with or without 0.4 μg/ml demecolcine for 45 min [chemically assisted handmade enucleation (CAHE) group vs polar body (PB) oriented handmade enucleation (OHE) group respectively]. After removal of the cumulus cells and partial digestion of the zona pellucida, oocytes with visible extrusion cones and/or polar bodies attached to the surface were subjected to oriented bisection. Putative cytoplasts without extrusion cones or PB were selected as recipients. Two cytoplasts were electrofused with one transgenic fibroblasts expressing green fluorescent protein (GFP), while non-transgenic fibroblasts were used as controls. Reconstructed embryos were cultured in Well of Wells (WOWs) with porcine zygote medium 3 (PZM-3) after activation. Cleavage and blastocyst rates were registered on day 2 and day 7 of in vitro culture respectively. Meanwhile, the total blastocyst cell number was counted on day 7. We found that the difference was only observed between blastocyst rates (38.6 ± 2% vs 48.1 ± 3%) of cloned embryos with GFP transgenic fibroblast cells after CAHE vs OHE. With adjusted time-lapse for zonae-free cloned embryos cultured in WOWs with PZM-3, it was obvious that in vitro developmental competence after CAHE was compromised when compared with the OHE method. OHE enucleation method seems to be a potential superior alternative method used for somatic cell nuclear transfer (SCNT) with transgenic fibroblast cells.     

The Importance of Having Zinc During In Vitro Maturation of Cattle Cumulus–Oocyte Complex: Role of Cumulus Cells

The aim of this study was to investigate the influence of zinc (Zn) on the health of cumulus–oocyte complex (COC) during in vitro maturation (IVM). Experiments were designed to evaluate the effect of Zn added to IVM medium on: DNA integrity, apoptosis, cumulus expansion and superoxide dismutase (SOD) activity of cumulus cells (CC). Also, role of CC on Zn transport during IVM was evaluated on oocyte developmental capacity. DNA damage and early apoptosis were higher in CC matured with 0 μg/ml Zn compared with 0.7, 1.1 and 1.5 μg/ml Zn (p < 0.05). Cumulus expansion did not show differences in COC matured with or without Zn supplementation (p > 0.05). Superoxide dismutase activity was higher in COC matured with 1.5 μg/ml Zn than with 0 μg/ml Zn (p < 0.05). Cleavage and blastocyst rates were recorded after IVM in three maturation systems: intact COCs, denuded oocytes with cumulus cells monolayer (DO + CC) and denuded oocytes (DO). Cleavage rates were similar when COC, DO + CC or DO were matured with 1.5 μg/ml Zn compared with control group (p > 0.05). Blastocyst rates were significantly higher in COC than in DO + CC and DO with the addition of 1.5 μg/ml Zn during IVM (p < 0.01). Blastocyst quality was enhanced in COC and DO + CC compared with DO when Zn was added to IVM medium (p < 0.001). The results of this study indicate that Zn supplementation to IVM medium (i) decreased DNA damage and apoptosis in CC; (ii) increased SOD activity in CC; (iii) did not modify cumulus expansion and cleavage rates after in vitro fertilization; (iv) improved subsequent embryo development up to blastocyst stage; and (v) enhanced blastocyst quality when CC were present either in intact COC or in coculture during IVM.     

Study of the efficiency of chemically assisted enucleation method for handmade cloning in goat (Capra hircus)

The present investigation was carried out to find an efficient chemically assisted procedure for enucleation of goat oocytes related to handmade cloning (HMC) technique. After 22-h in vitro maturation, oocytes were incubated with 0.5 μg/ml demecolcine for 2 h. Cumulus cells were removed by pipetting and vortexing in 0.5 mg/ml hyaluronidase, and zona pellucida were digested with pronase. Oocytes with extrusion cones were subjected to oriented bisection. One-third of the cytoplasm with the extrusion cone was removed with a micro blade. The remaining cytoplasts were used as recipients in HMC. Goat foetal fibroblasts were used as nuclear donors. The overall efficiency measured as the number of cytoplasts obtained per total number of oocytes used was significantly (p < 0.05) higher in chemically assisted handmade enucleation (CAHE) than oriented handmade enucleation without demecolcine (OHE) (80.02 ± 1.292% vs. 72.9 ± 1.00%, respectively, mean ± SEM). The reconstructed and activated embryos were cultured in embryo development medium (EDM) for 7 days. Fusion, cleavage and blastocyst development rate were 71.63 ± 1.95%, 92.94 ± 0.91% and 23.78 ± 3.33% (mean ± SEM), respectively which did not differ significantly from those achieved with random handmade enucleation and OHE. In conclusion, chemically assisted enucleation is a highly efficient and reliable enucleation method for goat HMC which eliminates the need of expensive equipment (inverted fluorescence microscope) and potentially harmful chromatin staining and ultraviolet (UV) irradiation for cytoplast selection.     

Effect of Physiologically Relevant Heat Shock on Development,Apoptosis and Expression of Some Genes in Buffalo (Bubalus bubalis) Embryos Produced In Vitro

For investigating the effects of physiologically relevant heat shock, buffalo oocytes/embryos were cultured at 38.5°C (control) or were exposed to 39.5°C (Group II) or 40.5°C (Group III) for 2 h once every day throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC). Percentage of oocytes that developed to 8‐cell, 16‐cell or blastocyst stage was lower (p < 0.05) and the number of apoptotic nuclei was higher (p < 0.05) for Group III > Group II > controls. At both 8–16‐cell and blastocyst stages, relative mRNA abundance of stress‐related genes HSP 70.1 and HSP 70.2 and pro‐apoptotic genes CASPASE‐3, BID and BAX was higher (p < 0.05) in Groups III and II than that in controls with the exception of stress‐related gene HSF1. Expression level of anti‐apoptotic genes BCL‐XL and MCL‐1 was also higher (p < 0.05) in Groups III and II than that in controls at both 8–16‐cell and blastocyst stages. Among the genes related to embryonic development, at 8–16‐cell stage, the expression level of GDF9 was higher (p < 0.05) in Group III than that in controls, whereas that of GLUT1, ZAR1 and BMP15 was not significantly different among the three groups. At the blastocyst stage, relative mRNA abundance of GLUT1 and GDF9 was higher (p < 0.05) in Group II than that in controls, whereas that of ZAR‐1 and BMP15 was not affected. The results of this study demonstrate that exposure of buffalo oocytes and embryos to elevated temperatures for duration of time that is physiologically relevant severely compromises their developmental competence, increases apoptosis and affects stress‐, apoptosis‐ and development‐related genes.     

Cilostazol Improves Developmental Competence of Pig Oocytes by Increasing Intraoocyte Cyclic Adenosine Monophosphate Level and Delaying Meiotic Resumption

Cilostazol (CLZ) is a cyclic adenosine monophosphate (cAMP) modulator that influences the steady state of the meiotic stage. This study was conducted to determine the effects of CLZ treatment during in vitro maturation (IVM) on developmental competence of pig oocytes. Immature oocytes were exposed to 0 (control), 0.5, 2 and 4 μm CLZ during the first 22 h of IVM. Nuclear maturation, intraoocyte glutathione content and embryo cleavage after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) were not influenced by CLZ at any concentrations. However, 4 μm CLZ significantly (p < 0.05) improved blastocyst formation after PA (52.1% vs 38.7–46.0%) and SCNT relative to other concentrations (40.8% vs 25.0–30.7%). The mean cell numbers of SCNT blastocysts were significantly increased by 4 μm CLZ compared to the control (42.6 cells vs 35.3 cells/blastocyst). CLZ treatment significantly increased the intraoocyte cAMP level and effectively arrested oocytes at the germinal vesicle (GV) and GV break down stages compared to the control (74.5% vs 45.4%). Our results demonstrated that improved developmental competence of PA and SCNT pig embryos occurred via better synchronization of nuclear and cytoplasmic maturation induced by increased cAMP and delayed meiotic resumption after CLZ treatment.     

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.     

Relationship among ovarian follicular status,developmental competence of oocytes,and anti‐Müllerian hormone levels: A comparative study in Japanese wild boar crossbred gilts and Large White gilts

The aim of this study was to investigate the ovarian follicular development, developmental competence of oocytes, and plasma anti‐Müllerian hormone (AMH) levels of Japanese wild boar crossbred (wild hybrid) gilts, whose litter size is inferior to that of European breeds. Ovary and plasma samples were collected from two different breeds of gilts (wild hybrid and Large White breeds). The ovaries from the wild hybrid gilts had a lower average numbers of secondary follicles and vesicular follicles in ovarian cross‐sections and of good quality oocytes collected from ovarian follicles as compared with those from Large White gilts (p < 0.05). The development rate to the blastocyst stage of good quality oocytes after in vitro maturation, fertilization and culture was also lower (p < 0.05) in wild hybrid gilts than in Large White gilts. Plasma AMH levels with >0.16 ng/ml were detected in 8.3% of the examined wild hybrid gilts and 33% of the Large White gilts. These results indicate that the low reproductive performance of wild hybrid breed may result in part from low numbers of vesicular follicles and good quality oocytes, and low developmental competence of oocytes. Moreover, plasma AMH levels may support low number of vesicular follicles in ovaries of wild hybrid gilts.     

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.