To improve culture system for in vitro maturation (IVM) of porcine oocytes, ghrelin, leptin or growth hormone (GH), at concentration of 0, 0.5, 5, 50 and 500 ng/ml were added to the porcine follicular fluid (pFF)-supplemented medium NCSU23, and their effects on the maturation and cytoskeletal distribution of the oocytes with or without cumulus cells were compared. In the cumulus-denuded oocytes, no significant changes were noted in the maturation rate by different hormone treatments due to a marked decline in the controls. Maturation of the cumulus intact oocytes was moderately interfered by ghrelin (0.5-50 ng/ml, p < 0.01), but not significantly affected by leptin and GH. Distribution density of the cytoplasmic microtubules was decreased significantly by addition of ghrelin (by approximately 30% in 50-500 ng/ml, p < 0.01), whereas no remarkable effect was noted by leptin supplementation. High concentration (500 ng/ml) of ghrelin or leptin decreased significantly the cytoplasmic microfilaments in density (by 43% and 38%, p < 0.01, respectively). GH did not affect cytoskeletal distribution. The results suggest, in the culture system using pFF-supplemented medium that (i) ghrelin may have some inhibitory effect on the organization of microtubules and microfilaments, probably being a factor in lowered maturation rate and (ii) the addition of higher concentration of leptin may decrease microfilaments in density with no effect on meiotic maturation of the porcine oocytes.

No abstract

Abstract. Selection of bovine cumulus-oocyte complexes (COCs) for in vitro embryo production (IVP) is generally based on the morphological characteristics of the cumulus cells surrounding the oocytes and the ooplasm under microscopic observation. The purpose of this study was to examine a simple method for selection of COCs by sedimentation with Percoll solutions. COCs were aspirated from ovaries derived from a local slaughterhouse, and the COCs were classified by the morphology of their cumulus cell layers, as follows: Class A, compact and thick; Class B, compact but thin; Class C, partially denuded and thin; and Class D, denuded. Percoll solutions were prepared by diluting Percoll to 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5 and 30% solutions, respectively. COCs were placed on the surface of the Percoll solution for 3 min, and the precipitated COCs were transferred to stepwise high density solution. The percentage of Percoll solution just before buoyancy was considered to the specific sedimentary value of the COC and oocyte. The mean sedimentary value of Class A COCs was higher than those of the other classes (P<0.01). The mean sedimentary values of denuded oocytes from Classes A and B were higher than those from Classes C and D (P<0.01). Our results show that sedimentation of COCs and denuded oocytes was generally related to the morphological quality of the COCs, although the sedimentary values ranged widely for one class of COCs and oocytes. The Percoll method can be used for simple selection of COCs. Key words: Bovine, Cumulus-oocyte complex, Percoll, Sedimentation (J. Reprod. Dev. 53: [971][972][973][974][975][976] 2007) n vitro maturation, fertilization and culture technologies for bovine oocytes are beneficial for increasing the number of embryos transferable to recipient cows. Various factors, including oocyte quality, culture media, protein source, growth factors and oxygen tension for the in vitro embryo production system (IVP), affect preimplantation embryo development in vitro [1][2][3]. Oocyte quality is an important factor affecting the success of an IVP system. Selection of oocytes for in vitro maturation is generally based on the quality of the cytoplasm and the characteristics of the cumulus cell investment around the oocyte [4,5]. These morphological criteria are routinely used to select the cumulus-oocyte complexes (COCs) with the most competent oocytes for embryo development. However, subjective evaluation of oocyte quality by morphological criteria under microscopic observation is thought to differ among evaluators. Recently, other means of characterizing bovine oocytes have been examined, such as evaluating the corona radiata density of cumulus-corona-oocyte complexes [6], distribution of cortical granules in oocytes [7] and expression of the LH receptor in

Nitrogen-doped and sulfur-doped mechanochemically synthesized multilayer graphene (N-doped and S-doped MSMG) were prepared by planetary ball-milling, and they were used in bifunctional gas diffusion electrodes (GDEs) for the oxygen reduction reaction (ORR) and oxygen evolution reaction (OER). Graphite, melamine, and elemental sulfur were used as raw materials. The surface area-normalized linear sweep voltammograms revealed that the N-doped and S-doped MSMG have higher intrinsic ORR/OER activity than the undoped MSMG. When the MSMG samples were used in GDEs, the N-doped and S-doped MSMG showed higher OER activity but lower ORR activity than the undoped MSMG. We analyzed the relationship between the specific surface area, intrinsic ORR/OER activity, and ORR/OER activity of GDEs and found that both the intrinsic ORR activity and surface area are important in the fabrication of GDEs with high ORR activity and that the intrinsic OER activity rather than the surface area is important in the fabrication of GDEs with high OER activity. The GDE fabricated from the S-doped MSMG showed the highest ORR/OER bifunctional activity among the MSMG-based GDEs, and its ORR/OER bifunctional activity was higher than the GDEs fabricated from other materials, such as reduced graphene oxide and electroconductive oxides.

A simple method to evaluate the quality of in vitro-matured bovine oocytes is available for development of an in vitro embryo production system. Oocyte quality relates closely to oocyte fatty acid composition and mitochondrial distribution. The purpose of this study was to examine the influence of the quality of cumulus–oocyte complexes (COCs) and serum supplementation in IVM medium on the distribution of bovine oocyte specific gravities by sedimentation with Percoll before and after IVM. COCs were aspirated from abattoir-derived ovaries and were classified as classes A to D by the morphology of their cumulus cell layers as follows: class A, compact and more than 3 layers thick; class B, compact but &lt;3 layers; class C, partially naked and &lt;3 layers; and class D, naked or expanded. The classified COCs were cultured in TCM-199 supplemented with 0.1% BSA, 5 µg mL−1 insulin, 10 µg mL−1 transferrin, and 10 ng mL−1 transforming growth factor-α (M199-BITT) for 22–24 h. To evaluate the influence of serum supplementation, oocytes from classes A and B were also incubated in M199-BITT as serum-free culture or TCM-199 supplemented with 10% fetal calf serum as serum-supplemented culture. Percoll solutions were prepared by diluting Percoll with PBS supplemented with 0.3% BSA, 1 mg mL−1 glucose, and 0.2 mM sodium pyrvate to 20, 17.5, 15, 12.5, 10, 7.5, and 5% solutions. After removal of cumulus cells, denuded oocytes were put on the surface of Percoll solution for 3 min, and the precipitated oocytes were transferred to stepwise high density solution. The percent of Percoll solution just before buoyancy was considered as the oocyte specific gravity value. Statistical analysis was performed by one-way ANOVA. Oocytes from class A had the highest specific gravities before and after IVM in all classes (Table 1). After IVM, oocyte specific gravities from classes A and C were higher than those of oocytes before IVM (class A: P &lt; 0.05, class C: P &lt; 0.001). The specific gravities of in vitro-matured oocytes cultured in serum-free medium were higher than those cultured in serum-supplemented medium (15.3 ± 0.3%, n = 71, and 14.0 ± 0.3%, n = 58; P &lt; 0.01). These results show that the specific gravity was affected by the morphological quality of COC, and the culture conditions for IVM may profile the metabolic activity of oocytes during IVM. Table 1.Specific gravities of the bovine oocytes classified by morphology of COC before and after IVM