Effects of oxygen tension and follicle cells on maturation and fertilization of porcine oocytes during in vitro culture in follicular fluid

The objective was to investigate the effects of oxygen tension and follicle cells (FCs) during in vitro maturation of porcine oocytes in only porcine (Sus scrofa domesticus) follicular fluid (pFF), using static and non-static (rotating) culture systems, on the nuclear maturation and subsequent in vitro fertilization of the oocytes. In the first experiment, cumulus-oocyte complexes (COCs) were matured for 48 h in pFF supplemented with (+) or without (−) FCs (5.2 × 10⁶ cells/mL), using the static (S) and rotating (R) culture systems (+FC/S, −FC/S, +FC/R, and −FC/R) under 5% or 20% O₂. Co-culture with FCs in the static culture system (+FC/S) had a detrimental effect on the meiotic competence of oocytes, whereas co-culture with FCs in the rotating culture system (+FC/R) increased maturation rates. In both culture systems, oxygen tension had no apparent effects on meiotic competence of oocytes, irrespective of culture system and FC addition. In the second experiment, COCs were matured under 5% or 20% O₂ using the −FC/S or +FC/R culture systems and then fertilized. Oxygen tension had no significant effects on fertilization parameters, irrespective of the culture system. The rotating culture system increased rates of sperm penetration and male pronuclear formation and decreased polyspermic fertilization compared with the static culture system (P < 0.05). In conclusion, both −FC/S and +FC/R culture systems supported meiotic competence, irrespective of oxygen tension. However, the +FC/R culture system may be superior to the −FC/S culture system for promoting fertilization.     

Effects of electric field strengths on fusion and in vitro development of domestic cat embryos derived by somatic cell nuclear transfer

The present study was conducted to determine the effect of electric field strength on the rate of membrane fusion between the somatic cell and cytoplast and on subsequent in vitro development of reconstructed embryos. Additionally, the in vitro developmental competence of cat oocytes artificially activated after 44 h of maturation culture was examined. An efficient fusion rate (64.2%) was obtained by applying a single pulse of 1.5 kV/cm for 50 micros, and the fusion rate remained almost constant at the higher field intensity (59.8 and 54.9% at 1.7 and 2.0 kV/cm, respectively). Although the cleavage rate of fused embryos increased with an increase of the electric field strength, there were no differences among the groups with respect to the proportion of development to the morula and blastocyst stages. In the additional experiment, oocytes at the metaphase II stage after culture for 44 h were activated by the combination of calcium ionophore (CaI) with cycloheximide (CHX). Some (11.8%) of activated oocytes developed to the blastocyst stage. Results from this study indicated that electric field strength affects the rates of fusion and cleavage but has no significant effects on the development to the blastocyst stage of reconstructed embryos. Prolonged maturation culture of cat oocytes (up to 44 h) decreased their ability to develop to the blastocyst stage.     

Knowledge and Perceptions of Highly Pathogenic Avian Influenza (HPAI) among Poultry Traders in Live Bird Markets in Bali and Lombok,Indonesia

Highly Pathogenic Avian Influenza (HPAI) has been prevalent in Indonesia since 2003 causing major losses to poultry production and human deaths. Live bird markets are considered high risk areas due to the density of large numbers of mixed poultry species of unknown disease status. Understanding trader knowledge and perceptions of HPAI and biosecurity is critical to reducing transmission risk and controlling the disease. An interview-administered survey was conducted at 17 live bird markets on the islands of Bali and Lombok in 2008 and 2009. A total of 413 live poultry traders were interviewed. Respondents were mostly male (89%) with a mean age of 45 years (range: 19–81). The main source of AI information was TV (78%), although personal communication was also identified to be an important source, particularly among female traders (60%) and respondents from Bali (43%). More than half (58%) of live poultry traders interviewed knew that infected birds can transmit HPAI viruses but were generally unaware that viruses can be introduced to markets by fomites. Cleaning cages and disposing of sick and dead birds were recognized as the most important steps to prevent the spread of disease by respondents. Two thirds (n = 277) of respondents were unwilling to report sudden or suspicious bird deaths to authorities. Bali vendors perceive biosecurity to be of higher importance than Lombok vendors and are more willing to improve biosecurity within markets than traders in Lombok. Collectors and traders selling large numbers (>214) of poultry, or selling both chickens and ducks, have better knowledge of HPAI transmission and prevention than vendors or traders selling smaller quantities or only one species of poultry. Education was strongly associated with better knowledge but did not influence positive reporting behavior. Our study reveals that most live poultry traders have limited knowledge of HPAI transmission and prevention and are generally reluctant to report bird deaths. Greater efforts are needed to engage local government, market managers and traders in education and awareness programs, regulatory measures and incentive mechanisms. Understanding and evaluating the social responses to such an integrated approach could lead to more effective HPAI prevention and control.     

Bacterial phospholipid molecular species analysis by ion-pair reversed-phase HPLC/ESI/MS

This work set out to optimize the detection and separation of several phospholipid molecular species on a reversed-phase column with the use of an electrospray ionization/mass spectrometry-compatible counter-ion. An application of this technique concerned a qualitative and quantitative analysis of bacterial membrane phospholipids extracted from Corynebacterium species strain 8. The phospholipid classes of strain 8 were identified as phosphatidylglycerol, phosphatidylinositol, diphosphatidylglycerol, and a peculiar lipid compound, acyl phosphatidylglycerol. Most of the molecular species structures were elucidated, and regarding phosphatidylglycerol, the fatty acid positions were clearly determined with the calculation of the sn-2/sn-1 intensity ratio of the fatty acyl chain fragments.     

Replanting reduces frog diversity in oil palm

A growing body of literature has demonstrated significant biodiversity losses for many taxa when forest is converted to oil palm. However, no studies have directly investigated changes to biodiversity throughout the oil palm life cycle, in which oil palm matures for 25–30 yr before replanting. This process leads to major changes in the oil palm landscape that likely influence species assemblages and ecosystem function. We compare frog assemblages between mature (21–27‐yr old) and recently replanted (1–2‐yr old) oil palm in Sumatra, Indonesia. Across eighteen 2.25‐ha oil palm plots, we found 719 frogs from 14 species. Frog richness was 31 percent lower in replanted oil palm (nine species) than mature oil palm (13 species). Total frog abundance was 47 percent lower in replanted oil palm, and frog assemblage composition differed significantly between the two ages of oil palm. The majority of frog species were disturbance‐tolerant, although we encountered four forest‐associated frog species within mature oil palm despite a distance of 28 km between our study sites and the nearest extensive tract of forest. Although it is clear that protection of forest is of paramount importance for the conservation of tropical fauna, our results indicate that management decisions within tropical agricultural landscapes also have a profound impact on biodiversity. Practices such as staggered replanting or maintenance of connectivity among mature oil palm patches could help maintain frog diversity in the oil palm landscape.     

Regulation of mucosal mast cell activation by short interfering RNAs targeting syntaxin4

Mucosal mast cells (MMCs) have an important role in allergic inflammation, and effective antagonists are required for their regulation. To discover a possible mechanism of controlling the activation of MMCs, we investigated the expression and function of syntaxin4, one of the soluble membrane N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, in RBL-2H3 cells, which is a rat mucosal mast cell line. Syntaxin4 silencing was induced by transfection of short interfering RNAs (siRNAs). Syntaxin4 was knocked down in mast cells at both the mRNA and protein levels. The release of granule contents that are involved in inflammation, such as histamine and hexosaminidase, was significantly suppressed by the gene silencing of syntaxin4. Silencing of this gene was also induced in the trachea and bronchi of rats by intratracheal application of the siRNAs using an atelocollagen delivery system. The activation of MMCs, which was monitored by the level of rat mast cell protease-II (RMCPII) in the bronchoalveolar lavage fluid (BALF), was inhibited, and asthmatic airway constriction was prevented by administration of the syntaxin/atelocollagen complex. These results indicate that siRNAs targeting syntaxin4 can stabilize mucosal mast cells and may have beneficial therapeutic effects on the asthmatic response.     

Influence of freezing with liquid nitrogen on whole-knee joint grafts and protection of cartilage from cryoinjury in rabbits

Improving survival rates for sarcoma patients are necessitating more functional and durable methods of reconstruction after tumor resection. Frozen osteoarticular grafts are utilized for joint reconstruction, but the joint may develop osteoarthritic change. We used a frozen autologous whole-rabbit knee joint graft model to investigate the influence of freezing on joint components. Thirty rabbit knee joints that had been directly immersed into liquid nitrogen (L) or saline (C) without use of cryoprotectants were re-implanted. Histological observations were made after 4, 8, and 12 weeks. Both groups had bone healing. In group L, despite restoration of cellularity to the menisci and ligaments, no live chondrocytes were observed and cartilage deterioration progressed over time. It was concluded that cryoinjury of chondrocytes caused osteoarthritic change. Then we tested whether a vitrification method could protect cartilage from cryoinjury. Full-thickness articular cartilage of rabbit knee was immersed into liquid nitrogen with and without vitrification. Histology, ultrastructure, and chondrocyte viability were examined before and after 24 h of culture. Vitrified cartilage cell viability was >85% compared with that of fresh cartilage. Transmission electron microscopy revealed preservation of original chondrocyte structure. Our vitrification method was effective for protecting chondrocytes from cryoinjury. Since reconstructing joints with osteoarticular grafts containing living cartilage avert osteoarthritic changes, vitrification method may be useful for storage of living cartilage for allografts or, in Asian countries, for reconstruction with frozen autografts containing tumors.     

Effects of oxygen tension on the development and quality of porcine in vitro fertilized embryos

The present study was conducted to examine the effect of oxygen tension during in vitro culture (IVC) of porcine oocytes/embryos on their development and quality using two different culture systems. Porcine cumulus oocyte complexes (COCs) were matured (IVM) and fertilized (IVF) in vitro, and subsequently cultured for 6 days in a simple and economical portable incubator or a standard CO(2) incubator. While the same temperature (38.5 degrees C) and CO(2) concentration (5%) were used in the both systems, the portable incubator was operated in a negative air pressure (- 300 mmHg) to create an O(2) level at 8-10% (low O(2) concentration), or in a positive air pressure (high O(2) concentration). To compare the two culture systems, IVM and IVF of COCs and subsequent IVC of in vitro produced (IVP) embryos were carried out in the portable incubator with a low O(2) concentration (Group I) or in the standard incubator with a high O(2) concentration (Group II). To assess the effect of O(2) concentration on IVC of IVP embryos, some oocytes that had been cultured in the standard incubator for IVM and IVF were subsequently cultured in the portable incubator with a low O(2) concentration (Group III) or a high O(2) concentration (Group IV). The occurrence of DNA fragmentation in the blastocysts produced under different culture conditions was examined by TUNEL staining to assess embryo quality. The rates of oocytes that reached MII and were penetrated by spermatozoa following IVF did not differ between the two incubation systems. In contrast, the proportions of development to blastocysts and the mean cell number of blastocysts in Group I were higher than those in Group II and Group IV. The index of DNA-fragmented nucleus in the blastocysts of Group I was significantly lower than that in the blastocysts of Group II. Therefore, low oxygen tension during IVM, IVF and IVC enhanced the subsequent development of IVP embryos to the blastocyst stage and improved their quality.     

Development of interspecies cloned embryos in yak and dog

Interspecies nuclear transfer (NT) could be an alternative to replicate animals when supply of recipient oocytes is limited or in vitro embryo production systems are incomplete. In the present study, embryonic development was assessed following interspecies NT of donor cumulus cells derived from yak and dog into the recipient ooplasm of domestic cow. The percentages of fusion and subsequent embryo development to the eight-cell stage of interspecies NT embryos were comparable to those of intraspecies NT embryos (cow-cow NT embryos). The percentage of development to blastocysts was significantly lower (p < 0.05) in yak-cow NT embryos than that in cow-cow NT embryos (10.9% vs. 39.8%). In dog-cow NT embryos, only one embryo (0.4%) developed to the blastocyst stage. These results indicate that interspecies NT embryos possess equally developmental competence to the eight-cell stage as intraspecies NT embryos, but the development to blastocysts is very low when dog somatic cells are used as the donor nuclei.