Journal of Microbiology and Biotechnology

1-8

Authors: Hong-Ye Zhao, Hong-Jiang Wei

The currently observed cloning efficiency is low and can be calculated using different methods. Therefore, a reasonable evaluation method for calculating the cloning efficiency in pigs is important for promoting the application of gene-modified miniature pigs in biomedical fields. In this study, we proposed a method for the scientific evaluation of cloning efficiency in pigs based on the production of cloned Diannan miniature pigs. Of 9 recipient gilts that were transferred with cloned embryos, 8 (88.9%) gilts became pregnant, and 7 (87.5%) of these delivered 37 piglets, 22 (59.5%) of which were alive and 17 survived to adulthood (over 1 year of age). Among the 15 stillborn piglets, 11 piglets experienced intrapartum death due to dystocia, and the other 4 died due to blastocolysis at the late stage of pregnancy. The greatest number of littermates was 11 piglets. The 3 piglets delivered by Caesarean section were alive and healthy. The procedure used for cloning pigs is a costly process in terms of both time and money; specifically, it requires 677.7-1265.4 min and $715.8-764.5 to transfer 50-400 cloned embryosto a recipient. The cloning efficiency defined as the total number of cloned piglets (including dead and mother piglets) divided by the total number of transferred embryos was 0.93%, whereas the cloning efficiency obtained based on the total number of live piglets divided by the total number of recipients was 2.44 piglets. The value is higher than those previously obtained using 19 different types of donor cells. These findings suggest that donor cells derived from Diannan miniature pigs are a valuable cell source for somatic cell nuclear transfer and the definition of cloning efficiency in pigs as “the total number of live cloned piglets divided by the total number of transferred recipient gilts” may be a reasonable scientific strategy.

1-8

Authors: Hongwei Rong1

To reveal the effect of dissolved oxygen (DO) on microorganism community structure during simultaneous nitrification and denitrification (SND) in sequencing biofilm batch reactor (SBBR), the molecular biological techniques of denaturing gradient gel electrophoresis (DGGE), cloning and real-time PCR were applied. DGGE sequence analysis showed some kinds of populations had been highly preponderance consistently, and cloning and phylogenetic tree of AOB gene revealed Nitrsospira was dominant species. The real-time PCR analysis indicated that the amount of the AOB and NOB both had most copy number in the lower surface, and the quantity relation of AOB and NOB population between three samples was “lower surface＞upper surface＞internal layer”. The result proved that DO concentration directly affected the proportion of aerobic fraction and anoxia fraction in the biofilm, which led to an effect on efficiency of SND.

1-9

Authors: Qingsen Chen

Objective: To explore the regulatory role of Casein glycomacropeptide (CGMP) in the fecal flora of mice with Ulcerative colitis (UC). Methods: The BALB/c mice were divided into a normal control group, a model group, CGMP groups with low doses (5 mg/kg·d), middle doses (50 mg/kg·d) and high doses (500 mg/kg·d), and a Sulfasalazine (SASP) group. Except the normal control group, all mice were treated with Oxazolone (OXZ) by perfusion to induce the UC model. In contrast, the mice in the normal control group and the model group were treated with an equal volume of saline for 7 consecutive days, which the changes in the fecal flora of the mouse intestines were evaluated by Fluorescence in situ hybridization (FISH) and DNA staining. Conclusion: CGMP can rectify the structural imbalances in the colon fecal flora in mice with UC and can improve the proportion of dominant flora in the intestine. CGMP at a dose of 50 mg/kg·d can significantly reduce the number of Enterobacteriaceae and Enterococci and promote the proliferation of Bifidobacteria, Lactobacilli, Bacteroides and Clostridium coccoides in the intestine.

1-8

Authors: Hong-Ye Zhao, Hong-Jiang Wei

The present study investigated the effect of delipation (lipid droplet removal) on the developmental competence of porcine oocytes. Delipated (+/−) and/or vitrified (+/−) oocytes were subjected to parthenogenetic activation, the viability of the early parthenogenetic embryos was recorded, and the embryos were then transferred to recipients for further development. The results showed that the cleavage and blastocyst rates of the parthenogenetic embryos from the early delipated oocytes were significantly lower than those of embryos from a control group (undelipated oocytes) (P<0.05). After transfer to recipient pigs, these embryos were able to further develop and to produce parthenogenetic fetuses. Using the minimum volume cooling (MVC) method, the parthenogenetic embryos from early delipated oocytes could be cryopreserved by vitrification at 1-cell to early blastocyst stages. After thawing, the early blastocyst stage was found to be optimal for vitrification. Also embryos vitrified at 2-4 cells derived from early delipated oocytes were transferred into two recipient pigs, and resulted in limb-bud stage fetuses. In conclusion, the results demonstrated that parthenogenetic fetuses can be produced from porcine embryos before or after vitrification by a strategic combination of in vitro-matured (IVM) oocyte delipation with vitrification at any early embryonic developmental stage. This approach may have application to cryopreservation of cloned and intracytoplasmic sperm injection (ICSI) embryos derived from delipated oocytes.

1-8

Authors: Luciana Konecny Kohn

The Hepatitis C virus (Flaviviridae family, Hepacivirus genus) represents a major public health problem worldwide and it is responsible for chronic infections in humans, which can develop to liver cirrhosis and hepatocellular carcinoma. As this virus does not replicate efficiently in cell culture and in animals, bovine viral diarrhea virus (BVDV) is used as a surrogate model for screening assays of antiviral activity, and mechanism of action assays. From marine invertebrates and their microorganisms isolated, we prepared extracts and fractions, and we isolated substances for assessment of their possible antiviral activity. Of the 71 tested, seven were considered promising presenting protection percentage of more than 80%. The best inhibition results were obtained from the extracts produced by the Gordonia bacteria samples with 99.9% inhibition and by Micrococcus with 99% inhibition. Furthermore, most of the extracts selected by the protection percentage showed selectivity index values considered promising, especially the extracts of the bacteria Williansia (SI=27) and Brachybacterium (SI=39). On the action mechanism, most of the promising extracts showed activity in the inhibition of intracellular replication steps, although it has been observed action of different extracts in several stages of viral replicative cycle. Thus, various extracts stood out and may lead to the development of drugs that ensure an alternative therapy for the treatment of hepatitis C.