The analysis of differences in gene expression, responding to cryopreservation may explain some of the observed differences in further development of the preimplantation stage embryos. The aim of this study was to create a link, for the first time, between morphological/developmental observations and gene activity changes following cryopreservation of embryos. Efficiency of two vitrification methods, solid surface and in-straw vitrifications for pronuclear-stage mouse zygotes and 8-cell stage mouse embryos was compared based on morphological survival, blastocyst formation, and changes in embryonic gene expression. Both stages of embryos were vitrified by SSV using 35% ethylene glycol (EG) for vitrification solution (VS) and in-straw vitrification using 40% EG for VS. No significant differences were found between immediate survival rates of embryos vitrified by SSV and in-straw vitrification in both stages. Blastocyst rates were significantly higher with SSV and not significantly different from that of control. These results showed that SSV was more efficient than in-straw vitrification. Treatment with cytochalasin-b did not improve cryosurvival during SSV. The quantification of selected gene transcripts from single embryo (6 embryos/treatment group) were carried out by quantitative real-time RT-PCR. It was performed by adding 1/8 of each embryo cDNA to the PCR mix containing the specific primers to amplify housekeeping gene (beta-actin), heat shock protein gene (Hsp70), genes related to oxidative stress (MnSOD and CuSOD), cold stress (CirpB, Rbm3), and cell-cycle arrest (Trp53). We found upregulation of all six stress-related genes at 3 hr post-warming in pronuclear stage embryos. Expression of these genes showed much higher level (2-33-fold) in in-straw vitrification than in in vitro control embryos. In SSV-treated embryos we could detect only slight changes (0.3-2-fold). At 10 hr post-warming, all genes were downregulated in embryos vitrified by in-straw method. In SSV-treated group expression of Hsp70 showed slight increase and Trp53 showed decrease. In contrast to pronuclear stage, there was no clear pattern of gene expression changes after vitrification in 8-cell stage embryos. Several genes were upregulated both at 3 and 10 hr post-warming. Moreover, we found upregulation of beta-actin gene which we expected to use as a reference gene in in-straw treated embryos in both 3 and 10 hr post-warming, while in pronuclear stage embryos and in SSV treatment there was no effect on beta-actin expression level. There was no difference in gene expression between blastocysts developed from fresh or vitrified embryos. In conclusion, the real-time RT-PCR method from single embryo opened new opportunities for the understranding of molecular events following cryopreservation. The upregulation of stress-related genes at 3 hr post-warming in pronuclear stage embryos might have been an early indicator of reduced viability following in-straw vitrification in good correlation with the developmental data to blastocyst stage.

The purpose of the present study was to investigate the effects of two vitrification procedures on developmental capacity and ultrastructural changes of matured swamp buffalo oocytes. In vitro-matured oocytes were vitrified by using 35 and 40% ethylene glycol as vitrification solution for solid surface vitrification (SSV) and in-straw vitrification (ISV), respectively. Survival rate of vitrified-warmed oocytes, evaluated on the basis of ooplasm homogeneity, oolemma integrity and zona pellucida intactness, as well as parthenogenetic blastocyst rates of vitrified-warmed oocytes were significantly higher with SSV (89.3 and 13.6%, respectively) than ISV (81.8 and 5.5%, respectively). However, they were still significantly lower than that of control oocytes (100 and 34.2%, respectively). For examining the ultrastructural changes, fresh, VS-exposed (ISV and SSV), and vitrified-warmed (ISV and SSV) oocytes were processed for transmission electron microscopy. In VS-exposed oocytes, reduction of microvilli abundance and damage of mitochondrial membrane were found only in the ISV group. In vitrified-warmed oocytes, however, it was clear that both methods of vitrification induced profound ultrastructural modifications to microvilli, mitochondria, oolemma and cortical granules as well as to the size and position of vesicles. Damaged mitochondria were, however, more abundant in ISV vitrified oocytes than in SSV vitrified oocytes, which correlated with the developmental data, showing the superiority of the SSV method. The present study demonstrated the feasibility of vitrification of in vitro-matured swamp buffalo oocytes.

BackgroundSpecies of the genus Borrelia are causative agents of Lyme disease and relapsing fever. Lyme disease is the most commonly reported vector-borne disease in the northern hemisphere. However, in some parts of the world Lyme borreliosis and relapsing fever may be caused by novel Borrelia genotypes. Herein, we report the presence of a Borrelia sp. in an Amblyomma varanense collected from Python reticulatus.MethodsTicks were collected from snakes, identified to species level and examined by PCR for the presence of Borrelia spp. flaB and 16S rRNA genes. Phylogenetic trees were constructed using the neighbour-joining method.ResultsThree A. varanense ticks collected from P. reticulatus were positive for a unique Borrelia sp., which was phylogenetically divergent from both Lyme disease- and relapsing fever-associated Borrelia spp.ConclusionThe results of this study suggest for the first time that there is a Borrelia sp. in A. varanense tick in the snake P. reticulatus that might be novel.

Long-tailed macaque (Macaca fascicularis) in the old town, Lopburi province rapid increase in their number as well nuisance activities. Strategies and management plans of Lopburi province, Thailand need to be developed and enacted in order to remedy some of the problems occurring in human-macaque interface zones. Therefore, this study aimed to investigate the macaque number and behavior, a survey on their population number and behavior was conducted from November, 2014 to May, 2015. Population estimation of long-tailed macaque was done by surveying seven macaque groups in the old town, Lopburi province: Prang Sam Yot shrine (GR1) Malai Rama theatre (GR2), Van station (GR3), Chayowanich building (GR4), Muang Thong hotel (GR5), Manora market (GR6) and Sengheng building (GR7). The identified macaque groups were visited during dusk or dawn and visual counts of each group were made carefully from close distance. One-way ANOVA, followed by Least Significant Difference (LSD) method was used for analysis of differences in macaque population number. Total estimated macaque population of seven groups range was between 1,920 and 2,080. Group size ranged from 70 to 700 individuals. Male and female sex ratio was estimated at 1:1.3. Behavior of M. fascicularis was observed by using scan sampling method for 216 hours during November, 2014 to May, 2015. The high levels frequency of behavior between macaque and macaque was feeding (20%) and inactive (29%). Food-related interaction (32%) was the highest frequency of behavior between macaque and human. Results suggested that factors related to macaque number and behavior for could be related to dietary resources. Moreover, feeding, playing, agonistic, moving, defecation and foodrelated interaction behavior were high frequency at period of 9 am. Conflicting, aggression and vocalization behavior were shown the most percentage of behavior frequency at 10 am and 4 pm. This data will be useful for management of respective locales was recommended to formulate practical strategies to avoid or decrease the interaction between macaque and human. The macaque population needs to be monitored and studied in depth to better understand their dynamics and behavioral adaptations to this old town.

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This study investigated the in vivo effect of Murraya paniculata leaf on gastrointestinal nematodes reduction, growth rates and haematological changes of goats. Four experimental groups (n = 6) of goats naturally acquired gastrointestinal nematodes were control (GI) (untreated), positive control (GII) (treated with albendazole, 6 mg/Kg BW) and GIII and GIV treated with 5 and 10 g/Kg BW of M. paniculata leaves. Fecal samples collections were performed weekly for assessment of fecal egg count. Number of nematode eggs in goat feces (egg per gram; EPG) was determined from week 0 (pre-treatment) to week 8 (post-treatment) by modified McMaster egg counting technique. EPG values of GIV goats were significantly lower than that of the controls and those receiving the albendazole (p<0.05) at week 3. GII goats showed highly effective at EPG reduction in 1-2 weeks after giving albendazole and then the efficiency decreased until the end of the experiment. The effect of M. paniculata leaves on growth rate was studied by examining goat weight on days 0, 30, 60 and 90. GIV group revealed significantly higher weight change than other groups (p<0.05). The feeding of M. paniculata leaves at different levels did not affect on haematological parameters, glucose, total protein, urea, albumin and globulin of goats. It was found that values of the haematological parameters were within the range of the standard values. Compound analysis of M. paniculata leaves revealed that M. paniculata leaves contained higher protein content than grass and the protein content was closed to the meal concentrate. This corresponds to the best growth rates of GIV goats. The results demonstrated that M. paniculata leaves reduce the gastrointestinal nematode eggs of goats efficiently, safely and environment-friendly.

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