Effects of the acid-tolerant engineered bacterial strain Megasphaera elsdenii H6F32 on ruminal pH and the lactic acid concentration of simulated rumen acidosis in vitro

The aim of this study was to examine the effects of the acid-tolerant engineered bacterial strain Megasphaera elsdenii H6F32 (M. elsdenii H6F32) on ruminal pH and the lactic acid concentrations in simulated rumen acidosis conditions in vitro. A mixed culture of ruminal bacteria, buffer, and primarily degradable substrates was inoculated with equal numbers of M. elsdenii H6 or M. elsdenii H6F32. The pH and lactic acid concentrations in the mixed culture were determined at 0, 2, 4, 6, 8, 10, 12, 14, 16, and 18 h of incubation. Acid-tolerant M. elsdenii H6F32 reduced the accumulation of lactic acid and increased the pH value. These results indicate that acid-tolerant M. elsdenii H6F32 could be a potential candidate for preventing rumen acidosis.     

Horizontal transmission dynamics of White spot syndrome virus by cohabitation trials in juvenile Penaeus monodon and P. vannamei

White spot syndrome virus (WSSV), a rod-shaped double-stranded DNA virus, is an infectious agent causing fatal disease in shrimp farming around the globe. Within shrimp populations WSSV is transmitted very fast, however, the modes and dynamics of transmission of this virus are not well understood. In the current study the dynamics of disease transmission of WSSV were investigated in small, closed populations of Penaeus monodon and Penaeus vannamei. Pair cohabitation experiments using PCR as a readout for virus infection were used to estimate transmission parameters for WSSV in these two species. The mortality rate of contact-infected shrimp in P. monodon was higher than the rate in P. vannamei. The transmission rate parameters for WSSV were not different between the two species. The relative contribution of direct and indirect transmission rates of WSSV differed between the two species. For P. vannamei the direct contact transmission rate of WSSV was significantly lower than the indirect environmental transmission rate, but for P. monodon, the opposite was found. The reproduction ratio R₀ for WSSV for these two species of shrimp was estimated to be above one: 2.07 (95%CI 1.53, 2.79) for P. monodon and 1.51 (95%CI 1.12, 2.03) for P. vannamei. The difference in R₀ between the two species is due to a lower host mortality and hence a longer infectious period of WSSV in P. monodon.     

Tembusu virus infection in Cherry Valley ducks: The effect of age at infection

Three groups of Cherry Valley ducks at 5 day, 2 week and 5 week of age were intranasally infected with the WFCL strain of Tembusu virus (TMUV) to investigate the effect of host age on the outcome of TMUV infection. For each age group, clinical signs, gross and microscopic lesions, viral copy numbers in tissues and serum neutralizing antibody titers were recorded. Age-related differences in the resistance to TMUV infection were observed with younger ducks being more susceptible. Some ducks infected at 5 day and 2 week of age developed severe clinical signs, including severe neurological dysfunction and death. However, subclinical signs and no mortality were observed in ducks infected at 5 week of age. A decline in the severity of gross and microscopic lesions was observed as ducks mature. Systemic infections were established in the three age groups post challenge. Higher viral copy numbers in the tissues, especially in vital organs such as the brain and the heart, were developed in the ducks infected at 5 day of age than older ducks, correlating with the severity of clinical signs and lesions in the tissues. Furthermore, ducks infected at 5 week of age developed significantly higher serum neutralizing antibody titers than ducks infected at 5 day of age as determined by serum neutralization test. Therefore, age-related differences in the resistance to TMUV infection should be considered when studying the pathogenicity, pathogenesis, formulation of the vaccination and therapy strategies of TMUV infection in ducks.     

Gene cloning of porcine adiponectin gene from adipose tissue and construction of its eukaryotic expression vector

To clone adiponectin (ADPN) gene from Shaziling porcine adipocyte and construct its eukaryotic expression vector, total RNA was extracted from subcutaneous fatty tissue. One pair of specific primers was designed by Primer 5.0 software according to the sequence of ADPN gene of porcine available in GenBank. The ADPN gene was amplified by PCR from cDNA and cloned into pMD18‐T vector to construct recombinant clonal vector pMD‐ADPN, sequenced and analysed. A recombinant expression plasmid pPICZaA‐ADPN was constructed by subcloning the cloned ADPN gene into the linearized pPICZaA vector. Then, the plasmid pPICZaA‐ADPN was expressed in Pichia pastoris (GS115) by electrotransformation. Western blot and Bradford analysis were used to determine the target protein induced by methanol. Results showed that the genome size of ADPN was 732 bp and encoded 244 amino acid, the nucleotide sequence of ADPN shared 100% identity with that of porcine available in GenBank. Western blot and Bradford analysis showed that the recombinant ADPN was expressed in GS115 correctly and has certain immune activity. The expression level of ADPN was 28.5 μg/ml. In conclusion, the recombinant ADPN could express in eukaryotic expression vector pPICZaA‐ADPN constructed in this study effectively.     

Apoptosis‐Like Events and In Vitro Fertilization Capacity of Sex‐Sorted Bovine Sperm

This study utilized three staining assays (Annexin V, mitochondrial membrane potential (JC‐1) and TUNEL) for flow cytometric analysis of apoptosis in sex‐sorted sperm from four different bulls (A, B, C and D). Correlations between sperm quality and IVF efficiency were then assessed to determine which assay provided the best prediction of IVF efficiency. The results of the Annexin V assays, as well as measures of viable sperm, early apoptosis, necrotic sperm and mitochondrial membrane potential (?ψm) showed that the sex‐sorted sperm collected from bull A significantly differed from those of the other three bulls (p < 0.05). In addition, the levels of DNA fragmentation in sex‐sorted sperm from bull A were significantly lower than those from bulls B and C (p < 0.05). The percentage of cells reaching the cleavage and blastocyst stages in sex‐sorted sperm from bull A were significantly greater than those from the other bulls (p < 0.05). A significant positive correlation was observed between viable sperm and the percentage of cells at the cleavage or blastocyst stages (p < 0.05). In contrast, a negative correlation was found between early apoptotic sperm and the percentage of cells at the cleavage or blastocyst stages (p < 0.05). In conclusion, these results indicated that the Annexin V assay was the most reliable technique for the prediction of the IVF success of sex‐sorted bovine sperm.     

Leucine markedly regulates pancreatic exocrine secretion in goats

Four goats (30.1 ± 1.3 kg) with common bile duct re‐entrant catheter and duodenal catheter were used to evaluate the effects of duodenal leucine infusion on pancreatic exocrine secretion and plasma parameters with two 4 × 4 Latin square design experiments. In the long‐term infusion experiment, goats were fed twice daily [700 g/day, dry matter (DM) basis] at 8:00 and 18:00 hours and were duodenally infused with 0, 3, 6, 9 g/day leucine for 14 days. Pancreatic juice and jugular blood samples were collected over 1‐h intervals for 6 h daily from d 11 to 14 days to encompass a 24‐h day. In the short‐term experiment, goats were infused leucine for 10 h continuously at the same infusion rate with Experiment 1 after feed deprivation for 24 h repeated every 10 days. Pancreatic juice and blood samples were collected at 0, 1, 2, 4, 6, 8 and 10 h of infusion. The results showed that the long‐term leucine infusion did not affect pancreatic juice secretion, protein output, trypsin and lipase secretion and plasma insulin concentration, but linearly increased α‐amylase secretion. No changes in pancreatic protein and lipase secretion were observed in the short‐term infusion. Pancreatic juice and α‐amylase secretion responded quadratically, with the greatest values observed in the 3 and 6 g/day leucine respectively. Trypsin secretion linearly decreased, while plasma insulin concentration increased linearly with increased leucine infusion. The results demonstrated that duodenal leucine infusion dose and time dependently regulated pancreatic enzyme secretion not associated with the change in plasma insulin concentration.     

Cryopreservation of marine microalgae and potential toxicity of cryoprotectants to the primary steps of the aquacultural food chain

Cryopreservation, a technique of high potential for culture collections, might offer a solution for reliable supply of microalgae in aquaculture units. Marine microalgae used in aquaculture were cryopreserved under 4, −20 and −80 °C using common cryoprotectants (methanol, dimethylsulfoxide (DMSO), propylene glycol and polyvinylpyrrolidone (PVP)) with promising results for Chlorella minutissima, Chlorella stigmatophora, Isochrysis galbana and Dunaliella tertiolecta. As cryoprotectants usually are toxic above certain concentrations and exposure time, and assuming that low amounts of cryoprotectants will remain in regenerated cultures, an experimental scheme was employed to explore the lower limits of safety for these algae and their primary consumers in hatchery food chains. Results showed that methanol was well tolerated by C. stigmatophora and D. tertiolecta up to a concentration of 1.6% (v/v) while I. galbana could not survive in culture at any concentration and C. minutissima exhibited some 30% of the control's yield at 0.2%. DMSO was highly tolerated up to 1.0% by all strains with the Chlorella strains surviving well up to 2%. Propylene glycol was not only tolerated up to 8% by Dunaliella but induced mixotrophic growth as well, while for Isochrysis it was lethal at any concentration. Among zooplanktonic consumers, brine shrimp Artemia nauplii could tolerate very high concentrations of the tested cryoprotectants, the rotifer Brachionus plicatilis was found sensitive to low amounts of PVP, while the nauplii of the shrimp Penaeus japonicus and the crab Eriocheir sinensis were in general very sensitive to all cryoprotectants and in several cases to much lower amounts than 1%. However, as long as the residues of cryoprotectants are kept below 1% in the regenerated cultures, there will be no problem with the animal consumers.