Cryopreservation of male and female gonial cells by vitrification in the critically endangered cyprinid honmoroko <Emphasis Type="Italic">Gnathopogon caerulescens</Emphasis>

We investigated the feasibility of cryopreservation of spermatogonia and oogonia in the critically endangered cyprinid honmoroko Gnathopogon caerulescens using slow-cooling (freezing) and rapid-cooling (vitrification) methods. Initially, we examined the testicular cell toxicities and glass-forming properties of the five cryoprotectants: ethylene glycol (EG), glycerol (GC), dimethyl sulfoxide (DMSO), propylene glycol (PG), and 1,3-butylene glycol (BG), and we determined cryoprotectant concentrations that are suitable for freezing and vitrification solutions, respectively. Subsequently, we prepared the freezing solutions of EG, GC, DMSO, PG, and BG at 3, 2, 3, 2, and 2 M and vitrification solutions at 7, 6, 5, 5, and 4 M, respectively. Following the cryopreservation of the testicular cells mainly containing early-stage spermatogenic cells (e.g., spermatogonia and primary spermatocytes), cells were cultured for 7 days and immunochemically stained against germ cell marker protein Vasa. Areas occupied by Vasa-positive cells indicated that vitrification led to better survival of germ cells than the freezing method, and the best result was obtained with 5 M PG, about 50% recovery of germ cells following vitrification. In the case of ovarian cells containing oogonia and stage I, II, and IIIa oocytes, vitrification with 5 M DMSO resulted the best survival of oogonia, with equivalent cell numbers to those cultured without vitrification. The present data suggest that male and female gonial cells of the endangered species G. caerulescens can be efficiently cryopreserved using suitable cryoprotectants for spermatogonia and oogonia, respectively.     

Control of Pseudomonas mastitis on a large dairy farm by using slightly acidic electrolyzed water

The disinfection effect of slightly acidic electrolyzed water (SAEW) use in a farm where Pseudomonas mastitis has spread was evaluated. Despite the application of antibiotic therapy and complete cessation of milking infected quarters, numerous new and recurrent Pseudomonas aeruginosa clinical mastitis infections (5.8–7.1% of clinical mastitis cases) occurred on the farm from 2003 to 2005. Procedural changes and equipment modifications did not improve environmental contamination or the incidence of Pseudomonas mastitis. To more thoroughly decontaminate the milking parlor, an SAEW system was installed in 2006. All milking equipment and the parlor environment were sterilized with SAEW (pH 5–6.5, available chlorine 12 parts per million) before and during milking time. After adopting the SAEW system, the incidence of clinical and subclinical Pseudomonas mastitis cases decreased significantly (P < 0.0001) and disappeared. These findings suggest that SAEW effectively reduced the incidence of mastitis in a herd contaminated by Pseudomonas species. This is the first report to demonstrate the effectiveness of disinfection by SAEW against mastitis pathogens in the environment.     

Cetacean Toll-like receptor 4 and myeloid differentiation factor 2, and possible cetacean-specific responses against Gram-negative bacteria

Toll-like receptor 4 (TLR4) and myeloid differentiation factor 2 (MD-2) are essential for recognizing the lipopolysaccharides (LPS) of Gram-negative bacteria. We determined the sequences of cDNAs encoding TLR4 and MD-2 from cetaceans and generated three-dimensional (3D) models for a better understanding of their modes of interaction and LPS recognition. The 3D reconstructions showed that cetacean TLR4 and MD-2 formed a horseshoe-like structure comprised of parallel β-strands and a β-cup structure consisting of two anti-parallel β-sheets, respectively. The (TLR4-MD-2)₂ duplex-heterodimer was shown to form a symmetrical structure. Comparison with the interfaces of the complexes in other mammals revealed that cetacean TLR4s have some amino acid residue substitutions involved in duplex-heterodimer formation and in species variation for LPS recognition. These substitutions in the changed amino acid residues may alter the interaction among TLR4, MD-2, and LPS and modify the TLR4/MD-2 immunological responses.     

Transmission of <Emphasis Type="Italic">Tomato chlorotic dwarf viroid</Emphasis> by bumblebees (<Emphasis Type="Italic">Bombus ignitus</Emphasis>) in tomato plants

Quantitative PCR revealed that Tomato chlorotic dwarf viroid (TCDVd) was present in substantial amounts in viroid-infected tomato flowers. Healthy tomato plants were arranged in two different glasshouses, and plants were mechanically inoculated with TCDVd. Bumblebees (Bombus ignitus) were then introduced into the glasshouses to reveal whether the viroid was transmitted from infected source plants to neighbouring healthy plants. TCDVd infection was found in neighbouring tomato plants more than 1 month after the introduction of the bees, some of which expressed symptoms, in both glasshouses. Thus, bumblebees transmitted TCDVd from tomato to tomato by pollination activities.     

Relationship between endogenous protein disulfide isomerase family proteins and glutenin macropolymer

The effects of endogenous protein disulfide isomerase (PDI) family proteins on the properties of gluten proteins in dough during breadmaking were determined using bacitracin, an inhibitor of PDI. Bread loaf volume in the presence of bacitracin was increased to 118% of that in the absence of bacitracin. The addition of bacitracin caused a decrease in the extension tolerance of the dough. The amount of sodium dodecyl sulfate (SDS)-insoluble glutenin macropolymer (GMP) in dough decreased to approximately 70% of that in flour during the 20 min of mixing for doughmaking. The addition of bacitracin to dough caused a dramatic GMP decrease, corresponding to ～20-30% of that in flour during the 20 min of mixing. The decrease in GMP was compensated by an increase in SDS-soluble glutenin polymer. Taken together, these results suggest that the endogenous PDI family proteins in flour suppress the depolymerization of GMP during dough mixing.     

Gut bacterial community profile in Pacific white shrimp Litopenaeus vannamei following 5‐aminolevulinic acid supplementation

Changes in the diet have been previously reported to alter the gut bacterial profile in several organisms, including shrimp. These shifts in microbial structure either promote beneficial effects to the host or cause diseases. Supplementation of 5‐aminolevulinic acid (5‐ALA), the precursor of tetrapyrroles, has been previously reported to enhance shrimp's immune response. To know whether 5‐ALA has effects on the gut bacterial structure in Pacific white shrimp (Litopenaeus vannamei), we investigated the bacterial communities in the intestine and stomach of L. vannamei using high‐throughput Illumina sequencing of 16S rRNA gene libraries. One week of 5‐ALA supplementation altered the bacterial community structure (beta diversity) in both tissues, as shown by the results of multi‐dimensional scaling (MDS) plots and analysis of similarities (ANOSIM). DESeq2 analysis revealed enrichment of differentially abundant taxa in the 5‐ALA group (e.g. Enhydrobacter and Oceaniovalibus) and control group (e.g. Tenacibaculum and Mycobacterium). Metagenomic predictions suggested that the control group had more KEGG pathways associated with ‘metabolism’ than the 5‐ALA group. This study suggests that 5‐ALA supplementation potentially promotes the formation of a beneficial bacterial community structure in shrimp. This is the first report on the effect of 5‐ALA supplementation on the bacterial community profile in any organism.     

Effects of high-K+, Na+-deficient solution on contractility of the smooth muscles of the bovine trachea

A high-K+, Na+-deficient (I-154 K+) solution induced contraction followed by gradual relaxation of the smooth muscles of the bovine trachea, while hyperosmotic addition of 65 mM KCl induced a large sustained contraction. Exposure of the muscle to the I-154 K+ solution induced an increase in the ratio of cellular water content and a sustained increase in oxidized flavoprotein fluorescence or reduced pyridine nucleotide fluorescence. The I-154 K+ solution also induced a sustained increase in [Ca2+]i level. Decreases in developed tension and increases in cellular water content were both prevented by the addition of sucrose or NaCl but not pyruvate. Substitution of KI for KCl in the I-154 K+ solution produced a greater inhibition of contraction, while substitution with K-propionate produced no inhibition of contraction. Moreover, decreases in developed tension and increases in cellular water content were both prevented by addition of 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), but not 10 microM bumetanide or 1 mM acetazolamide. In conclusion, I-154 K+ solution induced-relaxation in the bovine trachea may be due to swelling of smooth muscle cells and the mechanism of swelling is probably involved in DIDS-sensitive anion movement.     

Effects of 5'-uridylic acid feeding on postprandial plasma concentrations of metabolites and metabolic hormones in pre-weaning goats

5'-Uridylic acid (UMP), which is present at high concentrations in cow's colostrum, has been shown to cause a reduction in increased plasma levels of insulin and glucose after ingestion of milk replacer in pre-weaning calves. However, the precise mechanisms of UMP action have not been investigated, and its action has not been investigated in other pre-weaning ruminants. In order to demonstrate whether UMP causes changes in postprandial metabolic and hormonal parameters in pre-weaning goats, 11 Saanen kids were given milk replacer (twice a day) without ( n  = 5) or with ( n  = 6) UMP (1 g for each meal, 2 g/day for each head) for 14 days. Analysis of blood samples taken in the morning of day 14 demonstrated that the feeding of milk replacer with UMP abolished the significant changes in postprandial plasma glucose, NEFA, GH and insulin concentrations induced by feeding of milk replacer alone, and demonstrated a tendency to increase IGF-I levels. However, there was no significant difference between the two groups at any sampling time. We conclude that UMP feeding with milk replacer showed a tendency to blunt the postprandial changes in levels of some plasma metabolites and hormones that are induced by replacer alone in pre-weaning goats.     

Experimental infection of pigs with different doses of the African swine fever virus Armenia 07 strain by intramuscular injection and direct contact

We experimentally infected pigs with the African swine fever virus (ASFV) Armenia 07 strain (genotype II) to analyze the effect of different dose injections on clinical manifestations, virus-shedding patterns, histopathology, and transmission dynamics by direct contact. Each three pigs and four pigs were injected intramuscularly with 0.1 fifty percent hemadsorbing doses (HAD₅₀)/ml, 10¹ HAD₅₀/ml and 10⁶ HAD₅₀/ml of ASFV Armenia 07 strain, respectively. Each two of three pigs injected with 0.1 HAD₅₀/ml and 10¹ HAD₅₀/ml died by 10 days post inoculation. All pigs had a gross lesion of splenomegaly. Perigastric and renal lymph nodes were enlarged and resembled blood clots in nine of ten pigs. It was revealed that 0.1 HAD₅₀/ml of this ASFV was sufficient to infect healthy pigs by intramuscular injection and caused sub-acute lethal disease. For the transmission study, two 8-week-old pigs were injected intramuscularly with 10³ HAD₅₀/ml of the same virus. Each of the experimentally inoculated pigs was co-housed with two 8-week-old naive pigs. All contact pigs exhibited clinical manifestations at 6 or 7 days after the experimentally inoculated pigs developed pyrexia. These findings suggest that this strain may spread slowly within a herd. Histologically, lymph nodes resembled blood clots were formed by severe blood absorption and followed hemorrhage result of disruption of the lymphoid sinus filling with absorbed red blood cells. The severity of the gross and histological lesions depended on duration after infection, regardless of the difference of injection doses in this study.