Oxygen tension and medium supplements for in vitro maturation of bovine oocytes cultured individually in a chemically defined medium

The effects of adding cysteamine, EGF, and glucose as an energy substrate under low oxygen tension during in vitro maturation (IVM) were examined to find ways of improving the individual in vitro production (IVP) system in individually cultured bovine oocytes. The basic medium was mSOFaa containing 1 mg/ml polyvinyl alcohol. Immature oocytes were individually cultured in an IVM medium with 10 ng/ml EGF, 100 microM cysteamine, or EGF plus cysteamine under 20% or 5% O(2). Cleavage and blastocyst rates were significantly higher (P<0.05) in IVM culture was under 20% O(2) than in culture under 5% O(2). Under 5% O(2), neither EGF nor cysteamine improved embryonic development. The proportion of matured oocytes was significantly higher (P<0.05) in the presence of 1.5 mM glucose under 20% O(2) (68.6%), and 5.5 mM (66.7%) and 10 mM (65.5%) glucose under 5% O(2). The presence of 5.5 mM glucose significantly (P<0.05) increased the maturation rate compared with the absence of glucose, irrespective of addition of EGF and cysteamine. The addition of cysteamine alone in the maturation medium significantly (P<0.05) increased the intracellular GSH concentration in the oocytes. Also, under 5% O(2) cysteamine and/or EGF significantly (P<0.05) improved the proportions of penetrated oocytes, cleavage and blastocyst formation, which were similar levels to those of oocytes matured under 20% O(2). After vitrification, the re-expanding and hatching rates of blastocysts derived from the individual IVP system containing cysteamine under 5% O(2) were significantly (P<0.05) higher than those of blastocysts derived from the individual IVP system without cysteamine under 5% O(2) and the group IVP system under 20% O(2). The present study showed that a high glucose level (5.5 or 10 mM) was optimal in IVM culture under low (5%) oxygen tension. The addition of EGF and/or cysteamine to the maturation medium had no positive effect on nuclear maturation, but improved fertilizability, developmental competence and cryoresistance following vitrification, probably due to increased GSH synthesis during the IVM process.     

Translocation of intrauterine-infused bacterial lipopolysaccharides to the mammary gland in dexamethasone-treated goats

Our previous study showed that intrauterine-infused lipopolysaccharide (LPS) can be translocated to the mammary gland to induce weak inflammation. This study aimed to determine whether dexamethasone treatment facilitated the translocation of LPS from the uterus to the mammary gland to induce a heavy inflammatory response. Sixteen goats were divided into control and LPS groups, subjected to daily dexamethasone administration before saline or LPS infusion. Milk and blood samples were collected before and after LPS infusion to determine the milk yield and somatic cell count (SCC) and blood leucocyte count (BLC), cytokines, antimicrobial peptides and serum amyloid A (SAA) concentrations. Mammary gland tissues were collected from two goats before and 24 hr after LPS infusion for immunohistochemical analysis of LPS. The mean SCC in the LPS group was significantly higher, whereas the milk yield was significantly lower than that in the control group after LPS infusion. The mean BLC in the LPS group was significantly lower than in the control group after LPS infusion. Furthermore, milk concentrations of IL-1β, S100A8 and lactoferrin were higher in the LPS group than in the control group after infusion. LPS was detected in the connective tissues and inner alveolar spaces of the mammary glands 24 hr after LPS infusion. We concluded that dexamethasone administration facilitated the translocation of intrauterine-infused LPS to the mammary gland, where it induced an inflammatory response. Therefore, LPS translocated from other organs, such as the uterus, can induce heavy inflammation in the mammary gland under immunosuppressive conditions.     

Utilization patterns of estuarine and marine habitats by the halfbeak Zenarchopterus dunckeri at Iriomote Island,southern Japan,evaluated from otolith microchemistry

Estuarine and marine habitat use patterns in the halfbeak Zenarchopterus dunckeri were examined at Iriomote Island, southern Japan, by analyzing otolith Li/Ca and Sr/Ca ratios. The ranges of both Li/Ca and Sr/Ca ratios in juvenile Z. dunckeri from the maximum (30 psu) to minimum (0.5 psu) salinity levels of brackish water estimated from rearing experiments, were compared with those of wild individuals collected from upstream and downstream stations in the Urauchi River estuary. The majority of wild-caught individuals had invariable Li/Ca and Sr/Ca ratios along an otolith transect from the core to the posterior edge, which fell within the otolith Li/Ca and Sr/Ca ranges estimated for estuarine individuals in the rearing experiments, suggesting that such individuals developed within the estuary without migrating to a marine environment at any time, although some downstream-dwelling fish had higher otolith elemental ratios than the predetermined estuarine ranges in the mid transect section. The latter fish may have been accidentally flushed from the estuary into the sea by heavy flood events, subsequently returning to the estuary. The overall results suggested that Z. dunckeri is essentially an estuarine resident, completing its life cycle within an estuarine system.     

Number of nitrate- and nitrite-reducing Selenomonas ruminantium in the rumen, and possible factors affecting its growth

The cell number of Selenomonas ruminantium (S. ruminantium) that reduces nitrate and nitrite in the rumen was usually 8–10% of the total number of S. ruminantium (an order of 10⁶/mL). The percentage was not affected by the roughage/concentrate ratio or nitrate content of the diet in 2 weeks. However, feeding a high‐nitrate diet for 12 weeks increased the percentage. The percentage of lactate‐using S. ruminantium, such as the ssp. lactilytica, was less than 1% of the total number of S. ruminantium. No S. ruminantium was found that used formate as an electron donor for nitrate and nitrite reduction. Lactate and H₂ appeared to be important for nitrate and nitrite reduction by S. ruminantium. Nitrate reduction by S. ruminantium was enhanced by the coexistence of amylolytic bacteria in a medium containing starch, and as a result, nitrite accumulation increased. Coexistence of cellulolytic bacteria facilitated the growth of S. ruminantium in a medium containing cellulose, and consequently increased nitrite reduction. In order to suppress nitrite accumulation in the rumen, it may be important to enhance fiber digestion.     

Isolation and expression of five-amino-acid-deleted variant of feline hepatocyte growth factor (HGF) cDNA

Hepatocyte growth factor (HGF) is a pleiotropic cytokine that stimulates a wide array of cellular targets, including hepatocytes and other epithelial cells, melanocytes, endothelial and hematopoietic cells. We have cloned a different form of cDNA, with a deletion of 15 base pairs predicted to result in the loss of 5 amino acids from the first kringle domain. To investigate the biological activity, original and deleted variant of feline HGF cDNAs were transiently expressed in COS-7 cells. Both recombinant feline HGFs showed almost the same dose-response curves in the stimulation of the growth of BNL CL.2 cells (a mouse hepatocyte cell line) and scatter activity of Madin-Darby canine kidney (MDCK) cells. The findings reported here suggest that the deleted variant of feline HGF has almost the same biological activity as the original in terms of the proliferation and scatter activity.     

Axial correction of pes varus by transverse-opening wedge osteotomy and T-plate fixation with beta-tricalcium phosphate (beta-TCP) transplantation in dachshunds

Axial correction was performed surgically in two miniature dachshunds presenting with lateral patellar dislocation and limping caused by pes varus. Pes varus had resulted from asymmetric closure of the physis of the distal tibia. Prior to surgery, osteotomy was simulated by measuring X-ray films to determine the distance required for the wedge opening. Transverse-opening wedge osteotomy was performed on the medial side of the distal tibia, and beta-tricalcium phosphate (beta-TCP) was inserted in a wedge shape into the area created by the cuneiform osteotomy. Finally, the tibia was fixed by a veterinary 1.5/2.0-mm T-plate. Both dogs were able to walk a few days after surgery, and the lateral dislocation of the patella normalized almost completely in about one month. At two months, X-ray films showed that the implant had remained in position without any dislocation, and the beta-TCP had fused with the surrounding bone.     

Detection of cone dysfunction induced by digoxin in dogs by multicolor electroretinography

It is difficult to detect discrete cone function with the present conventional electroretinography (ERG) examination. In this study, we developed contact electrodes with a built-in color (red (644 nm), green (525 nm), or blue (470 nm)) light source (color LED-electrode), and evaluated an experimental model of digoxin in the dog. First, 17 normal Beagle dogs were used to determine which electrode works well for color ERG measurement on dogs. Then, color ERG was performed on seven normal Beagle dogs at various points during a 14-day period of digoxin administration. A single daily dose of 0.0125 mg/kg/day, which is within the recommended oral maintenance dosage range for dogs, was administered orally for 2 weeks. Ophthalmic examination, measurement of plasma concentration of digoxin, and color ERG examination were performed. On first examination, amplitudes of all responses were significantly (P < 0.01) lower with the red, than with the blue and green electrodes during ERG recording. In ERG using the red electrode, the standard deviation was large. According to these preliminary results, the red electrode was not used in the experimental dog model with digoxin. In the digoxin administrated animals, no significant change was observed in the ophthalmic examination findings. The digoxin level increased steadily throughout the dosing period but was always within the therapeutic range for dogs. In rod ERG, no abnormalities were detected with any electrode. In standard combined ERG, decreased amplitude of the a-wave was detected with every electrode. In single flash cone ERG, prolongation of implicit time was detected by color ERG with the blue and green electrodes. In 30-Hz flicker ERG, decreased amplitude was detected only by color ERG with the blue electrode. The decreased amplitude and prolonged implicit time recovered after termination of digoxin administration. Cone dysfunction induced by digoxin in the dog was revealed by multicolor ERG using blue and green LED-electrodes. Multi-color ERG was useful for detecting cone type-specific dysfunction in the dog.     

Effect of distance from early flowering pollinizers ‘Maypole’ and ‘Dolgo’ on ‘Fiji’ fruit set

An apple orchard consisting of a single cultivar under the condition of natural pollination must have suitable pollinizers such as Crab apples to ensure stable fruit production. We selected ‘Maypole’ and ‘Dolgo’ as pollinizers for the cultivar ‘Fuji’, and investigated the rate of fruit and seeds in ‘Fuji’ fruits produced by pollen of the pollinizers. We developed a method for tracing pollen flow based on the leaf color of progeny and S-RNase allele of ‘Maypole’, and on Simple Sequence Repeat (SSR) analyses of ‘Maypole’ and ‘Dolgo’. These were powerful tools for determining the distance insects (mainly Osmia cornifrons) carry pollen from the pollinizers to ‘Fuji’. Although the fruit set of ‘Fuji’ apples was not reduced with increasing distance between ‘Fuji’ apple trees and pollinizers (probably due to pollen flow from other commercial cultivars planted outside the area), the rate of ‘Fuji’ apple fruit produced by the pollen of the pollinizers decreased with increasing distance. The rate of fruit produced by the pollinizers was 84% and 77% when ‘Fuji’ was 2.5 m from ‘Maypole’ and ‘Dolgo’, respectively, and 71% and 64% when ‘Fuji’ was 5 m from ‘Maypole’ and ‘Dolgo’, respectively, but was reduced to 47% and 39% when ‘Fuji’ was 10 m from ‘Maypole’ and ‘Dolgo’, respectively. However, the spacing could cause reduced fruit size, and require extra fruit thinning for producing large fruits. It could also cause reduction of the yield for increasing the planting area of pollinizers. We recommend that pollinizers should be planted not more than 10 m from ‘Fuji’.     

Relationship between arginine vasopressin and high signal intensity in the pituitary posterior lobe on T1-weighted MR images in dogs

The posterior lobe of the pituitary gland (PL) normally shows characteristic high signal intensity (SI) on T1-weighted MR images (T1WI) in humans. The high SI is thought to represent storage of arginine vasopressin (AVP) in the PL. Normal dogs also show a high SI on T1WIs, but the origin is unclear. In the present study, we investigated whether the high SI in the PL on T1WIs in normal dogs is caused by AVP. We examined the SI in the PL on T1WIs, plasma AVP concentrations and plasma osmolality in normal dogs after excessive AVP secretion was induced by hypertonic saline overload. In addition, functional changes in the supraoptic nucleus and paraventricular nucleus of the hypothalamus under AVP secretion-stimulated conditions were examined immunohistologically. Under hypertonic saline overload, plasma osmolality and plasma AVP concentrations gradually increased, while the SI of the PL gradually decreased. This suggests that AVP secretion was stimulated by elevated osmolality. Moreover, there was a significant negative correlation between plasma AVP concentrations and the SI ratio of the PL. An immunohistochemical study of the hypothalamus nucleus revealed that AVP-immunopositive cells significantly increased in the hypertonic saline loaded dogs. We concluded that the high SI in the PL in T1WIs in normal dogs was caused by AVP stored at the site, and examination of the SI in the PL using MRI is useful for diagnosis of abnormal pituitary glands.