Comparative observation of freeze–thaw‐induced damage in pig,rabbit, and human corneal stroma

Objective Although variations exist between species with respect to outcomes after cryopreservation, little is known about the differences in the susceptibility of the corneal stroma to cryoinjury. We performed this study to investigate freeze–thaw‐induced damage in keratocytes and collagen in rabbit, pig, and human corneas. Animals studied Rabbit, pig, and human. Procedures We prepared 250‐μm‐thick anterior stroma from rabbit, pig, and human corneas after scraping off the epithelium and endothelium. Each 250‐μm‐thick corneal stroma without epithelium was placed in a 50‐mL tube, frozen with liquid N₂ for 15 min and taken out to thaw rapidly at 37 °C. This procedure of rapid freezing and thawing was repeated three times. Differences between the species with respect to cells and collagen structures were examined using hematoxylin–eosin (H&E) staining, terminal deoxynucleotidyl transferase‐mediated nick end labeling (TUNEL) assay, and transmission electron microscopy (TEM). We orthotopically transplanted the pig and rabbit corneal transplants after the triple freeze–thaw cycle into rabbit eyes and evaluated graft survival. Results On gross examination, rabbit corneas became opaque after the triple freeze–thaw procedure, while pig and human corneas remained transparent. Histologically, keratocytes were apoptotic on TUNEL assay and TEM in rabbit, pig, and human corneas. Collagen fibrils were fragmented and the arrangement of collagen fibrils was severely disturbed in rabbit corneas on H&E staining and TEM; collagen was well preserved in pig and human corneas. Rabbit corneal stroma underwent autolysis after transplantation, whereas the pig corneal stroma remained clear for 1 month. Conclusions Our study showed that rabbit corneal stroma was more susceptible to freeze–thaw injury than pig and human corneas.     

Effect of medetomidine and its antagonism with atipamezole on stress-related hormones, metabolites, physiologic responses, sedation, and mechanical threshold in goats

OBJECTIVE: To evaluate the effects of medetomidine and its antagonism with atipamezole in goats. STUDY DESIGN: Prospective randomized crossover study with 1 week between treatments. ANIMALS: Six healthy 3-year-old neutered goats (three male and three female) weighing 39.1-90.9 kg (60.0 +/- 18 kg, mean +/- SD). METHODS: Goats were given medetomidine (20 microg kg(-1), IV) followed, 25 minutes later, by either atipamezole (100 microg kg(-1), IV) or saline. Heart and respiratory rate, rectal temperature, indirect blood pressure, and mechanical threshold were measured, and sedation and posture were scored and blood samples obtained to measure epinephrine, norepinephrine, free fatty acids, glucose, and cortisol concentrations at baseline (immediately before medetomidine), 5 and 25 minutes after medetomidine administration, and at 5, 30, 60, and 120 minutes after the administration of antagonist or saline. Parametric and nonparametric tests were used to evaluate data; p < 0.05 was considered significant. RESULTS: Medetomidine decreased body temperature, heart rate, and respiratory rate and increased mean arterial blood pressure, cortisol, and glucose. Recumbency occurred 89 +/- 50 seconds after medetomidine administration. All goats were standing 86 +/- 24 seconds after atipamezole administration whereas all goats administered saline were sedate and recumbent at 2 hours. Tolerance to compression of the withers and metacarpus increased with medetomidine. From 5 to 120 minutes after saline or atipamezole administration, there were differences in body temperature, glucose, and cortisol but none in heart rate or blood pressure. Three of the six goats receiving saline developed bloat; five of six urinated. After atipamezole, four of six goats developed piloerection and all goats were agitated and vocalized. CONCLUSION: At the doses used, atipamezole antagonized the effects of medetomidine on recumbency, sedation, mechanical threshold, and the increase in glucose. Atipamezole increased the rate of return of cortisol toward baseline, and prevented further decline in rectal body temperature. CLINICAL RELEVANCE: Atipamezole may be used to antagonize some, but not all effects of medetomidine.     

Soil-Applied Nitrogen and Composted Manure Effects on Soybean Hay Quality and Grain Yield

ABSTRACT

Grain yield in many soybean experiments fails to respond to fertilizer nitrogen (N). A few positive responses have been reported when soybean were grown in the southern U.S., when N was applied near flowering and when biosolids were added. In a previous study, low N concentrations of soybean forage in north Texas on a high pH calcareous soil were reported and thus, we suspected a N nutrition problem. Consequently, we initiated this study to determine whether selected preplant N sources broadcast and incorporated into a Houston Black clay (fine, smectitic, thermic Udic Haplusterts) might increase forage N concentration, forage yield, or soybean grain yield. In 2003, N was applied as ammonium nitrate (NH₄NO₃, AN) up to 112 kg N ha^{? 1} and dairy manure compost (DMC) was applied at rates of 4.9, 9.9, 15.0, and 19.9 Mg ha^{? 1}. The DMC contained 5.9, 2.6, and 6.7 g kg^{? 1} of total N, P, and K, respectively; thus DMC added 29 to 116 kg N ha^{? 1}. In 2004, AN was applied at rates of 112 and 224 kg N ha^{? 1} and DMC was applied at 28 and 57 Mg ha^{? 1}; thus, DMC added 168 to 335 kg N ha^{? 1}. In another 2004 test, biosolids, a biosolids/municipal yard waste compost mixture (BYWC), and AN were compared. The biosolids contained 31, 18, and 2.9 g kg^{? 1} total N, P, and K, respectively. The BYWC mixture contained 8.8, 6.1, and 3.4 g kg^{? 1} of total N, P, and K, respectively. Biosolids were applied at 10 Mg ha^{? 1} (310 kg N ha^{? 1}), BYWC was applied at 58 Mg ha^{? 1} (510 kg N ha^{? 1}), and AN up to 224 kg N ha^{? 1}. None of the soil treatments increased soybean grain yield or forage yield although AN slightly increased forage N concentration in 2003.     

Production of stem cell factor in canine mast cell tumors

Mast cell tumor (MCT) is the most common cutaneous tumor in dogs. We recently revealed that production of stem cell factor (SCF) contributes to the proliferation of neoplastic mast cells in an autocrine/paracrine manner. The aim of the present study was to determine the contribution of the mechanism in clinical MCTs. In consequence, high SCF expression (>10 times compared to HRMC cells) was observed in 5 of 7 MCT samples used in the study regardless of KIT mutation, which was confirmed in immunohistochemical analysis. In addition, production of SCF was observed in Ki-67-positive cells in the MCT xenograft. These results indicate the broad contribution of SCF autocrine/paracrine mechanism on clinical MCTs, providing the rationale for the clinical use of KIT inhibitors regardless of KIT mutation.     

Reduced birth weight,cleft palate and preputial abnormalities in a cloned dog

The aim of the present study was to report a novel developmental abnormality in a cloned dog. A fibroblast cell line was established from an 8-year-old male German shepherd dog. In vivo matured oocytes were retrieved from a large breed dog, and the nucleus was removed from each oocyte. A donor cell was injected into an enucleated oocyte, and the oocyte-cell couplet was fused electrically. After chemical activation, the resulting embryos were transferred into a naturally estrus-synchronized recipient dog, and two cloned pups were delivered by Cesarean section 60 days later. One cloned pup (Clone 1) was healthy, but the other (Clone 2) had a birth weight of only 320 g and cleft palate, failure of preputial closure at the ventral distal part, and persistent penile frenulum. Clone 2 was raised by stomach feeding until Day 40 after birth, where palatoplasty was performed. The abnormalities in external genitalia in Clone 2 resulted in persistent penile extrusion that was surgically corrected. This complex developmental abnormality has not been reported in dogs previously.     

Impact of hydropattern disturbance on crayfish population dynamics in the seasonal wetlands of Everglades National Park,USA

1. The natural hydropattern in the seasonally‐flooded marl prairie wetlands of Everglades National Park has been severely disrupted by human water control activities, seriously impacting higher trophic organisms, e.g. wading birds, that depend on these wetlands. Less is known about the impacts on key aquatic fauna, such as crayfish Procambarus alleni, or how these populations might respond to proposed habitat restoration strategies. 2. Under severe environmental stress, populations of burrowing crayfish are predicted to have skewed size structure, low reproductive success, low survival, and widespread dispersal. As predicted for populations in stressed habitats, crayfish density was low, small dispersing adults were dominant, juvenile abundance was low, and survival was low in habitats where the hydroperiod (duration of flooding) was short and groundwater level was lowest. 3. Crayfish dispersed during flooding, but during the drydown, they burrowed rather than sought deeper water. This dispersal strategy may be adaptive for surviving in seasonal wetlands, but this had severe consequences on survival in disturbed habitats with shortened hydroperiods. Survival in burrows during the dry season was high in the longer‐hydroperiod habitats but was zero in the short‐hydroperiod habitat where the groundwater level fell more than 1 m. 4. Long‐hydroperiod marl prairie may function as sources, whereas short‐hydroperiod habitats act as population sinks. Our study suggests that the threshold conditions for preventing mass mortality of crayfish in these wetlands are hydroperiods >7 months and groundwater levels <0.5 m below the surface during the dry season. 5. Historical (pre‐drainage) hydroperiods appear to be restricted to the longest hydroperiod areas of the marl prairie. This indicates that much of the marl prairie wetlands now function as population sinks for crayfish and other invertebrates. The historical hydropatterns need to be re‐established throughout the marl prairie wetlands to achieve the restoration goal of increasing productivity in the aquatic faunal community. Copyright © 2001 John Wiley & Sons, Ltd.     

Sterol dynamics of heterotrophic Tetraselmis suecica and its nutritional implication in the bivalve aquaculture

Employment of heterotrophic culture of microalgal foods in the hatchery‐based seed production of oysters is still controversial because some algae produced using the method appear to loose sterols, a key nutritional factor for bivalve growth. We traced the changes in sterol content of Tetraselmis suecica growing under photoautotrophic and heterotrophic conditions with the aid of gas chromatography (GC) and GC–mass spectrometry. The photoautotrophic T. suecica at the mid‐logarithmic growth phase contained six major (cholesta‐5,22‐dien‐3β‐ol, ergost‐5‐en‐3β‐ol, cholest‐5‐en‐3β‐ol, 24‐methylcholesta‐5,22‐dien‐3β‐ol, 24‐methylcholesta‐5,24‐dien‐3β‐ol, and 24‐ethylchlolesta‐5,24‐dien‐3β‐ol) and two minor sterols (24‐methylcholesta‐5‐en‐3β‐ol and 24‐ethylchlolesta‐5en‐3β‐ol). In the comparison of algal growth and sterol level, photoautotrophic alga appeared to need higher amounts of major sterols for cell growth over heterotrophic alga. These findings, that heterotrophic alga needed less amount of sterols for growth, may have significant implications in the introduction of the method in bivalve hatcheries. We also reviewed the sterolic properties of the alga obtained from heterotrophic method with respect to bivalve aquaculture.     

The production and characterization of monoclonal antibodies against Photobacterium damselae ssp. piscicida and initial observations using immunohistochemistry

Five monoclonal antibodies (MAbs: F2B1, 1E4, 13B10, 4D4 and F3G12) were produced against lysed Photobacterium damselae ssp. piscicida ( Ph. d . ssp. piscicida ). The MAbs recognized three antigens of differing molecular weight on the Western blot of Ph. d . ssp. piscicida . They also cross-reacted with five different species of Vibrio . An enzyme linked immunosorbent assay (ELISA) with MAbs, F3G12 and 4D4 demonstrated differences between Ph. d . ssp. piscicida and three Ph. d . ssp. damselae type strains, indicating differences in the surface antigenicity between these two groups of bacteria. Antigen retrieval in conjunction with immunohistochemistry (IHC) using MAb 13B10, revealed colonies of bacteria in the kidney, spleen and liver of sea bass, Dicentrarchus labrax , infected with pasteurellosis. A number of positive colonies were observed around the mucosal layers of the intestinal tissue, especially within the lamina propria. In addition, a number of bacterial colonies were associated with red blood cells and blood vessels of the organs examined.     

In vitro digestion rate and estimated glycemic index of oat flours from typical and high β-glucan oat lines

The in vitro starch digestion rate and estimated glycemic index (GI) of oat flours and oat starches from typical and high β-glucan oat lines were evaluated along with the impact of heating on starch digestion. Flour from oat lines ('Jim', 'Paul', IA95, and N979 containing 4.0, 5.3, 7.4, and 7.7% β-glucan, respectively) was digested by pepsin and porcine pancreatin. To determine the impact of heating on starch digestion, oat slurries were prepared by mixing oat flour and water (1:8 ratio) and heating for 10 min prior to digestion. Viscosity, as measured on a Rapid Visco Analyzer, increased with increases in concentration and molecular weight of β-glucan. The in vitro starch digestion of oat flours and a control, white bread made from wheat flour, increased as the digestion time increased. Starch digestion of oat flour was slower than that of the control (p < 0.05). Heat treatment of oat-flour slurries increased the starch digestion from a range of 31-39% to a range of 52-64% measured after 180 min of in vitro digestion. There were no differences in starch digestibility among oat starches extracted from the different oat lines. The GI, estimated by starch hydrolysis of oat flours, ranged from 61 to 67, which increased to a range of 77-86 after heating. Oat-flour slurries prepared from IA95 and N979 lines with high β-glucan concentrations had lower GI values than did slurries made from Jim and Paul lines. Starch digestion was negatively correlated with β-glucan concentrations in heated oat-flour slurries (R(2) = 0.92). These results illustrate that the oat soluble fiber, β-glucan, slowed the rate of starch digestion. This finding will help to develop new food products with low GI by using oat β-glucan.