Plasma amino acid profiles at various reproductive stages in female rats

We measured the plasma levels of amino acids at various reproductive stages in female rats, including the estrous cycle, pregnancy and lactation, and compared the resulting amino acid profiles using two- or three-dimensional figures. These figures revealed that the amino acid profiles of pregnant and lactating dams differed considerably from those during the estrous cycle or in male rats. The plasma levels of individual amino acids were almost the same between proestrus, estrus, metestrus and diestrus, and their profiles did not differ significantly. However, the amino acid profiles changed during pregnancy and lactation in dams. The plasma Ser level decreased significantly in mid and late pregnancy, whereas Tyr, Gly and His decreased significantly in the late and end stages of pregnancy, and Trp and Lys significantly decreased and increased at the end of pregnancy, respectively. Much larger changes in amino acid profiles were observed during lactation, when the levels of many amino acids increased significantly, and none showed a significant decrease. Plasma Pro, Ser and Gly levels increased continuously from day 1 until day 15 of lactation, whereas Asn and Met increased significantly from days 1 and 5 respectively until the end of lactation. These results suggest that the profiles of plasma amino acids show characteristic changes according to reproductive stage and that it may be necessary to consider such differences when performing amino acid-based diagnosis.     

Comparison of tracheal aspirates before and after high-speed treadmill exercise in racehorses

OBJECTIVE: To determine whether percentages of neutrophils in tracheal aspirate (TA) samples collected from racehorses are increased after exercise and whether interpretation of results from TA samples taken before and after exercise agree. DESIGN: Case series of 40 young Thoroughbred and Standardbred racehorses in race training presented for evaluation of poor performance. PROCEDURE: TA samples were collected endoscopically from racehorses presented for poor performance 24 h before and 1 to 2 h after high speed treadmill exercise testing. Aliquots of the retrieved fluid were cytocentrifuged and smears were stained with Diff-Quik. Mean neutrophil counts were expressed as percentages of the total number of inflammatory cells counted and subsequently were categorised as either above or below an accepted cut-off of 20%. Comparisons between percentages of neutrophils before and after exercise were made. RESULTS: Percentage of neutrophils from TA samples obtained from racehorses after exercise was significantly higher than neutrophil percentages from TA samples collected from the same horse before exercise. In horses with TA specimens that were categorised as having < or = 20% neutrophils before treadmill exercise, the percentage of neutrophils in their TA specimens after exercise was, on average, significantly higher and was greater than the cut-off value of 20%. CONCLUSION: Recent strenuous exercise may change the proportion of neutrophils in lower airways of racehorses and practitioners should be aware of this when collecting and interpreting the results from TA samples. The most practical time for collection of a TA sample to obtain the most diagnostically useful information might be after a suitable washout period of at least 1 to 2 h post-exercise.     

Background data of 2-year-old male and female F344 gpt delta rats

Although gpt delta rats, as reporter gene-transgenic rats, were originally developed for in vivo mutation assays, they have also been used to evaluate chemical carcinogenesis and comprehensive toxicity. Therefore, it is necessary to accumulate background data on carcinogenicity and general toxicity in gpt delta rats. Here, we investigated the background data of 110-week-old male and female F344 gpt delta rats and wild-type rats. There was no effect of reporter gene transfection on animal survival rates and body weights during the experiment. The relative weight of male gpt delta rat adrenals was significantly higher than that of wild-type rats, possibly due to the higher incidence of pheochromocytoma. There were no intergenotype differences in the incidence of nonneoplastic lesions in both sexes, including chronic progressive nephropathy and focus of cellular alteration in the liver, which had a higher incidence in both genotypes. Additionally, the significantly higher incidence of adrenal pheochromocytoma in male gpt delta rats than that in wild-type rats was likely incidental because of the lack of differences in the incidences of preneoplastic (male and female) and neoplastic (female) adrenal lesions in both genotypes. Other neoplastic lesions in both sexes showed no intergenotype differences in incidence rates, although large granular lymphocytic leukemia in the spleen and Leydig cell tumors in the testes of males showed higher incidence rates. Overall, there were no effects of reporter gene transfection on the spectrum of spontaneous lesions in F344 gpt delta rats, thus supporting their applicability in evaluating chemical toxicity and carcinogenicity.     

Histopathological nerve and skeletal muscle changes in rats subjected to persistent insulin-induced hypoglycemia

New insulin analogues with a longer duration of action and a flatter pharmacodynamic profile are developed to improve convenience and safety for diabetic patients. During the nonclinical development of such analogues, safety studies must be conducted in nondiabetic rats, which consequently are rendered chronically hypoglycemic. A rat comparator model using human insulin would be valuable, as it would enable differentiation between effects related to either persistent insulin-induced hypoglycemia (IIH) or a new analogue per se. Such a model could alleviate the need for an in-study-comparator and thereby reduce the number of animals used during development. Thus, the aims of the present study were i) to develop a preclinical animal model of persistent hypoglycemia in rats using human insulin infusion for four weeks and ii) to investigate histopathological changes in sciatic nerves and quadriceps femoris muscle tissue, as little is known about the response to persistent hypoglycemia in these tissues. Histopathologic changes in insulin-infused animals included axonal degeneration and myofibre degeneration. To our knowledge, this is the first study to show that persistent IIH provokes peripheral nerve and skeletal myofiber degeneration within the same animals. This suggests that the model can serve as a nonclinical comparator model during development of long-acting insulin analogues.     

Analysis of L-type amino acid transporter in canine hepatocellular carcinoma

Analysis of L-type amino acid transport expression of hepatocellular carcinoma cells (HCCs) of the dog was performed. The leucine transport activity of canine HCCs was 0.628 ± 0.018 nmol/mg protein/min. The inhibitor of LAT 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) reduced 90% of the activity at 1 mM. The deduced amino acid sequences of canine LAT2, LAT3 and LAT4 were well conserved in mammalians, exhibiting 89, 88 and 77% homology, respectively. RT-PCR revealed distinct LAT1 expression compared with normal hepatocytes. Western blotting analysis confirmed the potent LAT1 expression in canine HCCs but not hepatocytes, and real-time RT-PCR analysis indicated that canine HCCs possessed 28 times higher LAT1 expression than hepatocytes. These results indicated that the leucine transport activity of canine HCCs was due to LAT1.     

Effect of methotrexate on rostral migratory stream in newborn rats

Two-day-old rats were treated with subcutaneous injections of methotrexate (MTX) 5 mg/kg and 150 mg/kg, and their rostral migratory streams (RMS) were examined time-dependently. MTX treatment increased pyknotic and TUNEL-positive cells and decreased mitotic and phospho-Histone H3-positive cells at almost all time points in the vertical arm, elbow and horizontal arm regions of the RMS. There were more TUNEL-positive cells ratio in the MTX 150 mg/kg group than in the MTX 5 mg/kg group. Treatment with MTX 150 mg/kg decreased the cellularity in the vertical arm region on Postnatal day (PD) 4, but that with the MTX 5 mg/kg did not. TUNEL-positive cells ratio was the highest in the vertical arm region, followed by elbow and horizontal regions in both MTX-treated groups. TUNEL-positive cells ratio in the vertical arm and elbow regions reached their peaks on PD 4 in both MTX-treated groups, and both MTX-treatments significantly decreased Phospho-Histone H3-positive cells ratio on PDs 2.5 and 3 in the vertical arm, elbow and horizontal arm regions. The phospho-Histone H3-positive cells ratio in the vertical arm region recovered on PD4 in the MTX 150 mg/kg group. These findings suggested that RMS required a great amount of folic acid on PD 2 and that the folic acid-requirement differed depending on the anatomical region of the RMS. To our knowledge, this is the first report demonstrating the effect of MTX on the RMS and the necessity of the folic acid metabolism on RMS development in newborn rats.     

Repeated restraint stress modifies fatty acid and amino acid metabolism in the mouse skin

In modern society, stress caused by relationships and emotions is one of the greatest social problems. Similar to humans, domestic and captive animals live under various stresses. Several stresses have been associated with skin disorders, such as atopic dermatitis, but there is a lack of reliable and objective indicators for the characterization of this association. This study aimed to define the changes in fatty acid composition and amino acid concentration in the skin following repeated restraint stress in ICR mice. Mice subjected to 30 min of daily restraint stress for 8 days showed changes in the composition of saturated fatty acids, such as an increase in palmitic acid content, which are the substrates of Δ-9 desaturase. Conversely, unsaturated fatty acids decreased with stress treatment, which appeared to be a result of these fatty acids being the substrate of Δ-6 desaturase. Changes in fatty acid composition after stress treatment may be one of the factors that cause skin inflammation. The water-retention capacity may have been lowered by stress treatment because histidine and leucine, which are natural moisturizing factors, were significantly decreased. The collagen content in the skin gradually decreased after repeated stress treatment. Our results indicate that repeated restraint stress may impact skin health through changes in both the fatty acid composition and amino acid concentration in mice.     

Effect of methotrexate on cerebellar development in infant rats

Six-day-old rats were treated intraperitoneal injections with methotrexate 1 mg/kg, and the cerebellum was examined. Both the length and width of the vermis decreased in the methotrexate-treated group instead of the control from 4 day after treatment (DAT) onward. A significant reduction in the width of the external granular layer was detected on 2 and 3 DAT in the methotrexate group. By 4 DAT, the width of the external granular layer of the methotrexate group was indistinguishable from the control, and by 8 DAT, it was greater than that of the control. The molecular layer of methotrexate group on 8 and 15 DAT was thinner than that of the control. On 1 DAT, in the methotrexate group, there were many TUNEL and cleaved caspase-3-positive granular cells throughout the external granular layer, and they decreased time-dependently. On 1 DAT, in the methotrexate group, phospho-histone H3-positive cells in the external granular layer were fewer than in the control and tended to increase on 2–4 DAT. The p21-positive-rate of the external granule cells in the MTX group was higher than in the control on 1–4 DAT. These results suggested that methotrexate exposure on postnatal day 6 induces a delay, slowing in the migration of external granular cells to the inner granular layer, attributed to decrease or inhibition in the production of external granular cells that had arisen from apoptosis and the decrease in cell proliferative activity, resulting in cerebellar hypoplasia.     

Effects of inactive parapoxvirus ovis on cytokine levels in rats

The aim of this study is to determine the effects of iPPOV on pro-inflammatory and anti-inflammatory cytokine levels in rats. iPPOV (1 ml/rat) was administered intraperitoneal route to 49 rats, except for 7 rats (Control, 0 group). Serum samples were collected from 7 rats at 1st, 2nd, 4th, 8th, 12th, 16th and 24th hr after treatments. Levels of TNF-α, IL-6, IL-12 and IL-10 were determined using ELISA. Administration of iPPOV stimulated TNF-α (16th and 24th hr) and IL-6 (12th, 16th and 24th hr) synthesis and caused fluctuations in IL-10 and IL-12 concentrations. In conclusion, increased cytokine levels could be attributed to immunomodulatory activity of iPPOV, however, detailed studies are required to fully understand effects of iPPOV on immune system.     

Imbalanced dietary methionine-to-sulfur amino acid ratio can affect amino acid profiles,antioxidant capacity,and intestinal morphology of piglets

Animal protein sources such as fishmeal and plasma powder are excellent and indispensable sources of energy, amino acids, and minerals in animal production. Amino acid imbalance, especially methionine-to-sulfur amino acid (Met:SAA) ratio, caused by an imbalance of animal protein meal leads to growth restriction. This study was conducted to evaluate the effects of imbalanced Met:SAA ratio supplementation of different animal protein source diets on growth performance, plasma amino acid profiles, antioxidant capacity and intestinal morphology in a piglet model. Twenty-four weaned piglets (castrated males; BW = 10.46 ± 0.34 kg), assigned randomly into 3 groups (8 piglets/group), were fed for 28 d. Three experimental diets of equal energy and crude protein levels were as follows: 1) a corn-soybean basal diet with a Met:SAA ratio at 0.51 (BD); 2) a plasma powder diet with a low Met:SAA ratio at 0.41 (L-MR); 3) a fishmeal diet with a high Met:SAA ratio at 0.61 (H-MR). Results revealed that compared to BD, L-MR significantly decreased (P < 0.05) the activities of plasma total antioxidant capacity and glutathione peroxidase, plasma amino acid profiles, and significantly reduced (P < 0.05) villus height and crypt depth in the duodenum and jejunum. Additionally, L-MR significantly reduced (P < 0.05) the mRNA expression level of solute carrier family 7 member 9 (SlC7A9) in the ileum, and significantly increased (P < 0.05) mRNA expression levels of zonula occludens-1 (ZO-1) in the duodenum, and Claudin-1, ZO-1, sodium-coupled neutral amino acid transporters 2 (SNAT2) and SlC7A7 in the jejunum. H-MR significantly increased (P < 0.05) plasma SAA levels, and significantly reduced (P < 0.05) average daily feed intake, villus height, and villus height-to-crypt depth (VH:CD) ratio in the ileum compared to BD. In conclusion, L-MR may result in oxidative stress and villous atrophy but proves beneficial in improving intestinal barrier function and the activity of amino acid transporters for compensatory growth. H-MR may impair intestinal growth and development for weaned piglets. The research provides a guidance on the adequate Met:SAA ratio (0.51) supplementation in diet structure for weaned piglets.     

Time-course comparison of pulmonary inflammation induced by intratracheal instillation of four different nickel oxide nanoparticles in male Fischer rats

Occupational exposure to nickel oxide (NiO) is an important cause of respiratory tract cancer. Toxicity is known to be associated with the dissociated component, i.e. nickel (II) ions. To address the relationship between physicochemical properties, including solubility in artificial lysosomal fluid, of NiO and time-course changes in the pulmonary response, we conducted an intratracheal instillation study in male Fischer rats using four different well-characterized NiO products, US3352 (NiO A), NovaWireNi01 (NiO B), I small particle (NiO C), and 637130 (NiO D). The NiOs were suspended in purified water and instilled once intratracheally into male F344 rats (12 weeks old) at 0 (vehicle control), 0.67, 2, and 6 mg/kg body weight. The animals were euthanized on days 3, 28, or 91 after instillation, and blood analysis, bronchoalveolar lavage fluid (BALF) testing, and histopathological examination were performed. The most soluble product, NiO B, caused the most severe systemic toxicity, leading to a high mortality rate, but the response was transient and surviving animals recovered. The second-most-soluble material, NiO D, and the third, NiO A, caused evident pulmonary inflammation, and the responses persisted for at least 91 days with collagen proliferation. In contrast, NiO C induced barely detectable inflammation in the BALF examination, and no marked changes were noted on histopathology. These results indicate that the early phase toxic potential of NiO products, but not the persistence of pulmonary inflammation, is associated with their solubility.     

Orbital malignant schwannoma and embryonal rhabdomyosarcoma in rats caused by leakage of locally injected nickel subsulfide to the orbit

Nickel subsulfide (Ni₃S₂) is known to induce intraocular neoplasms when injected intravitreally into the eyes of rats. Here, we found two extraocular orbital neoplasms in two different rat strains, presumably due to the leakage of locally injected Ni₃S₂ to the extraocular orbital tissues. In the F344/DuCrlCrlj rat, an orbital mass arose at 30 weeks after injection, and invaded into the cranium. Histologically, the orbital mass was composed of areas arranged in parallel bundles formed by densely packed elongated or spindle-shaped cells with indistinct cytoplasmic borders, and of areas of hypocellular arrangement consisting of round cells in eosinophilic myxoid-like substances. Metastases were observed in the right submandibular and cervical lymph nodes. The neoplastic cells were immunopositive for S-100 protein and vimentin. Transmission electron microscopy revealed that the neoplastic cells had cellular processes and pericytoplasmic basal laminae. In the RccHan^TM:WIST rat, an orbital mass arose at 36 weeks after injection. Histologically, the mass consisted of rhabdoid-like large round cells with proliferation of small round-to-polygonal cells, and these neoplastic cells infiltrated into the extraocular muscles. Immunohistochemically, the neoplastic cells were positive for desmin and vimentin. Transmission electron microscopy detected immature myofibrils with Z-band structures in the cytoplasm of these neoplastic cells. Consequently, the tumors were diagnosed as an orbital malignant schwannoma in an F344/DuCrlCrlj rat and an orbital embryonal rhabdomyosarcoma in a RccHan^TM:WIST rat. The results of this case report suggest that leakage of Ni₃S₂ to the orbit caused the induction of orbital malignant schwannoma or rhabdomyosarcoma in rats.     

Histopathological and immunohistochemical features of proliferative lesions in the pituitary pars distalis of rats

Pituitary proliferative lesions derived from the endocrine cells of the pars distalis are frequently encountered and adenomas/carcinomas are a common cause of death in standard 2-year carcinogenicity studies using various rat strains, especially Sprague-Dawley. This report describes the immunohistochemical characteristics of pituitary tumors derived from the pars distalis in rats. Prolactin (PRL)-containing tumors are the most common, with PRL/growth hormone (GH) dual positive tumor masses (PRL/GH co-positive tumor masses) being more prevalent than only PRL-positive tumor masses (PRL single-positive tumor masses). GH-containing tumors are relatively numerous and many of these are also PRL/GH co-positive tumor masses. TSH-containing tumors are common in females. PRL-containing tumors have been shown to increase the incidence of hyperlactation in males and mammary adenomas/adenocarcinomas in females, suggesting that these masses are functional tumors.     

Effects of eccentric exercise on branched‐chain amino acid profiles in rat serum and skeletal muscle

Supplementation of branched‐chain amino acid (BCAA) is often used to attenuate exercise‐induced skeletal muscle damage and promote adaptation, but no definitive conclusion on the benefits of BCAA on muscle recovery after injurious exercise can be drawn. Exploration of the systematic BCAA alteration in muscular injury‐repair stage per se without any BCAA supplement should provide some useful information in favour of BCAA application in muscle regeneration after injury. One bout of 90‐min downhill‐running exercise was performed to cause rat skeletal muscle injury. After exercise, myofibrillar BCAA concentrations showed minor changes compared with exercise before, while serum concentrations of BCAA were lower after exercise. Especially, serum leucine, isoleucine and total BCAA concentrations 2 weeks post‐run were significantly lower than normal values of exercise before (p = 0.008, p = 0.041, p = 0.015). The data demonstrate that a single eccentric exercise can significantly decrease the serum BCAA concentrations, which mean high utilization of BCAA for myogenesis after injurious exercise.     

Effects of rearing system on meat quality,fatty acid and amino acid profiles of Hu lambs

To determine the effects of early rearing system on meat quality traits, fatty acid and amino acid profiles of meat, 48 Hu lambs were selected and randomly divided into four treatments: control lambs were ewe‐reared from birth to 60 days of age (ER), while treatment lambs were segregated from their dams at 10, 20 and 30 days of age, then artificially reared with milk replacer until 60 days of age (EW10, EW20 and EW30, respectively). All lambs had no access to the milk replacer or their dams from 61 to 90 days. Creep feed was supplied ad libitum to all lambs from 15 to 90 days of age. Artificially reared lambs (EW10, EW20 and EW30) had higher (p < .05) creep feed intake, average daily gain, slaughter weight, hot carcass weight, meat ether extract content, bright meat color (lightness and yellowness) than ewe‐reared lambs. The content of unsaturated fatty acids and monounsaturated fatty acids in EW10 and EW20 were greater (p < .05) than that in ER, whereas the proportions of saturated fatty acids in EW10 and EW20 were lower than that in ER (p < .05). The percentages of C18:0, C18:2, C18:3 and the ratio of polyunsaturated fatty acids and saturated fatty acids of artificially reared lambs were greater than ewe‐reared lambs (p < .05). The meat proportions of leucine, alanine and proline were higher (p < .05) in artificially reared lambs than ewe‐reared ones. Finally, the artificially reared system increased meat production and changed the fatty acid and amino acid profiles of meat, and it would be appropriate to rear Hu lambs artificially from 10 days with milk replacer.     

Relation of soya bean meal level to the concentration of plasma free amino acids and body growth in white rats

Amino acid (AA) levels in plasma and body growth were determined in rats (n20) fed diets with different soya bean meal levels. Free AA in plasma was determined by reversed-phase high-pressure liquid chromatography. We have used four levels of protein diets like 8%, 15%, 23% and 35% in this trial. Rats which were fed the low-protein (8%) diet with low percentage of soya bean meal were found to be growth-retarded. The body weight gain of high protein group (35%) was lower than that of the 23% groups. In the rats fed with the low-soya bean meal diet, some nonessential AA (NEAA) in plasma like asparagine, aspartic acid, cysteine, glutamic acid and serine increased, whereas the essential AA (EAA), with the exception of arginine, methionine and valine decreased. Here, plasma EAA-to-NEAA ratios were not correlated to growth and experimental diet. We hypothesize that AA metabolism is associated to changes in growth in rats on different protein intake. This study has showed the sensitivity of body mass gain, feed intake, feed conversion rate of rats to four levels of protein in the diet under controlled experimental conditions.     

The neuroprotective effect of submicron and blended Lycium barbarum for experiment retinal ischemia and reperfusion injury in rats

The purpose of this study was to investigate the neuroprotective potential of submicron (milled) and blended Lycium barbarum (LB) in glaucomatous retinal neuropathy using a rat model of high intraocular pressure (HIOP) induced retinal ischemia. The rats were treated with 500, 250, 100 mg/kg LB (submicron or blended form) orally once daily for 56 days respectively after 1 week of retinal ischemia induction. We conducted electroretinography (ERG), histopathological analysis in retina and antioxidative level assays, such as total glutathione (GSH (glutathione) + reduced glutathione) + GSSH (glutathione disulfide), catalase activity, SOD (superoxide dismutase) activity, and lipid peroxidant malondialdehyde (MDA) in the retina and plasma of test rats. The results indicated that the amplitudes of a and b wave of ERG were preserved in rats treated with submicron and blended LB groups, the best protective effect on ERG b wave amplitudes was observed at the dosage of 250 mg/kg of both forms of LB. Retinal thickness was best preserved, particularly significant in the retinal inner nuclear layer in submicron 250 mg/kg LB group. The levels of antioxidant GSSH+GSH, SOD and catalase activity in the retina were higher in blended 500 mg/kg and submicron 250 mg/kg groups than other groups, while the MDA level was lower in submicron LB groups than that in blended LB and non-LB IR group. In the plasma, there was no significant difference in the levels of GSSH+GSH and catalase activity between treated groups, but higher levels of SOD and lower levels of MDA were observed in 250 mg/kg submicron and 500 mg/kg submicron LB groups than the blended LB and non-LB IR groups. Generally better antioxidative effects were observed in the submicron LB than blended LB among treated groups, especially the 250 mg/kg submicron LB, providing good retinal neuroprotection by preserving retinal structure and function with improved antioxidative capacity. The submicron LB may have clinical implication as an adjuvant therapy of oxidative stress and retinal damage caused by HIOP induced retinal ischemia and reperfusion injury.     

Effect of L‐theanine on meat quality,muscle amino acid profiles,and antioxidant status of broilers

This study investigated the effect of L ‐theanine on carcass traits, meat quality, muscle antioxidant capacity, and amino acid (AA) profiles of broilers. Three hundred 1‐day‐old Ross 308 male broilers were randomly allotted to five groups with six replicates. Birds were fed the basal diet or basal diet with 300, 600, 900, or 1,500 mg/kg L ‐theanine for 42 consecutive days. The results showed that L ‐theanine quadratically increased dressing percentage, eviscerated percentage, and leg muscle yield (p < .05). Meanwhile, drip loss, cooking loss, shear force, L*_24h, and muscle lactate content decreased quadratically in response to dietary L ‐theanine supplementation (p < .05), while pH_24h and muscle glycogen content were quadratically improved by L ‐theanine (p < .05). Notably, the contents of muscle malondialdehyde and protein carbonyl, and the activities of muscle total antioxidant capacity, catalase, and glutathione peroxidase decreased quadratically in response to dietary L ‐theanine supplementation (p < .05), suggesting that the oxidative stress level of muscle was decreased quadratically. Moreover, L ‐theanine quadratically increased the concentrations of most of muscle essential AA, nonessential AA, and flavor AA (p < .05). In conclusion, L ‐theanine can be used as a valuable feed additive to modulate carcass traits, meat quality, muscle antioxidant status, and AA profiles of boilers, and its optimum addition level is 600 mg/kg based on the present study.