Suspected white kidney bean (Phaseolus vulgaris) toxicity in horses and cattle

Thirty-four mixed breed horses from two separate farms showed signs of abdominal discomfort, pyrexia and dehydration after being exposed to a new batch of 14% complete horse feed. A new batch of cattle feed from the same manufacturer resulted in dairy cows showing depression, a drop in milk production and diarrhoea. Examination of both diets revealed the presence of white kidney beans (Phaseolus vulgaris). Inclusion of raw beans of this genus in animal feeds is to be avoided.     

Use of bean meal (Phaseolus vulgaris L.) in goat rations for meat production

Tropical Animal Health and Production - Bean meal (Phaseolus vulgaris L.) may be a viable alternative in ruminant feeding, mainly as a source of protein due to its high crude protein content. The...     

Use of post‐harvest common bean (Phaseolus vulgaris L.) residues in diet of lambs and its effect on finishing performance,rumen fermentation,protozoa population and meat characteristics

The aim of the experiment was to investigate the replacement value of common bean residues in the diet of finishing lambs as an inexpensive fodder instead of wheat straw or maize silage. Forty Arabi male lambs with average body weight of 18.90 ± 0.6 kg were randomly divided into five treatments with eight replications. The five experimental treatments consisted of control diet, and 50% and 100% replacement of bean residues with wheat straw or 25% and 50% replacement with maize silage in the control diet respectively. Lambs were slaughtered at the end of the experiment to evaluate carcass traits and meat characteristics. Dry matter intake, blood glucose, urea nitrogen, cholesterol and triglyceride concentration were not affected by the experimental treatments. Apparent digestibility of nutrients; total concentration of volatile fatty acids, acetate and butyrate; and total rumen protozoa population were linearly increased by increasing the levels of bean residues, as replacement with wheat straw (p < .05). Replacement of bean residues in control diets instead of wheat straw or maize silage caused a positive linear incremental trend for average daily gain and total weight gain. There was no significant difference in daily gain, final weight and feed conversion ratio among the treatments. Instead of wheat straw or maize silage, replacement bean residues linearly increased chewing, ruminating and eating activities per neutral detergent fibre intake (p < .05). Carcass traits and physicochemical characteristics of meat were not affected by the treatments. It is advisable to use bean residues in the diet of lambs as an alternative to the wheat straw or maize silage, because no adverse effects were observed on rumen fermentation, blood metabolites and performance; moreover, improvement in average daily gain, total weight gain and digestibility of nutrients, and the fact that bean residue is less expensive than wheat straw and maize silage.     

Roasted Full-fat Kidney Bean (Phaseolus vulgaris L.) and Soyabeans (Glycine max) Meals in Broiler Chicken Diet

An 8-week experiment was carried out to evaluate the performance of broiler chickens fed on diets containing either roasted full-fat soyabean meal (RSBM), roasted full-fat kidney bean meal (RKBM), cottonseed cake meal (CSM) or soyabean meal (SBM). A total of 240 unsexed, day-old-chicks were randomly assigned to four treatments so that there were 10 chicks per experimental unit in a randomized complete block design with three blocks and two replicates per block. Cumulative feed intake was depressed (p < 0.01) with the RSBM-based diet (71 g/bird per day) and was lowest with the RKBM-based diet (64 g/bird per day). Growth was closely related to feed intake, averaging 2122 g, 2094 g, 1960 g and 1601 g for CSM-, SBM-, RSBM- and RKBM-based diets, respectively. However, feed conversion ratio (g food intake/g body weight gain) was similar at 8 weeks of birds' age, although the values were highest (p<0.01) at the first 2 weeks of age for birds fed on RKBM-based diets. Mortality was not affected (p >0.05) by treatment effects. Feed cost efficiency (CFA Franc (Fcfa) per kilogram live chicken) showed that the SBM-based diet (494 Fcfa) and RKBM-based diet (493 Fcfa) were more expensive (p<0.01) than the RSBM-based diet (400 Fcfa). The CSM-based diet was the most economical (358 Fcfa) as a result of the lower market price of cottonseed cake. Despite suboptimal production performance and given current market prices, replacing imported SBM with locally produced RKBM, RSBM or CSM would allow farmers to save 1%, 19% and 28%, respectively, on feed cost efficiency. Roasting soyabeans in place of imported SBM allowed farmers to save about 19% for each CFA franc spent on a kilogram of live bird at 8 weeks of age.     

Ovine skeletal muscle multicatalytic proteinase complex (proteasome): purification, characterization, and comparison of its effects on myofibrils with mu-calpains.

The latent form of multicatalytic proteinase complex (MCP) was purified to homogeneity from ovine skeletal muscle. The MCP ran as a single band (M(r) 600,000) on nondenaturing polyacrylamide gel (PAGE) and dissociated to a number of subunits (M(r) 21,000 to 31,000) under denaturing and reducing conditions (SDS-PAGE). The proteinase complex was activated reversibly by heating at 60 degrees C and in the presence of SDS. Maximum activation (18-fold) was observed after 2 min at 60 degrees C and there was rapid inactivation beyond 2 min. Maximum proteolytic activity (12.8-fold) occurred in the presence of .25 mM SDS and diminished rapidly at higher SDS concentrations. The MCP was maximally active at pH 7.5 to 8.0 and 45 degrees C using radiolabeled alpha-casein. These and other results (e.g., proteinase inhibitor profiling) indicate that ovine skeletal muscle does indeed contain MCP and that its biochemical properties are the same as MCP isolated from other sources. By using [14C]-casein as a substrate, the specific activities (milligrams of protein degraded/milligrams of proteinase) for mu-, m-calpain, and MCP were 44.0, 59.7, and 2.0, respectively. Purified ovine myofibrils were incubated with mu-calpain or MCP. Classical effects of calpains, which include degradation of Z-disks, titin, desmin, troponin-T, and troponin-I and removal of alpha-actinin, were observed. However, only troponin-C and myosin light chains-2 and -3 were degraded by MCP. Morphologically, MCP had no detectable effect on myofibrils. Results suggest that MCP is not involved in the initial steps of myofibril disassembly. However, its involvement in the degradation of myofilaments remains to be determined.     

Heat-treated bean (Phaseolus vulgaris) residue meal as an alternative protein source in pelleted diets for Nile tilapia fingerlings: growth,body composition,and physical characteristics of diets

Tropical Animal Health and Production - The study aimed to evaluate heat-treated bean residue meal (Phaseolus vulgaris)—BRM—as an alternative protein source in diets for Nile tilapia...     

Performance of Mashona doelings supplemented with different levels of velvet bean (Mucuna pruriens L. DC. var. utilis) seed meal

The objective of the current study was to evaluate the effects of feeding increasing levels of velvet bean seed meal (VBM; 0, 12, 24, and 36 %) on the performance of Mashona doelings. Dry matter intake was lower (P?<?0.05) for the control diet compared to VBM diets, but linearly declined (P?<?0.05) with increasing levels of VBM. Average daily weight gain was significantly different between experimental groups. Doelings’ final live weights and average daily gains were slightly higher in control group than other three supplemented groups where they linearly declined (P?<?0.05) with increasing levels of VBM. The cost per kilogram of feed, however, decreased with high inclusion level of VBM. Result suggested that high inclusion level of VBM negatively influenced the growth of young goats probably due to the presence of some anti-nutritional factors which needs further investigation.     

Effects of field bean (Vicia faba L. var. minor) dietary supplementation on plasma thyroid hormones, insulin, insulin-like growth factor-1 concentrations and mohair characteristics in growing Angora goat kids

Twenty-two Angora kids were used to study the effect of a dietary supplementation with field bean (Vicia faba L. var. minor) on hair follicle activity, mohair characteristics and plasma total thyroxine (T4), triiodothyronine (T3), insulin (INS) and insulin-like growth factor-1 (IGF-1) concentrations. At birth, their mothers were divided in two groups (S: supplemented, C: control), the S group being supplemented with 300 g/head/day of whole field bean. At weaning, diet of kids from S group was supplemented with 80 g/head/day of whole field bean. At weaning, secondary hair follicle activity (S: 0.91 ± 0.01, C: 0.84 ± 0.02, p < 0.01) and staple length (S: 5.82 ± 0.13 cm, C: 5.16 ± 0.14 cm, p < 0.001) were greater in the S group. At 155 days of age, secondary fibre diameter (S: 16.54 ± 0.35 μm, C: 18.09 ± 0.31 μm, p < 0.01) was higher in the C kids. Concentrations of total plasma T4 and T3 were higher (p < 0.001) in S (120 ± 12 and 4.87 ± 0.92 ng/ml respectively) than in C kids (92 ± 8 and 2.97 ± 0.77 ng/ml respectively). At weaning, T4 values were negatively correlated (p < 0.05) with the secondary fibre diameter and the T3/T4 ratio was positively correlated (p < 0.05) with the secondary to primary hair follicle ratio. At 155 days of age, both T3 and T3/T4 values were positively correlated (p < 0.01) with staple length. Plasma INS (0.11 ± 0.03 ng/ml) and total IGF-1 concentrations (232 ± 13 ng/ml) were not affected by diet. Improvements of diet quality in kids positively affected fibre diameter and length and follicle activity rate. We suggest that increased circulating thyroid hormones concentrations, but not those of INS and IGF-1, may contribute to the beneficial actions of nutritional supplementation on anatomical growth and mohair production of kids.     

Effects of perinatal high dose dexamethasone on skeletal muscle development in rats.

Five litters of suckling rats were given either dexamethasone (DEX), 1 mg/kg, subcutaneously, three times daily (n = 4/litter) or vehicle control (n = 4/litter) from day 3 through day 7 after birth. Rats were weighed weekly and were weaned on day 30. On day 60, rats were killed and the soleus (SOL) and extensor digitorum longus (EDL) were removed for the following analyses: 1) wet weight, 2) light microscopic examination of hematoxylin and eosin stained transverse sections, 3) quantitative morphometric analysis of myosin ATPase stained transverse sections (fiber numbers, fiber type percentages and mean fiber diameters), and 4) DNA (total and mg/g wet weight). The following parameters were significantly reduced in treated rats: 1) body weight, 2) wet weight of SOL and EDL, and 3) mean diameter of SOL type I fibers. There was a trend for total DNA of SOL and EDL to be decreased in treated rats but this was not statistically significant. In a second experiment, pregnant rats (n = 4) were given DEX, 1 mg/kg, subcutaneously, twice daily, on days 17 and 18 of gestation. Two rats served as vehicle controls. The prenatally DEX-exposed rats weighed significantly less on weeks 3, 4, 6, 7 and 8. There were significant reductions in the following parameters for treated rats: 1) SOL wet weight, and 2) total number of SOL type I fibers. There was a trend for SOL DNA to be reduced but this was not statistically significant.     

Differential effects of dexamethasone and clenbuterol on rat growth and on beta2-adrenoceptors in lung and skeletal muscle

Beta-adrenergic agonists increase growth rate, but their efficacy is reduced over time as the number of beta2-adrenoceptors in muscle decreases. Dexamethasone increases beta2-adrenoceptor density in many tissues, but this effect has not been reported in skeletal muscle. In this study, male rats were treated daily for 10 d with either clenbuterol (4 mg/kg of feed), dexamethasone (.2 mg/kg BW, s.c.), or clenbuterol plus dexamethasone. Untreated rats served as controls. Dexamethasone caused a marked suppression of growth rate, which resulted in decreased (P < .001) body weight (-29%), carcass weight (-30%), hind-limb muscles (-22%), omental fat (-22%), and heart weight (-10%). Feed intake was reduced (-26%), but feed conversion efficiency was also impaired (P < .001). Clenbuterol caused a small increase in growth rate (+6%; P < .05), with an increase in leg muscle (+7%; P < .01) and heart mass (+8%; P < .05). Feed efficiency was improved (P < .001) by clenbuterol. Rats given the combined treatment still showed a reduction in growth rate (-81%). Clenbuterol caused only a mild attenuation of the effects of dexamethasone on feed intake, BW, and carcass weight, but reduced the catabolic effect of dexamethasone on hind-limb muscle to only -8%. Clenbuterol caused a slight increase in the affinity beta2-adrenoceptors in lung for binding to the radioligand (-)[125I]iodocyanopindolol. Relative to control values, the density of beta2-adrenoceptors in lung was +31% with dexamethasone treatment, -45% with clenbuterol, and -23% with the combined treatment. Clenbuterol also decreased beta2-adrenoceptors in skeletal muscle (-35%), but so did dexamethasone (-13%), so the effects of the beta-adrenergic agonist were not attenuated through use of the combined treatment (-40%). The results show that the inductive effect of glucocorticoids on beta2-adrenoceptors is tissue-specific and that glucocorticoid treatment is not a useful adjunct to beta-adrenergic agonist treatment in animal production.     

Dietary-induced changes of muscle growth rate in pigs: effects on in vivo and postmortem muscle proteolysis and meat quality

The effects of various growth rates in pigs induced by four different feeding strategies on the activity of the calpain system and on postmortem (PM) muscle proteolysis and tenderness development were studied. An increased growth rate may be caused by an increased protein turnover, which results in up-regulated levels of proteolytic enzymes in vivo that, in turn, possibly will affect PM tenderness development. It can be hypothesized that increased proteolytic activity pre-slaughter will increase the PM tenderization rate. From postnatal d 28 to d 90 (phase 1) the pigs were divided into two groups, given either ad libitum (A) or restricted (R, 60% of ad libitum) access to feed. The two groups were then divided into two subgroups, given either restricted or ad libitum access to feed from d 91 to slaughter at d 165 (phase 2). Measurements of the activity of mu-calpain, m-calpain, and calpastatin; concentrations of total collagen and the percent of soluble collagen; and RNA, DNA, and elongation factor-2 where made at slaugther. Myofibrillar fragmentation index (MFI) was determined at slaughter and 24 h PM. Warner-Braztler shear force was determined 1 d and 4 d PM. Pigs fed restricted diets in phase 1 and fed ad libitum in phase 2 (RA pigs) had increased growth rates in the last phase compared to pigs fed ad libitum during both phase 1 and phase 2 (AA pigs). The increased growth rate (compensatory growth) was followed by an increased proteolytic potential (mu-calpain:calpastatin ratio), increased MFI values, and higher tenderization rates. There was a positive correlation between the activities of m-calpain and growth rates (r = 0.35, P = 0.03), and between RNA levels and growth rates (r = 0.43, P = 0.006). The proposed hypothesis is largely supported by the results. The activities of both mu- and m-calpain at slaughter were highest in fast-growing pigs. The calpain activity was highest in RA pigs, which in turn also had the fastest growth rates prior tslaughter among the four groups. This implies that the synthesis of these enzymes was up-regulated during the second feeding period to a larger extent in RA pigs. The proteolytic potential and the MFI values indicate that the up-regulated in vivo calpain activity had an effect on PM protein degradation, which also is supported by the higher tenderization rate in RA pigs.     

肉质改良剂对大鳞鲃生长、肌肉结构及抗氧化能力的影响

为探讨长期投喂肉质改良剂对大鳞鲃生长、肌肉结构及抗氧化力的影响,选取初始体重为(311.32±2.54)g,初始体长(35.79±2.33)cm健康大鳞鲃成鱼240尾,随机分为4组(每组3个重复,每个重复20尾鱼),分别投喂添加0、6.5%、10.5%和14.5%肉质改良剂的等氮等能饲料,依次标记为Y1～Y4组,其中Y1组为对照组。饲养75 d后测定大鳞鲃生长指标,各组织(鳃、脑、肝胰脏、中肾、脾脏)及血清中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和丙二醛(MDA),背部生肌肉肌纤维直径和质构特性。结果显示:Y2、Y3组大鳞鲃增重率、蛋白质效率显著提高,其中Y3组达到最大值,Y2组次之(P<0.05);背部生肌肉硬度和咀嚼度最大值均出现在Y3组,Y4组次之,分别比Y1组提高了37.52%、24.82%、51.6%和39.7%(P<0.05);胶着度最大值出现在Y3组(P<0.05),比Y1组提高25.59%;各试验组肌纤维直径均有不同程度的降低,其中Y3组直径最小,比Y1组降低29.65%(P<0.05);肉质改良剂能提高大鳞鲃抗氧化能力,其中Y3组SOD、CAT活性相对高,而MDA含量下降也最为明显。总之,肉质改良剂可效提高大鳞鲃生长和抗氧化能力,改善肌肉结构。建议大鳞鲃饲料中肉质改良剂添加水平为10.5%。     

饲料组胺对美洲鳗鲡生长及肌肉品质的影响

为研究饲料组胺对美洲鳗鲡生长及肌肉品质的影响,将重量[(2 870.0±17.1)kg/池)]和规格(7.8±0.3)P接近的养殖美洲鳗鲡的4口精养池,随机分为2个组,分别投喂组胺水平为67 mg/kg和414 mg/kg的试验饲料,每组2个重复,试验期为2个月。结果表明,与低组胺水平组相比,高组胺水平组美洲鳗鲡的增重率和饲料效率有降低的趋势(P<0.1);肌肉中多种多不饱和脂肪酸比例显著降低(P<0.05),氨基酸水平无显著变化(P>0.05)。综上所述,饲料中的组胺可一定程度抑制美洲鳗鲡生长和降低肌肉品质。     

Comparison of the effects of supplemental Korean mistletoe (Viscum album var. coloratum) powder and antibiotic on growth performance,serum cholesterol profiles,and meat quality of broilers

Abstract

The effects of Korean mistletoe (KM) powder on the growth performance, serum cholesterol profiles, and meat quality of broiler chickens were assessed. A total of 150 male broilers (n = 30 birds/treatment) were randomly assigned to one of five groups (0.05% chlortetracycline or 0, 0.5%, 1%, or 2% KM). No significant differences were observed in growth performance, proximate composition of meat, and meat color, except for crude fat and L* values. The KM-treated groups showed significantly reduced serum cholesterol levels (but not high-density lipoprotein [HDL] cholesterol) as the amount of KM in the diet increased. Meat groups that received KM showed a decline in pH and thiobarbituric acid-reactive substances (TBARS) and an increase in total phenol and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging compared with the antibiotics and control groups. The addition of 1% and 2% KM at the optimal rate can help develop practical broiler diets and improve the antioxidant effects.     

Effects of low-protein diet supplementation with alpha-ketoglutarate on growth performance,nitrogen metabolism and mTOR signalling pathway of skeletal muscle in piglets

Excessive protein levels in diets result in incomplete digestion of nitrogenous nutrients that are excreted from the body, causing environment pollution. Alpha-ketoglutarate (AKG) has been reported to decrease dietary protein levels, promote intestinal health in piglets and reduce environmental pollution. However, the underlying mechanisms of AKG are largely unknown. The objective of this study was to determine the effects of low-protein diet supplementation of AKG on the growth performance, nitrogen metabolism, relative expression of amino acid transporter genes and mTOR signalling pathway of skeletal muscle in piglets. Forty-eight piglets with an initial weight of 11.53 ± 0.04 kg were randomly divided into four groups. Each group had four replicates, and each replicate had three pigs. A low-protein (LP) diet (crude protein was 14.96%) served as the control without AKG, while 0.5%, 1.0% and 1.5% AKG were added to the LP diet for the other experimental groups. The trial period lasted for 28 days. Compared with the LP group, the LP + 1.0%A and LP + 1.5%A groups increased the growth performance (p < .05);increased the mRNA levels of amino acid transporters in the duodenum, anterior jejunum and posterior jejunum (p < .05); and reduced faecal nitrogen and urine nitrogen emissions (p < .05). They also showed greater mRNA levels and phosphorylated protein levels for S6 kinase beta (S6K) (p < .05), mammalian target of rapamycin (mTOR) (p < .05) and 4E-binding protein 1 (4EBP1) (p < .05) in skeletal muscle. An LP diet supplemented with AKG activated the mTOR signalling and promoted the ability of the small intestine to absorb protein, thereby increasing protein deposition. Taken together, an LP diet supplemented with AKG provides a theoretical basis for the promotion and application of AKG in piglet production.     

Inhibitory effects of epidermal growth factor on progesterone production of ovarian granulosa cells in Tsaiya duck (Anas platyrhynchos var. domestica)

1.?The purpose of this study was to investigate the effects of epidermal growth factor (EGF) on progesterone (P₄) secretion from ovarian granulosa cells in Tsaiya ducks (Anas platyrhynchos var. domestica).

2.?We obtained the largest (F1) follicle from Tsaiya duck, the granulosa layer was separated and the cells were isolated according to their proximity to the germinal disc.

3.?The granulosa cells were cultured in vitro, the culture media and the cells were used to determine P₄ and steroidogenic enzyme concentrations, respectively.

4.?P₄ concentrations were decreased in cultured granulosa cells taken proximal to the germinal disc (GD) compared to those distal to the germinal disc (NGD).

5.?EGF inhibited both basal and ovine luteinising hormone (oLH)-induced P₄ concentrations. It also inhibited the P₄ secretion via protein kinase A (PKA) pathway when cultured with GD and NGD granulose cells (mixed together) in vitro.

6.?Western blot results showed decreased concentrations of cytochrome P450 side-chain cleavage (P450scc) enzyme, 3β-hydroxysteroid dehydrogenase (3β-HSD) enzyme and steroid acute regulatory (StAR) protein expression, when the cells were co-treated with EGF and oLH.

7.?The inhibitory effect of oLH-induced P₄ production was attenuated by EGF by the addition of MAP-erk kinase (MEK) inhibitor (PD98059) suggests EGF may inhibit P₄ production by affecting via the mitogen-activated protein kinase (MAPK) pathway.     

The effects of kale (Brassica oleracea ssp. acephala), basil (Ocimum basilicum) and thyme (Thymus vulgaris) as forage material in organic egg production on egg quality

1. In organic egg production, forage material as part of the diet for laying hens is mandatory. The purpose of the present study was to examine the effect of feeding with forage materials including maize silage, herbs or kale on egg production and various egg quality parameters of the shell, yolk colour, egg albumen, sensory properties, fatty acid and carotenoid composition of the egg yolk.

2. A total of 5 dietary treatments were tested for 5 weeks, consisting of a basal organic feed plus 120?g/hen.d of the following forage materials: 1) maize silage (control), 2) maize silage incl. 15?g/kg basil, 3) maize silage incl. 30?g/kg basil, 4) maize silage incl. 15?g/kg thyme, or 5) fresh kale leaves. Each was supplied to three replicates of 20 hens. A total of 300 hens was used.

3. Feed intake, forage intake and laying rate did not differ with treatment, but egg weight and egg mass produced increased significantly with the kale treatment.

4. The egg shell strength tended to be higher with the kale treatment, and egg yolk colour was significantly more red with the kale treatment and more yellow with basil and kale treatments. The albumen DM content and albumen gel strength were lowest with the thyme treatment. By sensory evaluation, the kale treatment resulted in eggs with less sulphur aroma, higher yolk colour score, and more sweet and less watery albumen taste. Furthermore, the eggs of the kale treatment had significantly higher lutein and β-carotene content. Also, violaxanthin, an orange xanthophyll, tended to be higher in kale and eggs from hens receiving kale.

5. In conclusion, forage material, especially basil and kale, resulted in increased egg production and eggs of high and differentiable quality.     

Relationship of thyroid status to growth hormone and insulin-like growth factor-I (IGF-I) in plasma and IGF-I mRNA in liver and skeletal muscle of cattle

Steers were made hyperthyroid or hypothyroid to study the effects of physiological alterations in thyroid hormone status on plasma growth hormone (GH) profiles, plasma insulin-like growth factor-I (IGF-I) concentrations, and relative abundance of IGF-I mRNA in skeletal muscle and liver. Eighteen yearling crossbred steers (360 to 420 kg) were randomly allotted to hyperthyroid (subcutaneous injection 0.6 μg/kg BW L-thyroxine for 10 d), hypothyroid (oral thiouracil; 0.25% diet plus 12.5 g capsule/d for 17 d), or control (subcutaneous injection 0.9% NaCl) treatment groups. Blood samples were taken for measurement of GH, IGF-I, thyroxine (T₄) and triiodothyronine (T₃) by RIA. Samples of liver and skeletal muscle were taken by biopsy for measurement of IGF-I mRNA by solution hybridization. Steers receiving thiouracil had 57 and 53% (P<.05) lower T₄ and T₃, respectively, than control steers (84.1 and 1.7 ng/ml). The hyperthyroid steers had 228 and 65% greater (P<.05) T₄ and T₃ than control steers. Neither increased nor decreased thyroid status had any significant effects on plasma GH profiles, liver IGF-I mRNA, or plasma concentration of IGF-I. There was no effect of thyroid hormone alteration on skeletal muscle IGF-I mRNA concentrations. The results of this study suggest that short-term changes in thyroid status of cattle had no major impact on the GH-IGF-I axis or skeletal muscle IGF-I mRNA.