NADPH-oxidase activity and lipid peroxidation in neutrophils from rats fed fat-rich diets

In order to investigate the effect of fat-rich diets on neutrophil functions, 21 day-aged rats were fed for 6 weeks with a control diet consisting of a regular laboratory rodent chow (4 per cent final fat content), a control diet supplied with soybean oil (15 per cent final fat content), or a control diet supplied with coconut oil (15 per cent final fat content). Glycogen-elicited peritoneal neutrophils from rats fed soybean and coconut oil-enriched diets presented a reduction in spontaneous and PMA-stimulated H2O2 generation relative to neutrophils from rats fed the control diet. The activity of superoxide dismutase, glutathione peroxidase and catalase did not change in animals fed fat-rich diets. In addition, the capacity to generate O2-, spontaneously or in response to PMA, did not change in neutrophils from animals fed fat-rich diets. Values attained matched those observed in animals fed the control diet, regardless of the method used to measure O2-, the superoxide dismutase-inhibitable reduction of cytochrome c or the lucigenin-dependent chemiluminescence. However, the initial rate of O2- generation both in resting neutrophils and in PMA-stimulated cells was significantly reduced when animals were fed with coconut or soybean oil-enriched diets due, at least in part, to a reduction in the activity of glucose-6-phosphate dehydrogenase. The concentration of thiobarbituric acid reactive substances, an index of lipid peroxidation, was increased in animals fed both fat-rich diets. This was accompanied by an increase in arachidonic acid content in these cells. Results presented suggest that lipid peroxidation in neutrophils from animals fed fat-rich diets may be associated with a consumption of H2O2 yielding more reactive oxygen-derived species such as the hydroxyl radical.     

Accumulation of histidine, 3-methylhistidine, and homocarnosine in the brains of protein-calorie deficient monkeys

Abstract— Seventeen monkeys (M. nemestrina and M. fascicularis) aged 10 months to about 5 yr were divided into two groups and fed either an adequate protein diet (20% casein) or a low-protein diet (2% casein). The diets were supplied to the animals in restricted amount (200 g/animal in two daily rations). In one experiment, the malnourished animals were initially fed a diet containiing 8 per cent protein and the protein content of the diet was gradually reduced over a period of 9 months, to 2 per cent. After about 3 months on the 2 per cent protein diet, the malnourished monkeys showed growth failure, severe anorexia, peri-ocular oedema, tremors of the head and limbs, atrophy of several visceral organs, fatty liver, hypoalbuminaemia, and depressed serum levels of many essential amino acids with an elevation of the ratio of non-essential to essential amino acids. These features are consistent with protein-calorie malnutrition. Examination of the brains revealed significant alterations in the levels of glycerophosphoethanolamine (—40 per cent), glutamic acid (—25 per cent), histidine (+230 per cent), homocamosine (+185 per cent), 3-methyl-histidine (+147 per cent), lysine (+55 per cent), phenylalanine (+33 per cent) and tyrosine (+26 per cent) in comparison to findings on the well-fed monkeys. The possible implications of elevated cerebral contents of homocarnosine in malnourished monkeys are discussed in the light of several reported human cases in whom neurological disorders are associated with increased histidine-containing dipeptides in the brain, CSF, blood and urine.     

Utilization of fatty acids by Pieris brassicae reared on artificial and natural diets

The fatty acid composition of Pieris brassicae was measured from larvae reared on four different diets. Pieris can alter the composition of fatty acids in the diet through selective incorporation and synthesis. Oleate is preferentially accumulated on artificial diets (15·9 per cent in diet, 43·8 per cent in neutral lipid (NL) of fifth instar larvae), but not equally on natural diets (18·1 per cent in Brassica napus, 25·6 per cent in the NL of fifth instar larvae). Incorporation of linolenate appears to depend on the concentration of both linolenate and linoleate in the diet. With dietary levels of 35·7% linolenate and 32·2% linoleate, fifth instar larvae contain 12·2 and 16·0 per cent, respectively, of these acids. With 45·8% linolenate and 12·5% linoleate in the diet, fifth instar larvae contain 44·1 and 11·6 per cent of these acids, respectively, in the NL. Palmitoleate is actively synthetized on the artificial diets; with trace amounts of dietary palmitoleate, fifth instar larvae have 9·3 per cent of this acid in the NL. Pieris regulates the uptake of linoleate from the diet at the intestinal wall as was shown by linoleic acid-1-¹⁴C, and is unable to convert dietary linoleate to any of the 18-carbon analogues. The female apparently accumulates linolenate into egg phospholipids on the artificial diet, but in general the fatty acid composition of the eggs resembles that of the fat body.     

The cellular composition of the blood and haematopoietic organs of turbot Scophthalmus maximus L.

The cellular composition of the blood, anterior kidney, spleen and thymus of turbot Scophrhalmus maximus L., aged 1 + was determined. Ninety-four per cent of blood cells belonged to the erythrocyte lineage of which 82% were mature erythrocytes. The leucocytes, which represented 4.5% of the blood cells, were mainly lymphocytes (50%). The presence of crythroblasts in the anterior kidney and the spleen demonstrated an erythropoietic activity in both organs. However, this activity appeared to be prevalent in the spleen which also appeared to act as a storage zone for erythrocytes and as the centre point for thrombopoiesis. Although 96% of the anterior kidney cells were leucocytes, the number of white cells per gram of organ was higher in the spleen.     

Postnatal changes in fatty acids composition of brown adipose tissue

It has been demonstrated that thermogenic activity of brown adipose tissue (BAT) is higher during the early postnatal period, decreasing towards a low adult level. The present study examined postnatal changes in the lipid composition of BAT. BAT from pre-weaning rats at 4 and 14 days old showed the following differences in lipid composition compared to that from adults of 12 weeks old. (i) Relative weight of interscapular BAT to body weight was markedly greater. (ii) BAT-triglyceride (TG) level was lower, while BAT-phospholipid (PL)level was higher. (iii) In TG fatty acids (FA) polyunsaturated fatty acids (PU; mol %), arachidonate index (AI), unsaturation index (UI) and PU/saturated FA (SA) were higher; rare FA such as eicosadienoate, bishomo--linolenic acid and lignoceric acid in mol % were also higher. (iv) In PL-FA monounsaturated FA (MU) in mol % was lower; PU mol %, AI and UI were higher. These features in BAT of pre-weaning rats resembled those in the cold-acclimated adults, suggesting a close relationship of the PL-FA profile to high activity of BAT.     

Effect of olive oil- and corn oil-enriched diets on the tissue mineral content in mice

The mineral content (zinc, iron, magnesium, and calcium) in the liver, spleen, and thymus of male Balb/C mice was analyzed. Animals were fed, over 21 d, diets enriched with corn oil (FCO diet) or olive oil (FOO diet) (5% addition to standard pellet, w/w). Olive oil with predominant oleic acid (C18:1, n-9) had a quite different composition than corn oil, in which linoleic acid (C18:2, n-6) prevails. The zinc and magnesium tissue concentrations were not changed in either group. The calcium concentration in liver as well as the calcium concentration in spleen increased in mice fed both the FCO and FOO diets. Furthermore, mice fed both the FOO and FCO diets had increased spleen iron concentration. Mice fed the FCO diet had increased thymus calcium concentration compared to controls. The results show the effect of diets with unsaturated, particularly polyunsaturated fatty acids, on the calcium and iron concentration in some organs.     

The fatty acid composition of sphingolipids from bovine CNS axons and myelin

Abstract— Cerebrosides, sulphatides and sphingomyelin were isolated from bovine CNS myelin and from myelin-free axons derived from myelinated axons. The fatty acid composition of each sphingolipid was determined by gas-liquid chromatography of the fatty acid methyl esters. In each case the fatty acids of the axonal sphingolipids were of shorter average chain length than those from the corresponding myelin lipids. These differences, however, were small and the fatty acids of the axonal cerebrosides and sulphatides were similar in average chain length to those reported previously for bovine myelin. The principal unsubstituted acid of both cerebroside and sulphatide from axons was 24: 1, with the total long chain acids (> C₁₈) amounting to 80 and 85 per cent, respectively. The corresponding figures for myelin galactolipids were 94 and 95 per cent long chain acids. The principal α-hydroxy acid of both axonal galactolipids was 24 h:0, with cerebroside having 80 per cent and sulphatide 92 per cent long chain acids, compared to the figures of 87 and 97 per cent for the corresponding myelin lipids. In axonal sphingomyelin the major acid was 18:0 (compared to 24:1 in myelin) and the long chain acids were 61 per cent of the total vs 76 per cent of the total for myelin sphingomyelin. The non-identity of axonal and myelin sphingolipid fatty acids substantiates the belief that they are intrinsic axonal constituents. These findings do not rule out the possibility of a close metabolic relationship between the sphingolipids of the axon and its myelin sheath.     

The effects of dietary (n-3) fatty acid supplementation on lipid dynamics and composition in rat lymphocytes and liver microsomes

Rats were fed diets devoid of (n-3) fatty acids (olive oil supplementation) or high in (n-3) fatty acids (fish oil supplementation) for a period of 10 days. In spleen lymphocytes and liver microsomes derived from animals fed fish oil diets, relatively high levels of (n-3) eicosapentaenoic (20:5), docosapentaenoic (22:5) and docosahexaenoic acids (22:6) were obtained compared to minimal levels when fed the olive oil diet. When the average lipid motional properties were examined by measuring the fluorescence anisotropy of diphenylhexatriene, no significant different was found between intact liver microsomes from animals fed the two diets. However, when lipid motion was examined in vesicles of phosphatidylcholine, isolated from the microsomes from fish oil fed animals (21.4% (n-3) fatty acids), the fluorescence anisotropy was significantly less than the corresponding phosphatidylcholine from olive oil fed animals (5.6% (n-3) fatty acids), indicating a more disordered or fluid bilayer in the presence of higher levels of (n-3) fatty acids. Phosphatidylethanolamine (n-3) fatty acids were also elevated after fish oil supplementation (41.3% of total fatty acids), compared to the level after olive oil supplementation (21.4%). The major effect of the fish oil supplementation was a replacement of (n-6) arachidonic acid by the (n-3) fatty acids and when this was 'modeled', using liposomes of synthetic lipids, 1-palmitoyl-2-arachidonyl(n-6) or docosahexaenoyl(n-3)-phosphatidylcholine, significant differences in lipid motional properties were found, with the docosahexaenoate conferring a more disordered or fluid lipid environment. Thus it appears that although lipid order/fluidity can be significantly decreased by increases in the highly unsaturated (n-3) fatty acid levels, alterations in membrane domain organization and/or phospholipid molecular species composition effectively compensated for the changes, at least as far as average lipid motional properties in the intact membranes was concerned.     

Extracellular release of free fatty acids by rat T lymphocytes is stimulus-dependent and is affected by dietary lipid manipulation

[(3)H]-Arachidonic acid-labelled rat T lymphocytes released radioactivity extracellularly when stimulated by the calcium ionophore A23187 or by monoclonal antibodies to some cell surface structures (CD2, CD5, CD11a, CD18, CD54, T-cell receptor) but not to others (CD49d, CD62L); release was greater with the calcium ionophore. Almost all of the radioactivity released from anti-CD2-stimulated lymphocytes was recovered in the free fatty acid fraction, whereas only about 50 per cent of that released after A23187 stimulation was recovered in this fraction. A23187 stimulation resulted in release of arachidonic acid from a variety of phospholipids (phosphatidylinositol, phosphatidylcholine and perhaps phosphatidylethanolamine), while the monoclonal antibody stimulation released arachidonic acid from phosphatidylinositol and perhaps phosphatidylcholine. Unstimulated lymphocytes released a range of fatty acids extracellularly, with palmitic acid accounting for 35-40 per cent and arachidonic acid for 5 per cent of released fatty acid. Stimulation of lymphocytes with either anti-CD2 or A23187 increased total fatty acid release 1.5- to 1.8-fold. In both cases palmitic acid remained the most predominant fatty acid released but the contribution of arachidonic acid increased. The type of lipid fed to the rats significantly influenced the amount and type of fatty acid released. Fish oil feeding significantly reduced extracellular fatty acid release by stimulated lymphocytes.     

Differential effects of unsaturated fatty acids on modulation of endotoxin-induced tissue factor activation in cultured human leukemia U937 cells.

We examined the direct effects of unsaturated fatty acids, oleic (18:1 n-9), linoleic (18:2 n-6), eicosapentaenoic (20:5 n-3) and docosahexaenoic (22:6 n-3) on tissue factor (TF) activity in the human leukemia monocytic U937 cell line. After exposing cells to fatty acids for 16 h, there were no significant effects on either TF activity or its activation induced by bacterial endotoxin (LPS). When the cells were primed with fatty acids for 24 h, 48 h or 72 h, the TF activity remained essentially unchanged. However, the extent of TF-activation induced by LPS depended on the length of priming, and the dose and the degree of unsaturation of the fatty acids to which cells were exposed. After a 72-h priming, 18:1 produced 40-60 per cent elevation in LPS-challenge. In contrast, approximately 20-50 per cent reduction in LPS-challenge was achieved by 18:2, 20:5 and 22:6 at high concentrations. The results suggest that chronic exposure of U937 cells to unsaturated fatty acids leads to modulation of the TF-activation in response to LPS.     

Dose-dependent effects of dietary gamma-linolenic acid on rat spleen lymphocyte functions.

Feeding rodents a diet rich in evening primrose oil (EPO), which contains 5-10 g gamma-linolenic acid (GLA)/100 g total fatty acids, has been shown to decrease lymphocyte proliferation and natural killer cell activity. However, EPO contains a very high level of linoleic acid which itself can affect lymphocyte functions and it is not clear to what extent the effects of EPO can be attributed to GLA. The current study investigated the effect of two levels of GLA in the rat diet upon immune cell functions; the level of linoleic acid was maintained below 30 g/100 g total fatty acids. Weanling rats were fed on high fat (178 g/kg) diets which contained 4.4 g or 10 g GLA/100 g total fatty acids in place of a proportion of linoleic acid. The total polyunsaturated fatty acid content and the n-6 to n-3 polyunsaturated fatty acid ratio of the diet were maintained at 35 g/100 g total fatty acids and 7, respectively. The fatty acid compositions of the serum and of spleen leukocytes were markedly influenced by that of the diet, with an increase in the proportions of GLA and dihomo-gamma-linolenic acid when the diets containing GLA were fed; these diets also increased the proportion of arachidonic acid in spleen leukocytes. Spleen lymphocyte proliferation in response to concanavalin A was significantly reduced (by 60%) by feeding the diet containing the higher level of GLA, but not by the diet containing the lower level of GLA. Spleen natural killer cell activity and prostaglandin E (PGE) production by spleen leukocytes were not significantly affected by inclusion of GLA in the diet, although there was a tendency towards decreased natural killer cell activity by cells from rats fed the high GLA diet. Thus, this study shows that dietary GLA is capable of altering the fatty acid composition of cells of the immune system and of exerting some immunomodulatory effects, but that the level of GLA in the diet must exceed 4.4 g/100 g total fatty acids for these effects to become apparent.     

Stability of the brain fatty acid pattern in adult rats during extreme starvation

Abstract— Adult rats were denied food for 7 days. As compared with a control group, this severe starvation reduced the mean total body weight by 44 per cent, the weight of the diaphragm by 33 per cent and liver by 67 per cent, the total lipid content of the diaphragm by 57 per cent and liver by 69 per cent, and the total lipid phosphorus content of the diaphragm by 19 per cent and liver by 68 per cent. The decrease in lipid phosphorus contents indicates that the diaphragm and liver catabolized membrane phospholipids as well as triglycerides. In contrast, the fresh weight of the brain and the total lipid content of the brain were not significantly altered by starvation. The fatty acid patterns of the total lipid of the diaphragm and liver (determined by GLC) were grossly altered by starvation. In the brain, however, 17 of the 21 fatty acids measured did not change significantly (P > 0.05) and the remaining four changed, with borderline statistical significance, by only 2 to 13 per cent. There was no significant effect of starvation of the pattern of the brain polyunsaturated fatty acids when measured by alkali isomerization. In contrast to the liver and diaphragm, the brain is apparently unable to utilize its lipids appreciably as an energy source. Presumably the lipids of the brain are preserved to permit this organ to function properly, even in the last stages of starvation.     

Rôle of labelled dietary fatty acids and acetate in phospholipids during the metamorphosis of Pieris brassicae

The incorporation of labelled dietary palmitic, linoleic, and linolenic acids into neutral (NL) and phospholipids (PL) during the metamorphosis of Pieris brassicae was studied, and the ability of the fat body to incorporate acetate into PL determined. Thirty-three per cent of total lipid in early fifth instar larvae (minus haemolymph) is PL, while the corresponding value in female 4-day pupae is 13·0 per cent and in the fat body of 4-day pupae 6·3 per cent. Incorporation of label into PL was studied more closely and in all cases the label was recovered from phosphatidylcholine (PTC) and phosphatidylethanolamine (PTE). The label from palmitate was also found in sphingomyelin and possibly phosphatidylserine. Specific activity of PL in the case of palmitic and linolenic acids was greatest in late fifth instar larvae. In early fifth instar larvae on palmitic acid-1-¹⁴C 39·0 per cent of label was in PTC, 52·8 per cent in PTE, and 2·0 per cent in sphingomyelin. In late fifth instar 45·0 per cent was in PTC, 45·5 per cent in PTE, and 6·5 per cent in sphingomyelin, while in 4-day female pupae 45·2 per cent was in PTC, 41·3 per cent in PTE, and 13·5 per cent in sphingomyelin. The label from linolenic acid only varied a little from early fifth instar to 4-day pupae, 51·8 per cent being in PTC and 48·2 per cent in PTE in early fifth instar larvae. The label from linoleic acid is incorporated in fat body PL almost exclusively in PTC and PTE, 55·8 and 43·2 per cent respectively in 4-day female pupae. Injected acetate is distributed after 1 hr between PTC (58·6 per cent), PTE (24·4 per cent), and sphingomyelin (17·0 per cent). It was concluded that the polyunsaturated acids are proportionately more common in PTE than in other PL types, and that the fatty acids of sphingomyelin are mainly those that the insect is capable of synthesizing from acetate. Palmitic acid is desaturated by Pieris to palmitoleic acid and the latter possibly utilized in PTE to compensate for a deficiency of linolenic acid in the artificial diet. No saturation of linoleic or linolenic acid was found. The rates of PL and NL synthesis during development and the rôle of the investigated fatty acids in the biosynthesis of PL are discussed.     

Brain cardiolipin: isolation and fatty acid positions

Abstract— Cardiolipin (diphosphatidyl glycerol) was isolated from bovine brain grey matter in pure form by column chromatography. A combination of a DEAE cellulose column, an acid-silicic acid column and a bicarbonate-treated silicic acid column was used for the isolation. Analytical data on cardiolipin (and lyso-cardiolipin), including phosphorus and fatty ester values, infrared and ultraviolet spectra, and chromatographic behaviour of intact cardiolipin, lyso-cardiolipin and their water-soluble hydrolysis products gave results which were consistent with the diphosphatidylglycerol structure proposed by MacFarlane. Cardiolipin constituted 1·2 per cent of the total lipids from grey matter, or 0·43 per cent of the dry weight. No aldehydes were detected in purified cardiolipin. The major fatty acids were 16:0, 16:1, 18:1, 18:2 and 20:4. Cardiolipin contained much higher concentrations of 18:2 (17·8 per cent) than any of the other major grey matter glycerophosphatides. Cardiolipin was hydrolysed by incubation with snake venom (Trimeresurus flavoviridis) phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4) to determine the distribution of fatty acids on the carbons of its diglyceride moieties. The fatty acids were not distributed randomly; 16:0, 18:0 and 18:2 were predominantly localized in the α and α positions, while 18:1 and 20:4 were predominantly localized in the β and β positions.     

Amino acid transport in thymic- and spleen-derived lymphocytes.

We have examined the transport of amino acids by the sodium-dependent "A" and "ASC" system in thymic- and splenic-derived lymphocytes from the Long-Evans rat. Lymphocytes derived from the thymus transport amino acids by both the "A" and "ASC" systems, whereas lymphocytes from the spleen transport amino acids by the "ASC" system only. Thymic lymphocytes are capable of establishing a steady state distribution ratio of 7.9 for 2-aminoisobutyric acid, but splenic lymphocytes can attain only 3.5. The steady state distribution ratio of alanine was the same in both cell types. Sodium-independent transport is also different in splenic and thymic lymphocytes. But both cells move amino acids by a Na+-independent system for mediated exchange-diffusion. The studies show that lymphocytes derived from the spleen and thymus transport amino acids differently, and that the "T" lymphocytes from the spleen have membrane transport systems different from "T" lymphocytes from the thymus.     

Polyunsaturated and saturated fatty acids-rich diets and immune tissues. 1. Fresh weight, DNA content, percentage of lipids and rate of lipogenesis from 3H2O in thymus, spleen and mesenteric lymph nodes

It has been widely reported that polyunsaturated fatty acids-rich diets (PUFA n-6) cause immunodeficiency. In this study, fresh weight, DNA and lipid content and rate of lipogenesis from 3H2O of thymus, spleen, mesenteric lymph nodes and liver were assessed in rats fed polyunsaturated (UC) or saturated (SC) fatty acids-rich chows. The results obtained indicate that both types of fatty acids caused similar increment of lipid content in the immune tissues. The rate of lipogenesis was decreased only in the thymus of (UC) rats and liver of both experimental groups as compared to control rats. The results of fresh weight and DNA content was decreased only by (UC) diet.     

Distribution of enzymes of purine metabolism in lymphocytes of horse, Equus caballus

A microassay requiring as few as 2 X 10(5) cells per assay was developed for systematic analysis of 9 purine enzymes in lymphocytes from equine peripheral blood, spleen, lymph node, thymus and bone marrow. The activities of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), adenosine kinase (AK), deoxyadenosine kinase (dAK), deoxycytidine kinase (dCK), 5'-nucleotidase (5'-N), AMP deaminase, hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), and adenine phosphoribosyl transferase (APRT) were measured by this microassay in lymphocytes from peripheral blood from four different breeds of horses (Arabian, Quarter Horse, Thoroughbred and Shetland Pony). There were no significant differences in the enzyme activities among the various breeds. Peripheral blood lymphocytes (PBL) from foals exhibited enzyme activities similar to those observed for adult animals. All lymphoid tissue contained similar levels of activity for each kinase (AK, dAK and dCK). Spleen had the highest activity for ADA, PNP, 5'-N, and HGPRT. The lowest activity for ADA, APRT, PNP and AMP deaminase was found in thymus. Enzymatic activities that varied the most among the tissue were 5'-N, ADA, APRT, HGPRT and AMP deaminase.     

Starvation-sensitive UCP 3 protein expression in thymus and spleen mitochondria

To date, UCP 3 has only been associated with skeletal muscle and brown adipose tissue (BAT). Using RT-PCR/PCR methodology, we show that human spleen and human thymus contain UCP 3. In addition, using peptide antibodies, previously demonstrated to be selective for UCP 3, we show that UCP 3 protein is present in mitochondria isolated from rat thymus and mitochondria isolated from reticulocytes, monocytes and lymphocytes of rat spleen. UCP 3 protein expression is also starvation-sensitive. UCP 3 abundance is augmented in mitochondria isolated from thymus and mitochondria isolated from lymphocytes of the spleen from fasted rats when compared to fed controls. The results are consistent with a role for UCP 3 in developing lymphocytes, thymus atrophy and fatty acid utilisation in spleen and thymus.     

Comparative studies of the cyclic AMP content and renewal of this nucleotide in thymic, splenic and lymph-node lymphocytes in adult rats]

Cyclic AMP (c-AMP) content and turnover were measured in pure preparations of lymphocytes obtained from thymus, spleen and lymph nodes in the Rat. The c-AMP content was determined by combining the methods of Krishna and of Thomson and Appleman, and its turnover was estimated from the activities of adenylcyclase and phosphodiesterase using 3H-adenine. The values, espressed per 10(8) cells, were the lowest for the thymus and the highest for the lymph nodes, while they were intermediary for the spleen. The differences in the c-AMP turnover between the three organs may be correlated with the extent of the mitotic activity of the corresponding lymphocytes, this activity being related inversely to the turnover of c-AMP.     

Nutritive value of lentil and vetch straws as compared with alfalfa hay and wheat straw for replacement ewe lambs

The objective of this study was to compare the nutritive value of lentil straw (LS) and vetch straws (VS) with alfalfa hay (AH) and wheat straw (WS). Forty Awassi ewes (body weight (BW)=56.2 kg) were assigned randomly to four dietary treatments (10 ewes per treatment) for 6 weeks using a complete randomize design. In each dietary treatment, ewes were fed 550 g per day of concentrate mix formulated to supply 40% of the metabolizable energy requirement. After the consumption of the concentrate mix, ewes had ad libtum access to AH, LS, VS, or WS. Forage intake, total dry matter intake (DMI), and organic matter intake (OMI) were higher for AH and LS and lowest for WS. Final weight of ewes fed AH and LS diets were higher (P<0.05) than VS and WS diets. However, ewes fed VS diet gained more weight (2.5 kg) than ewes on WS diet (0.2 kg). Dry matter digestibility was higher for the AH and LS diets (59.4%), and lowest for WS diet (49.6%). The VS diet had an intermediate value (54.4%). Ruminal passage rate was higher for AH and LS (3.4% per hour) compared with WS (1.9% per hour). Eating time for the AH diet was lowest compared with the rest of the forages. Eating time, expressed as minutes per kilogram NDF intake, was highest for WS and VS diets (680 min) compared with AH and LS diets (535 min). WS diet required more time for rumination (395 min per day) compared to AH, LS, and VS diets. However, ewes on VS diet spent more time ruminating per kilogram NDF consumed (954 min) compared with WS, LS, and AH diets (901, 617, and 597 min, respectively). These results were significant and demonstrate that the nutritive value of LS is greater than VS and close to the nutritive value of AH. VS nutritive value is slightly greater than WS.