Processed fava bean as a substitute for rapeseed meal with or without rumen-protected methionine supplement in grass silage-based dairy cow diets.

Fava bean offers a sustainable home-grown protein source for dairy cows, but fava bean protein is extensively degraded in the rumen and has low Met concentration. We studied the effects of protein supplementation and source on milk production, rumen fermentation, N use, and mammary AA utilization. The treatments were unsupplemented control diet, and isonitrogenously given rapeseed meal (RSM), processed (dehulled, flaked, and heated) fava bean without (TFB) or with rumen-protected (RP) Met (TFB+). All diets consisted of 50% grass silage and 50% cereal-based concentrate including studied protein supplement. The control diet had 15% of crude protein and protein-supplemented diets 18%. Rumen-protected Met in TFB+ corresponded to 15 g/d of Met absorbed in the small intestine. Experimental design was a replicated 4 × 4 Latin square with 3-wk periods. The experiment was conducted using 12 multiparous mid-lactation Nordic Red cows, of which 4 were rumen cannulated. Protein supplementation increased dry matter intake (DMI), and milk (31.9 vs. 30.7 kg/d) and milk component yields. Substituting RSM with TFB or TFB+ decreased DMI and AA intake but increased starch intake. There were no differences in milk yield or composition between RSM diet and TFB diets. Rumen-protected Met did not affect DMI, or milk or milk component yields but increased milk protein concentration in comparison to TFB. There were no differences in rumen fermentation except for increased ammonium-N concentration with the protein-supplemented diets. Nitrogen-use efficiency for milk production was lower for the supplemented diets versus control diet but tended to be greater for TFB and TFB+ versus RSM. Protein supplementation increased plasma essential AA concentration but there were no differences between TFB diets and RSM. Rumen-protected Met clearly increased plasma Met concentration (30.8 vs. 18.2 µmol/L) but did not affect other AA. Absence of differences between RSM and TFB in milk production together with limited effects of RP Met suggest that TFB is a potential alternative protein source for dairy cattle.

Effects of different barley and oat varieties on methane production, digestibility, and fermentation pattern in vitro.

The objective of this in vitro study was to determine the effects of different barley and oat varieties on CH4 production, digestibility, and rumen fermentation patterns in dairy cows. Our hypothesis was that oat-based diets would decrease CH4 production compared with barley-based diets, and that CH4 production would differ between varieties within grain species. To evaluate this hypothesis, we conducted an in vitro experiment using a fully automated gas production technique, in which the total gas volume was automatically recorded by the system. The experiment consisted of triplicate 48-h incubations with 16 treatments, including 8 different varieties of each grain. The grain varieties were investigated as a mix with an early-cut grass silage (1:1 ratio of grain to silage on a dry matter basis) and mixed with buffered rumen fluid. We estimated predicted in vivo total gas production and CH4 production by applying a set of models to the gas production data obtained by the in vitro system. We also evaluated in vitro digestibility and fermentation characteristics. The variety of grain species did not affect total gas production, CH4 production, or fermentation patterns in vitro. However, in vitro-determined digestibility and pH were affected by variety of grain species. Grain species affected total gas and CH4 production: compared with barley-based diets, oat-based diets decreased total gas production and CH4 production by 8.2 and 8.9%, respectively, relative to dry matter intake. Grain species did not affect CH4 production relative to in vitro true dry matter digestibility. Oat-based diets decreased digestibility and total volatile fatty acid production, and maintained a higher pH at 48 h of incubation compared with barley-based diets. Grain species did not affect fermentation patterns, except for decreased molar proportions of valerate with oat-based diets. These results suggest that replacing barley with oats in dairy cow diets could decrease enteric CH4 production.

The effect of partial substitution of rapeseed meal and faba beans by Spirulina platensis microalgae on milk production, nitrogen utilization, and amino acid metabolism of lactating dairy cows.

Alternative protein sources such as microalgae and faba beans may have environmental benefits over rapeseed. We studied the effects of rapeseed meal (RSM) or faba beans (FB) as a sole protein feed or as protein feeds partially substituted with Spirulina platensis (spirulina) microalgae on milk production, N utilization, and AA metabolism of dairy cows. Eight multiparous Finnish Ayrshire cows (113 ± 36.3 d in milk; mean ± SD) were used in a balanced, replicated 4 × 4 Latin square with 2 × 2 factorial arrangement of treatments and 21-d periods. Four cows in one Latin square were rumen cannulated. Treatments were 2 isonitrogenously fed protein sources, RSM or rolled FB, or one of these sources with half of its crude protein substituted by spirulina (RSM-SPI and FB-SPI). Cows had ad libitum access to total mixed rations consisting of grass silage, barley, sugar beet pulp, minerals, and experimental protein feed. The substitution of RSM with FB did not affect dry matter intake (DMI) but decreased neutral detergent fiber intake and increased the digestibility of other nutrients. Spirulina in the diet decreased DMI and His intake. Spirulina had no effect on Met intake in cows on RSM diets but increased it in those on FB diets. Energy-corrected milk (ECM) and protein yields were decreased when RSM was substituted by FB. Milk and lactose yields were decreased in cows on the RSM-SPI diet compared with the RSM diet but increased in those on FB-SPI compared with FB. The opposite was true for milk fat and protein concentrations; thus, spirulina in the diet did not affect ECM. Feed conversion efficiency (ECM:DMI) increased in cows on FB diets with spirulina, whereas little effect was observed for those on RSM diets. The substitution of RSM by FB decreased arterial concentration of Met and essential AA. Spirulina in the diet increased milk urea N and ruminal NH4-N and decreased the efficiency of N utilization in cows on RSM diets, whereas those on FB diets showed opposite results. Met likely limited milk production in cows on the FB diet as evidenced by the decrease in arterial Met concentration and milk protein yield when RSM was substituted by FB. The results suggest the potential to improve milk production response to faba beans with supplementation of Met-rich feeds such as spirulina. This study also confirmed spirulina had poorer palatability than RSM and FB despite total mixed ration feeding and lower milk production when spirulina partially replaced RSM.

Effects of dry period energy intake on insulin resistance, metabolic adaptation, and production responses in transition dairy cows on grass silage-based diets.

High energy intake in the dry period has reportedly had adverse effects on mobilization of body reserves, dry matter intake, and productivity of dairy cows. We investigated whether grass silage (GS) fed ad libitum (high energy intake, HEI; 141% of daily metabolizable energy requirements) in an 8-wk dry period affects metabolic adaptation-specifically, peripheral insulin resistance-compared with a total mixed ration consisting of GS, wheat straw, and rapeseed meal (55/40/5%; controlled energy intake, CEI; 108% of metabolizable energy/d) fed ad libitum. Multiparous Ayrshire dairy cows (n = 16) were used in a randomized complete block design until 8 wk after parturition. Commercial concentrates were fed 1 and 2 kg/d during the last 10 to 6 and 5 to 0 d before the expected calving date, respectively. Postpartum, a similar lactation diet with ad libitum access to GS and increasing concentrate allowance (maximum of 16 kg/d) was offered to all. The HEI group gained more body weight and had higher plasma insulin, glucose, and ß-hydroxybutyrate concentrations than the CEI group prepartum. Postpartal plasma glucose tended to be higher and milk yield was greater from wk 5 onward for HEI compared with CEI cows. An intravenous glucose tolerance test (IVGTT) was performed at -13 ± 5 d and 9 ± 1 d relative to calving. The HEI cows had greater insulin response to glucose load and smaller area under the response curve for glucose than CEI cows in prepartal IVGTT. Thus, compensatory insulin secretion adapted to changes in insulin sensitivity of the peripheral tissues, preserving glucose tolerance of HEI cows. Higher insulin levels were needed in HEI cows than in CEI cows to elicit a similar decrement of nonesterified fatty acid concentration in prepartal IVGTT, suggesting reduced inhibition of lipolysis by insulin in HEI cows before parturition. In conclusion, high energy intake of moderately digestible GS with low concentrate feeding in the close-up dry period did not have adverse effects on metabolic adaptation, insulin sensitivity, and body mobilization after parturition. Instead, this feeding regimen was more beneficial to early-lactation performance than GS-based total mixed ration diluted with wheat straw.

Effect of incremental amounts of camelina oil on milk fatty acid composition in lactating cows fed diets based on a mixture of grass and red clover silage and concentrates containing camelina expeller.

Camelina is an ancient oilseed crop that produces an oil rich in cis-9,cis-12 18:2 (linoleic acid, LA) and cis-9,cis-12,cis-15 18:3 (α-linolenic acid, ALA); however, reports on the use of camelina oil (CO) for ruminants are limited. The present study investigated the effects of incremental CO supplementation on animal performance, milk fatty acid (FA) composition, and milk sensory quality. Eight Finnish Ayrshire cows (91d in milk) were used in replicated 4×4 Latin squares with 21-d periods. Treatments comprised 4 concentrates (12kg/d on an air-dry basis) based on cereals and camelina expeller containing 0 (control), 2, 4, or 6% CO on an air-dry basis. Cows were offered a mixture of grass and red clover silage (RCS; 1:1 on a dry matter basis) ad libitum. Incremental CO supplementation linearly decreased silage and total dry matter intake, and linearly increased LA, ALA, and total FA intake. Treatments had no effect on whole-tract apparent organic matter or fiber digestibility and did not have a major influence on rumen fermentation. Supplements of CO quadratically decreased daily milk and lactose yields and linearly decreased milk protein yield and milk taste panel score from 4.2 to 3.6 [on a scale of 1 (poor) to 5 (excellent)], without altering milk fat yield. Inclusion of CO linearly decreased the proportions of saturated FA synthesized de novo (4:0 to 16:0), without altering milk fat 18:0, cis-9 18:1, LA, and ALA concentrations. Milk fat 18:0 was low (<5g/100g of FA) across all treatments. Increases in CO linearly decreased the proportions of total saturates from 58 to 45g/100g of FA and linearly enriched trans-11 18:1, cis-9,trans-11 18:2, and trans-11,cis-15 18:2 from 5.2, 2.6, and 1.7 to 11, 4.3, and 5.8g/100g of FA, respectively. Furthermore, CO quadratically decreased milk fat trans-10 18:1 and linearly decreased trans-10,cis-12 18:2 concentration. Overall, milk FA composition on all treatments suggested that one or more components in camelina seeds may inhibit the complete reduction of 18-carbon unsaturates in the rumen. In conclusion, CO decreased the secretion of saturated FA in milk and increased those of the trans-11 biohydrogenation pathway or their desaturation products. Despite increasing the intake of 18-carbon unsaturated FA, CO had no effect on the secretions of 18:0, cis-9 18:1, LA, or ALA in milk. Concentrates containing camelina expeller and 2% CO could be used for the commercial production of low-saturated milk from grass- and RCS-based diets without major adverse effects on animal performance.

Effects of replacing rapeseed meal with fava bean at 2 concentrate crude protein levels on feed intake, nutrient digestion, and milk production in cows fed grass silage-based diets.

The objective of this study was to evaluate the production and physiological responses of dairy cows to the substitution of fava bean for rapeseed meal at 2 protein supplementation levels in grass silage-based diets. We used 6 primiparous and 6 multiparous Finnish Ayrshire cows in a cyclic changeover trial with a 2×3 factorial arrangement of treatments. The experimental diets consisted of formic acid-treated timothy-meadow fescue silage and 3 isonitrogenous concentrates containing either rapeseed meal, fava bean, or a 1:1 mixture of rapeseed meal and fava bean at low and high inclusion rates, resulting in concentrate crude protein (CP) levels of 15.4 and 19.0% in dry matter. Silage dry matter intake decreased linearly when rapeseed meal was replaced with fava bean, the negative effect being more distinct at the high CP level than the low (-2.3 vs. -0.9kg/d, respectively). Similarly, milk and milk protein yields decreased linearly with fava bean, the change tending to be greater at the high CP level than the low. Yield of milk fat was lower for fava bean compared with rapeseed meal, the difference showing no interaction with CP level. Especially at the high CP level, milk urea concentration was higher with fava bean compared with rapeseed meal indicating better utilization of protein from the rapeseed meal. The apparent total-tract organic matter digestibility did not differ between treatments at the low CP level, but digestibility was higher for fava bean than for rapeseed meal at the high CP level. Plasma concentrations of essential amino acids, including methionine and lysine, were lower for fava bean than for rapeseed meal. Compared with rapeseed meal, the use of fava bean in dairy cow diets as the sole protein supplement decreased silage intake and milk production in highly digestible formic acid-treated grass silage-based diets.

Sense of coherence and health behaviour in adolescence.

AIM: To explore associations of Sense of coherence (SOC) with health behaviour and social competence among 15-year-old adolescents. METHODS: Study population is a prospective cohort of a randomized cluster sample of families and their first-born children from south-western Finland in 1986-1987. In this study, cross-sectional data of the 15-year-olds were used. The present data were based on mailed, pretested questionnaires. The outcome variable, SOC, was based on the 13-item scale of Antonovsky's Orientation to Life Questionnaire (OLQ). The principal explanatory variables were health behaviour, including experienced oral health, and social competence. The statistical analysis was performed using linear regression modelling. RESULTS: Strong SOC of adolescents associated significantly with lighter use of alcohol, being a non-smoker, better care of oral health and better social competence compared with the others. CONCLUSION: Sense of coherence is a useful tool for identifying adolescents in need of extra support and motivation for their health behaviour.

Effects of faba bean, blue lupin and rapeseed meal supplementation on nitrogen digestion and utilization of dairy cows fed grass silage-based diets.

There is increasing interest in using locally produced protein supplements in dairy cow feeding. The objective of this experiment was to compare rapeseed meal (RSM), faba beans (FBs) and blue lupin seeds (BL) at isonitrogenous amounts as supplements of grass silage and cereal based diets. A control diet (CON) without protein supplement was included in the experiment. Four lactating Nordic Red cows were used in a 4 × 4 Latin Square design with four 21 d periods. The milk production increased with protein supplementation but when expressed as energy corrected milk, the response disappeared due to substantially higher milk fat concentration with CON compared to protein supplemented diets. Milk protein output increased by 8.5, 4.4 and 2.7% when RSM, FB and BL were compared to CON. The main changes in rumen fermentation were the higher propionate and lower butyrate proportion of total rumen volatile fatty acids when the protein supplemented diets were compared to CON. Protein supplementation also clearly increased the ruminal ammonia N concentration. Protein supplementation improved diet organic matter and NDF digestibility but efficiency of microbial protein synthesis per kg organic matter truly digested was not affected. Flow of microbial N was greater when FB compared to BL was fed. All protein supplements decreased the efficiency of nitrogen use in milk production. The marginal efficiency (amount of additional feed protein captured in milk protein) was 0.110, 0.062 and 0.045 for RSM, FB and BL, respectively. The current study supports the evidence that RSM is a good protein supplement for dairy cows, and this effect was at least partly mediated by the lower rumen degradability of RSM protein compared to FB and BL. The relatively small production responses to protein supplementation with simultaneous decrease in nitrogen use efficiency in milk production suggest that economic and environmental consequences of protein feeding need to be carefully considered.

Perineurial cells coexpress genes encoding interstitial collagens and basement membrane zone components.

Perineurial cell cultures were established from the sciatic nerves of adult Wistar rats. Highly enriched cultures were studied with respect to the production of extracellular matrix components under conditions free from the influence of Schwann cells, axons, or the extracellular matrix of peripheral nerves. Indirect immunofluorescence staining revealed the presence of collagen type IV epitopes, and electron microscopy demonstrated patches of basement membrane on the perineurial cell surfaces. Collagenous fibrils with a diameter of 15-20 nm were also observed in the intracellular space. SDS-PAGE of radiolabeled medium proteins showed a pattern of bands suggesting the synthesis and secretion of fibronectin, and type I and IV collagens. Northern hybridizations revealed characteristic polymorphic mRNA transcripts corresponding to fibronectin, laminin B2 chain, as well as to the alpha-chain subunits of type I, III, and IV collagens. Furthermore, in situ hybridizations suggested expression of these genes by cultured perineurial cells without apparent heterogeneity within the cell populations. In situ hybridizations of sciatic nerve tissue from 2-wk-old rats also suggested that perineurial cells express alpha 1(I) and alpha 2(IV) collagen, as well as laminin B2 chain genes in vivo. This profile of matrix gene expression is different from that of Schwann cells, which do not synthesize fibronectin, or that of fibroblastic cells, which do not form a cell surface basement membrane. The capability of perineurial cells to express genes for the basement membrane zone and for interstitial collagens further adds to our understanding of the functional role of perineurial cells in developing and healing peripheral nerve, as well as in certain neoplastic lesions of neural origin, such as von Recklinghausen's neurofibromas.

Plasticity of integrin expression by nerve-derived connective tissue cells. Human Schwann cells, perineurial cells, and fibroblasts express markedly different patterns of beta 1 integrins during nerve development, neoplasia, and in vitro.

Strikingly selective expression patterns of beta 1, alpha 2, alpha 3, and alpha 5 integrin subunits were revealed in endoneurium, perineurium, and epineurium of fetal and adult human peripheral nerve by immunostaining with specific antibodies. The alpha 2 subunit was expressed only on Schwann cells both in fetal and adult nerve, whereas the alpha 3 epitopes were expressed exclusively in the adult tissue and were primarily present on perineurial cells. The alpha 5 epitopes were expressed only on the innermost cell layer of perineurium of fetal and adult nerve. The tumor cells within schwannomas and cutaneous neurofibromas expressed both alpha 2 and alpha 3 subunits, indicating that Schwann cells have the potential to express also the alpha 3 subunit in vivo. Cell cultures established from human fetal nerve and neurofibromas revealed expression of the alpha 2 and alpha 5 epitopes on Schwann cells, perineurial cells, and fibroblasts, whereas only Schwann cells contained the alpha 3 epitopes which were occasionally concentrated on the adjacent Schwann cells at cell-cell contacts. Our findings emphasize that nerve connective tissue cells change their profiles for expression of extracellular matrix receptors under conditions which have different regulatory control signals exerted by, for example, axons, humoral factors, or the extracellular matrix of the peripheral nerve. This plasticity may play an important role during nerve development and in neoplastic processes affecting the connective tissue compartments of peripheral nerve.

Type 1 neurofibromatosis: selective expression of extracellular matrix genes by Schwann cells, perineurial cells, and fibroblasts in mixed cultures.

Cutaneous neurofibromas, characteristic lesions of neurofibromatosis 1, are composed of an abundant extracellular matrix and nerve connective tissue-derived cell types: Schwann cells, perineurial cells, and fibroblasts. In this study, the extracellular matrix gene expression by these cells was examined under culture conditions that allowed them to be metabolically active and readily identifiable by morphologic and immunocytochemical criteria. Northern hybridizations demonstrated expression of genes for type I, III, IV, and VI collagens, as well as for fibronectin, laminin, and elastin. In situ hybridizations revealed that all three cell types expressed pro alpha 1 (I), pro alpha 2 (VI), and laminin B1 chain genes. However, fibroblasts did not contain [35S]cDNA-mRNA hybrids specific for type IV collagen, whereas both Schwann cells and perineurial cells expressed these genes. Perineurial cells and fibroblasts readily expressed the fibronectin gene whereas Schwann cells were essentially devoid of the corresponding mRNA. Perineurial cells also expressed the gene for laminin A chain. The results indicate that the extracellular matrix gene expression profiles of Schwann cells, perineurial cells, and fibroblasts are distinct: all three cell types are capable of expressing some of the genes for extracellular matrix components, such as type I and VI collagens, whereas Schwann cells and perineurial cells may have the primary role in synthesizing basement membrane zone components, type IV collagen and laminin. These observations potentially relate to the mechanisms of growth and development of human neurofibromas. The results attest to the applicability of the methodology utilized here to study other human tumors with mixed cell populations.

Collagen gene expression by cultured human skin fibroblasts. Abundant steady-state levels of type VI procollagen messenger RNAs.

Previous studies have suggested that procollagen types I and III are the major collagenous gene products of cultured human skin fibroblasts. In this study the expression of 10 different genes, encoding the subunit polypeptides for collagen types I-VI, by human skin fibroblasts in culture was analyzed by molecular hybridizations. Northern transfer analysis demonstrated the presence of specific mRNA transcripts for collagen types I, III, IV, V, and VI, but not for type II collagen. Quantitation of the abundance of these mRNAs by slot blot hybridizations revealed that type I, III, and VI procollagens were the major collagenous gene products of skin fibroblasts in culture. The mRNAs for type IV and V collagens represented only a small percentage of the total collagenous mRNA transcripts. Further analysis by in situ hybridization demonstrated that the majority of the cultured cells coexpressed the genes for type I, III, and VI procollagen pro-alpha chains. Further in situ hybridization analyses revealed the expression of type VI collagen genes in normal human skin. These data demonstrate that human skin fibroblast cultures can be used to study the transcriptional regulation of at least nine genetically distinct procollagen genes. The data further suggest that type VI collagen, in addition to types I and III, may be a major collagenous component of human skin.

Localization of integrin receptors for fibronectin, collagen, and laminin in human skin. Variable expression in basal and squamous cell carcinomas.

VLA integrins in human skin were examined by indirect immunofluorescence utilizing antibodies recognizing the beta 1, alpha 2, alpha 3, or alpha 5 subunits. Staining of fetal, newborn, or adult skin with antibodies to beta 1, alpha 2, or alpha 3 subunits gave essentially similar staining patterns: intense staining was associated with the basal layer of the epidermis, hair follicles, and blood vessel walls. The alpha 5 subunit could be detected only in epidermis and the inner root sheath of hair follicles in fetal skin. In epidermis, the staining reaction for the beta 1 subunit was not only found in sites interfacing with the basement membrane zone, but also around the entire periphery of these cells. We speculate that these receptors might have previously unrecognized functions in cell-cell interactions or that these findings may suggest the presence of previously unrecognized ligands in the intercellular spaces of keratinocytes. Examination of nine nodular basal cell carcinomas revealed a prominent staining reaction with anti-beta 1 and anti-alpha 3 antibodies at the periphery of the tumor islands. In contrast, staining of five squamous cell carcinomas revealed either the absence of integrins or altered and variable expression. Thus, matrix components and their receptors may participate in modulation of growth, development, and organization of human skin.

Effects of replacing grass silage with barley silage in dairy cow diets.

This study examined the effects of gradually replacing grass silage with whole-crop barley silage on feed intake, ruminal and total tract digestibility, and milk yield in lactating dairy cows. Four dairy cows in early lactation, equipped with rumen cannulas, were fed 4 diets over four 21-d periods. The diets consisted of 4 forage mixtures of grass silage and whole-crop barley silage supplemented with 8.9 kg/d of concentrates [dry matter (DM) basis]. The proportion of barley silage in the forage was adjusted to 0, 0.20, 0.40, and 0.60 kg/ kg of DM. Ruminal nutrient metabolism was measured on the basis of digesta flow entering the omasal canal. Ammonia concentrations and volatile fatty acid profiles were determined in the rumen fluid. Ruminal digestion and passage kinetics were assessed by the rumen evacuation technique. Replacement of grass silage with barley silage had no effect on DM, digestible organic matter, or neutral detergent fiber (NDF) intake, but starch intake increased, whereas nitrogen and digestible NDF (dNDF) intake decreased. Increases in the proportion of barley silage linearly decreased milk yield, and the molar proportion of acetate in the rumen, and increased that of propionate, butyrate, and valerate. Decreases in milk yield due to inclusion of barley silage were attributed to decreases in diet digestibility and nutrient supply to the animal. Barley silage linearly decreased organic matter digestibility in the total tract and NDF and dNDF digestibility in the rumen and the total tract, and decreased nonammonia N flow entering the omasal canal. No significant differences between diets were noted in the digestion rate of dNDF or passage rate of indigestible NDF from the rumen. Decreases in organic matter and NDF digestibility were attributed to the higher indigestible NDF concentration of barley silage compared with that of grass silage and to the smaller pool size of dNDF in the rumen.

Increased matrix gene expression by glucose in rat neural connective tissue cells in culture.

The effects of different glucose concentrations on the expression of extracellular matrix genes were examined in primary cell cultures initiated from sciatic nerves of nondiabetic Sprague-Dawley rats. The cells were incubated in medium containing 5.5, 15, or 25 mM D-glucose, and the expression of type I and IV collagens, laminin, and fibronectin genes was examined at mRNA steady-state levels by Northern hybridizations. Incubation of cell cultures, consisting of Schwann cells, perineurial cells, and fibroblasts, in high glucose concentrations (15 or 25 mM D-glucose) resulted in elevation of pro-alpha 1(IV), pro-alpha 2(IV), and pro-alpha 1(I) collagen chain and fibronectin mRNAs after only 3 days of incubation, whereas laminin B2 chain mRNA levels appeared unaltered. These observations suggest that hyperglycemia may partially contribute to basement membrane thickening in peripheral nerves of diabetic individuals by increasing the expression of genes coding for basement membrane components, e.g., type IV collagen.

Effects of plant species, stage of maturity, and level of formic acid addition on lipolysis, lipid content, and fatty acid composition during ensiling.

Forage type and management influences the nutritional quality and fatty acid composition of ruminant milk. Replacing grass silage with red clover (RC; L.) silage increases milk fat 18:3-3 concentration. Red clover has a higher polyphenol oxidase (PPO) activity compared with grasses, which has been suggested to decrease lipolysis and . The present study characterized the abundance and fatty acid composition of esterified lipid and NEFA before and after ensiling of grass and RC to investigate the influence of forage species, growth stage, and extent of fermentation on lipolysis. A randomized block design with a 2 × 3 × 4 factorial arrangement of treatments was used. Treatments comprised RC or a mixture of timothy ( L.) and meadow fescue ( Huds.) harvested at 3 growth stages and treated with 4 levels of formic acid (0, 2, 4, and 6 L/t). Lipid in silages treated with 0 or 6 L/t formic acid were extracted and separated into 4 fractions by TLC. Total PPO activity in fresh herbage and the content of soluble bound phenols in all silages were determined. Concentrations of 18:3-3 and total fatty acids (TFA) were higher ( < 0.001) for RC than for grass. For both forage species, 18:3-3 and TFA content decreased linearly ( < 0.001) with advancing growth stage, with the highest abundance at the vegetative stage. Most of lipid in fresh RC and grass herbage (97%) was esterified, whereas NEFA accounted for 71% of TFA in both silages. Ensiling resulted in marginal increases in TFA content and the amounts of individual fatty acids compared with fresh herbages. Herbage total PPO activity was higher ( < 0.001) for RC than grass (11 vs. 0.11 µkatal/g leaf fresh weight). Net lipolysis during ensiling was extensive for both forage species (660 to 759 g/kg fatty acid for grass and 563 to 737 g/kg fatty acid for RC). Formic acid application (0 vs. 6 L/t) resulted in a marked decrease ( = 0.026) in net lipolysis during the ensiling of RC, whereas the opposite was true ( = 0.026) for grass. In conclusion, results suggest that formic acid addition during the ensilage of RC decreases lipolysis . For both plant species, total PPO activity was not associated with the extent of lipolysis . However, bound phenols formed via PPO activity appear to have a role in protecting lipid and protein against degradation in grass and lowering proteolysis of RC during ensiling.

Expression of beta 4 integrins in human skin: comparison of epidermal distribution with beta 1-integrin epitopes, and modulation by calcium and vitamin D3 in cultured keratinocytes.

The expression of beta 4 integrins in adult and fetal human skin as well as in cultured keratinocytes was studied by immunodetection with monoclonal antibodies, and compared with that of beta 1 integrins. The distribution of the beta 1 and beta 4 integrin epitopes was entirely different in the adult epidermis. As reported previously by us (J Clin Invest 84:1916, 1989), the beta 1 epitopes were present most prominently at lateral cell-cell contact points, whereas staining with the beta 4 antibody demonstrated a linear staining pattern polarizing to the basal surface juxtaposed to the dermal-epidermal basement membrane. In contrast, in fetal skin, the staining patterns both with beta 1 and beta 4 antibodies were similar and demonstrated the presence of epitopes surrounding the entire cell surface of both basal and suprabasal keratinocytes. Distinct polarization of beta 4 integrin epitopes was noted in cultured keratinocytes maintained in low-calcium (0.15 mM) medium, and the expression of these integrins was also noted in human papilloma virus-transformed keratinocytes. Switch of the cultures to high-calcium (1.2 mM) medium resulted in redistribution of the epitopes to a pattern suggesting their location at under-surface of the cells adjacent to the substratum. This Ca(++)-induced redistribution of beta 4 integrin epitopes could be counteracted by 10(-7) M vitamin D3. Finally, incubation with anti-beta 4 integrin antibody reduced the capacity of keratinocytes to attach to plastic substratum. The results provide further evidence for the role of beta 4 integrins in cell-matrix interactions.

Fibronectin gene expression by epithelial tumor cells in basal cell carcinoma: an immunocytochemical and in situ hybridization study.

Previous observations have demonstrated that fibronectin is deposited in high abundance in basal cell carcinoma stroma. In this study, the nature of fibronectin and the site of its synthesis were explored in 10 basal cell carcinomas of the nodulo-ulcerative type by immunocytochemistry and in situ hybridization. First, simultaneous localization of epithelial tumor cell islands and fibronectin epitopes was carried out by double immunofluorescence staining with monoclonal anti-cytokeratin antibodies and polyclonal fibronectin antibodies, the latter recognizing both the cellular and plasma types of the protein. Large amounts of fibronectin were deposited in the basal cell carcinoma stroma, with the highest concentration present in the immediate proximity of the epithelial cell islands. Immunofluorescence with a monoclonal anti-fibronectin antibody, which is directed against the ED-domain of cellular fibronectin and does not recognize the plasma type of fibronectin, revealed essentially the same staining pattern as that obtained with the polyclonal anti-fibronectin antibody. This observation suggested that fibronectin in BCC was predominantly of the cellular type. Second, in situ hybridizations, utilizing a human fibronectin specific cDNA, demonstrated that the highest concentration of fibronectin mRNA was found in the most peripheral cell layer of the epithelial tumor islands. The presence of fibronectin mRNAs in the tumor cells of the central regions of the islands, as well as within occasional stromal cells, was also noted. Thus, two lines of evidence suggest that the epithelial tumor cells are predominantly responsible for the synthesis and deposition of fibronectin in basal cell carcinoma. The presence of fibronectin may explain the characteristic biologic behavior of basal cell carcinomas, including low degree of metastatic potential and local destructive nature of the tumors.

Evaluation of transforming growth factor beta and type I procollagen gene expression in fibrotic skin diseases by in situ hybridization.

Full thickness biopsies of affected skin and fascia from one patient with diffuse fasciitis and eosinophilia (DF), two patients with generalized morphea (GM), and five patients with progressive systemic sclerosis (PSS) of recent onset were examined for the expression of transforming growth factor beta 1 (TGF beta 1) and type I procollagen genes by in situ hybridization with human sequence-specific cDNA. An increased number of fibroblasts showing clearly detectable expression of pro alpha 1(I)collagen gene was found in all fibrotic lesions when compared with unaffected skin from the patient with DF and skin from two normal individuals examined in parallel. Expression of the TGF beta 1 gene was noted in a fibroblast subpopulation of the affected tissues from the patients with DF and GM. In contrast, the TGF beta 1 gene was not expressed at a detectable level in affected skin from the five patients with PSS. The results suggest that TGF beta 1 may play a role in the development of skin fibrosis in cases of DF and GM. However, from these studies, we cannot implicate TGF beta 1 in the pathogenesis of skin fibrosis in PSS.

Activation of collagen gene expression in keloids: co-localization of type I and VI collagen and transforming growth factor-beta 1 mRNA.

Untreated, clinically active keloids were examined as model system to study the spatial expression of extracellular matrix and transforming growth factor-beta 1 (TGF-beta 1) genes in fibrotic skin diseases. In situ hybridizations localized active expression of type I and VI collagen genes to the areas containing an abundance of fibroblasts and apparently representing the expanding border of the lesions. Within this zone, microvascular endothelial cells also expressed the type I collagen genes, as evaluated by simultaneous use of in situ hybridization for collagen gene expression and immunolocalization for factor VIII-related antigen, a marker for endothelial cell differentiation. Slot-blot hybridizations of RNA isolated from this zone suggested that the expression of type I and IV collagen genes was selectively enhanced, as compared to type III collagen gene expression. TGF-beta 1 protein and mRNA were also detected in areas active in type I and type VI collagen gene expression, indicating that TGF-beta 1 gene is transcribed and the corresponding protein is deposited in areas of elevated collagen gene expression, including microvascular endothelial cells. We conclude that the initial step in the development of fibrotic reaction in keloids involves the expression of the TGF-beta 1 gene by the neovascular endothelial cells, thus activating the adjacent fibroblasts to express markedly elevated levels of TGF-beta 1, as well as type I and VI collagen genes.