Fatty Acid Profile Of Milk Fat Research Articles

Milk fatty acid composition in Holstein x Gyr dairy cows fed chopped elephantgrass-based diets containing two types of sunflower oil associated with two methods of concentrate feeding

wo experiments were carried out in a 2 x 2 factorial arrangement with the objective of evaluating two methods of concentrate feeding for Holstein x Gyr cows fed 600 g kg-1 chopped elephantgrass-based diets supplemented at 45 g kg-1 DM with two types of sunflower oil (SO). The types of SO differed in the levels of oleic and linoleic fatty acids (FAs): high oleic/low linoleic acid – HO (73 and 10 g 100 g-1 FA, respectively) and medium oleic/medium linoleic acid – MO (43 and 34 g 100 g-1 FA, respectively). The concentrates containing HO SO or MO SO were supplied separately from the forage twice a day after the two milkings (TAD) or as part of a total mixed ration (TMR). In Experiment 1, a 4 x 4 Latin square design was used to evaluate the ruminal fermentation and degradation parameters in four rumen-cannulated cows (430±39 kg; 79±20 days in milk; 16.4±3.1 kg day-1 of milk). In Experiment 2, a randomized block design was used to evaluate the nutrient intake, plasma contents of metabolites and FAs, milk yield and composition, and FA profile of milk fat in 32 cows (444±84 kg; 75±31 days in milk; 15.4±4.8 kg day-1 of milk). The results were analyzed by mixed models (P ? 0.05). The TMR diets promoted higher nutrient intake and rumen fermentation (higher ammonia N, acetate, propionate and total volatile FA contents) without affecting milk, fat, protein and lactose yields. TAD-fed cows presented higher feed efficiency and produced milk fat with a more nutritionally desirable FA composition, with higher vaccenic and rumenic acid contents and lower trans-10 C18:1 and palmitic acid contents. The DM intake, parameters of rumen fermentation and milk, fat, protein and lactose yields were similar for the HO SO and MO SO diets. The most nutritionally positive characteristics for human health in the milk fat of HO SO-fed cows were the higher eicosapentaenoic (+34%) and oleic acid (+11%) contents and lower palmitic acid content (-10%). Higher contents of vaccenic (+71%) and rumenic (+74%) acids and lower trans-10 C18:1 (-10%), elaidic (-32%), lauric (-14%) and myristic (-11%) acid contents were the most positive aspects of the milk fat of MO SO-fed cows. Considering the magnitudes of the differences in the levels of these FAs, it is concluded that the milk fat of cows fed MO SO showed a healthier milk FA profile.

Study of Fatty-acid Composition of Goat and Sheep Milk and Its Transformation in the Production of Yogurt

The information on the structure of fatty acids is crucial for the production and promotion of goat and sheep milk in the manufacture of dairy products. The fatty-acid profile of milk fat can affect the nutritional value and market value of dairy products. The purpose of the research is to study the properties of fatty-acid composition of goat and sheep milk and its transformation in the process of yogurt production. The study of fatty-acid composition was performed using the method of gas chromatography. The milk of goats of Zaanen breed and sheep of North Caucasian breed, as well as yogurt produced from a mixture of goat and sheep milk in a ratio of 1:1 have become the objects of the study. It was found that goat’s milk contains 12% less saturated fatty acids than sheep’s milk. Oleic, stearic and palmitic acids are the main fatty acids found in the fat phase of milk and yogurt production. Changes in the concentration of individual fatty acids during milk processing and in the period of yogurt storage were noted. As a result of maturation and seven-day storage, the amount of saturated fatty acids in yogurt increased by 5% compared to the original milk mixture. The content of polyunsaturated fatty acids in yogurt decreased by 19.27 %. The highest ratio value of hypocholesterolemic and hypercholesterolemic fatty acids was noted in goat milk. It is proved that goat milk is characterized by the most acceptable fatty acid composition in terms of healthy nutrition and prevention of atherosclerosis and thrombosis. A tendency to reduce the amount of monounsaturated and polyunsaturated fatty acids, with a simultaneous increase in the content of saturated fatty acids is noted in the process of yogurt production and storage. It was found that the production of yogurt with the use of goat’s milk in the mixture allows reducing the values of atherogenicity and thrombogenicity indices. The results of the study provide an information basis for the production of qualitatively new fermented milk drinks with a fatty-acid profile favorable for human health.

Effect of dietary quebracho tannin extract on milk fatty acid composition in cows.

The aim of this study was to examine the capacity of quebracho tannin extract (QTE) to modulate the fatty acid (FA) profile in the milk fat of cows. Fifty Holstein cows yielding 33.2 ± 8.2 kg/d of milk were divided into 2 groups. The cows were fed a basal diet with a forage-concentrate ratio of 66:34 on a dry matter (DM) basis. Diets tested were control (CON, basal diet without QTE) and basal diet plus 15 or 30 g of QTE/kg of DM (QTE15 and QTE30, respectively). Two treatments could be tested simultaneously and were arranged along 6 periods. The milk FA profile was characterized by increments in the proportion of linoleic (LA) and α-linolenic acid (α-LNA) (QTE15 = 10 and 6.1%; QTE30 = 28 and 25%, respectively) compared to CON, which might indicate reduced ruminal biohydrogenation (BH) of both dietary LA and α-LNA. Vaccenic acid (VA) in the milk fat was reduced (QTE15 8.9% and QTE30 12%) compared to CON, which may be linked to inhibited BH of LA and α-LNA. Rumenic acid (RA), a conjugated LA (cis-9,trans-11 conjugated linoleic acid) and an important human health promoter, was unfortunately decreased (QTE15 8.3% and QTE30 16%) in the milk compared with CON, probably because of inhibited ruminal BH of LA. However, reduced RA in the milk was probably due to reduced availability of VA produced in the rumen and the consequently low VA available to be desaturated to RA in the mammary gland by Δ9-desaturase. The proportions of total polyunsaturated FA were increased with QTE15 and QTE30 by 4.7 and 15% compared to CON, respectively, and the long-chain FA proportions were also increased (QTE15 2.0% and QTE30 8.2%). Moreover, myristic and palmitic acid were reduced by QTE30 (9.6 and 3.3%, respectively) compared to CON, which also contributed to increasing the nutritional quality of milk because they are recognized to increase high-density lipoprotein in humans. Branched-chain FA in milk was reduced with QTE treatments, which indicates inhibited ruminal BH and microbial activity. In general, our findings suggest that dietary QTE have the potential to modulate FA profile of milk fat, and this effect is dosage dependent. Because QTE influenced the FA profile of milk fat both positively and negatively, further research is needed before concluding that QTE may improve the nutritional quality of cow milk fat in human diets.

Milk fatty acid profile in cows fed red clover- or alfalfa-silage based diets differing in rumen-degradable protein supply

Polyphenol oxidase in red clover (RC) silage reduces lipolysis and consequently protects its constituent fatty acids (FA) against biohydrogenation by ruminal microorganisms. Fatty acid biohydrogenation could be further inhibited by reducing the nitrogen (N) supply to ruminal bacteria. To compare the effects of RC and alfalfa (AL) silage fed in diets differing in rumen-degradable protein supply on the transfer efficiency of polyunsaturated FA from diet to milk, and on the resulting FA profile of milk fat, 8 multiparous Holstein dairy cows (72±17days in milk) were used in a replicated 4×4 Latin square design (21-day periods including 14days of adaptation). Four treatments were compared in a 2×2 factorial arrangement with AL or RC fed in diets formulated to provide 100% (RDP‐100) or 85% (RDP‐85) of calculated rumen-degradable protein requirements. Rumen-degradable protein concentrations were adjusted by varying the supplies of ground vs. steam-flaked corn grains, and untreated vs. heat-treated soybean meals. Feeding RC, as compared with AL diets, decreased ruminal ammonia-N concentration as well as urea-N in plasma and milk. Intakes of c9c12 18:2 and c9c12c15 18:3 were lower for cows fed RC, but their secretion in milk and their apparent transfer efficiency from diet were greater as compared to cows fed AL. As a result, feeding RC increased the concentrations of c9c12 18:2 and c9c12c15 18:3, but decreased the proportions of t11 18:1, c9t11 18:2, and t11c15 18:2 in milk fat. These results are consistent with the lower effective ruminal disappearance of c9c12 18:2 and c9c12c15 18:3 in RC as determined by a 96h in sacco incubation, and the lower abundance of ruminal bacteria capable of hydrolyzing dietary lipids and hydrogenating polyunsaturated FA (Ruminococcus albus and Ruminococcus flavefaciens) in the rumen content. The apparent transfer efficiency of c9c12 18:2 from diet to milk was further increased by feeding RDP‐85 as compared with RDP‐100 diet. Feeding RC decreased concentrations of linear odd-chain FA (11:0, 13:0, 15:0, 17:0, and c9 17:1) and increased proportions of branched-chain FA (iso13:0, iso14:0, iso15:0, iso16:0, iso17:0, anteiso15:0, and anteiso17:0) in milk fat as compared with AL. In conclusion, as compared with AL, cows fed RC produced milk with greater concentrations of major forage FA (c9c12 18:2 and c9c12c15 18:3) and lower proportions of intermediates in ruminal biohydrogenation of these FA. Variations in milk fat concentrations of odd- and branched-chain FA, which are synthesized in the rumen by various microbial populations, may be reflecting the effects of forage source on ruminal fermentation.

Lipid complex effect on fatty acid profile and chemical composition of cow milk and cheese

The effect of administration of lipid complex (LC) on cow milk and cheese characteristics was studied. Lipid complex was elaborated based on grapeseed oil with synthesized conjugated linoleic acid (CLA) and Atlantic mackerel oil enriched in n-3 fatty acids. The 4-wk experiment was conducted on 30 Polish Holstein Friesian cows. The experimental group cow diet was supplemented with 400 g/d of LC (containing 38% CLA, and eicosapentaenoic acid + docosahexaenoic acid in a relative amount of 36.5%) on a humic-mineral carrier. The chemical composition and fatty acid profile of milk and rennet cheese from raw fresh milk were analyzed. Lipid complex supplementation of the total mixed ration had no effect on milk yield and milk composition, except fat content, which decreased from 4.6 to 4.1%, a 10.9% decrease. Milk from cows treated with LC had greater relative amounts of unsaturated fatty acids, particularly polyunsaturated fatty acids, and lesser relative amounts of saturated fatty acids. Lipid complex addition changed milk fat fatty acid profile: C18:2 cis-9,trans-11 and trans-10,cis-12 isomer (CLA) contents increased by 278 and 233%, respectively, as did eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6) contents. Milk fat fatty acid profile changes were correlated with the modifications in rennet cheese fatty acid profile. Lipid complex supplementation of dairy cows produced considerable changes in the biological value of milk and cheese fat.

Near Infrared Spectroscopy (NIRS) for the determination of the milk fat fatty acid profile of goats

Milk fatty acid (FA) composition is important for the goat dairy industry because of its influence on cheese properties and human health. The aim of the present work was to evaluate the feasibility of NIRS reflectance (oven-dried milk using the DESIR method) and transflectance (liquid milk) analysis to predict milk FA profile and groups of fats in milk samples from individual goats. NIRS analysis of milk samples allowed to estimate FA contents and their ratios and indexes in fat with high precision and accuracy. In general, transflectance analysis gave better or similar results than reflectance mode. Interestingly, NIRS analysis allowed direct prediction of the Atherogenicity and Thrombogenicity indexes, which are useful for the interpretation of the nutritional value of goat milk. Therefore, the calibrations obtained in the present work confirm the viability of NIRS as a fast, reliable and effective analytical method to provide nutritional information of milk samples.

Effect of calcium salts of fish and palm oils on lactational performance of Holstein cows

Two experiments were conducted to determine the effect of source of fatty acids (FA) fed as tallow or calcium salts of fish and palm oils (CaSFO) on performance of dairy cows. In experiment 1, 738 multiparous Holstein cows were fed a diet with either 18 g/kg of tallow or 19 g/kg of CaSFO to supply equal amounts of FA in two replicates per treatment. In experiment 2, 331 multiparous cows were fed one of the two supplements described previously. In both experiments, all cows received a pre-treatment diet during the first 25 days in milk (DIM) consisting of half of each fat supplement (9 g/kg of tallow + 9.5 g/kg of CaSFO). Thereafter, cows received their respective diets for the first 145 DIM. Yields of milk and milk fat were not affected by dietary source of FA in both experiments. However, feeding CaSFO reduced concentration of true protein and solids-not-fat, as well as yields of true protein. Group and individual dry matter (DM) intakes and feed efficiency were not altered by treatment, but cows fed CaSFO had higher total tract apparent digestibility of DM. Cows fed CaSFO had increased concentrations of eicosapentaenoic, docosahexaenoic, conjugated linoleic acid ( cis 9, trans 11), C18:1 trans 9, and C18:1 trans 11 FA in milk fat. Treatment had no effect on blood parameters and estimated energy balance, but cows fed CaSFO lost more body condition. Feeding CaSFO at 19 g/kg of the diet modified the FA composition of milk fat compared with tallow, but did not improve lactational performance of dairy cows. The present findings suggest that supplementation with tallow or CaSFO results in similar lactational performance, with changes in FA profile of milk fat.

Response of lactating dairy ewes to various levels of dietary calcium soaps of fatty acids

Two experiments were completed to assess lactational effects of feeding calcium soaps of fatty acids (CSFA) to lactating sheep. In the first experiment, 50 Manchega ewes were blocked into groups of 10 to which the treatments were applied. Ewes grazed and were supplemented indoors with hay and a concentrate. Treatments consisted of different levels of CSFA in the concentrate, being: control, 50, 100, 150 or 200 g CSFA/kg as fed. Concentrates were fed individually at fixed amounts depending on lactation period: nursing (weeks 1–4; 1.0 kg/day), and early (weeks 5–7; 1.0 kg/day), mid (weeks 8–14; 0.8 kg/day) and late milking (weeks 15–21; 0.6 kg/day). Level of CSFA did not affect milk yield, and effects on milk composition varied by lacation period. The biggest effects occurred during nursing, when milk fat content (P<0.05) and yield (P<0.001), as well as milk total solids content (P<0.001) and yield (P<0.05), increased linearly with CSFA dose. Milk fat content increased at a decreasing rate (linear and quadratic P<0.05) with CSFA dose during milking, but milk protein content decreased (P<0.05) linearly in mid and late (P<0.01) lactation. Relative milk fat yield response to CSFA fat was bigger (P<0.01) during nursing than in milking (55% and 20%, respectively) and decreased linearly (P<0.001) with increasing CSFA feeding at the end of lactation. Findings from the first experiment were applied in a second experiment to the total mixed ration of a flock of 94 Manchega ewes. Treatments consisted of control and CSFA added (42 g CSFA/kg DM) total mixed rations. Diets were compared during mid lactation (weeks 7–16) by using a switch-back design of three periods of 20 days each (1, control; 2, CSFA; 3, control). Contents of milk fat and milk total solids, as well as their yields, increased (P<0.05) in the CSFA diet, but milk protein did not vary. Milk from CSFA fed ewes had less saturated fatty acids (FA) and more monounsaturated FA than the control. The C14:1/C14:0 Δ 9–desaturase index was not modified by CSFA. Overall, feeding CSFA to dairy ewes increased milk fat content and modified the FA profile of milk fat. Although FA from dietary CSFA were efficiently transferred to milk at the beginning of lactation, use of moderate amounts of CSFA is recommended, particularly at the end of lactation in order to avoid negative effects on milk protein concentration.

Influence of percentage of concentrate in combination with rolled canola seeds on performance, rumen fermentation and milk fatty acid composition in dairy goats

Abstract The effect of rolled canola seeds (RCS, 0% or 20% of the dry matter (DM) of the concentrate) combined with percentage of concentrate (45% (L) vs. 65% (H) of the diet DM) on rumen fermentation characteristics, production parameters and fatty acid (FA) profile of milk fat was studied in 20 entire goats and 12 goats fitted with ruminal and duodenal cannula. Goats were fed TMR ad libitum for 15 weeks with the final 12 weeks as the test period. Individual milk samples were taken in weeks 5 and 11 to determine the FA profile of milk fat. Feeding the H diets increased NE L intake, raw milk yield and yield of fat, protein and lactose. Feeding RCS increased fat corrected milk, milk fat content and fat yield. Combination of RCS and high percentage of concentrate increased fat corrected milk and fat yield. Milk protein content was not affected by dietary factors. Feeding the H diets increased the proportion of short-chain FA (C4:0–C8:0), each isomer of trans -C18:1 and c9,t11-C18:2 in milk at the expense of medium-chain FA with 11 to 17 C units. Feeding RCS decreased the proportion of medium-chain FA with 10 to 17 C units in milk and it increased that of C18:0, all isomers of trans -C18:1, c9-C18:1 and c9,t11-C18:2. Goats fed the H diets, and those fed the RCS had lower c9-C18:1/C18:0 and c9,t11-C18:2/t11-C18:1 ratios. Combination of RCS and high percentage of concentrate produced the highest proportion of all isomers of trans -C18:1 and c9,t11-C18:2 in milk, with the highest variability among the four diets for these FA. This was possibly related to between-goats differences in ruminal fermentation and to individual difference in desaturase activity in the mammary gland. It is concluded that feeding a high-concentrate diet combined with rolled canola seeds had a synergistic effect on the milk production and fat yield without alteration in milk protein content in dairy goats. This combination also resulted in changes in FA profile that may differently affect the nutritional value of goat milk for human health.

Milk production and fatty acid profile of milkfat by dairy cows fed a winter oats (Avena sativa L.) pasture only or a total mixed ration

Milk production and milk fatty acid (FA) composition were determined in 12 Holstein dairy cows in mid‐lactation fed winter oats pasture only (WOP) or total mixed ration (TMR) in a single crossover design. The WOP treatment cows received a high quality winter oats (Avena sativa L.) pasture rotationally grazed, while the TMR treatment cows were fed a corn silage‐based TMR feed ad libitum once daily. Milk production tended to be lower (14.9 versus 16.3 kg day‐1; P = 0.15), and milkfat (32.2 versus 35.5 gkg‐1; P < 0.01), and total protein (35.8 versus 37.8 g kg‐1; P < 0.01) concentration were significantly lower for the WOP fed cows than for the TMR fed cows. The cows fed WOP lost live‐weight and body condition score, but the cows fed TMR gained liveweight. Cows fed WOP had a lower saturated:unsaturated FA ratio (1.85 versus 2.22; P < 0.02). The cis‐9 trans‐11 CLA content was 2.6‐fold higher (1.41 versus 0.55% of total FA; P < 0.01) for the WOP cows. These results indicated that the nutritive value of the milk could be improved by feeding cows on high quality pasture; however, milk production was lower for grazing dairy cows.

Influence of Extruded Soybeans With or Without Bicarbonate on Milk Performance and Fatty Acid Composition of Goat Milk

The effects of extruded soybeans (ESB) included at 0, 10, or 20% of dry matter (DM) of the diet in combination with sodium bicarbonate (0 vs. 1% bicarbonate added to DM) on rumen fermentation characteristics, production parameters, and fatty acid (FA) profiles of milk fat were examined in 30 midlactation goats and 6 rumen-cannulated goats fed high-concentrate diets (30:70 forage-to-concentrate ratio) ad libitum in a 3×2 factorial design. Diets were fed as total mixed rations. The trial lasted 13 wk with the final 9 wk as the test period. Milk yield and composition were recorded each week throughout the trial. Individual samples of milk were taken in wk 4, 7, 10, 11, and 13 to determine FA profile of milk fat. Dry matter intake and intake of net energy for lactation were not affected by dietary treatments. Feeding ESB did not modify ruminal pH or volatile fatty acids concentration in the rumen fluid, but it increased the molar proportion of propionate. Feeding ESB increased fat-corrected milk, milk fat content, and fat yield compared with the control diets. There was no change in milk protein content when ESB were fed. Feeding ESB increased the proportions of oleic, linoleic, and linolenic acids in milk fat at the expense of most of the saturated FA. It also increased the n-6 to n-3 FA ratio of milk. The largest changes in milk yield and milk composition were generally obtained with ESB included at 20% of DM. The addition of sodium bicarbonate tended to increase ruminal pH, VFA concentrations in the rumen fluid, and the molar proportions of acetate. The addition of sodium bicarbonate increased milk fat content and fat yield, with no change in milk FA composition. It is concluded that during midlactation, the inclusion of ESB to 20% of DM prevented low milk fat content for goats fed high-concentrate diets, with no decrease in milk protein content. The addition of sodium bicarbonate may enhance the effects of ESB on milk fat content and fat yield.

Effect of diet on the fatty acid pattern of milk from dairy cows

Twelve dairy cows 130 days in milk were sorted by milk production and body weight and assigned to three feeding regimens in a 3 × 3 Latin-square design, in order to study the effects of diet on milk fatty acid (FA) composition. The cows were fed a total mixed ration (TMR) consisting of corn silage (60%) and concentrate (40%) on dry matter basis, or grazed pasture, without (P) or with 5 kg·d -1 concentrate as a supplement (SP). Supplemented grazing dairy cows produced signif- icantly more milk than the cows on the TMR and P diets (P < 0.05). The supplementation of grazing dairy cows with a low fat concentrate did not significantly affect the milk fat FA profile. The pasture diet, with a supplement or not, decreased the concentration of saturated FA (P < 0.05) and increased the concentration of unsaturated FA (P < 0.05), of milk fat as compared to the TMR diet. The reduc- tion in medium-chain FA was offset in large part by increases in long-chain FA (mainly oleic acid). The concentrations of conjugated linoleic acid (CLA) (P < 0.05) and trans-vaccenic acid were higher (P < 0.05) in the milk fat from the grazing cows. The results showed substantial variation among individual cows within treatments on milk fat content of CLA. Significant correlations were found for individual cow's milk fat CLA content across diets. Overall, this study indicates that the con- centration of CLA in milk fat is enhanced by the dietary intake of pasture and that moderate low fat concentrate supplementation of grazing dairy cows increases performance without compromising the FA profile of milk fat. conjugated linoleic acid / dairy cow / fatty acid / milk fat / pasture / total mixed ration

Flame‐ionization detector response to methyl, ethyl, propyl, and butyl esters of fatty acids

AbstractThe concept of theoretical response factors is not directly applicable to methyl esters of short‐chain fatty acids (FA), since their carbon deficiency is larger than expected from theory. Substituting the methyl group by an ethyl, propyl, or butyl group improved the flame‐ionization efficiency of fatty acid esters gradually, up to the point where the empirical response factors of the butyl esters were identical within experimental error to the theoretical values. Butyl esters of FA have a uniform flame‐ionization detection (FID) response irrespective of the number of carbon atoms contained in the FA. They exhibit a carbon deficiency of 1.0, i.e. the carbonyl carbon atom does not respond, as expected from theory. Compared to methyl esters, which have a carbon deficiency of 1.4–1.5 for short‐chain FA, use of butyl esters has the advantage that a precalculation of the FID response enables the analyst to judge whether the analytical system employed works properly and the data produced are accurate and reliable. Both acid (BF3 or H2SO4)‐and alkali (butoxide)‐catalyzed butyl ester preparation were equally effective, giving the analyst a choice of methods so that different analytical needs can be addressed efficiently. Computing response factors and comparing the theoretically expected values with those obtained experimentally gives the experimenter an indication whether the analytical system employed for FA profiling (transesterification plus the subsequent gasliquid chromatographic separation and quantitation by FID) works properly. This setup is particularly useful for an accurate analysis of the FA profile of milk fat.

Variation in the characteristics of milkfat from pasture‐fed dairy cows during late spring and the effects of grain supplementation.

Sources of variation in milkfat characteristics were determined after approximately 115 days‐in‐milk for Friesian cows that had calved in spring and grazed ryegrass‐white clover pastures. Three groups each of 15 multiparous cows were managed as separate herds. Each herd was subjected to one of 3 management strategies: pasture only, pasture only during the experimental period but previously supplemented with 2–3 kg/cow/day maize grain, and pasture plus 3–4 kg/cow/day maize grain. During a 3‐week period beginning in November 1994, milk samples were collected twice‐weekly from individual cows and analysed for total milkfat, protein and lactose, fatty acid profiles, and solid fat content (SFC). Repeat analyses were conducted on 39 samples to provide an estimation of analytical variation for fatty acid profiles. Estimates of between‐cow variation were greater than those of within‐cow variation for all parameters except fatty acids C6:0, C14:0, and C18:2. Analytical variation was small. SFC at 10°C of milkfat from individual cows was correlated significantly with total milkfat concentration, the protein: fat ratio, and the proportions of fatty acids C16:0, C17:0, C17:1, C18:1, and conjugated C18:2. Supplementing pasture with maize grain had minor effects on the fatty acid profile of milkfat, but had no significant effect on SFC. These data will enable increased efficiency of experimental design in this research area, and detail the extent of the natural variation in milkfat characteristics.