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The Journal of Nutrition Vol. 128 No. 9 September 1998,
pp. 1525-1532
, and
* École Nationale Vétérinaire, Département Élevage & Produits, Laboratoire d'Alimentation, 31076 Toulouse Cedex, France and École Nationale Supérieure Agronomique, Laboratoire d'Ingénierie Agronomique, 31076 Toulouse Cedex, France
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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The effect of dietary lipids on the fatty acid (FA) profile of cows' milk fat is mainly dependent on digestive processes and mammary gland uptake and metabolism of FA. The objective of this study was to determine the separate effects of high arterial concentrations of 16:0, 18:0 and cis-18:1(n-9) on uptake, synthesis and 18:0 desaturation rate in the mammary gland of lactating dairy cows, via arterio-venous differences and mammary gland balance of FA. In a 4 × 4 Latin square, four lactating Holstein cows with cannula in the proximal duodenum were infused duodenally with a mixture providing daily 0 (C treatment) or 500 g FA with mainly 16:0 (P treatment), 18:0 (S treatment) or cis-18:1(n-9) (O treatment). Significantly higher arterial concentrations of infused FA in arterial plasma nonesterified FA and triglycerides (NETGFA) were observed with P and O treatments, but the effect of the S treatment was much lower. Arterio-venous differences of NETGFA increased with arterial concentrations. The number of synthesized FA in the mammary gland was not significantly affected by duodenal infusion of FA. Mean chain length was significantly reduced by P and O treatments, suggesting an effect of mammary gland uptake of long-chain FA on the termination process of mammary gland synthesis of FA. Across all treatments, 4:0 mammary gland balance increased linearly (r = 0.67, P = 0.004) with mammary gland FA uptake. Mammary gland desaturation of 18:0 to cis-18:1(n-9) averaged 52% and was not significantly affected by treatments, but was reduced by trans-18:1 mammary gland uptake. Uptake, synthesis and desaturation of FA by the mammary gland of dairy cows are affected by arterial concentrations of 16:0, 18:0 and cis-18:1(n-9).
KEY WORDS: fatty acids · mammary gland metabolism · synthesis · desaturation · dairy cows
Fatty acids (FA)3 of cows' milk have two main origins. FA with 12 carbons or less, most of 14:0 and about 50% of 16:0 are synthesized in the mammary gland from acetate and butyrate produced by the ruminal degradation of dietary carbohydrates (Smith 1980 Modifications of the FA profile of cows' milk fat can be drawn through the diet, which provides both precursors of synthesis and part of plasmatic FA. However, large ruminal biohydrogenation of unsaturated FA results in strong differences between dietary and milk FA profiles (Steele and Moore 1968 Our experiment used arterio-venous differences of individual FA to specifically study their mammary gland balance via comparison between arterial FA uptake and FA exportation into milk fat. Modifications of arterial concentrations and equilibrium of FA were obtained by continuous duodenal infusion of 16:0, 18:0 or cis-18:1(n-9) in a nearly pure form to avoid interaction effects.
Cows, diets and experimental procedures.
All procedures for this study complied with the Guide for the Care and Use of Laboratory Animals (NRC 1985). Four lactating Holstein cows (body weight 650 kg, mean time in milk at the beginning of experiment 100 d) fitted with a duodenal cannula were used in a 4 × 4 Latin square. The T-type duodenal cannula was 100 to 150 mm distal to the pylorus. Surgery was performed in a sterile environment under general anesthesia (Xylazine; Rhône Mérieux, Lyon, France). Cows received antibiotic treatments during the 5 d following surgery.
Sample collection.
Milk was collected twice daily before feeding. On d 10 of each period, during the evening milking, a sample (200 mL) was collected and stored at Analytical procedures.
Feedstuffs were analyzed for dry matter (105°C, 48 h), N (AOAC 1996) and fiber (Goering and Van Soest 1970
Calculation of results and statistical analyses.
Extraction (%) from arterial plasma by the mammary gland was calculated as (arterial-venous concentration)/arterial concentration × 100. Mammary uptake (mmol/L milk) of each FA was calculated as (arterial-venous concentration) × plasma flow, with plasma flow (L/L milk) = milk concentration of [18:0 + cis-18:1(n-9)]/{arterial [18:0 + cis-18:1(n-9)] Dry matter intake, milk production and milk fat content.
Diet consumption was complete with all treatments. Duodenal infusion of FA did not modify milk production (23.3, 25.1, 24.5 and 21.3 L/d with treatments C, P, S and O respectively). Milk fat content tended (P = 0.086 to be higher with P and O treatments compared with C treatment (49.9, 49.9 and 41.1 g/L respectively), and S treatment resulted in intermediate value (45.5 g/L).
FA concentration in plasma.
P, S and O treatments generally produced (P = 0.067) higher arterial concentrations of NETGFA (Table 3), compared with values observed with C treatment. Individual NETGFA concentrations were greater when the corresponding FA was infused. The effects were much larger with P and O treatments (+186 µmol/L and +151 µmol/L, respectively) than with S treatment (+76 µmol/L). Venous concentrations of total NETGFA showed very little variability. Total NETGFA, cis-18:1(n-9) and 18:2(n-6) concentrations were higher with O treatment.
Mammary gland extraction of arterial NETGFA.
Compared with C, the extraction rates of total NETGFA, 16:0 and 18:0 were higher with P treatment, and extraction rate of 18:0 was higher with S treatment. O treatment did not significantly affect the extraction rates of plasma NETGFA. A general trend toward higher mammary gland extraction rates when FA were infused into the duodenum was observed. This trend was supported by the regression between arterial concentration of NETGFA as an independent variable and the arterio-venous difference of NETGFA. The relationship between these two variables had a quadratic component, with the slope representing the rate of extraction of arterial NETGFA increasing with arterial concentration (Fig. 1).
Mammary gland uptake.
Uptake of plasma total NETGFA was higher with duodenal infusion of FA (Table 4), but the effect was not significant with S treatment. The uptake of 16:0 was higher by 40.6 µmol/L of milk with P treatment, and the uptake of cis-18:1(n-9) was higher by 37.6 µmol/L of milk with O treatment. Effect of S treatment on 18:0 uptake was much lower (+10.9 µmol/L of milk, P = 0.055).
Mammary gland balance.
Effects of FA infusion were not significant because of great variability. When FA were infused into the duodenum, P treatment produced significantly higher 4:0 balance, but O and S treatments only resulted in a trend toward higher 4:0 balance (P = 0.108 and P = 0.126, respectively). O treatment tended to lower balance of 14:0 (P = 0.114) and 16:0 (P = 0.092). Lower mean chain length of synthesized FA was significant with P and O treatments. Balances of 18:0 and cis-18:1(n-9) were opposite, resulting from the mode of determination. These balances reflected the importance of the desaturation of 18:0 taken up in the plasma to cis-18:1(n-9), and the desaturation rate was not lowered (P = 0.58) with S treatment.
As expected, we observed a specific improvement of arterial concentration and mammary gland uptake of the infused FA for P, S and O treatments. This made possible the study of effects of each FA on the mammary gland metabolism of FA, without interactions, and validates our experimental model. Moreover, our methodology is independent of effects of digestibility or milk yield because we used only arterio-venous concentration differences and milk FA concentrations.
Plasma concentrations.
Arterial concentrations of NETGFA were in the range of values frequently reported. When results of literature separately express TG and NEFA, conversion using NETGFA = NEFA + 3TG is needed to compare with our findings. Previously reported values are 500-900 µmol/L (Choi and Palmquist 1996 Mammary gland uptake.
Arterio-venous differences of total TG and NEFA have been reported to depend on arterial concentrations, with very different slopes among authors. Cant et al. (1993b) Mammary gland synthesis of FA.
Biosynthesis of FA with less than 18 C can be measured through mammary gland balance, because oxidative degradation of FA is negligible in the mammary gland (Annison et al. 1967 Mammary gland desaturation.
Desaturation of 18:0 to cis-18:1(n-9) in the mammary gland was about 52% of uptake (Table 4, Fig. 4). Noble et al. (1969)
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
). A minor part of 14:0, about 50% of 16:0 and all 18-carbon FA are extracted from the arterial blood. Further, extracted or synthesized FA are incorporated into milk triglycerides (TG).
). These manipulations of FA can lead to better organoleptic properties such as butter spreadability (Banks et al. 1980) or better dietetic characteristics, by increasing cis-18:1(n-9) and decreasing 16:0 (O'Donnell 1993). Present knowledge on the mammary metabolism of FA in dairy cows is mostly based on the effects of dietary or infused lipids on milk fat composition, so that mammary gland effects can be confounded with digestion effects, or with effects of FA metabolism in other tissues. Moreover, most studies used natural sources of fat containing many different FA. Few papers concerned direct studies of the mammary gland metabolism of FA. In vitro, Hansen and Knudsen (1987a, 1987b) showed that FA extracted from the plasma interact with de novo synthesis. In vivo, using radioactive FA in goats, Annison et al. (1967) showed that FA that are taken up can be modified because desaturation of 18:0 to cis-18:1(n-9) can occur.
SUBJECTS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
Table 1.
Ingredient and chemical composition of diet (dry matter basis)
View this table:
Table 2.
Main fatty acids composition of fat mixtures infused
into the duodenum
). Diarrhea stopped immediately so that samples were collected from healthy cows.
20°C until analysis.
). On d 10 of each period, blood samples were collected at 0800, 1100, 1400 and 1700 h into 10-mL heparinized tubes (5000 USP units of heparin/L whole blood). Arterial and venous samples were obtained simultaneously. They were immediately centrifuged at 3000 × g for 15 min at 4°C, and plasma samples were stored at 4°C until the 1700-h blood collection. Then, the four samples for each cow were composited and kept frozen at 30°C. Thus, sampled blood was presumed to represent blood-providing substrates for the milk fat that was harvested at the evening milking.
) contents. FA in feed samples were extracted and methylated with a one-step procedure (Sukhija and Palmquist 1988) after addition of 19:0 (Sigma-Aldrich Chimie, Saint Quentin Fallavier Cedex, France) as an internal standard. They were quantified, according to the procedure of Wu et al. (1991), by gas-liquid chromatography (Dani GC 1000, equipped with flame-ionization detector and split injector; Viale Elvezia, Monza, Italy). The column was a fused silica capillary (SP-2340, 30 m × .32 mm i.d.; Supelco Inc., Bellefonte, PA). Temperature was 160°C for 10 min and then increased by 3°C/min until 180°C. This method does not allow separation of different positional isomers of trans-18:1.
, we measured together NEFA and FA from TG (NETGFA), in arterial and venous plasma.
with a solvent system consisting of petroleum ether/diethyl ether/acetic acid (80:20:1, v/v/v). After migration, silica was scraped on bands corresponding to NEFA and TG, and treated and quantified by gas-liquid chromatography in the same manner as feed samples.
View this table:
Table 3.
Concentration of fatty acids from triglycerides and nonesterified fatty acids in plasma and mammary gland extraction rate in cows duodenally infused with control and fatty acids enriched suspension1
venous [18:0 + cis-18:1(n-9)] concentration}, because mammary gland secretion and uptake of 18:0 + cis-18:1 are very close (Annison et al. 1967). Mammary gland balance (mmol/L milk) of each FA was calculated as milk concentration-mammary gland uptake. Desaturation (%) of extracted 18:0 was calculated as cis-18:1(n-9) mammary gland balance/18:0 uptake × 100. Mean chain length of synthesized FA was calculated from mammary gland balances of individual FA with 4 to 16 carbons.
). Regression was used to assess the relationship between mammary gland uptake and balance of FA. The regression equation representing the relationship between mammary gland uptake of 18:0 and mammary gland balance of cis-18:1(n-9) was forced through the origin. Significance was declared at P < 0.05.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View larger version (16K):
[in a new window]
Fig 1.
The regression equation y = 10.44 0.025 x + 0.00082 x2, r2 = 0.98, P < 0.001 represents the relationship between arterial plasma concentration of fatty acids from triglycerides and NEFA (NETGFA) in arterial plasma and arterio-venous difference of these fatty acids in cows duodenally infused with control and fatty acid-enriched suspensions (stearic, palmitic and oleic acids). Values are single determinations for four cows administered each treatment, n = 16.
View this table:
Table 4.
Mammary uptake and balance of fatty acids and desaturation rate of stearic acid in cows duodenally infused with control or fatty acids enriched suspension1
View this table:
Table 5.
Correlation coefficients between mammary gland uptake and balance of fatty acids or desaturation of 18:0 in cows duodenally infused with control or fatty acids enriched suspension1
View larger version (14K):
[in a new window]
Fig 2.
The regression equation, y = 7.34 + 0.167 x, r2 = 0.45, P = 0.004 represents the relationship between mammary uptake of fatty acids from arterial plasma and mammary balance of 4:0 in cows duodenally infused with control and fatty acid-enriched suspensions (stearic, palmitic and oleic acids). Values are single determinations for four cows administered each treatment, n = 16.
View larger version (15K):
[in a new window]
Fig 3.
The regression equation, y = 13.4 0.03 x, r2 = 0.55, P < 0.001 represents the relationship between mammary uptake of fatty acids from arterial plasma and mean chain length of synthesized fatty acids in cows duodenally infused with control and fatty acid-enriched suspensions (stearic, palmitic and oleic acids). Values are single determinations for four cows administered each treatment, n = 16.
View larger version (14K):
[in a new window]
Fig 4.
The regression equation, y = 0.52 x, r2 = 0.75, P < 0.001 represents the relationship between mammary uptake of 18:0 from arterial plasma and mammary balance of cis-18:1 (n-9) in cows duodenally infused with control and fatty acid-enriched suspensions (stearic, palmitic and oleic acids). Values are single determinations for four cows administered each treatment, n = 16.
View larger version (15K):
[in a new window]
Fig 5.
The regression equation, y = 61.2 4.03 x, r2 = 0.31, P = 0.02 represents the relationship between mammary uptake of trans-18:1 from arterial plasma and desaturation of 18:0 in cows duodenally infused with control and fatty acid-enriched suspensions (stearic, palmitic and oleic acids). Values are single determinations for four cows administered each treatment, n = 16.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
), 450-600 µmol/L (Cant et al. 1993b), or 500-700 µmol/L (Miller et al. 1991b). The effect of fat supplements on plasma TG and NEFA has been studied. Choi and Palmquist (1996) recently observed higher concentrations of TG and NEFA in the plasma of cows receiving 30 to 90 g/kg diet of supplemental fat as calcium soaps of FA. In the same manner, Gagliostro et al. (1991) observed significantly higher plasma TG and NEFA in midlactation cows after duodenal infusion of rapeseed oil. Such a rise in arterial NETGFA was observed in our trial, but it was not significant, because of substantial variability. We did not separate NEFA from TG, so it is difficult to ascertain whether this observed trend toward higher arterial concentration of NETGFA was due to NEFA, TG or both. Large concentrations of plasma NEFA can be expected when cows are in negative energy balance, which was not the case in our study because diet consumption was always complete due to distribution in a limited amount, and because milk production was not affected by FA infusion. Moreover, the trend toward higher milk fat content with P and O treatments compared with C treatment cannot be interpreted as a result of a negative energy balance, because fat addition to the diet of cows directly increases milk fat content (Yang et al. 1978b). Consequently, as reported in previous papers, both higher arterial NEFA and TG values were probable in our infused cows.
), so that cis-18:1(n-9) was not significantly higher in arterial plasma NETGFA with S treatment. Lower digestibility of infused 18:0 compared with cis-18:1(n-9) or 16:0 could explain low effects of S treatment on arterial 18:0. In most experiments, intestinal digestibility of 18:0 is high (Steele 1983, Wu et al. 1991), but when duodenal flow of 18:0 increases from 170 to about 400 g/d, digestibility decreases from 73 to 45% (Ferlay et al. 1993). Rapid incorporation of absorbed 18:0 into tissues could be another explanation. Such differential effects of fat on individual FA of plasma TG were observed by Marty and Block (1992), with supplemental calcium soaps of palm oil FA providing dietary 16:0 and cis-18:1 (n-9). Ruminal biohydrogenation of cis-18:1(n-9) from such salts is known to be substantial (Enjalbert et al. 1997) so that duodenal flow was mostly enriched with 16:0 and 18:0. However, plasma TG concentration of 16:0 was lowered, but 18:0 was not affected.
, Gagliostro et al. (1991) and Miller et al. (1991a), found slopes of 0.29, 0.68 and 0.81, respectively, for TG, and 0.21, 0.48 and 0.30 for NEFA, but these authors did not explore the quadratic relationship between arterial concentration and arterio-venous differences. Such a relationship could however be expected from the figure of Cant et al. (1993b), and high extraction rates when arterial concentrations are high were outlined by Gagliostro et al. (1991). This quadratic relationship can explain differences of slopes reported by different authors when only linear relationship was tested, because arterial concentrations of TG and NEFA were different among studies.
in cows receiving a diet without added fat. Extractions in this study were higher than in our experiment, but they were measured only on FA from TG. These different patterns of mammary gland uptake among FA may be due to positions of FA in plasma TG. Indeed, it was observed in sheep that TG from very low density lipoprotein show a marked accumulation of 18:0 and trans-18:1 in positions sn-1 and sn-3, whereas 14:0 and 16:0 are more prevalent in position sn-2 (Christie et al. 1984, Christie and Moore 1971). As observed for total NETGFA, extraction rates of 16:0 and 18:0 were higher when duodenal infusions increased their arterial concentration.
), but because neither TG nor NEFA was measured in this experiment, only a high NETGFA concentration can be hypothesized to be the cause.
). In our experiment, FA duodenal infusion did not affect the total number of FA synthesized by the mammary gland, suggesting no effect on initiation of FA synthesis.
, Beaulieu and Palmquist 1995) or in vitro on dispersed bovine mammary gland cells with palmitate or oleate addition (Hansen and Knudsen 1987b). Barbano and Sherbon (1980) suggested that a large diglyceride pool of high molecular weight resulting from incorporation of long-chain FA taken up in the plasma might result in a larger output of 4:0 to maintain fluidity of milk fat at body temperature. Such a compensatory effect is expected to be more important with 16:0 or 18:0 than with cis-18:1(n-9), which has a low melting point, as short- and medium-chain FA. This trend was confirmed by our study because correlations between 16:0 or 18:0 uptakes and 4:0 balance were higher than correlations between cis-18:1(n-9) uptake and 4:0 balance (Table 5). Therefore, lack of significant effects of S and O treatments on 4:0 mammary gland balance may have divergent explanations: low biological effect of cis-18:1(n-9) for O treatment, and insufficient augmentation of 18:0 uptake with S treatment.
). Consequently, comparison of our results, obtained with duodenal infusion, with most results of literature, obtained with dietary addition, is difficult. When results are converted to mol/L milk, abomasal infusion of FA tended to produce higher proportions of short- and medium-chain FA (Drackley et al. 1992) as in our work.
. These authors explained this effect by the proposal that cis-18:1(n-9) competes with newly synthesized short- or medium-chain acyl-CoA for esterification at the sn-2 and sn-3 positions of glycerol.
, Wonsil et al. 1994).
). Because desaturation of 18:1 to 18:2 is not possible in cows, this low apparent balance could be due to a slight mammary uptake of plasma cholesteryl esters by the mammary gland (Gagliostro et al. 1991). The cholesteryl esters contain much more 18:2 than 18:0 or 18:1 (Marchello et al. 1972). Consequently, their contribution to 18:0 and 18:1 mammary gland uptake is low, so that the validity of our method for determination of mammary gland uptake, based only on plasma TG and NEFA, remains good.
with dietary 18:0 showed that increase in total milk 18:1 was 1.5 times the change in 18:0, suggesting desaturation of about 60% of added 18:0. Because part of this desaturation is known to occur in the duodenal mucosa, these results are compatible with our findings. From these results, it is expected that, when mammary gland uptake of 18:0 is high, half of the additional uptake of 18:0 will be converted to cis-18:1(n-9), so that the ratio of cis-18:1(n-9) to 18:0 in milk fat will draw nearer to 1, when the usual ratio is more than 2 (Jensen et al. 1991).
who observed, in vitro on goat-isolated mammary tissue, that cis-18:1(n-9) strongly inhibited the desaturation of 18:0. However, Christensen et al. (1994) and LaCount et al. (1994) showed that abomasal infusion of large amounts of cis-18:1(n-9) results in insignificant reduction of 18:0 in milk fat, suggesting that desaturation of 18:0 is not reduced by cis-18:1(n-9) uptake. The inhibition of 18:0 desaturation observed in vitro by Bickerstaffe and Annison (1970) was relieved by glycerol-phosphate or particle-free supernatant, which could explain discrepancies between in vivo and in vitro results.
after abomasal infusion of trans-18:1 FA, but was observed in rat-liver microsomes (Mhafouz et al. 1980).
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FOOTNOTES |
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Manuscript received 29 Jan 98. Initial reviews completed 10 March 98. Revision accepted 16 June 98.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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