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Changes in mammary uptake of free fatty acids, triglyceride, cholesterol and phospholipid in relation to milk synthesis during lactation in goats
Camp. Biochem. Plr~“iol. Vol. 109A. No. 4. pp. 857-867, 1994 Copyright /(; 1994 Elsevier Science Ltd Printed in Great Britain. All rights resewed 0300-9629/94 $7.00 + 0.00
Pergamon 0300-9629(94)00136-7
Changes in mammary uptake of free fatty acids, triglyceride, cholesterol and phospholipid in relation to milk synthesis during lactation in goats M. 0. Nielsen* and K. Jakobsent *Department of Anatomy and Physiology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark; and TDepartment of Animal Physiology and Biochemistry, National Institute of Animal Science, P.O. Box 39, DK-8830 Tjele, Denmark Uptake of free fatty acids (FFA), triglycerides (TG), cholesterol (CHOL) and phospholipids (PL) was measured in both mammary glands of dairy goats during lactation. Arterial concentrations of TG, CHOL and PL as well as arteric+venous difference (AV) and extraction rate (E) for TG were highest in goats with the highest dietary feed intake. AV were linearly related to arterial concentrations for the four lipid classes, and arterial concentrations of CHOL were linearly related to output of lactose, protein and fat in milk. Arterial supply, and not mammary synthetic activity, is the main determinant of mammary FFA, TG and CHOL uptake. Key words: Mammary gland; Lipid uptake; FFA; Triglyceride; Cholesterol; Phospholipid; Lactation; Goat. Comp. Biochem. Physiol. 109A, 857-867,
1994.
Introduction Milk synthesis in the mammary gland changes markedly during the lactation period with a dramatic increase in early lactation and a gradual decrease after peak lactation. Much remains to be understood about the physiological background for these changes. Nutrients taken up by mammary cells for milk synthesis are supplied to the mammary gland by the blood, and mammary blood flow (MBF) has been assumed to play a key role in the regulation of mammary nutrient supply and thus milk to: M. 0. Nielsen, Department of Anatomy and Physiology,The Royal Veterinary
Correspondence
and Agricultural University, Thorvaldsensvej 40, DK- 1871 Frederiksberg C, Denmark. Received 23 January 1994; revised 4 July 1994; accepted 18 July 1994. CBPA 109,LD
857
synthesis (Davis and Collier, 1985). In lactating goats, it has been found that animals with a different capacity for milk synthesis also have a very different capacity for mammary uptake/extraction from the blood of the key nutrient glucose (Nielsen and Jakobsen, 1993), high-yielding animals being 40% more efficient than low-yielding in this respect. Metabolic activity may therefore also influence nutrient uptake in the mammary cells. It was the purpose of the present experiment to measure mammary uptake of preformed plasma lipids (free fatty acids, triglycerides, cholesterol and phospholipids) in relation to milk synthesis during the lactation period in goats with different capacities for milk synthesis, and in this way illuminate the role of nutrient supply
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versus the synthetic capacity of mammary cells in the regulation of milk production at different stages of lactation. Nutrient uptake was determined by arterio-venous difference measurements across the mammary gland in dairy goats. Results on MBF measurements (Nielsen et al., 1990) and mammary glucose and nitrogen uptake (Nielsen and Jakobsen, 1993) from the same experiment have been reported previously.
Materials and Methods Animals and experimental procedure
Three Norwegian (nos 53,64 and 67) and one Saanen (no. 73) dairy goats in their second and first lactation, respectively, and surgically prepared with one carotid artery exteriorized under the skin were used. Based on their peak milk yields, goats no. 53 and 73 will, in the following, be termed low-yielding, and 64 and 67 high-yielding. Arterio-venous (AV) differences across the mammary gland were measured on 1 day at approx. week 8, 11, 15, 25 and 37 post-partum. Feeding and housing of the experimental animals as well as the experimental procedure have been described previously (Nielsen and Jakobsen, 1993). On goat no. 64, measurements stopped after week 11 post -partum due to difficulties with insertion of catheters into the milk veins. Analyses
Milk lactose, protein and fat were determined as described previously (Nielsen and Jakobsen, 1993). Plasma free fatty acids (FFA) were determined using a NEFAC kit (Wako Chemicals GmbH, Neuss, Germany), and plasma triglycerides (TG), total cholesterol (CHOL) and phospholipids (PL) by Triglycerides GPO-PAP, Monotest@, Cholesterol, and Test Combination Phospholipids kits, respectively (all from Boehringer Mannheim GmbH, Mannheim, Germany). Calculations and statistical analyses
Mean concentrations of nutrients in the left and right milk veins were used for the calculation of AV differences, and extraction rates (E) derived as AV/A x lOO%, where A is arterial concentration. When values were missing for one of the milk
veins, the nutrient concentration in the other vein was used alone. This introduces a small error for goat no. 73, where concentrations of TG differed between the two milk veins (see Results). All values are expressed as mean + SEM, unless otherwise stated. The statistical models used to analyse data for concentrations of lipids in milk venous plasma, arterial concentrations, AV differences and E for the measured lipids have been reported previously (Nielsen and Jakobsen, 1993). The relationships between A, AV, E and A, AV, E, day of lactation or milk yield were investigated by linear regression and non-linear models. All analyses were performed using OLS (ordinary least square) software procedures (SAS, 1985).
Results Milk yield and composition
Milk production in the four experimental animals followed the normal pattern of the lactation curve, except for goat 64 which was dried off deliberately 20 weeks postpartum. Peak milk yields were obtained 6-9 weeks post-partum, and ranged from 1.9 and 1.2 kg/day in goats nos 53 and 73, respectively (low-yielding), and approx. 3.6 kg/day in goats nos 64 and 67 (highyielding). Milk yields declined gradually thereafter, and the goats were dried off 33-38 weeks post-partum (except goat 64, see above). Milk fat content, ranging from 2.3 to 4.2%, was generally highest in early lactation, decreased until after peak milk yields were reached and increased thereafter. Protein and lactose contents (%) were fairly constant during the lactation period. Daily yields of milk fat are presented in Fig. 1. Plasma concentrations and mammary extraction of lipids during lactation
The range and mean values of measured concentrations of FFA, TG, CHOL and PL in plasma obtained from the carotid artery and milk veins are presented in Table 1. No effect of udder half (milk vein) was found on venous concentrations of these nutrients, except for TG in goat no. 73, where concentrations were found to be higher
Mammary lipid uptake in goats
Table 1. Concentrations*
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of FFA, TG, CHOL and PL in arterial and milk venous plasma
Arterial concentration Nutrient
Mean + SEM
FFA (mM) TG (mgil) CHOL (mg/l) PL (mM)
(Min-max
0.24 If: 0.028 I38 + 12 680 * 39 1.32 f 0.059
Milk venous concentration
values)
(0.07-0.82) (23-270) (230-949) (0.59-1.91)
n
Mean + SEM
31 31 32 32
0.21 f 0.012 ll7k8.3 686 + 26 138 + 0.044
(Min-max
values)
(0.10-0.58) (15-230) (247-972) (0.72-2. IO)
n 54 54 57 57
*Means of values obtained from all goats during the lactation period. For abbreviations: see text.
(P < 0.01) in the right than in the left milk vein, 79 & 9 mg/l and 68 + 9 mg/l, respectively. In the following, the two venous sides have not been treated separately, and the means of venous concentrations in the left and right milk vein have been used to calculate AV differences and E across the mammary gland. Although this is not strictly correct in the case of TG for goat no. 73, only a minor error is introduced. Free fatty acids (FFA). Measured AV differences for FFA in the total material and 0.28 mM varied between -0.085 with an average of 0.025 f 0.014 mM and E varying between - 65 and 5 1%, averaging 0.48 f 4.5%. Arterial FFA concentrations were slightly higher in low-yielding than in high-yielding goats (P c 0.05). Net mammary FFA extractions were found to be Table 2. Effect of production
level and sampling time on A, AV and E for FFA, TG, CHOL and PL in the mammary gland Milk production
Nutrient FFA
TG
CHOL
PL
Parameter* A
@W
AV
(mM)
E
W)
iv
;:;;t;
E
WI
A
@x/U
AV
(mg/l)
E
(%I
A
@W
AV
(mM)
positive in the morning but negative in the afternoon (P < 0.05), and the same tendency was observed for arterial concentrations and E. Otherwise, goat, yield-level and sampling time had no influence on A, AV and E (see also Table 2). Figure 1 shows the development in arterial concentrations and mammary extractions of FFA during lactation as well as daily milk fat production. It can be seen from the figure that arterial FFA concentrations were high in early lactation when also milk fat and overall milk production was high, and declined thereafter with a tendency for a small increase again in late lactation. The changes were significant both with goats as a systematic (P < 0.001) and a random (P < 0.05) effect. AV differences and E also changed during lactation
Sampling time
level
Low
High$
Pt
0.25 + 0.043 0.021 &-0.019 - 1.55 + 5.62 (n = 18) 105 f 13.9 7.8 f 5.3 7.44 &-6.45 (n = 17) 559 * 50.2 -37.9 f 17.3 - 10.9 f 4.30 (n = 18) 1.16 + 0.074 - 0.056 + 0.045 (n = 18)
0.23 + 0.031 0.033 f 0.021 3.29 + 7.66 (n = 13) 177+ 15.4 27.8 f 5.5 17.0 k 3.16 (n = 14) 837 k 24.1 6.2 f 16.8 0.417 k 2.27 (n = 14) 1.52 & 0.062 -0.065 f 0.07 1 (n = 14)
$ NS NS 7 /j NS 7 ** ** 7 NS
Morning
Afternoon
0.26 + 0.047 0.22 f 0.033 0.043 f 0.024 0.0091 + 0.00011 5.81 f 5.81 -4.52 k 6.79 (n = 15) (n = 16) 135 &-15.9 141 f 18.8 11.9 * 5.1 22.0 f 6.6 7.54 + 5.89 16.2 + 4.84 (n = 16) (n = 15) 697 + 57.5 673 f 53.6 - 19.2 k 18.5 - 18.0 + 17.7 -6.57 f 3.99 - 5.29 f 3.96 (n = 16) (n = 16) 1.27 + 0.080 1.36 f 0.087 -0.1 I f 0.67 -0.0094 k 0.039 (n = 16) (n = 16)
*Means of values obtained during the lactation period. iData analyses performed with parameter goat as a systematic effect (see Materials and Methods). $After week I I post-partum, data are derived from only one goat. 9, 11,7, **: significant at 0.05, 0.01, 0.001 and 0.06 level, respectively. NS: non-significant. For abbreviations: see text.
Pi
NS NS $ NS NS NS NS NS NS 9: NS
M. 0. Nielsen and K. Jakobsen
860
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-
od&
P<0.001)
-
l
/
I
10
Weeka
I
I
20
30
pa&-partam
Fig. 1. Changes in arterial FFA concentration (A), mammary arterio-venous difference (AV) of FFA and daily milk fat production (FP) during the lactation period. 0, 0, 0, v: Goat nos 53 and 73 (low-yielding), 64 and 67 (high-yielding). Each point represents means of the two udder-halves (AV), and means of morning and afternoon values (A and AV). n : Each point represents means of the two udder halves (AV). Solid lines represent average of all measured values in the panels showing changes in A or AV during lactation, and the regression line in the panel showing the relation between AV and A.
(P < 0.06 and P < 0.001, respectively). Changes in arterial concentrations and AV differences could be described by the following equations: A (mM) = 0.655 0.0065.day + 0.0000195.day2 (R2 = 0.31, P < 0.01) and AV (mM) = 0.182 0.00266.day + O.O000084~day* (R2 = 0.20, P < 0.05). AV followed the arterial concentrations closely as a linear function of A : AV (mM) = - 0.068 + 0.390.A (mM) (R* = 0.65, P < 0.001). Generally, AV and E became negative when arterial FFA concentrations fell below 0.2 mM, as in mid-late lactation. Triglyceride (TG). AV differences for TG varied between -29.0 and 70.0 mg/l with an average of 16.8 + 4.2 mg/l. E varied between -39 and 59% averaging 11.7 f 3.9%. E was not very high but was still significantly higher than 0 (P < 0.05). A
and AV differences for TG differed between goats (P -C 0.001 and P < 0.05, respectively). As can be seen from Table 2, the arterial TG concentration was 70% higher in the high- than in the low-yielding goats, leading also to higher AV differences. Sampling time had no effect on A, AV-differences and E for TG (see Table 2). Arterial concentration was dependent on the stage of lactation (P < 0.001). This was not the case for AV and E (P > O.S), and AV and A were only poorly correlated: AV (mg/l) = 0.062 + 0.118.A (mg/l) (R’ = 0.12, P < 0.06), as also evident from Fig. 2. Arterial concentrations decreased in the later part of lactation, as shown in Fig. 2, whereas the development in early lactation varied from goat to goat, the interaction effect being significant (P < 0.001).
Mammary
lipid uptake
in goats
-30
861
1 0
I , JO 20 Week8 pod-partum
I 10
,
60 ,”
-
(nil/l)
(a*- 0.13;
o.oaa + 0.118
-
P
l
*
ha/o
’
.*
-20
1
0
I
I
8
t
I
,
10
20
SO
40
0
100
W*eh
post-partum
Merid
’
l
I
200 uoncantration (mr/l)
1
300
Fig. 2. Changes in arterial TG concentration (A), and mammary arterbvenous difference (AV) and extraction (E) of TG during the lactation period. 0, 0, 0, V: Goat nos 53 and 73 (low-yielding), 64 and 67 (high-yieiding). Each point represents means of the two udder halves (AV and E) and means of morning and afternoon values (A, AV and E ). n : See legend to Fig. 1. Solid lines represent average of all measured values in the panels showing changes in A, AV or E during lactation, and the regression line in the panel showing the relation between AV and A.
Total c~o~e~ter~~(CHUL). Measured AV differences for CHOL varied between - 195.5 and 89.Omg/l with an average of - 18.6 + 12.6 mg/l and E varied between - 50.6 and 12.1% with an average of - 5.92 &-2.76%. Arterial CHOL concentrations were 50% higher in the highcompared with the low-yielding goats (P c 0.00 1, see Table 2). AV differences and mammary E of CHOL tended to be negative in low-yielding, and slightly positive in high-yielding goats (P c 0.06). No effect of sampling time could be demonstrated, but the interactions between the time and the goat were all significant for A, AV and E, since goat 73 in contrast with all other goats yielded the lowest values in the morning. As shown in Fig. 3, arterial concentrations and AV differences for CHOL
changed during the lactation period (P < 0.001 and P < 0.05, respectively). Arterial concentrations were relatively constant during early-mid lactation and decreased thereafter, following the general pattern of milk fat and overall milk production. The pattern of individual goats was, however, different, resulting in a significant interaction between the goat and the week of lactation (P < 0.001). AV differences and E across the mammary gland were high in the beginning of lactation and gradually decreased with progressing lactation, except for a marked around week 10 post -partum drop (P < 0.05). The same pattern was observed for E (P = 0.12). Both AV and E were linearly related to arterial concentrations: AV (mg/l) = -133+0.168.A tmg/lI,
862
M. 0. Nielsen and K. Jakobsen
a 0 IO
0
100
10
20 30 Toeke poBt-putum
I
10
40
t
AV 0
100 -
5
$
(a*-
- -100 + 0.100
o*
0
l
A (r/r)
P
.
.
-100
1
1
40
1
200
.
.
.=
-200 I
*
0
f?J 20 30 weeks pod-putum
.l
l
z0’B
,a
40
woeke post-puwlB
r
10
I
30
20
. I
I
I
1
400 000 600 1000 Artenl oonoontr~tlon (mu/t)
Fig. 3. Changes in arterial CHOL concent~tion (A) and mammary arterio-venous difference (AV) and extraction (E) of CHOL during the lactation period. 0, c], 0, 0, a, and solid lines: see legend to Fig. 2.
(R2 = 0.27, P < 0.001) and E (%) = -37.8 + 0.468.A (mg/l) (R2 = 0.43, P < 0.001). Phospho~ipid (PL). Measured AV differ-
ences for phosphohpid varied between -0.64 and 0.42 mM with an average of -0.060 + 0.039 mM. AV differences for PL were unaffected by goat, yield-level, sampling time (see Table 2) as well as the stage of lactation, and were not significantly different from 0 (P > 0.1), and calculations have therefore not been performed for E. AV and A were only poorly correlated (see Fig. 4): AV (mM) = -0.396 +0.255-A (mM) (R2 = 0.14, P < 0.05). Arterial concentrations were found to be approx. 30% higher in high-yielding compared with low-yielding goats (P < 0.001) and slightly higher values were found in the afternoon than in the morning samples (P c 0.05), as shown in Table 2. Arterial concentrations generally increased in the first part of lactation and decreased in
the later part of lactation (P < O.OOl), as shown in Fig. 4, but interaction effects were found for goats and the week of lactation (P c 0.01) as well as for sample time and week of lactation (P < 0.05). The development in arterial concentration during lactation could be described by the equation: A (mM) = 1.14 + 0.00491 *day 0.0000209~day2 (R2 = 0.18, P < 0.05). Mammary extraction milk synthesis
of lipids in relation to
No relationship was found between the yield of milk constituents and A, AV or E for FFA, TG and PL, and AV and E for CHOL. However, production of lactose, milk protein and milk fat was found to be related to mammary supply (arterial concentration) of CHOL (P < 0.05 in all cases). An increased yield of milk constituents was associated with higher arterial CHOL concentrations, as shown in
Mammary
lipid uptake in goats
863
Fig. 5. Due to a strong effect of goat and a strong interaction between goat and yield of milk constituents on arterial CHOL concentrations (P < O.Ol), simple correlations of A with yield of lactose, milk protein and milk fat only ranged from 0.20 to 0.30.
Discussion Triglycerides normally account for 97-99% of total lipids secreted into goat and cow milk. They are excreted from mammary cells as milk lipid droplets, which are enveloped in the plasma membrane before being expelled into the alveolar lumen (Nickerson and Akers, 1984), and synthesis of plasma membranes must therefore be considered of importance for overall milk secretion in milk. The major lipids in plasma membranes from goat and cow
0
Ar(mM)--o.ooo+O.8oo*A(mx)
00
00
100 (r/d)
9. .
.
g 400 -
140
. .
t
,
~~~~~;““~”
0
20
40
Proteln
produotion
20
40 Fat production
.
120
.
i
0 -
40
Laotona prodrotlon
%
1 ‘“:
0.4
20
,
60
80
100
80
100
(g/d)
80 (g/d)
(P’ - 0.11; P
Fig. 5. Changes in arterial CHOL concentration in relation to daily lactose (LP), milk protein (PP) and milk fat (FP) production. The regressions between arterial concentration and LP, PP or FP are depicted as solid lines. P
*-0.4 -0.6
0.50
.
.
n
. . t I I I I 0.75 1.00 1.25 1.10 1.75 Artmrhl aonc.ntration (mY)
I 2.00
Fig. 4. Changes in arterial PL concentration (A) during the lactation period and relationship of mammary arterio-venous difference (AV) to A. 0, 0, 0, V, H and solid lines: see legend to Fig. 1.
mammary glands are TG, CHOL and PL. CHOL and PL accounts for approximately 0.5% and 1% (Bitman et al., 1984) of total milk lipids, respectively. In the present experiment, the purpose was to investigate how mammary uptake of TG, CHOL and PL in circulating plasma lipids changes during the lactation period in
864
M. 0. Nielsen and K. Jakobsen
relation to changes in milk production, and to study if a high mammary synthetic activity also is associated with a high capacity for extraction of plasma lipids, as seems to be the case for glucose (Nielsen and Jakobsen, 1993). In other words, what is the role of mammary substrate supply versus mammary synthetic activity in the regulation of mammary uptake of these lipids? The observed levels of arterial concentrations (A), arterio-venous differences (AV) and extraction rates (E) for FFA (Annison et al., 1967; Miller et al., 1991a; West et al., 1972), TG (Bickerstaffe et al., 1974; Miller et al., 1991a; West et al., 1972), CHOL (Annison et al., 1967; Bickerstaffe et al., 1974; Miller et al., 1991a) and PL (Bickerstaffe et al., 1974; Linzell, 1974; West et al., 1972) were generally in agreement with those reported in the literature. AV and E for TG and AV for CHOL were, however, in the low range, and measured values of A for total CHOL very low compared with those reported by others (see above). Free fatty acids (FFA)
FFA are known to be extracted by the mammary gland as verified by isotopic tracer studies (Annison et al., 1967), but during hydrolysis of lipoprotein TG, FFA are released into the blood stream, and positive values of AV differences for FFA are therefore only found in situations where arterial FFA concentrations are high: above 0.3 mM as reported by Kronfeld (1965) or 0.2 mM, as observed in the present experiment. Concentrations were generally highest in early lactation as expected, since loss of body weight (fat mobilization) is normally associated with high plasma levels of FFA. There was a close positive correlation between AV and FFA and arterial concentrations, as also reported by others (Miller et al., 1991a), suggesting that FFA is taken up by mammary cells by passive diffusion, and extracellular concentration must be the main determinant of the concentration gradient across the cell membrane, which drives uptake. Triglyceride (TG)
were Arterial TG concentrations generally highest in early lactation, and
lactation. decreased with progressing Others have found the lowest levels in early lactation (Marcos et al., 1990; Miller et al., 1991b), where mammary nutrient uptake is maximal. The diet fed to the goats in the present experiment contained a fairly large amount of fish meal, which is rich in fat containing long-chain polyunsaturated fatty acids (PUFA). The high fat intake may explain the high arterial concentrations in early lactation. AV differences were positively correlated with arterial concentation, although the correlation was poor (r2 = 0.12) compared with previously reported values (Miller et al., 1991a: r2 = 0.25), but AV differences were generally quite low in the present experiment as discussed above, and in some cases negative, which, however, must be ascribed to analytical error. The high dietary content of long-chain PUFA may explain the low values observed for AV and E for TG, since a high dietary intake of PUFAs is associated with increased formation of trans-fatty acids during hydrogenation in the rumen. Trans-fatty acids and long-chained (C2,,-C22) PUFAs in plasma are believed to have an inhibitory effect on the activity of lipoprotein lipase (Rook and Thomas, 1983), and inhibit de nouo fatty acid synthesis (Banks et al., 1990; Hansen and Knudsen, 1987) resulting in lowered uptake of fatty acids from lipoprotein-TG and reduced milk fat synthesis. In agreement with this, milk fat content was fairly low in our study. Annison et al. (1974) have reported data, which also indicate that feeding-induced low milk fat content is associated with low values for TG-AV, a lower lipoprotein lipase activity would result in lower release of fatty acids from lipoprotein TG, and this could also explain why AV differences for FFA as mentioned above became positive at lower arterial concentrations of FFA than previously reported. As is the case for FFA, TG uptake in the mammary gland seems, therefore, to be determined primarily by arterial supply, at least at arterial plasma concentrations of TG below approx. 300 mg/l. At higher concentrations, the mammary capacity for uptake (e.g. lipoprotein lipase activity and mammary cellular activity) may, according to Baldwin and Smith (1983), become
Mammary lipid uptake in goats
limiting. In our experiment, high-yielding goats seemed to have significantly higher AV differences for TG than low-yielding goats. However, this could be explained by higher arterial concentrations of TG in the high-yielding animals, due to higher dietary fat intake (Christie, 1979). It must be emphasized that differences between high- and low-yielding goats must be taken with some caution, since one of the two high-yielding goats was excluded from the experiment after week 11 post partum. Despite the low AV differences measured in the present experiment, it was possible to account for the output of preformed fatty acids in the milk of the highyielding goats, utilizing the average AV differences for TG and FFA in Table 2 and the previously reported values for mammary blood flow (Nielsen et al., 1990). However, TG-AV differences may have been underestimated in the low-yielding goats. Cholesterol (CHOL) In humans, a high dietary intake of longchain PUFA (e.g. fish oils) may have a lowering effect on plasma CHOL concentration (Hornstra, 1989). A relatively high absorption of PUFA must be expected on a diet as for that in the present experiment, and perhaps the low arterial CHOL concentrations generally observed in our goats may, in part, be the result of a high PUFA absorption. Arterial concentrations were relatively constant through most of the lactation period, but decreased in late lactation. Similar observations have been reported by Raphael et al. (1973) and Marcos et al. (1990), and probably reflect a high metabolism of TG-rich lipoproteins (very low density lipoprotein) in lactating animals depending on the milk yield. AV differences for CHOL were linearly related to (Y’= 0.25) and followed arterial concentrations, except for a drop around week 10 post-partum, for which we have no immediate explanation. CHOL is taken up by the mammary gland (More and Christie, 1979) but even so, AV differences are normally reported not to be significantly different from zero (Annison et al., 1967; Peeters et al., 1979). Arterial CHOL
865
concentrations are very high compared with AV differences (E around - 5- + 4%), and it is therefore difficult to demonstrate a significant uptake by the AV difference technique. The differences in AV for CHOL between high- and low-yielding goats (P < 0.06) can again be ascribed to differences in arterial concentrations, as was the case for TG. Interestingly, yields of milk constituents were found to be positively correlated with arterial CHOL concentration. Considering the importance of CHOL in membrane synthesis, and the role of plasma membranes in the excretion of milk (see above), it is tempting to speculate that CHOL supply to the mammary gland is a factor of importance for the formation of plasma membranes and overall milk synthesis and excretion. In rat mammary acini, fatty acid and CHOL synthesis have been found to be inversely regulated (Smith et al., 1986), which means that in periods, where de nouo fatty acid synthesis is very high, CHOL synthesis will be low, and plasma supply of CHOL may be important for the maintenance of normal plasma membrane synthesis. Low mammary CHOL synthesis would then be expected in early-midlactation, and positive values for CHOLAV were mainly observed in this period (see Fig. 3). Phospholipids (PL) Previous investigations, including radioisotopic tracer studies, have shown that PL circulating in plasma lipoproteins are not taken up by the mammary gland (Annison et al., 1967; Bickerstaffe et al., 1974), but rather synthesized de nouo (Bitman and Wood, 1990; Moore and Christie, 1979). It was, therefore, expected that AV differences for PL would be small and not significantly different from zero, but despite this, a positive (although weak (r* = 0.14)) relationship was found between arterial concentration and AV difference. Perhaps, at high arterial concentrations, the mammary gland is capable of extracting small amounts of PL? In conclusion, mammary uptake of FFA, TG and CHOL seems to be regulated primarily by arterial supply, and there is no evidence that synthetic activity of mammary cells and thus production capacity is
866
M. 0. Nielsen and K. Jakobsen
a factor determining the efficiency of uptake, as is the case for glucose (Nielsen and Jakobsen, 1993). It should be emphasized that there is a great variability in the data reported in the literature on mammary lipid uptake and metabolism, and much remains to be clarified. In light of the findings in the present experiment, the role of CHOL in regulation of mammary membrane synthesis and milk excretion, and the effect of dietary fish oils on mammary lipid metabolism in the ruminant are areas which deserve to be studied in more detail. Acknowledgements-The present study was supported by a grant from the Danish Agricultural and Veterinary Research Council. Special thanks are due to Mrs Anne Mette Buhl and Mr Jens Randrup for expert technical assistance and care of the animals, respectively, and to MS Anne Mette Kjeldsen (M.Sc.Agric.) for statistical evaluation of the data.
References Annison E. F., Bickerstaffe R. and Linzell J. L. (1974) Glucose and fatty acid metabolism in cows producing milk of low fat content. J. agric. Sci., Camh. 82, 87-95. Annison E. F., Linzell J. L., Fazakerley S. and Nichols B. W. (1967) The oxidation and utilization of palmitate, stearate, oleate and acetate by the mammary gland of the fed goat in relation to their overall metabolism, and the role of plasma phospholipids and neutral lipids in milk-fat synthesis. Biochem. J. 102, 637-647. Baldwin R. L. and Smith N. E. (1983) Adaptation of metabolism to various conditions: Milk production. In Dynamic Biochemistry of Animal Production (Edited by Riis P. M.), pp. 359-388. World Animal Science, Vol. A3. Elsevier Science, Amsterdam. Banks W., Clapperton J. L. and Girdler A. K. (1990) Effect of unsaturated fatty acids in various forms on the de nouo synthesis of fatty acids in the bovine mammary gland. J. Dairy Res. 57, 179-185. Bickerstaffe R. M., Annison E. F. and Linzell J. L. (1974) The metabolism of glucose, acetate, lipids and amino acids in lactating dairy cows. J. Agric. Sci. 82, 71-85. Bitman J. and Wood D. L. (1990) Changes in milk fat phospholipids during lactation. J. Dairy Sri. 73, 1208-1216. Bitman J., Wood D. L., Tyrrell H. F., Bauman D. E., Peel C. J., Brown A. C. G. and Reynolds P. J. (1984) Blood and milk lipid responses induced by growth hormone administration in lactating cows. J. Dairy Sci. 67, 2873-2880. Christie W. W. (1979) The effects of diet and other factors on the lipid composition of ruminant tissues and milk. Prog. Lipid Res. 17, 245-277.
Davis S. R. and Collier R. J. (1985) Mammary blood flow and regulation of substrate supply for milk synthesis. J. Dairy Sci. 68, 1041-1058. Hansen H. 0. and Knudsen J. (1987) Effect of exogenous long-chain fatty acids on lipid biosynthesis in dispersed ruminant mammary gland epithelial cells: Esterification of longchain exogenous fatty acids. J. Dairy Sci. 70, 1344-l 349. Hornstra G. (1989) The significance of fish and fish-oil enriched food for prevention and therapy of ischaemic cardiovascular disease. In The Role of Fats in Human Nutrition (Edited by Vergroesen A. J. and Crawford M.), pp. 151-236. Academic Press, London. Kronfeld D. S. (1965) Plasma non-esterified fatty acid concentrations in the dairy cow: Responses to nutritional and hormonal stimuli, and significance in ketosis. Vet. Rec. 72, 30-35. Linzell J. L. (1974) Mammary blood flow and methods of identifying and measuring precursors of milk. In Lactation-A Comprehensive Treatise (Edited by Larson B. L. and Smith V. R.), Vol. 1, pp. 143-225. Academic Press, New York. Marcos E., Mazur A., Cardot P. and Rayssiguier Y. (1990) The effect of pregnancy and lactation on serum lipid and apolipoprotein B and A-I levels in dairy cows. J. Anim. Physiol. Anim. Nutr. 64, 133-138. Miller P. S., Reis B. L., Calvert C. C., dePeters E. J. and Baldwin R. L. (1991a) Patterns of nutrient uptake by the mammary glands of lactating dairy cows. J. Dairy Sci. 74, 3791-3799. Miller P. S., Reis B. L., Calvert C. C., dePeters E. J. and Baldwin R. L. (1991b) Relationship of early lactation and bovine somatotropin on nutrient uptake by cow mammary gland. J. Dairy Sci. 74, 3800-3806. Moore J. H. and Christie W. W. (1979) Lipid metabolism in the mammary gland of ruminant animals. Prog. Lipid Res. 17, 347-395. Nickerson S. C. and Akers R. M. (1984) Biochemical and ultrastructural aspects of milk synthesis and secretion. Int. J. Biochem. 16, 855-865. Nielsen M. 0. and Jakobsen K. (1993) Changes in mammary glucose and protein uptake in relation to milk synthesis during lactation in high- and lowyielding goats. Comp. Biochem. Physiol. 106A, 359-365. Nielsen M. O., Jakobsen K. and Jorgensen J. N. (1990) Changes in mammary blood flow during the lactation period in goats measured by the ultrasound Doppler principle. Camp. Biochem. Physiol. 97A, 5 19-524. Peeters G., Houvenaghel A., Roets E., Massert-Leen A. M., Verbeke R., Dhondt G. and Verschooten F. (1979) Electromagnetic blood flow recording and balance of nutrients in the udder of lactating cows. J. Anim. Sri. 48, 1143-1153. Raphael B. C., Dimick P. S. and Puppione L. (1973) Lipid characterization of bovine serum lipoproteins throughout gestation and lactation. J. Dairy Sci. 56, 1025-1032. Rook J. A. F. and Thomas P. C. (1983) Milk secretion and its nutritional regulation. In
Mammary lipid uptake in goats
Nutritional Physiology of Farm Animals (Edited by Rook J. A. F. and Thomas P. C.), pp. 314-368. Longman Group Ltd, Essex. SAS (1985) SAS User’s Guide: Statistics. 5th edition. SAS Institute, Cary, North Carolina, U.S.A. Smith R. A. W., Middleton B. and West D. W. (1986) Cholesterol synthesis and fatty acid synthesis may
867
be inversely regulated in rat mammary acini. Biothem. Sot. Transacf. 14, 901-902. West C. E., Bickerstaffe R., Annison E. F. and Linzell J. L. (1972) Studies on the mode of uptake of blood triglycerides by the mammary gland of lactating goats. Biochem. J. 126, 477-490.
Pergamon 0300-9629(94)00136-7
Changes in mammary uptake of free fatty acids, triglyceride, cholesterol and phospholipid in relation to milk synthesis during lactation in goats M. 0. Nielsen* and K. Jakobsent *Department of Anatomy and Physiology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark; and TDepartment of Animal Physiology and Biochemistry, National Institute of Animal Science, P.O. Box 39, DK-8830 Tjele, Denmark Uptake of free fatty acids (FFA), triglycerides (TG), cholesterol (CHOL) and phospholipids (PL) was measured in both mammary glands of dairy goats during lactation. Arterial concentrations of TG, CHOL and PL as well as arteric+venous difference (AV) and extraction rate (E) for TG were highest in goats with the highest dietary feed intake. AV were linearly related to arterial concentrations for the four lipid classes, and arterial concentrations of CHOL were linearly related to output of lactose, protein and fat in milk. Arterial supply, and not mammary synthetic activity, is the main determinant of mammary FFA, TG and CHOL uptake. Key words: Mammary gland; Lipid uptake; FFA; Triglyceride; Cholesterol; Phospholipid; Lactation; Goat. Comp. Biochem. Physiol. 109A, 857-867,
1994.
Introduction Milk synthesis in the mammary gland changes markedly during the lactation period with a dramatic increase in early lactation and a gradual decrease after peak lactation. Much remains to be understood about the physiological background for these changes. Nutrients taken up by mammary cells for milk synthesis are supplied to the mammary gland by the blood, and mammary blood flow (MBF) has been assumed to play a key role in the regulation of mammary nutrient supply and thus milk to: M. 0. Nielsen, Department of Anatomy and Physiology,The Royal Veterinary
Correspondence
and Agricultural University, Thorvaldsensvej 40, DK- 1871 Frederiksberg C, Denmark. Received 23 January 1994; revised 4 July 1994; accepted 18 July 1994. CBPA 109,LD
857
synthesis (Davis and Collier, 1985). In lactating goats, it has been found that animals with a different capacity for milk synthesis also have a very different capacity for mammary uptake/extraction from the blood of the key nutrient glucose (Nielsen and Jakobsen, 1993), high-yielding animals being 40% more efficient than low-yielding in this respect. Metabolic activity may therefore also influence nutrient uptake in the mammary cells. It was the purpose of the present experiment to measure mammary uptake of preformed plasma lipids (free fatty acids, triglycerides, cholesterol and phospholipids) in relation to milk synthesis during the lactation period in goats with different capacities for milk synthesis, and in this way illuminate the role of nutrient supply
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M. 0. Nielsen and K. Jakobsen
versus the synthetic capacity of mammary cells in the regulation of milk production at different stages of lactation. Nutrient uptake was determined by arterio-venous difference measurements across the mammary gland in dairy goats. Results on MBF measurements (Nielsen et al., 1990) and mammary glucose and nitrogen uptake (Nielsen and Jakobsen, 1993) from the same experiment have been reported previously.
Materials and Methods Animals and experimental procedure
Three Norwegian (nos 53,64 and 67) and one Saanen (no. 73) dairy goats in their second and first lactation, respectively, and surgically prepared with one carotid artery exteriorized under the skin were used. Based on their peak milk yields, goats no. 53 and 73 will, in the following, be termed low-yielding, and 64 and 67 high-yielding. Arterio-venous (AV) differences across the mammary gland were measured on 1 day at approx. week 8, 11, 15, 25 and 37 post-partum. Feeding and housing of the experimental animals as well as the experimental procedure have been described previously (Nielsen and Jakobsen, 1993). On goat no. 64, measurements stopped after week 11 post -partum due to difficulties with insertion of catheters into the milk veins. Analyses
Milk lactose, protein and fat were determined as described previously (Nielsen and Jakobsen, 1993). Plasma free fatty acids (FFA) were determined using a NEFAC kit (Wako Chemicals GmbH, Neuss, Germany), and plasma triglycerides (TG), total cholesterol (CHOL) and phospholipids (PL) by Triglycerides GPO-PAP, Monotest@, Cholesterol, and Test Combination Phospholipids kits, respectively (all from Boehringer Mannheim GmbH, Mannheim, Germany). Calculations and statistical analyses
Mean concentrations of nutrients in the left and right milk veins were used for the calculation of AV differences, and extraction rates (E) derived as AV/A x lOO%, where A is arterial concentration. When values were missing for one of the milk
veins, the nutrient concentration in the other vein was used alone. This introduces a small error for goat no. 73, where concentrations of TG differed between the two milk veins (see Results). All values are expressed as mean + SEM, unless otherwise stated. The statistical models used to analyse data for concentrations of lipids in milk venous plasma, arterial concentrations, AV differences and E for the measured lipids have been reported previously (Nielsen and Jakobsen, 1993). The relationships between A, AV, E and A, AV, E, day of lactation or milk yield were investigated by linear regression and non-linear models. All analyses were performed using OLS (ordinary least square) software procedures (SAS, 1985).
Results Milk yield and composition
Milk production in the four experimental animals followed the normal pattern of the lactation curve, except for goat 64 which was dried off deliberately 20 weeks postpartum. Peak milk yields were obtained 6-9 weeks post-partum, and ranged from 1.9 and 1.2 kg/day in goats nos 53 and 73, respectively (low-yielding), and approx. 3.6 kg/day in goats nos 64 and 67 (highyielding). Milk yields declined gradually thereafter, and the goats were dried off 33-38 weeks post-partum (except goat 64, see above). Milk fat content, ranging from 2.3 to 4.2%, was generally highest in early lactation, decreased until after peak milk yields were reached and increased thereafter. Protein and lactose contents (%) were fairly constant during the lactation period. Daily yields of milk fat are presented in Fig. 1. Plasma concentrations and mammary extraction of lipids during lactation
The range and mean values of measured concentrations of FFA, TG, CHOL and PL in plasma obtained from the carotid artery and milk veins are presented in Table 1. No effect of udder half (milk vein) was found on venous concentrations of these nutrients, except for TG in goat no. 73, where concentrations were found to be higher
Mammary lipid uptake in goats
Table 1. Concentrations*
859
of FFA, TG, CHOL and PL in arterial and milk venous plasma
Arterial concentration Nutrient
Mean + SEM
FFA (mM) TG (mgil) CHOL (mg/l) PL (mM)
(Min-max
0.24 If: 0.028 I38 + 12 680 * 39 1.32 f 0.059
Milk venous concentration
values)
(0.07-0.82) (23-270) (230-949) (0.59-1.91)
n
Mean + SEM
31 31 32 32
0.21 f 0.012 ll7k8.3 686 + 26 138 + 0.044
(Min-max
values)
(0.10-0.58) (15-230) (247-972) (0.72-2. IO)
n 54 54 57 57
*Means of values obtained from all goats during the lactation period. For abbreviations: see text.
(P < 0.01) in the right than in the left milk vein, 79 & 9 mg/l and 68 + 9 mg/l, respectively. In the following, the two venous sides have not been treated separately, and the means of venous concentrations in the left and right milk vein have been used to calculate AV differences and E across the mammary gland. Although this is not strictly correct in the case of TG for goat no. 73, only a minor error is introduced. Free fatty acids (FFA). Measured AV differences for FFA in the total material and 0.28 mM varied between -0.085 with an average of 0.025 f 0.014 mM and E varying between - 65 and 5 1%, averaging 0.48 f 4.5%. Arterial FFA concentrations were slightly higher in low-yielding than in high-yielding goats (P c 0.05). Net mammary FFA extractions were found to be Table 2. Effect of production
level and sampling time on A, AV and E for FFA, TG, CHOL and PL in the mammary gland Milk production
Nutrient FFA
TG
CHOL
PL
Parameter* A
@W
AV
(mM)
E
W)
iv
;:;;t;
E
WI
A
@x/U
AV
(mg/l)
E
(%I
A
@W
AV
(mM)
positive in the morning but negative in the afternoon (P < 0.05), and the same tendency was observed for arterial concentrations and E. Otherwise, goat, yield-level and sampling time had no influence on A, AV and E (see also Table 2). Figure 1 shows the development in arterial concentrations and mammary extractions of FFA during lactation as well as daily milk fat production. It can be seen from the figure that arterial FFA concentrations were high in early lactation when also milk fat and overall milk production was high, and declined thereafter with a tendency for a small increase again in late lactation. The changes were significant both with goats as a systematic (P < 0.001) and a random (P < 0.05) effect. AV differences and E also changed during lactation
Sampling time
level
Low
High$
Pt
0.25 + 0.043 0.021 &-0.019 - 1.55 + 5.62 (n = 18) 105 f 13.9 7.8 f 5.3 7.44 &-6.45 (n = 17) 559 * 50.2 -37.9 f 17.3 - 10.9 f 4.30 (n = 18) 1.16 + 0.074 - 0.056 + 0.045 (n = 18)
0.23 + 0.031 0.033 f 0.021 3.29 + 7.66 (n = 13) 177+ 15.4 27.8 f 5.5 17.0 k 3.16 (n = 14) 837 k 24.1 6.2 f 16.8 0.417 k 2.27 (n = 14) 1.52 & 0.062 -0.065 f 0.07 1 (n = 14)
$ NS NS 7 /j NS 7 ** ** 7 NS
Morning
Afternoon
0.26 + 0.047 0.22 f 0.033 0.043 f 0.024 0.0091 + 0.00011 5.81 f 5.81 -4.52 k 6.79 (n = 15) (n = 16) 135 &-15.9 141 f 18.8 11.9 * 5.1 22.0 f 6.6 7.54 + 5.89 16.2 + 4.84 (n = 16) (n = 15) 697 + 57.5 673 f 53.6 - 19.2 k 18.5 - 18.0 + 17.7 -6.57 f 3.99 - 5.29 f 3.96 (n = 16) (n = 16) 1.27 + 0.080 1.36 f 0.087 -0.1 I f 0.67 -0.0094 k 0.039 (n = 16) (n = 16)
*Means of values obtained during the lactation period. iData analyses performed with parameter goat as a systematic effect (see Materials and Methods). $After week I I post-partum, data are derived from only one goat. 9, 11,7, **: significant at 0.05, 0.01, 0.001 and 0.06 level, respectively. NS: non-significant. For abbreviations: see text.
Pi
NS NS $ NS NS NS NS NS NS 9: NS
M. 0. Nielsen and K. Jakobsen
860
W..ka pod-partum
Ar(mll)-4.oea+O.a*.A(mM) (E’
-
od&
P<0.001)
-
l
/
I
10
Weeka
I
I
20
30
pa&-partam
Fig. 1. Changes in arterial FFA concentration (A), mammary arterio-venous difference (AV) of FFA and daily milk fat production (FP) during the lactation period. 0, 0, 0, v: Goat nos 53 and 73 (low-yielding), 64 and 67 (high-yielding). Each point represents means of the two udder-halves (AV), and means of morning and afternoon values (A and AV). n : Each point represents means of the two udder halves (AV). Solid lines represent average of all measured values in the panels showing changes in A or AV during lactation, and the regression line in the panel showing the relation between AV and A.
(P < 0.06 and P < 0.001, respectively). Changes in arterial concentrations and AV differences could be described by the following equations: A (mM) = 0.655 0.0065.day + 0.0000195.day2 (R2 = 0.31, P < 0.01) and AV (mM) = 0.182 0.00266.day + O.O000084~day* (R2 = 0.20, P < 0.05). AV followed the arterial concentrations closely as a linear function of A : AV (mM) = - 0.068 + 0.390.A (mM) (R* = 0.65, P < 0.001). Generally, AV and E became negative when arterial FFA concentrations fell below 0.2 mM, as in mid-late lactation. Triglyceride (TG). AV differences for TG varied between -29.0 and 70.0 mg/l with an average of 16.8 + 4.2 mg/l. E varied between -39 and 59% averaging 11.7 f 3.9%. E was not very high but was still significantly higher than 0 (P < 0.05). A
and AV differences for TG differed between goats (P -C 0.001 and P < 0.05, respectively). As can be seen from Table 2, the arterial TG concentration was 70% higher in the high- than in the low-yielding goats, leading also to higher AV differences. Sampling time had no effect on A, AV-differences and E for TG (see Table 2). Arterial concentration was dependent on the stage of lactation (P < 0.001). This was not the case for AV and E (P > O.S), and AV and A were only poorly correlated: AV (mg/l) = 0.062 + 0.118.A (mg/l) (R’ = 0.12, P < 0.06), as also evident from Fig. 2. Arterial concentrations decreased in the later part of lactation, as shown in Fig. 2, whereas the development in early lactation varied from goat to goat, the interaction effect being significant (P < 0.001).
Mammary
lipid uptake
in goats
-30
861
1 0
I , JO 20 Week8 pod-partum
I 10
,
60 ,”
-
(nil/l)
(a*- 0.13;
o.oaa + 0.118
-
P
l
*
ha/o
’
.*
-20
1
0
I
I
8
t
I
,
10
20
SO
40
0
100
W*eh
post-partum
Merid
’
l
I
200 uoncantration (mr/l)
1
300
Fig. 2. Changes in arterial TG concentration (A), and mammary arterbvenous difference (AV) and extraction (E) of TG during the lactation period. 0, 0, 0, V: Goat nos 53 and 73 (low-yielding), 64 and 67 (high-yieiding). Each point represents means of the two udder halves (AV and E) and means of morning and afternoon values (A, AV and E ). n : See legend to Fig. 1. Solid lines represent average of all measured values in the panels showing changes in A, AV or E during lactation, and the regression line in the panel showing the relation between AV and A.
Total c~o~e~ter~~(CHUL). Measured AV differences for CHOL varied between - 195.5 and 89.Omg/l with an average of - 18.6 + 12.6 mg/l and E varied between - 50.6 and 12.1% with an average of - 5.92 &-2.76%. Arterial CHOL concentrations were 50% higher in the highcompared with the low-yielding goats (P c 0.00 1, see Table 2). AV differences and mammary E of CHOL tended to be negative in low-yielding, and slightly positive in high-yielding goats (P c 0.06). No effect of sampling time could be demonstrated, but the interactions between the time and the goat were all significant for A, AV and E, since goat 73 in contrast with all other goats yielded the lowest values in the morning. As shown in Fig. 3, arterial concentrations and AV differences for CHOL
changed during the lactation period (P < 0.001 and P < 0.05, respectively). Arterial concentrations were relatively constant during early-mid lactation and decreased thereafter, following the general pattern of milk fat and overall milk production. The pattern of individual goats was, however, different, resulting in a significant interaction between the goat and the week of lactation (P < 0.001). AV differences and E across the mammary gland were high in the beginning of lactation and gradually decreased with progressing lactation, except for a marked around week 10 post -partum drop (P < 0.05). The same pattern was observed for E (P = 0.12). Both AV and E were linearly related to arterial concentrations: AV (mg/l) = -133+0.168.A tmg/lI,
862
M. 0. Nielsen and K. Jakobsen
a 0 IO
0
100
10
20 30 Toeke poBt-putum
I
10
40
t
AV 0
100 -
5
$
(a*-
- -100 + 0.100
o*
0
l
A (r/r)
P
.
.
-100
1
1
40
1
200
.
.
.=
-200 I
*
0
f?J 20 30 weeks pod-putum
.l
l
z0’B
,a
40
woeke post-puwlB
r
10
I
30
20
. I
I
I
1
400 000 600 1000 Artenl oonoontr~tlon (mu/t)
Fig. 3. Changes in arterial CHOL concent~tion (A) and mammary arterio-venous difference (AV) and extraction (E) of CHOL during the lactation period. 0, c], 0, 0, a, and solid lines: see legend to Fig. 2.
(R2 = 0.27, P < 0.001) and E (%) = -37.8 + 0.468.A (mg/l) (R2 = 0.43, P < 0.001). Phospho~ipid (PL). Measured AV differ-
ences for phosphohpid varied between -0.64 and 0.42 mM with an average of -0.060 + 0.039 mM. AV differences for PL were unaffected by goat, yield-level, sampling time (see Table 2) as well as the stage of lactation, and were not significantly different from 0 (P > 0.1), and calculations have therefore not been performed for E. AV and A were only poorly correlated (see Fig. 4): AV (mM) = -0.396 +0.255-A (mM) (R2 = 0.14, P < 0.05). Arterial concentrations were found to be approx. 30% higher in high-yielding compared with low-yielding goats (P < 0.001) and slightly higher values were found in the afternoon than in the morning samples (P c 0.05), as shown in Table 2. Arterial concentrations generally increased in the first part of lactation and decreased in
the later part of lactation (P < O.OOl), as shown in Fig. 4, but interaction effects were found for goats and the week of lactation (P c 0.01) as well as for sample time and week of lactation (P < 0.05). The development in arterial concentration during lactation could be described by the equation: A (mM) = 1.14 + 0.00491 *day 0.0000209~day2 (R2 = 0.18, P < 0.05). Mammary extraction milk synthesis
of lipids in relation to
No relationship was found between the yield of milk constituents and A, AV or E for FFA, TG and PL, and AV and E for CHOL. However, production of lactose, milk protein and milk fat was found to be related to mammary supply (arterial concentration) of CHOL (P < 0.05 in all cases). An increased yield of milk constituents was associated with higher arterial CHOL concentrations, as shown in
Mammary
lipid uptake in goats
863
Fig. 5. Due to a strong effect of goat and a strong interaction between goat and yield of milk constituents on arterial CHOL concentrations (P < O.Ol), simple correlations of A with yield of lactose, milk protein and milk fat only ranged from 0.20 to 0.30.
Discussion Triglycerides normally account for 97-99% of total lipids secreted into goat and cow milk. They are excreted from mammary cells as milk lipid droplets, which are enveloped in the plasma membrane before being expelled into the alveolar lumen (Nickerson and Akers, 1984), and synthesis of plasma membranes must therefore be considered of importance for overall milk secretion in milk. The major lipids in plasma membranes from goat and cow
0
Ar(mM)--o.ooo+O.8oo*A(mx)
00
00
100 (r/d)
9. .
.
g 400 -
140
. .
t
,
~~~~~;““~”
0
20
40
Proteln
produotion
20
40 Fat production
.
120
.
i
0 -
40
Laotona prodrotlon
%
1 ‘“:
0.4
20
,
60
80
100
80
100
(g/d)
80 (g/d)
(P’ - 0.11; P
Fig. 5. Changes in arterial CHOL concentration in relation to daily lactose (LP), milk protein (PP) and milk fat (FP) production. The regressions between arterial concentration and LP, PP or FP are depicted as solid lines. P
*-0.4 -0.6
0.50
.
.
n
. . t I I I I 0.75 1.00 1.25 1.10 1.75 Artmrhl aonc.ntration (mY)
I 2.00
Fig. 4. Changes in arterial PL concentration (A) during the lactation period and relationship of mammary arterio-venous difference (AV) to A. 0, 0, 0, V, H and solid lines: see legend to Fig. 1.
mammary glands are TG, CHOL and PL. CHOL and PL accounts for approximately 0.5% and 1% (Bitman et al., 1984) of total milk lipids, respectively. In the present experiment, the purpose was to investigate how mammary uptake of TG, CHOL and PL in circulating plasma lipids changes during the lactation period in
864
M. 0. Nielsen and K. Jakobsen
relation to changes in milk production, and to study if a high mammary synthetic activity also is associated with a high capacity for extraction of plasma lipids, as seems to be the case for glucose (Nielsen and Jakobsen, 1993). In other words, what is the role of mammary substrate supply versus mammary synthetic activity in the regulation of mammary uptake of these lipids? The observed levels of arterial concentrations (A), arterio-venous differences (AV) and extraction rates (E) for FFA (Annison et al., 1967; Miller et al., 1991a; West et al., 1972), TG (Bickerstaffe et al., 1974; Miller et al., 1991a; West et al., 1972), CHOL (Annison et al., 1967; Bickerstaffe et al., 1974; Miller et al., 1991a) and PL (Bickerstaffe et al., 1974; Linzell, 1974; West et al., 1972) were generally in agreement with those reported in the literature. AV and E for TG and AV for CHOL were, however, in the low range, and measured values of A for total CHOL very low compared with those reported by others (see above). Free fatty acids (FFA)
FFA are known to be extracted by the mammary gland as verified by isotopic tracer studies (Annison et al., 1967), but during hydrolysis of lipoprotein TG, FFA are released into the blood stream, and positive values of AV differences for FFA are therefore only found in situations where arterial FFA concentrations are high: above 0.3 mM as reported by Kronfeld (1965) or 0.2 mM, as observed in the present experiment. Concentrations were generally highest in early lactation as expected, since loss of body weight (fat mobilization) is normally associated with high plasma levels of FFA. There was a close positive correlation between AV and FFA and arterial concentrations, as also reported by others (Miller et al., 1991a), suggesting that FFA is taken up by mammary cells by passive diffusion, and extracellular concentration must be the main determinant of the concentration gradient across the cell membrane, which drives uptake. Triglyceride (TG)
were Arterial TG concentrations generally highest in early lactation, and
lactation. decreased with progressing Others have found the lowest levels in early lactation (Marcos et al., 1990; Miller et al., 1991b), where mammary nutrient uptake is maximal. The diet fed to the goats in the present experiment contained a fairly large amount of fish meal, which is rich in fat containing long-chain polyunsaturated fatty acids (PUFA). The high fat intake may explain the high arterial concentrations in early lactation. AV differences were positively correlated with arterial concentation, although the correlation was poor (r2 = 0.12) compared with previously reported values (Miller et al., 1991a: r2 = 0.25), but AV differences were generally quite low in the present experiment as discussed above, and in some cases negative, which, however, must be ascribed to analytical error. The high dietary content of long-chain PUFA may explain the low values observed for AV and E for TG, since a high dietary intake of PUFAs is associated with increased formation of trans-fatty acids during hydrogenation in the rumen. Trans-fatty acids and long-chained (C2,,-C22) PUFAs in plasma are believed to have an inhibitory effect on the activity of lipoprotein lipase (Rook and Thomas, 1983), and inhibit de nouo fatty acid synthesis (Banks et al., 1990; Hansen and Knudsen, 1987) resulting in lowered uptake of fatty acids from lipoprotein-TG and reduced milk fat synthesis. In agreement with this, milk fat content was fairly low in our study. Annison et al. (1974) have reported data, which also indicate that feeding-induced low milk fat content is associated with low values for TG-AV, a lower lipoprotein lipase activity would result in lower release of fatty acids from lipoprotein TG, and this could also explain why AV differences for FFA as mentioned above became positive at lower arterial concentrations of FFA than previously reported. As is the case for FFA, TG uptake in the mammary gland seems, therefore, to be determined primarily by arterial supply, at least at arterial plasma concentrations of TG below approx. 300 mg/l. At higher concentrations, the mammary capacity for uptake (e.g. lipoprotein lipase activity and mammary cellular activity) may, according to Baldwin and Smith (1983), become
Mammary lipid uptake in goats
limiting. In our experiment, high-yielding goats seemed to have significantly higher AV differences for TG than low-yielding goats. However, this could be explained by higher arterial concentrations of TG in the high-yielding animals, due to higher dietary fat intake (Christie, 1979). It must be emphasized that differences between high- and low-yielding goats must be taken with some caution, since one of the two high-yielding goats was excluded from the experiment after week 11 post partum. Despite the low AV differences measured in the present experiment, it was possible to account for the output of preformed fatty acids in the milk of the highyielding goats, utilizing the average AV differences for TG and FFA in Table 2 and the previously reported values for mammary blood flow (Nielsen et al., 1990). However, TG-AV differences may have been underestimated in the low-yielding goats. Cholesterol (CHOL) In humans, a high dietary intake of longchain PUFA (e.g. fish oils) may have a lowering effect on plasma CHOL concentration (Hornstra, 1989). A relatively high absorption of PUFA must be expected on a diet as for that in the present experiment, and perhaps the low arterial CHOL concentrations generally observed in our goats may, in part, be the result of a high PUFA absorption. Arterial concentrations were relatively constant through most of the lactation period, but decreased in late lactation. Similar observations have been reported by Raphael et al. (1973) and Marcos et al. (1990), and probably reflect a high metabolism of TG-rich lipoproteins (very low density lipoprotein) in lactating animals depending on the milk yield. AV differences for CHOL were linearly related to (Y’= 0.25) and followed arterial concentrations, except for a drop around week 10 post-partum, for which we have no immediate explanation. CHOL is taken up by the mammary gland (More and Christie, 1979) but even so, AV differences are normally reported not to be significantly different from zero (Annison et al., 1967; Peeters et al., 1979). Arterial CHOL
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concentrations are very high compared with AV differences (E around - 5- + 4%), and it is therefore difficult to demonstrate a significant uptake by the AV difference technique. The differences in AV for CHOL between high- and low-yielding goats (P < 0.06) can again be ascribed to differences in arterial concentrations, as was the case for TG. Interestingly, yields of milk constituents were found to be positively correlated with arterial CHOL concentration. Considering the importance of CHOL in membrane synthesis, and the role of plasma membranes in the excretion of milk (see above), it is tempting to speculate that CHOL supply to the mammary gland is a factor of importance for the formation of plasma membranes and overall milk synthesis and excretion. In rat mammary acini, fatty acid and CHOL synthesis have been found to be inversely regulated (Smith et al., 1986), which means that in periods, where de nouo fatty acid synthesis is very high, CHOL synthesis will be low, and plasma supply of CHOL may be important for the maintenance of normal plasma membrane synthesis. Low mammary CHOL synthesis would then be expected in early-midlactation, and positive values for CHOLAV were mainly observed in this period (see Fig. 3). Phospholipids (PL) Previous investigations, including radioisotopic tracer studies, have shown that PL circulating in plasma lipoproteins are not taken up by the mammary gland (Annison et al., 1967; Bickerstaffe et al., 1974), but rather synthesized de nouo (Bitman and Wood, 1990; Moore and Christie, 1979). It was, therefore, expected that AV differences for PL would be small and not significantly different from zero, but despite this, a positive (although weak (r* = 0.14)) relationship was found between arterial concentration and AV difference. Perhaps, at high arterial concentrations, the mammary gland is capable of extracting small amounts of PL? In conclusion, mammary uptake of FFA, TG and CHOL seems to be regulated primarily by arterial supply, and there is no evidence that synthetic activity of mammary cells and thus production capacity is
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M. 0. Nielsen and K. Jakobsen
a factor determining the efficiency of uptake, as is the case for glucose (Nielsen and Jakobsen, 1993). It should be emphasized that there is a great variability in the data reported in the literature on mammary lipid uptake and metabolism, and much remains to be clarified. In light of the findings in the present experiment, the role of CHOL in regulation of mammary membrane synthesis and milk excretion, and the effect of dietary fish oils on mammary lipid metabolism in the ruminant are areas which deserve to be studied in more detail. Acknowledgements-The present study was supported by a grant from the Danish Agricultural and Veterinary Research Council. Special thanks are due to Mrs Anne Mette Buhl and Mr Jens Randrup for expert technical assistance and care of the animals, respectively, and to MS Anne Mette Kjeldsen (M.Sc.Agric.) for statistical evaluation of the data.
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Davis S. R. and Collier R. J. (1985) Mammary blood flow and regulation of substrate supply for milk synthesis. J. Dairy Sci. 68, 1041-1058. Hansen H. 0. and Knudsen J. (1987) Effect of exogenous long-chain fatty acids on lipid biosynthesis in dispersed ruminant mammary gland epithelial cells: Esterification of longchain exogenous fatty acids. J. Dairy Sci. 70, 1344-l 349. Hornstra G. (1989) The significance of fish and fish-oil enriched food for prevention and therapy of ischaemic cardiovascular disease. In The Role of Fats in Human Nutrition (Edited by Vergroesen A. J. and Crawford M.), pp. 151-236. Academic Press, London. Kronfeld D. S. (1965) Plasma non-esterified fatty acid concentrations in the dairy cow: Responses to nutritional and hormonal stimuli, and significance in ketosis. Vet. Rec. 72, 30-35. Linzell J. L. (1974) Mammary blood flow and methods of identifying and measuring precursors of milk. In Lactation-A Comprehensive Treatise (Edited by Larson B. L. and Smith V. R.), Vol. 1, pp. 143-225. Academic Press, New York. Marcos E., Mazur A., Cardot P. and Rayssiguier Y. (1990) The effect of pregnancy and lactation on serum lipid and apolipoprotein B and A-I levels in dairy cows. J. Anim. Physiol. Anim. Nutr. 64, 133-138. Miller P. S., Reis B. L., Calvert C. C., dePeters E. J. and Baldwin R. L. (1991a) Patterns of nutrient uptake by the mammary glands of lactating dairy cows. J. Dairy Sci. 74, 3791-3799. Miller P. S., Reis B. L., Calvert C. C., dePeters E. J. and Baldwin R. L. (1991b) Relationship of early lactation and bovine somatotropin on nutrient uptake by cow mammary gland. J. Dairy Sci. 74, 3800-3806. Moore J. H. and Christie W. W. (1979) Lipid metabolism in the mammary gland of ruminant animals. Prog. Lipid Res. 17, 347-395. Nickerson S. C. and Akers R. M. (1984) Biochemical and ultrastructural aspects of milk synthesis and secretion. Int. J. Biochem. 16, 855-865. Nielsen M. 0. and Jakobsen K. (1993) Changes in mammary glucose and protein uptake in relation to milk synthesis during lactation in high- and lowyielding goats. Comp. Biochem. Physiol. 106A, 359-365. Nielsen M. O., Jakobsen K. and Jorgensen J. N. (1990) Changes in mammary blood flow during the lactation period in goats measured by the ultrasound Doppler principle. Camp. Biochem. Physiol. 97A, 5 19-524. Peeters G., Houvenaghel A., Roets E., Massert-Leen A. M., Verbeke R., Dhondt G. and Verschooten F. (1979) Electromagnetic blood flow recording and balance of nutrients in the udder of lactating cows. J. Anim. Sri. 48, 1143-1153. Raphael B. C., Dimick P. S. and Puppione L. (1973) Lipid characterization of bovine serum lipoproteins throughout gestation and lactation. J. Dairy Sci. 56, 1025-1032. Rook J. A. F. and Thomas P. C. (1983) Milk secretion and its nutritional regulation. In
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