- Email: [email protected]
Feeding Oleamide to Lactating Jersey Cows. 2. Effects on Nutrient Digestibility, Plasma Fatty Acids, and Hormones1
Feeding Oleamide to Lactating Jersey Cows. 2. Effects on Nutrient Digestibility, Plasma Fatty Acids, and Hormones1 D. D. DeLuca2 and T. C. Jenkins3 Clemson University, Clemson, SC 29634
ABSTRACT
INTRODUCTION
Six lactating Jersey cows were used in a 6 × 6 Latin square with 14-d periods to evaluate different ratios of canola oil and oleamide on nutrient digestibility, plasma fatty acids, and plasma hormones. The control diet contained no added fat. All other diets contained 3.5% added fat consisting of 0, 25, 50, 75, and 100% as oleamide and the remainder as canola oil. Data were collected during the final 4 d of each period. Dry matter intake was reduced by the addition of canola oil to the diet, and further reduced by replacing canola oil with oleamide. Milk yield was not affected by diet but increasing oleamide proportion in the fat supplement caused linear increases in cis-C18:1 and linear decreases in C4 to C16 fatty acids in milk. Adding canola oil reduced total tract digestibilities of fiber and fatty acids, but had no effect on the digestibilities of dry matter or protein. Replacing canola oil with oleamide increased protein digestibility linearly, and increased digestibility of fiber (quartic relationship) and fatty acids (quadratic relationship). Oleic acid concentration in plasma increased by adding canola oil to the diet, and was further increased by replacing canola oil with oleamide. Diet had no effect on plasma concentrations of insulin or IGF-I. Oleamide fed to Jersey cows in this study was highly digestible and had no deleterious effects on total tract digestibility of fiber or protein. Increasing oleic acid concentration in plasma lipids while maintaining a constant level of added fat in the ration had no effect on circulating concentrations of insulin or IGF-I in Jerseys. (Key words: oleamide, Jerseys, digestion, plasma hormones)
Oleamide reduces the rate of biohydrogenation of oleic acid by ruminal microbes (17) and increases oleic acid content of milk when fed to lactating Holstein cows (10). The oleamide was highly digestible and had no adverse effects on the digestibilities of fiber or protein when fed to Holsteins (10). Similar to Holsteins, oleamide fed to lactating Jerseys (11) also substantially elevated oleic acid content of milk. However, increases in milk oleic acid concentration were lower for Jerseys than for Holsteins. Adding a commercial oleamide to the diet of Jerseys increased milk oleic acid 72% (11), compared with a 108% increase for Holsteins (10). Additional fat fed to lactating dairy cows has had variable effects on plasma hormones. Blood insulin concentrations were lower for cows fed added fat in 8 out of 17 studies summarized by Staples et al. (20). Some of this variability may result from ruminal biohydrogenation and the inconsistent delivery of unsaturated fatty acids to the small intestine (20). A positive association between unsaturated fatty acids and insulin release was reported in nonruminants (14). Based on the increase in milk oleic acid concentration reported in the previous study (11), oleamide would be expected to increase unsaturated fatty acids in blood, which may have effects on circulating insulin levels. This study had two objectives. The first was to determine if the digestibility of fatty acids and other nutrients in Jerseys fed oleamide were as complete as reported previously for Holsteins. A second objective was to determine if the concentrations of plasma insulin and IGF-I were altered when unsaturated fatty acids were elevated in plasma without a concomitant increase in total dietary fat.
Received June 21, 1999. Accepted October 29, 1999. Corresponding author: T. C. Jenkins; e-mail: [email protected]. 1 Technical Contribution Number 4504 of the South Carolina Agricultural Experiment Station, Clemson University. Additional financial support was provided by Church & Dwight, Inc., Princeton, NJ. 2 Present address: Purina Mills, Inc., P.O. Box 46, Montgomery City, MO 63361. 3 Department of Animal & Veterinary Sciences, 151 Poole Agricultural Center, Clemson University, Clemson, SC. 2000 J Dairy Sci 83:569–576
MATERIALS AND METHODS Cows and Diets Six lactating Jersey cows (four multiparous and two primiparous) were fed six diets in a 6 × 6 Latin square with 14-d periods. At the start of the study, DIM for all cows averaged 60 d (43, 54, 63, 64, 67, and 68 d). Cows were housed individually in tie stalls and bedded on wood shavings. Water was available at all times.
569
570
DELUCA AND JENKINS Table 1. Chemical composition of the total mixed diets. % Amide in fat supplement Item
Control
0
25
NDF CP Fatty acids Amide
36.3 17.0 2.8
35.0 16.5 5.9
35.2 16.1 5.8 0.5
50
75
100
34.5 16.5 6.1 1.2
34.5 16.5 6.1 1.7
35.0 16.8 6.0 2.4
% of DM
Cows were fed twice per day (0800 and 1600 h) and had ad libitum access to a TMR of 47% corn silage and 53% concentrate (DM basis). Ingredients comprising the TMR were described previously (11). Chemical composition of the TMR is shown in Table 1. Feed refusals (orts) were collected and weighed each day prior to the a.m. feeding. Cows were milked daily at 1000 and 2200 h. The control diet contained no added fat and the other five diets contained 3.5% added fat consisting of blends of canola oil (Cargill Oilseeds Division, Minneapolis, MN) and oleamide (Table 2). Oleamide comprised 0, 25, 50, 75, 100% of the added fat portion and the remainder was canola oil. Oleamide was synthesized from canola fatty acids (EMERY 790, Henkel Corporation, Cincinnati, OH) as described previously (11). Fatty acid composition of the canola fatty acids as reported by the manufacturer was 5% C16:0, 2% C18:0, 59% cis-C18:1, 22% C18:2, 10% C18:3, 1% C20:0, and 1% C22:0. Sample Collection and Analysis Ort samples for each diet were collected during the last 4 d of each period. Fecal grab samples were collected the last 3 d of each period (0800 and 2300 h on d 12; 1100, 1700, and 0200 h on d 13; and 1400, 2000,
Table 2. Fatty acid composition of the canola oil and oleamide supplements. Canola oil1
Oleamide2
% of total fatty acids C16:0 C18:0 cis-C18:1 C18:2 C18:3 Other3
4.4 2.2 61.8 21.1 7.4 3.1
5.3 2.7 62.6 22.1 6.4 0.3
1 As reported by the supplier (Cargill Oilseeds Division, Minneapolis, MN). Additional reported specifications of the oil were 0.05% FFA maximum as oleic acid, 1.00 meq/kg maximum peroxide value, and no additives. 2 Determined by GLC. 3 One or more of the following fatty acids were present at ≤ 2% of total fatty acids; trans-C18:1, C20:0, and C20:1.
Journal of Dairy Science Vol. 83, No. 3, 2000
and 0500 h on d 14) in a time sequence that gave a sample every 3 h over a 24-h period. Chromic oxide (10 g) in gelatin capsules was orally administered the last 10 d of each period prior to each feeding. Chromic oxide was used as an indigestible marker to determine nutrient digestibility. Silage, ort, and fecal samples were dried at 55°C and, along with samples of the concentrate, were ground in a Wiley mill through a 2-mm sieve. Ground samples were analyzed for DM (100°C), Kjeldahl N (1), and NDF (22, procedure A). Fatty acids in samples were methylated in 5% methanolic HCl for 15 h at 80°C. The higher temperature and longer incubation time than normally used were required to ensure complete hydrolysis of the amide bond (10) but had no effect on recovery of a fatty acid standard (12). Fatty acid methyl esters were separated by GLC (11). Fecal samples were analyzed for Cr (8). Milk samples were collected the last eight milkings (4 d) of each period and composited daily in proportion to milk yield. Milk samples (preserved in bronopol) were analyzed for fat and protein concentrations by mid infrared spectroscopy (Southeast DHIA laboratory, McDonough, GA). Milk fatty acid composition was determined by GLC (11). Blood samples were collected hourly for 4 h after the morning feeding on d 14 of each period. Blood was taken by jugular venipuncture into tubes containing EDTA as an anticoagulant. Tubes were centrifuged for 20 min at 1500 × g (5°C). Plasma was removed and frozen until later analyzed for insulin (Coat-A-Count Insulin kits, Diagnostic Products Corporation, Los Angeles, CA) and IGF-I (13). Blood taken at 3-h postprandial on d 14 of each period also was analyzed for fatty acid composition of various lipid fractions. Lipids were extracted from plasma with chloroform/methanol (2:1, vol/vol). Triglycerides, phospholipids, and cholesterol esters in the plasma extract were separated by TLC on 20- × 20-cm preparative plates coated with silica gel. The mobile phase consisted of hexane/diethyl ether/formic acid (16:4:1, by vol). Samples were scraped into tubes and analyzed for fatty acids by GLC. Samples were methylated in 5% methanolic HCl as described previously for feed and fecal
NUTRIENT DIGESTION OF JERSEY COWS FED OLEAMIDE
samples. Methyl esters of fatty acids were separated by GLC on a 30-m × 0.25-mm i.d. fused silica capillary column with a 0.25-µm film thickness (Supelco, Bellefonte, PA). The injector and detector temperatures were maintained at 250°C. Initial column temperature was 150°C (held for 2 min) and programmed to increase 2°C/min to a final temperature of 220°C. Amide Analysis Feed and fecal samples were analyzed for amide content by HPLC. A 10-ml extraction mixture (chloroform/ methanol, 1:1 vol/vol) was added to 1 g of dried sample and placed on a wrist-action shaker for 1 h. Samples were centrifuged for 15 min at 500 × g and the supernatant was transferred to another 50-ml tube. This procedure was repeated with another 5 ml of extraction mixture added to the pellet and placed on the shaker for an additional 30 min. Distilled water (4.5 ml) was added to the supernatant, mixed, and centrifuged at 500 × g for 5 min to separate the top layer (aqueous/methanol) from the chloroform layer which contained the amide. A 2-ml sample of the chloroform extract was loaded onto a preconditioned (5 ml of chloroform) silica extraction column and washed through the column with 20 ml of chloroform into a 25-ml volumetric flask. Amide content was determined by HPLC and quantified using stearamide as an external standard (10). Statistics All data, except for plasma hormones, were analyzed as a 6 × 6 Latin square design by ANOVA by the general linear models procedure of SAS (18). Diet, period, and cow were included in the model as sources of variation. The effect of adding canola oil to the diet was determined by a linear contrast that compared the control diet to the 0% oleamide diet. The effect of replacing canola oil with oleamide was determined by orthogonal polynomials among the equally spaced levels of oleamide that included linear or nonlinear (quadratic, cubic, and quartic) components. Data for plasma insulin and IGF-I were analyzed as a split plot in time ANOVA using the general linear models procedure of SAS (18). Cow, period, and diet were whole-plot factors tested against the main plot error term (interaction of cow, period, and diet). Time and all interactions with time were included in the model as sub-plot error terms tested against residual error. The diet × time interaction was not significant (P > 0.15) for either insulin or IGF-I. RESULTS AND DISCUSSION Adding canola oil to the diet reduced (P < 0.05) DMI of the cows from 15.7 to 14.6 kg/d (Table 3). Replacing
571
canola oil with oleamide further reduced (P < 0.01) DMI in a linear fashion. Depressed DMI from feeding fatty acyl amides was reported for Jerseys (11), and also for Holsteins (10). Despite the decline in DMI, milk yield was not reduced in this study. Oleamide fed to Holsteins similarly had no effect on milk yield (10), but feeding oleamide to lactating Jerseys (11) reduced milk yield 2.4 kg/d compared with feeding the same quantity of canola oil. Milk fat percentage was lower (P < 0.05) for the 0 diet compared with the control diet. The depressed milk fat percentage was maintained as oleamide replaced canola oil in the fat supplement. In Holsteins, milk fat was not affected by feeding oleamide but was reduced by canola oil (10). In the previous Jersey study (11), milk fat percentage tended to be reduced by the oleamide supplement, which was suggested as possibly being due to the 5 to 6% trans-C18:1 in the oleamide fatty acids. The oleamide supplement in this study contained lower (<2%) transC18:1 but still depressed milk fat compared to the control diet. Alternate explanations to account for the depressed milk fat percentage include inhibition of ruminal fermentation by either the amide or by the unreacted fatty acids contaminating the amide supplement. The amides fed to Jersey cows in this study and the previous study (11) were not identical, and differed mainly in their fatty acid compositions. Although the amides were synthesized in both studies by the same method, the source of fatty acid in the previous study was technical grade oleic acid (76% C18:1 and 0.5% C18:2) while canola fatty acids (59% C18:1 and 22% C18:2) were used in the present investigation. Milk fat yield (kg/d) was lower (P < 0.05) for the 0 diet than for the control diet with no effect of replacing canola oil with amide. Yield of 3.5% FCM also was reduced (P < 0.05) by the 0 diet, which declined (P = 0.06) quadratically as the amide replaced canola oil. Most of the decline in FCM occurred for the 100 diet. Results were similar for energy-corrected milk, which was not affected by adding canola oil but was reduced (P = 0.12) quadratically as amide increased in the fat supplement. Diet had no effect on energy-corrected milk when expressed per kilogram of DMI. The 0 diet reduced (P < 0.05) milk protein percentage, but no further change in protein concentration occurred as amide replaced canola oil in the fat supplement. Opposite results occurred in the previous Jersey study (11) in which the amide but not canola oil reduced milk protein percentage. Yield of milk protein (kg/d) was not affected by canola oil or any proportion of amide in the fat supplement. The effects of feeding canola oil and oleamide to lactating cows on milk fatty acid composition (Table 4) Journal of Dairy Science Vol. 83, No. 3, 2000
572
DELUCA AND JENKINS Table 3. Dry matter intake, milk yield and milk composition for Jersey cows fed combinations of canola oil and oleamide. Probabilities1
% Amide in fat supplement
DMI, kg/d Milk, kg/d 3.5% FCM, kg/d ECM, kg/d Fat, % Fat, kg/d Protein, % Protein, kg/d ECM/DMI, kg
Control
0
25
50
75
100
SEM
L
NL
15.7 21.0 25.0 24.9 4.66 0.98 3.60 0.76 0.77
14.6* 21.6 23.8* 23.8 4.15* 0.90* 3.49* 0.75 0.77
13.9 22.2 24.3 24.2 4.12 0.91 3.42 0.75 0.83
13.9 21.5 24.0 23.8 4.25 0.92 3.40 0.73 0.79
13.3 21.4 23.2 23.1 4.04 0.86 3.37 0.72 0.82
13.4 20.7 21.9 22.0 3.84 0.80 3.40 0.71 0.82
0.34 0.49 0.6 0.5 0.13 0.03 0.06 0.02 0.03
0.01 NS 0.01 <0.01 NS 0.01 NS NS NS
NS NS 0.06 0.12 NS 0.06 NS NS NS
1 Probabilities for a linear (L) or nonlinear (NL) effect of increasing the percentage of amide in the fat supplement. The NL effects were quadratic for FCM, ECM, and fat yield. Probabilities > 0.15 are noted as not significant (NS). *The 0% oleamide and control diets differed (P < 0.05).
were similar to the results reported in the previous Jersey study (11). Milk cis-C18:1 concentration in this study increased (P < 0.05) from 19.8 to 23.6% of total fatty acids when canola oil was added to the diet. As oleamide replaced canola oil in the fat supplement, cisC18:1 in milk linearly increased (P < 0.01) from 23.6 to 28.4% of total fatty acids. The synthesized oleamide in the previous Jersey study (11) increased cis-C18:1 22% over the canola oil diet, compared with a 20% increase in this study. The previous study with Jersey cows (11) showed that oleamide increased milk cis-C18:1 concentration more (36%) when the oleamide was a commercial source of higher purity.
The effects of increasing oleamide in the fat supplement on other fatty acids in milk also were similar to oleamide effects on milk fatty acids in the previous lactation study (11). Increasing oleamide concentration in the fat supplement linearly reduced (P < 0.04) the concentration of all C4 to C16 milk fatty acids, increased (P = 0.08) milk C18:0 up to 75% oleamide in the fat supplement, but had no effects on the concentrations of trans-C18:1, C18:2, or cis-9, trans-11 C18:2. Increasing oleamide proportions in the fat supplement reduced (P = 0.09) milk C18:3 concentration in a quadratic fashion, although its concentration never exceeded 0.4% of total milk fatty acids.
Table 4. Milk fatty acid composition for Jersey cows fed combinations of canola oil and oleamide. Probabilities1
% Amide in fat supplement
C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C14:1 C16:0 C16:1 C17:0 C18:0 cis-C18:1 trans-C18:1 C18:2 C18:3 CLA2
Control
0
25
50
75
2.70 2.77 1.68 3.69 3.97 12.21 1.38 34.51 1.61 0.51 10.72 19.83 1.53 2.32 0.35 0.21
2.73 2.53 1.48 3.15* 3.28* 10.88* 1.19* 28.29* 1.35 0.47 15.18* 23.59* 2.94* 2.22 0.39* 0.34*
% of total fatty acids 2.84 2.72 2.43 2.58 2.49 2.16 1.45 1.43 1.24 2.91 3.01 2.63 2.98 3.07 2.77 10.12 10.03 9.35 1.10 1.10 1.01 26.91 25.82 26.21 1.44 1.36 1.48 0.48 0.49 0.50 15.24 15.72 16.17 25.08 25.84 28.41 3.94 3.89 2.81 2.18 2.26 2.16 0.37 0.36 0.35 0.38 0.40 0.32
100
SEM
L
NL
2.56 2.31 1.30 2.71 2.83 9.65 1.10 25.63 1.52 0.50 14.65 28.35 3.94 2.19 0.36 0.38
0.09 0.11 0.07 0.14 0.15 0.34 0.04 0.63 0.10 0.01 0.46 0.99 0.36 0.07 0.01 0.03
0.02 0.02 0.02 0.02 0.03 <0.01 0.04 <0.01 NS 0.09 NS <0.01 NS NS 0.03 NS
NS NS NS NS NS NS 0.09 NS NS NS 0.08 NS NS NS 0.09 NS
1 Probabilities for a linear (L) or nonlinear (NL) effect of increasing the percentage of amide in the fat supplement. The NL effects were quadratic for C14:1, C18:0, and C18:3 concentrations in milk. Probabilities > 0.15 are noted as not significant (NS). 2 Conjugated linoleic acid. *The 0% oleamide and control diet differed (P < 0.05).
Journal of Dairy Science Vol. 83, No. 3, 2000
573
NUTRIENT DIGESTION OF JERSEY COWS FED OLEAMIDE Table 5. Apparent nutrient digestibilities in Jersey cows fed combinations of canola oil and oleamide. Probabilities1
% Amide in fat supplement Control
0
25
58.8 59.4 37.6 79.1
56.1 61.3 28.8* 72.1*
56.7 60.3 28.5 77.1
50
75
100
SEM
L
NL
56.9 63.4 25.5 76.3
61.4 66.0 38.0 77.5
60.0 65.5 35.1 72.2
1.4 1.4 2.6 1.8
0.01 0.01 0.02 NS
NS NS 0.04 0.01
% DM CP NDF Fatty acids
1 Probabilities for a linear (L) or nonlinear (NL) effect of increasing the percentage of amide in the fat supplement. The NL effects were quartic for NDF and quadratic for fatty acids. Probabilities > 0.15 are noted as not significant (NS). *The 0% oleamide and control diet differed (P < 0.05).
Digestibility of DM was not affected by adding canola oil to the diet (Table 5). However, total tract digestibility of fiber was reduced (P < 0.05) by feeding canola oil. Replacing canola oil with oleamide linearly increased (P = 0.01) DM digestibility. Changes in NDF digestibility were inconsistent and led to a quartic effect of replacing canola oil with oleamide. Fiber digestibilities were highest when oleamide comprised 75% or more of the fat supplement. Fats added to ruminant diets, including dairy cows, will decrease the extent of fiber digestion depending on several factors including the amount of fat added, the degree of unsaturation of the fat source, and the nature of the basal diet (7). Depending on the combination of these factors, fat supplements have had variable effects on fiber digestibility in studies with lactating cows. Other examples of reduced fiber digestion include lower NDF digestion in the rumen of cows fed animal-vegetable fat (15) and lower total tract digestibility of NDF when cows were infused intraruminally with yellow grease (4). Similar fiber digestibilities for the control and 100 diets suggests that the oleamide was rumen-inert. The slow rate of amide hydrolysis in the rumen presumably prevented accumulation of antimicrobial fatty acids. However, higher fiber digestibility might also be attributable to the lower DMI, and perhaps slower turnover of ruminal contents when cows were fed the amide. Reduced DMI can only be a partial explanation because NDF digestibility for the 75 diet was 49% higher than the 50 diet but DMI intake of the 75 diet was only 4% lower. Holstein cows fed oleamide also had higher fiber digestibilities compared with cows fed canola oil (10). Digestibility of CP was not affected by the 0 diet but increasing the proportion of amide in the fat supplement linearly increased (P = 0.01) protein digestibility. Similar results were reported for Holsteins fed oleamide (10). Lower DMI of diets containing amide, combined with a high digestibility of amide N, may account for this effect. Amide that escapes hydrolysis by ruminal microbes is believed to undergo hydrolysis postru-
minally via action of intestinal amidases (19) or proteolytic enzymes in pancreatic fluid (3). This would cause release of the free fatty acid and ammonia, making both available for absorption or utilization by hindgut microbes. Digestibility of fatty acids was improved when amide replaced canola oil, except for the 100 diet. Fatty acid digestibility was markedly improved by feeding oleamide to Holstein cows accompanied by low fecal excretion of the amide supplement (10). Therefore, utilization of fatty acids in the form of fatty acyl amides equals or exceeds digestive efficiencies of other fat supplements in both Jerseys and Holsteins. The percentages of palmitic acid (C16:0), stearic acid (C18:0), oleic acid (cis-C18:1), and linoleic acid (C18:2) in plasma phospholipid, triglyceride, and cholesterol ester fractions are shown in Table 6. These four fatty acids accounted for 75 to 95% of the total fatty acids present in the three lipid fractions. Jersey cows fed the control diet in this study and in the study by Cook et al. (6) had percentages of these four fatty acids within 8 percentage units of each other for all plasma lipid fractions including phospholipids, triglycerides, and cholesterol esters. For instance, Cook et al. (6) reported that plasma phospholipids in Jerseys were comprised of approximately 10% C16:0, 20% C18:0, 20% C18:1, and 25% C18:2 compared with 18% C16:0, 25.6% C18:0, 11.7% cis-C18:1, and 29.9% C18:2 in this study. The only disparity was for plasma triglycerides that had higher C18:0 and lower C18:1 by 15 percentage units in this study compared with the Cook et al. (6) study. The reason for the higher triglyceride C18:0/C18:1 ratio in this study was not clear, although part of the explanation may be that only the cis isomer of C18:1 was reported. Feeding canola oil increased (P < 0.05) cis-C18:1 in plasma phospholipids, triglycerides, and cholesterol esters. This indicates some ability of dietary lipid supplements to influence plasma fatty acid composition in ruminants. The elevation of plasma cis-C18:1 was probably a combination of escape of oleic acid in the canola Journal of Dairy Science Vol. 83, No. 3, 2000
574
DELUCA AND JENKINS
oil from ruminal biohydrogenation combined with the action of tissue desaturases that convert stearic acid to oleic acid (21). As the amide replaced canola oil in the fat supplement, there were linear increases in cis-C18:1 in all plasma lipid fractions including phospholipids (P = 0.01), triglycerides (P = 0.05), and cholesterol esters (P = 0.03). The cis-C18:1 concentration increased from 14.8 to 20.3% in phospholipids, from 6.5 to 8.7% in triglycerides, and 6.6 to 9.8% in cholesterol esters as oleamide increased from 0 to 100% of the fat supplement. The increases in plasma cis-C18:1, although significant, were relatively small in magnitude, which may be attributable to low purity of the amide supplement. The 100 diet with 3.5% added amide product contained 2.4% actual amide content by HPLC analysis. This indicates 68% purity of the amide product with the primary contaminant presumably being unreacted canola fatty acids. The increases in cis-C18:1 in plasma as cows were fed increasing amide in the diet support previous results that amides of unsaturated fatty acids resist ruminal biohydrogenation (17). The ability of dietary oleamide to increase cis-C18:1 in plasma and milk (11) shows that unsaturated fatty acids can be fed to lactating Jerseys as fatty acyl amides as a way to significantly increase their delivery to body and mammary tissues. The only other fatty acid in plasma affected by the percentage of amide in the fat supplement was C18:2. As amide replaced canola oil, the percentage of C18:2 declined linearly (P = 0.01) in plasma phospholipids
and declined quadratically (P = 0.05) in plasma triglycerides. The percentage of C18:2 in plasma cholesterol esters was not affected by amide level in the fat supplement. Insulin and IGF-I concentrations for all diet and time after feeding combinations are shown in Table 7. Diet × time interactions did not occur (P > 0.15) for either insulin or IGF-I, so main effects of time and diet were included in Table 7. Diet had no effect on plasma insulin, showing that insulin concentrations in plasma were not changed by either canola oil or by replacing canola oil with oleamide. Plasma insulin concentrations also were not affected by dietary lipid in previous studies when cows were fed calcium salts of fatty acids (9) or prilled lipid (2). Staples et al. (20) reviewed data on blood or plasma insulin from 17 published studies and found eight studies where insulin was significantly depressed when cows were fed additional dietary fat. Including diet energy density as a covariate in the model eliminated diet influences on plasma insulin, suggesting to Staples et al. (20) that lower insulin was caused by improved energy status rather than a unique effect of the fat supplement. This study evaluated the effect of increasing oleic acid in plasma while holding total dietary fat and energy density constant. Plasma insulin concentrations did not change under these conditions which supports the findings by Staples et al. (20) that lowered insulin in fat feeding studies is the result of enhanced energy status. The lack of insulin response in this study might also be influenced by the DMI depression as oleamide
Table 6. The percentages of four major fatty acids in phospholipid, triglyceride, and cholesterol ester fractions isolated from plasma of Jersey cows fed combinations of canola oil and oleamide. Probabilities1
% Amide in fat supplement Control
0
25
50
75
100
SEM
L
NL
% of total fatty acids Phospholipid C16:0 C18:0 cis-C18:1 C18:2 Triglyceride C16:0 C18:0 cis-C18:1 C18:2 Cholesterol ester C16:0 C18:0 cis-C18:1 C18:2
18.0 25.6 11.7 29.9
16.5 28.3* 14.8* 30.6
17.4 28.5 15.6 30.1
16.7 28.3 16.8 28.4
17.8 28.5 18.2 26.2
17.2 27.7 20.3 25.5
0.8 1.0 0.6 0.7
NS NS 0.01 0.01
NS NS NS NS
23.3 45.0 5.1 1.0
18.7* 51.0* 6.5* 2.5
20.6 57.2 7.1 0.9
21.2 55.0 7.9 0.2
21.5 54.2 8.9 0.6
20.7 53.4 8.7 1.2
1.4 3.0 1.0 0.7
NS NS 0.05 NS
NS NS NS 0.05
7.2 0.5 6.6 53.2
6.9 3.9 7.9* 56.5
6.4 1.0 7.7 57.1
6.3 1.7 8.0 61.6
7.4 3.0 9.2 55.6
7.1 4.5 9.8 60.2
0.7 2.1 0.7 3.4
NS NS 0.03 NS
NS NS NS NS
1 Probabilities for a linear (L) or nonlinear (NL) effect of increasing the percentage of amide in the fat supplement. All NL effects were quadratic. Probabilities > 0.15 are noted as not significant (NS). *The 0% oleamide and control diets differed (P < 0.05).
Journal of Dairy Science Vol. 83, No. 3, 2000
575
NUTRIENT DIGESTION OF JERSEY COWS FED OLEAMIDE
Table 7. Insulin and IGF-I concentrations in plasma taken hourly for 4 h after feeding from Jersey cows fed combinations of canola oil and oleamide. % Amide in fat supplement Control
0
25
50
75
100
Time meansa
IGF-I, ng/mL 1h 2h 3h 4h Diet meansb
47.3 54.3 52.7 46.3 50.2 ± 4.4
58.7 55.0 51.7 48.7 53.5 ± 4.4
54.3 49.3 50.0 43.7 49.3 ± 4.4
59.3 58.7 52.7 52.7 55.8 ± 4.4
48.0 46.3 48.3 47.0 47.4 ± 4.4
55.7 52.3 51.0 47.3 51.6 ± 4.4
53.9 52.7 51.1 47.6
± ± ± ±
1.2 1.2 1.2 1.2
Insulin, µIU/ml 1h 2h 3h 4h Diet effect
6.7 10.2 12.6 9.4 9.7 ± 0.6
6.4 8.1 10.9 9.3 8.7 ± 0.6
5.3 7.5 10.9 9.3 8.3 ± 0.6
6.7 10.0 9.2 8.8 8.7 ± 0.6
6.2 8.7 9.0 8.3 8.0 ± 0.6
5.9 9.4 11.8 9.9 9.3 ± 0.6
6.2 9.0 10.7 9.2
± ± ± ±
0.4a 0.4 0.4 0.4
Main effects of time after feeding were different (P < 0.01) for both insulin and IGF-I. Main effect of diet was not different for either insulin or IGF-I.
a b
increased in the ration, or by increasing oleic acid in plasma rather than increasing other unsaturated fatty acids. Feeding calcium salts of palm oil fatty acids reduced insulin in a previous study (5) even though the fat supplement reduced DMI and contained mostly monounsaturated fatty acid. Insulin concentration in plasma increased (P < 0.01) with time after feeding until reaching a peak at 3 h post-feeding. In a study by Phuntsok et al. (16) where plasma samples were collected at 30-min intervals from 0.5 h before to 4 h after feeding, insulin in plasma of cows increased 1 to 2 h after feeding and remained at basal levels at all other sampling times. Results for IGF-I were similar to insulin. Replacing canola oil with the amide had no effect on plasma concentrations of IGF-I in the cows. However, as was the case with insulin, IGF-I changed with time after feeding. Plasma IGF-I declined (P < 0.01) from 53.9 to 47.6 ng/ml through 4 h postfeeding. The greatest numerical decline in IGF-I occurred from 3 to 4 h postprandial. CONCLUSIONS Oleamide fed to lactating Jersey cows in this study was highly digestible and had no adverse effects on nutrient digestibility, similar to results reported previously for Holsteins (10). Despite a decline in dry matter intake with feeding the amide, milk yield was maintained during this short-term study. Increasing oleamide at the expense of canola oil in the fat supplement increased the concentration of oleic acid in milk and plasma lipids. However, this study showed that higher oleic acid concentration in plasma lipids and maintaining constant added fat and energy density in the ration has no effect on circulating concentrations of insulin or IGF-I.
ACKNOWLEDGMENTS Thanks is extended to Cargill Oilseeds Division (Minneapolis, MN) for their donation of the canola oil, and to Church & Dwight, Inc. (Princeton, NJ) for partial financial support of this project. We also thank Evanne Thies for assistance with laboratory analysis, and Jim Hampton for analysis of plasma hormones. REFERENCES 1 Association of Official Analytical Chemists. 1990. Official Methods of Analysis Vol. I. 15th ed. AOAC, Arlington, VA. 2 Beam, S. W., and W. R. Butler. 1998. Energy balance, metabolic hormones, and early postpartum follicular development in dairy cows fed prilled lipid. J. Dairy Sci. 81:121–131. 3 Buttery, P. J., S. Manomai-Udom, and D. Lewis. 1977. Preliminary investigations on some potential sources of protected methionine derivatives for ruminant rations. J. Sci. Food Agric. 28:481–485. 4 Cant, J. P., E. J. DePeters, and R. L. Baldwin. 1991. Effect of dietary fat and postruminal casein administration on milk composition of lactating dairy cows. J. Dairy Sci. 74:211–219. 5 Choi, B., and D. L. Palmquist. 1996. High fat diets increase plasma cholecystokinin and pancreatic polypeptide, and decrease plasma insulin and feed intake in lactating cows. J. Nutr. 126:2913. 6 Cook, L. J., T. W. Scott, and Y. S. Pan. 1972. Formaldehydetreated casein-safflower oil supplement for dairy cows. II. Effect on the fatty-acid composition of plasma and milk lipids. J. Dairy Res. 39:211–218. 7 Doreau, M., and Y. Chilliard. 1997. Digestion and metabolism of dietary fat in farm animals. Br. J. Nutr. 78, Suppl. 1, S15–S35. 8 Fenton, T. W., and M. Fenton. 1979. An improved method for the determination of chromic oxide in feed and feces. Can. J. Anim. Sci. 59:631–634. 9 Garcia-Bojalil, C. M., C. R. Staples, C. A. Risco, J. D. Savio, and W. W. Thatcher. 1998. Protein degradability and calcium salts of long-chain fatty acids in the diets of lactating dairy cows: productive responses. J. Dairy Sci. 81:1374–1384. 10 Jenkins, T. C. 1998. Fatty acid composition of milk from Holstein cows fed oleamide or canola oil. J. Dairy Sci. 81:794–800. 11 Jenkins, T. C. 2000. Feeding oleamide to lactating Jersey cows. 1. Effects on lactation performance and milk fatty acid composition. J. Dairy Sci. 83:332–337. Journal of Dairy Science Vol. 83, No. 3, 2000
576
DELUCA AND JENKINS
12 Jenkins, T. C., and E. Thies. 1997. Plasma fatty acids in sheep fed hydroxyethylsoyamide, a fatty acyl amide that resists biohydrogenation. Lipids 32:173. 13 Lee, C. Y., and D. M. Henricks. 1990. Comparisons of various acidic treatments of bovine serum on IGF-I immunoreactivity and binding activity. J. Endocrinol. 127:139–148. 14 Opara, E. C., M. Garfinkel, V. S. Hubbard, W. M. Burch, and O. E. Akwari. 1994. Effects of fatty acids on insulin release: role of chain length and degree of unsaturation. Am. J. Physiol. 266:E635–E639. 15 Pantoja, J., J. L. Firkins, M. L. Eastridge, and B. L. Hull. 1996. Fatty acid digestion in lactating dairy cows fed fats varying in degree of saturation and different fiber sources. J. Dairy Sci. 79:575–584. 16 Phuntsok, T., M. A. Froetschel, H. E. Amos, M. Zheng, and Y. W. Huang. 1998. Biogenic amines in silage, apparent postruminal passage, and the relationship between biogenic amines and digestive function and intake by steers. J. Dairy Sci. 81:2193–2203.
Journal of Dairy Science Vol. 83, No. 3, 2000
17 Reeves, L. M., M. L. Williams, and T. C. Jenkins. 1998. In vitro biohydrogenation of oleamide and total tract digestibility of oleamide by sheep. J. Sci. Food Agric. 77:187–192. 18 SAS System for Windows. Release 6.12. 1996. SAS Inst., Inc., Cary, NC. 19 Schmid, H.H.O., P. C. Schmid, and V. Natarajan. 1990. N-Acylated glycerophospholipids and their derivatives. Prog. Lipid Res. 29:1–43. 20 Staples, C. R., J. M. Butrke, and W. W. Thatcher. 1998. Influence of supplemental fats on reproductive tissues and performance of lactating cows. J. Dairy Sci. 81:856–871. 21 St. John, L. C., D. K. Lunt, and S. B. Smith. 1991. Fatty acid elongation and desaturation enzyme activities of bovine liver and subcutaneous adipose tissue microsomes. J. Anim. Sci. 69:1064–1073. 22 Van Soest, P. J., J. B. Robertson, and B. A. Lewis. 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74:3583– 3597.
ABSTRACT
INTRODUCTION
Six lactating Jersey cows were used in a 6 × 6 Latin square with 14-d periods to evaluate different ratios of canola oil and oleamide on nutrient digestibility, plasma fatty acids, and plasma hormones. The control diet contained no added fat. All other diets contained 3.5% added fat consisting of 0, 25, 50, 75, and 100% as oleamide and the remainder as canola oil. Data were collected during the final 4 d of each period. Dry matter intake was reduced by the addition of canola oil to the diet, and further reduced by replacing canola oil with oleamide. Milk yield was not affected by diet but increasing oleamide proportion in the fat supplement caused linear increases in cis-C18:1 and linear decreases in C4 to C16 fatty acids in milk. Adding canola oil reduced total tract digestibilities of fiber and fatty acids, but had no effect on the digestibilities of dry matter or protein. Replacing canola oil with oleamide increased protein digestibility linearly, and increased digestibility of fiber (quartic relationship) and fatty acids (quadratic relationship). Oleic acid concentration in plasma increased by adding canola oil to the diet, and was further increased by replacing canola oil with oleamide. Diet had no effect on plasma concentrations of insulin or IGF-I. Oleamide fed to Jersey cows in this study was highly digestible and had no deleterious effects on total tract digestibility of fiber or protein. Increasing oleic acid concentration in plasma lipids while maintaining a constant level of added fat in the ration had no effect on circulating concentrations of insulin or IGF-I in Jerseys. (Key words: oleamide, Jerseys, digestion, plasma hormones)
Oleamide reduces the rate of biohydrogenation of oleic acid by ruminal microbes (17) and increases oleic acid content of milk when fed to lactating Holstein cows (10). The oleamide was highly digestible and had no adverse effects on the digestibilities of fiber or protein when fed to Holsteins (10). Similar to Holsteins, oleamide fed to lactating Jerseys (11) also substantially elevated oleic acid content of milk. However, increases in milk oleic acid concentration were lower for Jerseys than for Holsteins. Adding a commercial oleamide to the diet of Jerseys increased milk oleic acid 72% (11), compared with a 108% increase for Holsteins (10). Additional fat fed to lactating dairy cows has had variable effects on plasma hormones. Blood insulin concentrations were lower for cows fed added fat in 8 out of 17 studies summarized by Staples et al. (20). Some of this variability may result from ruminal biohydrogenation and the inconsistent delivery of unsaturated fatty acids to the small intestine (20). A positive association between unsaturated fatty acids and insulin release was reported in nonruminants (14). Based on the increase in milk oleic acid concentration reported in the previous study (11), oleamide would be expected to increase unsaturated fatty acids in blood, which may have effects on circulating insulin levels. This study had two objectives. The first was to determine if the digestibility of fatty acids and other nutrients in Jerseys fed oleamide were as complete as reported previously for Holsteins. A second objective was to determine if the concentrations of plasma insulin and IGF-I were altered when unsaturated fatty acids were elevated in plasma without a concomitant increase in total dietary fat.
Received June 21, 1999. Accepted October 29, 1999. Corresponding author: T. C. Jenkins; e-mail: [email protected]. 1 Technical Contribution Number 4504 of the South Carolina Agricultural Experiment Station, Clemson University. Additional financial support was provided by Church & Dwight, Inc., Princeton, NJ. 2 Present address: Purina Mills, Inc., P.O. Box 46, Montgomery City, MO 63361. 3 Department of Animal & Veterinary Sciences, 151 Poole Agricultural Center, Clemson University, Clemson, SC. 2000 J Dairy Sci 83:569–576
MATERIALS AND METHODS Cows and Diets Six lactating Jersey cows (four multiparous and two primiparous) were fed six diets in a 6 × 6 Latin square with 14-d periods. At the start of the study, DIM for all cows averaged 60 d (43, 54, 63, 64, 67, and 68 d). Cows were housed individually in tie stalls and bedded on wood shavings. Water was available at all times.
569
570
DELUCA AND JENKINS Table 1. Chemical composition of the total mixed diets. % Amide in fat supplement Item
Control
0
25
NDF CP Fatty acids Amide
36.3 17.0 2.8
35.0 16.5 5.9
35.2 16.1 5.8 0.5
50
75
100
34.5 16.5 6.1 1.2
34.5 16.5 6.1 1.7
35.0 16.8 6.0 2.4
% of DM
Cows were fed twice per day (0800 and 1600 h) and had ad libitum access to a TMR of 47% corn silage and 53% concentrate (DM basis). Ingredients comprising the TMR were described previously (11). Chemical composition of the TMR is shown in Table 1. Feed refusals (orts) were collected and weighed each day prior to the a.m. feeding. Cows were milked daily at 1000 and 2200 h. The control diet contained no added fat and the other five diets contained 3.5% added fat consisting of blends of canola oil (Cargill Oilseeds Division, Minneapolis, MN) and oleamide (Table 2). Oleamide comprised 0, 25, 50, 75, 100% of the added fat portion and the remainder was canola oil. Oleamide was synthesized from canola fatty acids (EMERY 790, Henkel Corporation, Cincinnati, OH) as described previously (11). Fatty acid composition of the canola fatty acids as reported by the manufacturer was 5% C16:0, 2% C18:0, 59% cis-C18:1, 22% C18:2, 10% C18:3, 1% C20:0, and 1% C22:0. Sample Collection and Analysis Ort samples for each diet were collected during the last 4 d of each period. Fecal grab samples were collected the last 3 d of each period (0800 and 2300 h on d 12; 1100, 1700, and 0200 h on d 13; and 1400, 2000,
Table 2. Fatty acid composition of the canola oil and oleamide supplements. Canola oil1
Oleamide2
% of total fatty acids C16:0 C18:0 cis-C18:1 C18:2 C18:3 Other3
4.4 2.2 61.8 21.1 7.4 3.1
5.3 2.7 62.6 22.1 6.4 0.3
1 As reported by the supplier (Cargill Oilseeds Division, Minneapolis, MN). Additional reported specifications of the oil were 0.05% FFA maximum as oleic acid, 1.00 meq/kg maximum peroxide value, and no additives. 2 Determined by GLC. 3 One or more of the following fatty acids were present at ≤ 2% of total fatty acids; trans-C18:1, C20:0, and C20:1.
Journal of Dairy Science Vol. 83, No. 3, 2000
and 0500 h on d 14) in a time sequence that gave a sample every 3 h over a 24-h period. Chromic oxide (10 g) in gelatin capsules was orally administered the last 10 d of each period prior to each feeding. Chromic oxide was used as an indigestible marker to determine nutrient digestibility. Silage, ort, and fecal samples were dried at 55°C and, along with samples of the concentrate, were ground in a Wiley mill through a 2-mm sieve. Ground samples were analyzed for DM (100°C), Kjeldahl N (1), and NDF (22, procedure A). Fatty acids in samples were methylated in 5% methanolic HCl for 15 h at 80°C. The higher temperature and longer incubation time than normally used were required to ensure complete hydrolysis of the amide bond (10) but had no effect on recovery of a fatty acid standard (12). Fatty acid methyl esters were separated by GLC (11). Fecal samples were analyzed for Cr (8). Milk samples were collected the last eight milkings (4 d) of each period and composited daily in proportion to milk yield. Milk samples (preserved in bronopol) were analyzed for fat and protein concentrations by mid infrared spectroscopy (Southeast DHIA laboratory, McDonough, GA). Milk fatty acid composition was determined by GLC (11). Blood samples were collected hourly for 4 h after the morning feeding on d 14 of each period. Blood was taken by jugular venipuncture into tubes containing EDTA as an anticoagulant. Tubes were centrifuged for 20 min at 1500 × g (5°C). Plasma was removed and frozen until later analyzed for insulin (Coat-A-Count Insulin kits, Diagnostic Products Corporation, Los Angeles, CA) and IGF-I (13). Blood taken at 3-h postprandial on d 14 of each period also was analyzed for fatty acid composition of various lipid fractions. Lipids were extracted from plasma with chloroform/methanol (2:1, vol/vol). Triglycerides, phospholipids, and cholesterol esters in the plasma extract were separated by TLC on 20- × 20-cm preparative plates coated with silica gel. The mobile phase consisted of hexane/diethyl ether/formic acid (16:4:1, by vol). Samples were scraped into tubes and analyzed for fatty acids by GLC. Samples were methylated in 5% methanolic HCl as described previously for feed and fecal
NUTRIENT DIGESTION OF JERSEY COWS FED OLEAMIDE
samples. Methyl esters of fatty acids were separated by GLC on a 30-m × 0.25-mm i.d. fused silica capillary column with a 0.25-µm film thickness (Supelco, Bellefonte, PA). The injector and detector temperatures were maintained at 250°C. Initial column temperature was 150°C (held for 2 min) and programmed to increase 2°C/min to a final temperature of 220°C. Amide Analysis Feed and fecal samples were analyzed for amide content by HPLC. A 10-ml extraction mixture (chloroform/ methanol, 1:1 vol/vol) was added to 1 g of dried sample and placed on a wrist-action shaker for 1 h. Samples were centrifuged for 15 min at 500 × g and the supernatant was transferred to another 50-ml tube. This procedure was repeated with another 5 ml of extraction mixture added to the pellet and placed on the shaker for an additional 30 min. Distilled water (4.5 ml) was added to the supernatant, mixed, and centrifuged at 500 × g for 5 min to separate the top layer (aqueous/methanol) from the chloroform layer which contained the amide. A 2-ml sample of the chloroform extract was loaded onto a preconditioned (5 ml of chloroform) silica extraction column and washed through the column with 20 ml of chloroform into a 25-ml volumetric flask. Amide content was determined by HPLC and quantified using stearamide as an external standard (10). Statistics All data, except for plasma hormones, were analyzed as a 6 × 6 Latin square design by ANOVA by the general linear models procedure of SAS (18). Diet, period, and cow were included in the model as sources of variation. The effect of adding canola oil to the diet was determined by a linear contrast that compared the control diet to the 0% oleamide diet. The effect of replacing canola oil with oleamide was determined by orthogonal polynomials among the equally spaced levels of oleamide that included linear or nonlinear (quadratic, cubic, and quartic) components. Data for plasma insulin and IGF-I were analyzed as a split plot in time ANOVA using the general linear models procedure of SAS (18). Cow, period, and diet were whole-plot factors tested against the main plot error term (interaction of cow, period, and diet). Time and all interactions with time were included in the model as sub-plot error terms tested against residual error. The diet × time interaction was not significant (P > 0.15) for either insulin or IGF-I. RESULTS AND DISCUSSION Adding canola oil to the diet reduced (P < 0.05) DMI of the cows from 15.7 to 14.6 kg/d (Table 3). Replacing
571
canola oil with oleamide further reduced (P < 0.01) DMI in a linear fashion. Depressed DMI from feeding fatty acyl amides was reported for Jerseys (11), and also for Holsteins (10). Despite the decline in DMI, milk yield was not reduced in this study. Oleamide fed to Holsteins similarly had no effect on milk yield (10), but feeding oleamide to lactating Jerseys (11) reduced milk yield 2.4 kg/d compared with feeding the same quantity of canola oil. Milk fat percentage was lower (P < 0.05) for the 0 diet compared with the control diet. The depressed milk fat percentage was maintained as oleamide replaced canola oil in the fat supplement. In Holsteins, milk fat was not affected by feeding oleamide but was reduced by canola oil (10). In the previous Jersey study (11), milk fat percentage tended to be reduced by the oleamide supplement, which was suggested as possibly being due to the 5 to 6% trans-C18:1 in the oleamide fatty acids. The oleamide supplement in this study contained lower (<2%) transC18:1 but still depressed milk fat compared to the control diet. Alternate explanations to account for the depressed milk fat percentage include inhibition of ruminal fermentation by either the amide or by the unreacted fatty acids contaminating the amide supplement. The amides fed to Jersey cows in this study and the previous study (11) were not identical, and differed mainly in their fatty acid compositions. Although the amides were synthesized in both studies by the same method, the source of fatty acid in the previous study was technical grade oleic acid (76% C18:1 and 0.5% C18:2) while canola fatty acids (59% C18:1 and 22% C18:2) were used in the present investigation. Milk fat yield (kg/d) was lower (P < 0.05) for the 0 diet than for the control diet with no effect of replacing canola oil with amide. Yield of 3.5% FCM also was reduced (P < 0.05) by the 0 diet, which declined (P = 0.06) quadratically as the amide replaced canola oil. Most of the decline in FCM occurred for the 100 diet. Results were similar for energy-corrected milk, which was not affected by adding canola oil but was reduced (P = 0.12) quadratically as amide increased in the fat supplement. Diet had no effect on energy-corrected milk when expressed per kilogram of DMI. The 0 diet reduced (P < 0.05) milk protein percentage, but no further change in protein concentration occurred as amide replaced canola oil in the fat supplement. Opposite results occurred in the previous Jersey study (11) in which the amide but not canola oil reduced milk protein percentage. Yield of milk protein (kg/d) was not affected by canola oil or any proportion of amide in the fat supplement. The effects of feeding canola oil and oleamide to lactating cows on milk fatty acid composition (Table 4) Journal of Dairy Science Vol. 83, No. 3, 2000
572
DELUCA AND JENKINS Table 3. Dry matter intake, milk yield and milk composition for Jersey cows fed combinations of canola oil and oleamide. Probabilities1
% Amide in fat supplement
DMI, kg/d Milk, kg/d 3.5% FCM, kg/d ECM, kg/d Fat, % Fat, kg/d Protein, % Protein, kg/d ECM/DMI, kg
Control
0
25
50
75
100
SEM
L
NL
15.7 21.0 25.0 24.9 4.66 0.98 3.60 0.76 0.77
14.6* 21.6 23.8* 23.8 4.15* 0.90* 3.49* 0.75 0.77
13.9 22.2 24.3 24.2 4.12 0.91 3.42 0.75 0.83
13.9 21.5 24.0 23.8 4.25 0.92 3.40 0.73 0.79
13.3 21.4 23.2 23.1 4.04 0.86 3.37 0.72 0.82
13.4 20.7 21.9 22.0 3.84 0.80 3.40 0.71 0.82
0.34 0.49 0.6 0.5 0.13 0.03 0.06 0.02 0.03
0.01 NS 0.01 <0.01 NS 0.01 NS NS NS
NS NS 0.06 0.12 NS 0.06 NS NS NS
1 Probabilities for a linear (L) or nonlinear (NL) effect of increasing the percentage of amide in the fat supplement. The NL effects were quadratic for FCM, ECM, and fat yield. Probabilities > 0.15 are noted as not significant (NS). *The 0% oleamide and control diets differed (P < 0.05).
were similar to the results reported in the previous Jersey study (11). Milk cis-C18:1 concentration in this study increased (P < 0.05) from 19.8 to 23.6% of total fatty acids when canola oil was added to the diet. As oleamide replaced canola oil in the fat supplement, cisC18:1 in milk linearly increased (P < 0.01) from 23.6 to 28.4% of total fatty acids. The synthesized oleamide in the previous Jersey study (11) increased cis-C18:1 22% over the canola oil diet, compared with a 20% increase in this study. The previous study with Jersey cows (11) showed that oleamide increased milk cis-C18:1 concentration more (36%) when the oleamide was a commercial source of higher purity.
The effects of increasing oleamide in the fat supplement on other fatty acids in milk also were similar to oleamide effects on milk fatty acids in the previous lactation study (11). Increasing oleamide concentration in the fat supplement linearly reduced (P < 0.04) the concentration of all C4 to C16 milk fatty acids, increased (P = 0.08) milk C18:0 up to 75% oleamide in the fat supplement, but had no effects on the concentrations of trans-C18:1, C18:2, or cis-9, trans-11 C18:2. Increasing oleamide proportions in the fat supplement reduced (P = 0.09) milk C18:3 concentration in a quadratic fashion, although its concentration never exceeded 0.4% of total milk fatty acids.
Table 4. Milk fatty acid composition for Jersey cows fed combinations of canola oil and oleamide. Probabilities1
% Amide in fat supplement
C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C14:1 C16:0 C16:1 C17:0 C18:0 cis-C18:1 trans-C18:1 C18:2 C18:3 CLA2
Control
0
25
50
75
2.70 2.77 1.68 3.69 3.97 12.21 1.38 34.51 1.61 0.51 10.72 19.83 1.53 2.32 0.35 0.21
2.73 2.53 1.48 3.15* 3.28* 10.88* 1.19* 28.29* 1.35 0.47 15.18* 23.59* 2.94* 2.22 0.39* 0.34*
% of total fatty acids 2.84 2.72 2.43 2.58 2.49 2.16 1.45 1.43 1.24 2.91 3.01 2.63 2.98 3.07 2.77 10.12 10.03 9.35 1.10 1.10 1.01 26.91 25.82 26.21 1.44 1.36 1.48 0.48 0.49 0.50 15.24 15.72 16.17 25.08 25.84 28.41 3.94 3.89 2.81 2.18 2.26 2.16 0.37 0.36 0.35 0.38 0.40 0.32
100
SEM
L
NL
2.56 2.31 1.30 2.71 2.83 9.65 1.10 25.63 1.52 0.50 14.65 28.35 3.94 2.19 0.36 0.38
0.09 0.11 0.07 0.14 0.15 0.34 0.04 0.63 0.10 0.01 0.46 0.99 0.36 0.07 0.01 0.03
0.02 0.02 0.02 0.02 0.03 <0.01 0.04 <0.01 NS 0.09 NS <0.01 NS NS 0.03 NS
NS NS NS NS NS NS 0.09 NS NS NS 0.08 NS NS NS 0.09 NS
1 Probabilities for a linear (L) or nonlinear (NL) effect of increasing the percentage of amide in the fat supplement. The NL effects were quadratic for C14:1, C18:0, and C18:3 concentrations in milk. Probabilities > 0.15 are noted as not significant (NS). 2 Conjugated linoleic acid. *The 0% oleamide and control diet differed (P < 0.05).
Journal of Dairy Science Vol. 83, No. 3, 2000
573
NUTRIENT DIGESTION OF JERSEY COWS FED OLEAMIDE Table 5. Apparent nutrient digestibilities in Jersey cows fed combinations of canola oil and oleamide. Probabilities1
% Amide in fat supplement Control
0
25
58.8 59.4 37.6 79.1
56.1 61.3 28.8* 72.1*
56.7 60.3 28.5 77.1
50
75
100
SEM
L
NL
56.9 63.4 25.5 76.3
61.4 66.0 38.0 77.5
60.0 65.5 35.1 72.2
1.4 1.4 2.6 1.8
0.01 0.01 0.02 NS
NS NS 0.04 0.01
% DM CP NDF Fatty acids
1 Probabilities for a linear (L) or nonlinear (NL) effect of increasing the percentage of amide in the fat supplement. The NL effects were quartic for NDF and quadratic for fatty acids. Probabilities > 0.15 are noted as not significant (NS). *The 0% oleamide and control diet differed (P < 0.05).
Digestibility of DM was not affected by adding canola oil to the diet (Table 5). However, total tract digestibility of fiber was reduced (P < 0.05) by feeding canola oil. Replacing canola oil with oleamide linearly increased (P = 0.01) DM digestibility. Changes in NDF digestibility were inconsistent and led to a quartic effect of replacing canola oil with oleamide. Fiber digestibilities were highest when oleamide comprised 75% or more of the fat supplement. Fats added to ruminant diets, including dairy cows, will decrease the extent of fiber digestion depending on several factors including the amount of fat added, the degree of unsaturation of the fat source, and the nature of the basal diet (7). Depending on the combination of these factors, fat supplements have had variable effects on fiber digestibility in studies with lactating cows. Other examples of reduced fiber digestion include lower NDF digestion in the rumen of cows fed animal-vegetable fat (15) and lower total tract digestibility of NDF when cows were infused intraruminally with yellow grease (4). Similar fiber digestibilities for the control and 100 diets suggests that the oleamide was rumen-inert. The slow rate of amide hydrolysis in the rumen presumably prevented accumulation of antimicrobial fatty acids. However, higher fiber digestibility might also be attributable to the lower DMI, and perhaps slower turnover of ruminal contents when cows were fed the amide. Reduced DMI can only be a partial explanation because NDF digestibility for the 75 diet was 49% higher than the 50 diet but DMI intake of the 75 diet was only 4% lower. Holstein cows fed oleamide also had higher fiber digestibilities compared with cows fed canola oil (10). Digestibility of CP was not affected by the 0 diet but increasing the proportion of amide in the fat supplement linearly increased (P = 0.01) protein digestibility. Similar results were reported for Holsteins fed oleamide (10). Lower DMI of diets containing amide, combined with a high digestibility of amide N, may account for this effect. Amide that escapes hydrolysis by ruminal microbes is believed to undergo hydrolysis postru-
minally via action of intestinal amidases (19) or proteolytic enzymes in pancreatic fluid (3). This would cause release of the free fatty acid and ammonia, making both available for absorption or utilization by hindgut microbes. Digestibility of fatty acids was improved when amide replaced canola oil, except for the 100 diet. Fatty acid digestibility was markedly improved by feeding oleamide to Holstein cows accompanied by low fecal excretion of the amide supplement (10). Therefore, utilization of fatty acids in the form of fatty acyl amides equals or exceeds digestive efficiencies of other fat supplements in both Jerseys and Holsteins. The percentages of palmitic acid (C16:0), stearic acid (C18:0), oleic acid (cis-C18:1), and linoleic acid (C18:2) in plasma phospholipid, triglyceride, and cholesterol ester fractions are shown in Table 6. These four fatty acids accounted for 75 to 95% of the total fatty acids present in the three lipid fractions. Jersey cows fed the control diet in this study and in the study by Cook et al. (6) had percentages of these four fatty acids within 8 percentage units of each other for all plasma lipid fractions including phospholipids, triglycerides, and cholesterol esters. For instance, Cook et al. (6) reported that plasma phospholipids in Jerseys were comprised of approximately 10% C16:0, 20% C18:0, 20% C18:1, and 25% C18:2 compared with 18% C16:0, 25.6% C18:0, 11.7% cis-C18:1, and 29.9% C18:2 in this study. The only disparity was for plasma triglycerides that had higher C18:0 and lower C18:1 by 15 percentage units in this study compared with the Cook et al. (6) study. The reason for the higher triglyceride C18:0/C18:1 ratio in this study was not clear, although part of the explanation may be that only the cis isomer of C18:1 was reported. Feeding canola oil increased (P < 0.05) cis-C18:1 in plasma phospholipids, triglycerides, and cholesterol esters. This indicates some ability of dietary lipid supplements to influence plasma fatty acid composition in ruminants. The elevation of plasma cis-C18:1 was probably a combination of escape of oleic acid in the canola Journal of Dairy Science Vol. 83, No. 3, 2000
574
DELUCA AND JENKINS
oil from ruminal biohydrogenation combined with the action of tissue desaturases that convert stearic acid to oleic acid (21). As the amide replaced canola oil in the fat supplement, there were linear increases in cis-C18:1 in all plasma lipid fractions including phospholipids (P = 0.01), triglycerides (P = 0.05), and cholesterol esters (P = 0.03). The cis-C18:1 concentration increased from 14.8 to 20.3% in phospholipids, from 6.5 to 8.7% in triglycerides, and 6.6 to 9.8% in cholesterol esters as oleamide increased from 0 to 100% of the fat supplement. The increases in plasma cis-C18:1, although significant, were relatively small in magnitude, which may be attributable to low purity of the amide supplement. The 100 diet with 3.5% added amide product contained 2.4% actual amide content by HPLC analysis. This indicates 68% purity of the amide product with the primary contaminant presumably being unreacted canola fatty acids. The increases in cis-C18:1 in plasma as cows were fed increasing amide in the diet support previous results that amides of unsaturated fatty acids resist ruminal biohydrogenation (17). The ability of dietary oleamide to increase cis-C18:1 in plasma and milk (11) shows that unsaturated fatty acids can be fed to lactating Jerseys as fatty acyl amides as a way to significantly increase their delivery to body and mammary tissues. The only other fatty acid in plasma affected by the percentage of amide in the fat supplement was C18:2. As amide replaced canola oil, the percentage of C18:2 declined linearly (P = 0.01) in plasma phospholipids
and declined quadratically (P = 0.05) in plasma triglycerides. The percentage of C18:2 in plasma cholesterol esters was not affected by amide level in the fat supplement. Insulin and IGF-I concentrations for all diet and time after feeding combinations are shown in Table 7. Diet × time interactions did not occur (P > 0.15) for either insulin or IGF-I, so main effects of time and diet were included in Table 7. Diet had no effect on plasma insulin, showing that insulin concentrations in plasma were not changed by either canola oil or by replacing canola oil with oleamide. Plasma insulin concentrations also were not affected by dietary lipid in previous studies when cows were fed calcium salts of fatty acids (9) or prilled lipid (2). Staples et al. (20) reviewed data on blood or plasma insulin from 17 published studies and found eight studies where insulin was significantly depressed when cows were fed additional dietary fat. Including diet energy density as a covariate in the model eliminated diet influences on plasma insulin, suggesting to Staples et al. (20) that lower insulin was caused by improved energy status rather than a unique effect of the fat supplement. This study evaluated the effect of increasing oleic acid in plasma while holding total dietary fat and energy density constant. Plasma insulin concentrations did not change under these conditions which supports the findings by Staples et al. (20) that lowered insulin in fat feeding studies is the result of enhanced energy status. The lack of insulin response in this study might also be influenced by the DMI depression as oleamide
Table 6. The percentages of four major fatty acids in phospholipid, triglyceride, and cholesterol ester fractions isolated from plasma of Jersey cows fed combinations of canola oil and oleamide. Probabilities1
% Amide in fat supplement Control
0
25
50
75
100
SEM
L
NL
% of total fatty acids Phospholipid C16:0 C18:0 cis-C18:1 C18:2 Triglyceride C16:0 C18:0 cis-C18:1 C18:2 Cholesterol ester C16:0 C18:0 cis-C18:1 C18:2
18.0 25.6 11.7 29.9
16.5 28.3* 14.8* 30.6
17.4 28.5 15.6 30.1
16.7 28.3 16.8 28.4
17.8 28.5 18.2 26.2
17.2 27.7 20.3 25.5
0.8 1.0 0.6 0.7
NS NS 0.01 0.01
NS NS NS NS
23.3 45.0 5.1 1.0
18.7* 51.0* 6.5* 2.5
20.6 57.2 7.1 0.9
21.2 55.0 7.9 0.2
21.5 54.2 8.9 0.6
20.7 53.4 8.7 1.2
1.4 3.0 1.0 0.7
NS NS 0.05 NS
NS NS NS 0.05
7.2 0.5 6.6 53.2
6.9 3.9 7.9* 56.5
6.4 1.0 7.7 57.1
6.3 1.7 8.0 61.6
7.4 3.0 9.2 55.6
7.1 4.5 9.8 60.2
0.7 2.1 0.7 3.4
NS NS 0.03 NS
NS NS NS NS
1 Probabilities for a linear (L) or nonlinear (NL) effect of increasing the percentage of amide in the fat supplement. All NL effects were quadratic. Probabilities > 0.15 are noted as not significant (NS). *The 0% oleamide and control diets differed (P < 0.05).
Journal of Dairy Science Vol. 83, No. 3, 2000
575
NUTRIENT DIGESTION OF JERSEY COWS FED OLEAMIDE
Table 7. Insulin and IGF-I concentrations in plasma taken hourly for 4 h after feeding from Jersey cows fed combinations of canola oil and oleamide. % Amide in fat supplement Control
0
25
50
75
100
Time meansa
IGF-I, ng/mL 1h 2h 3h 4h Diet meansb
47.3 54.3 52.7 46.3 50.2 ± 4.4
58.7 55.0 51.7 48.7 53.5 ± 4.4
54.3 49.3 50.0 43.7 49.3 ± 4.4
59.3 58.7 52.7 52.7 55.8 ± 4.4
48.0 46.3 48.3 47.0 47.4 ± 4.4
55.7 52.3 51.0 47.3 51.6 ± 4.4
53.9 52.7 51.1 47.6
± ± ± ±
1.2 1.2 1.2 1.2
Insulin, µIU/ml 1h 2h 3h 4h Diet effect
6.7 10.2 12.6 9.4 9.7 ± 0.6
6.4 8.1 10.9 9.3 8.7 ± 0.6
5.3 7.5 10.9 9.3 8.3 ± 0.6
6.7 10.0 9.2 8.8 8.7 ± 0.6
6.2 8.7 9.0 8.3 8.0 ± 0.6
5.9 9.4 11.8 9.9 9.3 ± 0.6
6.2 9.0 10.7 9.2
± ± ± ±
0.4a 0.4 0.4 0.4
Main effects of time after feeding were different (P < 0.01) for both insulin and IGF-I. Main effect of diet was not different for either insulin or IGF-I.
a b
increased in the ration, or by increasing oleic acid in plasma rather than increasing other unsaturated fatty acids. Feeding calcium salts of palm oil fatty acids reduced insulin in a previous study (5) even though the fat supplement reduced DMI and contained mostly monounsaturated fatty acid. Insulin concentration in plasma increased (P < 0.01) with time after feeding until reaching a peak at 3 h post-feeding. In a study by Phuntsok et al. (16) where plasma samples were collected at 30-min intervals from 0.5 h before to 4 h after feeding, insulin in plasma of cows increased 1 to 2 h after feeding and remained at basal levels at all other sampling times. Results for IGF-I were similar to insulin. Replacing canola oil with the amide had no effect on plasma concentrations of IGF-I in the cows. However, as was the case with insulin, IGF-I changed with time after feeding. Plasma IGF-I declined (P < 0.01) from 53.9 to 47.6 ng/ml through 4 h postfeeding. The greatest numerical decline in IGF-I occurred from 3 to 4 h postprandial. CONCLUSIONS Oleamide fed to lactating Jersey cows in this study was highly digestible and had no adverse effects on nutrient digestibility, similar to results reported previously for Holsteins (10). Despite a decline in dry matter intake with feeding the amide, milk yield was maintained during this short-term study. Increasing oleamide at the expense of canola oil in the fat supplement increased the concentration of oleic acid in milk and plasma lipids. However, this study showed that higher oleic acid concentration in plasma lipids and maintaining constant added fat and energy density in the ration has no effect on circulating concentrations of insulin or IGF-I.
ACKNOWLEDGMENTS Thanks is extended to Cargill Oilseeds Division (Minneapolis, MN) for their donation of the canola oil, and to Church & Dwight, Inc. (Princeton, NJ) for partial financial support of this project. We also thank Evanne Thies for assistance with laboratory analysis, and Jim Hampton for analysis of plasma hormones. REFERENCES 1 Association of Official Analytical Chemists. 1990. Official Methods of Analysis Vol. I. 15th ed. AOAC, Arlington, VA. 2 Beam, S. W., and W. R. Butler. 1998. Energy balance, metabolic hormones, and early postpartum follicular development in dairy cows fed prilled lipid. J. Dairy Sci. 81:121–131. 3 Buttery, P. J., S. Manomai-Udom, and D. Lewis. 1977. Preliminary investigations on some potential sources of protected methionine derivatives for ruminant rations. J. Sci. Food Agric. 28:481–485. 4 Cant, J. P., E. J. DePeters, and R. L. Baldwin. 1991. Effect of dietary fat and postruminal casein administration on milk composition of lactating dairy cows. J. Dairy Sci. 74:211–219. 5 Choi, B., and D. L. Palmquist. 1996. High fat diets increase plasma cholecystokinin and pancreatic polypeptide, and decrease plasma insulin and feed intake in lactating cows. J. Nutr. 126:2913. 6 Cook, L. J., T. W. Scott, and Y. S. Pan. 1972. Formaldehydetreated casein-safflower oil supplement for dairy cows. II. Effect on the fatty-acid composition of plasma and milk lipids. J. Dairy Res. 39:211–218. 7 Doreau, M., and Y. Chilliard. 1997. Digestion and metabolism of dietary fat in farm animals. Br. J. Nutr. 78, Suppl. 1, S15–S35. 8 Fenton, T. W., and M. Fenton. 1979. An improved method for the determination of chromic oxide in feed and feces. Can. J. Anim. Sci. 59:631–634. 9 Garcia-Bojalil, C. M., C. R. Staples, C. A. Risco, J. D. Savio, and W. W. Thatcher. 1998. Protein degradability and calcium salts of long-chain fatty acids in the diets of lactating dairy cows: productive responses. J. Dairy Sci. 81:1374–1384. 10 Jenkins, T. C. 1998. Fatty acid composition of milk from Holstein cows fed oleamide or canola oil. J. Dairy Sci. 81:794–800. 11 Jenkins, T. C. 2000. Feeding oleamide to lactating Jersey cows. 1. Effects on lactation performance and milk fatty acid composition. J. Dairy Sci. 83:332–337. Journal of Dairy Science Vol. 83, No. 3, 2000
576
DELUCA AND JENKINS
12 Jenkins, T. C., and E. Thies. 1997. Plasma fatty acids in sheep fed hydroxyethylsoyamide, a fatty acyl amide that resists biohydrogenation. Lipids 32:173. 13 Lee, C. Y., and D. M. Henricks. 1990. Comparisons of various acidic treatments of bovine serum on IGF-I immunoreactivity and binding activity. J. Endocrinol. 127:139–148. 14 Opara, E. C., M. Garfinkel, V. S. Hubbard, W. M. Burch, and O. E. Akwari. 1994. Effects of fatty acids on insulin release: role of chain length and degree of unsaturation. Am. J. Physiol. 266:E635–E639. 15 Pantoja, J., J. L. Firkins, M. L. Eastridge, and B. L. Hull. 1996. Fatty acid digestion in lactating dairy cows fed fats varying in degree of saturation and different fiber sources. J. Dairy Sci. 79:575–584. 16 Phuntsok, T., M. A. Froetschel, H. E. Amos, M. Zheng, and Y. W. Huang. 1998. Biogenic amines in silage, apparent postruminal passage, and the relationship between biogenic amines and digestive function and intake by steers. J. Dairy Sci. 81:2193–2203.
Journal of Dairy Science Vol. 83, No. 3, 2000
17 Reeves, L. M., M. L. Williams, and T. C. Jenkins. 1998. In vitro biohydrogenation of oleamide and total tract digestibility of oleamide by sheep. J. Sci. Food Agric. 77:187–192. 18 SAS System for Windows. Release 6.12. 1996. SAS Inst., Inc., Cary, NC. 19 Schmid, H.H.O., P. C. Schmid, and V. Natarajan. 1990. N-Acylated glycerophospholipids and their derivatives. Prog. Lipid Res. 29:1–43. 20 Staples, C. R., J. M. Butrke, and W. W. Thatcher. 1998. Influence of supplemental fats on reproductive tissues and performance of lactating cows. J. Dairy Sci. 81:856–871. 21 St. John, L. C., D. K. Lunt, and S. B. Smith. 1991. Fatty acid elongation and desaturation enzyme activities of bovine liver and subcutaneous adipose tissue microsomes. J. Anim. Sci. 69:1064–1073. 22 Van Soest, P. J., J. B. Robertson, and B. A. Lewis. 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74:3583– 3597.