J Sci Food Agric 1998, 77, 187È192

Biohydrogenation and Total TractIn VitroDigestibility of Oleamide by Sheep¤LaChanda M Reeves, Mervin L Williams and Thomas C Jenkins*

Department of Animal, Dairy and Veterinary Sciences, 151 Poole Agricultural Building, ClemsonUniversity, Clemson, SC 29634, USA

(Received 27 March 1997 ; revised version received 25 June 1997 ; accepted 6 October 1997)

Abstract : Disappearance of cis-18 : 1(n-9) from ruminal in vitro cultures supple-mented with either oleic acid or oleamide was measured over 48 h to determine ifthe amide resisted biohydrogenation. Oleamide added to the substrate main-tained higher concentrations of cis-18 : 1(n-9) in the microbial cultures at 24 and48 h of incubation compared to substrates with added oleic acid. Disappearancerates of cis-18 : 1(n-9) from the cultures, which were calculated as a measure ofbiohydrogenation, were 0É064 and 0É025 h~1 for the oleic acid and oleamidesupplements, respectively. Four sheep were fed four diets (control, 42 g kg~1oleic acid, 23 g kg~1 oleamide, and 45 g kg~1 oleamide) in a 4] 4 Latin squareto determine how the amide a†ected fatty acid digestibility. Total tract digest-ibilities of protein and Ðbre were not a†ected (P[ 0É05) by either oleic acid oroleamide compared to the control diet. Fatty acid and energy digestibilities werenot changed (P[ 0É05) by oleic acid, but were increased (P\ 0É05) when olea-mide was added to the sheep diets at 45 g kg~1. These results show that olea-mide resists ruminal biohydrogenation without impairing fatty acid digestibility.

1998 SCI.(

J Sci Food Agric 77, 187È192 (1998)

Key words : fatty acyl amides ; oleamide ; biohydrogenation ; digestibility ; sheep

INTRODUCTION

Biohydrogenation of unsaturated fatty acids by ruminalmicrobes only occurs when the carboxyl group on thefatty acyl moiety is unesteriÐed (Jenkins 1993). Fattyacids esteriÐed to glycerol in plant oil triglycerides arequickly released by microbial lipases in the rumen thusmaking them susceptible to biohydrogenation. There-fore, supplementing ruminant diets with unsaturatedplant oils has only a limited capacity to increase unsatu-rated fatty acids in body tissues.

Exposure of the carboxyl group can be reduced byconverting the unsaturated plant oils to fatty acylamides. The amide bond was shown to resist microbialdegradation (Steen and Collette 1989) resulting in less

* To whom correspondence should be addressed.¤ Technical contribution number 4260 of the South CarolinaAgricultural Experiment Station, Clemson University.

biohydrogenation and greater accumulation of linoleicacid in ruminal contents (Fotouhi and Jenkins 1992).When soybean oil was reacted with either butylamineor ethanolamine, the amide products fed to cattle andsheep increased unsaturated fatty acids in blood andmilk more than feeding soybean oil alone (Jenkins et al1996 ; Jenkins and Thies 1997). Fatty acid digestibilities,however, were low for butylsoyamide indicating thatsome amides may overprotect fatty acids to the point ofreducing their availability in the intestines.

This study was conducted to determine if oleamidesimilarly reduced biohydrogenation of oleic acid byruminal microbes. Equal quantities of oleic acid aseither the free acid or as oleamide were added to in vitrocultures to determine their e†ects on fatty acid composi-tion with time and their rates of biohydrogenation.Also, to determine if overprotection was a problem foroleamide, a metabolic study was conducted with sheep

1871998 SCI. J Sci Food Agric 0022È5142/98/$17.50. Printed in Great Britain(

188 L M Reeves, M L W illiams, T C Jenkins

to quantify amide and fatty acid excretion for diets withadded oleamide or oleic acid.

MATERIALS AND METHODS

Lipid supplements

Oleamide used for the microbial culture and sheepmetabolism trials was supplied by Church & DwightInc (Princeton, NJ, USA). Purity of the oleamide deter-mined by HPLC (procedure described below) averaged89É7 ^ 4É4% for eight measurements. Technical gradeoleic acid (90%, catalogue no 36452-5) was purchasedfrom Aldrich Chemical Co Inc (Milwaukee, WI, USA).Fatty acid compositions of the oleic acid and oleamidesupplements are shown in Table 1. Oleamide containedless cis-18 : 1 and more saturated and polyunsaturatedfatty acids than the oleic acid supplement.

Microbial cultures

Cultures contained 2É0 g ground hay substrate, 40 mlruminal inoculum, and 160 ml medium (Goering andVan Soest 1970). Ruminal inoculum was taken from aruminally cannulated Holstein cow fed grass hay. Sub-strates were the ground (1 mm) control, oleic acid andoleamide (45 g kg~1) diets described in Table 2. Incu-bation Ñasks were maintained under anaerobic condi-tions at 39¡C in a water bath.

Two in vitro trials were run on di†erent days, butruminal inocula were always obtained from the sameÐstulated cow fed only hay. The Ðrst in vitro trial wasconducted to determine how the lipid supplementsa†ected fatty acid composition of culture contents asincubation time progressed, and to quantify bio-hydrogenation of oleic acid and oleamide. There weretwo Ñasks per treatment. While vigorously mixing the

TABLE 1Fatty acid composition of the oleic acid and oleamide supple-

ments

Oleamide Oleic acid

Total (g kg~1 supplement) 876 931Individual (g kg~1 fatty acids)

12 : 0 0É1514 : 0 3É0916 : 0 7É82 0É6718 : 0 2É74 3É09cis-18 : 1 68É28 92É48trans-18 : 1 7É7518 : 2 5É02 3É6218 : 3 0É40 0É1420 : 0 0É48

TABLE 2Composition of diets used for the in vitro and sheep digest-

ibility trialsa

Oleamide

Item Control Oleic L ow High

Ingredients (g kg~1 diet DM)Soy hulls 493 493 493 493Corn 384 330 355 326Soybean meal 103 115 109 116Limestone 10 10 10 10Oleic acid 42Oleamide 23 45TM salt 5 5 5 5Ammonium chloride 5 5 5 5

By analysis (g kg~1 diet DM)CP 150 156 166 166NDF 355 342 328 349ADF 227 229 233 240Fatty acids 21 61 39 55

a Abbreviations : CP, crude protein ; DM, dry matter ; NDF,neutral detergent Ðbre ; ADF, acid detergent Ðbre.

Ñask contents under samples (5 ml) were taken atCO2 ,0 (immediately after ruminal inoculum was added), 3, 6,9, 12, 24 and 48 h for determination of long-chain fattyacids. Samples were immediately frozen to inhibit fer-mentation. The frozen samples containing both liquidand particulate matter were then freeze-dried, followedby conversion of the fatty acids to methyl esters byreaction of the sample for 15 h at 80¡C in acetylchloride/methanol (1 : 10) (Jenkins and Thies 1997). Aknown amount (0É5 mg) of internal standard (17 : 0) wasadded prior to methylation to quantify methyl esters.Methyl esters were separated by gasÈliquid chromatog-raphy as described by Jenkins and Thies (1997). Fattyacids were expressed as g kg~1 total fatty acids in theculture contents, or as g kg~1 of the supplement fattyacids by subtracting control fatty acids from the totalfatty acids.

The second in vitro trial was conducted to determineamide content in the microbial cultures over time. Con-ditions were the same as described for the Ðrst in vitrotrial except that there were three Ñasks per treatment.Samples (10 ml) of mixed culture contents were taken at0, 12, 24 and 48 h, freeze-dried, and then analysed foramide content by HPLC (Jenkins and Thies 1997).

Sheep metabolism trial

Four Romonov sheep, averaging 15 kg body weight atthe start of the study, were fed four diets in a 4 ] 4Latin square for 14-day periods. The control diet (Table2) contained no added fat, and the other three diets

Biohydrogenation and digestibility of oleamide by sheep 189

were supplemented with either 42 g kg~1 oleic acid,23 g kg~1 oleamide or 45 g kg~1 oleamide. Diets weremixed and o†ered to sheep without pelleting or furtherprocessing. Oleamide diets were higher in crude proteincontent because N contributions from the amide wereignored. This approach was taken to avoid a proteindeÐciency for the oleamide diets, in the event thatdigestibility of amide N was low. The crude proteincontent of the amide diets only exceeded the oleic aciddiet by 10 g kg~1.

Feed boxes for the metabolism cages were designedto minimise spillage but had to be removed after a mealto allow free movement and comfort of the animals.Therefore, the sheep were given access to feed for two1 h feeding periods starting at 11 :00 and 19 :00 h. Feednot consumed by the end of the second feeding periodwas weighed. Feed intake appeared to be only slightlyrestricted by this approach since animals normally Ðn-ished eating within 20È30 min, and intake of the controldiet exceeded 4% of body weight. During the Ðnal 5days of each period, feed refusals were subsampled andsaved for later analysis. Sheep had ad libitum access towater. Animal research procedures were approved bythe Institutional Animal Care and Use Committee.

Sheep were housed in stainless-steel metabolism cagesdesigned for the separation and collection of faeces andurine. Urine was discarded throughout the study. Thesheep were acclimated to diets for the Ðrst 9 days ofeach period, with total collection of faeces over the last5 days of each period. Faecal samples were oven-driedat 55¡C, then ground, along with feed and refusalsamples, in a centrifugal mill with a 1 mm sieve.Ground samples were analysed for dry matter (100¡C),Kjeldahl nitrogen (AOAC 1984), and neutral detergentand acid detergent Ðbres (Goering and Van Soest 1970).Energy content of samples was determined by bombcalorimetry.

Statistical analysis

All data were analysed by ANOVA using the generallinear models procedure of SAS (1985). Least squaresmeans are reported in tables and graphs.

In the sheep trial, the model for the 4 ] 4 Latinsquare accounted for variation due to period, animaland diet. The sheep assigned to the oleic acid diet inperiod 4 died from circumstances unrelated to treat-ment. Means were separated by least signiÐcant di†er-ence protected by a signiÐcant (P\ 0É05) F-value fordiet in the ANOVA (SAS 1985).

Data from the in-vitro trials were analysed as a split-plot design with substrate used as the main plot testedagainst replicate within treatment as the error term.E†ects of incubation time and the time by treatmentinteraction were tested against residual error. Meanswere separated by least signiÐcant di†erence protected

by an overall signiÐcant (P\ 0É05) F-value for treat-ment in the ANOVA (SAS 1985).

RESULTS AND DISCUSSION

Biohydrogenation

In the Ðrst in vitro trial, total fatty acid (sum of 14 : 0,16 : 0, 16 : 1, 18 : 0, cis-18 : 1, trans-18 : 1, 18 : 2 and18 : 3) concentrations in the culture contents werea†ected by the amount of lipid supplement, but not bythe source of lipid supplement or by incubation time.From 0 to 48 h, fatty acids in the control culturestended (P\ 0É09) to increase (0É27È0É31 g litre~1) whichis attributable to de novo fatty acid synthesis by theruminal microbes (Fulco 1983). Fatty acid concentra-tion, however, did not change (P[ 0É10) from 0 to 48 hwhen substrates contained either oleic acid (0É80È0É79 glitre~1) or oleamide (0É78È0É79 g litre~1). Fatty acidconcentration was constant over time because of negli-gible catabolism of long-chain fatty acids as energysources by ruminal microorganisms maintained underanaerobic conditions (Wu and Palmquist 1991).

Although total fatty acid content changed only slight-ly with incubation time, there was a dramatic shift inthe proportions of C-18 fatty acids characteristic of bio-hydrogenation. Concentration of 18 : 2(n-6) declined toless than 50 g kg~1 total fatty acids by 48 h for alltreatments (Fig 1). At the same time, there was a largeincrease in 18 : 0 (Fig 2) indicating its accumulation inculture contents as a biohydrogenation end-product.

The proportion of cis-18 : 1(n-9) also declined inmicrobial cultures with time indicating bio-hydrogenation of this fatty acid for all treatments (Fig3). However, for the cultures supplemented with oleicacid, cis-18 : 1(n-9) declined more quickly and exten-sively, reaching control values by 24 h. Oleamide addedto the cultures maintained higher (P\ 0É05) concentra-tions of cis-18 : 1(n-9) than the control cultures even at

Fig 1. Changes in concentration of 18 : 2(n-6) with time ofincubation in ruminal in vitro cultures containing oleic acid(42 g kg~1 substrate) or oleamide (45 g kg~1 substrate). Eachpoint is the mean of two observations (SEM \ 0É69 g kg~1).

190 L M Reeves, M L W illiams, T C Jenkins

Fig 2. Changes in concentration of 18 : 0 with time of incu-bation in ruminal in vitro cultures containing oleic acid(42 g kg~1 substrate) or oleamide (45 g kg~1 substrate). Eachpoint is the mean of two observations (SEM \ 0É69 g kg~1).

48 h. Higher 18 : 0 in oleic acid cultures compared tooleamide cultures at 24 and 48 h (Fig 2) is further evi-dence of reduced biohydrogenation of fatty acids in theamide.

Total cis-18 : 1(n-9) in the cultures consisted of contri-butions from the lipid supplements as well as the basalsubstrate ingredients. Supplement fatty acids weredetermined by subtracting cis-18 : 1(n-9) in the controlculture from its concentration in the oleic acid andamide cultures at each incubation time. Linear regres-sions were calculated from the natural logarithm of sup-plement cis-18 : 1(n-9) concentration vs incubation time.The slope was an estimate of the disappearance rate forcis-18 : 1(n-9) and, since biohydrogenation was the onlyroute of loss of unsaturated fatty acids (assuming negli-gible fatty acid catabolism), it was also an estimate ofthe rate of biohydrogenation for the supplements. Fromthese slopes, biohydrogenation of oleic acid was0É064 ^ 0É0037 h~1 (r \ 0É97) compared to only0É025 ^ 0É0014 h~1 (r \ 0É97) for oleamide. Therefore,biohydrogenation of cis-18 : 1(n-9) was reduced 61% byadding it to microbial cultures as oleamide rather thanas the free acid.

Fig 3. Changes in concentration of cis-18 : 1(n-9) with time ofincubation in ruminal in vitro cultures containing oleic acid(42 g kg~1 substrate) or oleamide (45 g kg~1 substrate). Eachpoint is the mean of two observations (SEM \ 0É88 g kg~1).

Amide content in microbial cultures declined withincubation time, showing that the amide bond was sus-ceptible to microbial degradation (Fig 4). Amidecontent declined to 74 and 60% of its original amountby 24 and 48 h, respectively. As amide declined, pre-sumably cis-18 : 1(n-9) was released as the free acid andwas susceptible to biohydrogenation. However, asshown in Fig 3, breakdown of the amide was slowenough to maintain higher concentration of cis-18 : 1 (n-9) in microbial cultures at 24 and 48 h.

Digestibility

The concentration of oleamide in the feed actually con-sumed (Table 3) was lower than in the diet o†ered(Table 2) indicating that the sheep were able to selec-tively choose feed ingredients and lower their consump-tion of amide. The actual amide consumed averaged 9É1and 20É4 g day~1 for the sheep fed the low and highlevels of oleamide, respectively. Amide was not detectedin the diet or faeces of sheep fed the control or oleic aciddiets. Fatty acids and cis-18 : 1(n-9) consumed (g kg~1DM intake) by sheep fed oleamide were higher(P\ 0É05) than the control diet but lower (P\ 0É05)than the oleic acid diet.

Faecal excretion of amide was less than 1% of amideconsumed. Consequently, faecal excretion of fatty acidsand cis-18 : 1(n-9) for the oleamide and control dietswere similar. However, their excretion was higher(P\ 0É05) for the oleic acid diet compared to the otherdiets. These results are consistent with previous reportsthat fatty acids in fatty acyl amides are released in thesmall intestine by the action of intestinal amidases(Schmid et al 1990) or proteolytic enzymes in pancreaticÑuid (Buttery et al 1977). The low digestibility (\50%)of butylsoyamide reported previously (Jenkins 1995 ;Jenkins and Thies 1997) suggests only limited post-ruminal cleavage of this amide.

Adding oleic acid to the diet had no e†ect on drymatter or digestible energy intake by the sheep (Table4). The low level of oleamide reduced dry matter intake

Fig 4. Changes in amide concentration with time of incu-bation in ruminal in vitro cultures containing oleamide

(45 g kg~1 substrate).

Biohydrogenation and digestibility of oleamide by sheep 191

TABLE 3Amide, total fatty acids and cis-18 : 1(n-9) consumed andexcreted in faeces of sheep fed diets supplemented with oleic

acid or oleamideahc

Oleamide

Item Control Oleic L ow High SEMd

Consumed (g kg~1 DMI)Amide ND ND 16É1b 31É0a 2É6FA 24É6d 66É6a 34É8c 45É1b 1É9cis-18 : 1 5É5d 46É4a 15É4c 24É1b 1É2(n-9)

Faecal excretion (g kg~1 DMI)Amide ND ND 0É16 0É06 0É06FA 1É0b 3É5a 1É4b 0É9b 0É36cis-18 : 1 0É07b 0É19a 0É07b 0É06b 0É009(n-9)

a Means with unlike following letters di†er at P\ 0É05.b Abbreviations : DMI, dry matter intake ; FA, fatty acids ;ND, not detected.c Diets fed to sheep contained added oleic acid at 42 g kg~1diet, or added oleamide at 23 (Low) or 45 (High) g kg~1 dietas described in Table 1.d Pooled standard errors for the control and oleamide diets.Multiply this value by 1É29 to obtain the SEM for the oleicacid diet.

relative to the control and oleic acid diets. Becausedigestible energy intake was also reduced (P\ 0É05) bythe low oleamide diet, the depression in intake cannotbe explained simply by physiological attempts to main-tain constant energy intake. Depressed feed intake for

the low oleamide diet occurred mainly in period 1.When period 1 data was omitted, average intake for thelow amide diet was 662 g day~1. Intake for the highlevel of oleamide was intermediate between the othertwo fat-supplemented diets, and was 14É6% lower thanthe control diet.

Other amides have been shown to have variablee†ects on feed intake by ruminants. Diets containingbutylsoyamide were readily consumed by sheep (Jenkins1995) and cattle (Jenkins et al 1996). However, intakewas severely reduced when an amide made fromsoybean oil and ethylenediamine was added to sheepdiets (Jenkins and Thies 1997). Amides made fromsoybean oil and ethanolamine appeared to reduceintake in a similar way to the oleamide in this study(Jenkins and Thies 1997).

Total tract digestibilities of protein and Ðber were nota†ected by either oleic acid or oleamide added to thesheep diets (Table 4). Fats and oils added to ruminantdiets often inhibit ruminal fermentation and Ðbredigestion (Jenkins 1993). However, across the entiredigestive tract, hindgut fermentation often lessens oreliminates this Ðbre digestibility depression (Jenkins1993).

Digestibilities of fatty acids and energy were nota†ected by adding oleic acid to the diet. However,digestibility of fatty acids was increased (P\ 0É05) byfeeding oleamide, which also increased (P\ 0É05)energy digestibility for the high oleamide treatment.This may partially be attributable to lower fatty acidintakes for the amide diets as true fatty acid digestibilitydeclines with increasing intake in ruminants (Bauchart1993). These results, along with the low excretion of

TABLE 4Dry matter and digestible energy intakes, and total tract digestibilities by sheep fed diets supple-

mented with oleic acid or oleamideahc

Oleamide

Item Control Oleic L ow High SEMd

DMI (g day~1) 769É8a 698É7a 562É7b 657É1ab 37É5DEI (Mcal day~1) 2É39a 2É38ab 1É88b 2É24ab 0É15

Digested (g kg~1 nutrient consumed)DM 741b 768ab 764ab 774a 8CP 708 721 703 746 18ADF 468 565 567 524 32NDF 571 633 605 570 31Fatty acids 919b 915b 954a 967a 10Energy 733b 755ab 758ab 772a 11

a Means with di†erent following letters di†er at P\ 0É05.b Abbreviations : DEI, digestible energy intake ; DM, dry matter ; DMI, dry matter intake ; CP,crude protein ; NDF, neutral detergent Ðbre ; ADF, acid detergent Ðbre.c Diets fed to sheep contained added oleic acid at 42 g kg~1 diet, or added oleamide at 23 (Low) or45 (High) g kg~1 diet as described in Table 1.d See footnote d to Table 3.

192 L M Reeves, M L W illiams, T C Jenkins

amide in faeces (Table 3), clearly show that fatty acids inoleamide were digested postruminally without difficulty.

CONCLUSIONS

Oleamide reduced the rate of cis-18 : 1(n-9) bio-hydrogenation by mixed ruminal microbes. Faecal exc-retion of oleamide was low and digestibility of dietaryfatty acids and other nutrients was not impaired byadding oleamide to sheep diets. Therefore, oleamide canbe used as a source of protected cis-18 : 1(n-9) in rumi-nant diets to increase the intestinal absorption of mono-unsaturated fatty acids.

ACKNOWLEDGEMENTS

Partial Ðnancial support and donation of the oleamidewere generously supplied by Church and Dwight Inc,Princeton, NJ. The authors thank Evanne Thies for herassistance with analytical methods.

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