The term conjugated linoleic acid (CLA) is used to designate a mixture of positional and geometric isomers of linoleic acid in which the double bonds are conjugated. Considerable attention has been paid to the potential, beneficial health effects of dietary CLA. CLA was found to act as a growth factorReference Chin, Storkson, Albright, Cook and Pariza1, as a fat-to-lean repartitioning agentReference Pariza, Park, Cook, Albright and Liu2–Reference Terpstra, Beynen, Everts, Kocsis, Katan and Zock7, and it has anticarcinogenicReference Schulz, Chew, Seaman and Luedecke8, Reference Ip9, hypocholesterolaemic and antiatherogenicReference Lee, Kritchevsky and Pariza10, Reference Nicolosi, Rogers, Kritchevsky, Scimeca and Huth11 properties. Food intake is usually not affected by incorporation of CLA in the diets and, therefore, the body fat-lowering effect of CLA is most likely mediated by enhanced energy expenditure. Measuring the energy expenditure of mice in metabolic chambers fed CLA indeed demonstrated an increase of energy expenditureReference West, Truett, Delany and Scimeca12.
Szymczyk et al. Reference Szymczyk, Pisulewski, Szczurek and Hanczakowski13 showed that feeding CLA to broiler chickens resulted in subtantial incorporaton of CLA isomers into their tissue lipids, thus providing a potential CLA-rich source for human consumption. In their study, feeding CLA significantly decreased feed intake during the starter (8–21 d) period, but no effect was noted during the grower–finisher (22–42 d) period. Abdominal fat deposition was significantly reduced whereas the relative proportion of breast muscles was unaffected and that of leg muscles significantly increasedReference Szymczyk, Pisulewski, Szczurek and Hanczakowski13. It could be suggested that CLA feeding influences body composition and energy metabolism of broiler chickens.
The objective of the present study was to test whether the earlier observed CLA-induced reduction of abdominal fat in chickensReference Szymczyk, Pisulewski, Szczurek and Hanczakowski13 is associated with enhanced energy expenditure by investigating the influence of dietary CLA on growth, body composition and energy balance in broilers. In addition, the fatty acid composition of total carcass lipid was evaluated.
Experimental methods
The experimental protocol was approved by the animal experiments committee of the Faculty of Veterinary Medicine, Utrecht University, The Netherlands.
Animals and diets
One-day-old male broiler chickens (Ross 308) were purchased from a local hatchery. On arrival, they were wing-banded, weighed and housed in wire-floored, suspended cages. The temperature of the animal house was controlled and continuous lighting used throughout the entire experimental period. There were two dietary treatments, each consisting of eight replicates. A replicate was identical to a cage with four birds so that each treatment had thirty-two animals. Ten birds were killed at the beginning of the study to determine pre-experimental body composition. Sixty-four broilers were used for the feeding trial. The base diet was in pelleted form (Table 1) and fed for 7 d. To produce the experimental diet, sunflower oil was replaced by 1 g CLA per 100 g diet. The fatty acid composition of the diets is given in Table 2. CLA was purchased from Lipid Nutrition B.V. (Wormerveer, The Netherlands). It came in the form of a Clarinol G-80™ preparation that contained 79·5 % of CLA as TAG. The CLA preparation consisted of cis-9, trans-11 and trans-10, cis-12 CLA in equal amounts. The birds were fed the experimental diet for a period of 21 d. Feed and water were provided ad libitum. Individual body weight and feed intake per replicate were monitored weekly. From day 7 on, excreta were collected quantitatively.
Composition of the diets (g/kg)†
cp, crude protein.
† The diets were prepared by Research Diets Services (Wijk bij Duurstede, The Netherlands).
‡ The 5 g Premix consisted of 12 000 IU vitamin A (4·1 mg retinol acetate); 2400 IU vitamin D3 (0·06 mg cholecalciferol); 30 mg vitamin E; 1·5 mg vitamin K3; 2·0 mg vitamin B1; 7·5 mg vitamin B2; 3·5 mg vitamin B6; 20 μg vitamin B12; 35 mg niacin; 10 mg d-pantothenate; 460 mg choline chloride; 1·0 mg folic acid; 0·2 mg biotin; 80 mg Fe; 12 mg Cu; 85 mg Mn; 60 mg Zn; 0·4 mg Co; 0·8 mg I; 0·1 mg Se; 200 mg Ca; 125 mg anti-oxidant (Oxytrap PXN).
Selected fatty acids (% of FAME) in the diets†
† Total fat content of the diets: 7·53 and 7·32 % for the control and the CLA-containing diet, respectively.
CLA, conjugated linoleic acid.
Carcass analysis
At the end of the experiment, the birds were weighed and killed by cervical dislocation. Carcasses were cut in pieces and ground (Retsch, SM 2000, Haan, Germany) and the carcasses for each replicate were mixed, sampled, weighed and then dried in a forced-hot air oven at 60°C for a period of 3 d. The dried carcasses were weighed again and the percentage of water was calculated. Subsequently, the dried carcasses were ground in a coffee grinder and the homogenised samples were stored in plastic containers until analysed. The excreta were collected during the experimental period of 3 weeks and were also dried for a period of 3 d and homogenised in a coffee grinder.
Total lipids in the dried, homogenised carcasses and excreta were extracted as described previouslyReference Javadi, Everts, Hovenier, Kocksis, Lankhorst, Lemmens, Schonewille, Terpstra and Beynen14. Total lipids were saponified and methylated according to Metcalfe et al. Reference Metcalfe, Schmitz and Pelka15 followed by GLC for determination of the fatty acid composition of feed and carcasses. The protein content of the dried carcasses was determined with the macro Kjeldahl method16. For the determination of the ash content, about 0·5 g dried, homogenised carcass was added to a small porcelain crucible and put in an oven that was programmed as follows: 1 h at 200°C, 2 h at 300°C, 3 h at 400°C and 10 h at 500°C17.
Bomb calorimetry
The gross energy content in dried, homogenised carcasses, faeces, diets and oils was determined with a bomb calorimeter (IKA Calorimeter C4000 Adiabatic, IKA Analystechnik, Heitersheim, Germany). As a thermochemical standard benzoic acid (BDH Ltd, Poole, UK) was usedReference McLean and Tobin18. The total amount of energy that was lost as heat (heat production or energy expenditure) was calculated with the formula: energy lost as heat = energy in food − energy in excreta − energy stored in body. Energy stored in the body was determined as total energy at the end of the 21 d feeding period minus energy in the body at the beginning ( = mean body weight × energy content) of the 21 d feeding period. The same procedure was used to calculate the retention of water, protein, fat and ash.
Statistical analysis
Four birds in a cage were considered as one experimental unit. This resulted in eight experimental units per dietary treatment. Mean data per cage were used in a one-way ANOVA with diet (sunflower oil v. CLA) as an independent variable. The level of statistical significance was preset at P < 0·05.
Results
Body weight and body composition
Food intake was lower in CLA-fed birds than in controls, the lowering almost reaching statistical significance (Table 3). There was no difference in body weight gain and feed conversion rate between CLA-fed birds and controls. The proportion of body fat was higher in the CLA-fed group than in the control group (P = 0·044). There were no differences in the proportions of body water, protein and ash between the two groups.
(Mean values with their standard errors for eight units with each unit including four birds)
Energy balance
Energy intake was lower in CLA-fed birds, lowering almost reaching statistical significance (Table 3). Apparent fat digestibility and energy metabolisability were higher in the control group (P = 0·026 and 0·003, respectively). Energy expenditure was calculated as the difference between the energy intake and the energy stored and excreted in the excreta. The higher heat production calculated for the control group differed from that for the CLA group (P = 0·002). Energy storage was not affected by CLA feeding. The proportion of energy intake that was stored in the body was lower in controls than in the CLA-fed group (0·34 (sem 0·02) and 0·37 (sem 0·02), respectively; P = 0·007).
Feed and carcass fatty acid composition and feed efficiency
As CLA was added to the experimental diet at the expense of sunflower oil, the ingested amounts of SFA, MUFA, PUFA-n-6 and PUFA-n-3 dropped (Table 4). The amounts of fatty acid stored in the carcasses are shown in Table 5. The amount of SFA in carcasses was increased in the CLA-fed group (P < 0·001) and the amount of MUFA and PUFA were decreased (P = 0·003 and P < 0·001, respectively).
(Mean values with their standard errors for eight units with each unit including four birds)
† Total fatty acid contents were calculated as follows: total fat measured × 0·95 × percentage of selected fatty acid.
(Mean values with their standard errors for eight units with each unit including four birds)
† Total fatty acid contents were calculated as follows: total fat measured × 0·95 × percentage of selected fatty acid.
CLA consumption markedly increased the efficiency of incorporation (fatty acid deposited/fatty acid ingested) of SFA and decreased the incorporation of PUFA-n-3 (Table 6). Taking into account the amounts of fatty acids in the body at the start of the experiment, the ingested amounts of fatty acids and the amounts of fatty acids at the end of the experiment, one can estimate the minimal rate of de novo fatty acid synthesis during 21 d or the maximal rate of fatty acid degradation/disappearance in that period. The data indicated that CLA feeding preferentially induced SFA synthesis and that degradation/disappearance of PUFA is unaffected (Table 6).
(Mean values for eight determinations per treatment group)
Mean values were significantly different from those of the control diet: *P < 0·05, **P < 0·01, ***P < 0·001.
† Efficiency is expressed as the ratio of fatty acid deposited in the body and dietary fatty acid.
‡ The values for the minimum amount of fatty acid synthesised are obtained by subtracting the amount of intake from the amount of retained.
§ The values for the maximum amount of fatty acid disappearing are obtained by subtracting the amount of retained from the amount of intake.
Discussion
The effect of CLA on body composition and energy expenditure was studied in broiler chickens fed 1 g CLA/100 g diet. CLA feeding depressed feed intake, fat digestibility and energy metabolisability. This must result in a lower amount of metabolisable energy in the CLA treated group. However, weight gain during the experimental period did not differ between the dietary treatments. Moreover, deposition of fat, water, protein, ash and energy was not different (Table 3). CLA feeding had no negative effect on body fat deposition. Feed conversion was non-significantly lower in the CLA-fed birds, which is consistent with the finding by Szymczyk et al. Reference Szymczyk, Pisulewski, Szczurek and Hanczakowski13. The fat proportion, however, was higher in the body of birds fed CLA when compared to controls. This result is consistent with the finding by Du & AhnReference Du and Ahn19 who found that feeding a diet containing 0·5 % CLA to broilers at 3 weeks of age, for a period of 3 weeks, resulted in an increase in abdominal fat content. Several studies have shown that incorporation of 1 % or less CLA in the diet can substantially reduce the proportion of body fat in miceReference Terpstra, Beynen, Everts, Kocsis, Katan and Zock7, Reference West, Truett, Delany and Scimeca12, Reference Delany, Blohm, Truett, Scimeca and West20, ratsReference Rahman, Wang, Yotsumoto, Cha, Han, Inoue and Yanagita21, Reference Koba, Akahoshi, Yamasaki, Tanaka, Yamada, Iwata, Kamegai, Tsutsumi and Sugano22, chickensReference Szymczyk, Pisulewski, Szczurek and Hanczakowski13 and manReference Basu, Riserus, Turpeinen and Vessby23, Reference Blankson, Stakkestad, Fagertun, Thom, Wadstein and Gudmundsen24. The effects in mice appear more striking than in other speciesReference Terpstra25. Badinga et al. Reference Badinga, Selberg, Dinges, Comer and Miles26 found that feeding CLA at the level of 5 % to 1-d-old broiler chickens for a period of 21 d significantly lowered the proportion of body fat and increased the proportion of body water. Szymczyk et al. Reference Szymczyk, Pisulewski, Szczurek and Hanczakowski13 found lower abdominal fat in the body when they fed birds a diet with 1 % CLA. As mentioned earlier, Du & AhnReference Du and Ahn19 observed an increase in abdominal fat in broilers fed CLA. Thus, experimental conditions such as age, genotype and metabolic status of the animal, as well as the level, the type of isomer and duration of CLA treatment may play an integral role in how CLA affects body composition. The lack of agreement between previous works suggests mechanisms involved are complicated and multiple.
Much to our surprise, the energy balance indicated that the calculated heat production was about 20 % lower in CLA-fed birds compared to the controls. This is opposite to what happens in mice after CLA consumptionReference Terpstra, Beynen, Everts, Kocsis, Katan and Zock7. The present study does not give any information on the mechanism responsible for the decrease in energy expenditure in broilers fed CLA. However, it is tempting to speculate that the decrease in energy expenditure is related to the increase in the proportion of body fat. Another possibility is an effect of CLA on non-shivering thermogenesis, which is quite different in birds as compared to mammals where brown adipose tissue is the site for non-shivering thermogenesis. Birds lack brown adipose tissueReference Talbot, Duchamp, Rey, Hanuise, Rouanet, Sibille and Brand27.
Lee et al. Reference Lee, Storkson and Pariza28 observed that CLA has the ability to alter the fatty acid composition of tissues by reducing the levels of MUFA which is consistent with the present findings. Choi et al. Reference Choi, Kim, Han, Park, Pariza and Ntambi29 observed that the ratio of SFA to MUFA in mice fed CLA was increased and indicated that this was related to a lipid-lowering effect of CLA. Studies in ratsReference Szymczyk, Pisulewski, Szczurek and Hanczakowski30, Reference Sisk, Hausman, Martin and Azain31 and chickensReference Szymczyk, Pisulewski, Szczurek and Hanczakowski13, Reference Du, Ahn, Nam and Sell32 have shown that the percentage of SFA in the body increases whereas those of MUFA and PUFA decrease. The same was true for egg yolks of eggs produced by hens fed CLAReference Watkins, Feng, Strom, DeVitt, Yu and Li33, Reference Muma, Palander, Nasi, Pfeiffer, Keller and Griinari34. In the present study, we also found a marked increase in SFA, but no lipid-lowering effect of CLA was observed. Carcasses of rats fed CLA also contained a higher proportion of SFA and less PUFAReference Stangle35. The reduction in MUFA (oleic acid) may be the result of a reduced Δ-9 desaturase activity due to feeding CLAReference Lee, Pariza and Ntambi36–Reference Bretillon, Chardigny, Gregorie, Berdeaux and Sebedio38. The arachidonic acid concentration decreased in the carcasses. The present results are consistent with those of Belury & Kempa-SteczkoReference Belury and Kempa-Steczko39 who proposed that CLA, acting as a substrate for Δ-6 desaturase, inhibited the conversion of linoleic acid into arachidonic acid. Consistent with the present observations is the finding that CLA dramatically reduced the percentages of MUFA in all tissues investigated through inhibition of Δ-9 desaturaseReference Choi, Kim, Han, Park, Pariza and Ntambi29, Reference Lee, Pariza and Ntambi36, Reference Park, Storkson, Ntambi, Cook, Sih and Pariza40, Reference Choi, Park, Pariza and Ntambi41. The trans-10, cis-12 CLA isomer has been shown to have the highest biological activity in this respect, whereas cis-9, trans-11 CLA does not reduce the activity of Δ-9 desaturaseReference Park, Storkson, Ntambi, Cook, Sih and Pariza40, Reference Eder, Slomma and Becker42.
The changes in fatty acid composition greatly increase the melting point for fat retained in the CLA-fed group (from 21 to around 35°C). What kind of effects this will trigger in the broilers is unknown yet. A similar change in fatty acid composition results in complete loss of hatchability of eggs from CLA-fed chickensReference Watkins, Feng, Strom, DeVitt, Yu and Li33, Reference Muma, Palander, Nasi, Pfeiffer, Keller and Griinari34. In the broilers such a change in fatty acid composition makes chicken meat harder and drierReference Du and Ahn19.
When we calculate the amounts of fatty acids stored in the body during the experimental period and also the amounts of fatty acids ingested, we can determine the efficiency of fatty acid incorporation into the body. Calculation revealed a dramatically higher efficiency for SFA and a lower efficiency for PUFA-n-3 (Table 6). The CLA-induced differences in efficiency of incorporation might be related to preferential effects on synthesis or degradation of certain fatty acids. If the incorporation ratio was higher than 1·0, then the minimum amount of de novo synthesis of a specific fatty acid was calculated as deposited amount (g/21 d) minus the ingested amount (g/21 d). If the incorporation ratio was lower than 1·0, then the maximum amount of oxidation (or degradation) of a specific fatty acid was calculated as the ingested amount (g/21 d) minus the deposited amount (g/21 d). Both calculations can only indicate the lower and upper limit, respectively, because actual information about digestibility of individual fatty acids and the efficiency of incorporation of dietary fatty acids in deposited fatty acids is not available in the present experiment. The calculations show that CLA feeding preferentially induced SFA synthesis. This may explain the CLA-induced increase in body fat, which was statistically significant when expressed as percentage of the body. Much to our surprise the oxidation of PUFA was unaffected. This is contrary to many observations indicating preferential oxidation of PUFAReference Belury and Kempa-Steczko39, Reference Banni, Angioni, Casu, Melis, Carta, Corongiu, Thompson and Ip43, Reference Poulos, Sisk, Hausman, Azain and Hausman44. In contrast, some studies have shown that CLA may have a modest enhancing effect on the level of PUFAReference Kavanaugh, Liu and Belury45, Reference Banni, Carta, Angioni, Murru, Scanu, Melis, Bauman, Fisher and Ip46. Yet other studies, like the present one, show no effect of CLA on PUFA levelsReference Sisk, Hausman, Martin and Azain31, Reference Li, Seifert, Ney, Grahn, Grant, Allen and Watkins47–Reference Petrik, McEntee, Johnson, Obukowicz and Whelan49. It appears that the ability of CLA to alter PUFA levels is tissue and species dependent. Consistent with the present results on fatty acid synthesis and degradation are our earlier observations showing CLA-induced activity of the lipogenic pathway in mice as evidenced by enhanced activities of acetyl-CoA carboxylase and fatty acid synthaseReference Javadi, Beynen, Hovenier, Lankhorst, Lemmens, Terpstra and Geelen50. In that same study it was shown that CLA did not alter the activities of 3-hydroxy-acyl-CoA dehydrogenase and citrate synthase, suggesting that fatty acid oxidation was not affected by CLA feedingReference Javadi, Beynen, Hovenier, Lankhorst, Lemmens, Terpstra and Geelen50.
It might be argued that differences in fatty acid composition between the control and CLA-containing diet may have caused differences in fatty acid deposition. There are indeed differences in fatty acid intake between the two experimental diets, as can be seen from Table 2 with the analysed fatty acid composition of the experimental diets. These differences are due to the fact that CLA was added at the expense of sunflower oil. The differences are minor except for linoleic acid. However, earlier workReference Javadi, Everts, Hovenier, Kocksis, Lankhorst, Lemmens, Schonewille, Terpstra and Beynen14 indicates that the difference in linoleic acid intake cannot have caused the diet effects observed in the present study. The difference in ingested fatty acids as calculated in Table 4 is mainly caused by the difference in feed intake between the treatments.
Several studies have shown that the specific mechanisms by which dietary CLA reduces the body fat content are likely to vary from one animal species to another. Whether reduced accumulation of liver lipid in broilers fed CLA as observed by Badinga et al. Reference Badinga, Selberg, Dinges, Comer and Miles26 reflected enhanced β-oxidation or reduced de novo lipid synthesis warrants further investigation, but the present observation indicates higher de novo synthesis and lower desaturase activity. Measurement of enzyme expression and/or activity would complement the present data.
Acknowledgements
This study was supported in part by the Stichting Toxicologisch Onderzoek Utrecht. The CLA used in this research was supplied by Loders Croklaan b.v, Wormerveer, The Netherlands. The technical assistance of Jan van der Kuilen is gratefully acknowledged.
