The conversion of short-chain PUFA to long-chain PUFA is rate limiting, and competition exists for the Δ6-desaturase enzyme(Reference Calder1). The extent of conversion varies between species(Reference Dunbar and Bauer2), and a study(Reference Bauer, Dunbar and Bigley3) has shown that canines have a differential metabolising ability compared with rodents. Within the canine species, it is not known whether breeds have a distinct differential ability.
Fish oil, known to be rich in n-3 long-chain PUFA, has been reported to have health benefits in human subjects(Reference Calder and Yaqoob4). In dogs, less inflammatory mediators were produced when fed diets with fatty acid ratios (n-6:n-3) of 5:1 and 10:1 in comparison with being fed an n-6-rich diet with a fatty acid ratio of 100:1(Reference Vaughn, Reinhart and Swaim5). The need for a vegetarian source of n-3 due to the reduction of global marine sources(Reference Brunner, Jones and Friel6) has led to studies in dogs(Reference Bauer, Dunbar and Bigley3, Reference Dunbar, Bigley and Bauer7) using plant sources such as flaxseed.
The present study evaluates the plasma composition of fatty acids following supplementation with flaxseed oil in two dog breeds and investigates the time course following oil supplementation to determine an effective supplementation period for future experiments.
Experimental methods
Animals
For the study, five beagles and five greyhounds were used. All were females and aged between 2 and 9 years. The dogs weighed an average of 12·4 (sd 1·2) kg (beagles) and 25·6 (sd 2·0) kg (greyhounds) at the start of the study. All dogs were borrowed with written consent from their owners and returned at the end of the trial. Health checks were performed at the start of the trial, and only healthy dogs were accepted. Dogs were housed at the University of New England dog research facilities at Armidale, NSW, Australia. The care and use of animals followed the guidelines set by the University of New England Animal Ethics Committee (authority no. AEC09/072), in accordance with section 25 of the Animal Research Act (1985).
Basal diet and flaxseed supplementation
A nutritionally complete and balanced commercial dog food (substantiated by the Association of American Feed Control Officials feeding protocol) was fed for 4 months before supplementation. Beagles and greyhounds were fed once a day, and ad libitum water was available at all times. From day 1 of the supplementation period, beagles were fed 300 g and greyhounds were fed 600 g of the basal diet daily to which Melrose® (Mitcham, VIC, Australia) flaxseed oil (57 % α-linolenic acid (ALA) and 17 % linolenic acid (LA)) was added at the rate of 100 ml/kg food, which equates to 2·4 (sd 0·2) ml/kg body weight for 3 weeks (30 ml/beagle and 60 ml/greyhound). The supplementation represented a fifty-two times increase in ALA and a two times increase in LA compared with the basal diet alone. The basal and supplemented diet compositions are summarised in Table 1.
Composition of the basal and supplemented diets per kg
ME, metabolisable energy; LA, linolenic acid; AA, arachidonic acid; ALA, α-linolenic acid.
Sample analyses
Whole blood (5 ml) was collected on three occasions: day 0 (before supplementing oil) and on days 15 and 22 post-supplementation. Plasma was extracted by centrifugation at 1900 g for 15 min. Fatty acid components in plasma were determined by GC ( < 6 % CV for the analysis).
Statistics
Initial analysis with generalised linear model showed no significant interaction between the breed effect and the day effect. Hence, for each individual fatty acid constituent in the plasma, the breed effect and the day effect were analysed using fully nested ANOVA, wherein the breed effect was the fixed effect and the day effect was the random variable. Windows MINITAB version 14 (Minitab Limited, Coventry, WMD, UK) was used to analyse the data. The level of significance was considered at P < 0·05. Significant differences between means were detected by the t test. The relationship between body-weight gain and plasma fatty acid concentrations was determined using regression analysis.
Results
Dogs remained healthy throughout the 3-week study, and there were no refusals of food or supplements. Average body-weight gains over this period were 2·4 % for the beagles and 3·1 % for the greyhounds. Regression analysis showed no relationship between body-weight gain and concentrations of plasma fatty acid constituents (R 2 values: ALA, 0·29; EPA, 0·002; DHA, 0·16; LA, 0·03; arachidonic acid, 0·43).
Plasma ALA, EPA and LA increased steadily and significantly from days 0 to 22 with no significant breed difference. Plasma arachidonic acid showed neither a significant breed effect nor a day effect.
Plasma DHA, on the other hand, showed no significant changes over time, but a significant breed effect (P = 0·02) was observed at all time points, including at day 0 which is before supplementing flaxseed oil (P values: day 0, 0·008; day 15, 0·036; day 22, 0·002). Beagles maintained a higher plasma level of DHA than greyhounds throughout the study. Plasma fatty acid data are presented in Table 2.
(Mean values and standard deviations)
ALA, α-linolenic acid; LA, linolenic acid; AA, arachidonic acid.
* There were no significant diet × breed interaction.
Discussion
Following supplementation with flaxseed oil, a steady rise in the concentrations of ALA and EPA was observed but not of DHA, which agrees with previous studies in dogs(Reference Bauer, Dunbar and Bigley3) as well as in human subjects(Reference Burdge and Calder8). It was observed that fifty-two times more ALA was consumed with the supplemented diet than with the basal diet, which explains the increase in ALA and EPA in plasma. The extent of efficiency of the canine system in elongating C18 PUFA to C20 PUFA was also demonstrated. Greyhounds showed a consistent numerically higher increase in ALA and EPA from days 0 to 22 compared with beagles, but this difference was not significant.
Bauer et al. (Reference Bauer, Dunbar and Bigley3) explained from their study that the unchanged level of DHA could be due to some resistance exhibited in the conversion of ALA to DHA in the liver, which is a rate-limiting step. Most of the DHA is produced in the neurological tissue, which could explain why the plasma levels of DHA remain not elevated in the present study and previous reports.
Similarly, LA levels rose significantly, which may be due to the increased levels in the supplemented diet. Arachidonic acid levels showed no significant change, perhaps resulting from competition for the Δ6-desaturase enzyme, from the conversion of ALA to EPA.
Breed differences were observed with respect to DHA. Dunbar et al. (Reference Dunbar, Bigley and Bauer7) concluded that hepatic conversion of docosapentaenoic acid to DHA is slow in the canine, and that even after docosapentaenoic acid is produced from ALA, it is likely to be relocated to the neurological tissues for DHA synthesis as stated previously. This is similar in other species such as cats as reported by Pawlosky et al. (Reference Pawlosky, Barnes and Salem9). It is possible that different breeds may have a different rate at which this conversion and transportation to the neurological tissue occurs. Extensive studies are required to confirm how different breeds metabolise PUFA and what this means in terms of dietary recommendations for specific breeds.
Levels of ALA and EPA were still rising significantly between days 15 and 22, indicating that plasma levels had not stabilised in 3 weeks. In the study of Bauer et al. (Reference Bauer, Dunbar and Bigley3), plasma fatty acid enrichment attained a steady state after 4 weeks. This needs to be taken into account when choosing time frames of supplementation in future studies. In conclusion, flaxseed oil could be used as an alternative source to fish oil for some essential fatty acids, but not DHA, in dog foods. There was an apparent breed difference between greyhounds and beagles in their plasma DHA levels. Further breed studies are warranted, as are fatty acid measurements in tissues or erythrocytes, for further clarification.
Acknowledgements
The present study was funded by the University of New England Postgraduate Research Fund. We thank the dog owners Shirley Fraser for providing the beagles and Greg Nordstrom for providing the greyhounds. We acknowledge Professor Manohar Garg, Director, Nutraceuticals Research Group, University of Newcastle, Australia, for conducting the fatty acid analysis. We also thank M. L. A. N. R. Deepani Mlanr for the statistical support. The abstract of the present study was presented at the Waltham International Nutritional Sciences Symposium 2010 in Cambridge, UK. All authors contributed to the data analysis and the writing and editing of the final version. The present study was conducted as part of doctoral programme for D. P. under the supervision of W. Y. B., S.-B. W. and B. V. The authors declare that there is no conflict of interest in the present study.
