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Supplement: WALTHAM International Science Symposium: Nature, Nurture, and the Case for Nutrition

Maternal Diet -Linolenic Acid during Gestation and Lactation Does Not Increase Docosahexaenoic Acid in Canine Milk^1,2

John E. Bauer^3,*,, Kimberly M. Heinemann^*,, Karen E. Bigley^*,, George E. Lees and Mark K. Waldron^**

^* Comparative Nutrition Laboratory and Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4474, and ^** Nestle-Purina PetCare, St. Louis, MO 63102

³ To whom correspondence should be addressed. E-mail: jbauer{at}cvm.tamu.edu.

KEY WORDS: • canine • milk • fatty acids • diet • -linolenic acid • lactation • gestation

Both (n-3) and (n-6) classes of long-chain polyunsaturated fatty acids (LCPUFA),⁴ which have 20 carbon atoms and >2 double bonds, are important in perinatal development. Brain and retinal functions depend on the (n-3) polyunsaturate, docosahexaenoic acid [DHA, 22:6(n-3)] not only during gestational development but also postnatally. Although species differences among mammals are likely, maximal brain growth begins in the third trimester of gestation and continues throughout the first few months of neonatal life (1–3). During this crucial period, accumulation of both DHA and arachidonic acid [AA, 20:4(n-6)] in brain and retina occurs 10 times faster than incorporation of their respective precursors, linoleic acid [LA, 18:2(n-6)] and -linolenic acid [ALA 18:3(n-3)] (4,5). In accordance with this knowledge, several authors (6–8) have demonstrated that plasma DHA is the preferred substrate for retinal uptake in early developmental stages when the demand for DHA is greatest.

Canine neurologic development

DHA is highly conserved in the retina and has a role in neurologic function in this tissue (7). Canine retina is capable of synthesizing DHA from its 22-carbon precursor, docosapentaenoic acid [DPA, 22:5(n-3)] (9). Bauer et al. (10) reported the accumulation of DPA but not DHA in canine plasma phospholipids when the precursor, ALA, was fed. It is therefore likely that canine retina and presumably other nervous system tissues synthesize and utilize DHA in a manner similar to other mammalian species, and that plasma DPA provides a likely substrate for such synthesis. Thus, a dietary source of preformed DHA or one of its precursors may be necessary during gestation and lactation for normal neurodevelopment in dogs.

It is possible that ALA is sufficient as a dietary precursor for the synthesis of requisite amounts of DHA during pre- and postnatal development. However, the quantity of ALA that is needed to optimize neural development in companion animals is presently unknown. Additionally, because both (n-6) and (n-3) fatty acids compete for the same enzyme systems, it also is unclear what relative amounts may be needed.

Canine milk composition

Early studies of canine milk composition were limited to macronutrient analysis, and fatty acid analyses typically were not performed. Thus, few reports exist regarding the fatty acid composition of canine milk (11,12). An effect of dietary LCPUFA intake during gestation and lactation on milk composition is expected; however, this effect has not been specifically investigated, nor has a dose-response relationship been established. The purpose of the present study was to document the dietary effects of both 18-carbon (n-3) precursors as well as LCPUFAs on canine milk when these fatty acids are included in gestation and lactation diets, and to evaluate the possibility of a dose-response relationship with respect to dietary amounts of these fatty acids.

	MATERIALS AND METHODS

TOP MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
An existing breeding colony of dogs provided bred bitches and their puppies for this study. The colony contains a kindred of dogs with hereditary nephritis (13), but all dogs used in this study were clinically healthy. Twelve bitches (three dogs each per diet group) were fed one of four complete and balanced, extruded-type diets from the time of insemination throughout gestation, parturition, and lactation. Sufficient quantities of the diets were fed to maintain weight gain of the bitches in the latter stages of gestation by adjusting the amount fed as necessary. The diets were formulated using typical pet food ingredients and varying fat sources (Nestle-Purina PetCare, St. Louis, MO). They contained 15% total fat using either beef tallow, linseed oil, or higher and lower amounts of menhaden fish oil as primary fat source The final diets thus differed in their (n-3) fatty acid composition and contained LA in concentrations ranging from 1.2 to 3.5% dry matter (DM) (Table 1). Diets were designated according to their ALA/(n-3) LCPUFA contents: Lo/Lo, Hi/Lo, Lo/Mod, and Lo/Hi (Table 1). All other dietary ingredient sources and amounts including total protein, nitrogen-free extract, vitamins, and minerals were formulated to be identical in the diets to result in similar nutrient profiles and energy densities (Table 2).

	RESULTS

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Canine milk contained 8.0 ± 2.0% total fat (on an as-is basis; mean ± SD), and no significant differences due to diet or day of lactation were found. Also, no main time effects or time x diet interactions were observed with respect to the individual fatty acids. However, highly significant dietary effects were observed among the polyunsaturated fatty acid concentrations in individual milk samples.

Among the (n-6) fatty acids, a dose-response relationship was seen for LA as a function of its dietary concentration. Differences due to diet were statistically significant, especially as dietary LA concentrations increased from 1.2 to 2.6 and 3.5% DM in the Lo/Hi, Lo/Mod, and Hi/Lo diets, respectively (Fig. 1). However, despite the doubling of dietary LA contents and a modest increase in dietary AA, milk AA concentrations remained unchanged in all groups. Dose responses of (n-3) fatty acids in milk were also observed as a function of increasing dietary (n-3) LCPUFA contents, especially when dietary ALA concentrations were low and nearly constant (i.e., in the Lo/Lo, Lo/Mod, and Lo/Hi groups; Fig. 2). Thus, as dietary eicosapentaenoic acid (EPA) and DHA concentrations increased, highly statistically significant elevations of these fatty acids in milk were noted (P < 0.001). For DPA, nominal increases were also seen in these three groups, but these differences did not attain statistical significance. A statistically significant difference for DPA was found in the Hi/Lo group compared with the other groups, but only at P < 0.05. Reasons for this finding are unknown, but may be due to the fact that the fatty acid profile of the Hi/Lo diet contained a very low DPA amount, markedly elevated ALA concentration, and the highest LA content of any of the diets studied. As such, it was most dissimilar to the other diets, contained the highest amount of LA, and had a markedly greater ALA concentration.

View larger version (22K): [in this window] [in a new window]   FIGURE 1  Concentration of LA and AA fatty acids in milk of dogs fed the various gestation and/or lactation diets (n = 3 dogs/group). Dietary concentrations of LA and AA are indicated as percent of dry matter. Diets are designated as Lo/Lo, Lo/Mod, Lo/Hi, and Hi/Lo based on their ALA/(n-3) LCPUFA contents. Different letters indicate significant difference; P < 0.001.

View larger version (24K): [in this window] [in a new window]   FIGURE 2  Concentrations of selected (n-3) polyunsaturated fatty acids in canine milk in dogs fed the various gestation and/or lactation diets (n = 3 dogs/group). Different letters indicate significant difference; P < 0.001; different numbers indicate significant difference; P < 0.05.

	DISCUSSION

TOP MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
It is interesting that neither (n-6) nor (n-3) LCPUFAs (i.e., AA, EPA, DPA, and DHA) become enriched in milk when their respective 18-carbon precursors are fed throughout gestation and lactation in dogs. Small contributions of dietary LA to milk AA composition have likewise been observed in humans using stable isotope techniques (14). With respect to the (n-3) fatty acids, only ALA quantity was significantly increased in milk in response to dietary sources, and no changes were seen in any of the derived (n-3) LCPUFAs. This finding is also similar to that reported for humans by Francois et al. (15) in which seven lactating mothers took dietary flaxseed oil supplements at the rate of 20 g of oil (10.7 g of ALA)/d for 4 wk during lactation. In that study, milk sample analyses were reported at baseline, 2 and 4 wk of supplementation, and 4 wk after supplementation. In the present canine study, dogs were fed larger amounts of ALA that approximated 18–37 g of ALA/d depending on the stage of gestation and during lactation. Nevertheless, no significant enrichment of milk DHA occurred at any time. Francois et al. (15) did report that a modest yet statistically significant trend toward increased EPA and DPA was found over time. However, inspection of their data show that the mean values of both EPA and DPA appeared to modestly increase from baseline after 2 wk and then decrease from this value by the 4-wk time period. It is possible that the milk samples of these subjects at 2 wk were not representative of a diet-supplement–induced metabolic steady state. Instead, they may have been a reflection (to varying extents) of the mothers' previous diets, DHA status, or tissue stores immediately before the dietary supplements were started (14). Indeed, our studies show that it generally requires >2 wk (21–28 d) of dietary lipid modification to result in steady-state plasma fatty acid concentrations (10). Also, Francois et al. (15) supplemented human mothers only during lactation. In the present study, dogs were fed the high linseed–oil diet (Hi/Lo diet) from the onset of breeding and throughout gestation (63 d) and lactation periods (28 d). These differences may partly explain the modest elevations of EPA and DPA contents seen in human but not canine milk even though more ALA was fed to dogs in the present study.

The observations that dietary LA did not increase milk AA content and that dietary ALA did not increase milk (n-3) LCPUFA amounts support the possibility that canine milk-fat biosynthetic pathways that specifically relate to desaturation and chain elongation are poorly developed in mammary tissue. An alternative explanation is that these pathways are competitively inhibited in the presence of either small dietary amounts of LCPUFAs or their existing tissue stores. Thus, supplementation of gestation and lactation diets with LA or ALA does not appear to be an effective method of increasing milk-fat LCPUFAs in developing canines.

In summary, dietary ALA supplementation during gestation and lactation is an ineffective means of increasing milk DHA content to supply dietary amounts of this LCPUFA for neonatal nutritional modification. Whether sources of preformed dietary (n-3) LCPUFAs are necessary to support puppy development during suckling or whether puppies are themselves capable of synthesizing sufficient (n-3) and (n-6) LCPUFAs from 18-carbon precursors is a question that we are pursuing presently. Finally, the consistency of the milk AA concentration, independent of dietary LA content and the dose responses seen with the (n-3) LCPUFAs, will assist in future efforts to approximate dietary PUFA amounts needed to support specific milk PUFA concentrations for puppies during suckling. However, it should be noted that exact amounts that are most beneficial for puppies themselves remain undetermined.

	FOOTNOTES

 
¹ Presented as part of the WALTHAM International Science Symposium: Nature, Nurture, and the Case for Nutrition held in Bangkok, Thailand, October 28–31, 2003. This symposium and the publication of the symposium proceedings were sponsored by the WALTHAM Centre for Pet Nutrition, a division of Mars, Inc. Symposium proceedings were published as a supplement to The Journal of Nutrition. Guest editors for this supplement were D'Ann Finley, James G. Morris, and Quinton R. Rogers, University of California, Davis.

² This work is supported in part by Nestle-Purina PetCare and the Mark L. Morris Professorship in Clinical Nutrition at Texas A&M University.

⁴ Abbreviations used: AA, arachidonic acid; ALA, -linolenic acid; DHA, docosahexaenoic acid; DM, dry matter; DPA, docosapentaenoic acid; EPA, eicosapentaenoic acid; LA, linoleic acid; LCPUFA, long-chain polyunsaturated fatty acids.

	LITERATURE CITED

TOP MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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6. Scott, B. L. & Bazan, N. G. (1989) Membrane docosahexaenoate is supplied to the developing brain and retina by the liver. Proc. Natl. Acad. Sci. USA 86: 2903–2907.[Abstract/Free Full Text]

7. Anderson, G. J., Connor, W. E. & Corliss, J. D. (1990) Docosahexaenoic acid is the preferred dietary n-3 fatty acid for the development of the brain and retina. Pediatr. Res. 27: 89–97.[Medline]

8. Chen, H., Wiegand, R. D., Koutz, C. A. & Anderson, R. E. (1992) Docosahexaenoic acid increases in frog retinal pigment epithelium following rod photoreceptor shedding. Exp. Eye Res. 55: 93–100.[Medline]

9. Alvarez, R. A., Aguirre, G. D., Acland, G. M. & Anderson, R. E. (1994) Docosapentaenoic acid is converted to docosahexaenoic acid in the retinas of normal and prcd-affected miniature poodle dogs. Invest. Ophthalmol. Vis. Sci. 35: 402–408.[Abstract/Free Full Text]

10. Bauer, J. E., Dunbar, B. L. & Bigley, K. E. (1998) Dietary flaxseed in dogs results in differential transport and metabolism of n-3 polyunsaturated fatty acids. J. Nutr. 128: 2641S–2644S.[Free Full Text]

11. Iverson, S. J., Kirk, C. L., Hamosh, M. & Newsome, J. (1991) Milk lipid digestion in the neonatal dog. The combined actions of gastric and bile salt-stimulated lipases. Biochim. Biophys. Acta 1083: 109–119.[Medline]

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13. Cox, M. L., Lees, G. E., Kashtan, C. E. & Murphy, K. E. (2003) Genetic cause of x-linked Alport syndrome in a family of domestic dogs. Mamm. Genome 14: 396–403.[Medline]

14. Demmelmair, H., Baumheur, M., Koletzko, B., Dokoupil, K. & Kratl, G. (2001) Investigation of long chain polyunsaturated fatty acid metabolism in lactating women by means of stable isotope techniques. Adv. Exp. Med. Biol. 501: 169–177.[Medline]

15. Francois, C. A., Conner, S. L., Bolewicz, L. C. & Conner, W. E. (2003) Supplementing lactating women with flaxseed oil does not increase docosahexaenoic acid in their milk. Am. J. Clin. Nutr. 77: 226–233.[Abstract/Free Full Text]

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