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© 2005 The American Society for Nutritional Sciences J. Nutr. 135:1960-1966, August 2005

Nutritional Neurosciences

Long-Chain (n-3) Polyunsaturated Fatty Acids Are More Efficient than {alpha}-Linolenic Acid in Improving Electroretinogram Responses of Puppies Exposed during Gestation, Lactation, and Weaning1

Kimberly M. Heinemann*, Mark K. Waldron{dagger}, Karen E. Bigley*, George E. Lees and John E. Bauer*,2

Department of Small Animal Clinical Sciences, * Comparative Nutrition Laboratory, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843 and {dagger} Nestle-Purina Pet Care, St. Louis, MO

2To whom correspondence should be addressed. E-mail: jbauer{at}cvm.tamu.edu.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Long-chain PUFAs (LCPUFAs) are essential for proper neural and retinal development in many mammalian species. We investigated puppies born to dogs fed diets containing varying amounts of vegetable and marine (n-3) fatty acids during gestation/lactation. The fatty acid compositions of dogs’ milk and puppy plasma phospholipids were evaluated, and electroretinographic responses of the young dogs were determined after they were weaned to the same diets. Dogs’ milk fatty acid composition reflected the diets fed during gestation/lactation. The milk of dogs fed a high {alpha}-linolenic acid (ALA) diet was enriched in ALA but not docosahexaenoic acid (DHA). Puppies fed this ALA-enriched milk accumulated more plasma phospholipid DHA than the low (n-3) fatty acid group. However, this accumulation was less than that obtained in puppies fed preformed DHA during development and suckling (P < 0.05). Electroretinograms (ERGs) of 12-wk-old puppies revealed significantly improved visual performance in dogs fed the highest amounts of (n-3) LCPUFAs (P < 0.05). These puppies demonstrated improved rod response (improved amplitude and implicit time of the a-wave, P < 0.05). Puppies from the low (n-3) fatty acid group exhibited the poorest ERG responses compared with the high-marine or high-vegetable (n-3) groups. A novel parameter devised in this study, the initial intensity at which the a-wave was detectable (i.e., threshold intensity), also demonstrated that retinal response of puppies consuming the (n-3) LCPUFA-containing diets occurred at lower light intensity, thereby exhibiting greater rod sensitivity, than the other diet groups. These findings indicate that preformed dietary (n-3) LCPUFA is more effective than ALA in enriching plasma DHA during perinatal development and results in improved visual performance in developing dogs.

KEY WORDS: • (n-3) long-chain PUFA • dogs • electroretinogram • vision • development

In the last 20 years, the importance of (n-3) fatty acids in brain and retinal development has become increasingly evident. Questions remain, however, one of which is whether {alpha}-linolenic acid (ALA)3 provided as a precursor is sufficient for optimal development or whether preformed docosahexaenoic acid (DHA) is required. The greatest concern is for premature infants, whose intrauterine development is shortened, often by several weeks (1).

Neural development in primates begins in the 3rd trimester of gestation, peaks about the time of birth, and continues for about 18–24 mo after parturition (2,3). During this developmental period, arachidonic acid (AA) and DHA are rapidly incorporated into the neural tissues (4,5). The high amounts of DHA in the brain and especially in the retina suggest a functional role in these tissues (6). Deficiency of (n-3) PUFAs during the developmental phase of neural tissues can result in irreversible functional abnormalities. Electroretinogram (ERG) data from humans and monkeys indicate decreased amplitudes and increased implicit times of both the a- and b-waves in response to (n-3) PUFA insufficiency (7,8). Reduced a- and b-wave amplitudes were also reported in (n-3)–deficient rats; however, retinal function was restored when the rats were fed (n-3)–replete diets (9).

DHA is highly conserved in the retina, and it has a role in neurological function in this tissue (7). The canine retina is capable of synthesizing DHA from its 22-carbon precursor, docosapentaenoic acid [22:5 (n-3), DPA] (10). Bauer et al. (11) reported the accumulation of DPA, but not DHA, in plasma phospholipids in dogs when the precursor ALA was fed. It is likely, therefore, that the dog retina, and presumably other nervous tissues, synthesizes and utilizes DHA in a manner similar to other mammalian species and that plasma DPA provides the substrate for such synthesis. Thus, a dietary source of preformed DHA or one of its precursors may be necessary during gestation and suckling for normal neural development in dogs.

It is possible that ALA may be sufficient as a dietary precursor for the synthesis of requisite amounts of DHA during pre- and postnatal development. However, what quantity of ALA may be needed to optimize neural development in companion animals is not known at present. Additionally, because both (n-6) and (n-3) precursor fatty acids compete metabolically for the same enzyme systems, it also is unclear what relative amounts may be needed.

Electroretinography is a sensitive and quantitative measure of retinal function in humans and animals (12,13). The ERG recording represents photoreceptor responses, and their subsequent postsynaptic signals, to a series of flash stimuli of varying intensity. It is a summation of responses across the retina and includes the responses of many retinal cell types. Major ERG components have been studied in dogs (14), and studies support using dogs as a suitable model for studies of human retinal physiology and pathology (15).

The goal of the present study was to assess the effect of maternal and perinatal dietary (n-3) fatty acid supply on retinal function in neonatal dogs. To our knowledge, such a study has not been reported.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Animals and diets. An existing breeding colony of dogs provided bred hound/Labrador retriever dogs and their puppies for this study, as described previously (16). Dogs were individually maintained in kennels according to the American Physiological Society Guidelines for Animal Research, and protocols were approved by the Texas A&M University Animal Care and Use Committee. Clinically normal, sexually intact female dogs (n = 12) from this colony, 2–4 y old, were randomly assigned to 1 of 4 diet groups. The diets were fed from the time of estrus, breeding, artificial insemination, and throughout gestation, parturition, and lactation (n = 3/diet group) (16). The number of puppies available for study in each group varied due to differences in the litter size obtained from each breeding.

All diets contained sufficient amounts of linoleic acid (LA), ranging from 1.8 to 3.5% dry matter (DM), and differed in their fatty acid composition. Each diet consisted of ~15% total fat and contained one of the following as its primary fat source: beef tallow, "low" amounts of Menhaden fish oil, "high" amounts of Menhaden fish oil, or linseed oil. Each diet was designed to have a unique ratio of ALA to (n-3) LCPUFA content. Based on these relative amounts, the diets were designated as low ALA/low (n-3) LCPUFA (Lo/Lo; tallow); low ALA/moderate (n-3) LCPUFA (Lo/Mod; low Menhaden fish oil); low ALA/high (n-3) LCPUFA (Lo/Hi; high Menhaden fish oil); and high ALA/low (n-3) LCPUFA (Hi/Lo; linseed oil) (Table 1). The expected nutrient composition of the diets (by weight) was 71.8% moisture, 27.8% protein, 15.1% fat, 9.1% moisture, 5.7% ash, 2.0% crude fiber, and the remainder carbohydrate. The diets were analyzed by Purina Laboratories and were within the expected analytical variance of these targets (Table 2).


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  		TABLE 1 Experimental diet fatty acid concentration1

 

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  		TABLE 2 Experimental diet nutrient profiles1, 2

 
After parturition, puppies were allowed to suckle ad libitum. Milk samples were collected from all bitches by manual expression during lactation on d 4, 10, 16, and 28 for lipid extraction and fatty acid profile analyses, as described previously (16). At 21 d postpartum, a gruel consisting of the mothers’ respective diets and water was offered to the puppies 3 times a day in addition to suckling. Gradually, the time the puppies spent suckling was decreased until they were completely weaned by d 42. Upon weaning, puppies continued to consume the same diets as their mothers until 12 wk of age. The puppies were weighed daily until 6 wk of age and 2 times/wk thereafter to monitor proper growth and development. Puppies that failed to thrive were removed from the study and supplemented to ensure proper nutrition. Generally this occurred only when litter size was large; small puppies were the most likely to be removed. Venous blood samples were collected from the puppies into EDTA-containing tubes on d 4, 10, 16, 28, 70, and 84 of age. The puppies were removed from their mothers 3 h before blood collection on d 4, 10, 16, and 28, and food was withheld overnight on d 70 and 84. Blood (2 mL) was taken on each sample day through d 16. On d 28, 4 mL was collected, and on d 70 and 84, 7 mL was obtained. Plasma and milk total lipids were extracted with chloroform:methanol (2:1, v:v) using a modified Folch procedure (17). Total plasma phospholipids (PLs) were separated using TLC (18). FAMEs of the plasma PLs and milk total lipids were prepared and fatty acid profiles were determined using capillary GC (11).

At 12 wk of age, retinal function of the puppies was assessed via electroretinography. ERGs of both eyes of all puppies studied were recorded using a computer-based ERG acquisition system that had been custom designed and built at Cornell University, using Windows-based software (Microsoft). It was acquired from colleagues at the Cornell University veterinary hospital (Dr. Ellis Loew).

On the day ERGs were performed, 12-wk-old puppies were adapted to the dark for 2 h before measurement. Before the procedure, 0.04 mg/kg atropine sulfate (Sparhawk Laboratories) was administered subcutaneously as a preanesthetic. This was followed by subcutaneous injection of 20 mg/kg ketamine hydrochloride (Ketaset®, Fort Dodge Animal Health) and 2.0 mg/kg xylazine (Vedo). When the puppies were sufficiently sedated, they were positioned in lateral recumbency on an examination table, and subdermal platinum-iridium needle electrodes were placed on the muzzle (indifferent) and in an ear flap (ground). The eye to be tested was then exposed using an ocular speculum inserted underneath the lids and the nictitating membrane. Local anesthesia was achieved using 1 drop of 0.5% solution of proparacaine hydrochloride (Ophthetic®, Allergan America) applied to the cornea, and pupillary mydriasis was induced by the addition of 1 drop of 1.0% tropicamide (Mydriacyl®, Alcon Laboratories) before the active contact lens electrode (ERG-Jet, LKC Technology) was placed on the eye.

Each eye was tested separately using a series of square-wave flash stimuli, 50 ms in duration, with an interflash interval of 5 s, from a white-light-emitting diode placed ~1 cm from the cornea. The ERGs were obtained at 10 settings of increasing intensity (0.5-log-unit steps) up to b-wave saturation. Earlier, the highest intensity setting had been shown to saturate the rod response in dogs (E. Loew, Cornell University, personal communication). The parameters used to assess ERG characteristics were a- and b-wave amplitude and a- and b-wave implicit time. A typical ERG series obtained with the equipment used in this study is shown in Figure 1.



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  		FIGURE 1 A representative ERG series from a puppy in the Lo/Hi group. The intensity of the stimulus increased in half log-unit steps, eliciting ERGs starting from the bottom of the figure.

 
An additional parameter, ä, was derived from the slope of the a-wave (19). The ERG software used in the study calculated the slope of the descending limb of the a-wave for the 3 highest-intensity responses; the increase in magnitude of this slope as a function of intensity was reported as the parameter ä. The slope for each of the 3 intensities was then plotted against flash intensity. The resultant data were modeled with linear regression to yield ä as the calculated slope parameter (20). The slope ä represents the increase in initial response with increasing light intensity (21).

    Statistical methods. Data are expressed as means ± SD. The effects of diet and time on individual plasma PL fatty acid data were evaluated using repeated-measures ANOVA with "litter" as the experimental unit (n = 12). For the milk data, "dam" was the experimental unit (n = 3). Significance was set at P < 0.05. Where appropriate, multiple comparisons for main effects of diet, time, and diet x time interactions were performed at P < 0.05 (Statistix 7.0, Analytical Software). Where there was a significant interaction of group and time, contrasts were made for each plasma PL fatty acid using Bonferroni’s test to determine where the difference occurred. An experiment-wide type I error of 0.05 was maintained. Statistical analyses were performed on ERG parameters using data obtained from the 8th flash intensity using 1-way ANOVA with multiple comparisons performed at P < 0.05. Because each eye was tested separately, sample sizes were twice that of the number of puppies in each group. Statistical analyses were performed on these data as well as on the mean values of both eyes together. All data followed a normal distribution at P < 0.05 using the Shapiro-Wilk test. If variances were nonhomogeneous, log10 transformed data were analyzed.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Because no time effects were present for any fatty acid in either milk or plasma samples, values reported in the text and presented in the figures were combined for all 4 sample days during suckling and both sample days during the postweaning period. The ERG data are presented as means ± SD of each eye evaluated separately using 18 puppies in group Lo/Lo, 13 in group Lo/Mod, 10 in group Lo/Hi, and 15 in group Hi/Lo. The measurements on both eyes were also averaged and statistics were calculated. Significant differences found were the same in both cases.

    Suckling period. There was a dose response of dietary LA (from milk) in neonatal plasma (Fig. 2A). Milk from the Hi/Lo group contained the highest concentration of LA of any group (P < 0.05) (Table 3); consequently, so did the plasma PL from neonates in this group. Plasma PL arachidonate levels in neonates from the Lo/Lo group were higher than those in all other groups (Fig. 2A).



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  		FIGURE 2 Proportions of plasma PL LA and AA (A) and major (n-3) fatty acids (B) in puppies during suckling. Because no time effects were observed for any fatty acid, each litter’s mean value from all 4 sample days during suckling was pooled using repeated-measures ANOVA and analyzed for main diet effects. Values are means ± SD, n = 12. Means for a fatty acid without a common letter differ, P < 0.05.

 

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  		TABLE 3 Major (n-6) and (n-3) PUFAs in milk of dogs during suckling1, 2

 
Dose responses occurred for plasma PL ALA, eicosapentaenoic acid (EPA), and DHA. Puppies in the Hi/Lo group received the largest relative amount of ALA in their diets (Table 3) and their plasma PLs also contained more ALA than the other groups (P < 0.05, Fig. 2B). Milk EPA and DHA levels were higher in the 2 fish-oil diet groups (Lo/Mod and Lo/Hi) than in the Lo/Lo and Hi/Lo groups (P < 0.05, Table 3). Similarly, neonatal plasma from these groups contained higher amounts of EPA and DHA in the PL fraction (P < 0.05, Fig. 2B). In addition, both milk and neonatal plasma PL EPA in the Lo/Hi group were higher than in the Lo/Mod group (P < 0.05, Table 3 and Fig. 2B). Despite a 2-fold difference between milk EPA in the Lo/Mod and Hi/Lo groups (P < 0.05), plasma PL EPA from neonates in these groups did not differ (P = 0.11). However, milk ALA was markedly greater in the Hi/Lo group than in the other groups (P < 0.05).

Puppy dietary (milk) DPA levels were lower in the Hi/Lo group than in the Lo/Hi group (P < 0.05, Table 3). However, plasma DPA concentrations were lower in the Lo/Lo group than in the other groups (P < 0.05).

A DHA dose response also occurred in the plasma PLs of puppies fed the (n-3) LCPUFA diets (Fig. 2B). Puppies in the Hi/Lo group received the greatest amount of ALA and lowest relative amounts of DHA in milk, yet their plasma PL DHA levels were higher than in the low (n-3) diet group but lower than in the marine oil groups (P < 0.05) (Fig. 2B). Plasma PL DHA concentrations in all diet groups differed from each other (P < 0.05).

    Postweaning period. Plasma PL fatty acid levels in the puppies consuming the dry diets were similar to those observed during suckling. A dose response again occurred for LA, and AA concentrations in the Lo/Lo group were higher than in all other groups (P < 0.05, Fig. 3A). The Hi/Lo group had significantly greater plasma PL ALA levels compared with the other 3 groups (P < 0.05, Fig. 3B). Plasma PL EPA in both the Hi/Lo and Lo/Hi groups was greater than in the Lo/Lo group (P < 0.05, Fig. 3B).



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  		FIGURE 3 Proportions of plasma PL LA and AA (A) and major (n-3) fatty acids (B) in puppies after weaning. Because no time effects were observed for any fatty acid, each litter’s mean value from both sample days after weaning was pooled using repeated-measures ANOVA and analyzed for main diet effects. Values are means ± SD, n = 12. Means for a fatty acid without a common letter differ, P < 0.05.

 
During the postweaning period, a dose response of fish oil was not as apparent in plasma in the Lo/Lo, Lo/Mod, and Lo/Hi puppies, in contrast to the suckling period, during which a dose effect of DHA was clearly observed. Nevertheless, plasma PL DHA concentrations in both fish-oil groups remained higher than in the Hi/Lo and Lo/Lo diet groups after weaning (P < 0.05).

    Electroretinography. The puppies in the Lo/Hi and Hi/Lo groups demonstrated higher a-wave responses (a-amp) than those in the Lo/Lo and Lo/Mod groups (P < 0.05, Table 4). Mean a-wave implicit time (ai) in the Lo/Hi group was lower than that in the Lo/Lo group (P < 0.05), indicating a quicker response in the Lo/Hi group.


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  		TABLE 4 ERG parameters of 12-wk-old puppies obtained at the 8th light intensity (a-amp, b-amp, ai, and bi) and threshold intensity (It)1, 2

 
The ä parameter followed a pattern similar to that obtained for the a-amp data, again demonstrating improved visual performance in the Lo/Hi group compared with the Lo/Lo and Lo/Mod groups (P < 0.05). The ä value of the Hi/Lo group was intermediate between the other values and not significantly different from them (P = 0.116).

The Lo/Hi group showed a higher b-wave response (b-amp) compared with the Lo/Mod group (P < 0.05), and puppies in the Hi/Lo group elicited a quicker b-wave response (lower bi) than those in the Lo/Lo and Lo/Mod groups (P < 0.05).

Not all dogs responded equally to all intensities of light. In some puppies, an a-wave response was not elicited until the 7th or 8th flash intensity, whereas others responded as early as the 4th flash intensity. Based on this observation, mean values were obtained for the threshold intensity (It), defined as the intensity at which the a-wave was first detected. To our knowledge, such a parameter was not reported previously. It values in the Lo/Hi group were lower than those in the Lo/Lo and Hi/Lo groups (P < 0.05, Table 4).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
    Plasma phospholipids. For the first 21 d of life during suckling, maternal milk was the sole source of nutrition for the neonates. By 42 d of age, the puppies had been weaned completely onto the same experimental diets as their mothers had been fed. With few exceptions, the plasma PL fatty acid profiles of puppies in the suckling and postweaning periods did not differ. Dose responses occurred for plasma PL LA, ALA, and DHA in the Lo/Lo, Lo/Mod, and Lo/Hi groups during suckling. However, this response was not as prominent after weaning.

The Hi/Lo and Lo/Lo groups received approximately the same amounts of EPA from mothers’ milk, yet plasma PL EPA concentrations in the Hi/Lo puppies were significantly higher than in the Lo/Lo puppies. By comparison, the Lo/Mod group milk contained significantly higher EPA levels than the Hi/Lo group milk, yet the PL EPA levels in the 2 groups of puppies did not differ. Furthermore, milk DHA levels in the Hi/Lo group were the lowest among all 4 groups, yet plasma PLs in these puppies contained markedly elevated concentrations of DHA. From these data, it is apparent that neonatal dogs are capable of synthesizing both EPA and DHA from ALA. This finding is consistent with results from studies in human infants reporting the ability of both fetuses and neonates to synthesize LCPUFAs from their 18-carbon precursors (21–25). It is thus of interest that when adult dogs are fed higher amounts of ALA, there is little additional accumulation of PL DHA in plasma (11), yet neonates appear to have considerable PL DHA in plasma under similar dietary conditions. The reasons for this observation are unknown. However, it appears that neonatal dogs may preferentially synthesize DHA at a time of life when demand for this fatty acid is especially high. Although not examined in this study, this observation suggests that peroxisomal conversion of DPA to DHA in young dogs may be increased compared with adults.

The significantly higher plasma PL AA levels in the Lo/Lo group compared with the other groups were likely due to substitution of AA by increased concentrations of (n-3) LCPUFAs found in the Lo/Mod, Lo/Hi, and Hi/Lo diets. Numerous studies report that when dietary (n-3) PUFA content is high, EPA is substituted for AA in PLs. It is also possible that low concentrations of dietary ALA in the Lo/Lo diet may have facilitated increased rates of elongation and desaturation of LA by means of reduced competition for {Delta}6 desaturase. Our laboratory previously reported that the Km of hepatic {Delta}6 desaturase for LA in dogs is twice as large as that for ALA. However, because most dog diets are especially replete in LA, its conversion to AA readily occurs, especially in the absence of high dietary ALA (26). Competition for {Delta}6 desaturation is of particular concern in developing neonates. Therefore, careful consideration must be taken when formulating milk replacers, whether for dogs or humans, to ensure adequate tissue enrichment of both (n-6) and (n-3) LCPUFAs.

Noted differences in the plasma distribution of EPA were present between the suckling and postweaning periods. During suckling, the plasma PL EPA content in the high-ALA group was significantly lower than in the fish-oil diet groups. However, after weaning, the EPA content in the high-ALA group was equivalent to that in the Lo/Hi group and greater than that in the Lo/Mod group. Lower plasma EPA during suckling may indicate a more efficient conversion of ALA to DPA and DHA in younger puppies, when the demand for DHA is greatest. After weaning, demand for DHA by neural tissues is decreased, allowing EPA to accumulate. These findings are consistent with studies of human infants suggesting that LCPUFA formation is a function of gestational age, and that the elongation/desaturation pathways are more active at earlier gestational ages (27).

The enrichment of plasma PL DHA in the Hi/Lo group did not parallel the increase in EPA in this group after weaning and is consistent with previous studies. Bauer et al. (11) reported the accumulation of plasma EPA and DPA, but not DHA, in adult dogs fed flaxseed-enriched diets. Although DHA is not enriched in plasma under this condition, tissue needs may still be met by the further conversion of DPA in those tissues per se. In humans and guinea pigs, conversion of ALA is also far less efficient at enriching plasma and cellular DHA than when preformed DHA is supplied in the diet (28–31). Although the accumulation of DHA in neural tissues was not measured in the above studies, other investigations in guinea pigs and baboons showed that dietary DHA was 10 and 7 times more effective, respectively, as a substrate for neural DHA than dietary ALA (5,32–34). These findings, in combination with the data presented here, underscore the concept that dietary ALA, unless markedly increased, is not an equivalent substitute for dietary DHA. These data also show that a diet high in ALA supported plasma PL accumulation of DHA during suckling but not after weaning and that young dogs are capable of converting ALA to DHA during the suckling period.

    Electroretinography. Numerous investigations have evaluated the effects of breast milk or (n-3) LCPUFA-supplemented formula on retinal development in neonatal humans and other primates (8,35–46). Studies in humans and nonhuman primates indicate that LCPUFA synthesis may be more active in late-term and premature fetuses than in older neonates (28,46). All puppies in this study were born at full term, and all received milk, rather than formula, during suckling. Beneficial effects of dietary (n-3) PUFAs on ERG response occurred in the puppies in the present study.

ERG data obtained at the 8th light intensity were used for analysis of all ERG parameters because the highest intensity (i.e., 10th) saturates the b-wave response in dogs. Such a saturation effect could possibly mask measurable differences in retinal function among diet groups.

The descending segment of the a-wave represents photoreceptor activity, whereas the b-wave is the composite postsynaptic response of the bipolar and Müller cells (20,47,48). The ä parameter is related to the time course of activation for the phosphodiesterase cascade in the rod outer segments (ROSs) (19).

In the present study, marked ERG differences due to diet (n-3) fatty acids were found, especially between the Lo/Hi and Lo/Lo groups. Differences between the Lo/Hi and Lo/Mod groups were present but less pronounced.

The data suggest that the b-wave response in breast-fed dogs may be independent of retinal DHA content and that other factors may be involved. Diau et al. reported in 4-wk-old baboons that improvement of the b-wave response occurs independently of retinal DHA content and that "DHA per se is not the limiting factor in the development of the b-amplitude in formula-fed neonates" (19). Also, only modest differences in b-wave implicit time were present. Puppies in the Hi/Lo group had shorter bi compared with the Lo/Lo and Lo/Mod groups (P < 0.05), and intermediate values were seen in the Lo/Hi group. A reduction in bi may indicate increased efficiency of postsynaptic signal transduction of the visual response in the presence of increased retinal DHA. Further studies, however, should be conducted using reliable biomarkers of retinal DHA status.

Puppies in the Lo/Lo group had poorer retinal performance as measured by the a-amp and ai compared with the Lo/Hi group. The a-amp in the Hi/Lo puppies did not differ from the Lo/Hi group, but was superior to both the Lo/Mod and Lo/Lo groups. Although the improved rod response (highest a-amp, lowest ai) occurred in those dogs that received the highest amounts of dietary DHA, it was not significantly better than that of dogs fed the markedly ALA-enriched diet. Similarly, there was no difference in rod photoreceptor function in monkeys fed either 8% ALA or 0.6% DHA (8) or between monkeys fed either ALA as the sole (n-3) PUFA or a combination of AA and DHA (43). Furthermore, a study in guinea pigs reported the highest ERG amplitudes in the group fed ALA alone rather than in the group fed ALA in combination with (n-3) LCPUFAs (49). Consistent with these findings, it can be concluded from the present study that gestation diets containing 5.5 g/kg DM DHA and milk containing 25 g DHA/kg milk increased photoreceptor activity in young dogs. In addition, it appears that gestation diets and milk high in ALA are able to induce adequate DHA synthesis and neural enrichment such that photoreceptor activity is at least moderately improved. However, the formulation of high-ALA diets, such as the one in this study, may be of little practical use due to the undesirable odor and variable palatability associated with diets of this type.

It should be noted that a recent study using rats found desensitization of visual signaling in (n-3) fatty acid–deficient ROS membranes to be correlated with reduced rhodopsin activation, rhodopsin-transducin (Gt) coupling, cGMP phosphodiesterase activity, and slower formation of metarhodopsin II (MII) and the MII-G(t) complex relative to (n-3) fatty acid–adequate ROS. These findings provide an explanation for the reduced amplitude and delayed ERG a-waves that occurred in (n-3) fatty acid–deficient rats (50). They may also help explain similar ERG findings in other species studied to date, including dogs.

A decrease in the ä parameter indicates a reduction in the initial amplification cascade induced when a photon is absorbed by rhodopsin (19). Therefore, a higher ä represents an increase in the initial amplification, although it does not distinguish whether such an increase is due to increased rhodopsin content or increased efficiency of photon absorption. However, greater amplification demonstrates a favorable response when the overall value of this parameter is elevated. In light of the higher ä in the Lo/Hi group, we concluded that DHA conveys an additional beneficial effect (i.e., an increase) on the initial amplification of the photoreceptor response.

A novel parameter devised in this study is the ERG threshold intensity, the light intensity at which the initial a-wave occurred. Puppies that consumed the highest amounts of (n-3) LCPUFAs (Lo/Hi and Lo/Mod diets) elicited the earliest photoreceptor response, and at a lower intensity than those of the Lo/Lo and Hi/Lo groups. Thus, puppies whose diets contained (n-3) LCPUFAs had lower rod thresholds (i.e., greater rod sensitivity) than puppies in the other groups. Studies in both term and preterm human infants have reported an association between DHA status and retinal sensitivity (51,52).

Taken together, the data from this study indicate an advantage of dietary DHA for retinal function in young dogs. Puppies consuming the highest concentrations of DHA in both milk and dry diet consistently demonstrated improved rod sensitivity (as measured by a-amp, ai, and It) and elicited the greatest increase in the amplification of the phosphodiesterase cascade. Although visual performance in puppies fed the high-ALA diet was not significantly lower than in those fed DHA, it was not generally equivalent to the level of retinal function observed in the DHA-fed puppies.

Thus, when data from previous studies and the present work are considered collectively, the likelihood of dietary DHA in dogs resulting in retinal enrichment and its associated improvement in ERG-related measures helps confirm and extend the importance of DHA in fetal and neonatal development comparatively among mammalian species. New data reported here on the relation of dietary PUFAs and milk fatty acid composition will also aid in the development of the most appropriate diets for gestation, lactation, and weaning in dogs.


   ACKNOWLEDGMENTS
 
The authors wish to thank Dr. Ellis Loew, Cornell University, for providing the electroretinogram hardware and software for this study and for his helpful discussions, and Mary Sanders for technical assistance.


   FOOTNOTES
 
1 Funded by Nestlé-Purina PetCare, St. Louis, MO and the Mark L. Morris Professorship in Clinical Nutrition, Texas A&M University. Back

3 Abbreviations used: AA, arachidonic acid; a-amp, a-wave response; ai, a-wave implicit time; ALA, {alpha}-linolenic acid; b-amp, b-wave response; bi, b-wave implicit time; DHA, docosahexaenoic acid; DM, dry matter; DPA, docosapentaenoic acid; EPA, eicosapentaenoic acid; ERG, electroretinogram; Gt, transducin; Hi/Lo, high {alpha}-linolenic acid/low (n-3) LCPUFA; It, threshold intensity; LA, linoleic acid; LCPUFA, long-chain PUFA; Lo/Lo, low {alpha}-linolenic acid/low (n-3) long-chain PUFA; Lo/Hi, low {alpha}-linolenic acid/high (n-3) long-chain PUFA; Lo/Mod, low {alpha}-linolenic acid/moderate (n-3) long-chain PUFA; MII, metarhodopsin II; PL, phospholipid; ROS, rod outer segment. Back

Manuscript received 2 February 2005. Initial review completed 16 March 2005. Revision accepted 18 May 2005.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

1. Hornstra, G. (2000) Essential fatty acids in mothers and their neonates. Am. J. Clin. Nutr. 7:1262S-1269S.

2. Menard, C. R., Goodman, K. J., Corso, T. N., Brenna, J. T. & Cunnane, S. C. (1998) Recycling of carbon into lipids synthesized de novo is a quantitatively important pathway of alpha-[U-13C]linolenate utilization in the developing rat brain. J. Neurochem. 71:2151-2158.[Medline]

3. Martinez, M. (1992) Tissue levels of polyunsaturated fatty acids during early human development. J. Pediatr. 120:S129-S138.[Medline]

4. Sinclair, A. J. (1975) Long chain polyunsaturated fatty acids in the mammalian brain. Proc. Nutr. Soc. 34:287-291.[Medline]

5. Greiner, R. C., Winter, J., Nathanielsz, P. W. & Brenna, J. T. (1997) Brain docosahexaenoate accretion in fetal baboons: bioequivalence of dietary alpha-linolenic and docosahexaenoic acids. Pediatr. Res. 42:826-834.[Medline]

6. Litman, B. J., Niu, S. L., Polozova, A. & Mitchell, D. C. (2001) The role of docosahexaenoic acid containing phospholipids in modulating G protein-coupled signaling pathways. J. Mol. Neurosci. 16:237-242.[Medline]

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. Jeffrey, B. G., Mitchell, D. C., Gibson, R. A. & Neuringer, M. (2002) n-3 Fatty acid deficiency alters recovery of the rod photoresponse in rhesus monkeys. Investig. Ophthalmol. Vis. Sci. 43:2806-2814.[Abstract/Free Full Text]

9. Bourre, J. M., Francois, M., Youyou, A., Dumont, O., Piciotti, M., Pascal, G. & Durand, G. (1989) The effects of dietary {alpha}-linolenic acid on the composition of nerve membranes, enzymatic activity, amplitude of electrophysiological parameters, resistance to poisons and performance of learning tasks in rats. J. Nutr. 119:1880-1892.

10. Fliesler, S. J. & Anderson, R. E. (1983) Chemistry and metabolism of lipids in the vertebrate retina. Prog. Lipid Res. 22:79-131.[Medline]

11. 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(suppl.):2641S-2644S.[Free Full Text]

12. Armington, J. C. (1974) Dietary flaxseed in dogs results in differential transport and metabolism of (n-3) polyunsaturated fatty acids. The Electroretinogram Academic Press New York, NY.

13. Gouras, P. (1970) Electroretinography: some basic principles. Investig. Ophthalmol. 9:557-569.[Abstract/Free Full Text]

14. Yanase, J., Ogawa, H. & Ohtsuke, H. (1996) Scotopic threshold response of the electroretinogram of dogs. Am. J. Vet. Res. 57:361-366.[Medline]

15. Kommonen, B., Dawson, W. W. & Parmer, R. (1991) Pigment epithelial function in canine retina. Am. J. Vet. Res. 52:1341-1344.[Medline]

16. Bauer, J. E., Heinemann, K. M., Bigley, K. E., Lees, G. E. & Waldron, M. K. (2004) Maternal diet {alpha}-linolenic acid during gestation and lactation does not increase canine milk docosahexaenoic acid. J. Nutr. 134:2035S-2038S.[Free Full Text]

17. Folch, J., Lees, M. & Sloane-Stanley, G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509.[Free Full Text]

18. Bauer, J. E., McAlister, K. G., Rawlings, J. M. & Markwell, P. (1997) Molecular species of cholesteryl esters formed via plasma lecithin:cholesterol acyltransferase in fish oil supplemented dogs. Nutr. Res. 17:861-872.

19. Diau, G. Y., Loew, E. R., Wijendran, V., Sarkadi-Nagy, E., Nathanielsz, P. W. & Brenna, J. T. (2003) Docosahexaenoic and arachidonic acid influence on preterm baboon retinal composition and function. Investig. Ophthalmol. Vis. Sci. 44:4559-4566.[Abstract/Free Full Text]

20. Breton, M. E., Schueller, A. W., Lamb, T. D. & Pugh, E. N. (1994) Analysis of ERG a-wave amplification and kinetics in terms of the G-protein cascade of phototransduction. Investig. Ophthalmol. Vis. Sci. 35:295-309.[Abstract/Free Full Text]

21. Li, Z., Kapan, M. L. & Hachey, D. L. (2000) Hepatic microsomal and peroxisomal docosahexaenoate biosynthesis during piglet development. Lipids 35:1325-1333.[Medline]

22. Demmelmair, H., von Schenck, U., Behrendt, E., Sauerwald, T. U. & Koletzko, B. (1995) Estimation of arachidonic acid synthesis in full term neonates using natural variation of 13C content. J. Pediatr. Gastroenterol. Nutr. 21:31-36.[Medline]

23. Salem, N., Jr, Wegher, B., Mena, P. & Uauy, R. (1996) Arachidonic and docosahexaenoic acids are biosynthesized from their 18-carbon precursors in human infants. Proc. Natl. Acad. Sci. U.S.A. 93:49-54.[Abstract/Free Full Text]

24. Sauerwald, T. U., Hachey, D. L., Jensen, C. L., Chen, H., Anderson, R. E. & Heird, W. C. (1996) Effect of dietary {alpha}-linolenic acid intake on incorporation of docosahexaenoic and arachidonic acids into plasma phospholipids of term infants. Lipids 31(suppl.):S131-S135.

25. Sauerwald, T. U., Hachey, D. L., Jensen, C. L., Chen, H., Anderson, R. E. & Heird, W. C. (1997) Intermediates in endogenous synthesis of C22:6 omega 3 and C20:4 omega 6 by term and preterm infants. Pediatr. Res. 41:183-187.[Medline]

26. Dunbar, B. L. & Bauer, J. E. (2002) Conversion of essential fatty acids by {Delta}-6 desaturase in dog liver microsomes. J. Nutr. 132:1701S-1703S.[Abstract/Free Full Text]

27. Uauy, R., Mena, P., Wegher, B., Nieto, S. & Salem, N., Jr (2000) Long chain polyunsaturated fatty acid formation in neonates: effect of gestational age and intrauterine growth. Pediatr. Res. 47:127-135.[Medline]

28. Fu, Z. & Sinclair, A. J. (2000) Increased {alpha}-linolenic acid intake increases tissue {alpha}-linolenic acid content and apparent oxidation with little effect on tissue docosahexaenoic acid in the guinea pig. Lipids 35:395-400.[Medline]

29. Gerster, H. (1998) Can adults adequately convert {alpha}-linolenic acid to eicosapentaenoic acid and docosahexaenoic acid?. Int. J. Vitam. Nutr. Res. 68:159-173.[Medline]

30. Li, D., Sinclair, A., Wilson, A., Nakkote, S., Kelly, F., Abedin, L., Mann, N. & Turner, A. (1999) Effect of dietary {alpha}-linolenic acid on thrombotic risk factors in vegetarian men. Am. J. Clin. Nutr. 69:872-882.[Abstract/Free Full Text]

31. Mantizioris, E., James, M. J., Gibson, R. A. & Cleland, L. G. (1994) Dietary substitution with {alpha}-linolenic acid-rich vegetable oil increases eicosapentaenoic acid concentration in tissues. Am. J. Clin. Nutr. 59:1304-1309.[Abstract/Free Full Text]

32. Abedin, L., Lien, E. L., Vingrys, A. J. & Sinclair, A. J. (1999) The effects of dietary {alpha}-linolenic acid compared with docosahexaenoic acid on brain, retina, liver, and heart in the guinea pig. Lipids 34:475-482.[Medline]

33. Su, H. M., Bernardo, L., Mirmiran, M., Ma, X. H., Nathanielsz, P. W. & Brenna, J. T. (1999) Dietary 18:3 n-3 and 22:6 n-3 as sources of 22:6 n-3 accretion in neonatal baboon brain and associated organs. Lipids 34(suppl.):S347-S350.

34. Woods, J., Ward, G. & Salem, N., Jr (1996) Is docosahexaenoic acid necessary in infant formulas? evaluation of high linolenate diets in the neonatal rat. Pediatr. Res. 40:687-694.[Medline]

35. Neuringer, M., Connor, W. E., van Petten, C. & Barstad, L. (1984) Dietary omega-3 fatty acid deficiency and visual loss in infant rhesus monkeys. J. Clin. Investig. 73:272-276.

36. Innis, S. M., Nelson, C. M., Rioux, M. F. & King, D. J. (1994) Development of visual acuity in relation to plasma and erythrocyte omega-6 and omega-3 fatty acids in healthy term gestation infants. Am. J. Clin. Nutr. 60:347-352.[Abstract/Free Full Text]

37. Auestad, N., Montalto, M. B., Hall, R. T., Fitzgerald, K. M., Wheeler, R. E., Connor, W. E., Neuringer, M., Connor, S. L., Taylor, J. A. & Hartmann, E. E. (1997) Visual acuity, erythrocyte fatty acid composition, and growth in term infants fed formulas with long chain polyunsaturated fatty acids for one year. Pediatr. Res. 41:1-10.[Medline]

38. Auestad, N., Halter, M. A., Hall, R. T., Blatter, M., Bogle, M. L., Burks, W., Erickson, J. R., Fitzgerald, K. M. & Dobson, V., et al (2001) Growth and development in term infants fed long-chain polyunsaturated fatty acids: a double-masked, randomized, parallel, prospective, multivariate study. Pediatrics 108:372-381.[Abstract/Free Full Text]

39. Makrides, M., Simmer, K., Goggin, M. & Gibson, R. A. (1993) Erythrocyte docosahexaenoic acid correlates with the visual response of healthy, term infants. Pediatr. Res. 33:425-427.[Medline]

40. Makrides, M., Neumann, M. A., Jeffrey, B., Lien, E. L. & Gibson, R. A. (2000) A randomized trial of different ratios of linoleic to {alpha}-linolenic acid in the diet of term infants: effects on visual function and growth. Am. J. Clin. Nutr. 71:120-129.[Abstract/Free Full Text]

41. Makrides, M., Neumann, M. A. & Gibson, R. A. (2001) Perinatal characteristics may influence the outcome of visual acuity. Lipids 36:897-900.[Medline]

42. Carlson, S. E., Werkman, S. H., Rhodes, P. G. & Tolley, E. A. (1993) Visual-acuity development in healthy preterm infants: effect of marine oil supplementation. Am. J. Clin. Nutr. 58:35-42.[Abstract/Free Full Text]

43. Jeffrey, B. G., Mitchell, D. C., Hibbeln, J. R., Gibson, R. A., Chedester, A. L. & Salem, N. J. (2002) Visual acuity and retinal function in infant monkeys fed long-chain PUFA. Lipids 37:839-848.[Medline]

44. Benolken, R. M., Anderson, R. E. & Wheeler, T. G. (1973) Membrane fatty acids associated with the electrical response in visual excitation. Science (Washington, DC) 182:1253-1254.[Abstract/Free Full Text]

45. Wheeler, T. G., Benolken, R. M. & Anderson, R. E. (1975) Visual membranes: specificity of fatty acid precursors for the electrical response to illumination. Science (Washington, DC) 188:1312-1314.[Abstract/Free Full Text]

46. Su, H. M., Huang, M. C., Saad, N. M., Nathanielsz, P. W. & Brenna, J. T. (2001) Fetal baboons convert 18:3 n-3 to 22:6 n-3 in vivo: a stable isotope tracer study. J. Lipid. Res. 42:581-586.[Abstract/Free Full Text]

47. Karwoski, C. (1991) Introduction to the origins of electroretinographic components. Principles and Practice of Clinical Electrophysiology of Vision :87-90 Mosby St. Louis, MO.

48. Neuringer, M. (2000) Infant vision and retinal function in studies of dietary long-chain polyunsaturated fatty acids: methods, results, and implications. Am. J. Clin. Nutr. 71:256S-267S.[Abstract/Free Full Text]

49. Weisinger, H. S., Vingrys, A. J. & Sinclair, A. J. (1996) Effect of docosahexaenoic acid on the electroretinogram of the guinea pig. Lipids 31:65-70.[Medline]

50. Niu, S. L., Mitchell, D. C., Lim, S. Y., Wen, Z. M., Kim, H. Y., Salem, N., Jr & Litman, B. J. (2004) Reduced G protein-coupled signaling efficiency in retinal rod outer segments in response to n-3 fatty acid deficiency. J. Biol. Chem. 279:31098-31104.[Abstract/Free Full Text]

51. Malcolm, C. A., Hamilton, R., McCulloch, D. L., Montgomery, C. & Weaver, L. T. (2003) Scotopic electroretinogram in term infants born of mothers supplemented with docosahexaenoic acid during pregnancy. Investig. Ophthalmol. Vis. Sci. 44:3685-3691.[Abstract/Free Full Text]

52. van Houwelingen, A. C., Srensen, J. D., Hornstra, G., Simonis, M. M., Boris, J., Olsen, S. F. & Secher, N. J. (1995) Essential fatty acids in neonates after fish oil supplementation during late pregnancy. Br. J. Nutr. 74:723-731.[Medline]

53. Blank, C., Neumann, M. A., Makrides, M. & Gibson, R. A. (2002) Optimizing DHA levels in piglets by lowering the linoleic to {alpha}-linolenic acid ratio. J. Lipid Res. 43:1537-1543.[Abstract/Free Full Text]

54. Sarkadi-Nagy, E., Wijendran, V., Diau, G.-Y., Chao, A. C., Hsieh, A. T., Turpeinen, A., Nathanielsz, P. W. & Brenna, J. T. (2003) The influence of prematurity and long chain polyunsaturate supplementation in 4-week adjusted age baboon neonate brain and related tissues. Pediatr. Res. 54:244-252.[Medline]




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