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The Journal of Nutrition Vol. 128 No. 4 April 1998,
pp. 740-743
Department of Pediatrics, Division of Neonatology and * Department of Anatomy, University of South Florida College of Medicine, Tampa, FL 33612
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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The effect of pre- and postnatal maternal dietary fatty acid composition on neurodevelopment in rat pups was studied. Timed pregnant dams were fed, beginning on d 2 of gestation and throughout lactation, either nonpurified diet (reference) or a purified diet whose fat source (22% of energy) was either corn oil or menhaden fish oil. On postnatal d 3, pups were randomly cross-fostered among dams of the same diet group and culled to 10 pups per dam. Milk was removed from stomachs of culled pups for fatty acid analyses. From postnatal d 4 to 30, pups were assessed daily for the appearance of neurodevelopmental reflexes. Auditory brainstem conduction times were measured on postnatal d 23 and 29. Pups were killed on postnatal d 30, and cerebrums were removed for fatty acid analyses. The fatty acid composition of maternal milk and pup cerebrums reflected maternal diet with higher levels of (n-3) and (n-6) fatty acids in the fish oil and corn oil groups, respectively. The time of appearance of auditory startle was significantly delayed (P = 0.004), and auditory brainstem conduction times on postnatal d 23 and 29 were significantly longer in pups of the fish oil- than corn oil-fed dams (P 
0.05). A delay in the appearance of the auditory startle reflex and longer auditory brainstem conduction times in pups of dams fed fish oil-supplemented diet may be due to negative effects on myelination of the auditory brainstem pathway.
Fatty acids are constituents of phospholipids, which make up the structural matrix of cell and subcellular membranes. The 20- and 22-carbon long-chain polyunsaturated fatty acids are also precursors of eicosanoids. Alterations in tissue fatty acid composition can affect tissue function by modifying membrane fluidity or membrane thickness, by changing properties of the lipid phase, by specific interactions with membrane proteins or by altering the balance of eicosanoids synthesized (Stubbs and Smith 1993 Large quantities of long-chain polyunsaturated fatty acids, particularly arachidonic and docosahexanoic acids, are deposited in the central nervous system during brain growth (Arbuckle and Innis 1993 Cerebral cortex levels of docosahexanoic acid are reported to be higher in breast- than in formula-fed infants, which may relate to higher levels of this fatty acid in human milk (Farquharson et al. 1992 The role of dietary fatty acids in infant neurodevelopment remains controversial. In this study, we assessed the functional effects of pre- and postnatal dietary fatty acid composition on neurodevelopment in rats as measured by neurodevelopmental reflex appearance and auditory brainstem conduction times. The time of appearance of specific neurodevelopmental reflexes in rats has been established (Smart and Dobbing 1971 Animals and diets.
Beginning on d 2 of gestation and continuing throughout lactation, Sprague-Dawley rats (Zivic Miller, Pittsburg, PA) were fed either a reference diet (Laboratory Rodent Diet 5001, Purina Mills, Richmond, IN)6 or a synthetic diet (Purina Basal Diet 5755)7 whose fat source was either corn oil or menhaden fish oil. Diets were stored at Neurodevelopmental reflex assessments.
Smart and Dobbing (1971)
Auditory brainstem conduction times.
Auditory brainstem conduction times were measured on postnatal d 23 and 29. Pups were lightly sedated with intraperitoneal sodium pentobarbital (0.05 mg/g body weight) and 5.4 µg atropine to prevent respiratory congestion. Silver-chloride cup electrodes were placed on the vertex of the scalp and the right and left mastoid areas. A tubal insert earphone was inserted into the ear canal and 33/s, 0-70 dBpSPL, 8 kHz Blackman tonepips were delivered. The electroencephalogram was filtered (5 Hz to 3 kHz) and amplified (×100,000). Responses were averaged over 500-1000 samples, and the latencies of the eighth nerve and brainstem auditory components were measured digitally. Data are presented as brainstem conduction time from wave I, originating in the eighth cranial nerve, to wave III, generated by the nuclei of the brainstem auditory pathway (Jewett and Romano 1972 Fatty acid analyses.
All solvents were glass distilled, with 50 mg/L of butylated hydroxytoluene added to extraction solvents to minimize auto-oxidation. Homogenates of pup cerebrums were prepared with normal saline at 4°C using a glass homogenizer. Total lipid extracts of cerebrum homogenates, milk and diets were prepared. Lipid extracts of diet, maternal milk and pup cerebrum homogenates were prepared, methylated, layered with nitrogen and analyzed within 24 h of extraction (Rayon et al. 1997 Statistical analyses.
Statistical analyses were performed using the SPSS Statistical Package (Chicago, IL). Comparisons were made between the fish oil and corn oil group using Levene's test for equality of variance and Student's t test. Analysis of covariance (ANCOVA) with diet as the main effect and pup weight as covariant was also used to control for the effect of pup body weight upon the time of appearance of neurodevelopmental reflexes and auditory brainstem conduction times (Kleinbaum et al. 1988 Length of gestation, dam weight gains, number of pups per litter and pups weights on postnatal d 3 did not differ among diet groups. Weights of pups of the corn oil-fed dams were slightly but significantly greater than pups of fish oil-fed dams at several time points (data not shown).
Pups of dams fed fish oil-supplemented diet throughout pregnancy and lactation had slower rates of growth and a delay in neurodevelopment as assessed by auditory brainstem conduction times and the time of appearance of the auditory startle reflex. Diet effects upon neurodevelopment remained significant after controlling for differences in body weights.
The scientific and editorial guidance of Lewis A. Barness is gratefully acknowledged.
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
, Zurier 1993).
, Clandinin et al. 1980, Jumpsen and Clandinin 1995). The fatty acid composition of developing neural tissues can be altered in animals by changes in pre- and/or postnatal dietary fatty acid composition (Arbuckle and Innis 1992, Carlson et al. 1986, Yonekubo et al. 1993). These alterations are associated with effects on several measures of neurodevelopment including reflex development (Wainwright et al. 1991), visual acuity (Neuringer et al. 1986), brightness discrimination (Yamamoto et al. 1987) and exploratory activity (Enslen et al. 1991).
, Makrides et al. 1994). Although the functional consequences of these differences are unknown, it is suggested that enhanced neurodevelopment reported in breast-fed infants, including higher scores on tests of cognitive (Lucas et al. 1992, Rogan and Gladen 1993, Temboury et al. 1994) and psychomotor (Agostino et al. 1995) development, may relate to differences in dietary fatty acid composition.
), and dietary lipids are known to affect the deposition of myelin (DiBiase and Salvati 1997, Salvati et al. 1993 and 1996), a primary determinant of the speed of conduction through the auditory brainstem pathway (Bunge 1968). The rat provides a particularly useful model because the development of auditory function is a postnatal event and is complete within 4 wk of birth (Church et al. 1984).
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
20°C, and contained 125 µg/g ethoxyquin to minimize oxidation. All diets provided 17.4 kJ/g. Dams were caged individually and had free access to diets and water. Fresh diet was provided every 48 h.
, Rayon et al. 1997, Stockard et al. 1996). Culled pups were killed by lethal injection of pentobarbital (75 mg/kg, intraperitoneal); cerebrums and milk from stomachs of culled pups were removed and stored at 70°C. Pups were weaned on postnatal d 21 and were then fed the corresponding maternal diets for the duration of the study. Pups were weighed every other day.
have established the time at which neurodevelopmental reflexes are expected to appear in healthy rat pups (Table 1). From postnatal d 4 through 28, pups were assessed daily for the appearance of these reflexes. During testing, pups were removed from dams and were kept in a 37°C chamber. Testing was conducted between 1000 and 1400 h by one of two investigators. In the case of testing for cliff avoidance and negative geotaxis appearance, a time limit criterion of 30 s was employed by both investigators.
View this table:
Table 1.
Description of reflex tests used to assess the effect of diet on neurodevelopment in rat pups1
).
).
). A P-value of 0.05 was considered significant. Data are presented as means ± SD.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
Table 2.
Fatty acid composition of diets fed to rats throughout pregnancy and lactation
View this table:
Table 3.
Fatty acid composition of maternal milk and cerebrum of pups whose dams were fed fish oil or corn oil diets
during pregnancy and lactation1,2
View this table:
Table 4.
Postnatal day of neurodevelopmental reflex appearance in rat pups of dams fed fish oil, corn oil or nonpurified (reference) diets during pregnancy and lactation1
View this table:
Table 5.
Auditory brainstem conduction times in rat pups whose dams were fed fish oil, corn oil or nonpurified (reference) diets during pregnancy and lactation1
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
, Uauy et al. 1990) and animals (Weisinger et al. 1996). However, that technique has limited use as a measure of neural development because the response is dominated by receptor cells of the peripheral visual pathway (Weinstein et al. 1991). Auditory brainstem responses consist of a series of evoked electrical potentials that reflect the sequential activation of the eighth nerve and brainstem auditory nuclei. Contributions from the peripheral auditory pathway can be removed by subtracting the latency of the eighth nerve action potential from that of a brainstem component of the response. Auditory brainstem conduction times are determined primarily by the degree of myelination of the pathway (Bunge 1968), and dietary lipids can affect myelin development in the postweaning period (DiBiase and Salvati 1997). Salvati et al. (1993) reported that whole-brain homogenates of rats fed diets supplemented with fish oil have lower levels of myelin basic protein and lower activity of 2
-3-cyclic nucleotide 3-phosphodiesterase (Salvati et al. 1996), an enzyme used as an indicator of myelination. They suggested that reduced enzyme activity may be due to a delay in myelin deposition and/or instability in myelin structure. In these studies, longer brainstem conduction times in pups of the fish oil group suggest that fish oil-supplemented diets have negative effects on myelination within the auditory brainstem pathway.
), structures that are also essential to the transmission and generation of the auditory brainstem response (Jewett and Romano 1972). The startle is readily observable and modifiable by neuropharmacologic treatments, including administration of N-methyl-D-aspartate (NMDA) and non-NMDA antagonists (Miserendino and Davis 1993). Its short latency suggests that the reflex is mediated by simple myelinated pathways with only a few synapses. The startle reflex has often been used as a neurobiological test for studying neural mechanisms of sensorimotor reactivity and plasticity at the brainstem level (Pellet 1990). Our finding of a significant delay in the appearance of this reflex among pups of the fish oil group further supports an effect of dietary fatty acid composition on the brainstem auditory pathway.
ACKNOWLEDGMENT
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FOOTNOTES |
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-tocopherol, 50 µg/g.
Manuscript received 24 June 1997. Initial reviews completed 5 August 1997. Revision accepted 4 December 1997.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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-linolenate formula with and without fish oil.
Lipids
1992;
27:89-93
[Medline]
-3-cyclic nucleotide 3-phosphodiesterase and acetylcholinesterase) and muscarinic receptor sites in growing rats.
Nutr. Biochem.
1996;
7:113-117 This article has been cited by other articles:
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