**2. Methods**

#### **2.1 Animals and diets**

Female pregnant *Wistar* rats weighing 200-250 g were obtained from the colony of Department of Nutrition of the Federal University of Pernambuco. Twenty-four hours after the birth of the entire mothers´ nestling, born on the same day, were contained in a large group and randomly each litter was culled to six males and two female pups. The litters

Among the various organic systems, the nervous system plays the main role in controlling several physiological processes. During development, this system presents a rapid growth spurt period or a vulnerable period which corresponds to the highest rate of cellular migration and differentiation, neurogenesis, synaptogenesis, myelinization and maturation of neurotransmitter pathways. Depending on the animal species, this critical period of intense neural development occurs at different time points early in life (Dobbing, 1968). In human, for example, it occurs during the last trimester of gestation until the second year of life, while in rats it corresponds the lactation period. During this period, the nutrition is one of the essential environmental factors to a normal development because it provides

The classical studies about malnutrition show that nutritional deficiency in macro or micronutrients has deleterious effects on the brain (Winick & Rosso, 1969). In rats, malnutrition induces functional and developmental failures, as well as reduction in brain size (Morgane et al., 1992;1993). In children, malnutrition showed influence both short and long term problems of cognition and behavior (Grantham-McGregor & Baker-Henningham,

Essential fatty acids are important constituents of structural lipids in nervous membranes cell and signaling molecules, as such, are involved in many brain functions. Around 30–40% of the total phospholipids in these structures are docosahexaenoic acid molecules (Young et al., 2000), which appear to be specifically concentrated in membranes surrounding synapses (Carlson, 2001). Changes in the quantity and quality of the dietary fatty acids are often

At the cellular level, an -linolenic deficient diet can induce less complex patterns of dendritic branching (Wainwright, 2002), smaller neurite growth in hippocampal neurons (Calderon & Kim 2004) and reduced neuronal soma size in some brain regions (Ahmad et al., 2002). Modification in the fatty acid composition of rat brain cell membranes of neurons, astrocytes, oligodendrocytes, and of subcellular fractions, such as myelin and synaptosomes, are also induced by a diet with reduced levels of n-3 fatty acids (Bourre et al., 1984). It has been shown that essential fatty acids imbalance as well as specific fatty acid deficiencies in the maternal diet can affect the neuromotor development of pups, including the ability to respond to environmental stimulation (Lamptey & Walker, 1976; Wainwright, 2002;

Fats and oils as they exist in nature must be processed before they are suitable for human consumption (González et al., 2007). On the other hand, only a few studies have described the effects of the ingestion of trans fatty acids early in life on the rat brain development. In this study we investigated the replacement of soybean oil in the diet by partially hydrogenated vegetable oil, rich in trans fatty acids, from the beginning of gestation

Female pregnant *Wistar* rats weighing 200-250 g were obtained from the colony of Department of Nutrition of the Federal University of Pernambuco. Twenty-four hours after the birth of the entire mothers´ nestling, born on the same day, were contained in a large group and randomly each litter was culled to six males and two female pups. The litters

nutrients without them the neurodevelopment would be impaired (Walker, 2005).

associated with developmental and functional alterations in the nervous system.

**1.2 The role of lipids on the development of nervous system** 

2005; Benton, 2008).

Anselmo et al., 2006).

**2. Methods** 

**2.1 Animals and diets** 

through lactation on reflex ontogeny.

were also randomly assigned to isocaloric diets containing as lipid source 7% soybean oil (control group-C; n=32) or 7% of hydrogenated vegetal fat (experimental group-E; n=39), since gestation until weaning (21 days old). Both diets (table 1) were formulated based on recommendations of the American Institute of Nutrition-AIN-93 (Reeves et al., 1993). The animals were kept on a 12:12-h light –dark photoperiod at 24o C temperature during the whole period. The animals were maintained according to recommendations from the National Institute of Health (USA) and approved by the Ethics Committee for Use Animal, Federal University of Pernambuco (CEUA, protocol.23076.020339/2010-24). After delivery, from P1 until 21d, the pups were weighted at 1, 7, 14 and 21days of age. Indicators of somatic maturation and reflex ontogeny were studied daily from P1 to P21 between 07:00 am and 09:00 am. Daily it was observed the occurrence of the reflex responses, being considered the consolidation day to be the first one, of a series of three consecutive days where the reply was verified. For each reflex it was established a maximum observation time of 10s according to the experimental model established by Smart & Dobbing (1971).


Table 1. Composition of the diets. Vitamin mixture (Rhoster Ind.Com. LTDA. SP. Brazil) containing (m%): folic acid (20); niacin (300); biotin (2); calcium pantothenate 160; pyridoxine 70; riboflavin 60; thiamine chloride 60; vitamin B12 0.25; vitamin K1 7.5. Additionally containing (UI%): vitamin A 40.000; vitamin D3 10.000; vitamin E 750. 2 Mineral mixture (Rhoster Ind. Com. LTDA. SP. Brazil) containing (m%): CaHP04 (38); K2HP04 (24); CaCO3 (18.1); NaF (0.1); NaCl(7.0); MgO (2.0); MgS04 7H20 (9.0); FeS04 7H20 (0.7); ZnS04 H20 (0.5); MnSO+ H20 (0.5); CuS04 5H20 (0.1); Al2 (S04)3K2S04 24H20 (0.02); Na2SeO3 5H20 (0.001); KCl (0.008).

#### **2.2 Indicators of somatic maturation**

The following indicators of somatic maturation, as illustrated in figure 1, were analyzed in order to study whether the replacement of soybean oil by vegetable hydrogenated fat in the diet influenced the development of physical features of the rat pups.

#### **2.3 Indicators of reflex ontogeny**

The following indicators of the reflex ontogeny investigated at the present study are described and illustrated below (figure 2):

*Palmar Grasp (PG)-* This reflex is present at birth and consists of a dorso flexion of the digits ('grasping') in response to the stimulation of the hand-palm with a small metallic stick. The

Lipids, Nutrition and Development 101

expected response is the disappearance of the palmar grasping response, as the organism

*Righting (R)-* The newborn is placed on its back on a flat surface and the expected mature response is to turn over on the ventral surface, resting in the normal position with the four

*Vibrissa Placing (VP)-* The pup is held by the tail, with the head facing the edge of a table and the vibrissae just touching the vertical surface of the table. The expected response is to lift

*Cliff Avoidance (CA) -* The newborn is placed on the edge of a 'cliff' (for instance, on the edge of a table), with the forepaws and face just over the edge. The expected response is to move

*Negative Geotaxis (NG)-* The pup is placed on an inclined ramp (45º slope) with its head pointing to the ground. The expected mature response is to turn around and crawl up the

*Free-Fall Righting (FFR)-* The pup is held with the back downwards 35cm above a cotton pad

*Auditory Startle Response (ASR)-* The newborn is exposed suddenly to a loud, sharp noise. The expected response is a prompt extension of the head and the limbs, followed by

The body weight was analyzed by Student's t-test. Results are presented as means±standard error of the mean (SEM). Differences were significant when p< 0.05. Results of the somatic maturation and reflex ontogeny were evaluated by Mann-Whitney test. Results are

The figure 3 shows that pups fed an experimental diet did not exhibited significant differences in the body weight at the 1st, 7th, 14th and 21th day, during the lactation period when compared to the controls. (C = 6.7 g ± 0.12; 16.3 g ± 0.49; 29.6 g ± 1.18; 46.4 g ±1.52; E =

The effect of the experimental diet on somatic development is shown in Figure 4, where the results are expressed in median (min. – max.) and compared with the control group. There was no difference between the number of days for ear unfolding, eye opening and the eruptions of superior and inferior incisors. However, the opening of the external auditory canal was significantly delayed in the experimental group as compared to the control (C: 12-

As can be seen in Figure 5, the development of the early reflexes which appear in the first postnatal week such as righting, cliff avoidance and vibrissa placing did not differ between control and experimental pups. In the second postnatal week, the negative geotaxis was the only reflex which was delayed in the experimental group when compared to the control (C:

and dropped. The expected response is to turn in mid-air to land on its four paws.

expressed as median interquartile. Statistical significance was set at p < 0.05.

the head and to extend the fore legs in direction of the table.

away from the cliff, to avoid dropping.

withdrawal of the limbs and a crouching posture.

6.6 g ± 0.11; 16.5 g ± 0.22; 30.3 g ± 0.48; 47 g ± 0.86).

**3.2 Indicators of somatic maturation** 

**3.3 Indicators of reflex ontogeny** 

matures.

slope.

feet on the ground.

**2.4 Statistical analysis** 

**3. Results** 

**3.1 Body weight** 

1; E: 14-2.5; p<0.05).

11-3; E: 9-1.5).

Fig. 1. Indicators of somatic maturation. A) Eruption of the Upper Incisors (EUI) and Eruption of the Lower Incisors (ELI); B) Ear Unfolding (EU); C) Eye Opening (EO); D) Auditory Conduit Opening (ACO).

Fig. 2. The figure represents the indicators of reflex ontogeny observed during lactation period.

Fig. 1. Indicators of somatic maturation. A) Eruption of the Upper Incisors (EUI) and Eruption of the Lower Incisors (ELI); B) Ear Unfolding (EU); C) Eye Opening (EO); D)

Fig. 2. The figure represents the indicators of reflex ontogeny observed during lactation

Auditory Conduit Opening (ACO).

period.

expected response is the disappearance of the palmar grasping response, as the organism matures.

*Righting (R)-* The newborn is placed on its back on a flat surface and the expected mature response is to turn over on the ventral surface, resting in the normal position with the four feet on the ground.

*Vibrissa Placing (VP)-* The pup is held by the tail, with the head facing the edge of a table and the vibrissae just touching the vertical surface of the table. The expected response is to lift the head and to extend the fore legs in direction of the table.

*Cliff Avoidance (CA) -* The newborn is placed on the edge of a 'cliff' (for instance, on the edge of a table), with the forepaws and face just over the edge. The expected response is to move away from the cliff, to avoid dropping.

*Negative Geotaxis (NG)-* The pup is placed on an inclined ramp (45º slope) with its head pointing to the ground. The expected mature response is to turn around and crawl up the slope.

*Free-Fall Righting (FFR)-* The pup is held with the back downwards 35cm above a cotton pad and dropped. The expected response is to turn in mid-air to land on its four paws.

*Auditory Startle Response (ASR)-* The newborn is exposed suddenly to a loud, sharp noise. The expected response is a prompt extension of the head and the limbs, followed by withdrawal of the limbs and a crouching posture.

#### **2.4 Statistical analysis**

The body weight was analyzed by Student's t-test. Results are presented as means±standard error of the mean (SEM). Differences were significant when p< 0.05. Results of the somatic maturation and reflex ontogeny were evaluated by Mann-Whitney test. Results are expressed as median interquartile. Statistical significance was set at p < 0.05.
