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Hydroxylase Affect Piglet Cerebrum Weight, Cholesterol Concentration, and Behavior1,2
3 USDA-Agricultural Research Service, Children's Nutrition Research Center, Baylor College of Medicine, Houston, TX 77030; 4 Pork Industry Institute, Department of Animal and Food Sciences, Texas Tech University, Lubbock, TX 79409-2141; and 5 Beckman Institute, University of Illinois, Urbana, IL 61801
* To whom correspondence should be addressed. E-mail: wgp3{at}cornell.edu.
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ABSTRACT |
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hydroxylase (CYP7) gene locus and dietary cholesterol (C) on cerebrum C in neonatal pigs fed sow's milk formulas. Thirty-six pigs (18 male and 18 female) genetically selected for high (HG) or low (LG) plasma total C were weaned at 24–36 h after birth and assigned in a 2 x 2 x 2 factorial arrangement of treatments with 2 diets (0 or 0.5% C), 2 sexes, and 2 genotypes (HG and LG). Individually housed pigs consumed diets ad libitum for 42 d. Open-field behavior was tested at wk 2 and 4. All pigs were killed at 42 d of age, the cerebrum was weighed, and C content and concentration measured. All data were analyzed by general linear model ANOVA. Cerebrum weight was greater in HG than LG pigs (P < 0.03) but was not affected by diet or sex. Pigs fed C tended to have a higher cerebrum C concentration than those deprived (P = 0.12). At 2 wk, LG pigs explored a novel open-field environment less often (P < 0.001) than did HG pigs. At 4 wk, some LG pigs explored the open field but fewer (P < 0.001) vs. HG pigs retreated back to the safe area. There were no genotype x diet, genotype x sex, or diet x sex interactions affecting cerebrum weight, or C content or concentration. Polymorphism in the CYP7 gene locus affected cerebrum weight and behavior and dietary C tended to increase cerebrum C concentration in neonatal pigs. These findings in neonatal pigs have considerable potential importance in human infant nutrition and behavioral development.
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Introduction |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Lower cerebrum C concentration has been reported (5,6) in pigs deprived of dietary C during neonatal life, suggesting the possibility that C may be required in the neonatal pig for brain growth and/or myelination. Behavioral differences have also been observed in a cohort of neonatal pigs deprived of dietary C compared with pigs fed C (5).
The genetic diversity of the pig provides an opportunity to study interrelationships between genetic background and nutritional manipulations. Specifically, plasma C is genetically controlled in pigs, as in humans. Estimates of paternal half-sib heritability for plasma total C in swine range from 0.25 to 0.40 (7–9).
In 1987, genetic selection within a crossbred (Chester White x Landrace x Large White x Yorkshire) population was started with a base of 388 animals, not previously selected for plasma total C concentration, at the age of 8 wk (mean = 2.23 mmol/L). In generation 3 of selection, the mean plasma C of 173 pigs in the line selected for high plasma C (HG) (previously designated HC until generation 4) was 2.74 mmol/L compared with 1.68 mmol/L in 219 pigs in the line selected for low plasma C (LG) (previously designated LC until generation 4) (10). Liver and jejunum activity of 3-hydroxyl-3-methylglutaryl-CoA reductase, the rate-limiting enzyme in C synthesis, did not differ between LG and HG pigs, and Taq1 restriction fragment length polymorphism (RFLP) analysis indicated no polymorphism in this enzyme system in these pigs. Also, Taq1 RFLP analysis of the LDL receptor gene, a regulator of LDL-C transport in plasma and uptake by liver, revealed no polymorphism. Plasma 7-dehydrocholesterol, which is increased in plasma of children who cannot convert plasma 7-dehydrocholesterol to C (Smith-Lemli-Opitz Syndrome), did not differ in LG and HG pigs (5). However, Taq1 RFLP analysis of the cholesterol 7- hydroxylase (CYP7) gene locus in 8th generation HG and LG pigs revealed 2 alleles: a 2.8-kb fragment associated with the former and a 5.0-kb fragment associated with the latter (11).
C homeostasis involves a balance between C synthesis, absorption, transport via lipoproteins, uptake via lipoprotein receptors in liver and other tissues, and degradation to bile acids. CYP7 catalyzes C degradation to bile acids. Variation in C degradation, controlled by CYP7, appears to be responsible for some or all of the difference in plasma C concentration between HG and LG pigs in this selected population. When 3-hydroxyl-3-methylglutaryl-CoA reductase is downregulated by C ingestion (12,13), CYP7 activity is upregulated (14).
This experiment was designed to confirm the previously observed increase in cerebrum C and altered behavior of female and male neonatal pigs fed C (5,6). The experiment also tested the hypothesis that polymorphism in the CYP7 gene locus interacts with neonatal dietary C to modulate plasma total C level and weight and C concentration of cerebrum in neonatal pigs fed sow's milk replacer formulas containing 0 or 0.5% C.
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Materials and Methods |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Blood processing. Blood was collected in heparinized tubes, centrifuged at 5°C, and plasma removed and stored at –20°C. Plasma was analyzed for total C and HDL-C (Corning clinical analyzer). Buffy coat at the cell-plasma interface of the blood sample collected at d 42 was used for Taq1 RFLP analysis of the CYP7 alleles in all pigs. DNA from each sample was extracted, purified, and cut into restriction fragments using Taq1 endonuclease. The restriction fragments were then separated according to length by agarose gel electrophoresis enhanced by Southern blot.
Behavioral testing. At d 14 and 28, each pig underwent behavioral testing. Behavioral testing measures were adapted from those reported earlier (5). Behavioral measures assessed relative levels of fear/anxiety and emotionality. Pigs showing less fear or anxiety will explore a novel environment longer and with a lower latency to begin exploration. Pigs were placed in a portable kennel, which was placed on the floor in the corner of an observation room (3 x 2.5 m) in which a grid had been painted on the floor. The kennel door was opened to allow piglets to voluntarily leave the kennel and explore the novel, open field. Pig movements were observed for 10 min (600 s) by viewing the pig through a 1-way glass window. Three behavioral indices were quantified: 1) amount of time (s) elapsed before the pig exited the kennel; 2) total amount of time (s) the pig was in the kennel (after leaving the kennel, pigs would sometimes re-enter it); and 3) total number of points accrued during the 10-min observation period. Pigs received 1 point when they entered (all 4 legs) the square that contained the kennel, 2 points when they entered any of the 3 squares adjacent to the kennel, and 3 points whenever they entered any of the 5 remaining squares. Observations were done in the morning, 30–60 min after feeding. To minimize the effects of known variation in brain neurochemistry during a 24-h period, behavioral testing was restricted to a time window beginning at 0800 and ending at 1000 each test day, during which pigs from each genetic group and diet group were tested at 2 and 4 wk. In addition, observers recorded the percentage of pigs that vocalized, explored the open field, and explored, then retreated back into the kennel.
Tissue collection and analysis. Pigs were killed with an overdose of ketamine and acepromazine acetate and exsanguinated at d 42 of age. Brain was removed and the cerebrum weighed and immediately frozen in liquid N2, sealed in a plastic bag, and stored frozen at –70°C until analysis. For analysis, the cerebrum was homogenized in a Waring blender and a composite sample was used for C analysis. Lipid extracts were saponified with potassium hydroxide and C concentration was determined according to Rhee et al. (16) as modified from Searcy and Berquist (17) in a Beckman DU-7 spectrophotometer (Beckman Instruments).
Statistical analysis. Most data were analyzed by general linear model ANOVA (body weight, cerebrum weight, cerebrum total C, and C concentration) and by repeated measures ANOVA (plasma total C, HDL-C, and total C:HDL-C ratio). All interactions were tested in the model. Chi-square analyses were used to assess treatment effects on percentage of pigs that vocalized, explored, or explored and then retreated in the open-field test. Values in the text are means ± SEM. Statistical significance was set at P < 0.05 or as designated in specific marginal effects (P < 0.12).
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Results |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Body weight gain was not affected by diet, genotype, or sex, in agreement with earlier results in HG and HG pigs from the 4th generation of selection (5). Daily gain of pigs fed C tended to be higher than in pigs fed no C (P = 0.08). Overall final body weight (42 d) was 9.9 kg. Cerebrum weight (Table 2) was greater (P < 0.03) among HG pigs (43.4 g) in those homozygous for the 2.8-kb CYP7 allele and 42.7 g in heterozygotes) than in LG pigs (homozygous for the 5.0-kb CYP7 allele, 40.4 g). Neither diet nor sex had a significant effect on cerebrum weight (Table 2), in agreement with previous results (5). Heart weight, as in the case of cerebrum weight, tended to be heavier (P = 0.10) among HG pigs (56.5 g in both homozygotes and heterozygotes) than in LG pigs (49.3 g) (Table 2).
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Plasma total C (Table 2) was not affected by genotype at either 4 or 6 wk. We previously found (5) higher plasma total C in HG than in LG pigs at 4 wk of age (P < 0.05). The failure to detect significant differences in the present experiment may have been due to the large variability in this trait in this pig population. As expected, pigs fed the 0.5% C diet in our study had a higher plasma C at 4 wk (P = 0.06) and 6 wk (P = 0.003) of age. There was an effect of time (P < 0.05) and a time x diet interaction (P = 0.02) but no other significant interactions. Plasma HDL-C (Table 2) tended to be higher in HG than in LG pigs at wk 4 (P < 0.10) but not at wk 6 (P = 0.26) of age. Males had higher plasma HDL-C than females at wk 4 and 6 combined (P < 0.05) and pigs fed C had higher plasma HDL-C than those fed no C at wk 4 and 6 combined (P < 0.03). The ratio of plasma total C:HDL-C concentration (Table 2) was not affected by genotype or sex but was higher (P < 0.001) in pigs fed C than in pigs fed no C (wk 4 and 6 combined).
Behavioral testing results (Table 3) showed no effects of genotype, diet, age, or their interactions in amount of time elapsed before the pig exited the kennel, total amount of time the pig was in the kennel, or total number of points (general activity) accrued during the 10-min observation period (data not shown). However, the percentage of pigs vocalizing at 4 wk of age was affected by genotype and diet. Sixty percent of LG pigs fed C vocalized during testing, whereas only 12.5% of LG pigs fed no C and 25–33.3% of HG pigs fed either diet vocalized. Because vocalization is a sign of emotionality, LG pigs fed C exhibited more emotionality than pigs in other treatment groups (P < 0.01).
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A small percentage (11.1%) of 2-wk-old LG pigs extended their head out of the kennel and retreated, and none of the 4-wk-old LG pigs retreated. The measure of retreat back into the kennel, which could be a measure of fear, could not be assessed in LG pigs, because so few of them explored the open field. Rather, they remained in the kennel at a high rate and those fed C vocalized at a high rate.
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Discussion |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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The physiological basis for the difference in cerebrum and heart weight related to CYP7 genotype is unclear. Gale et al. (18) reported that the brain volume of children at 1 y of age, based on head circumference measurements (corrected for other variables, e.g. body size), helped determine later intelligence. The larger cerebrum of HG pigs in the present experiment is suggestive of some metabolic relationship differentiating the 2 CYP7 alleles with respect to cerebrum growth and function.
CYP7 is the first and limiting enzyme in bile acid synthesis from C and there are 2 isoenzymes: CYP7A1 is liver specific and is induced by C, whereas CYP7B1 is present in brain (and other tissues) and is involved in synthesis of neurosteroids (19). The potential importance of polymorphism of CYP7 in normal brain development of the neonate should be confirmed in pigs as a model for studies in humans.
Behavioral testing results showed that genotype, diet, age, or their interactions did not differ in amount of time elapsed before the pig exited the kennel, total amount of time the pig was in the kennel, or total number of points (general activity) accrued during the 10-min observation period (data not shown). However, the percentage of pigs vocalizing at 4 wk of age was affected by genotype and diet (Table 3). Because vocalization is a sign of emotionality, LG piglets receiving dietary C exhibited more emotionality than pigs in other treatment groups. This is the first report, to our knowledge, of a diet effect on emotionality that is dependent on the CYP7 gene polymorphism.
We demonstrate here, for the first time, to our knowledge, an interaction between genotype and diet in behavioral measures. That certain genotypes may behave differently when fed different diets has interesting possibilities in the management of emotional behaviors such as fear, anxiety, and emotionality. This is a potentially fruitful area of future investigation.
The ontogeny of brain development in pigs resembles that of the human infant (3,20–22); the shape of the growth curves for cerebrum and myelination (estimated by C accretion) are similar and the peak of the brain growth spurt occurs perinatally in both species. Therefore, the pig is widely used as an animal model in studies of lipid nutrition in human brain development.
This work indicates that selection in a pig population for HG or LG plasma total C, now known to be related to polymorphism in the CYP7 gene locus (23,24), does not interact with dietary C content to affect cerebrum C concentration in neonatal pigs. This work confirms the earlier observations (5,6) that dietary C given to neonatal pigs is associated with increased cerebrum C concentration. Endogenous C synthesis within the brain is believed by most to meet the C requirements for optimum growth and development of the brain. There is evidence that some C crosses the blood-brain barrier (1,25–29). A small amount of C (0.3–0.4 mg) may cross the blood-brain barrier daily compared with a much larger amount (7–8 mg) synthesized daily within the brain via LDL (30,31). However, most researchers have concluded that all brain C is synthesized within the brain (4,32–35). The mechanisms involved in the increased cerebrum C in pigs fed C during the neonatal period are unknown. They must be related to either a small amount of plasma C crossing the blood-brain barrier during the brain growth spurt or to an unidentified signal in response to a change in cerebrum C synthesis or cerebrum C exit.
Compelling evidence exists for a relationship between dietary fatty acid composition and development of the central nervous system in the infant (36–42). Human milk contains large amounts of long chain PUFA, including docosahexaenoic acid, an (n-3) fatty acid. Term infant formulas now are supplemented with docosahexaenoic acid in response to published evidence for a beneficial effect on brain development and cognition. Infant formulas contain no C, although breast milk contains 0.2–0.5% C. Research is needed to confirm and understand the role of dietary C in neonatal brain development and to ascertain and quantify possible interactive responses to dietary C and PUFA fed separately and in combination. Such research may provide important new insights into the role of dietary C and PUFA in the neonate.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Author disclosures: W. G. Pond, H. J. Mersmann, D. Su, J. J. McGlone, M. B. Wheeler, and E. O'Brian Smith, no conflicts of interest.
6 Abbreviations used: C, cholesterol; CYP7, cholesterol 7- hydroxylase; HG, high cholesterol genetic line; LG, low cholesterol genetic line; RFLP, restriction fragment length polymorphism.
Manuscript received 19 September 2007. Initial review completed 22 October 2007. Revision accepted 28 November 2007.
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