Nutrition Research Methodology 313replaced on an isonitrogenous basis with the test
protein source. Otherwise the diets are identical. After
several weeks of ad libitum feeding a blood sample is
taken and the results show that blood cholesterol rose
with the experimental diet. Then the researcher begins
to look at other data and observes that growth rates
in the control rats were far higher than in the experi-
mental group because the latter had a much lower
food intake. Quite simply, the new protein source was
unpalatable. The experiment will now have to be
carried out as a pair-fed study. The food intake of rats
given the experimental diet will be measured each
day. On the following day the control rats will be
rationed to that amount. Food intakes and probably
growth rates are identical. Only the protein source
differs. Now the researcher can truly reach conclu-
sions as to the effect of the new protein source on
LDL cholesterol metabolism. The intake or supply of
nutrients may be administered orally, intravenously,
intraperitoneally, or by means of some specifi c tools
(gavage, stereotaxis, etc.).
The experimental techniques available
The outcome or variables of interest to be assessed
condition the experimental techniques to be applied,
which may include growth curves, nutrient and
energy balance, nutrient utilization and signalling,
etc., using cellular, molecular or other strategies.
Another approach to investigate nutritional pro-
cesses is to overexpress, inactivate, or manipulate
specifi c genes playing a role in body metabolism
(Campión et al. 2004). These new technologies allow
the study of the regulation and function of different
genes. The current standard methods for manipulat-
ing genes in nutrition research depend on the method
of introducing/blocking genes. Thus, genetic manip-
ulation can be sustained for generations by creating
germline transmission. In this way, there are examples
of transgenic animals, overexpressing or knocking out
genes, but still controlling this gene manipulation in
a spatial or temporal manner. However, when the aim
is not to transfer genetic information to subsequent
generations, the most usual method is gene transfer
to somatic cells. Different viral and nonviral vectors
are used for the in vivo gene transfer, allowing a tran-
sient or permanent overexpression of the gene of
interest. The RNAi (interference) approach allows the
creation of new in vivo models by transient ablation
of gene expression by degrading target mRNA. More-
over, by inserting RNAi encoding sequences in theTable 13.6 An example of more refi ned experimental diets to examine the cholesterolemic effects
of fats in transgenic CETP miceControl diet Low-fat diet High-fat diet
(g/100 g) (g/MJ) (g/100 g) (g/MJ) (g/100 g) (g/MJ)
Casein 20 12 19 12 24 12
L-Cystine 0.03 0.18 0.28 0.18 0.36 0.18
Maize starch 40 24 48 31 13 6
Dextrinized starch 13 8 12 8 16 8
Sucrose 10 6 9 6 12 6
Cellulose 5 3 5 3 6 3
Soybean oil 7 4 0 0 0 0
Saffl ower oil 0 0 2 1.2 2.4 1.2
Experimental oila 0 0 0 02211
Mineral mix 3.5 2.1 3.3 2.1 4.2 2.1
Vitamin mix 1 0.6 0.9 0.6 1.2 0.6
Energy (%)
Total fat 16.9 5.7 48.6
Sugar 10.1 10.9 10.5
Starch 54.1 22.3 22.1
Protein 20.2 20.9 20.7
MJ/kg 16.7 15.9 20.1
a Three different experimental oils were used (butter, saffl ower, high oleic saffl ower) for three
different high-fat diets varying in types of fatty acids.
From Chang and Snook (2001). Reproduced with permission.