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What you should know about reducing experimental variation through proper selection of diet for laboratory rats and mice.

Researchers from all disciplines want to reduce variability in their studies, but they may not recognize that diet is a source of variability^1,2 This article describes some of the factors that personnel involved in diet selection for an institution or for a specific study should be aware of, and how the potential to confound experimental results can be avoided or minimized.

Types of Diets
Diets fed to laboratory animals can be generally classified as one of two types — purified or natural ingredient. Purified diets are made of refined ingredients such as casein, amino acids, sucrose, fructose, corn starch, various fats or oils, cellulose, vitamins, and minerals. Such mixtures are well suited to minimize nutrient variation, certain environmental contaminants,³ and the presence of various non-nutritive biologically active compounds naturally occurring in plants.^4,5 Purified diets are important research tools because they can be manipulated to contain very high or low levels of specific macro nutrients (i.e. 60% of kcal from fat or 6% protein) and micro nutrients (i.e. 2% calcium or vitamin A deficient).Many studies do not require such precise control and an appropriately selected natural ingredient diet is suitable.

The largest volume of laboratory animal diet produced is comprised of agricultural commodities like corn, wheat, plant by-products, soybean meal, oats, alfalfa meal, and animal derived ingredients such as fish meal and meat and bone meal. Unfortunately there is no consistent terminology used across the industry, and these diets might be called “standard” or “grain-based” or “chow.” In this article these types of diets are most correctly referred to as natural ingredient diets, which denotes the relative lack of processing the ingredients have been subjected to.

Natural ingredient diets support reproduction, growth, and maintenance of laboratory animals. There are a variety to choose from, designed for various life stages or a more specific application. A major source of information about a natural ingredient diet is the technical data sheet that a manufacturer makes available, usually via their website or printed materials. While these data sheets are an essential resource, it is also important to have reasonable expectations about the accuracy and precision of the nutrient information. The nutrient levels are estimates, derived from a variety of sources including published commodity compendium data, wet chemistry testing of raw materials, and finished product testing (the latter particularly for protein, fat, and crude fiber). Nutrient losses due to heat treatment and mechanical processes during manufacturing, or post-production effects of irradiation or autoclaving are not routinely taken into consideration in these estimates.

As a general rule, the degree of variation, calculated as the coefficient of variation (CV) for the nutrient classes will be as follows: protein –CVless than 5%; fat –CVup to 10%;minerals –CVgenerally less than 10%, although some such as iron can be more variable; vitamins – CV ranges from 5%to 20% or more. This wide range is due to both variable manufacturing loss and, in some cases, fairly sizeable analytical variation. If the interpretation of an experiment depends on accurate knowledge of a nutrient level, it should be measured; this is advisable whether using a natural ingredient or purified diet.

Looking at the list of ingredients is also instructive. While the actual recipe is rarely disclosed, the major commodity ingredients present in a diet will be listed in descending order of inclusion. When comparing diets on paper, reviewing the ingredient listing and the nutrient information results in a more meaningful evaluation than either alone.

Diet Formulation Philosophy
Diets for laboratory animals can be classified as open or closed. Open formulas are in the public domain, meaning that any manufacturer could produce it and researchers can report a full formula. Examples of these include natural ingredient diets created by government agencies like NIH and NTP. Also, the majority of purified diets are considered open, as most manufacturers of these diets fully disclose these formulations to the end-user. Closed formulas are proprietary, meaning that while the ingredients are disclosed, the exact recipe is known only to the manufacturer; the end-user relies on the technical datasheet for details about the diet. Open formulas are by definition fixed; closed formulas can be either fixed or variable.

Fixed formula diets use the same ingredients, in the same proportion, each time the diet is produced. In contrast, both ingredients and ingredient inclusion rates may be adjusted in variable formula diets. The justification for the use of variable formulas is that adjustments are necessary because raw material macronutrient variability can be of a magnitude to cause significant variation in the finished product. However, the effects of geographical and seasonal variation on ingredient nutrient levels are typically overstated. Let’s take the example of protein since it is on this basis that formula adjustments are likely taking place. In a recent report on quality and variation in soybean meal sources,⁶ the maximum difference in the average protein content of soybean meal from six highly varied countries was 2.8%. To put in the context of finished feed, this would mean an average difference in protein in the finished product of about 0.14% to 0.7%, considering that soybean meal is probably going to be present in many laboratory diets at about 5 to 25%. This slight difference would furthermore be balanced by the protein variation in other ingredients.

The within and between year variation for protein content of soybean meal, corn gluten meal (CGM), and wheat sourced by Harlan for production of Teklad Diets is shown in Figure 1. Soybean meal and corn gluten meal are concentrated protein sources. Wheat, while lower in protein, quantitatively contributes a significant amount of protein to our Global Rodent Diets by virtue of its inclusion rate. The columns show the annual mean as a percentage of the grand mean over the period of nine years (grand mean is set at 100%), along with the standard deviation (vertical bars) within a year. Deviations from 100% within and between years rarely exceed 5%.

When ingredient nutrient content is relatively stable, it stands to reason that the diet will show a similar level of consistency. Table 1 compares literature values for nutrient variation^7,8in a variable, closed formula diet, two fixed, open formulas with contemporary data obtained by an independent testing laboratory for two fixed, closed formula diets, and two variable, closed formula diets. When comparing the CVs for protein, fat, and selected micronutrients, both types of formulation strategies (fixed and variable) can result in similar nutrient stability in the finished diet.

However, it is important to appreciate that a change in the inclusion rate of an ingredient or an addition or deletion of an ingredient can impact the levels of those nutrients that are not monitored during the reformulation process. Additionally, batch specific re-formulation may also affect the amount and type of non-nutrients in the diet, which may have considerable impact on study results. ^9,10 Manufacturers that use fixed formulas, coupled with careful selection and monitoring of raw materials, do so because they recognize that diet has significant effects on research studies not only through the content of nutrients but also by the presence of non-nutrients.

(Click Image For A Larger Version)

(Click Image For A Larger Version)

Effects of Phytoestrogens
The significant effect of certain non-nutrients such as phytoestrogens on research studies emphasizes the importance of using fixed-formula diets that remove or reduce ingredients containing non-nutrients that are reported to have confounding effects. For instance, soybean meal and alfalfa meal are the major sources of phytoestrogens in laboratory rodent diets. Phytoestrogens of relevance to laboratory animal diets include the isoflavones daidzein and genistein found in soybean meal, and coumestrol in alfalfa meal.

Phytoestrogens are naturally-occurring chemicals in plants that have estrogenic structures (Figure 2) and can affect research results, primarily through interaction with estrogen receptors which are widely distributed among body tissues. While 17β-estradiol is a significantly more potent estrogen, rodents consuming diets containing soybean meal have phytoestrogens circulating at nmol or μmol levels compared to pmol levels for 17β-estradiol. This greater access to estrogen receptors helps to explain the wide variety of research effects (Table 2). Most of these effects occur at levels of phytoestrogens found in traditional laboratory animal diets.

The range of isoflavones found in diets containing soybean meal extends from ~75 to over 600mg/kg (ppm); those diets without soybean meal contain less than ~20mg/kg and are considered minimal phytoestrogen (provided they do not contain alfalfa meal) or minimal isoflavone diets. The literature describes batch to batch variation of two fold or more in the isoflavone content of diets containing soybean meal.^10,12 This can be attributed to both the natural flucuation in soybean meal, and to soybean meal inclusion rate changes in variable formula diets.

Effects of soy and/or isoflavones have been noted in animal models used to study reproductive and endocrine disruptors, cancer, bone metabolism, behavior, and diabetes (Table 2 contains a more comprehensive list). The reader is referred to some excellent review papers, ^5,9,11-18 and the following examples demonstrate a range of effects described in the literature.

• Dietary phytoestrogens influence the onset of puberty, and have corresponding effects on markers of puberty such as uterine weight and vaginal opening. This has led to concern that dietary phytoestrogens might adversely impact studies designed to assess reproductive toxicity or evaluate potential endocrine disruptors. ^9,19,20
• The effects of phytoestrogens may be difficult to predict. Depending on the presence of other estrogens, phytoestrogens may have an agonistic or antagonistic effect. A modest level of soy in the diet (7%) had a stimulatory effect on uterine weight but the combination of dietary soy and a potent synthetic estrogen (diethylstilbestrol; DES) resulted in a reduced uterine weight relative to a soy-free diet plus DES,²¹
• A further complication may arise from variation in sensitivity of rodent stocks and strains to dietary isoflavones, with some showing little or no response, while others show significant responses. Variation in isoflavone content (98, 223, 431 mg/kg diet) between different mill dates of the same diet led to differences in the time of vaginal opening in F344 rats but not Sprague Dawley rats, ¹⁰
• Numerous studies have found that consumption of diets containing soy or purified diets supplemented with isoflavones (particularly genistein) reduced the numbers of tumors and increased tumor latency time in rats exposed to chemical carcinogens, ¹⁴
• Isoflavones, particularly genistein, have been shown to have a dose-dependent (125-1000 mg/kg diet) stimulatory effect on estrogen-dependent MCF-7 cells implanted in athymic ovariectomized mice, ²²
• Even at low levels (100 mg/kg diet), genistein reduced the development of spontaneous prostate tumors in the Transgenic Adenocarcinoma Mouse Prostate (TRAMP) model, ²³
• Dietary concentrations of just 25 mg genistein/kg diet had effects on mammary gland hypertrophy in male rats, ²⁴
• Daidzein (150 mg/kg diet) was as effective as oral estrogen in restoring the decrease in vertebral bone mineral density brought about by ovariectomy in 12 month old female Wistar rats, ²⁵
• In Long Evans rats, exposure to phytoestrogens (minimal level vs. 200 and 600 mg/kg diet) decreased anxiety, as assessed by performance in the elevated plus maze, ¹⁶
• Glucose tolerance was improved in obese female Zucker rats consuming diet with soy protein isolate with isoflavones (~700 mg/kg diet).²⁶

While it may be tempting to consider whether phytoestrogens are beneficial or not with respect to the system being studied, it is essential to consider:

1. The variation in experimental data that can be caused by the inevitable variation in phytoestrogen levels in different batches of the same product;
2. There is no simple absolute threshold for the physiological effects of phytoestrogens, as many effects show a dose response down to very low levels of phytoestrogens;
3. Their presence may substantially decrease the effectiveness of animal models by suppressing the end points for which the model was selected. The solution to minimizing the impact of dietary phytoestrogens on biomedical research is to minimize their presence in diet.

References

1. Newberne P and Sotnikov A. 1996. Diet: The neglected variable in chemical safety evaluations. Toxicological Pathology 24:746-756.
2. Warden C and Fisler J. 2008. Comparisons of diets used in animal models of high fat feeding. Cell Metab 7:277.
3. Kozul CD, Nomikos AP, Hampton TH, Warnke LA, Gosse JA, Davey JC, Thorpe JE, Jackson BP, Ihnat MA, Hamilton JW. 2008. Laboratory diet profoundly alters gene expression and confounds genomic analysis in mouse liver and lung. Chem Biol Interact 173:129-140.
4. Thigpen JE, Setchell KD, Ahlmark KB, Locklear J, Spahr T, Caviness GF, Goelz MF, Haseman JK, Newbold RR, Forsythe DB. 1999. Phytoestrogen content of purified, open- and closed-formula laboratory animal diets. Lab Anim Sci 49:530-536.
5. Brown NM, Setchell KD. 2001. Animal models impacted by phytoestrogens in commercial chow: implications for pathways influenced by hormones. Lab Invest 81:735-747.
6. Thakur M, Hurburgh CR. 2007. Quality of US Soybean Meal Compared to the Quality of Soybean Meal from Other Origins. Journal of the American Oil Chemists' Society 84:835-843.
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8. Oller WL, Kendall DC, Greenman DL. 1989. Variability of selected nutrients and contaminants monitored in rodent diets: a 6-year study. J Toxicol Environ Health 27:47-56.
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10. Thigpen JE, Setchell KD, Padilla-Banks E, Haseman JK, Saunders HE, Caviness GF, Kissling GE, Grant MG, Forsythe DB. 2007. Variations in Phytoestrogen Content between Different Mill Dates of the Same Diet Produces Significant Differences in the Time of Vaginal Opening in CD-1 Mice and F344 Rats but Not in CD Sprague-Dawley Rats. Environ Health Perspect 115:1717-1726.
11. Setchell KD. 1998. Phytoestrogens: the biochemistry, physiology, and implications for human health of soy isoflavones. Am J Clin Nutr 68:1333S-1346S.
12. Jensen MN, Ritskes-Hoitinga M. 2007. How isoflavone levels in common rodent diets can interfere with the value of animal models and with experimental results. Lab Anim 41:1-18.
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19. Thigpen JE, Haseman JK, Saunders HE, Setchell KD, Grant MG, Forsythe DB. 2003. Dietary phytoestrogens accelerate the time of vaginal opening in immature CD-1 mice. Comp Med 53:607-615.
20. Owens W, Ashby J, Odum J, Onyon L. 2003. The OECD program to validate the rat uterotrophic bioassay. Phase 2: dietary phytoestrogen analyses. Environ Health Perspect 111:1559-1567.
21. Makela SI, Pylkkanen LH, Santti RS, Adlercreutz H. 1995. Dietary soybean may be antiestrogenic in male mice. J Nutr 125:437-445.
22. Ju YH, Allred CD, Allred KF, Karko KL, Doerge DR, Helferich WG. 2001. Physiological concentrations of dietary genistein dose-dependently stimulate growth of estrogen-dependent human breast cancer (MCF-7) tumors implanted in athymic nude mice. J Nutr 131:2957-2962.
23. Mentor-Marcel R, Lamartiniere CA, Eltoum IE, Greenberg NM, Elgavish A. 2001. Genistein in the diet reduces the incidence of poorly differentiated prostatic adenocarcinoma in transgenic mice (TRAMP). Cancer Res 61:6777-6782.
24. Delclos KB, Bucci TJ, Lomax LG, Latendresse JR, Warbritton A, Weis CC, Newbold RR. 2001. Effects of dietary genistein exposure during development on male and female CD (Sprague- Dawley) rats. Reprod Toxicol 15:647-663.
25. Picherit C, Coxam V, Bennetau- Pelissero C, Kati-Coulibaly S, Davicco MJ, Lebecque P, Barlet JP. 2000. Daidzein is more efficient than genistein in preventing ovariectomy-induced bone loss in rats. J Nutr 130:1675-1681.
26. Mezei O, Banz WJ, Steger RW, Peluso MR, Winters TA, Shay N. 2003. Soy isoflavones exert antidiabetic and hypolipidemic effects through the PPAR pathways in obese Zucker rats and murine RAW 264.7 cells. J Nutr 133:1238-1243.

Barbara Mickelson, PhD is a Nutritionist with Harlan Laboratories, in the Research Models and Services Division and provides technical support for Teklad Diets. Harlan Laboratories, 8520 Allison Pointe Blvd, Suite 400, Indianapolis, IN 46250. www.harlan.com; 800.483.5523 ext. 2198; bmickelson@harlan.com.