Effects of Autoclaving Soy-Free and Soy-Containing Diets for Laboratory Rats on Protein and Energy Values Determined In Vitro and In Vivo

Abstract

Autoclaving diminishes the nutritional value of rat diets, depending on the duration and temperature of the process and the type of dietary protein. We evaluated in vivo and in vitro the effects of autoclaving on the protein and energy values of soy-free and soy-containing rat diets. The true digestibility and biological value of the dietary protein were determined in a 10-d experiment involving 28-d-old Wistar Crl:WI(Han) male rats fed casein- or soy-containing diet that was autoclaved for 20 min at 121 °C (T1), 10 min at 134 °C (T2), or not autoclaved (T0). The apparent protein digestibility and metabolizable energy concentration of experimental diets were assayed during an 18-d trial involving 6-wk-old Wistar-Crl:WI(Han) male rats and compared with a commercial diet. The neutral detergent fiber (NDF) content, amount of protein bound to NDF, protein solubility, and in vitro ileal protein digestibility were determined. Autoclaving decreased protein solubility, with the T2 condition having a greater effect than that of T1, and decreased the protein parameters determined in vivo, except for the apparent digestibility of the standard rat diet. Autoclaving decreased metabolizable energy slightly. The Atwater formula yielded higher values than those determined in rats, in vitro, and calculated according to the pig equation. We conclude that autoclaving diets according to the T1 program was less detrimental to dietary protein than was T2 and that the NDF content and protein solubility may be helpful in assessing the effect of autoclaving. The pig formula and in vitro method appear to be valid for estimating the metabolizable energy of rat diets.

The extensive use of SPF rats in biomedical research has increased the demand for sterilized feeds for breeding and maintenance purposes. Many breeding units preferentially use autoclaving as an inexpensive and convenient method for sterilizing diets. Autoclaving, which combines heat, pressure, and steam treatment, decreases the nutritional value of diets, particularly the contents of heat-labile vitamins and the availability of nutrients. Autoclavable diets are, therefore, fortified with increased amounts of vitamins or with their heat-stable forms,¹³ but the effect of autoclaving on the diets’ nutritional value in terms of energy and protein availability has not been established satisfactorily. The protein quality of sterilized diets likely depends on the duration and temperature of autoclaving and on the type of protein in the diet.^15,17

The common use of soybean meal as a protein source in diets for laboratory rodents has fallen into disfavor because soy is a rich source of estrogenic isoflavones, which affect the hormonal status of animals and may distort the outcome of experiments.^11,33 The use of low-phytoestrogen diets is recommended, and soy-free diets are commercially available, yet information on the protein components is not always disclosed, and the effects of sterilization of such diets have not been determined.

Studies addressing the effects of heat treatment on protein-containing feeds for monogastric animals have identified Maillard reactions and protein denaturation as the main factors responsible for decreasing their nutritional value.⁴⁰ Several analytical methods for monitoring the effects of feed processing have been proposed. Determining the protein solubility in sodium hydroxide or sodium borate and the in vitro ileal digestibility are some of the methods used most often, and the results of these assays correlate with those from animal experiments.^{3,8,14,26,30,40}

The in vitro analysis of protein and amino-acid digestibility at the ileal level was developed for pigs as an alternative to experiments performed on cannulated animals. This method simulates digestive processes in monogastric animals and consists of step-by-step treatment of feed with proteolytic enzymes.⁸ Similar procedures have been developed for in vitro metabolizable energy (ME) determination as an alternative to experiments involving animals.⁹ We assume that the methods developed for the evaluation of pig diets will be effective for rat diets as well, given that protein and energy digestibility values measured in growing pigs and rats are in good agreement.¹⁶

As a reference method for protein evaluation in rats, the classic Thomas–Mitchell method is still commonly used to compare the protein value of different feeds or foods and to assess the effect of thermal treatments.^18,20,32 The method is based on measurement of nitrogen balance in growing animals fed diets containing the evaluated protein feed as the only source of this nutrient. The obtained results represent so-called ‘true’ (actual) measurements, given that the method takes into account the amount of endogenous nitrogen excreted by animals fed a protein-free diet or calculated according to respective equations.²⁷

Nutritional and biomedical studies analyzing the effect of feed restriction on metabolic processes, obesity, and longevity or in cancer research require knowledge about feed and energy consumption. Therefore, an exact evaluation of the energy content of experimental and breeding diets is an important, although often underestimated, issue. The ME values declared by commercial manufacturers of breeding diets may differ from the actual energy concentration, depending on the system used for energy calculation, which is not always indicated.

The energy content of any diet or feed is the sum of the energy of energy-yielding nutrients and can by estimated from its chemical composition. Available (metabolizable) energy determined in vivo represents the difference between energy consumed and excreted in feces and urine. Usually the ME concentration in rat and mouse diets is calculated according to the Atwater formula,²⁵ which uses so-called ‘fuel values’ as the energy value of protein, fat, and carbohydrates. Atwater's factors are based on the assumption that the digestibility of nutrients is high and uniform, which is not the case in natural-ingredient or thermally processed products.²⁵ Atwater's factors are thought to predict with adequate accuracy the ME of purified diets, whereas for natural diets, empirical equations developed for pig feeds have been proposed and validated.⁷ Compared with the Atwater formula, these pig-feed equations involve more chemically determined dietary components and take into account the negative effect of fiber on ME. Several companies recently have used the pig equations to calculate the energy value of diets for laboratory rats and mice,³⁵ but their validity for autoclaved diets has not been verified. The in vitro method developed for the determination of ME contents in pig diets appears to be promising for calculating the ME in rat diets as well.⁹ To our knowledge, a comparison of the energy values of thermally treated rat diets as determined by in vivo, in vitro, and various methods of calculation has not been reported to date.

The objective of this study was to assess the effects of thermal sterilization according to 2 common autoclaving programs on the protein and energy values of soy-free and soy-containing diets. Another objective was to determine whether various in vitro analyses can be considered as supplementary or alternative tests for the evaluation of the nutritional value of autoclaved diets for laboratory rats and mice.

Materials and Methods

Composition and processing of the diets.

Two diets containing either casein or soybean meal as the main protein source (HC and HS diets, respectively), were produced by the Morawski Feed Production Plant (Kcynia, Poland). The diets were fortified with greater amounts of vitamins than conventional diets, and had slightly higher protein concentrations to make up for the expected decreased protein availability. The autoclavable SSNIFF 1324-3 diet (Ssniff Spezialdiaten, Soest, Germany) was used as the standard (SN) diet. The composition of the diets is shown in Figure 1.

Figure 1.

Composition (g/kg) of soy-free (HC) and soy-containing (HS) diets in the current study.

Pelleted (10 mm × 20 mm) diets were packed into autoclavable perforated 10-kg paper bags and steam-autoclaved (model 9612-2ED SteriVap SP HP, BMT Medical Technology, Brno, Czech Republic) at 121 °C for 20 min (T1 condition) or at 134 °C for 10 min (T2 condition), followed by cooling and drying for 15 min. One portion of each diet was not autoclaved (T0 condition).The efficiency of sterilization was verified by using a biologic test (Sporal A, IBSS Biomed, Krakow, Poland).

Animal tests.

Two experiments were conducted according to protocols approved by the Third Local Ethics Commission in Warsaw and in compliance with the principles of laboratory animal care according to European Union and Polish Law on Animal Protection. Both experiments used male Wistar-Crl:WI(Han) rats (Center for Experimental Medicine, Medical University of Białystok, Bialystok, Poland). The vendor's sentinel program, which included serology, PCR assays, and microbiologic evaluation, confirmed that the rats were free of pathogenic bacteria, viruses, and ecto- and endoparasites. The rats were maintained individually in wire-bottom polycarbonate metabolic cages (Tecniplast, Buguggiate, Italy) in a temperature-controlled (22 ± 1 °C) room with 50% ± 10% humidity and a 12:12-h light:dark cycle. The rats had free access to feed and municipal drinking water. Daily inspection and all manipulations were performed by the same qualified personnel.

In experiment 1, the apparent digestibility of the macronutrients and energy in the nonautoclaved and autoclaved HC, HS, and SN diets, and their ME contents were determined. The experiment was performed over 18 d on 9 groups of 6-wk-old rats (n = 8; mean initial body weight, 150 g). After 4 d of adaptation, feces and urine were collected daily for 14 d and refrigerated at 4 °C until analysis. Urine was analyzed fresh after collection; feces were analyzed after drying at 60 °C. The apparent digestibility (as %) of nutrients and energy was calculated as the difference between intake and fecal excretion, and the ME concentration (as MJ/kg) was estimated as the difference between the gross energy intake and the combined excretion in feces and urine.

In experiment 2, the protein value of the nonautoclaved and autoclaved HC and HS diets were determined according to the Thomas–Mitchell method. The experiment was performed over 10 d on 6 groups of 28-d-old male rats (n = 8). The rats were fed on semisynthetic diets containing the HC and HS diets as the only source of protein at a 10% level. The composition of the diets designated as HC_TM and HS_TM is given in Table 1. After 4 d of adaptation, feces and urine were collected for 6 d and analyzed for nitrogen content. True digestibility, biologic value, and net protein utilization were computed according to the equations on page 3 under Table 2.

Table 1.

Composition (g/kg) of soy-free (HC_TM) and soy-containing (HS_TM) diets used in experiment 2 (Thomas–Mitchell method)

	HCTM			HSTM
	T0	T1	T2	T0	T1	T2
Basal dieta	422.0	424.0	420.0	383.0	378.0	374.0
Mineral mix	18.0	18.0	18.0	18.0	18.0	18.0
Vitamin mix	5.0	5.0	5.0	5.0	5.0	5.0
Sucrose	120.0	120.0	120.0	120.0	120.0	120.0
Cellulose	17.0	17.0	16.0	31.0	27.0	30.0
Rapeseed oil	16.0	16.0	17.0	19.0	17.0	18.0
Choline chloride	1.0	1.0	1.0	1.0	1.0	1.0
Corn starch	401.0	399.0	403.0	423.0	434.0	434.0

^aHC and HS diets—nonautoclaved (T0), autoclaved at 121 °C for 20 min (T1), and autoclaved at 134 °C for 10 min (T2)—were the only protein source in the study and provided 10% crude protein.

All parameters were calculated as true values (expressed as nitrogen equivalents), because they were corrected for metabolic fecal and endogenous urinary N excretion relative to body weight.²⁷

In vitro analyses.

The total tract digestibility of energy and the metabolizable energy contents of the nonautoclaved and autoclaved HC, HS, and SN diets were determined according to a method simulating digestion in the gut that was developed for the evaluation of pig diets.^9,10 The method involves a 3-step incubation, with porcine pepsin (2000 FIP U/g, Merck, Darmstadt, Germany) at pH 2.0 for 2 h, pancreatin from porcine pancreas (grade VI, 4× USP; Sigma, Poznań, Poland) at pH 6.8 for 4 h, and a mixed multienzyme complex (100 FBG/g, Viscozyme L, Novozymes, Bagsvaerd, Denmark) containing a wide range of microbial carbohydrases, including arabinase, cellulase, β-glucanase, hemicellulase, xylanase, and pectinase, at pH 4.8 for 18 h.⁹ All incubations were performed under continuous magnetic stirring at 39 °C. Undigested residues were collected by filtration and analyzed for organic matter content. The in vitro digestibility of organic matter was calculated as the difference between the organic matter content in the sample and undigested residues. The digestibility of energy and content of digestible and metabolizable energy in the diets were calculated as previously described.^9,10

The ileal digestibility of protein was assayed by using a 2-step incubation procedure, with porcine pepsin at pH 2.0 for 6 h and pancreatin at pH 6.8 for 18 h.⁸ Both incubations were performed under continuous magnetic stirring at 39 °C. Undigested residues were collected by filtration and analyzed for protein content. The apparent in vitro ileal digestibility of protein was calculated as previously described.⁸

The protein solubility in potassium hydroxide and in sodium borate was determined as described previously.^3,26 Briefly, feed samples (approximately 1 g) were stirred for 20 min at room temperature or at 40 °C in 0.5% potassium hydroxide or 0.1 M sodium borate solution (pH 9.2). After centrifugation (10 min, 1655 × g), the nitrogen content in the supernatant was analyzed by using the Kjeldahl method.²

Analytical methods.

The chemical composition of diets, feces, urine, and products from the in vitro determinations (insoluble residues) was analyzed according to standard methods.² The gross energy of diets and excreta was measured in a Parr adiabatic oxygen bomb calorimeter (model KL10, Precyzja, Bydgoszcz, Poland).

Calculation of ME.

The ME concentration of the diets was estimated according to the method of Atwater⁷ and that developed for analyzing pig diets.⁷

Statistics.

Data are presented as means and 95% confidence intervals. Results of animal tests were analyzed by using Statgraphics Centurion version XVI software (Statpoint Technologies, Warrenton, VA). The effects of diet and autoclaving treatments were studied by using 2-factor ANOVA followed by the Tukey post hoc test, at a 5% significance level. Pearson correlation coefficients and regression equations were calculated for parameters related to protein quality analyzed in vitro (protein solubility, protein bound to NDF, in vitro ileal digestibility) and in vivo (apparent digestibility, true digestibility, biologic value).

Results

Chemical composition of the diets.

Chemical composition of the nonautoclaved diets was fairly uniform, except for slightly lower contents of protein, ether extract, and starch in the commercial SN diet than in the HC and HS and greater concentrations of crude fiber, acid detergent fiber, and starch in the HC diet than in HS (Table 2).

Table 2.

Chemical composition (% air-dried matter) of soy-free (HC), soy-containing (HS), and standard (SN) diets before and after autoclaving

	HC			HS			SN
	T0	T1	T2	T0	T1	T2	T0	T1	T2
Dry matter	88.8	88.1	88.2	88.6	88.0	87.9	89.8	89.9	90.1
Crude protein	22.9	22.0	22.4	23.4	23.3	23.4	20.7	21.1	22.0
Crude ash	6.2	6.1	6.0	7.1	7.0	7.1	6.6	6.5	6.4
Ether extract	5.7	5.7	5.5	5.4	6.0	5.9	4.1	3.4	3.1
Crude fiber	5.4	5.3	5.6	2.5	3.5	2.8	4.9	4.7	5.1
ADF	9.0	9.7	9.8	4.7	5.6	5.3	6.5	7.1	7.3
NDF	14.4	15.7	17.2	11.0	15.5	19.6	15.4	25.1	29.0
Nitrogen bound to NDF	0.2	0.3	0.4	0.2	0.6	1.0	0.2	1.0	1.5
Starch	41.7	41.8	39.7	37.9	37.1	35.7	33.3	34.9	34.0
Sugars	3.7	3.4	4.7	5.0	3.9	4.7	5.5	4.4	5.0

ADF, acid detergent fiber; NDF, neutral detergent fiber; T0, nonautoclaved; T1, autoclaved at 121 °C for 20 min; T2, autoclaved at 134 °C for 10 min

Regardless of the conditions used, autoclaving did not influence the nutrient contents univocally but caused an increase in NDF and the amount of protein bound to NDF. This effect was greater after shorter autoclaving at a higher temperature (T2 condition) than after longer treatment at a lower one (T1 condition) and was more prominent for SN than HC and HS. The NDF content in the nonautoclaved diets ranged from 11.0% to 15.4%, compared with 17.2%, 19.6%, and 29.0% in the T2-autoclaved HC, HS, and SN, respectively. The proportion of protein bound to the NDF, expressed as a percentage of the total dietary protein content, was rather uniform among the nonautoclaved diets (range, 5.3% to 6.0%) and increased considerably after autoclaving, was the lowest for HC and greatest for SN, and was greater after T2 than T1 autoclaving. Specifically, autoclaving according to program T1 increased the protein bound to NDF (as a percentage of total protein) to 8.5%, 16.0%, and 29.6% in HC, HS, and SN, respectively, compared with 11.2%, 26.7%, and 42.7% after T2 autoclaving.

Parameters of protein quality.

The effects of autoclaving on protein quality are presented in Table 3. Protein solubility in both solvents was greater in nonautoclaved than autoclaved diets and was the highest for HC across all treatments. Autoclaving considerably decreased the solubility of protein in all diets and in both solvents, with the effect after T2 autoclaving greater than that after T1. The decrease in protein solubility was the smallest for HC and greatest for SN. Ileal protein digestibility assayed in vitro was slightly lower in SN than for the other diets and was not affected by autoclaving.

Table 3.

In vivo and in vitro indices of protein quality of soy-free (HC), soy-containing (HS), and standard (SN) diets

	HC				HS				SN			P
	T0	T1	T2	T0	T1	T2	T0	T1	T2	Diet	Treatment	Interaction
In vivo (n = 8)
Apparent digestibility, %	87.6e 87.0–88.2	86.4d,e 85.9–87.0	85.8d 85.3–86.4	84.3c 83.7–84.8	83.6c 83.0–84.2	81.8b 81.4–82.3	78.4a 77.7–79.1	78.1a 77.7–78.6	78.2a 77.8–78.7	<0.0001	<0.0001	<0.0001
True digestibility, %	90.4c 89.6–91.2	88.2b 87.4–89.0	88.3b 87.5–89.1	88.9b,c 88.1–89.7	88.3b 87.5–89.1	86.1a 85.3–86.9				0.001	<0.0001	0.018
Biological value	83.7 81.1–86.3	80.6 78.0–83.2	81.0 78.3–83.6	79.6 77.0–82.2	78.0 75.4–80.6	75.5 72.9–78.2				0.001	0.038	0.557
Net protein utilization	75.7 73.1–78.2	71.1 68.5–73.7	71.5 68.9–74.1	70.7 68.2–73.3	68.9 66.3–71.5	65.0 62.5–67.6				<0.0001	0.001	0.255
In vitrof
Solubility in KOH, %	89.6 87.7–91.4	67.4 65.5–69.3	60.0 58.1–61.9	80.6 78.7–82.4	51.5 49.6–53.4	38.5 36.7–40.5	83.4 81.5–85.3	37.7 35.8–39.6	33.6 31.7–35.5
Solubility in sodium borate, %	52.4 51.2–53.7	30.9 29.6–32.1	26.2 24.9–27.4	38.2 26.9–39.4	22.8 21.5–24.0	18.7 17.4–20.0	30.1 28.8–31.4	16.7 15.4–18.0	12.3 11.1–13.6
Apparent ileal digestibility, %	84.6 83.9–85.3	83.8 83.0–84.4	83.9 83.2–84.6	83.5 82.7–84.1	84.8 84.1–85.5	83.8 83.1–84.5	79.3 78.6–80.0	80.4 79.6–81.1	80.8 80.1–81.5

T0, nonautoclaved; T1, autoclaved at 121 °C for 20 min; T2, autoclaved at 134 °C for 10 min

Data are presented as mean and 95% confidence interval.

^a–eDifferent superscripted letters indicate values that differ significantly and show Tukey contrasts for interaction.

^fn = 4 except for apparent ileal digestibility (n = 3)

The apparent digestibility of protein determined in experiment 1 was significantly (P < 0.05) affected by diet, autoclaving, and the interaction between these factors. The protein in HC diet had the highest digestibility, whereas that of SN was the lowest. The small negative effect of autoclaving on the apparent protein digestibility was significant (P < 0.05) for HC and HS, whereas the protein digestibility of diet SN did not decrease after autoclaving.

True protein digestibility, as determined in experiment 2 for HC and HS only, was greater in HC than in HS; autoclaving decreased this parameter in both diets. However the diets responded differently to treatments T2 and T1 (P = 0.038 for interaction). For HC, both T1 and T2 decreased protein digestibility to similar levels, whereas only T2 autoclaving altered the protein digestibility of HS. The biologic value of protein was higher in HC than in HS; autoclaving decreased this parameter in both diets. Consequently, net protein utilization was greater for HC than HS, and T2 autoclaving decreased the value by 4.2 and 5.7 units in HC and HS, respectively.

The relationships between digestibility and the biological value of protein determined in rats and the chemical calculation and in vitro methods are shown in Figures 2 through 5. The apparent digestibility of protein in HC, HS, and SN was not correlated with protein solubility in potassium hydroxide (Figure 2 A, P = 0.145) but was positively correlated with solubility in sodium borate (Figure 3 A, P = 0.036) and with ileal in vitro digestibility (Figure 5 A, P = 0.001). True digestibility of diets HC and HS was positively correlated with protein solubility both in potassium hydroxide (Figure 2 B, P = 0.010) and in sodium borate (Figure 3 B, P = 0.015) but not with ileal in vitro protein digestibility (Figure 5 B, P = 0.274). The biological value of protein was positively correlated with protein solubility in potassium hydroxide (Figure 2 C, P = 0.026) and in sodium borate (Figure 3 C, P = 0.036). All parameters of protein value determined in vivo were negatively correlated with the amount of protein bound to NDF (Figure 4 A through C).

Figure 2.

Relationships between protein solubility in potassium hydroxide and (A) apparent (AD) and (B) true (TD) protein digestibility and (C) biological value (BV) in vivo. Protein solubility in potassium hydroxide strongly correlates with true digestibility and biological value of protein in a positive manner. Correlation between protein solubility in potassium hydroxide and apparent digestibility of protein is not significant.

Figure 5.

Relationships between ileal in vitro and (A) apparent (AD) and (B) true (TD) protein digestibility in vivo. Ileal protein digestibility measured in vitro strongly correlates, in a positive manner, with apparent protein digestibility determined in vivo and is not correlated with true digestibility of protein.

Figure 3.

Directly proportional relationships between protein solubility in sodium borate and (A) apparent (AD) and (B) true (TD) protein digestibility and (C) biological value (BV) in vivo. Protein solubility in sodium borate moderately correlates with apparent digestibility of protein and strongly correlates with biological value and true digestibility of protein.

Figure 4.

Relationships between protein bound to neutral detergent fiber (N-NDF contents) and (A) apparent (AD) and (B) true (TD) protein digestibility and (C) biological value (BV) in vivo. Content of protein bound to neutral detergent fiber strongly correlates with true digestibility and biological value of protein in a negative manner. Correlation between content of protein bound to neutral detergent fiber and apparent digestibility is not significant.

Nutrient digestibility and metabolizable energy content.

The apparent digestibility of all nutrients determined in experiment 1 differed among the diets (Table 4). The digestibility of energy and nutrients, except crude fiber and ash, were the lowest in nonautoclaved SN; the digestibility of ash and fiber were the lowest in HC. Autoclaving (both T1 and T2) decreased fat digestibility, but only in SN (P < 0.0001 for interaction) and slightly increased the digestibility of ash in all 3 diets. Autoclaving inconsistently affected fiber digestibility (P = 0.014 for the interaction), which was greatly increased by T2 for HC but by T1 for HS.

Table 4.

Apparent digestibility of nutrients and dietary energy concentration of soy-free (HC), soy-containing (HS) and standard (SN) diets determined in vivo, in vitro and calculated

	HC			HS			SN			P
	T0	T1	T2	T0	T1	T2	T0	T1	T2	Diet	Treatment	Interaction
Digestibility, % (n = 8)
Protein	87.6e	86.4d,e	85.8d	84.3c	83.6c	81.8b	78.4a	78.1a	78.2a	<0.0001	<0.0001	<0.0001
	87.0–88.2	85.9–87.0	85.3–86.4	83.7–84.8	83.0–84.2	81.4–82.3	77.7–79.1	77.7–78.6	77.8–78.7
Crude fat	93.5e	92.3d,e	92.5d,e	91.2d	91.5d	91.8d	84.7c	77.7b	75.4a	<0.0001	<0.0001	<0.0001
	92.8–94.3	91.3–93.3	91.5–93.0	90.4–91.9	90.5–92.4	91.1–92.6	84.0–85.5	76.9–78.4	74.7–76.1
Crude fiber	10.3a	9.5a	17.3a,b	36.2c,d	49.4d	37.1c,d	29.8b,c	28.4b,c	32.2c	<0.0001	0.285	0.014
	3.9–16.8	3.4–15.6	11.1–23.4	30.2–42.3	43.3–55.5	31.2–43.1	23.8–35.9	22.8–34.1	26.8–37.6
N-free extractives	88.9	89.7	89.4	89.7	90.1	90.4	87.6	87.6	87.5	<0.0001	0.230	0.434
	88.5–89.4	89.2–90.3	88.8–89.9	89.1–90.2	89.1–91.0	89.8–90.9	87.0–88.2	87.0–88.1	86.9–88.0
Crude ash	30.6	31.2	33.9	42.5	46.4	44.5	46.3	48.5	47.3	<0.0001	0.002	0.062
	28.8–32.3	29.5–33.0	32.3–35.6	40.9–44.2	44.8–48.1	42.9–46.1	44.7–48.0	47.0–50.0	45.8-48.8
Gross energy, %
In vivo (n = 8)	84.3	84.2	83.9	86.7	86.7	85.9	81.5	81.0	80.3	<0.0001	<0.0001	0.490
	83.7–84.8	83.6–84.7	83.4–84.4	86.4–87.1	86.0–87.3	85.5–86.4	80.9–82.0	80.4–81.5	79.6–80.9
In vitro (n = 3)	83.7	83.1	83.5	87.6	87.0	86.4	83.2	82.0	82.6	<0.0001	0.233	0.783
	82.4–84.9	81.8–84.3	82.3–84.7	86.4–88.9	85.8–88.3	85.1–87.6	82.0–84.5	80.8–83.3	81.4–83.9
Metabolizable energy, MJ/kg
In vivo (n = 8)	14.46	14.30	13.97	15.17	14.72	14.60	13.83	13.66	13.35
	14.37–14.56	14.20–14.41	13.87–14.08	15.03–15.30	14.62–14.83	14.50–14.71	13.74–13.93	13.57–13.76	13.27–13.44
In vitro (n = 3)	14.31	14.07	13.83	15.22	14.74	14.68	14.15	13.89	13.80
	14.09–14.53	13.84–14.29	13.61–14.05	15.00–15.44	14.53–14.97	14.47–14.91	13.93–14.37	13.66–14.11	13.58–14.03
Calculated Af (n = 1)	16.00	16.02	15.95	16.26	16.24	16.32	15.67	15.58	15.46
Calculated Pg (n = 1)	14.41	14.23	14.12	14.61	14.35	14.40	13.15	13.13	13.10

T0, nonautoclaved; T1, autoclaved at 121 °C for 20 min; T2, autoclaved at 134 °C for 10 min.

Data are presented as mean and 95% confidence interval.

^a–eDifferent superscripted letters within a row indicate that the values differ significantly and show Tukey contrasts for interaction.

^fEnergy calculated according to the Atwater formula.

^gEnergy calculated according to the pig formula.

The ME concentration determined in rats in experiment 1 was the highest in HS and lowest in SN (Table 4). Autoclaving, regardless of treatment, produced a small but consistent depression of the ME content in all of the diets. The ME values determined by using the in vitro method were very close to those determined in rats and showed that the HS diet had the highest ME content and that autoclaving had a small but consistent negative effect. The ME values computed according to the pig formula were nearly identical with those determined in vivo and confirmed the slight negative effect of autoclaving. The ME values calculated according to the Atwater formula ranged from about 1.5 to 2.0 MJ/kg and were higher than those determined in rats, in vitro, and calculated according to the pig formula.

Discussion

The composition of the diet fed to laboratory animals affects their physiologic and metabolic responses and may consequently affect experimental outcomes.¹³ In view of the effects of soy phytoestrogens on the utility of rats and mice as animal models, soybean meal—which is the most common protein source in diets for laboratory animals—is recommended to be replaced by other protein sources.^11,36 Among alternatives, casein is considered one of the most suitable, given its high nutritional value.³⁸ However, because thermal sterilization might decrease the quality of dietary protein, optimization of the time and temperature parameters likely will benefit the quality and uniformity of the diets.

In the current study, all indices of protein quality determined in rats, including digestibility, biologic value, and net protein utilization, were higher for casein than soy, in agreement with our previous results.³¹ This finding may facilitate formulating well-defined low-phytoestrogen diets, which are indispensable in studies on metabolic pathways influenced by hormones.¹¹ Commenting on the lower protein value of the commercial SN diet is difficult, in view of its closed formula.

Overall, autoclaving had a moderately negative effect on the protein quality of HC and HS determined in rats, but autoclaving for a longer time at a lower temperature had less of a negative effect on all of the in vivo parameters, protein solubility, and chemically determined indices than did shorter treatment at a higher temperature. We previously found that, compared with T2, the T1 program caused smaller losses of vitamins and lower levels of potentially harmful compounds, suggesting that the T1 program might be suitable for implementation in practice.³⁹

Our results concerning protein value are not strictly comparable with those found in other studies because of differences among autoclaving parameters,^15,17 but they are in line with general observations regarding decreased protein solubility, protein utilization, and animal performance due to raising the temperature or prolonging the time of heating of animal feeds.^1,3,19,26,30 One of the reasons for the lower protein value of heat-treated feeds is the increase of the NDF and protein bound to NDF, a consistent finding in our studies and other experiments, and its negative influence on protein and amino acid availability in monogastric animals.^12,21,23 Contrary to our expectations, autoclaving had no effect on the in vitro measurement of ileal (enzymatic) protein digestibility. This result thus calls into question the reliability of this method in rat studies.

The energy digestibility of the HS diet determined in rats and in vitro was greater than of the HC diet mainly due to a markedly greater digestibility of fiber (36.2% compared with 10.3% in nonautoclaved diets, respectively). The difference in fiber digestibility was a consequence of more intensive fermentation of the intrinsic soy fiber than of weakly fermented cellulose added to the HC diet.^6,22,24 In other studies, the fiber digestibility of rat diets was far more variable than was the digestibility of other nutrients and ranged from 6.7% to 65.6%; this broad range points to vast differences in the extent of microbial degradation of this component.⁷ Both our results and those reported in the literature ^6,7,22,24 point to the importance of the source of fiber in diets for laboratory animals. Both the content and the characteristics of dietary fiber affect the rate of microbial fermentation in the hindgut and the profile of short-chain fatty acids. Because the type of fiber can influence protein, fat, carbohydrate, and mineral metabolism and affect the health status of animals,^4,34,41 inconsistency regarding the dietary fiber in natural-ingredient diets may contribute to the variability of animal responses in experiments and should be monitored.

The concentrations of metabolizable energy in HS and HC diets determined in rats, in vitro, and calculated according to the pig formula were close to the recommended concentration of 15.0 MJ/kg for growing rats,²⁸ whereas the determined and calculated energy value of SN was close to the 13.3 MJ/kg, which the producer reported as estimated according to the pig formula. Autoclaving decreased the ME concentration in all diets by 1% to 4%, with the effect of T1 being less than that of T2. The slightly decreased ME value likely will not negatively affect the performance of growing and adult animals, because they can increase feed intake to compensate for its smaller energy density, but the ME level may be marginal for lactating female rats, particularly those simultaneously pregnant, because these animals have a very high energy requirement.^28,37

We conclude that applying a less detrimental autoclaving program highly preserves the protein and energy values of the treated diets and, accordingly, likely leads to more economical use of feed, due to decreased consumption. However, our earlier studies did not reveal any negative effects of either of the 2 programs on animal performance, physiologic functions, or health.⁵

The results of the present study show that the concentration of metabolizable (available) energy in natural-ingredient diets can be estimated highly precisely according to the in vitro method and from their chemical composition by using the formula developed for pig feeds. The in vitro determined ME values were very similar to the respective ME values determined in vivo and revealed a small negative effect of autoclaving under the tested conditions. Physiologically available energy values calculated according to the Atwater equation was more than 1.5 MJ/kg greater than the values determined experimentally and calculated according to the pig formula and did not show an effect of autoclaving. The better predictive value of the pig formula than of the Atwater equation is in agreement with the results of other researchers, who concluded that Atwater's system can be applied to purified diets containing highly digestible nutrients, whereas the ME concentration in natural-ingredient diets should be calculated according to the pig formula.⁷

Despite the limited number of data, the current study demonstrated the utility of chemical and in vitro measurements as predictors of the nutritional value of processed diets. Actual protein digestibility as well as its biologic value, representing utilization of the absorbed protein, were negatively correlated to the amount of protein bound to the NDF and positively to protein solubility in both solvents. These results are in agreement with relationships found for rapeseed meal and cake heated at different intensities.²⁹ However, the incorporation of these parameters into practice should be preceded by studies encompassing more diets and a wider range of treatment parameters. Contrary to our expectation, the in vitro method for determining ileal protein digestibility, developed to imitate enzymatic digestion in pigs, appeared to have limited predictive value for rats, because this technique seems to depend on the age of the animals: the method approximated protein digestibility in older, but not younger, rats.

In addition, our current results point to the high accuracy of the chemical and in vitro methods developed for pigs for assessing the ME concentration of diets for rats. These methods might be useful for estimating the energy value of experimental nonpurified diets or for verifying the energy content reported by a feed producer, especially when the system used for calculation is not indicated.