DOI: 10.1111/jpn.12272
ORIGINAL ARTICLE
Growth performance and physiological parameters of conventional and specified pathogen-free rats fed autoclaved diets with different protein sources ska-Furas2, M. Barszcz1, J. Paradziej-Łukowicz2, M. Taciak1, A. Tusnio1, Ł. Staskiewicz1, B. Muszyn 2 1 1 A. Lewandowska , B. Pastuszewska and J. Skomiał 1 The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Jabłonna, Poland, and sk, Tri-City Animal Laboratory Research and Service Center, Gdan sk, Poland 2 Medical University of Gdan
Summary The effects of feeding autoclaved commercial SSNIFF (SN) diet and diets containing soya bean (S) and casein (C) to growing conventional (CON) and specified pathogen-free (SPF) rats were determined. Diets S, C and SN, autoclaved at 121 °C during 20 min (T1), at 134 °C during 10 min (T2) and non-autoclaved (T0), were fed during four weeks, each to 8 CON males and 8 females of mean initial body weight 56 g, kept individually. Diets S, C and SN, autoclaved at T1, were fed during two months, each to 20 SPF males and 20 females of mean initial body weight 58 g, kept in group of 5 animals per cage. In CON rats, autoclaving did not affect feed intake and weight gain, decreased thyroid and stomach weight, increased caecal tissue and digesta weight, and concentrations of isobutyric, isovaleric and valeric acid in caecal digesta. Among biochemical blood parameters, autoclaving decreased only total protein concentration and aspartate aminotransferase activity. Feeding C diet resulted in lower feed intake and weight gain in CON and SPF males. Diet affected organ weights and the greatest differences were found in rats on SN diet for weights of stomach, caecum and female reproductive organs. Diet affected concentration of all short-chain fatty acids, pH and weight of caecal digesta, the most important being the greatest butyric acid concentration on SN diet and isoacids on C diet. It is concluded that autoclaving of both soyacontaining and soya-free diets does not affect negatively animal performance and physiology. Keywords rat, diets, autoclaving, protein source, blood parameters, fermentation Correspondence Marcin Barszcz, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, 05-110 Jabłonna, Instytucka 3, Poland. Tel: +48 22 76 53 300; Fax: +48 22 76 53 302; E-mail: [email protected] Received: 18 April 2014; accepted: 15 October 2014
Autoclaving is a convenient and commonly applied method of sterilization of diets for specified pathogenfree (SPF) rodents, and the autoclavable commercial diets for breeding and maintenance purposes are now available. These diets are usually fortified with additional amounts of vitamins to compensate for their anticipated losses and should also contain more protein to overcome its lower availability. As a thermal processing, autoclave sterilization leads to formation of Maillard reaction products, decreases protein digestibility and availability of essential amino acids (Seiquer et al., 2006). Diet is a major factor affecting all vital processes such as growth, reproduction and longevity, but also response to harmful agents such as pathogens, toxins and chemicals (Rao, 1997). Composition of the diet is
therefore essential for the validity of biomedical research conducted on laboratory animals and particularly on the SPF rodents. The SPF animals require diet which is designed for sterilization and does not disturb outcome of the experiments. Most of the natural-ingredient diets for laboratory animals contain soya bean meal as the main protein source. Soya bean products contribute to oestrogenic activity of the diet because of the presence of phyto-oestrogens (Rao, 1996); therefore, the use of such components is not recommended (Brown and Setchell, 2001). The lowphyto-oestrogen diets, containing other protein sources, are available at the market, but their exact composition is often not disclosed. It has been established long ago that the negative effects of autoclaving, determined in conventional animals, depend on the temperature and duration of the treatment (Eggum, 1969; Ford, 1976), but the
Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
1
Introduction
M. Barszcz et al.
Response of rats to dietary protein in autoclaved diets
well-founded recommendations on the autoclaving parameters of commercial diets are lacking. As autoclaving reduces nutritional value of the diets, including decrease of protein availability, losses of vitamins
and formation of potentially toxic compounds, determination of the overall impact of this treatment on animal performance and physiology seems to be of particular importance. The objective of the study was to evaluate the effect of autoclaved diets differing in source of supplementary protein on growth performance and physiology of conventional and SPF rats. Feeding non-autoclaved diets in SPF breeding units is not allowed; therefore, the effect of autoclaved diets was determined on conventional animals and then, after choosing less detrimental procedure of sterilization, on SPF rats.
Table 1 Composition of experimental diets Ingredients (g/kg)
S*
C1†
C2‡
Cereals§ Soya bean meal Casein Sodium caseinate Fibre preparation Rapeseed oil Dried brewer yeast Minerals¶ Vitamins** Amino acids††
605.3 200.0 50.0 – – 40.0 40.0 50.0 10.0 4.7
665.1 – 140.0 – 50.0 40.0 40.0 50.0 10.0 4.9
665.1 – – 140.0 50.0 40.0 40.0 50.0 10.0 4.9
Materials and methods Diets and experimental design
Three cereal-based diets containing soya bean meal (S), casein or sodium caseinate (C) as the main source of supplementary protein were formulated to meet requirement of growing rats according to NRC (1995) and fortified with greater amounts of heat labile vitamins. Composition of the diets is shown in Table 1. The autoclavable SSNIFF diet (SN), not declared as low phyto-oestrogen (catalogue No. 1324-3; SSNIFF, 2007), was used as a control diet. According to producer, the SN diet contained grain and grain by-products, oil seed products, minerals, brewer yeast, vegetable oils, vitamins and trace elements. The S, C and SN diets for experiment 1 on conventional rats were autoclaved in a steam autoclave STERIVAP SP HP 9612-2ED (BMT, Czech Republic) at 121 °C for
*Diet with soya bean meal. †Diet with casein used in experiment 1 on conventional rats. ‡Diet with sodium caseinate used in experiment 2 on SPF rats. §Wheat and maize. ¶Minerals added (per kg diet): Ca, 12.6 g; Na, 2.7 g; P, 5.7 g; Fe, 160 mg; Mn, 50 mg; Zn, 60 mg; I, 1.4 mg; Se, 0.3 mg; Co, 2.0 mg; Cu, 13 mg. **Vitamins added (per kg diet): vitamin A, 30 000 IU; vitamin D3, 1700 IU; vitamin K, 6 mg; vitamin E, 100 mg; ascorbic acid, 60 mg; thiamine, 108 mg; riboflavin, 20 mg; pyridoxine, 17 mg; cobalamin, 0.14 mg; niacin, 170 mg; Ca-pantothenate, 51 mg; folic acid, 6 mg; biotin, 0.25 mg; choline chloride, 1600 mg; inositol, 130 mg. ††Amino acids added (per kg diet): L-lysine, 1.5 g in S and 1.1 g in C1 and C2 diets; DL-methionine, 2.0 g; L-threonine, 0.7 g in S and 1.0 g in C1 and C2 diets; L-tryptophan, 0.5 g in S and 0.8 g in C1 and C2 diets.
Table 2 Nutrient content in experimental diets SN*
S
C1
C2
Nutrient content (% dry matter)
T0†
T1‡
T2§
T0
T1
T2
T0
T1
T2
Dry matter Crude protein Crude ash Ether extract Crude fibre ADF¶ NDF** N-NDF†† Starch Sugars
89.8 20.7 6.6 4.1 4.9 6.5 15.4 0.2 33.3 5.5
89.9 21.1 6.5 3.4 4.7 7.1 25.1 1.0 34.9 4.4
90.1 22.0 6.4 3.1 5.1 7.3 29.0 1.5 34.0 5.0
88.6 23.4 7.1 5.4 2.5 4.7 11.0 0.2 37.9 5.0
88.0 23.3 7.0 6.0 3.5 5.6 15.5 0.6 37.1 3.9
87.9 23.4 7.1 5.9 2.8 5.3 19.6 1.0 35.7 4.7
88.8 22.9 6.2 5.7 5.4 9.0 14.4 0.2 41.7 3.7
88.1 22.0 6.1 5.7 5.3 9.7 15.7 0.3 41.8 3.4
88.2 22.4 6.0 5.5 5.6 9.8 17.2 0.4 39.7 4.7
91.8 21.8 6.2 6.3 6.9 9.7 24.2 0.6 48.8 4.7
*Commercial SSNIFF diet. †Not autoclaved diet. ‡Diet autoclaved at 121 °C for 20 min. §Diet autoclaved at 134 °C for 10 min. ¶Acid-detergent fibre. **Neutral-detergent fibre. ††Protein bound to NDF.
2
Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
M. Barszcz et al.
20 min or at 134 °C for 10 min (programmes denoted as treatment T1 and T2 respectively), followed by cooling and drying during 15 min. Efficiency of sterilization was verified using biological test SPORAL A. One portion of each diet was not autoclaved (T0). The S, C and SN diets for experiment 2 on SPF rats were autoclaved only at 121 °C for 20 min. Sodium caseinate was chosen for SPF rats as a protein source easier available on the market than casein. Experiment 1 was performed as a block design with a 2 9 3 9 3 arrangement of factors comprising gender, diet (SN, S and C) and autoclaving programme (T0, T1 and T2), whereas experiment 2 was conducted in a 2-factorial design with gender and diet (SN, S and C) as the experimental factors. Experimental procedures were approved by the Local Ethical Commission. Animals Experiment 1
The experiment was performed on 144 three-weekold Crl:W (Han) Wistar rats divided into 9 groups (8 females and 8 males per group) of mean initial body weight 56 g. Rats were housed individually in wirebottom metabolic cages, in a room with a 12-h dark– light cycle, at 22 1 °C. Rats were given free access to feed and water. Body weight and feed intake were measured weekly. After 28 days, rats were sacrificed and blood was collected into heparinized tubes by intracardiac puncture, and plasma was stored at 20 °C for further analyses. Internal organs (thymus, thyroid, heart, spleen, liver, pancreas, stomach, small intestine, caecum, kidneys, testicles, ovaries, oviducts and uterus) were excised and weighed. Caecal digesta was also weighed and stored until analyses at 20 °C. Experiment 2
The experiment was performed on 120 Crl:W (Han) Wistar rats of SPF status confirmed according to recommendations of Federation of European Laboratory Animal Science Associations (Nicklas et al., 2002). Four-week-old rats of mean body weight 58 g were randomly assigned to three experimental groups (20 females and 20 males per group). Animals were housed for 2 months in polypropylene cages, five rats of one gender per cage, in constant conditions of temperature 22 1 °C, humidity 55 10%, 12-h dark– light cycle, with air exchanged 12 times or more per hour. Rats were fed ad libitum and had free access to water. Body weight and feed intake were measured weekly. At the end of the experiment, 6 females and 6 males from each group were sacrificed and blood was Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
Response of rats to dietary protein in autoclaved diets
taken for biochemical analyses as in experiment 1. For morphological analyses, blood was collected into tubes with ethylenediamine tetraacetic acid (EDTA). Analytical procedures
Dry matter, crude protein, ether extract, crude ash, crude fibre, acid-detergent fibre (ADF), neutral-detergent fibre (NDF), protein bound to NDF (N-NDF), starch and sugars contents in diets were determined according to Association of Official Analytical Chemists (AOAC) (2000). Caecal digesta pH was measured on WTW pH/340 pH-meter (WTW GmbH, Weilheim, Germany), and short-chain fatty acids (SCFA) concentrations were analysed using HP 5890 Series II gas chromatograph (Hewlett-Packard, Waldbronn, Germany) as described earlier (Barszcz et al., 2011). Biochemical parameters of blood plasma were determined spectrophotometrically on MAXMAT PL multidisciplinary diagnostic platform (Erba Diagnostics France SARL, Montpellier, France). Blood glucose concentration was measured colorimetrically based on
Table 3 Growth performance of conventional rats (experiment 1)
Diet Males SN
S
C
Females SN
S
C
SEM p-values* Diet Programme Gender Diet 9 Gender
Programme
Feed intake (g)
Weight gain (g)
Feed efficiency (g/g)
T0 T1 T2 T0 T1 T2 T0 T1 T2
540 550 543 509 520 503 518 507 503
169 172 166 169 175 177 167 161 163
3.2 3.2 3.3 3.0 3.0 2.9 3.1 3.2 3.1
T0 T1 T2 T0 T1 T2 T0 T1 T2
422 424 437 429 423 419 438 408 419 4.9
102 101 100 112 104 104 111 104 107 2.8
4.2 4.2 4.4 3.8 4.1 4.1 4.0 3.9 3.9 0.05
0.004 0.699
ORIGINAL ARTICLE
Growth performance and physiological parameters of conventional and specified pathogen-free rats fed autoclaved diets with different protein sources ska-Furas2, M. Barszcz1, J. Paradziej-Łukowicz2, M. Taciak1, A. Tusnio1, Ł. Staskiewicz1, B. Muszyn 2 1 1 A. Lewandowska , B. Pastuszewska and J. Skomiał 1 The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Jabłonna, Poland, and sk, Tri-City Animal Laboratory Research and Service Center, Gdan sk, Poland 2 Medical University of Gdan
Summary The effects of feeding autoclaved commercial SSNIFF (SN) diet and diets containing soya bean (S) and casein (C) to growing conventional (CON) and specified pathogen-free (SPF) rats were determined. Diets S, C and SN, autoclaved at 121 °C during 20 min (T1), at 134 °C during 10 min (T2) and non-autoclaved (T0), were fed during four weeks, each to 8 CON males and 8 females of mean initial body weight 56 g, kept individually. Diets S, C and SN, autoclaved at T1, were fed during two months, each to 20 SPF males and 20 females of mean initial body weight 58 g, kept in group of 5 animals per cage. In CON rats, autoclaving did not affect feed intake and weight gain, decreased thyroid and stomach weight, increased caecal tissue and digesta weight, and concentrations of isobutyric, isovaleric and valeric acid in caecal digesta. Among biochemical blood parameters, autoclaving decreased only total protein concentration and aspartate aminotransferase activity. Feeding C diet resulted in lower feed intake and weight gain in CON and SPF males. Diet affected organ weights and the greatest differences were found in rats on SN diet for weights of stomach, caecum and female reproductive organs. Diet affected concentration of all short-chain fatty acids, pH and weight of caecal digesta, the most important being the greatest butyric acid concentration on SN diet and isoacids on C diet. It is concluded that autoclaving of both soyacontaining and soya-free diets does not affect negatively animal performance and physiology. Keywords rat, diets, autoclaving, protein source, blood parameters, fermentation Correspondence Marcin Barszcz, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, 05-110 Jabłonna, Instytucka 3, Poland. Tel: +48 22 76 53 300; Fax: +48 22 76 53 302; E-mail: [email protected] Received: 18 April 2014; accepted: 15 October 2014
Autoclaving is a convenient and commonly applied method of sterilization of diets for specified pathogenfree (SPF) rodents, and the autoclavable commercial diets for breeding and maintenance purposes are now available. These diets are usually fortified with additional amounts of vitamins to compensate for their anticipated losses and should also contain more protein to overcome its lower availability. As a thermal processing, autoclave sterilization leads to formation of Maillard reaction products, decreases protein digestibility and availability of essential amino acids (Seiquer et al., 2006). Diet is a major factor affecting all vital processes such as growth, reproduction and longevity, but also response to harmful agents such as pathogens, toxins and chemicals (Rao, 1997). Composition of the diet is
therefore essential for the validity of biomedical research conducted on laboratory animals and particularly on the SPF rodents. The SPF animals require diet which is designed for sterilization and does not disturb outcome of the experiments. Most of the natural-ingredient diets for laboratory animals contain soya bean meal as the main protein source. Soya bean products contribute to oestrogenic activity of the diet because of the presence of phyto-oestrogens (Rao, 1996); therefore, the use of such components is not recommended (Brown and Setchell, 2001). The lowphyto-oestrogen diets, containing other protein sources, are available at the market, but their exact composition is often not disclosed. It has been established long ago that the negative effects of autoclaving, determined in conventional animals, depend on the temperature and duration of the treatment (Eggum, 1969; Ford, 1976), but the
Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
1
Introduction
M. Barszcz et al.
Response of rats to dietary protein in autoclaved diets
well-founded recommendations on the autoclaving parameters of commercial diets are lacking. As autoclaving reduces nutritional value of the diets, including decrease of protein availability, losses of vitamins
and formation of potentially toxic compounds, determination of the overall impact of this treatment on animal performance and physiology seems to be of particular importance. The objective of the study was to evaluate the effect of autoclaved diets differing in source of supplementary protein on growth performance and physiology of conventional and SPF rats. Feeding non-autoclaved diets in SPF breeding units is not allowed; therefore, the effect of autoclaved diets was determined on conventional animals and then, after choosing less detrimental procedure of sterilization, on SPF rats.
Table 1 Composition of experimental diets Ingredients (g/kg)
S*
C1†
C2‡
Cereals§ Soya bean meal Casein Sodium caseinate Fibre preparation Rapeseed oil Dried brewer yeast Minerals¶ Vitamins** Amino acids††
605.3 200.0 50.0 – – 40.0 40.0 50.0 10.0 4.7
665.1 – 140.0 – 50.0 40.0 40.0 50.0 10.0 4.9
665.1 – – 140.0 50.0 40.0 40.0 50.0 10.0 4.9
Materials and methods Diets and experimental design
Three cereal-based diets containing soya bean meal (S), casein or sodium caseinate (C) as the main source of supplementary protein were formulated to meet requirement of growing rats according to NRC (1995) and fortified with greater amounts of heat labile vitamins. Composition of the diets is shown in Table 1. The autoclavable SSNIFF diet (SN), not declared as low phyto-oestrogen (catalogue No. 1324-3; SSNIFF, 2007), was used as a control diet. According to producer, the SN diet contained grain and grain by-products, oil seed products, minerals, brewer yeast, vegetable oils, vitamins and trace elements. The S, C and SN diets for experiment 1 on conventional rats were autoclaved in a steam autoclave STERIVAP SP HP 9612-2ED (BMT, Czech Republic) at 121 °C for
*Diet with soya bean meal. †Diet with casein used in experiment 1 on conventional rats. ‡Diet with sodium caseinate used in experiment 2 on SPF rats. §Wheat and maize. ¶Minerals added (per kg diet): Ca, 12.6 g; Na, 2.7 g; P, 5.7 g; Fe, 160 mg; Mn, 50 mg; Zn, 60 mg; I, 1.4 mg; Se, 0.3 mg; Co, 2.0 mg; Cu, 13 mg. **Vitamins added (per kg diet): vitamin A, 30 000 IU; vitamin D3, 1700 IU; vitamin K, 6 mg; vitamin E, 100 mg; ascorbic acid, 60 mg; thiamine, 108 mg; riboflavin, 20 mg; pyridoxine, 17 mg; cobalamin, 0.14 mg; niacin, 170 mg; Ca-pantothenate, 51 mg; folic acid, 6 mg; biotin, 0.25 mg; choline chloride, 1600 mg; inositol, 130 mg. ††Amino acids added (per kg diet): L-lysine, 1.5 g in S and 1.1 g in C1 and C2 diets; DL-methionine, 2.0 g; L-threonine, 0.7 g in S and 1.0 g in C1 and C2 diets; L-tryptophan, 0.5 g in S and 0.8 g in C1 and C2 diets.
Table 2 Nutrient content in experimental diets SN*
S
C1
C2
Nutrient content (% dry matter)
T0†
T1‡
T2§
T0
T1
T2
T0
T1
T2
Dry matter Crude protein Crude ash Ether extract Crude fibre ADF¶ NDF** N-NDF†† Starch Sugars
89.8 20.7 6.6 4.1 4.9 6.5 15.4 0.2 33.3 5.5
89.9 21.1 6.5 3.4 4.7 7.1 25.1 1.0 34.9 4.4
90.1 22.0 6.4 3.1 5.1 7.3 29.0 1.5 34.0 5.0
88.6 23.4 7.1 5.4 2.5 4.7 11.0 0.2 37.9 5.0
88.0 23.3 7.0 6.0 3.5 5.6 15.5 0.6 37.1 3.9
87.9 23.4 7.1 5.9 2.8 5.3 19.6 1.0 35.7 4.7
88.8 22.9 6.2 5.7 5.4 9.0 14.4 0.2 41.7 3.7
88.1 22.0 6.1 5.7 5.3 9.7 15.7 0.3 41.8 3.4
88.2 22.4 6.0 5.5 5.6 9.8 17.2 0.4 39.7 4.7
91.8 21.8 6.2 6.3 6.9 9.7 24.2 0.6 48.8 4.7
*Commercial SSNIFF diet. †Not autoclaved diet. ‡Diet autoclaved at 121 °C for 20 min. §Diet autoclaved at 134 °C for 10 min. ¶Acid-detergent fibre. **Neutral-detergent fibre. ††Protein bound to NDF.
2
Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
M. Barszcz et al.
20 min or at 134 °C for 10 min (programmes denoted as treatment T1 and T2 respectively), followed by cooling and drying during 15 min. Efficiency of sterilization was verified using biological test SPORAL A. One portion of each diet was not autoclaved (T0). The S, C and SN diets for experiment 2 on SPF rats were autoclaved only at 121 °C for 20 min. Sodium caseinate was chosen for SPF rats as a protein source easier available on the market than casein. Experiment 1 was performed as a block design with a 2 9 3 9 3 arrangement of factors comprising gender, diet (SN, S and C) and autoclaving programme (T0, T1 and T2), whereas experiment 2 was conducted in a 2-factorial design with gender and diet (SN, S and C) as the experimental factors. Experimental procedures were approved by the Local Ethical Commission. Animals Experiment 1
The experiment was performed on 144 three-weekold Crl:W (Han) Wistar rats divided into 9 groups (8 females and 8 males per group) of mean initial body weight 56 g. Rats were housed individually in wirebottom metabolic cages, in a room with a 12-h dark– light cycle, at 22 1 °C. Rats were given free access to feed and water. Body weight and feed intake were measured weekly. After 28 days, rats were sacrificed and blood was collected into heparinized tubes by intracardiac puncture, and plasma was stored at 20 °C for further analyses. Internal organs (thymus, thyroid, heart, spleen, liver, pancreas, stomach, small intestine, caecum, kidneys, testicles, ovaries, oviducts and uterus) were excised and weighed. Caecal digesta was also weighed and stored until analyses at 20 °C. Experiment 2
The experiment was performed on 120 Crl:W (Han) Wistar rats of SPF status confirmed according to recommendations of Federation of European Laboratory Animal Science Associations (Nicklas et al., 2002). Four-week-old rats of mean body weight 58 g were randomly assigned to three experimental groups (20 females and 20 males per group). Animals were housed for 2 months in polypropylene cages, five rats of one gender per cage, in constant conditions of temperature 22 1 °C, humidity 55 10%, 12-h dark– light cycle, with air exchanged 12 times or more per hour. Rats were fed ad libitum and had free access to water. Body weight and feed intake were measured weekly. At the end of the experiment, 6 females and 6 males from each group were sacrificed and blood was Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
Response of rats to dietary protein in autoclaved diets
taken for biochemical analyses as in experiment 1. For morphological analyses, blood was collected into tubes with ethylenediamine tetraacetic acid (EDTA). Analytical procedures
Dry matter, crude protein, ether extract, crude ash, crude fibre, acid-detergent fibre (ADF), neutral-detergent fibre (NDF), protein bound to NDF (N-NDF), starch and sugars contents in diets were determined according to Association of Official Analytical Chemists (AOAC) (2000). Caecal digesta pH was measured on WTW pH/340 pH-meter (WTW GmbH, Weilheim, Germany), and short-chain fatty acids (SCFA) concentrations were analysed using HP 5890 Series II gas chromatograph (Hewlett-Packard, Waldbronn, Germany) as described earlier (Barszcz et al., 2011). Biochemical parameters of blood plasma were determined spectrophotometrically on MAXMAT PL multidisciplinary diagnostic platform (Erba Diagnostics France SARL, Montpellier, France). Blood glucose concentration was measured colorimetrically based on
Table 3 Growth performance of conventional rats (experiment 1)
Diet Males SN
S
C
Females SN
S
C
SEM p-values* Diet Programme Gender Diet 9 Gender
Programme
Feed intake (g)
Weight gain (g)
Feed efficiency (g/g)
T0 T1 T2 T0 T1 T2 T0 T1 T2
540 550 543 509 520 503 518 507 503
169 172 166 169 175 177 167 161 163
3.2 3.2 3.3 3.0 3.0 2.9 3.1 3.2 3.1
T0 T1 T2 T0 T1 T2 T0 T1 T2
422 424 437 429 423 419 438 408 419 4.9
102 101 100 112 104 104 111 104 107 2.8
4.2 4.2 4.4 3.8 4.1 4.1 4.0 3.9 3.9 0.05
0.004 0.699
