Effect of dietary crude protein and energy content on nitrogen utilisation, water intake and urinary output in growing pigs Jarmo Valaja Agricultural Research Centre ofFinland, Animal Production Research, Animal Nutrition, FIN-31600 Jokioinen. Finland, e-mail: jarmo.valaja@mtt.fi Hilkka Siljander-Rasi Agricultural Research Centre ofFinland, Animal Production Research, Pig Husbandry, Tervamdentie 179, FIN-05840 Hyvinkää, Finland A digestibility and balance trial was carried out with four intact castrated male pigs (live weight 33- 82 kg) to study the effects of dietary crude protein and energy content on nutrient digestibility, nitro- gen metabolism, water intake and urinary output. In a 4 x 4 Latin square design, four barley-oats- soya bean meal based diets were arranged 2x2 factorially. The corresponding factors were dietary crude protein (CP) content: high (180 g/kg CP) or low protein diet (140 g/kg CP) supplemented with free lysine, methionine and threonine; and dietary net energy content; high (1.05 feed units (FU)/kg) (feed unit=9.3 MJ net energy) or low net energy content (0.95 FU/kg). Lowering dietary CP content (mean values of 189 to 152 g/kg dry matter, respectively) by supple- mentation of free amino acids decreased urinary nitrogen (N) excretion by 6.9 g/day (32%) (PcO.Ol) with no effect on faecal N excretion. Dietary protein reduction also increased the proportion of N retained per unit intake (PcO.lO) and absorption (P<0.05). Daily N retention of pigs tended to decline with reduced dietary CP content (PcO.lO). Dietary energy intake had no effect on N metabolism. Water intake and urinary output of pigs was unaffected by variations in protein and energy content which may be due to limitations in change-over experimental designs. The present experiment clear- ly demonstrated benefical effects of lowering dietary protein supply by free amino acid supplementa- tion on N excretion. Key words: amino acids, digestibility, lysine, methionine, nitrogen excretion, protein, threonine ntroduction have altered feeding strategies. Formerly, feed- ing strategies aimed at maximising production performance without special concern for protein and amino acid supply. Environmental con- straints have directed pig feeding towards low- In pig production environmental problems relat- ed to nitrogen (N) excretion in animal manure © Agricultural and Food Science in Finland Manuscript received March 1998 381 Vol. 7(1998): 381-390. AGRICULTURAL AND FOOD SCIENCE IN FINLAND Valaja, J. & Siljander-Rasi, H. Effect ofdietary protein and energy on nitrogen utilisation in pigs est possible levels of nitrogen output by adjust- ment of protein and amino acid supply relative to requirements (Henry and Dourmad 1993). Availability of free amino acids, lysine, methio- nine, threonine and tryptophan, for practical feed formulation has given new opportunities to match amino acid and protein supply closer to requirements based on the ideal protein concept. Feeding of pigs with free amino acid supple- ments to low protein diets based on barley (Näsi 1985, Gatel and Grosjean 1992) or corn (Pierce et al. 1994, Kerr and Easter 1995) have clearly decreased N excretion in urine. In many experi- ments, however, daily N retention of pigs has simultaneusly been depressed indicating a defi- ciency of certain amino acids or total protein supply (Näsi 1985, Lee et al. 1993, Pierce et al. 1994, Kerr and Easter 1995). Lowering of pro- tein supply of pigs has also decreased water in- take and urine production (Wahlström et al. 1970, Close et al. 1983) and can therereby affect slur- ry output (Kay and Lee 1997). N retention is related to energy intake if an adequate amount of protein is available (Campbell et al. 1985,Dunkin et al. 1986)and can threrefore influence N excre- tion of pigs. However, quantitative effects of varying both energy and protein supply on N excretion and water intake of pigs have not been assessed in balance studies. The aim of this study was to investigate the effects of dietary crude protein (CP) and energy content on nutrient digestibility and N metabo- lism of growing pigs. In addition, the effects of dietary treatments on water intake and urinary output were studied. Dietary CP was reduced by supplementation of free lysine, threonine and methionine in order to maintain an equal dietary content of ileal digestible amino acids. Material and methods The study was conducted on four castrated male pigs (Landrace x Finnish Yorkshire) between an average weight range of 33.5 (SE 1.03) and 82.0 (SE 0.91) kg. Experimental design was a 4 x 4 latin square, where four experimental diets were arranged 2x2 factorially. The corresponding factors were dietary CP content: high protein diet (180 g/kg CP) without amino acid supplementa- tion or low protein diet (140 g/kg CP) supple- mented with free lysine, methionine and threo- nine; and dietary net energy content: high (1.05 feed units (FU)/kg) (feed unit = 9.3 MJ net en- ergy) or low net energy content (0.95 FU/kg). Animals were kept individually in pens of 1.43 m x 1.23 m with a slattedplastic floor throughout the study. The four 10-day experimental periods com- prised of preliminary feeding for 5 days followed by 5 days of total faeces and urine collection. Faeces was collected using plastic bags attached around the anus with glued adhesive tape and snap-fasteners (Van Kleef et al. 1994). Bags were replaced after the pigs had defecated. Collected faeces was weighed and stored at -20°C until the end of experimental period. Urine was quan- titatively collected via collection trays installed under the pens into 40 ml of 10 N H 2S04 , sub- sampled and frozen at -20°C. Four experimental diets consisted of barley, oats, soya bean meal and wheat syrup (Table 1). Diets were supplemented with minerals and vi- tamins to meet the requirements of growing pigs (Tuori et al. 1995). Ileal digestible amino acid balance (lysine, threonine and sulphur contain- ing methionine and cystine) of experimental di- ets was calculated according to suggested ideal protein balance ofLenis (1996). Mean calculat- ed dietary contents ofileal digestible lysine, thre- onine and methionine and cystine were 8.1, 5.1 and 4.8 g/kg, respectively. CP content of diets 1 and 3 was reduced by substitution of soya bean meal with oats, while dietary content of ileal di- gestible lysine, threonine and sulphur-contain- ing amino acids was maintained by supplements of L-lysine-HCI, DL-methionine and L-threo- nine-premix. Net energy content of diets 3 and 4 was increased by the inclusion of animal fat. All diets were expanded and pelleted. Net ener- gy content and feed unit values of experimental diets were calculated with the Finnish energy 382 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Table 1.Dietary ingredients (g/kg) and chemical composition (g/kg DM). Diet 12 3 4 Dietary ingredients Barley 657.4 631.2 630.3 603.2 Oats 170.0 74.0 144.0 50.0 Soya bean meal 110.0 242.0 123.0 254.0 Animal fat - - 40.0 40.0 Wheat syrup 20.0 20.0 20.0 20.0 Limestone 12.0 12.5 12.0 12.5 Monocalcium phosphate 12.5 10.0 13.0 10.0 Natrium cloride 4.0 4.0 4.0 4,0 Vitamins and trace elements 1 - 2 6.3 6.3 6.3 6.3 L-lysine-HCI 3.9 - 3.6 DL-Methionine 1.1 - 1.1 - L-Threonine-premix (50% L-threonine) 2.8 - 2.7 - Chemical composition Feed units (calculated) FU/kg 0.95 0.95 1.05 1.05 Dry matter, g/kg 898.2 895.7 902.2 905.1 Organic matter 948.1 952.1 952.6 951.8 Ash 51.9 47.9 47.4 48.2 Crude protein 151.7 186.7 153.2 190.7 Ether extract 39.2 36.7 74.7 73.8 Crude fibre 53.1 49.3 54.9 47.5 N-free extracts 704.1 679.4 669.8 639.8 Amino acids Lysine 8.9 9.6 9.1 9.4 Threonine 6.1 6.9 6.2 7.1 Methionine 2.9 2.6 2.8 2.8 Cystine 3.0 3.4 3.0 3.4 Alanine 6.3 7.7 6.3 7.8 Arginine 8.8 11.1 8.5 11.2 Aspargic acid 12.4 16.7 12.4 17.3 Glutamic acid 30.8 37.6 30.3 37.4 Glycine 6.1 7.4 6.1 7.5 Histidine 3.4 4.2 3.5 5.2 Isoleucine 5.3 6.8 5.2 7.0 Leucine 10.3 12.4 10.0 12.8 Phenylalanine 7.1 8.3 6.8 8.7 Serine 6.8 8.5 6.7 8.8 Tyrosine 3.9 4.6 3.5 4.9 Valine 6.6 7.9 6.3 8.0 1 Vitamins and trace elements provided (per kg diet): vitamin A, 5000IU; vitamin D, 1000IU;vitamin E, 47 mg; vitamin K, 0.3 mg; vitamin 8,, 1 mg; vitamin 82,B 2, 3.5 mg; vitamin 86,B 6 , 3.5 mg; vitamin 812,B 12 , 0.018 mg; biotin, 0.12 mg; calcium pantothenate, 12 mg; folic acid, 1.2 mg; nicotinic acid, 18 mg; choline cloride, 125 mg; Mn, 40 mg; Fe, 90 mg; Zn, 160 mg; Cu, 105 mg; Co, 2.4 mg; J, 0.6 mg; Se, 0.16 mg. 2 Vitamin and trace element mixture contains (g/kg): barley 740.1, iron sulphate 71.4, copper sulphate 69.4, zinc oxide 39.7, potassium iodine 0.1, sodium selenite 0.1 and vitamin products 78.4. 383 Vol. 7(1998): 381-390. AGRICULTURAL AND FOOD SCIENCE IN FINLAND Valaja, J. & Siljander-Rasi, H. Effect ofdietary protein and energy on nitrogen utilisation in pigs evaluation system for pigs using constant digest- ibility coefficients for feed ingredients (Tuori et al. 1995). Pigs were fed twice daily, at 0600 and 1600. Daily feed intake was maintained at 93 g/kg W° 75 during the experiment and was adjusted for each period according to the increase in body weight (from 1512 to 2382 g DM/day). Water was add- ed to the diets prior to feeding in a ratio of 2:1. Between meals water was offered ad libitum from troughs. Water intake was measured dur- ing the collection period. Feeds and faeces were freeze-dried and ground through a 1-mm mesh before analysis. Proximate analysis of feeds and faeces was per- formed using standard methods (AOAC 1984). Ether extract was determined after acid hydrol- ysis with 4 N HCI. N content of urine was ana- lysed by Kjeldahl method and that of freeze- dried feeds and faeces by the methodof Sweeney (1989) on a Leco FP 428 nitrogen analyser. Amino acid content of diets was determined with a Beckman 6300 amino acid analyser after a 23 h hydrolysis with 6.0 N HCI at 110°C. Methio- nine and cystine were oxidated with performic acid to methionine sulphone and cysteic acid prior hydrolysis. All analyses were performed in duplicate. Apparent total tract digestibility of diets was calculated by totalcollection method. Data were subjected to analysis of variance by the GLM procedure of SAS (1985) using the following model: y,jk i = M- +p,+a j + dk +eiJk i where y is the dependent variable; pis the overall mean; p, is period effect; a, is animal ef- fect; dk is diet effect; and e, jld is a normally dis- tributed residual error. Three orthogonal con- trasts were formed to test the following effects: high CP vs. low CP diets (diets 1 and 3 vs. diets 2 and 4); high energy vs. low energy diets (diets 1 and 2 vs. diets 3 and 4); and interaction be- tween CP and energy level (diets 1 and 4 vs. di- ets 2 and 3). Results and discussion Chemical composition Analysed composition of the experimental diets was in close agreement with calculated values. CP contents of the low protein (Diets 1 and 3) and high protein diets (Diets 2 and 4) were very close to each other (Table 1). The mean CP con- tent of high protein diets was 37 g/kg DM high- er than that of low protein diets. Similarly, the analysed contents of lysine, threonine, methio- nine and cystine were maintained at similar lev- els in all diets.The contents of lysine and threo- nine were slightly higher for high than low pro- tein diets, because lower ileal amino acid digest- ibilities were used for dietary ingredients than for free amino acids during diet formulation. Due to animal fat inclusion dietary ether extract con- tent was 36 g/kg DM higher in high than low energy diets. Digestibility Pigs completed the experiment successfully with an average daily weight gain of 1121 g/day. Gen- erally, pigs ate their diets readily except minor diet refusals of two pigs during the last two pe- riods, which resulted in slightly higher average feed intake of the pigs on low protein diets (PcO.Ol). Apparent total tract nutrient digesti- bility was higher in high (1 and 3) than low pro- tein diets (2 and 4) (Table 2) being significant for the digestibility of dry matter (P<0.05), or- ganic matter (P<0.05), ash (PcO.10) and CP (P<0.05). Apparent total tract digestibility of ether extract was higher in high than low energy diets (P<0.001). These differences in digestibil- ity of nutrients can be explained by variations in diet composition and nutrient intake. Slightly lower dry matter and organic matter digestibili- ty of low protein diets mainly resulted from in- creased amounts of oat inclusion. In the earlier 384 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Table 2. Total tract nutrient digestibility, nitrogen metabolism, water intake and urinary output of pigs fed diets containing different amounts of crude protein and feed units. Diet 1 2 3 4 SEM Contrasts Crude protein, g/kg 140 180 140 180 High vs. High vs. CP/energy Feed unit, FU/kg 0.95 0.95 1.05 1.05 low CP low energy interaction n 4 4 4 4 Dry matter 81.9 84.2 82.5 85.0 0.68 * ns ns Organic matter 83.6 85.9 84.2 86.6 0.66 * ns ns Ash 53.3 54.8 51.6 55.3 1.31 o ns ns Crude protein 83.7 85.8 83.1 85.9 0.75 * ns ns Ether extract 68.4 67.4 78.5 78.6 1.17 ns *** ns N intake, g/day 54.0 64.9 54.8 65.6 0.58 *** ns ns N in faeces, g/day 8.9 9.2 9.0 9.2 0.54 ns ns ns N in urine, g/day 14.1 21.5 15.7 22.2 1.47 ** ns ns Total N excretion, g/day 22.9 30.7 24.7 31.3 1.65 ** ns ns N retained, g/day 31.1 34.2 30.0 34.3 1.59 o ns ns % of intake 58.7 52.7 56.4 51.6 2.33 o ns ns % of absorbed 70.0 61.4 67.7 60.1 2.49 * ns ns Feed intake, g DM/day 2000 1946 2016 1946 17.3 * ns ns Daily weight gain, g/day 1067 1065 1247 1106 49.9 ns o ns Water intake, g/day 11700 11350 11600 11951 1026.3 ns ns ns Urinary output, g/day 8171 7554 8174 8532 1016,4 ns ns ns Significance: ns=non significant, o =P