Materials and methods rooster was placed on its back on the surgery table

P . van Leeuwen et al. Livestock Production Science 67 2000 101 –111 103

2. Materials and methods rooster was placed on its back on the surgery table

and the area ventral of the pubis was cleaned with a 2.1. Experimental protocol general disinfectant. Laparotomy was performed by a 4-cm straight incision at the ventral side of the right 2.1.1. Birds and housing pubis. The ileo–ceacal junction was positioned at the Ten adult roosters Lohmann brown with an incision. The intestine was closed with two absorb- average body weight BW of 2.8 kg were indi- able sutures Polysorb GL-181 CV-25 around the vidually housed in metabolic galvanized wire mesh terminal ileum, with a distance of 3 mm between the cages 40 3 75 3 60 cm, width 3 height 3 depth sutures. The ileum was transected between the with a feed and water bowl. No bedding was sutures. A purse string suture Polysorb GL-181 CV- provided. The cages were placed in an environmen- 25 was placed at the antimesenteric side of the tally controlled room with an air temperature of proximal part of the transsected intestine. An inci- 19–21 8C. Birds were maintained under a 16 h light sion was made in the intestine between the purse with 8-h twilight cycle throughout. string suture, the flange of the cannula was inserted and fixed immediately with the ligature. A second 2.1.2. Surgical procedure for ileostomy ligature was placed around the cannula. The cannula The principle of the surgical procedure used in the was then exteriorized through a stab incision in the present experiment was previously described by body wall about 2 cm ventral of the first incision Schutte et al. 1991. From 3 weeks prior to the Fig. 2. After a routine closure of the laparotomy, surgeries and for 3 weeks post-surgery the roosters the cannula was fixed externally with tape, so that were fed a highly digestible diet with soy flour meal the intestine and the abdominal wall just came in 410 g kg, maize starch 180 g kg, glucose 200 light contact with each other. Too much pressure g kg, cellulose Arbocel, 100 g kg and a premix of would increase local necrosis. After a period of 3 vitamins and minerals mixed with maize 110 g kg. weeks, the digestibility trial started. Time needed for The cannulas consisted of a barrel with a flange Fig. surgery was, included the introduction of the anaes- 1, both segments of the same medical grade silicon thesia, about 1 h. The number of animals alive after tubing, Type SR 16 Maxxim B.V., s’Hertogenbosch, 2 months was over 90. The Netherlands, with an 8 mm inner diameter ID and an 11 mm outside diameter OD. The barrel and 2.2. Experimental diets the flange were glued together with a silicon ad- hesive, Elastosil E41 Wacker-Chemie GmbH, After a pre-test period of 3 weeks, the roosters ¨ Munchen, Germany. were fed maize starch based diets with maize diet Feed was removed 24 h prior to surgery, but water 1; wheat gluten diet 2; faba beans with a low was always freely available. Each rooster was pre- tannin content LT diet 3; lupins, angustifolius, medicated with an intramuscular injection of 1 mg of white diet 4; soybean meal diet 5 and casein diet  Ketamin Nimatec , Eurovet, Bladel, The Nether- 6 as main protein sources Tables 1 and 2. The lands; given as a sedative, 10 mg of flunixine– incorporation rates of the feedstuffs were calculated  meglumine Finadyne , Schering Plough Animal in order to obtain diets containing 160 g kg CP. In Health, USA; as an analgestic, 0.3 ml of diet 1, 94 g kg wheat gluten meal was added to  Depomycine 20 20 Mycofarm, De Bilt, The elevate the CP content of the diet to 160 g kg. The Netherlands; as a wide spectrum antibiotic and 0.05 same batch of the diets were used in previously mg of atropinsulphate Eurovet, Bladel, The Nether- conducted digestibility experiments with pigs van lands; as a cholinergic blocker. Oxygen O with Leeuwen et al., 1996a,b. Diets were stored at 2 isoflurane as anaesthesia was then given with a 2 208C for 2 years until the use in the present rooster mask. After sedation, each rooster was intubated experiment. For the present experiment with the OD of the silicon rubber trachea tube was 3 mm. ileostomised roosters, the meal was more finely When the anaesthesia relaxed the muscles, the ground over a 1-mm screen and not pelleted. The 104 P . van Leeuwen et al. Livestock Production Science 67 2000 101 –111 Fig. 1. A T-shaped silastic cannula for collection of ileal digesta; B the cannula connected with a digesta collection bottle. feed intake was restricted to 80 g day, equivalent to After 11 days of adaptation to the rations, without a semi ad libitum level. Water was freely available. fasting periods, digesta were collected over 3 succes- sive days 72 h via a plastic bottle of about 10 g 2.3. Determination of ileal digestibility in connected to the cannula Fig. 1. Digesta were ileostomised roosters removed hourly over the day 8.00–20.00 and each 6 h during the night and immediately frozen The ileal digestibility experiment consisted of four 2208C. Digesta was more frequently collected periods P1, P2, P3 and P4 of 14 days each. Diets during the day rather than in the night. This was 1–6 were assigned randomly with no rooster receiv- done 1 to have the possibility to compare digesta ing the same diet twice. The digestibility coefficients composition for the two different procedures separate of each diet were determined in at least five different and 2 to keep the roosters quiet during the night, roosters. maintaining the normal bio rhythm of the roosters as P . van Leeuwen et al. Livestock Production Science 67 2000 101 –111 105 much as possible. Unpublished results showed no systematically differences. After each experimental period, digesta of the 3 day collections were thawed and pooled per bird. 2.4. Analytical procedures Feedstuffs, diets and freeze-dried digesta were ground through a 1-mm screen using a Retsch AM 1 grinder prior to chemical analysis. Nitrogen N was analyzed by the Kjeldahl method and crude protein CP was calculated as N 3 6.25. Dry matter DM contents were determined after drying at 80 8C over- night. Amino acids were determined according to Bech-Anderson et al. 1990. 2.5. Calculations The apparent digestibility for DM, CP and AA of the diets were calculated from nutrient intake and the total collected amounts of digesta over periods of 3 days 72 h. The crystaline amino acids included were assumed to be completely absorbed. The data set for the calculation of the correlations between ileal aDC CP and AA were derived from the cannulated roosters in the present experiment and from cannulated pigs from the previous experiment van Leeuwen et al., 1996b. Fig. 2. A A cannulated rooster with an indication of the incision; 2.6. Statistical analysis B the ileo–caecal junction and the place of the cannula. Data were subjected to analysis of variance using the SPSS PC 1 V5.0 software Norusis, 1992. The Table 1 diet type was treated using the following model: Dry matter DM, crude protein CP; N 36.25 and crude fibre CFi as fed of the used feedstuffs y 5 T 1 a 3 D 1 e i, j i i, j Feedstuffs DM CP CFi where y 5response measurements, D 5diet type i, j i Maize 88.5 9.5 2.6 d i 5126, T5mean value and e 5residual error. i , j Wheatgluten 92.1 82.2 n.d. a The number of periods per diet was similar and a Faba beans LT 89.3 32.0 8.4 b Lupins 92.1 28.4 14.9 first statistical analysis showed no significant effect c Soybean meal 90.2 48.9 4.4 of periods. Therefore the period was not included in Casein 91.2 86.4 n.d. the final model. a Low tannins, ,0.05 tannins expressed as cat. eq. Kuhla Treatment means were tested for difference by use and Ebmeier, 1981, 1.9 mg g trypsin inhibitor activity TIA of the Least Significant Difference test Snedecor van Oort et al., 1989. b and Cochran, 1980. All statements of significance 0.4 Alkaloids European Commission, 1971. c are based on a probability of P ,0.05. 3.5 mg g TIA. d n.d 5Not determined. Correlations were calculated between ileal aDC 106 P . van Leeuwen et al. Livestock Production Science 67 2000 101 –111 Table 2 Composition of the maize starch based diets with maize, wheat gluten, faba beans, lupins soybean meal and casein as substituting feedstuffs as fed Diet 1 2 3 4 5 6 Substituted feedstuff Maize Wheat glutenmeal Faba beans Lupins Soy-bean meal Casein Content substituted 82.82 17.90 51.00 54.00 33.00 18.00 feedstuff Wheatgluten meal 9.40 Maize starch 51.90 26.78 24.98 43.30 53.35 Glucose 15.00 15.00 15.00 15.00 15.00 Soybean oil 1.50 1.00 1.00 1.00 Cellulose 5.00 2.00 5.00 CaCO 0.90 0.80 0.90 0.80 0.85 1.20 2 CaHPO ?2H O 2.00 2.25 1.85 1.60 1.75 1.60 4 2 NaCl 0.50 0.50 0.50 0.50 0.50 0.50 MgO 0.20 0.20 KHCO 1.30 1.80 0.20 0.40 1.50 3 NaHCO 0.30 0.40 0.30 0.30 0.35 0.40 3 L -Lysine ?HCl 0.50 0.50 L -Threonine L -Tryptophan 0.03 0.02 0.04 DL -Methionine 0.20 0.13 Vitamin-trace element 2.25 2.25 2.25 2.25 2.25 2.25 a mixture Analyzed content CP N 36.25 16.0 15.3 16.2 15.8 16.1 15.4 a Contributed the following vitamin sources, additional minerals and trace elements per kg of ration: vitamin E, 37.5 mg; riboflavin, 6 mg; niacin, 30 mg; D -pantothenic acid, 15 mg; choline chloride, 120 mg; vitamin B 0.45 mg; vitamin K , 3 mg; vitamin A, 9000 IU; vitamin 12 3 D , 1800 IU; KI, 0.81 mg and CoSO ?7H O, 7 mg; FeSO ?7H O, 0.4 g; CuSO ?5H O, 0.1 g; MnO , 0.07 g; ZnSO ?H O, 0.3 g. This 3 4 2 4 2 4 2 2 4 2 mixture was supplied with 20 ppm Tylosine as an antibiotic. determined in the present experiment using roosters additional experiments not presented. During this and from previously determined aDC data using pigs period, the weights of the animals were slightly van Leeuwen et al., 1996b. The model used to higher or unchanged compared to the weight prior to correlate the aDC CP and aDC AA determined in the surgery. roosters and pigs was y 5 ax 1 c where y5aDC CP Results of the amino acid analysis of the diets determined in roosters and x 5aDC CP determined in have been previously reported van Leeuwen et al., pigs. 1996b. The ileal aDC CP and aDC AA measured in The calculations were conducted using SPSS roosters are presented in Table 3. The aDC CP of the PC 1V5.0 software Norusis, 1992. maize–wheat gluten based diet diet 1, the wheat- gluten diet diet 2 and the casein diet diet 6 were significantly P ,0.05 different from those of the

3. Results faba bean diet diet 3, lupin diet diet 4 and