Arch.Geflügelk., 73 (1). S. 21–28, 2009, ISSN 0003-9098. © Verlag Eugen Ulmer, Stuttgart

Determination of the appetite of laying hens for methionine in drinking water by using colour cue Bestimmung der Bedarfsergänzung von Methionin über das Trinkwasser bei Legehennen anhand von Farbmarkierungen S. Cadirci1,2, W.K. Smith1 and R.M. Mc Devitt1 Manuskript eingegangen am 4. März 2008, angenommen am 12. April 2008

Introduction Methionine is the first limiting amino acid in most commercial feeds for laying hens SCHUTTE et al. (1994), in addition to its main role as a component of proteins it also acts in various metabolic pathways. There are several factors (e.g. feed energy and protein content, age, genotype, sex and various environmental factors) to take into account when balancing the feed formulation EMMANS and FISHER (1986), therefore amino acids provided by compounded feeds will meet a variable proportion of the requirements of the hens in a flock BOORMAN and BURGESS (1986). Moreover, once the compound feed is mixed, both its amino acid content and also the extent to which it will meet the requirements of all the hens in any given flock are fixed. However, the methionine requirements of a greater proportion of a flock of hens could be met if it could be demonstrated that the domestic fowl had an appetite for methionine and could express it when experiencing a marginal deficiency by consuming, as a choice, water supplemented with methionine. Relatively few attempts have been made to supply amino acids in drinking water. GRIGGS et al. (1971) succeeded in administering various nutrients, including amino acids to poults through the drinking water and observed early weight gain. DAMRON and GOODSON-WILLIAMS (1987) conducted a study with broilers where 0.05% liquid DL-methionine was added to drinking water while the birds received a low-methionine diet. They found neither reductions of feed or water intake, nor any change in levels of mortality. Moreover, as a result of practising the above feeding regimen, lower mortality and reduced stress have been reported ANONYMOUS (1984). When 2-hydroxy-4 (methylthio) butanoic acid was used in drinking water DAMRON and FLUNKER (1992), the results were similar to those of DAMRON and GOODSON-WILLIAMS (1987), in that no adverse effects on feed intake, water intake, and liveability were observed.

1Avian

Science Research Centre, Scottish Agricultural College, Auchincruive Campus, Ayr, Scotland of Animal Science, Faculty of Agriculture, Harran University, Sanliurfa, Turkey

2Department

Arch.Geflügelk. 1/2009

Research investigating the expression of a specific appetite for amino acids when given as an additive in drinking water has not been conducted. However, it has been shown (e.g. ROTH et al., 1990; STEINRUCK et al., 1990) that laying hens have the ability to self-select a diet supplemented with methionine in the feed when offered a choice. BOORMAN (1979) suggested that if a diet imbalanced for amino acids is absorbed from the digestive tract, then the bird's metabolism can be disturbed to the extent that food intake is reduced in proportion to the degree of amino acid deficiency or imbalance. This is thought to be due to the metabolic cost of deaminating the excess of those amino acids which can not be utilised because of the deficiency (relative or absolute) of others. It is therefore possible that the appetites for individual amino acids can be determined. The aim of this study was to determine whether hens have a specific appetite for methionine that can be met via drinking water when given a feed deficient in methionine. Two experiments were carried out to investigate the conditions of this way of methionine delivery.

Materials and Methods For both experiments, ISA Brown laying hens were taken from a 1000-hen commercial laying flock and placed singly in cages in the same house. The cages were 43 cm wide, 43 cm high and 46 cm deep. The birds were caged individually with one empty cage between neighbouring birds. The sides of the cages were closed with 3-ply wood so that the birds could not see their neighbours. This separation was to ensure that social influence did not affect the water bottle selection. The birds were housed in cages of the top tier of a three-tier battery system. Plastic water bottles (1000 ml) were used to supply water. They were fitted with nipples at the base (Val Watering System 2599 Old Philadelphia Pike, Bird – In – Hand, PA – USA). To collect and measure water waste, plastic cups were placed under the bottles. One feed-trough, two water bottles, and two waste-water collector cups were located at the front of each cage. The bottle-cup pairs were located at the cage front with a 20 cm gap between the nipples from which hens were to select water. The house temperature control system was set to maintain a daily minimum of 21°C at the middle tier level of the main banks of cages by controlling the ventilation rate. Temperature was measured at the middle tier level using maximum-minimum thermometers. Table 1 indicates the mean temperature reading for the middle tiers for both

22

Cadirci et al.: Application of methionine to layers by drinking water

Table 1. The temperatures over the duration of the two experiments Umgebungstemperaturen während der Durchführung der Versuche

Table 2. The ingredient- and calculated nutrient composition of Diets 1 and 2 Zusammensetzung und kalkulierte Nährstoffgehalte der Versuchsrationen 1 und 2

Temperature [°C] Maximum Minimum

Ingredient composition

Experiment 1 2

20.8 ± 0.13 21.8 ± 0.30

16.8 ± 0.22 17.6 ± 0.22

Data are means ± SEM.

experiments. Day length was 16 hours. Light was supplied by 40 Watt tungsten bulbs in all experiments. The amino acid content of the feed was designed in both experiments so that when the only source of methionine was that provided by the main ingredients (wheat and soya), hens producing eggs at a rate of 80 to 90% would experience a substantial methionine deficiency. It was determined in a preliminary experiment CADIRCI (2001) that a wheat-soya diet of 140 g/kg crude protein (CP) with supplemental methionine (providing a total of 3.7 g/kg methionine) caused a slight depression in egg mass output of hens from the same flock as that sampled for the present study. The same nutrient specification was used here. This level of CP and methionine was taken to be sufficient to induce a methionine deficiency when DL-methionine was not included as an ingredient. Two diets were formulated: for Diet 1 (+MET) the nutrient specifications were set to meet or exceed NRC (1994) nutrient requirements where level of methionine in the feed was 3.7 g/kg; Diet 2 (-MET) was essentially Diet 1 without supplemental methionine so that the calculated level of methionine in the feed was 2.1 g/kg. Before starting an experiment, a one-week period was allowed for the birds to adapt to the new diets. The ingredients used and the calculated nutrient content of the two diet formulations used in this study are shown in Table 2. Each day the hens were allocated enough feed (250 g) to exceed the expected daily food intake for hens of this age and strain. The normal daily feed intake would be approximately 120 g/day ISABROWN (1996). According to the design of the experiments, birds received Normal water (i.e. plain tap water) (-MET) or a water solution of methionine (+MET). For this, commercially available, water soluble crystalline DL-methionine (dl-2-Amino-4(methylthio)-butanoic acid) was used WEAST (1975). The water remaining in the bottles were discarded daily and replaced with fresh water. Daily feed intake and water consumption were measured gravimetrically every 24 hours. Methionine intake was calculated from the amounts consumed via the feed and water. All data were obtained on an individual hen basis. Experimental data were subjected to statistical analysis using the analysis of variance procedures of the statistical programme Genstat-5 (release 4.2, copyright 1994) Lawes Agricultural Trust (Rothamsted Experimental Station). Significant differences were tested further using a LeastSignificant Difference (LSD) multiple range test to determine the differences among treatments.

Experiment 1 Eighteen birds of 49 weeks old were selected randomly and used for this experiment.

Diet 1 (+MET) [g/kg]

Diet 2 (-MET) [g/kg]

Wheat (10.4% CP) H.P.1 Soya (46.2% CP) Limestone Maize Oil Dicalcium phosphate NaCl Vit/Min. Premix2 Yolk Colour A3 DL-Methionine L-Lysine HCl

714.0 137.3 90.3 36.7 11.4 3.7 2.5 1.0 1.6 1.5

712.8 139.8 90.3 37.1 11.4 3.7 2.5 1.0 – 1.4

Calculated nutrient composition4 Crude protein Calcium Total Phosphorus Sodium Arginine Isoleucine Leucine Lysine Methionine Methionine + cystine Threonine Tryptophan AME [MJ/kg]

140 37.5 5.5 1.8 8.3 5.3 9.9 7.2 3.7 6.4 4.7 1.7 12.14

140 37.5 5.5 1.8 8.3 5.3 10.0 7.2 2.1 4.8 4.7 1.7 12.14

1 H.P. = high protein 2 The composition of vitamins and minerals in the premix provided

the following amounts per kilogram of diet: vitamin A, 2400000 IU; vitamin D3, 1200000 ICU; vitamin E (α-tocopherol acetate), 1600 IU; nicotinic acid, 4000 mg; pantothenic acid, 1600 mg; vitamin B2 1000 mg; hetrazeen, 800 mg; iron (FeSO4), 0.40%; cobalt (CoSO4), 100 mg; manganese (MnO), 3.20%; copper (CuSO4), 0.20%; zinc (ZnO), 2.00%; iodine (CaI2), 400 mg; selenium (Na2SeO3), 60 mg. 3 Contains: canthoxanthin, ethyl ester of β-apo-8-carotenoic acid, citronaxanthin. 4 Based on NRC, 1994 values for wheat and soybean meal.

Based on a preliminary experiment in which the birds` water consumption level was determined CADIRCI (2001), the methionine content of the drinking water in this experiment was 0.1% (w/v). Bottles containing methionine-treated water were positioned so that one half of the hens received treated water on their right and the other half on their left. This positioning was to ensure that location did not affect the water selection. All bottles and cups were the same colour throughout the regimens of the experiment. All birds were subjected to three feeding regimens (Regimens A, B, C); each regimen period was 15 days. In regimen A, birds were fed Diet 1 (+MET) and Normal water (-MET) was provided from both bottles. In regimen B, birds were fed Diet 1 (+MET), and one bottle had Normal water (-MET), while the other one contained 0.1% (w/v) methionine-treated water (+MET). In regimen C, birds were fed Diet 2 (-MET) and one bottle had Normal water (-MET) the other one contained 0.1% (w/v) methioArch.Geflügelk. 1/2009

Cadirci et al.: Application of methionine to layers by drinking water nine-treated water (+MET). The sufficiency of methionine throughout the regimens of the experiment is shown in Table 3. Body weights were recorded at the beginning and at the end of each regimens.

Experiment 2 This experiment was designed to investigate the hens’ choice for methionine. For this, colour cue was introduced, that is treated (+Met) and Normal water (-Met) was given in differently coloured bottles (red or yellow). For this experiment, the hens were monitored for egg production for two weeks, then the 10 best egg producers, which were also fully feathered and of similar body weight (mean ± SEM = 2067 g ± 46 g), were chosen at 55 weeks of age. They were divided into two of five birds each (Group 1 and 2). One feed-trough, two water bottles, and two waste-water collector cups were located at the front of the cage of each hen in such way that the birds could only see the painted part of the bottles. The plastic bottles were painted on the bottom and sides in one colour. Ten bottles were red, ten were yellow. The waste collector cups under the bottles matched with the bottle in colour. Based on the findings of Experiment 1, treated drinking water contained an increased amount of methionine (0.15% w/v) in an attempt to shorten the birds’ response to treated water (+MET). All birds were subjected to five feeding regimens (Regimens A, B, C, D, E). In regimen A, only Normal water was given. Bottles were yellow for Group 1 and red for Group 2. After regimen A, hens in Group 1 were given Normal water in yellow bottles and Treated water in red bottles, while for Group 2 Normal water was given in red bottles and Treated water in yellow bottles. The regimens of the experiment were as follows: To determine normal levels of feed intake, methionine intake and water consumption for hens of this strain and age, the hens were fed Diet 1 (+MET) and were given Normal water (-MET) for the first 7 days of the experiment (Regimen A). Then followed a four-day cycle where birds received Diet 2 (-MET) and Normal water (-MET) for 2 days (Regimen B), then Diet 2 (-MET) and treated water (+MET) for 2 days (Regimen C). The cycle (Regimens B and C) was repeated once. Subsequently, birds received Diet 2 (-MET) and Normal water (-MET) for an additional 2 days (Regimen B). During this training period (a total of 10 days) the two types of diet were given to the hens alternately every two days in order to allow them to become accustomed to the colour cue and physiological effect of the two diets that were either adequate or deficient in methionine. Then followed a “free choice” part of the experiment during which the birds were fed Diet 2 (-MET) and were offered a choice between Normal (-MET) and treated water (+MET) for five days (Regimen D). The position of

the bottles was then swapped, and the water was offered for another five days (Regimen E) to ensure that the position of the bottle at the cage front did not bias water intake. The grouping of birds was necessary in order to eliminate the colour effect of the drinking bottles on their subsequent choice of drinking water. In addition, the position of the drinking bottle in the cage was controlled by including both regimens D and E in the experimental design. Every effort, therefore, was made to create an experimental condition where the methionine-treated water was the only variable. The experimental plan is summarized in Table 4. Each bird was weighed at the beginning and end of the experiment.

Results Experiment 1 Table 5 shows the effects of feeding regimens on feed-, water- and methionine intake, and body weight of laying hens. Feed intake of the birds did not change (p > 0.05) when their methionine requirement was met by the diet (regimens A and B). However, during regimen C feed intake was depressed with an average of 9% compared to regimens A (p < 0.01) and B (p < 0.01). A similar change in total water intake was not found; it remained unaffected throughout the three regimens of the experiment (p > 0.05). In contrast, total methionine intake was significantly (p < 0.05) different between the regimens (increased in regimen B and decreased in regimen C compared to regimen A). Body weight of the birds did not change significantly during the experiment (p > 0.05), although an average of 40 g decrease was found in regimen C when compared to regimens A and B. During regimens B and C, 61.0% and 56.7% respectively of the water consumed contained methionine. The observed proportions did not differ significantly (p > 0.05) from 50% (i.e. random choice). In addition, there was no significant difference (p > 0.05) in preferences between the two regimens.

Experiment 2 The average body masses (mean ± SEM) at the beginning and at the end of the experiment were 2067 g ± 46 g and 1995 g ± 57 g, respectively, the difference was not significant (p > 0.05). The daily feed and water intake during each of the 27 days of the experiment responded to alterations in the provision of methionine in the diet and in the drinking water (Figure 1). Each data point is the mean ± SEM of the daily feed and water intake from ten birds. Five distinct phases can be observed, corresponding to the different experi-

Table 3. Feeding regimens of birds in Experiment 1 Behandlungen in Versuch 1 Regimens

Methionine in Diet

A B C

Arch.Geflügelk. 1/2009

adequate adequate deficient

23

in Water bottle 1

bottle 2

– – –

– 0.1% 0.1%

Methionine adequacy when drinking from bottle 1 bottle 2 adequate adequate deficient

adequate excessive adequate

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Cadirci et al.: Application of methionine to layers by drinking water

Table 4. Experimental plan for Experiment 2 Behandlungen in Versuch 2 Control period Regimen A

Regimen B

Regimen C

1 (+MET) Normal (-MET) 7 Y

2 (-MET) Normal (-MET) 2 R

2 (-MET) Treated (+MET) 2 Y

2 (-MET) Normal (-MET) 2 R

R

Y

R

Y

Diet Water Length [days] Group 1 nb = 10 Group 2 nb = 10

Training Period Regimen B Regimen C

Regimen B

Choice Period Regimen D Regimen E

2 (-MET) Treated (+MET) 2 Y

2 (-MET) Normal (-MET) 2 R

2 (-MET) 2 (-MET) Normal (-MET) Normal (-MET) Treated (+MET) Treated (+MET) 5 5 Y R

R

Y

R

Y

nb = number of bottles; Y = yellow bottle, R = red bottle In order to establish the initial production parameters (feed and water intake), hens were initially maintained on a diet adequate in methionine (+MET) and were given Normal drinking water (-MET) (Regimen A). This was followed by a sequence of alternating periods of being fed a diet deficient in methionine (-MET), whilst simultaneously being given either Normal (-MET) drinking water (Regimen B) or with methionine-treated (+MET) water (Regimen C). The purpose of this phase was to train the hens to associate the relevant colour cue of their drinking bottles with Normal (-MET) or treated water (+MET). The experiment was completed with two choice tests (Regimens D and E) during which the hens were fed a methionine-deficient diet (-MET) whilst being offered the choice between Normal (-MET) and treated water (+MET). These last two regimens differed only in the position of the colour-cued drinking bottles.

Table 5. Feed-, water-, and methionine intake, and body weight during the regimens of the experiment 1 Futter-, Wasser- und Methioninaufnahme sowie Lebendgewichte in Versuch 1

Feed intake [g/hen/day] Water intake [ml/hen/day] Methionine intake [mg/hen/day] Body weight [g]

A (15 days)

Regimens B (15 days)

108.2b

107.7b

146.8

144.0

139.1

400.3b

486.2c

288.0a

1931

1930

C (15 days) 98.2a

1887

abc Values within a row with different superscripts differ signifi-

cantly (p < 0.01 for Feed intake; p < 0.001 for Methionine intake). Values are mean of n = 17.

mental regimens. During the first 7 days of the experiment, when the birds received Diet 1 (+MET) and Normal water (-MET), the standard errors are small and the birds’ appetite for water and feed was without dramatic change. On days 8 and 9, when the birds received Diet 2 (-MET), and Normal water (-MET) they lost their appetite for food and water, and the decrease was more apparent by the second day of this regimen. When the birds received methionine-treated water (+MET), even in the absence of supplemental methionine in the diet, their appetite for feed and water increased up to the level of regimen A. When repeating the treatment, the same responses were observed. During the days when hens were on regimen B, feed intake had a greater variability which diminished when they proceeded to regimen C, that is when they received methionine-treated water (+MET). After this training period, the hens practically ignored Normal water (-MET) and consumed almost exclusively the supplemented water (+MET), when they were able to choose between

the two types of drink. As would be predicted, there was a significant correlation (r = 0.776; p < 0.05) observed between feed intake and water consumption. The pattern of the calculated methionine intake, derived from the known quantity of methionine present in both diets and in the supplemented drinking water (Figure 2) closely paralleled the food and water intake pattern (Figure 1). In addition to the daily changes presented above, the effects of experimental regimen on overall feed-, waterand calculated methionine intakes during each of the five experimental regimens were determined (Table 6). Experimental regimen had a significant effect on all three parameters measured. The average daily feed intake of the hens decreased significantly from 111 g/day to 79 g/day (p < 0.001) when progressing from regimen A to B, then returned to initial level in regimen C (106 g/day), and was not different from that measured during regimen A for the remainder of the experiment (Table 6). Changes in water intake during the experiment were similar to those in feed intake, i.e. there was a significant difference (p < 0.01) between the average water intake during regimen B (117.7 g/day) and that recorded during the other experimental regimes (approximately 156 g/day). As there were no significant differences in feed and water intake between regimens C and A (p > 0.05), this suggests that the birds’ appetite for feed and water was restored during regimen C. This indicates that the source of methionine, either in feed or in the drinking water is insignificant for maintaining a normal appetite. The calculated methionine intake was significantly different (p < 0.001) between regimens B and A, decreasing from an average daily intake of approximately 415 g/day to 170 g/day. In addition, the methionine intake in these two regimens was significantly lower (p < 0.001) than in the rest of the regimens (C to E) where the hens had free access to drinking water containing methionine (Table 6). When the hens had the choice of consuming water either with or without methionine, the majority of the water consumed contained methionine (p < 0.001). If the hens were choosing water on a purely random basis then the likelihood of consuming either water type would be 50%. However, a comparison of the birds’ preference for treated Arch.Geflügelk. 1/2009

Cadirci et al.: Application of methionine to layers by drinking water

25

190 170

Intake [g]

150 130 110 90 70

Feed In take

50

Water Intake

30 1

2

3

Regimens Days Diet Water

4

5

6

7

A 7 +MET -MET

8

9

B 2 -MET -MET

10

11

C 2 -MET +MET

12

13

B 2 -MET -MET

14

15

C 2 -MET +MET

16

17

18

B 2 -MET -MET

19

20

21

22

D 5 -MET -MET and +MET

23

24

25

26

27

E 5 -MET -MET and +MET

Data are the mean ± SEM of n = 10.

Figure 1. Daily changes in the average feed intake and water consumption of laying hens in association with the five regimens in Experiment 2 Tägliche Veränderungen der Futter- und Wasseraufnahme der Legehennen in Abhängigkeit von den 5 Behandlungen in Versuch 2

600

Intake [mg]

500 400 300 200 100

Methionine Intake

0 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

Days

Regimens Days Diet Water

A 7 +MET -MET

B 2 -MET -MET

C 2 -MET +MET

B 2 -MET -MET

C 2 -MET +MET

B 2 -MET -MET

D 5 -MET -MET and +MET

E 5 -MET -MET and +MET

Data are the mean ± SEM of n = 10.

Figure 2. Daily changes in the calculated methionine intake of laying hens in association with the five regimens in Experiment 2 Tägliche Veränderungen der kalkulierten Methioninaufnahme der Legehennen in Abhängigkeit von den 5 Behandlungen in Versuch 2 Table 6. The average feed-, water-, and calculated methionine intake during the feeding regimens of the experiment 2 Futter-, Wasser- und kalkulierte Methioninaufnahme in Versuch 2

Feed intake [g/day] Water intake [ml/day] Methionine intake [mg/day]

A (7 days)

B (6 days)

C (4 days)

111.3b 153.5b 414.6b

79.1a 117.7a 170.0a

106.0b 156.5b 462.3c

Regimens D (5 days) 110.6b 160.6b 475.9c

E (5 days) 111.0b 154.8b 457.7c

SED

LSD

5.10 11.42 20.64

10.3 23.2 41.9

abc Values within a row with different superscript differ significantly (p < 0.001 for feed intake and methionine intake; p < 0.01 for water

intake). Values are mean of n = 10.

water to 50% consistently showed significant difference (p < 0.001) in the period examined. During regimens D and E, 98.9% and 93.9% respectively of the water consumed contained methionine. Thus, when offered a choice, an average of 96.4% of the water consumed was Arch.Geflügelk. 1/2009

supplemented with methionine. In addition, there was no evidence for colour preference. The choice made for red bottles was 93.4%, whereas for yellow ones was 99.4% (p > 0.05). The experimental regimen (i.e., either regime D or E) had no significant effect on the proportion of

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Cadirci et al.: Application of methionine to layers by drinking water

methionine supplemented water consumed (p > 0.05), indicating that the position of drinking bottles had no effect on water preference.

Discussion and conclusion Birds (laying hens or broilers) are able to compensate for a marginal deficiency of methionine by consuming more feed in an attempt to meet their requirement SCHUTTE and VAN WEERDEN (1978); SCHUTTE et al. (1983), (1984), (1994). Day-old broiler chicks are already able to make the appropriate selection between amino acid-deficient and balanced feeds PICARD et al. (1993), and the selection is made within the first day. However, older broilers (until 8 days of age) need only a few hours for the adjustment in feed selection if they are previously given adequate feeds and then allowed to choose between amino acid deficient and adequate feeds PICARD et al. (1993). Laying hens were also reported to make appropriate selections (in response to methionine deficiencies) when given a choice between methionine-deficient and adequate feeds UZU et al. (1993). However, because of their slower growth rate, feeding an amino acid deficient feed to layers requires more time to induce a metabolic effect NOBLE et al. (1993), therefore they respond slower than broilers. The present study has demonstrated that adult layers were also able to choose between Normal water and methionine-treated water. In order to obtain some preliminary information about their selection for methionine when supplied in water, the birds were subjected to the combinations of two types of diet (adequate or deficient in methionine) and Normal- or methionine-treated (0.1%) water in Experiment 1. Thus, in the regimens of experiment 1 they received methionine either in adequate (Regimens A, B and C), or excess (in Regimen B), or deficient amount (Regimen C). It became apparent that the birds could not express their appetite for methionine under the conditions of the experiment, therefore a cue (colour) was introduced in Experiment 2 to assist self-selection. It is known KUTLU and FORBES (1993) that hens can be trained to express their appetite for individual nutrients (Vitamin C) if there is some discernible sensory difference between two foods, one of which contains too little of it for the birds’ requirements and the other too much of it. However, such visualisation of methionine appetite, when methionine is offered in drinking water, has not been previously reported. Experiment 2 has demonstrated that, after a sufficient training period, adult layers are able to choose between plain water and methionine-treated water. If the methionine requirement of the hens could not be obtained from the food alone, the hens voluntarily selected water containing methionine to meet their requirements. Several pieces of additional information were also gained from the two experiments. Both experiments showed that methionine deficiency results decrease in feed intake. Previously, several investigators (e.g. ALMQUIST, 1954; GOUS and KLEYN, 1989; ROTH et al., 1990; UZU et al., 1993) noted that as the deficiency in methionine or other amino acids becomes severe, feed intake declines. In Experiment 1, feed intake was depressed when the diet did not meet the birds’ methionine requirement. Similarly, in Experiment 2 feed intake declined already at the first day of supplying the deficient feed, and became more apparent by the next day of the regimen. When the option of restoring methionine level through supplemented drinking water was offered, the birds responded quickly, and the feed intake returned to normal within a day. A similar observation was reported by ALMQUIST (1954) when withdrawing

and restoring crystalline indispensable amino acids to the feed. The results of Experiment 1 suggest that when the birds receive a diet containing adequate methionine NRC (1994), the intake from water containing an additional 0.1% methionine has no adverse effect on the body weight of birds. The mean water intake of the birds followed closely the changes in feed intake in Experiment 2. This phenomenon can be explained by the strong correlation between food and water intake HILL et al. (1979). In contrast, total water intake of the hens was affected neither by the excess nor the deficiency of methionine, in Experiment 1. This apparent disagreement in the results could be explained by the different magnitudes of methionine deficiency that birds experienced in the two experiments; in Experiment 1, methionine intake during regimen C was reduced by 28% from regimen A, whereas in Experiment 2, the intake of methionine was depressed by 60% during regimen B when compared with regimen A. A further observation in Experiment 2 was that once the methionine intake of the birds had exceeded that achieved in regimen A, they did not stop drinking the treated water, thus increasing the intake of methionine by 40 to 60 mg more than that achieved when consuming the methionine supplemented feed. This might be because the birds associated the plain water with methionine deficiency (i.e. an adverse effect) as plain water was previously always paired with deficient feed. This explanation is supported by EL BOUSHY and KENNEDY (1987) who reported that birds will not usually like a feed the second time once it has previously caused digestive disturbance or discomfort. If the birds could be trained in a way that they do not associate plain water with deficiency, they may start drinking it once their methionine appetite (requirement) from treated water was satisfied. The results of both experiments suggest that the source of methionine does not influence its metabolic effect. Thus it seems that methionine from the water is “as good as” when supplied wholly from the feed. In addition, Experiment 2 showed that the addition of methionine to drinking water, rather than to the feed, does not adversely effect water or feed intake. Similar observations were reported by DAMRON and GOODSON-WILLIAMS (1987) and DAMRON and FLUNKER (1992). However, BAKER (1977, cited by DAMRON and GOODSON-WILLIAMS, 1987) reported that water consumption of growing chicks was reduced by 50% when a low level of methionine was added in the drinking water. The results of Experiment 1 suggest that, without a cue, feeding regimens have no effect on choice of water supplier, i.e. birds follow a pattern of choice associated to the position rather than the metabolic effect of the diet. On the other hand, when colour cue assisted the selection (in Experiment 2), the birds were able to express their appetite for methionine. This was indicated by the observation that when the position of the bottles was changed, the proportioned choice for treated water was the same in both regimens, moreover, they were significantly different from 50% in both regimens. In addition, the observations that water intake increased during regimen C to the level similar in regimen A, and the methionine-treated water was preferentially selected during the choice part of the study (Experiment 2) show that there was no palatability problem. In conclusion, when colour cue assisted selection, the birds were able to express their appetite for methionine. This was indicated by the observation that when the position of the bottles was changed, the proportioned choice for treated water was the same in both regimens, moreover they were significantly different from 50% (i.e. random Arch.Geflügelk. 1/2009

Cadirci et al.: Application of methionine to layers by drinking water choice) in both regimens. The results of choice regimens in Experiment 2 (Regimens D and E) also showed that birds did not select water by the position of the bottles but, instead, they made their choice according to the effects of the water associated with colour-codes. The hypothesis that hens would preferentially select drinking water supplemented by methionine when fed a diet deficient in methionine was proven. This offers the potential opportunity for methionine to be offered in drinking water to hen flocks so that individuals whose methionine requirements were not being met via the diet, might meet their requirements from the water. Based on the above results further study is necessary in order to learn what the optimum methionine deficiency is in feed which would generate an appetite for methionine, and what the optimum methionine concentration is in drinking water for which layers can express an appetite.

Acknowledgement One of us (S.C.) wishes to thank the University of Harran, Turkey for financial support through a postgraduate scholarship. The Scottish Executive’s Environment and Rural Affairs Department (SEERAD) are thanked for their financial support of RMcD and WKS. We also thank I. Newison (Biomathematics and Statistics Scotland, BioSS) for statistical advice, D. Brown, L. Armstrong and S. Biggar for their technical support, and J. Savory for his general comments and advice during the project.

Summary The appetite of laying hens for methionine in drinking water was investigated in two experiments. Birds were subjected to the combinations of diet adequate or deficient in methionine and normal or methionine-treated water. The concentration of methionine in treated water was 0.1% (w/v) in Experiment 1, and 0.15% (w/v) in Experiment 2. In both experiments, first information was obtained on the birds’ normal intake of feed, water and methionine. For this, the hens were fed diet adequate in methionine and Normal water. In Experiment 1 hens were subsequently exposed to excess and deficiency of methionine. It became apparent that the birds could not express their appetite for methionine under the experimental conditions. In order to enable laying hens to differentiate between the water-supply bottles containing Normal and methionine-treated water, colour cues and training of the birds were introduced in Experiment 2. The colours were associated with the two types of water. Hens were exposed to methionine deficiency, by being fed a diet deficient in methionine. The birds were allowed to become accustomed to the colour cue of their water supply bottles, and to the physiological effects of their diet and drinking water. Feeding a diet deficient in methionine resulted in a substantial reduction in the intake of both feed and water. When the drinking water was then supplemented with methionine, both feed and water intake was restored to the previous (normal) level, moreover, methionine consumption equalled or exceeded that attained when methionine was supplied in the feed alone. Finally, the hens were fed a methionine-deficient diet and were offered a choice of both Normal and methionine-treated water. The birds showed a clear preference for methionine-treated water even after changing the position of colour-cued drinking bottles. Arch.Geflügelk. 1/2009

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Key words Laying hen, appetite, methionine, drinking water, colour cue

Zusammenfassung Bestimmung der Bedarfsergänzung von Methionin über das Trinkwasser bei Legehennen anhand von Farbmarkierungen In zwei Versuchsdurchgängen wurde untersucht, inwieweit Legehennen einen nicht durch das Futter abgedeckten Bedarf an Methionin über das Trinkwasser decken können. Hierzu wurden die Hennen mit Rationen mit Bedarfsdeckendem Gehalt an Methionin bzw. mit Methioninmangel gefüttert und normales bzw. mit Methionin ergänztes Trinkwasser angeboten. Der Methioninzusatz zum Trinkwasser betrug in Versuch 1 0,1% und in Versuch 2 0,15%. Für beide Versuche wurde zunächst die normale Aufnahme an Futter, Wasser und Methionin bestimmt. Hierzu wurde den Hennen die bedarfsdeckende Ration gefüttert und normales Trinkwasser verabreicht. In Versuch 1 wurden die Hennen nacheinander einem Überschuss bzw. Mangel an Methionin ausgesetzt. Hier zeigte sich, dass die Hennen ihren Ausgleichsbedarf für Methionin nicht realisieren konnten. In Versuch 2 wurden daher die Tränkwasserflaschen farbig markiert und die Legehennen entsprechend trainiert, um den Hennen die Wahl zwischen den Flaschen ohne und mit Methioninzusatz zu ermöglichen. Hierbei wurden die Farben jeweils einer Behandlung zugeordnet. Der Methioninmangel wurde induziert, indem die Hennen zunächst die Ration mit geringerem Methioningehalt erhielten. Ferner wurde den Hennen Zeit gegeben, sich an die Farbe der Wasserflaschen zu gewöhnen und auf die physiologischen Auswirkungen der Ration und das Trinwassers einzustellen. Die Fütterung der Methionin-armen Ration führte zu einer deutlichen Reduzierung der Futter- und Wasseraufnahme. Das Angebot von Trinkwasser mit Methioninzusatz führte zu einer Normalisierung der Futter- und Wasseraufnahme auf das vorherige Niveau. Die Methioninaufnahme war so hoch und z.T. sogar höher als bei Verabreichung des Methionins ausschließlich über das Futter. Bei Fütterung der Hennen mit der Methioninmangelration und Anbieten von Methionin in entsprechend farblich gekennzeichneten Tränkflaschen zeigte sich, dass die Hennen eine klare Präferenz für das mit Methionin ergänzte Wasser entwickelt haben, die auch beim Austausch der Position der Wasserflaschen bestehen geblieben ist.

Stichworte Legehenne, Appetit, Methionin, Trinkwasser, Farbmarkierung

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Correspondence: Sahin Cadirci, University of Harran, Faculty of Agriculture, Department of Animal Science, 63200 Sanliurfa, Turkey; E-mail: [email protected]

Arch.Geflügelk. 1/2009