Effects of Induced Molting on the Well-Being of Egg-Laying Hens

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1 JOURNAL OF APPLIED ANIMAL WELFARE SCIENCE, 9(1), 9 23 Copyright 2006, Lawrence Erlbaum Associates, Inc. Effects of Induced Molting on the Well-Being of Egg-Laying Hens Brenda McCowan, Joan Schrader, Ann Marie DiLorenzo, Carol Cardona, and Donald Klingborg Department of Population Health & Reproduction School of Veterinary Medicine University of California at Davis Induced molting in egg-laying hens is an important method for maximizing hen egg production and quality as well as hen health in commercial settings; however, there is growing societal concern over its effects on hen well-being. Using individual hens as their own controls, this research examined the behavior of hens subjected to different treatments of induced molting under premolt, molt, and postmolt conditions. Cage pecking increased in fast-induced subjects and aggression increased in fast-induced and nonfast-induced subjects during the molt. Gakel calling and several aspects of its acoustic structure were much higher during the molt condition in fast-induced subjects only. These data suggest that nonfast-induced molting treatments provide an effective method for inducing molting in hens and improving their well-being by minimizing discomfort due to food deprivation. In addition, these data further support that gakel vocalizations in hens may serve as an effective indicator for assessing well-being in a species otherwise behaviorally stoic in expressing stress or discomfort. There is growing societal concern that the induced molting of egg-laying hens compromises their well-being. This management practice is widespread in the commercial industry of California and other states because it provides significant benefits to both the producer and the consumer. Because both the number and quality of eggs are enhanced significantly by this practice and fewer hens are required to be hatched and reared through the investment period of development prior to reaching the laying stage, consumers and producers currently are reliant economically on molting. Several interest groups have criticized the practice of induced molting because hens are forced to fast during this process. Pre- Correspondence should be sent to Brenda McCowan, Department of Population Health & Reproduction, School of Veterinary Medicine, University of California at Davis, Davis, CA bmccowan@vmtrc.ucdavis.edu

2 10 MCCOWAN, SCHRADER, DILORENZO, CARDONA, KLINGBORG vious research has suggested that fast-induced molting diminishes hen well-being, causing frustration and/or discomfort in fasted subjects (Duncan & Mench, 2000; Webster, 1995, 2000, 2003; Zimmerman & Koene, 1998; Zimmerman, Koene, & van Hooff, 2000a, 2000b). Nonfast-molting methods have been described in the literature and include the feeding of nonnutritive diets (Bar, Razaphkovsky, Shinder, & Vax, 2003; Biggs, Persia, Koelkebeck, & Parsons, 2004; Landers, Howard, Woodward, Birkhold, & Ricke, 2005) and the feeding of high-salt diets (Breeding, Brake, Garlich, & Johnson, 1992; Goodman, Norton, & Diambra, 1986; Park, Birkhold, Kubena, Nisbet, & Ricke, 2004). These methods do not induce consistent molts in all hens in a flock, resulting, therefore, in adequate egg production in the subsequent egg production cycle to make them economically feasible alternatives. Consequently, commercial egg producers have frequently dismissed them. A novel, nonfast-molt program was developed recently (Biggs et al., 2004) and has had promising production results with commercial egg flocks. This method seems a reasonable alternative to the fast-induced molting programs; yet, results on the effects of this nonfast-molt program compared to feed removal on hen social behavior and psychological well-being were insignificant (Biggs et al., 2004). In this study, we examined the effects of induced molting on hen behavioral, vocal, and physiological parameters under three types of molting (fast-induced, nonfast-induced, no-molt) in both molting and control conditions. The objective of this research was to examine differences in hen behavior subjected to treatments of fast-induced molting; nonfast-induced molting; and no molting under premolt, molt, and postmolt conditions using individual hens as their own controls. Study Subjects and Treatments METHOD The university s institutional animal care and use committee approved all animal care procedures. Experiments were conducted with eight groups of three Single Comb White Leghorn hens of the Dekalb White strain (60 weeks of age) at the Veterinary Medicine Teaching & Research Center (Tulare, CA). Hens were housed in a standard size layer cage (12 16 in.) with a feeder space of 4 in. per hen and were provided with water ad libitum. Four experimental groups of three hens each were subjected to fast-induced molting. Two experimental groups of three hens each were subjected to nonfast-induced molting in which a low-caloric density diet (Table 1) was given to the hens. Two control groups of three hens each were unmanaged and allowed to undergo natural molting. All hens were tagged with color-coated numbered leg bands to identify subjects individually. The purpose of studying replicate groups of each treatment was to account for any differences due to individual and group dynamics. In addition,

3 EFFECTS OF INDUCED MOLTING ON LAYING HENS 11 TABLE 1 Nutritional Composition of Regular and Molt Diets Feed Type Nutrient Name Unit Regular Molt Weight LBS Met energy P CAL/LB 1, , Crude protein PCT Arginine PCT Lysine PCT Methionine PCT Meth and cystine PCT Tryptophane PCT Glycine PCT Isoleucine PCT Phenylalanine PCT Phenyl and tyro PCT Threonine PCT Valine PCT Crude fat PCT Crude fiber PCT Dry matter PCT Ash PCT Calcium PCT Phosphorus PCT Phosphorus T PCT Salt PCT Sodium PCT Xanthophyll MG/LB Vitamin D 3 KICU/LB Choline MG/LB Chloride PCT Linoleic acid PCT the fast-induced experimental groups were housed separately from the natural and nonfast-induced molt experimental groups during all stages of the study; they were physically, visually, and acoustically isolated from any feeding behavior during the fasting stage (Stage 2 of the study). Data Collection Stage 1 was a 30 day period in which the following oc- Stage 1: Premolt. curred:

4 12 MCCOWAN, SCHRADER, DILORENZO, CARDONA, KLINGBORG 1. All hens were subjected to a lighting program conducive to normal egg production and common in the commercial egg production industry with a 17-hr daily photoperiod (lights on at 0600 and off at 2300; nonmolt condition). 2. Regular full feed at 150 g per hen each day was provided during this period (Table 1). 3. Behavior and vocal data were collected remotely on each group onto videotape using Sony digital handycams (mounted on tripods directly facing each cage placed approximately 2.5 ft away) and Audiotechnica ATX directional microphones for 60 min twice on 3 days per week (1 session in the morning at 0900 or 1100 and 1 session in the afternoon at 1300 or 1500) to examine the baseline levels of aggressive, stereotypic, and alarm behaviors by subjects (Webster, 1995, 2000, 2003; see Table 2). The second stage lasted 26 days. During this time, the follow- Stage 2: Molt. ing occurred. TABLE 2 Ethogram of Hen Behaviors Used in This Study Behavior Aggressive peck Alert Bob Body shake Bowl peck Cage peck Displace Escape Feather peck Grab Groom Head down Head flick Head shake Mute Pace Preen Rest Shove Stand on Still Walk Wing up Description Pecks another cage mate (not on feathers) Neck extended and stiff head toward source of environmental disturbance Repetitive head and neck movement up and down Moves body from side to side (90 ) with at least slight ruffle Pecks food bowl when no food is present Pecks wall or floor of cage Causes a cage mate to move from present location Attempts to leave cage Pecks a feather on another cage mate Grabs a cage mate with its claw Preens another cage mate Moves head down without pecking (tends to be a submissive posture) Sharp or quick movement of the head other than bobbing Moves head from side to side (90 ) at least once with at least slight ruffle Extends neck, opens mouth, no vocalization Walks across cage at least twice Uses beak to clean feathers/body Eyes closed whether standing or sitting Pushes or shoves another cage mate Stands on top of another cage mate Stands or sits with head under feathers Moves more than two steps One or more wings raised and extended at least at a 45 angle

5 EFFECTS OF INDUCED MOLTING ON LAYING HENS Hens were subjected to the different treatments and behavioral data were collected as described earlier. 2. Experimental groups were molted by withdrawing regular feed for 10 days (fast-induced) or by a low-caloric diet feeding at 50 g per hen each day (nonfast-induced) for 26 days (Table 1) under the housing and lighting conditions typical of an induced molting program used by the poultry industry with an 8-hr photoperiod (lights on at 0830 and off at 1630). 3. On Day 11, fast-induced subjects were provided with 50 g of molt diet per hen for 3 days, then 150 g of molt diet per hen for an additional 10 days. 4. The control groups were also subjected to molt lighting conditions, but feed was not withdrawn or modified. Stage 3: Postmolt. The third stage consisted of an additional 30 day period. 1. On Day 22, we returned both the fast-induced (and nonfast-induced; Day 27) subjects to a layer diet. 2. On Day 26, we increased photoperiod to 12 hr, then, on Day 33, to 14 hr. 3. We then incrementally increased the photoperiod by 15 min per week until the end of the study. 4. During this additional 30 day period, we continued to monitor all hens. To monitor molting status for all groups and stages, data were collected on egg production, body weight, feather status (as a measure of body condition), and body temperature. Blood samples (5 ml samples) were collected from subjects between 0900 to 1000 on sampling days to measure corticosterone levels as a physiological indicator of stress during the various stages of the molting process. Blood samples were collected in less than 1 min by venipuncture of the brachial vein. Egg production was estimated on a per-group (cage) basis as the number of eggs collected each day. Body temperature was estimated using a standard thermometer that was administered anally. Feather status was evaluated on a 5-point scale ranging from 1(poor; few and weak feathers) to5(excellent; full body of robust feathers) and agreed on by five experimenters during sampling sessions. Physiological data were collected weekly on a day on which birds were not videotaped to avoid disruptions in behavior due to handling. Data Analysis Behavioral data were analyzed from a random subset of the videotapes with a total of 288 hr comprised of four 1-hr sessions from each of the 24 subjects for each of the three stages using a focal animal and one-zero sampling on a 10-sec interval for 15-min periods (Altmann, 1974). The observer effectively was blind

6 14 MCCOWAN, SCHRADER, DILORENZO, CARDONA, KLINGBORG to the identity of the treatment groups as the tapes were coded with only the group number and not the treatment. Each behavior was scored for the identity of the actor executing the behavior and the identity of the recipient who received the behavior. This regime permitted us to analyze both the types and rates of aggressive, alarm, and other stress-related behaviors under control and experimental conditions each hen acting as her own control. Vocal data from a subset of the analyzed tapes (N =72 hr) were processed using ISHMAEL software (Mellinger, National Oceanic and Atmospheric Association) to obtain an estimate of the number of gakel calls, a whining, elongated note often followed by shorter notes (Zimmerman et al., 2000a, 2000b) produced during the various stages of the molting process in all subjects. ISHMAEL contains a detection algorithm known as matched filtering that allows investigators to use an example acoustic file of the call as a detector algorithm for an entire sound file. ISHMAEL places detected sounds into separate files that can be used for subsequent acoustic analysis. Gakel calls, as opposed to other vocalization types, were our focus because previous research has shown that this calling behavior is associated with frustration in hens (Collias & Joos, 1953; Zimmerman & Koene, 1998; Zimmerman et al., 2000a, 2000b). A template consisting of a representative gakel recorded approximately 2 ft in front of a specific cage was used to filter the acoustic files. These data were analyzed on a group-by-group basis. We could determine the identity of the group because of amplitude differences in the vocal recordings due to the directional properties of the microphone. Vocalizations that did not match the gakel template or were of low amplitude based on template parameters were filtered during the detection phase of the analysis. In addition to rate of calling, Praat 4.3 acoustic software (Paul Boersma and David Weenink, Summer Institute of Linguistics) was used with each file of detected calls to measure several different aspects of call acoustic structure (Table 3) to determine if differences in acoustic structure of gakels might be found among treatments and conditions. Blood samples were allowed to clot at room temperature for 30 min. Once clotted, the sample was centrifuged for 10 min at 2000 rpm. Serum was extracted and stored frozen (below 15 C) until the corticosterone assay was performed. Corticosterone levels were determined (Davis, Anderson, & Carroll, 2000) from serum samples using an ImmunChem Double Antibody 125/RIA kit. A total of 204 samples from 24 individuals (8 to 10 samples per individual) was collected and analyzed as a part of this study. Behavioral, vocal, and physiological data were analyzed statistically using mixed effects linear regression with individual and cage (or group) as nested random effects in S Plus statistical software (Pinheiro & Bates, 2000). Pinheiro and Bates developed statistical models designed to protect against both Type 1 and Type 2 errors that are superior to traditional methods such as repeated measures

7 EFFECTS OF INDUCED MOLTING ON LAYING HENS 15 TABLE 3 Acoustic Variables Measured Using Praat 4.3 Variable Description Duration Time of signal in seconds Min frequency Minimum fundamental frequency Max frequency Maximum fundamental frequency M frequency Mean fundamental frequency SD in frequency Standard deviation in fundamental frequency Peak position Position of maximum frequency in call, expressed as a percentage of call Min position Position of minimum frequency in call, expressed as a percentage of call Slope Overall change in frequency across the duration of call divided by its duration % voiced Percentage of samples that were identified as tonal (vs. pulsed) characteristics Jitter Amount of frequency modulation in a call Shimmer Amount of amplitude modulation in a call Min HNR Minimum harmonic-to-noise ratio Max HNR Maximum harmonic-to-noise ratio M HNR Mean harmonic-to-noise ratio SD in HNR Standard deviation in harmonic-to-noise ratio Note. HNR = harmonic-to-noise ratio. analysis of variance, which protect against only Type I errors. The efficiency of this model is reflected both in the value of the t test and in the calculation of the degrees of freedom (Pinheiro & Bates, 2000). The former is modified by the amount of correlation in the grouped, random effect (i.e., how significant the random effect or repeated measure actually is) and the latter is calculated depending on the order of nesting of the grouped, random effect or repeated measure in relation to the variable of interest. The degrees of freedom can be closer either to the overall sample size or to the number of groups in the repeated measure, depending on whether the variable is nested by the repeated measure or the repeated measure is nested by the variable (Pinheiro & Bates, 2000, provided a detailed mathematical description). In addition, linear, mixed-effect models have advanced models for adjusting for different variance structures that frequently correct problems with deviations from normality. All statistical tests were evaluated for normality and variance; none violated these assumptions. Behavioral Indicators RESULTS Of the 29 behavioral indicators that we collected, only cage pecking and aggression differed among treatments (fast-induced, nonfast-induced, no-molt) and

8 16 MCCOWAN, SCHRADER, DILORENZO, CARDONA, KLINGBORG conditions (premolt, molt, postmolt). Cage pecking included pecking at the floor or walls of the cage, whereas aggression included aggressive pecking, standing on, displacing, grabbing, and shoving. Figure 1 presents the pattern of cage-pecking behavior for fast-induced, nonfast-induced, and no-molt subjects by condition (premolt, molt, postmolt). Fast-induced subjects exhibited significantly higher rates of cage pecking during the molting period, fast-induced: t(118) = 4.30, p < As seen in Figure 2, fast-induced and nonfast-induced subjects also exhibited significantly higher rates of aggression during the molting period than the FIGURE 1 Average frequency of cage pecking per session by condition (premolt, molt, and postmolt) for fast-induced, nonfast-induced, and no-molt subjects. FIGURE 2 Average frequency of aggression per session by condition (premolt, molt, and postmolt) for fast-induced, nonfast-induced, and no-molt subjects.

9 EFFECTS OF INDUCED MOLTING ON LAYING HENS 17 premolting period, fast-induced: t(118) = 2.40, p <.02; nonfast-induced: t(64) = 3.07, p <.003. Aggression during the molting stage was significantly higher in nonfast-induced subjects compared to aggression in fast-induced subjects, t(21) = 3.83, p <.009. However, neither fast-induced nor nonfast-induced significantly differed in rates of aggression from no-molt subjects, t(21) =.94, p =.48; t(21) = 3.84, p =.07. Vocal Indicators Figure 3 presents the rate of gakel calling by treatment and condition. As seen in this figure, hens subjected to fast-induced molting exhibited significantly higher rates of gakel calling during molt and postmolt as compared to the premolt period, fast-induced: t(13) = 2.34, p <.04; whereas subjects under the nonfast-induced molt and no-molt conditions showed no significant changes in calling behavior over time. Similarly, when changes in acoustic structure were examined, three acoustic variables shimmer, max frequency, and standard deviation in frequency were significantly higher and duration was significantly lower under the molt condition than under the premolt condition, but only in the fast-induced subjects, shimmer: t(913) = 6.06, p <.0001; max frequency: t(913) = 3.43, p <.0007; M harmonic to noise ratio: t(915) = 6.39, p <.0001; duration: t(915) = 5.92, p <.0001; (see Figure 4). Physiological Indicators Egg production (Figure 5) and weight (Figure 6) declined for fast-induced and nonfast-induced subjects during the molt as compared to premolt, egg production: fast-induced: t(474) = 7.94, p <.0001; nonfast-induced: t(236) = 6.09, p <.0001 FIGURE 3 Average rate of gakel calling per session by condition (premolt, molt, and postmolt) for fast-induced, nonfast-induced, and no-molt subjects.

10 FIGURE 4 Changes in the acoustic structure of gakel calls for fast-induced subjects by condition (premolt, molt, and postmolt). FIGURE 5 Changes in physiological measures of molt status by egg production for fast-induced, nonfast-induced, and no-molt subjects. 18

11 EFFECTS OF INDUCED MOLTING ON LAYING HENS 19 (Figure 5); weight: fast-induced: t(94) = 10.26, p <.0001; nonfast-induced: t(40) = 2.64, p <.02 (Figure 6), but not for no-molt subjects. Body temperature also declined during the molt as compared to premolt but for fast-induced subjects only, t(40) = 2.89, p <.006 (Figure 7). Feather status (Figure 8) vastly improved during the postmolt period for both fast-induced t(94) = 6.72, p <.0001, and nonfast-induced subjects t(40) = 2.98, p <.005 (Figure 7). However, feather status neither declined nor improved in the no-molt subjects. Feather status was significantly higher for both fast-induced t(174) = 2.49, p <.02, and nonfast-induced t(174) = 2.64, p <.01, subjects than for no-molt subjects during the postmolt period. In addition, we analyzed serum corticostereone levels to determine if changes in stress occurred during the molting process. Corticosterone levels actually decreased during the molting FIGURE 6 Changes in physiological measures of molt status by weight for fast-induced, nonfast-induced, and no-molt subjects. FIGURE 7 Changes in physiological measures of molt status by body temperature for fast-induced, nonfast-induced, and no-molt subjects.

12 20 MCCOWAN, SCHRADER, DILORENZO, CARDONA, KLINGBORG FIGURE 8 Changes in physiological measures of molt status by feather status for fast-induced, nonfast-induced, and no-molt subjects. FIGURE 9 Average level of corticosterone by condition (premolt, molt, and postmolt) for fast-induced, nonfast-induced, and no-molt subjects. stage for fast-induced and nonfast-induced subjects, fast-induced: t(94) = 5.66, p <.0001; nonfast-induced: t(40) = 2.27, p <.03, but not for no-molt subjects(figure 9). DISCUSSION The results of this study support previous research on the effects of food deprivation on egg-laying hen behavioral and vocal patterns (Duncan & Mench, 2000; Webster, 1995, 2000, 2003; Zimmerman & Koene 1998; Zimmerman et al., 2000a, 2000b). Hens generally appear to be stoic in expressing discomfort or stress as relatively few behavioral changes were observed under molting conditions. We found that fast-induced subjects exhibited higher cage pecking during the molting stage than prior to molting and that both nonfast-induced and

13 EFFECTS OF INDUCED MOLTING ON LAYING HENS 21 fast-induced subjects were more aggressive with other cage mates during the molting stage than during the premolt stage. These results indicate that a trade-off may exist between frustration resulting from food deprivation and discomfort resulting from aggressive dominance interactions (~47% aggressive pecking, ~35% displacement, ~18% other). Although nonfast-induced subjects were permitted at least to engage in the activity of feeding, thus perhaps inhibiting cage-pecking behavior, competition among cage mates over the low-caloric food source might have led to a larger increase in aggressive behavior by these subjects compared to that of fast-induced subjects. However, a closer look at premolt rates of aggressive behavior (Figure 3) reveals that the magnitude of change in aggressive behavior from the premolt to molt stages was similar between fast-induced and nonfast-induced subjects. Indeed, the rate of aggression during the molt treatment was approximately 80% higher than that during the premolt treatment both in fast-induced and nonfast-induced subjects. Further support that frustration was associated more frequently with food deprivation than with low-caloric intake can be found in the vocal results. A higher gakel-calling rate and changes in vocal, acoustic structure were found in fast-induced subjects but not in nonfast-induced or no-molt subjects. Indeed, all changes that occurred in the acoustic structure of calls increases in shimmer (amplitude fluctuations), max frequency, and harmonic-to-noise ratio based on the motivational-structural rules of animal vocalizations (Owings & Morton, 1998) moved in the direction one would expect. Motivation-structural rules predict that higher frequency and more tonal call structure should be associated with fear and lower frequency and more harsh call structure with aggression. Stress-induced modifications would be expected to follow the pattern of fear (often accompanying stress), which is supported by our results. For many of the behavioral indexes collected, nonfast-induced subjects and no-molt subjects showed similar results. One explanation may be due to social facilitation because nonfast-induced and no-molt subjects were housed in the same room whereas fast-induced subjects were housed in a separate room. This explanation is probably invalid, however, because the grouped, random effect of individual was a significant term in all analyses, indicating that individual birds acted independently from one another with respect to social and vocal behavior. Rather, the similarity in results between nonfast and no-molt subjects is more likely due to both sets of subjects having some form of food available to them during the molting stage of the study. Corticosterone levels declined during the molting stage in our fast-induced and nonfast-induced subjects. Given that previous studies have reported an increase in corticosterone during food deprivation (Hocking, Maxwell, & Mitchell, 1996; Hocking, Maxwell, Mitchell, & Robertson, 1998; Webster, 1995; Webster, 2000; Webster, 2003), this decrease in corticosterone at first seems puzzling. Two alter-

14 22 MCCOWAN, SCHRADER, DILORENZO, CARDONA, KLINGBORG natives may explain this finding. First, Webster (2003) reported that a temporary increase in corticosterone will occur within a few days of Phase 1 of the fast-induced molt but, because all energy expenditure derives from fat metabolism, corticosterone levels will be low during Phase 2 of a molt. Second, corticosterone levels may have declined in the fast-induced and nonfast-induced subjects (but not in the no-molt subjects) because the fast-induced and nonfast-induced subjects are not exhibiting the corticosterone peak normative during egg laying (Beuving & Vonder, 1977; Sharp & Beuving, 1978) due to the cessation of egg laying during the molt stage. Other physiological indicators of molting status indicate that the nonfast-induced molt method and the fast-induced molt method may be equally effective in promoting the molting process. This may be seen in weight loss and decline in egg production during the molt stage as well as an increase in feather quality during postmolt under both induced-molting regimes. That feather status greatly improved under the induced-molting regimes is important because feather loss during the molt and feather replacement postmolt often lead to reduced parasitic load (Clayton, 1999) and thus may enhance the long-term health of the hens, indicating that some type of induced-molting regime promotes hen health and, therefore, well-being. Indeed, hens left to undergo a natural molt, subjected only to the lighting conditions of the molting procedure, did not appear to undergo (nor garner the benefits of) a true molt, because egg production did not decline, body temperature and weight did not drop, and feather status did not improve as would be expected during a true molt (Figures 5, 6, 7, and 8). In conclusion, our results demonstrate that nonfast-induced methods, using a low-caloric diet, provide a good alternative for inducing molt in hens by minimizing discomfort due to food deprivation, thus improving well-being. In addition, these data further support that gakel vocalizations in hens may serve as an effective indicator for assessing well-being in a species that otherwise is behaviorally stoic in expressing stress or discomfort. ACKNOWLEDGMENTS The project was funded by grants from the Veterinary Medicine Extension and the Center for Food Animal Health at the University of California at Davis. The research was reviewed and approved by the institutional animal care and use committee under Protocol No We thank Karen Tonooka, Christina VanWorth, Brigid McCrea, and Kelly Weaver for their help on this project. REFERENCES Altmann, J. (1974). Observational study of behavior: Sampling methods. Behaviour, 49,

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