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Dustbathing behavior: Do ectoparasites matter?
Applied Animal Behaviour Science 169 (2015) 93–99
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Applied Animal Behaviour Science journal homepage: www.elsevier.com/locate/applanim
Dustbathing behavior: Do ectoparasites matter? Giuseppe Vezzoli a,b,1 , Bradley A. Mullens c , Joy A. Mench a,∗ a
Department of Animal Science and Center for Animal Welfare, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA Animal Biology Graduate Group, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA c Department of Entomology, University of California, Riverside, 900 University Avenue, Riverside, CA 92521, USA b
a r t i c l e
i n f o
Article history: Received 20 February 2015 Received in revised form 21 May 2015 Accepted 6 June 2015 Available online 19 June 2015 Keywords: Dustbathing Laying hen Northern fowl mite Astroturf Sand
a b s t r a c t A presumed function of dustbathing behavior is to remove ectoparasites. Providing dustbathing substrates in furnished cages for laying hens might therefore offer an alternative to pesticide use to reduce ectoparasite populations. We investigated the effectiveness of dustbathing substrates for controlling northern fowl mites in individually caged beak-trimmed White Leghorn hens (N = 32). Each cage contained a 32 cm × 32 cm plastic tray that was either: (1) filled with 1200 g of sand (SAND); (2) empty (CONTROL); (3) covered with Astroturf (AT); or (4) covered with AT on to which 150 g of feed was delivered daily (ATF). AT and ATF are used in the dustbathing/foraging area of many newer commercial furnished cages. Hens were infested with approximately 35 mites at 25 weeks of age. Mite numbers were estimated weekly. Time spent dustbathing and dustbathing bout numbers and lengths in the tray and on the wire cage floor were determined from video recordings made for 2 consecutive days from 12:00 to 20:00 h immediately before and after infestation and at weeks 1, 3, 5, and 7 post-infestation. Data were analyzed using a repeated-measures ANOVA in SAS. Treatment did not influence the total time spent dustbathing (average across substrates: 11.3 min). However, there were treatment effects on the time spent dustbathing in the trays (F2,21 = 3.67, P = 0.043) and on wire (F2,21 = 7.68, P = 0.031). SAND and ATF hens spent more time dustbathing in the trays (11.4 and 9.1 min, respectively) than AT (2.3 min), and CONTROL and AT hens spent more time dustbathing on wire (11.6 and 4.7 min, respectively) than ATF (0.4 min). There was a treatment effect on infestation (F3,28 = 3.08, P = 0.04), with ATF having more mites (back-transformed mean = 1500) than AT (330), and with SAND (460) and CONTROL (447) intermediate. This study confirmed that the substrate type affected dustbathing behavior. SAND was a preferred dustbathing substrate but was not effective for controlling mite numbers, nor was the time spent dustbathing in any substrate or in total influenced by infestation levels. Our data also suggest that adding feed to the AT pad in furnished cages might lead to increased mite numbers in infested hens. The reason for this effect is unclear, but could be due to feed particles contributing to a change in feather structure that creates a more favorable mite habitat. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Egg producers in a number of countries are adopting furnished cages (larger versions of which are also called enriched colonies) as an alternative to conventional cages. These cages include a nest, perches and a substrate to encourage foraging, scratching
∗ Corresponding author at: Department of Animal Science, University of California Davis, One Shields Avenue, Davis, CA 95616, USA. Tel.: +1 530 752 7125; fax: +1 530 752 0175. E-mail addresses: [email protected] (G. Vezzoli), [email protected] (B.A. Mullens), [email protected] (J.A. Mench). 1 Current address: Department of Math and Science, Kirkwood Community College, 6301 Kirkwood Blvd. SW, Cedar Rapids, Iowa, IA 52404, USA. http://dx.doi.org/10.1016/j.applanim.2015.06.001 0168-1591/© 2015 Elsevier B.V. All rights reserved.
and dustbathing behavior. The most common material used for this substrate is an Astroturf (AT) pad onto which loose material like feed may be distributed (Scholz et al., 2010; Alvino et al., 2013). Finer substrates like sand can be also dispensed onto this pad but create problems if they occlude the overhead dispenser or abrade the mechanical delivery system (Scholz et al., 2010). However, materials with a fine structure like sand are preferred by hens for dustbathing over substrates consisting of large particles (Olsson and Keeling, 2005) and are also preferred to substrates like AT or AT and feed (Alvino et al., 2013). One function of dustbathing is to maintain the integument in good condition by reducing excess feather lipids (Olsson and Keeling, 2005). It has long been suggested that another function of dustbathing is to help remove ectoparasites (Rothschild and
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Clay, 1952; de Jong et al., 2007; Clayton et al., 2010), however there is only one study evaluating this presumed function. Martin and Mullens (2012) provided hens housed on litter with dustboxes filled with sand and sulphur, sand and kaolin (clay) or sand and diatomaceous earth. Sulphur and inert dusts like kaolin and diatomaceous earth have been demonstrated to increase ectoparasite mortality (Creighton et al., 1943; Furman, 1952; Kilpinen and Steenberg, 2009). Martin and Mullens (2012) found that the hens that used these dustboxes had significantly fewer chicken body lice (Menacanthus stramineus) and northern fowl mites (Ornithonyssus sylviarum) than hens that did not use them, and also fewer ectoparasites than caged hens without access to dustboxes. Two older studies (Hoffman and Hogan, 1967; Hoffman and Gingrich, 1968) evaluated the effect of a chemical (Zytron) and a microbial (Bacillus thuringengis) insecticide mixed with sand in dustboxes on three species of lice in infested hens in a non-cage system. They reported that there was a reduction in the number of lice when the insecticides were included in the boxes but not when sand was the only substrate. However, they did not actually record dustbathing behavior. To our knowledge there are no published studies evaluating the relationship between ectoparasites and the performance of dustbathing behavior. Moreover, there is no information on whether hens that dustbathe in substrates that have not been treated with inert dusts or insecticides have reduced ectoparasite populations. If dustbathing does function to remove ectoparasites, providing the types of dustbathing substrates that are typically used in furnished cages might offer an alternative to pesticide use. Therefore, there is an opportunity to evaluate the effectiveness of dustbathing behavior for controlling ectoparasite populations using a preferred dustbathing substrate like sand (Shields et al., 2004; van Liere et al., 1990; Olsson and Keeling, 2005), or even a non-preferred substrate like Astroturf (AT). Sand might abrade the ectoparasite cuticle, leading to desiccation, while the AT could mechanically dislodge parasites, an effect that could be potentiated when feed is added to make the AT more desirable for dustbathing. It is also possible that dustbathing in feed could directly affect ectoparasite survival. Scholz et al. (2014) found that the feathers of hens that dustbathed in feed compared to lignocellulose had a higher lipid content. Moyer et al. (2003) found that preen oil from the uropygial gland negatively affected the survival of rock dove lice in vitro, although experimental removal of the uropygial gland did not impact louse populations in vivo. There have been no studies of the effects of feather lipids on mite infestation. Finally, it is not known whether the presence of ectoparasites affects dustbathing behavior and whether infested hens increase the time they spend dustbathing in non-preferred substrates like feed or AT in an attempt to reduce their infestation. The northern fowl mite (NFM) is the most common and serious poultry ectoparasite in North America (Axtell and Arends, 1990). It spends its entire life cycle on the host, feeding on blood and laying its eggs at the base of the feathers, particularly in the vent area. The aims of our experiment were to evaluate: (1) the role of dustbathing behavior in controlling NFM populations; (2) the effectiveness of commercially used dustbathing substrates in reducing NFM levels on infested hens; (3) the effect of NFM on dustbathing behavior. We hypothesized that dustbathing behavior plays a role in controlling ectoparasite populations and that different dustbathing substrates would have different effects on NFM populations. We predicted that NFM populations would be lower in hens that dustbathed, and that the provision of a highly preferred substrate would reduce NFM populations more than the provision of non-preferred substrates. We also hypothesized that infestation with NFM would increase the total time spent dustbathing by increasing both the length and the number of dustbathing bouts.
2. Materials and methods 2.1. Animals and Husbandry This experiment was conducted at the Avian Science Facility at the University of California, Davis. Thirty-two hens were randomly selected from a flock of 168 CV20/W36 beak-trimmed pullets that had been previously used in a study of early rearing effects on dustbathing substrate preference (Alvino, 2013). The day-old chicks had been obtained from a commercial hatchery (JS West and Companies, Modesto CA) and kept in a Petersime brooder, where they were raised either completely on wire or exposed to either AT plus feed (ATF) or sand as dustbathing substrates. At 11 weeks of age, the pullets were moved in pairs to grower cages, and the previous treatments were maintained until they were 18 weeks of age. The pullets were then singly housed in 90 cm × 46 cm × 46 cm cages in two experimental rooms for the current study. From 14 to 18 weeks of age the light was increased 1 h per week to reach a 16L:8D photoperiod, which was maintained for the duration of the experiment. Each experimental room contained 4 racks of cages and each rack held 4 cages. Each cage had a 32 cm × 32 cm plastic tray (Akro Mils SRO12500A34) that was either: (1) empty (CONTROL); (2) filled with 1200 g of sand (SAND) (Sakrete Natural Play Sand, Dixon, California); (3) lined with Astroturf (AT) (GrassWorx XPSP, 14 mm pile height); or (4) lined with AT on to which 150 g of feed was delivered daily (ATF). This amount of feed covered the entire surface of the pad. Pullets were assigned to the treatments most similar to their rearing substrate exposure (i.e., chicks reared with ATF were assigned either to AT or ATF, wire to CONTROL, and sand to SAND); the four treatments were balanced in the two rooms. All the trays and the AT pads were removed from the cages and washed and dried daily between 9:30 and 11:30 h. The trays were then lined with the clean pads or fresh sand and reintroduced into the cages. Feed was dispensed on to the ATF pads immediately after they were cleaned and dried. Although there was abundant sand and feed in the trays and on the pads at the end of a 24 h period, they were cleaned daily to remove feces and ensure that feed and sand availability and cleanliness were comparable each day. Cleaning was carried out in the morning so as not to disturb the hens during the video recording sessions. There was a 45 cm long feeder on the front of each cage which was filled every morning with a pelleted diet formulated for highproducing laying hens (Purina Layena, Turlock California 16% CP, high calcium ration, 2.5% fat). Running water was provided ad libitum via a trough placed along the back of the cages. The pullets/hens were housed and managed according to the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (FASS, 2010). All experimental procedures were approved by the University of California, Davis, Institutional Animal Care and Use Committee. 2.2. Ectoparasite infestation Because we were interested in determining how providing dustbathing opportunities affected the course of infestation, it was necessary to experimentally infest the hens. Approximately 35 northern fowl mites were placed on the abdominal feathers of each hen when the hens were 25 weeks of age. The mites came from naturally infested source hens housed in a building separate from the experimental room at the UC Davis Avian Science Facility. Miteinfested feathers were cut from the source hens and put in a plastic bag. A glass Pasteur pipette was then used to aspirate (Owen et al., 2008) and transfer the mites from the plastic bag to the vent feathers of each experimental hen. Prior to infestation (week −1) each hen was removed from her cage and the feathers and skin of her vent area were checked to verify that they did not harbor any mites;
G. Vezzoli et al. / Applied Animal Behaviour Science 169 (2015) 93–99
after infestation (week 0) each hen was removed weekly from her cage and the vent feathers and vent skin were visually scored for the number of mites. One person (GV) did the scoring. 2.3. Behavioral observations Cameras (Veilux SVB-70IR48, 3.6 mm Board Lens, 1/3 in. Sony CCD) mounted centrally in front of each cage were used to film the hens in real-time. Each cage was video recorded for two consecutive days from 12:00 to 20:00 h immediately before (week −1) and after (week 0) infestation, and at weeks 1, 3, 5 and 7 post-infestation. The videos were analyzed using the V 8.5 GeoVision software program (GeoVision V-Series Surveillance System, Vision Systems Inc., Irvine CA). The data collected over the two-day period were summed for analysis. Time spent dustbathing, dustbathing bout numbers and bout lengths in two locations, in the trays and on the wire floor, were recorded and analyzed throughout the entire video recorded period. The total time spent dustbathing was calculated by adding the time spent dustbathing in the trays and on the wire floor, and the total dustbathing bouts were calculated by adding the bouts performed in the trays to the bouts performed on the wire floor. The first vertical wing shake was used as the measure of the beginning of a dustbathing bout. A bout was considered to end when the hen showed vigorous body-shaking or when more than 2 min elapsed between dustbathing episodes. One observer (GV) coded the videos.
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To determine the effects of treatment and week on the behavioral data we used a repeated measures analysis with the number of mites as a covariate. Homogeneity among slopes was tested by including a two and a three-way interaction between the covariate and the fixed effects. When the interactions were non-significant we concluded that the slopes were homogenous and that we could remove the interaction terms from the model (Engqvist, 2005). The dependent variables were: time spent dustbathing, dustbathing bouts and bout lengths both in the trays and on the wire, total time spent dustbathing, total dustbathing bouts and total bout lengths. Since CONTROL hens were not observed dustbathing in the trays they were not included in the analysis of dustbathing in this part of the cage. Similarly, SAND hens were not observed to dustbathe on wire and were therefore not included in that analysis. Square root transformations were needed to meet the assumptions of ANOVA for all of the behavioral data; therefore the back-transformed and the transformed means and standard errors (SE) are reported. To analyze mite populations, we used a repeated measures analysis with a similar model as the one used for the behavioral data, with week and treatment as fixed effects and hen as the random effect. The dependent variable for this analysis was the number of mites. Week 0 was not included in the analysis because it was the week of infestation and the mite scores were therefore the same for each hen. Log transformations were needed to meet the assumption of ANOVA. Back-transformed and transformed means and standard errors (SE) are reported. One-way GLM (general linear model) ANOVA was used to analyze feather lipid levels, with treatment as the independent variable.
2.4. Feather lipids We analyzed feather lipids immediately prior to infestation to determine whether the rearing treatments had affected these levels, which could in turn differentially affect the infestation profile by changing the suitability of the habitat for the mites. For feather lipid analysis, approximately 1.2 g of breast feathers were collected from each hen the day before infestation. The feathers were cut at the base of the rachis and placed in plastic bags, and were then frozen at −20 ◦ C. For analysis, 1 g of feathers per hen was dried in a convection oven at 100 ◦ C for 2 h. Feather surface lipids were then extracted using a Soxtec system (Tecator Soxtec System HT 1043 Extraction Unit, Foss Tecator, Denmark). Aluminum extraction cups were weighed and the pre-extraction weights recorded. Lipids were extracted using anhydrous ethyl ether (Fisher Scientific, Fair Lawn, NJ). After 50 min of boiling, the samples were rinsed for 50 min, with the lipids from the samples retained in the extraction cups. After the solvent was allowed to evaporate for 10 min, the extraction cups were placed in a 100 ◦ C drying oven for 30 min, then in a desiccator for an additional 30 min. The difference in weight between the cups containing lipids and their original weight was the weight of the extracted lipids (expressed as mg of lipid per g of feathers). 2.5. Statistical analysis The Statistical Analysis System (SAS, 9.3) was used to analyze the data. The Shapiro-Wilk test was used to assess normality and graphical analysis of the residuals was used to assess homogeneity of variance. The level of statistical significance was set at P < 0.05, with 0.05 ≤ P < 0.10 values considered as showing a trend towards significance. The Tukey post hoc test was used when significant differences were found. Each hen served as her own control for all analyses. For the initial models, hen (cage) was the experimental unit, treatment and week were the fixed effects, room and hen were the random effects, and week was the repeated measure. Since the analyses revealed no differences between the two experimental rooms, room was removed from the subsequent models.
3. Results 3.1. Number of mites As expected there was a week effect (F6,168 = 69.50, P < 0.0001) on the number of mites (Fig. 1). Tukey comparisons revealed that mites increased until weeks 4–6, then started declining. There was also a main effect of treatment (F3,28 = 3.08, P = 0.04; Fig. 2), with ATF having more mites than AT (P = 0.03) and with SAND and CONTROL intermediate. There were no treatment × week interactions. 3.2. Dustbathing behavior Neither the main effects nor the interactions co-varied with the number of mites, indicating that dustbathing behavior was not influenced by infestation levels. Further results are therefore reported without the covariate. 3.2.1. Treatment effects Table 1 shows the treatment effects on the time spent dustbathing, bout numbers and bout lengths in both the trays and on wire. Treatment did not affect total dustbathing, since there were no effects on the total (tray and wire combined) time spent dustbathing (pooled back-transformed means 11.36 min) or the total bout numbers (pooled back-transformed mean = 1.75 bout). However, there were treatment differences related to the location of dustbathing within the cage. For dustbathing in the trays, treatment affected both the time spent dustbathing (F2,21 = 3.67, P = 0.043) and bout lengths (F2,21 = 8.51, P = 0.002). AT spent less time dustbathing and also had shorter bouts in the trays than SAND and ATF. There was also a treatment effect on the time spent dustbathing (F2,21 = 7.68, P = 0.003) and on bout numbers (F2,21 = 5.69, P = 0.01) and bout lengths (F2,21 = 9.69, P = 0.001) on wire. CONTROL and AT spent more time dustbathing and performed more dustbathing bouts on wire than ATF. CONTROL also performed longer
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A
4.0
d
Number of Mites
3.5
d
c
d
Table 1 Treatment effects on time spent dustbathing (min) and on dustbathing bout numbers and lengths in both locations, on wire and in trays. Back-transformed means are reported, with square root transformed means and standard errors in parentheses. Different letters indicate significant differences (P < 0.05) among treatments for each measure. CONTROL hens did not dustbathe in the trays and SAND did not dustbathe on wire (N/A). ATF (Astroturf plus feed) and AT (Astroturf).
c
3.0 b
2.5 a
2.0 1.5
Measure
Location Tray
Wire
Time spent dustbathing (min)
ATF: 9.06 (3.01 ± 0.51)a AT: 2.34 (1.53 ± 0.51)b SAND: 11.36 (3.37 ± 0.51)a CONTROL: N/A
ATF: 0.38 (0.62 ± 0.50)a AT: 4.71 (2.17 ± 0.50)b SAND: N/A CONTROL: 11.63 (3.41 ± 0.50)b
Dustbathing bouts
ATF: 0.85 (0.92 ± 0.16) AT: 0.36 (0.60 ± 0.15) SAND: 0.69 (0.83 ± 0.15) CONTROL: N/A
ATF: 0.10 (0.32 ± 0.29)a AT: 1.35 (1.16 ± 0.29)b SAND: N/A CONTROL: 2.76 (1.66 ± 0.29)b
Dustbathing bout length (min/bout)
ATF: 6.05 (2.46 ± 0.37)a AT: 1.04 (1.02 ± 0.37)b SAND: 9.67 (3.11 ± 0.37)a CONTROL: N/A
ATF: 0.19 (0.44 ± 0.21)a AT: 1.19 (1.09 ± 0.21)b SAND: N/A CONTROL: 3.03 (1.74 ± 0.21)c
1.0 0.5 0.0 0
1
2
3
4
5
6
7
Week
B
Number of Mites
2500 2000 1500 1000 500 0 0
1
2
3
4
5
6
7
Week Fig. 1. Main effect of week on number of mites. Letters indicate significant differences (P < 0.05) among weeks. Transformed means and SE are reported in (A) and back-transformed means are reported in (B).
Fig. 3. Week effect on the total (in the trays and on wire) time spent dustbathing. Letters indicate significant differences (P < 0.05) among weeks. Transformed means and SE are reported in (A) and back-transformed means are reported in (B).
bouts on wire than both ATF and AT. Similarly, AT performed longer bouts on wire than ATF.
Fig. 2. Main effect of treatment on number of mites. Letters indicate significant differences (P < 0.05) between treatments. Transformed means and SE are reported in (A) and back-transformed means are reported in (B). AT (Astroturf), ATF (Astroturf plus feed).
3.2.2. Week and interaction effects 3.2.2.1. Total dustbathing (wire plus tray). There was a week effect on the total time spent dustbathing (F5,140 = 4.35, P = 0.001) and total bout numbers (F5,140 = 2.64, P = 0.03). At week 7 hens spent more time dustbathing (Fig. 3) than they did prior to infestation (week −1) and at weeks 1, 3, and 5. There was a trend for a treatment × week interaction for this measure (F15,140 = 1.64, P = 0.07), with CONTROL tending to spend more time dustbathing at week 7 [back-transformed means in min, with transformed means ± SE in parenthesis: 33.76 (5.81 ± 0.85)] than they did prior to infestation
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3.3. Feather lipids There was no rearing substrate effect on the lipid content of the feathers (pooled means = 7.57 mg/g of feathers) collected immediately prior to infestation.
4. Discussion
Fig. 4. Week effect on the total dustbathing bout numbers. Letters indicate significant differences (P < 0.05) among weeks. Transformed means and SE are reported in (A) and back-transformed means are reported in (B).
[1.00 (1.00 ± 0.85)] and at weeks 1 [6.76 (2.60 ± 0.85)] and 3 [6.86 (2.62 ± 0.85)]. For dustbathing bout numbers (Fig. 4), hens at week 7 performed more bouts than they did prior to infestation and at weeks 1, 3 and 5.
3.2.2.2. Dustbathing in the trays. There were no week effects on the time spent dustbathing (pooled back-transformed means 6.7 min) or on the bout numbers (pooled back-transformed means 0.6) or bout lengths (pooled back-transformed means 4.9 min/bout) in the trays. There were also no treatment × week interactions.
3.2.2.3. Dustbathing on wire. There was a week effect on the time spent dustbathing (F5,105 = 3.51, P = 0.005), although this interacted with treatment. During week 7, hens spent more time dustbathing on wire [back-transformed means in min, with transformed means ± SE in parenthesis: 9.49 (3.08 ± 0.41)] than they did prior to infestation [2.40 (1.55 ± 0.41)] or at weeks 0 [2.10 (1.45 ± 0.43)], 1 [3.50 (1.87 ± 0.41)], 3 [2.34 (1.53 ± 0.41)] and 5 [4.45 (2.11 ± 0.41)]. There were treatment × week interactions for both time spent dustbathing (F10,105 = 4.11, P < 0.0001) and bout lengths (F10,105 = 3.01, P = 0.002) as well as a trend for bout numbers (F10,105 = 1.79, P = 0.07). Table 2 shows the treatment × week interactions for CONTROL hens. At week 7, CONTROL spent more time dustbathing and performed more dustbathing bouts on wire than they did during the preceding weeks. At week 7 CONTROL had longer bouts than before infestation and than at weeks 1 and 3. Posthoc test also showed that at week 7 AT hens tended to spend more time dustbathing [back-transformed means in min, with transformed means ± SE in parenthesis: 9.99 (3.16 ± 0.71)] than they did at week 5 [(2.56 (1.60 ± 0.71) P = 0.055] and 3 [(2.89 (1.70 ± 0.71) P = 0.06].
We showed for the first time that the presence of mites did not influence dustbathing behavior in laying hens, although the skin irritation caused by mite infestation may have stimulated dustbathing behavior in the CONTROL hens, as we discuss below. While substrate type did affect dustbathing behavior, NFM populations were not decreased by providing sand and/or Astroturf for dustbathing. As has been shown in previous studies (Alvino et al., 2013; Shields et al., 2004; van Liere et al., 1990), substrate type affected dustbathing behavior. SAND was an attractive dustbathing substrate, and SAND hens dustbathed exclusively in the trays. In addition, both SAND and ATF hens spent more time dustbathing in the trays and had longer bouts in the trays than AT, and spent less time dustbathing and performed fewer and shorter bouts on wire than CONTROL and AT. Our results show that hens used ATF, which could therefore be a good alternative to sand in terms of stimulating dustbathing behavior. This contrasts with the findings of Alvino et al. (2013) that hens given sand spent more time dustbathing and had longer bouts than hens given ATF. The hens in our study were raised with access to substrates similar to the treatments provided during the experiment, whereas the hens in Alvino et al. (2013) were raised in wire floor cages. Our findings therefore suggest that the provision of feed as dustbathing substrate during rearing might be crucial in motivating dustbathing behavior by adult hens on a non-preferred substrate like ATF. Our data support the results of Vestergaard and Hogan (1992), who found that the type of dustbathing substrate provided to chickens during rearing affects the choice of dustbathing substrate later in life. Although there was a decline in mite populations in all treatments at week 7 post-infestation, CONTROL hens at week 7 spent more time dustbathing, and performed more dustbathing bouts, than they did prior to infestation and at weeks 1, 3 and 5, with all of this dustbathing activity occurring on the wire floor. AT hens also tended to increase the time they spent dustbathing on wire at week 7 compared to weeks 3 and 5. This suggests that skin condition after infestation could play a role in eliciting dustbathing behavior. Owen et al. (2009) observed an immune/inflammatory response in the skin of hens infested with NFM with a consequent formation of scabs that became cracked and flaky during recovery. We found (Vezzoli, 2014; Vezzoli et al., 2015a) that the hens in the current study had scabs at weeks 5 and 7 post-infestation. Sheep infested with the mange mite Psoroptes ovis spend more time rubbing when they have more, larger and older scabs (Berriatua et al., 2001). Berriatua et al. (2001) suggested that histamine, which is a potent pruritogen (Greaves and Wall, 1996), was the cause. To our knowledge no one has evaluated whether the presence, age, or condition of scabs, or the histamine response to tissue injury, are factors that influence dustbathing behavior in birds. Berriatua et al. (2001) suggested that a “strong mechanical stimulus” like rubbing results in central nervous inhibition of the sensation of itchiness, although the mechanism for this is unknown (Davidson et al., 2009). Perhaps substrates like sand or feed reduce itchiness due to their mechanical contact with the hen’s skin. In our experiment, CONTROL hens did not have access to a substrate. AT hens did have access to substrate, but preferentially dustbathed on the wire instead, which like CONTROL may not have been ideal to reduce itchiness. This, coupled with the presence of cracked and flaky
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Table 2 Treatment × week interaction effects for CONTROL hens on dustbathing performances on wire. Back-transformed means are reported, with square root transformed means and standard errors in parentheses. Different letters indicate significant differences (P < 0.05) among weeks for each measure. Measure
Week −1
Time spent dustbathing Dustbathing bouts Dustbathing bout length (min/bout)
0
1.00 (1.00 ± 0.71) 0.77 (0.88 ± 0.35)a 0.46 (0.68 ± 0.38)a a
1
17.47 (4.18 ± 0.71) 3.28 (1.81 ± 0.35)b 4.49 (2.12 ± 0.38)b b
scabs, might explain the increase in dustbathing behavior observed in these treatments at week 7. Our study showed that ATF had a higher level of mites than AT. While there were no significant differences between ATF, CONTROL and SAND, ATF harbored three times more mites than either of these treatments. Dustbathing on ATF could have affected mite populations because the lipids in feed can be deposited on the feathers during dustbathing (Scholz et al., 2014). As discussed above, preen oil has been shown to reduce louse survival in vitro (Moyer et al., 2003). However, lipid deposition could also potentially make the feathers more suitable as a mite habitat by affecting the arrangement of barbs and barbules on the feathers, thereby changing microhabitat humidity, or by altering feather temperature (van Liere, 1991). However, we did not find that ATF hens had higher feather lipid levels prior to infestation than hens in the other treatments, possibly because the feed we used had a lower lipid (2.5%) content than that (3.5%) used by Scholz et al. (2014). We could not analyze the feathers post-infestation because the mites themselves have lipids which would have confounded the lipid level analysis. Further analysis of feather structure using a scanning electron microscope might elucidate whether and how different dustbathing substrates affect the feather physical structure and the habitat in on which the mites live. It is surprising that hens given sand, which is a preferred and effective dustbathing substrate (van Liere and Bokma, 1987; van Liere et al., 1990; Shields et al., 2004), had a similar number of mites as CONTROL, AT and ATF. The mite populations and the course of infestation in our study were comparable to those seen in naturally infested caged laying hens (Mullens et al., 2009). It has been suggested for decades that one function of dustbathing is removal of ectoparasites (Rothschild and Clay, 1952; de Jong et al., 2007; Clayton et al., 2010). Our study showed for the first time that dustbathing in sand or on the Astroturf pads that are commonly provided in enriched colony cages are not effective for controlling NFM. Hoffman and Hogan (1967) demonstrated that sand did not affect lice populations when it was available for dustbathing, although they did not observe whether the hens actually used the dustbox. Martin and Mullens (2012) showed that hens that used dustboxes filled with sand and inorganic inert dusts like kaolin or diatomaceous earth, or with sand and sulphur, had reduced northern fowl mite and chicken body louse populations compared to hens that did not use the dustboxes. Kilpinen and Steenberg (2009) and Martin and Mullens (2012) suggested that, due to their fine size and oil absorption capacity, inorganic inert dusts like kaolin clay and diatomaceous earth might abrade the cuticle, or more likely absorb the lipid on the cuticle leading to desiccation of the ectoparasites. Perhaps either the size or nature of sand grains causes sand to be ineffective for in abrading or absorbing oil from the mite cuticle. In addition, it is possible that dustbathing in natural soil might affect ectoparasites more than dustbathing in the substrates that we used for the present experiment. The soil is a “complex living system” with physical, chemical and biological components (Karlen et al., 1997). Kaolin is an abundant constituent of the soil, and Martin and Mullens (2012) suggested that birds might be able to suppress ectoparasite populations by dustbathing in kaolin-like soils. Perhaps the soil also contains components like essential oils,
3
6.76 (2.60 ± 0.71) 2.25 (1.50 ± 0.35)b 1.42 (1.19 ± 0.38)a c
5
6.86 (2.62 ± 0.71) 2.96 (1.72 ± 0.35)b 1.72 (1.31 ± 0.38)a c
7
17.98 (4.24 ± 0.71) 2.82 (1.68 ± 0.35)b 5.66 (2.38 ± 0.38)b b
33.76 (5.81 ± 0.71)d 5.71 (2.39 ± 0.35)c 7.73 (2.78 ± 0.38)b
which are known to increase the mortality of some poultry ectoparasites like the red mite (Dermanyssus gallinae) (George et al., 2009, 2010; Kim et al., 2004). An alternative potential effect of dustbathing that might help to reduce ectoparasites is dislodging (Clayton et al., 2010). However, in our study hens were housed in cages and therefore mites that were dislodged by a hen could have simply re-infested her. An evaluation of dislodging of mites or other chicken ectoparasites might help to understand if this mechanism really occurs during dustbathing. Although dustbathing behavior was not affected by NFM, it is possible that a different parasite, for example the chicken body louse, which is the second most important poultry ectoparasite in North America (Axtell and Arends, 1990), would have a different effect on dustbathing. If infestation with different kinds of parasites does not change dustbathing behavior, it would strengthen the hypothesis that dustbathing is not functionally related to the removal of ectoparasites. In addition, it is known that hens with an intact beak are able to reduce their ectoparasite loads (Mullens et al., 2010) via preening directed to the body areas that are most severely infested (Vezzoli et al., 2015b). However, to our knowledge there have been no studies evaluating the interactive effects of preening and dustbathing in beak-intact hens on ectoparasite loads. This comparison might further elucidate the evolutionary functions of these behaviors with respect to ectoparasite control. 5. Conclusions Although we did not find increases in dustbathing in NFMinfested hens, future work should investigate how diverse parasites affect dustbathing behavior. The idea that scabs or histamine might also affect dustbathing behavior should be explored. Further research is also necessary to understand if feed used as a dustbathing substrate creates a more favorable habitat for the mites and to better understand which substrate(s) can be most effective against NFM and more suitable for use in furnished cages. Acknowledgements We gratefully acknowledge the infrastructure support of the Department of Animal Science, the College of Agricultural and Environmental Sciences, and the California Agricultural Experiment Station of the University of California, Davis. We also thank GrassWorx, LLC for donating the AT pads. Giuseppe Vezzoli was partly supported by fellowships from the Pacific Egg and Poultry Association (PePa), the Olivera Memorial Fund, and Jastro-Shields. We also thank all of the following individuals at University of California, Davis, for their contributions to the project: Gina Alvino for caring for the chicks/pullets, Emily Bloom for caring for the hens, Margaret de Luz for feather lipid analysis, Gregory Archer and Richard Blatchford for helping with video equipment installation, the undergraduates who helped with the different phases of this project, the staff of Hopkins Avian Research Facility for their help with animal care, and Cassandra Tucker for her helpful comments on an earlier version of this manuscript.
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Martin, C.D., Mullens, B.A., 2012. Housing and dustbathing effects on northern fowl mites (Ornithonyssus sylviarum) and chicken body lice (Menacanthus stramineus) on hens. Med. Vet. Entomol. 26, 323–333. Moyer, B.R., Rock, A.N., Clayton, D.H., 2003. Experimental test of the importance of preen oil in rock doves (Columba livia). Auk 120, 490–496. Mullens, B.A., Owen, J.P., Kuney, D.R., Szijj, C.E., Klingler, K.A., 2009. Temporal changes in distribution, prevalence and intensity of northern fowl mite (Ornithonyssus sylviarum) parasitism in commercial caged laying hens, with a comprehensive economic analysis of parasite impact. Vet. Parasitol. 160, 116–133. Mullens, B.A., Chen, B.L., Owen, J.P., 2010. Beak condition and cage density determine abundance and spatial distribution of northern fowl mites, Ornithonyssus sylviarum, and chicken body lice, Menacanthus stramineus, on caged laying hens. Poult. Sci. 89, 2565–2572. Olsson, I.A.S., Keeling, L.J., 2005. Why in earth? Dustbathing behaviour in jungle and domestic fowl reviewed from a Tinbergian and animal welfare perspective. Appl. Anim. Behav. Sci. 93, 259–282. Owen, J.P., Delany, M.E., Mullens, B.A., 2008. MHC haplotype involvement in avian resistance to an ectoparasite. Immunogenetics 60, 621–631. Owen, J.P., Delany, M.E., Cardona, C.J., Bickford, A.A., Mullens, B.A., 2009. Host inflammatory response governs fitness in an avian ectoparasite, the northern fowl mite (Ornithonyssus sylviarum). Int. J. Parasitol. 39, 789–799. Rothschild, M., Clay, T., 1952. Fleas, Flukes and Cuckoos. A Study of Bird Parasites. Collins, London. Statistical Analysis Software® (SAS) Institute, 2010. Guide for Personal Computers, Version 9.3. SAS Institute, Cary, NC, USA. Scholz, B., Urselmans, S., Kjaer, J.B., Schrader, L., 2010. Food, wood, or plastic as substrates for dustbathing and foraging in laying hens: a preference test. Poult. Sci. 89, 1584–1589. Scholz, B., Kjaer, J.B., Petow, S., Schrader, L., 2014. Dustbathing in food particles does not remove feather lipids. Poult. Sci. 93, 1877–1882. Shields, S., Garner, J.P., Mench, J.A., 2004. Dustbathing by broiler chickens: a comparison of preference for four different substrates. Appl. Anim. Behav. Sci. 86, 291–298. van Liere, D.W., 1991. Function and organization of dustbathing in laying hens. In: PhD Dissertation. Wageningen, Netherlands. van Liere, D.W., Bokma, S., 1987. Short-term feather maintenance as a function of dust-bathing in laying hens. Appl. Anim. Behav. Sci. 18, 197–204. van Liere, D.W., Kooijman, J., Wiepkema, P.R., 1990. Dustbathing behaviour of laying hens as related to quality of dustbathing material. Appl. Anim. Behav. Sci. 26, 127–141. Vestergaard, K., Hogan, J.A., 1992. The development of a behavior system: dustbathing in the Burmese red junglefowl. III. Effects of experience on stimulus preference. Behaviour 121, 215–230. Vezzoli, G., 2014. Evaluation of the effects of lice and mite infestation on the grooming behavior, health and egg quality of laying hens. In: PhD Dissertation. University of California, Davis, Davis CA, USA. Vezzoli, G., King, A.J., Mench, J.A., 2015a. The effect of northern fowl mite (Ornithonyssus sylviarum) infestation on hen physiology, physical condition and egg quality. Poult. Sci. (submitted for publication). Vezzoli, G., Mullens, B.A., Mench, J.A., 2015b. Relationships between beak condition, preening behavior and ectoparasite infestation levels in laying hens. Poult. Sci., http://dx.doi.org/10.3382/ps/pev171.
Contents lists available at ScienceDirect
Applied Animal Behaviour Science journal homepage: www.elsevier.com/locate/applanim
Dustbathing behavior: Do ectoparasites matter? Giuseppe Vezzoli a,b,1 , Bradley A. Mullens c , Joy A. Mench a,∗ a
Department of Animal Science and Center for Animal Welfare, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA Animal Biology Graduate Group, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA c Department of Entomology, University of California, Riverside, 900 University Avenue, Riverside, CA 92521, USA b
a r t i c l e
i n f o
Article history: Received 20 February 2015 Received in revised form 21 May 2015 Accepted 6 June 2015 Available online 19 June 2015 Keywords: Dustbathing Laying hen Northern fowl mite Astroturf Sand
a b s t r a c t A presumed function of dustbathing behavior is to remove ectoparasites. Providing dustbathing substrates in furnished cages for laying hens might therefore offer an alternative to pesticide use to reduce ectoparasite populations. We investigated the effectiveness of dustbathing substrates for controlling northern fowl mites in individually caged beak-trimmed White Leghorn hens (N = 32). Each cage contained a 32 cm × 32 cm plastic tray that was either: (1) filled with 1200 g of sand (SAND); (2) empty (CONTROL); (3) covered with Astroturf (AT); or (4) covered with AT on to which 150 g of feed was delivered daily (ATF). AT and ATF are used in the dustbathing/foraging area of many newer commercial furnished cages. Hens were infested with approximately 35 mites at 25 weeks of age. Mite numbers were estimated weekly. Time spent dustbathing and dustbathing bout numbers and lengths in the tray and on the wire cage floor were determined from video recordings made for 2 consecutive days from 12:00 to 20:00 h immediately before and after infestation and at weeks 1, 3, 5, and 7 post-infestation. Data were analyzed using a repeated-measures ANOVA in SAS. Treatment did not influence the total time spent dustbathing (average across substrates: 11.3 min). However, there were treatment effects on the time spent dustbathing in the trays (F2,21 = 3.67, P = 0.043) and on wire (F2,21 = 7.68, P = 0.031). SAND and ATF hens spent more time dustbathing in the trays (11.4 and 9.1 min, respectively) than AT (2.3 min), and CONTROL and AT hens spent more time dustbathing on wire (11.6 and 4.7 min, respectively) than ATF (0.4 min). There was a treatment effect on infestation (F3,28 = 3.08, P = 0.04), with ATF having more mites (back-transformed mean = 1500) than AT (330), and with SAND (460) and CONTROL (447) intermediate. This study confirmed that the substrate type affected dustbathing behavior. SAND was a preferred dustbathing substrate but was not effective for controlling mite numbers, nor was the time spent dustbathing in any substrate or in total influenced by infestation levels. Our data also suggest that adding feed to the AT pad in furnished cages might lead to increased mite numbers in infested hens. The reason for this effect is unclear, but could be due to feed particles contributing to a change in feather structure that creates a more favorable mite habitat. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Egg producers in a number of countries are adopting furnished cages (larger versions of which are also called enriched colonies) as an alternative to conventional cages. These cages include a nest, perches and a substrate to encourage foraging, scratching
∗ Corresponding author at: Department of Animal Science, University of California Davis, One Shields Avenue, Davis, CA 95616, USA. Tel.: +1 530 752 7125; fax: +1 530 752 0175. E-mail addresses: [email protected] (G. Vezzoli), [email protected] (B.A. Mullens), [email protected] (J.A. Mench). 1 Current address: Department of Math and Science, Kirkwood Community College, 6301 Kirkwood Blvd. SW, Cedar Rapids, Iowa, IA 52404, USA. http://dx.doi.org/10.1016/j.applanim.2015.06.001 0168-1591/© 2015 Elsevier B.V. All rights reserved.
and dustbathing behavior. The most common material used for this substrate is an Astroturf (AT) pad onto which loose material like feed may be distributed (Scholz et al., 2010; Alvino et al., 2013). Finer substrates like sand can be also dispensed onto this pad but create problems if they occlude the overhead dispenser or abrade the mechanical delivery system (Scholz et al., 2010). However, materials with a fine structure like sand are preferred by hens for dustbathing over substrates consisting of large particles (Olsson and Keeling, 2005) and are also preferred to substrates like AT or AT and feed (Alvino et al., 2013). One function of dustbathing is to maintain the integument in good condition by reducing excess feather lipids (Olsson and Keeling, 2005). It has long been suggested that another function of dustbathing is to help remove ectoparasites (Rothschild and
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Clay, 1952; de Jong et al., 2007; Clayton et al., 2010), however there is only one study evaluating this presumed function. Martin and Mullens (2012) provided hens housed on litter with dustboxes filled with sand and sulphur, sand and kaolin (clay) or sand and diatomaceous earth. Sulphur and inert dusts like kaolin and diatomaceous earth have been demonstrated to increase ectoparasite mortality (Creighton et al., 1943; Furman, 1952; Kilpinen and Steenberg, 2009). Martin and Mullens (2012) found that the hens that used these dustboxes had significantly fewer chicken body lice (Menacanthus stramineus) and northern fowl mites (Ornithonyssus sylviarum) than hens that did not use them, and also fewer ectoparasites than caged hens without access to dustboxes. Two older studies (Hoffman and Hogan, 1967; Hoffman and Gingrich, 1968) evaluated the effect of a chemical (Zytron) and a microbial (Bacillus thuringengis) insecticide mixed with sand in dustboxes on three species of lice in infested hens in a non-cage system. They reported that there was a reduction in the number of lice when the insecticides were included in the boxes but not when sand was the only substrate. However, they did not actually record dustbathing behavior. To our knowledge there are no published studies evaluating the relationship between ectoparasites and the performance of dustbathing behavior. Moreover, there is no information on whether hens that dustbathe in substrates that have not been treated with inert dusts or insecticides have reduced ectoparasite populations. If dustbathing does function to remove ectoparasites, providing the types of dustbathing substrates that are typically used in furnished cages might offer an alternative to pesticide use. Therefore, there is an opportunity to evaluate the effectiveness of dustbathing behavior for controlling ectoparasite populations using a preferred dustbathing substrate like sand (Shields et al., 2004; van Liere et al., 1990; Olsson and Keeling, 2005), or even a non-preferred substrate like Astroturf (AT). Sand might abrade the ectoparasite cuticle, leading to desiccation, while the AT could mechanically dislodge parasites, an effect that could be potentiated when feed is added to make the AT more desirable for dustbathing. It is also possible that dustbathing in feed could directly affect ectoparasite survival. Scholz et al. (2014) found that the feathers of hens that dustbathed in feed compared to lignocellulose had a higher lipid content. Moyer et al. (2003) found that preen oil from the uropygial gland negatively affected the survival of rock dove lice in vitro, although experimental removal of the uropygial gland did not impact louse populations in vivo. There have been no studies of the effects of feather lipids on mite infestation. Finally, it is not known whether the presence of ectoparasites affects dustbathing behavior and whether infested hens increase the time they spend dustbathing in non-preferred substrates like feed or AT in an attempt to reduce their infestation. The northern fowl mite (NFM) is the most common and serious poultry ectoparasite in North America (Axtell and Arends, 1990). It spends its entire life cycle on the host, feeding on blood and laying its eggs at the base of the feathers, particularly in the vent area. The aims of our experiment were to evaluate: (1) the role of dustbathing behavior in controlling NFM populations; (2) the effectiveness of commercially used dustbathing substrates in reducing NFM levels on infested hens; (3) the effect of NFM on dustbathing behavior. We hypothesized that dustbathing behavior plays a role in controlling ectoparasite populations and that different dustbathing substrates would have different effects on NFM populations. We predicted that NFM populations would be lower in hens that dustbathed, and that the provision of a highly preferred substrate would reduce NFM populations more than the provision of non-preferred substrates. We also hypothesized that infestation with NFM would increase the total time spent dustbathing by increasing both the length and the number of dustbathing bouts.
2. Materials and methods 2.1. Animals and Husbandry This experiment was conducted at the Avian Science Facility at the University of California, Davis. Thirty-two hens were randomly selected from a flock of 168 CV20/W36 beak-trimmed pullets that had been previously used in a study of early rearing effects on dustbathing substrate preference (Alvino, 2013). The day-old chicks had been obtained from a commercial hatchery (JS West and Companies, Modesto CA) and kept in a Petersime brooder, where they were raised either completely on wire or exposed to either AT plus feed (ATF) or sand as dustbathing substrates. At 11 weeks of age, the pullets were moved in pairs to grower cages, and the previous treatments were maintained until they were 18 weeks of age. The pullets were then singly housed in 90 cm × 46 cm × 46 cm cages in two experimental rooms for the current study. From 14 to 18 weeks of age the light was increased 1 h per week to reach a 16L:8D photoperiod, which was maintained for the duration of the experiment. Each experimental room contained 4 racks of cages and each rack held 4 cages. Each cage had a 32 cm × 32 cm plastic tray (Akro Mils SRO12500A34) that was either: (1) empty (CONTROL); (2) filled with 1200 g of sand (SAND) (Sakrete Natural Play Sand, Dixon, California); (3) lined with Astroturf (AT) (GrassWorx XPSP, 14 mm pile height); or (4) lined with AT on to which 150 g of feed was delivered daily (ATF). This amount of feed covered the entire surface of the pad. Pullets were assigned to the treatments most similar to their rearing substrate exposure (i.e., chicks reared with ATF were assigned either to AT or ATF, wire to CONTROL, and sand to SAND); the four treatments were balanced in the two rooms. All the trays and the AT pads were removed from the cages and washed and dried daily between 9:30 and 11:30 h. The trays were then lined with the clean pads or fresh sand and reintroduced into the cages. Feed was dispensed on to the ATF pads immediately after they were cleaned and dried. Although there was abundant sand and feed in the trays and on the pads at the end of a 24 h period, they were cleaned daily to remove feces and ensure that feed and sand availability and cleanliness were comparable each day. Cleaning was carried out in the morning so as not to disturb the hens during the video recording sessions. There was a 45 cm long feeder on the front of each cage which was filled every morning with a pelleted diet formulated for highproducing laying hens (Purina Layena, Turlock California 16% CP, high calcium ration, 2.5% fat). Running water was provided ad libitum via a trough placed along the back of the cages. The pullets/hens were housed and managed according to the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (FASS, 2010). All experimental procedures were approved by the University of California, Davis, Institutional Animal Care and Use Committee. 2.2. Ectoparasite infestation Because we were interested in determining how providing dustbathing opportunities affected the course of infestation, it was necessary to experimentally infest the hens. Approximately 35 northern fowl mites were placed on the abdominal feathers of each hen when the hens were 25 weeks of age. The mites came from naturally infested source hens housed in a building separate from the experimental room at the UC Davis Avian Science Facility. Miteinfested feathers were cut from the source hens and put in a plastic bag. A glass Pasteur pipette was then used to aspirate (Owen et al., 2008) and transfer the mites from the plastic bag to the vent feathers of each experimental hen. Prior to infestation (week −1) each hen was removed from her cage and the feathers and skin of her vent area were checked to verify that they did not harbor any mites;
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after infestation (week 0) each hen was removed weekly from her cage and the vent feathers and vent skin were visually scored for the number of mites. One person (GV) did the scoring. 2.3. Behavioral observations Cameras (Veilux SVB-70IR48, 3.6 mm Board Lens, 1/3 in. Sony CCD) mounted centrally in front of each cage were used to film the hens in real-time. Each cage was video recorded for two consecutive days from 12:00 to 20:00 h immediately before (week −1) and after (week 0) infestation, and at weeks 1, 3, 5 and 7 post-infestation. The videos were analyzed using the V 8.5 GeoVision software program (GeoVision V-Series Surveillance System, Vision Systems Inc., Irvine CA). The data collected over the two-day period were summed for analysis. Time spent dustbathing, dustbathing bout numbers and bout lengths in two locations, in the trays and on the wire floor, were recorded and analyzed throughout the entire video recorded period. The total time spent dustbathing was calculated by adding the time spent dustbathing in the trays and on the wire floor, and the total dustbathing bouts were calculated by adding the bouts performed in the trays to the bouts performed on the wire floor. The first vertical wing shake was used as the measure of the beginning of a dustbathing bout. A bout was considered to end when the hen showed vigorous body-shaking or when more than 2 min elapsed between dustbathing episodes. One observer (GV) coded the videos.
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To determine the effects of treatment and week on the behavioral data we used a repeated measures analysis with the number of mites as a covariate. Homogeneity among slopes was tested by including a two and a three-way interaction between the covariate and the fixed effects. When the interactions were non-significant we concluded that the slopes were homogenous and that we could remove the interaction terms from the model (Engqvist, 2005). The dependent variables were: time spent dustbathing, dustbathing bouts and bout lengths both in the trays and on the wire, total time spent dustbathing, total dustbathing bouts and total bout lengths. Since CONTROL hens were not observed dustbathing in the trays they were not included in the analysis of dustbathing in this part of the cage. Similarly, SAND hens were not observed to dustbathe on wire and were therefore not included in that analysis. Square root transformations were needed to meet the assumptions of ANOVA for all of the behavioral data; therefore the back-transformed and the transformed means and standard errors (SE) are reported. To analyze mite populations, we used a repeated measures analysis with a similar model as the one used for the behavioral data, with week and treatment as fixed effects and hen as the random effect. The dependent variable for this analysis was the number of mites. Week 0 was not included in the analysis because it was the week of infestation and the mite scores were therefore the same for each hen. Log transformations were needed to meet the assumption of ANOVA. Back-transformed and transformed means and standard errors (SE) are reported. One-way GLM (general linear model) ANOVA was used to analyze feather lipid levels, with treatment as the independent variable.
2.4. Feather lipids We analyzed feather lipids immediately prior to infestation to determine whether the rearing treatments had affected these levels, which could in turn differentially affect the infestation profile by changing the suitability of the habitat for the mites. For feather lipid analysis, approximately 1.2 g of breast feathers were collected from each hen the day before infestation. The feathers were cut at the base of the rachis and placed in plastic bags, and were then frozen at −20 ◦ C. For analysis, 1 g of feathers per hen was dried in a convection oven at 100 ◦ C for 2 h. Feather surface lipids were then extracted using a Soxtec system (Tecator Soxtec System HT 1043 Extraction Unit, Foss Tecator, Denmark). Aluminum extraction cups were weighed and the pre-extraction weights recorded. Lipids were extracted using anhydrous ethyl ether (Fisher Scientific, Fair Lawn, NJ). After 50 min of boiling, the samples were rinsed for 50 min, with the lipids from the samples retained in the extraction cups. After the solvent was allowed to evaporate for 10 min, the extraction cups were placed in a 100 ◦ C drying oven for 30 min, then in a desiccator for an additional 30 min. The difference in weight between the cups containing lipids and their original weight was the weight of the extracted lipids (expressed as mg of lipid per g of feathers). 2.5. Statistical analysis The Statistical Analysis System (SAS, 9.3) was used to analyze the data. The Shapiro-Wilk test was used to assess normality and graphical analysis of the residuals was used to assess homogeneity of variance. The level of statistical significance was set at P < 0.05, with 0.05 ≤ P < 0.10 values considered as showing a trend towards significance. The Tukey post hoc test was used when significant differences were found. Each hen served as her own control for all analyses. For the initial models, hen (cage) was the experimental unit, treatment and week were the fixed effects, room and hen were the random effects, and week was the repeated measure. Since the analyses revealed no differences between the two experimental rooms, room was removed from the subsequent models.
3. Results 3.1. Number of mites As expected there was a week effect (F6,168 = 69.50, P < 0.0001) on the number of mites (Fig. 1). Tukey comparisons revealed that mites increased until weeks 4–6, then started declining. There was also a main effect of treatment (F3,28 = 3.08, P = 0.04; Fig. 2), with ATF having more mites than AT (P = 0.03) and with SAND and CONTROL intermediate. There were no treatment × week interactions. 3.2. Dustbathing behavior Neither the main effects nor the interactions co-varied with the number of mites, indicating that dustbathing behavior was not influenced by infestation levels. Further results are therefore reported without the covariate. 3.2.1. Treatment effects Table 1 shows the treatment effects on the time spent dustbathing, bout numbers and bout lengths in both the trays and on wire. Treatment did not affect total dustbathing, since there were no effects on the total (tray and wire combined) time spent dustbathing (pooled back-transformed means 11.36 min) or the total bout numbers (pooled back-transformed mean = 1.75 bout). However, there were treatment differences related to the location of dustbathing within the cage. For dustbathing in the trays, treatment affected both the time spent dustbathing (F2,21 = 3.67, P = 0.043) and bout lengths (F2,21 = 8.51, P = 0.002). AT spent less time dustbathing and also had shorter bouts in the trays than SAND and ATF. There was also a treatment effect on the time spent dustbathing (F2,21 = 7.68, P = 0.003) and on bout numbers (F2,21 = 5.69, P = 0.01) and bout lengths (F2,21 = 9.69, P = 0.001) on wire. CONTROL and AT spent more time dustbathing and performed more dustbathing bouts on wire than ATF. CONTROL also performed longer
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A
4.0
d
Number of Mites
3.5
d
c
d
Table 1 Treatment effects on time spent dustbathing (min) and on dustbathing bout numbers and lengths in both locations, on wire and in trays. Back-transformed means are reported, with square root transformed means and standard errors in parentheses. Different letters indicate significant differences (P < 0.05) among treatments for each measure. CONTROL hens did not dustbathe in the trays and SAND did not dustbathe on wire (N/A). ATF (Astroturf plus feed) and AT (Astroturf).
c
3.0 b
2.5 a
2.0 1.5
Measure
Location Tray
Wire
Time spent dustbathing (min)
ATF: 9.06 (3.01 ± 0.51)a AT: 2.34 (1.53 ± 0.51)b SAND: 11.36 (3.37 ± 0.51)a CONTROL: N/A
ATF: 0.38 (0.62 ± 0.50)a AT: 4.71 (2.17 ± 0.50)b SAND: N/A CONTROL: 11.63 (3.41 ± 0.50)b
Dustbathing bouts
ATF: 0.85 (0.92 ± 0.16) AT: 0.36 (0.60 ± 0.15) SAND: 0.69 (0.83 ± 0.15) CONTROL: N/A
ATF: 0.10 (0.32 ± 0.29)a AT: 1.35 (1.16 ± 0.29)b SAND: N/A CONTROL: 2.76 (1.66 ± 0.29)b
Dustbathing bout length (min/bout)
ATF: 6.05 (2.46 ± 0.37)a AT: 1.04 (1.02 ± 0.37)b SAND: 9.67 (3.11 ± 0.37)a CONTROL: N/A
ATF: 0.19 (0.44 ± 0.21)a AT: 1.19 (1.09 ± 0.21)b SAND: N/A CONTROL: 3.03 (1.74 ± 0.21)c
1.0 0.5 0.0 0
1
2
3
4
5
6
7
Week
B
Number of Mites
2500 2000 1500 1000 500 0 0
1
2
3
4
5
6
7
Week Fig. 1. Main effect of week on number of mites. Letters indicate significant differences (P < 0.05) among weeks. Transformed means and SE are reported in (A) and back-transformed means are reported in (B).
Fig. 3. Week effect on the total (in the trays and on wire) time spent dustbathing. Letters indicate significant differences (P < 0.05) among weeks. Transformed means and SE are reported in (A) and back-transformed means are reported in (B).
bouts on wire than both ATF and AT. Similarly, AT performed longer bouts on wire than ATF.
Fig. 2. Main effect of treatment on number of mites. Letters indicate significant differences (P < 0.05) between treatments. Transformed means and SE are reported in (A) and back-transformed means are reported in (B). AT (Astroturf), ATF (Astroturf plus feed).
3.2.2. Week and interaction effects 3.2.2.1. Total dustbathing (wire plus tray). There was a week effect on the total time spent dustbathing (F5,140 = 4.35, P = 0.001) and total bout numbers (F5,140 = 2.64, P = 0.03). At week 7 hens spent more time dustbathing (Fig. 3) than they did prior to infestation (week −1) and at weeks 1, 3, and 5. There was a trend for a treatment × week interaction for this measure (F15,140 = 1.64, P = 0.07), with CONTROL tending to spend more time dustbathing at week 7 [back-transformed means in min, with transformed means ± SE in parenthesis: 33.76 (5.81 ± 0.85)] than they did prior to infestation
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3.3. Feather lipids There was no rearing substrate effect on the lipid content of the feathers (pooled means = 7.57 mg/g of feathers) collected immediately prior to infestation.
4. Discussion
Fig. 4. Week effect on the total dustbathing bout numbers. Letters indicate significant differences (P < 0.05) among weeks. Transformed means and SE are reported in (A) and back-transformed means are reported in (B).
[1.00 (1.00 ± 0.85)] and at weeks 1 [6.76 (2.60 ± 0.85)] and 3 [6.86 (2.62 ± 0.85)]. For dustbathing bout numbers (Fig. 4), hens at week 7 performed more bouts than they did prior to infestation and at weeks 1, 3 and 5.
3.2.2.2. Dustbathing in the trays. There were no week effects on the time spent dustbathing (pooled back-transformed means 6.7 min) or on the bout numbers (pooled back-transformed means 0.6) or bout lengths (pooled back-transformed means 4.9 min/bout) in the trays. There were also no treatment × week interactions.
3.2.2.3. Dustbathing on wire. There was a week effect on the time spent dustbathing (F5,105 = 3.51, P = 0.005), although this interacted with treatment. During week 7, hens spent more time dustbathing on wire [back-transformed means in min, with transformed means ± SE in parenthesis: 9.49 (3.08 ± 0.41)] than they did prior to infestation [2.40 (1.55 ± 0.41)] or at weeks 0 [2.10 (1.45 ± 0.43)], 1 [3.50 (1.87 ± 0.41)], 3 [2.34 (1.53 ± 0.41)] and 5 [4.45 (2.11 ± 0.41)]. There were treatment × week interactions for both time spent dustbathing (F10,105 = 4.11, P < 0.0001) and bout lengths (F10,105 = 3.01, P = 0.002) as well as a trend for bout numbers (F10,105 = 1.79, P = 0.07). Table 2 shows the treatment × week interactions for CONTROL hens. At week 7, CONTROL spent more time dustbathing and performed more dustbathing bouts on wire than they did during the preceding weeks. At week 7 CONTROL had longer bouts than before infestation and than at weeks 1 and 3. Posthoc test also showed that at week 7 AT hens tended to spend more time dustbathing [back-transformed means in min, with transformed means ± SE in parenthesis: 9.99 (3.16 ± 0.71)] than they did at week 5 [(2.56 (1.60 ± 0.71) P = 0.055] and 3 [(2.89 (1.70 ± 0.71) P = 0.06].
We showed for the first time that the presence of mites did not influence dustbathing behavior in laying hens, although the skin irritation caused by mite infestation may have stimulated dustbathing behavior in the CONTROL hens, as we discuss below. While substrate type did affect dustbathing behavior, NFM populations were not decreased by providing sand and/or Astroturf for dustbathing. As has been shown in previous studies (Alvino et al., 2013; Shields et al., 2004; van Liere et al., 1990), substrate type affected dustbathing behavior. SAND was an attractive dustbathing substrate, and SAND hens dustbathed exclusively in the trays. In addition, both SAND and ATF hens spent more time dustbathing in the trays and had longer bouts in the trays than AT, and spent less time dustbathing and performed fewer and shorter bouts on wire than CONTROL and AT. Our results show that hens used ATF, which could therefore be a good alternative to sand in terms of stimulating dustbathing behavior. This contrasts with the findings of Alvino et al. (2013) that hens given sand spent more time dustbathing and had longer bouts than hens given ATF. The hens in our study were raised with access to substrates similar to the treatments provided during the experiment, whereas the hens in Alvino et al. (2013) were raised in wire floor cages. Our findings therefore suggest that the provision of feed as dustbathing substrate during rearing might be crucial in motivating dustbathing behavior by adult hens on a non-preferred substrate like ATF. Our data support the results of Vestergaard and Hogan (1992), who found that the type of dustbathing substrate provided to chickens during rearing affects the choice of dustbathing substrate later in life. Although there was a decline in mite populations in all treatments at week 7 post-infestation, CONTROL hens at week 7 spent more time dustbathing, and performed more dustbathing bouts, than they did prior to infestation and at weeks 1, 3 and 5, with all of this dustbathing activity occurring on the wire floor. AT hens also tended to increase the time they spent dustbathing on wire at week 7 compared to weeks 3 and 5. This suggests that skin condition after infestation could play a role in eliciting dustbathing behavior. Owen et al. (2009) observed an immune/inflammatory response in the skin of hens infested with NFM with a consequent formation of scabs that became cracked and flaky during recovery. We found (Vezzoli, 2014; Vezzoli et al., 2015a) that the hens in the current study had scabs at weeks 5 and 7 post-infestation. Sheep infested with the mange mite Psoroptes ovis spend more time rubbing when they have more, larger and older scabs (Berriatua et al., 2001). Berriatua et al. (2001) suggested that histamine, which is a potent pruritogen (Greaves and Wall, 1996), was the cause. To our knowledge no one has evaluated whether the presence, age, or condition of scabs, or the histamine response to tissue injury, are factors that influence dustbathing behavior in birds. Berriatua et al. (2001) suggested that a “strong mechanical stimulus” like rubbing results in central nervous inhibition of the sensation of itchiness, although the mechanism for this is unknown (Davidson et al., 2009). Perhaps substrates like sand or feed reduce itchiness due to their mechanical contact with the hen’s skin. In our experiment, CONTROL hens did not have access to a substrate. AT hens did have access to substrate, but preferentially dustbathed on the wire instead, which like CONTROL may not have been ideal to reduce itchiness. This, coupled with the presence of cracked and flaky
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Table 2 Treatment × week interaction effects for CONTROL hens on dustbathing performances on wire. Back-transformed means are reported, with square root transformed means and standard errors in parentheses. Different letters indicate significant differences (P < 0.05) among weeks for each measure. Measure
Week −1
Time spent dustbathing Dustbathing bouts Dustbathing bout length (min/bout)
0
1.00 (1.00 ± 0.71) 0.77 (0.88 ± 0.35)a 0.46 (0.68 ± 0.38)a a
1
17.47 (4.18 ± 0.71) 3.28 (1.81 ± 0.35)b 4.49 (2.12 ± 0.38)b b
scabs, might explain the increase in dustbathing behavior observed in these treatments at week 7. Our study showed that ATF had a higher level of mites than AT. While there were no significant differences between ATF, CONTROL and SAND, ATF harbored three times more mites than either of these treatments. Dustbathing on ATF could have affected mite populations because the lipids in feed can be deposited on the feathers during dustbathing (Scholz et al., 2014). As discussed above, preen oil has been shown to reduce louse survival in vitro (Moyer et al., 2003). However, lipid deposition could also potentially make the feathers more suitable as a mite habitat by affecting the arrangement of barbs and barbules on the feathers, thereby changing microhabitat humidity, or by altering feather temperature (van Liere, 1991). However, we did not find that ATF hens had higher feather lipid levels prior to infestation than hens in the other treatments, possibly because the feed we used had a lower lipid (2.5%) content than that (3.5%) used by Scholz et al. (2014). We could not analyze the feathers post-infestation because the mites themselves have lipids which would have confounded the lipid level analysis. Further analysis of feather structure using a scanning electron microscope might elucidate whether and how different dustbathing substrates affect the feather physical structure and the habitat in on which the mites live. It is surprising that hens given sand, which is a preferred and effective dustbathing substrate (van Liere and Bokma, 1987; van Liere et al., 1990; Shields et al., 2004), had a similar number of mites as CONTROL, AT and ATF. The mite populations and the course of infestation in our study were comparable to those seen in naturally infested caged laying hens (Mullens et al., 2009). It has been suggested for decades that one function of dustbathing is removal of ectoparasites (Rothschild and Clay, 1952; de Jong et al., 2007; Clayton et al., 2010). Our study showed for the first time that dustbathing in sand or on the Astroturf pads that are commonly provided in enriched colony cages are not effective for controlling NFM. Hoffman and Hogan (1967) demonstrated that sand did not affect lice populations when it was available for dustbathing, although they did not observe whether the hens actually used the dustbox. Martin and Mullens (2012) showed that hens that used dustboxes filled with sand and inorganic inert dusts like kaolin or diatomaceous earth, or with sand and sulphur, had reduced northern fowl mite and chicken body louse populations compared to hens that did not use the dustboxes. Kilpinen and Steenberg (2009) and Martin and Mullens (2012) suggested that, due to their fine size and oil absorption capacity, inorganic inert dusts like kaolin clay and diatomaceous earth might abrade the cuticle, or more likely absorb the lipid on the cuticle leading to desiccation of the ectoparasites. Perhaps either the size or nature of sand grains causes sand to be ineffective for in abrading or absorbing oil from the mite cuticle. In addition, it is possible that dustbathing in natural soil might affect ectoparasites more than dustbathing in the substrates that we used for the present experiment. The soil is a “complex living system” with physical, chemical and biological components (Karlen et al., 1997). Kaolin is an abundant constituent of the soil, and Martin and Mullens (2012) suggested that birds might be able to suppress ectoparasite populations by dustbathing in kaolin-like soils. Perhaps the soil also contains components like essential oils,
3
6.76 (2.60 ± 0.71) 2.25 (1.50 ± 0.35)b 1.42 (1.19 ± 0.38)a c
5
6.86 (2.62 ± 0.71) 2.96 (1.72 ± 0.35)b 1.72 (1.31 ± 0.38)a c
7
17.98 (4.24 ± 0.71) 2.82 (1.68 ± 0.35)b 5.66 (2.38 ± 0.38)b b
33.76 (5.81 ± 0.71)d 5.71 (2.39 ± 0.35)c 7.73 (2.78 ± 0.38)b
which are known to increase the mortality of some poultry ectoparasites like the red mite (Dermanyssus gallinae) (George et al., 2009, 2010; Kim et al., 2004). An alternative potential effect of dustbathing that might help to reduce ectoparasites is dislodging (Clayton et al., 2010). However, in our study hens were housed in cages and therefore mites that were dislodged by a hen could have simply re-infested her. An evaluation of dislodging of mites or other chicken ectoparasites might help to understand if this mechanism really occurs during dustbathing. Although dustbathing behavior was not affected by NFM, it is possible that a different parasite, for example the chicken body louse, which is the second most important poultry ectoparasite in North America (Axtell and Arends, 1990), would have a different effect on dustbathing. If infestation with different kinds of parasites does not change dustbathing behavior, it would strengthen the hypothesis that dustbathing is not functionally related to the removal of ectoparasites. In addition, it is known that hens with an intact beak are able to reduce their ectoparasite loads (Mullens et al., 2010) via preening directed to the body areas that are most severely infested (Vezzoli et al., 2015b). However, to our knowledge there have been no studies evaluating the interactive effects of preening and dustbathing in beak-intact hens on ectoparasite loads. This comparison might further elucidate the evolutionary functions of these behaviors with respect to ectoparasite control. 5. Conclusions Although we did not find increases in dustbathing in NFMinfested hens, future work should investigate how diverse parasites affect dustbathing behavior. The idea that scabs or histamine might also affect dustbathing behavior should be explored. Further research is also necessary to understand if feed used as a dustbathing substrate creates a more favorable habitat for the mites and to better understand which substrate(s) can be most effective against NFM and more suitable for use in furnished cages. Acknowledgements We gratefully acknowledge the infrastructure support of the Department of Animal Science, the College of Agricultural and Environmental Sciences, and the California Agricultural Experiment Station of the University of California, Davis. We also thank GrassWorx, LLC for donating the AT pads. Giuseppe Vezzoli was partly supported by fellowships from the Pacific Egg and Poultry Association (PePa), the Olivera Memorial Fund, and Jastro-Shields. We also thank all of the following individuals at University of California, Davis, for their contributions to the project: Gina Alvino for caring for the chicks/pullets, Emily Bloom for caring for the hens, Margaret de Luz for feather lipid analysis, Gregory Archer and Richard Blatchford for helping with video equipment installation, the undergraduates who helped with the different phases of this project, the staff of Hopkins Avian Research Facility for their help with animal care, and Cassandra Tucker for her helpful comments on an earlier version of this manuscript.
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