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    Rearing experience with ramps improves specific learning and behaviour and welfare on a commercial laying farm

    Experimental designOver 3 years, six paired organic British Blacktail flocks with intact beaks (i.e. not beak-trimmed) were visited between 1 and 40 weeks of age. Within each pair, one flock was ramp reared (RR) and one flock was control reared without ramps (CR). All flocks were kept on one farm which possessed two rearing houses and six laying sheds of approximately 2000 birds per flock. The site was multi-age, meaning that of the six laying sheds there were three different ages on the site at one time.The availability of this commercial facility enabled us to design an experiment whereby we allocated two rearing treatments, one with ramps provided to access elevated structures and a control with elevated structures but no ramps and to alternate these treatments between the two rearing houses available to avoid treatment x house confounds. Each rearing flock was moved independently to a laying house with no mixing, so we were able to continue data collection and examine any long-term effects of the rearing treatment during the laying period. Rearing flocks were systematically allocated so that each laying house received one RR flock and one CR flock during the experiment.Observations were made in the mornings at three time points during the rearing period at 1, 3 and 15–16 weeks, and three in the laying period at 16–17, 24 and 40 weeks of age. See Table 5 for a summary of experimental design, flock and housing information.Table 5 Experimental design for each ramp reared and control reared flock for the 6 replicates. There were two rearing sheds used, Rear1 (R1) and Rear2 (R2), with 6 different laying sheds named A1, A2, B1, B2, C1 and C2.Full size tableThe rearing sheds were static with 142.7 m2 of floor space covered with wood shavings. Rearing sheds were both set up with feed tracks giving mini pellet feed up to 11 weeks of age then pellet grower feed and 7 nipple drinker lines. The lighting schedule was 23 h light in the first day reducing gradually over the rearing period to 10 h light at 7 weeks of age. A minimum light intensity of 10 lx is required, but with windows and pop-holes light intensity was higher in the houses. The temperature was maintained at 30 °C during the first few days then slowly reduced to match the temperature in the laying sheds. Shed heating was provided by gas spot lamps, whole shed heating through hot pipes running along the length of the shed and hot air fans run by a biomass boiler. All flocks had access to the outside range by 10 weeks of age through two pop holes (each L: 2 m by H: 0.4 m). Flocks were moved the short distance from the rearing to the laying house at between 15 to 16 weeks of age in one night using transport modules.All rearing flocks had access to six elevated structures (ES) (see Fig. 3) from four days of age when the chicks were released from the brooding circles. Each ES comprised nine metal perches (length 302 cm, width 3.5 cm), with three perches (25 cm apart) at three different heights (43 cm, 73 cm and 103 cm). Two plastic grids (width 60 cm, length 115 cm) were fixed within the ES to provide platforms at different heights (Fig. 3). In each replicate, the RR flock had one ramp attached to each ES. Three of the ES were fitted with plastic grid ramps (width 60 cm, length 74 cm, angle 35.5°) leading up to the low perch and three ES had ramps (width 60 cm, length 115 cm, angle 40°) leading up to the middle perch. The CR flock had six ES without ramps.Figure 3Elevated structure dimensions used in the ramp reared sheds, (a) shows the high ramp (b) shows the low ramp. The control sheds elevated structures were identical to these but without ramps.Full size imageThe six single-tier laying houses on-site were mobile organic units with approximately 345m2 of floor space. See Fig. 4 for a schematic plan of their layout. All had a raised area comprising plastic slats over supports (approx. 70 cm from the litter) and a ground-level litter area covered with wood chip. Four of the sheds (Fig. 4a) were set up with the slatted area spanning the whole width of the shed and halfway down the length. In two of the sheds (Fig. 4b) the litter area was either side of the elevated slatted area. Nest boxes ran down the centre of the slatted area, dividing this into two sections. Intermittent ramps were installed at the level change, resulting in 4 m of ramp access and 4 m without ramps in the shed with litter at the end and 8 m of ramp access and 13 m without ramps in the shed with litter at the sides. In sheds A1 and A2 the height of the slatted area resulted in a steeper ramp angle of 45° compared to 30° in the other sheds. There were four pop holes at ground level with two on each side of the house (L: 2.35 m by H: 0.4 m) leading to the range from the litter area on both sides of the sheds. All sheds had aerial perches at 1 m high with 18 cm of perching space per bird resulting in approximately 360 m of perch length running the length of the slatted area. Feed tracks and drinker lines matched those in the rearing sheds. The lighting schedule was 16 h of light and varied between summer and winter with the lights set to turn off at the same time as natural dusk. The birds were fed on organic mini pellets throughout lay. Enrichment was provided to the flocks in the form of pecking objects such as buckets and boots. Replicates 5 and 6 were provided with pecking blocks and alfalfa hay nets hung on the litter area.Figure 4Plan view of the laying house layout (a) for replicates 1, 2, 4 and 5 and (b) for replicates 3 and 6. Images not to scale.Full size imageAssessments of behaviourObservations were made at three time points during the rearing period at 1, 3 and 15–16 weeks. On the first visit at 1 week of age, the total number of chicks on each ES was counted once in spot counts in the morning. At 3 and 15–16 weeks, observations of the movements up and down the ES were made. Three of the 6 structures were chosen at random in each shed. The number of chicks present on the different parts of the ES was counted at the beginning and end of the recording period to allow a comparison with the 1-week counts. The recordings involved 5-min continuous sampling where all movements down the ES were recorded and the area the chicks moved down from was noted. This was then repeated for movements up the ES. Focal bird recordings were taken at 3 and 15–16 weeks of age. Records were made for each of 3 randomly selected ES. When 10 focal birds had been observed (approximately 30 birds per flock), or 10 min had passed recordings stopped. A focal bird was chosen if it was performing orientation behaviour, indicating a downwards or upwards transition. This was described as the bird rotating its head to look in the direction of movement. Behaviours performed after the orientation behaviour were tallied, thus recorded as counts per behaviour (see Table 6). Recordings were stopped if birds completed a transition or moved away from transitioning.Table 6 An ethogram of behaviours of focal birds during up and down movements.Full size tableAt 15–16 weeks of age, three types of interactions were recorded for feather pecking. These included severe feather pecking (SFP), gentle feather pecking (GFP) and aggressive pecks (AP)28. A quadrat area 2 m by 2 m was randomly selected, with the number of birds in each quadrat counted at the beginning and end of the recording period. The number of SFP, GFP and AP were recorded over three minutes of continuous recordings in three different areas of the house, selected randomly at each end and the middle of the shed. Feather pecks and aggressive pecks were recorded as bouts: a series of pecks not separated by more than 5 s28. Rates of pecking were calculated as the number of pecks per bird per second.In the laying shed around 16–17 and at 24 weeks of age 3-min continuous sampling and focal bird recordings were taken for transitions between the slats and litter. Four recordings were made at 2-m lengths along the elevated slatted area: two areas with ramps (RA) and two areas without ramps (NRA) were selected. Separate recordings were taken for upwards and downwards movements and the number of birds in the recording area were counted at the start and end of the scans. At 16–17 and 24 weeks of age, feather pecking observations were taken using the same procedure as for the 15–16-week observations during rear.Welfare assessments and production data rearing phaseFeather scores of 20 birds per flock were recorded at 16–17 weeks of age by walking in a straight line down the centre of the shed, selecting a bird at random then counting two birds to the left of this and visually feather scoring that bird. Birds were not handled to minimise disturbance and plumage was scored using the method from Bright et al.33. The neck, back, rump, tail and wings were scored using a four-point scale 0 (best) to 4 (worst). Data were obtained from the farm records for percentage cumulative mortality and body weight.Welfare assessments and production data laying phaseAt 16–17 and 24 weeks of age, the attitude of the flocks was assessed using the approach distance and reactions to novel objects methodology developed by Whay et al.34. Distance to approach birds before they moved away was recorded by walking through the house selecting a bird at random and counting two birds to the left. The bird had to be standing up and facing the researcher, who approached the bird at a steady pace and recorded the distance before the bird moved away. This was repeated on 20 birds in each flock. Reactions to a novel object (blue folder at 17 weeks of age and a white and blue tub at 24 weeks of age) were assessed by placing a novel object on the ground and recording the time taken for the first bird to interact with it and then how many birds were within a 30 cm radius after 60 s. The novel object test was repeated in 4 areas per flock. Range use was recorded by counting the number of birds near to the house (5 m) in the middle range (5–20 m) and far (the rest of the range). Feather scores of 20 birds per flock were recorded at 17 and 24 weeks of age using the same procedure as for the 16-week assessment for birds at rear.At 40 weeks of age, feather cover and keel bone fractures were scored. Up to 100 birds per shed were caught from four different locations (25 litter, 25 slats, 25 perches, 25 nest boxes). In four sheds only 50 birds were caught as the birds were fearful and showed signs of distress. Feather cover was scored by picking the bird up and scoring the body and flight feathers separately using a the AssureWel three-point scale 0 (best) to 2 (worst)35. The keel damage was then scored using a 0 (no damage) to 2 scale based on the technique used by Wilkins et al.36. Validation for keel bone palpations was conducted. A score of 94% matched scores compared to an experienced gold standard assessor and 85% match at dissection for scoring a break. At 24 weeks of age, the number of floor eggs were counted over 1 day.Data were collected from the farm records on laying house percentage of daily eggs, average egg weight (grams), average hen body weight and feed conversion ratio.During the 16 week recordings in the final rearing flocks, the lighting inside the shed was considerably reduced compared to previous flocks. This resulted in poor visibility for feather cover and feather pecking observations, so these were not taken during this visit. Data were not obtained on keel fractures and feather cover scores at 40 weeks for the first laying flocks visited as their sheds were destroyed by strong winds.Statistical analysisData were analysed using SPSS 24 (IMB) or MLwiN 3.0. The statistical package MLwiN was chosen as it is designed for multilevel modelling and can therefore accommodate data nested within levels with repeated measures. Such models account for dependence between responses caused by grouping of birds within sheds, and repeated measures taken from the same sheds on different visits within and between replicates. Including visit and replicate as nested effects ensures that dependences (e.g. due to differing times of year when data were collected) are accounted for. All residuals were checked for normal distributions using a Shapiro-Wilks test or plotted graphically and no transformations were needed to meet the assumptions of the tests. All results are reported in the format mean ± SD unless when stated as the percentage of birds performing a behaviour during transitions.Assessments of behaviourAt rear, from the counts of chicks on structures and counts of transitions up and down the structures, a normal model (generalised linear model) was used with a four-level hierarchy (bird within shed within visit within replicate). The same normal model and four level hierarchy were used for the counts of transitions in the laying shed.For the focal bird behaviours of birds transitioning at rear and lay, the data were presented as the percentage of each behaviour calculated for the birds in the recording session for the two rearing treatments. The direction (up or down) was analysed separately. For the focal birds at lay, all were included in the analysis for the pre-transitioning behaviours, only birds that attempted a transition were included for analysis of the transitioning behaviours. Pre-transition behaviours for birds that moved-away and did not transition were analysed separately. Owing to the low occurrence of behaviours during the focal recordings for transitions up and down the ramps, data were coded as yes or no, and a Binomial model was used for analysis for both the rear and lay focal transition data with four hierarchical levels (bird within shed within visit within replicate).Welfare and production dataFor the Novel object test, human approach, feather pecking and feather cover data a normal model was used in MLwiN with four-levels (Bird within Shed within visit within replicate). Floor eggs were analysed using a two-tailed t-test in SPSS, due to limited data. Ordinal data such a keel bone fracture scores and feather cover recorded at 40 weeks of age were converted to binomial data due to a lack of data for some scores, these were therefore analysed using a binomial model in MLwiN with two levels (Bird within shed).Production data at rear (body weight in grams) and lay (% eggs daily, egg weight in grams, body weight in grams and feed conversion ratio) were obtained from farm records and analysed in SPSS using a general linear model with treatment (CR and RR) as a fixed factor and age (3, 8 and 14 weeks at rear and 20, 30 and 70 weeks at lay) as a random factor to account for repeated results. Cumulative percentage mortality was analysed at 14 weeks of age using a t-test to compare the treatment groups.Ethical approvalEthical approval for this project was granted by the University of Bristol’s Animal Welfare and Ethical review body under UIN: UB/16/040 and all methods were conducted in accordance with the review body and UK legislation. 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    Non-additive microbial community responses to environmental complexity

    Selection and initial metabolic profiling of organismsIn order to maximize the chance of obtaining communities with diverse taxonomic profiles from different environmental compositions, the organisms selected were drawn from a number of bacterial taxa known to employ varying metabolic strategies. In addition, given the growing relevance of synthetic microbial communities to industrial and biotechnological applications73,74,75,76, we chose to employ bacterial species that have previously been used as model organisms and have well-characterized metabolic capabilities. This criterion, paired with the availability of flux-balance models associated with a majority of these organisms, allows us to explore the metabolic mechanisms observed in our various experimental conditions with higher confidence. These selection principles resulted in a set of 15 candidate bacterial organisms (Acinetobacter baylyi, Bacillus licheniformis, Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, Lactococcus lactis, Methylobacterium extorquens, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Salmonella enterica, Streptomyces coelicolor, Shewanella oneidensis, Streptococcus thermophilus, and Vibrio natriegens) spanning three bacterial phyla (Actinobacteria, Firmicutes, and Proteobacteria, Supplementary Table 1, Supplementary Fig. 1a).A microtiter plate-based phenotypic assay was used to assess the metabolic capabilities of each of the 15 candidate organisms. Each organism, stored in glycerol at −80 °C, was initially grown in 3 mL of Miller’s LB broth (Sigma–Aldrich, St. Louis, MO) for 18 h with shaking at 300 rpm at each organism’s recommended culturing temperature (Supplementary Table 1). To maximize oxygenation of the cultures and prevent biofilm formation, culture tubes were angled at 45° during this initial growth phase. Candidate organism Streptococcus thermophilus was found to have produced too little biomass in this time period and was grown for an additional 8 h. Each culture was then separately washed three times by centrifuging at 6000 × g for 2 min, removing the supernatant, suspending the pellet in 1 mL of M9 minimal medium with no carbon source, and vortexing or triturating to homogenize. The cultures were then diluted to OD600 0.5 ± 0.1 as read by a microplate reader (BioTek Instruments, Winooski, VT) and distributed into each well of three PM1 Phenotype MicroArray Plates (Biolog Inc., Hayward, CA) per organism at final OD600 of 0.05 ± 0.01. The carbon sources in the PM1 plates (Supplementary Table 2, Supplementary Fig. 1b) were resuspended in 150 µl of M9 minimal media prepared from autoclaved M9 salts (BD, Franklin Lakes, NJ) and filter-sterilized MgSO4 and CaCl2 prior to inoculation. The cultures in each PM1 plate were incubated at each organism’s recommended culturing temperature with shaking at 300 rpm for 48 h. After this growing period, the OD600 of each culture was measured by a microplate reader to quantify growth. To account for evaporation in the outer wells of the plates, which could yield in inflated OD readings, three ‘evaporation control’ plates with no carbon source were inoculated with bacteria at a final OD600 of 0.05 and incubated at 30 °C for 48 h. The averaged OD600 readings of these plates were subtracted from the readings of the bacterial growth plates to correct for evaporation. A one-tailed t-test was performed using these corrected OD600 values to determine significance of growth above the value of the negative controls (p  More