Does energy intake influence diet selection of novel forages by horses?

Livestock Science ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Does energy intake influence diet selection of novel forages by horses? M. van den Berg a,n, C. Lee b, W.Y. Brown a, G.N. Hinch a a b

School of Environmental and Rural Science, University of New England, Armidale, NSW 2351, Australia CSIRO Agriculture Flagship, Armidale, NSW 2350, Australia

art ic l e i nf o

a b s t r a c t

Article history: Received 4 January 2015 Received in revised form 19 June 2015 Accepted 27 July 2015

While it has been shown that diet selection by equids can be influenced by nutritional factors, it is presently unclear how diet choices by horses are modulated when animals are faced with a familiarnovel dichotomy. Therefore, this study aimed to investigate the effect of energy intake on foraging behaviour in horses and the selection of familiar (FF) and nutritious novel (NF) forages. Twelve adult mares were maintained in yards during feeding and were housed in two groups in barren paddocks when not feeding. The experiment lasted for 8 weeks; in weeks 0, 4 and 8 horses received a maintenance diet and in weeks 1–3 and 5–7 horses were switched to a low energy (LE; 80% RDI) or high energy (HE; 120% RDI) diet. In weeks 3 and 7 a two-choice test was presented in a split-plot design, with 12 horses being the main-plot units. The LE and HE diets were applied at the main-plot level (n ¼6) while the subplots were the 10 min feeding intervals (combined over 3 consecutive days) where horses (one at a time) were introduced to a forage preference test (1 to 4). The tests were made up as pairs, based on the nutritional profile, with one being FF (oaten or lucerne chaff) and the other feed was NF (bamboo, tagasaste, willow or saltbush leaf chaff) and presented in a checkerboard design. In wk 8 the preference of all forages together was examined. Forage intake, the number of visits to each zone/bucket and time spent foraging or moving toward each zone/bucket were recorded. Horses demonstrated a greater preference for FF, but there were no differences between the LE and HE diet groups in the proportions of intake of NF. There was a higher acceptance of NF on Day 1, which declined on Day 2 and 3 for both diet groups (P o0.001). The LE group had a marginally higher proportion of zones visits to the NF compared to the HE group (P ¼0.009). In the final preference test, horses showed a greater acceptance of NF willow, bamboo and saltbush compared with tagasaste (P ¼0.01). Some possible explanations for the neophobic response seen in this study are discussed. & 2015 Elsevier B.V. All rights reserved.

Keywords: Horses Foraging behaviour Novel food Diet selection Energy intake

1. Introduction Herbivores typically forage on a diverse range of plant species that vary spatially and temporally in nutrient concentration and plant secondary compounds. Conditioned food preferences and aversions have evolved as important mechanisms by which herbivores can optimise diet selection by learning to associate the sensory properties of food plants with the metabolic consequences (negative and positive) of consuming them through sampling (Provenza, 1996). In herbivores, the role of post-ingestive feedback mechanisms in food selection has been primarily studied in ruminants (e.g. cattle, sheep and goats) (Burritt and Provenza, 1989; du Toit et al., 1991; Kyriazakis et al., 1998). There has been limited research into diet selection in horses (hindgut fermenters) and the ability of horses to discriminate foods based on their nutritional n

Corresponding author. E-mail address: [email protected] (M. van den Berg).

content. Like other ungulate herbivores, horses typically display patch foraging behaviour by taking a few bites of plants before walking a few steps to a new position, which allows them greater opportunity to select a better than average diet from the plants on offer. Archer (1971,1973) who studied food preferences of pastured horses, observed that bouts of grazing or browsing on the most preferred species of herbs and grasses were repeatedly interrupted by regular movement away from these sites, to areas where less preferred species were consumed. This suggests that while horses may have ranked preferences, they clearly sample all foods available, which may be a result of a change in palatability and/or satiety for particular flavours, nutrients or secondary compounds. Nonetheless, when choice is provided, horses seem to have a higher acceptance of forages with a lower cell wall content (Dulphy et al., 1997a) or a higher intake of digestible organic matter (van Wieren, 1996). This suggests that preference and digestibility are positively correlated in the equine and that forage selection may be driven by digestible energy and/or macronutrient content.

http://dx.doi.org/10.1016/j.livsci.2015.07.022 1871-1413/& 2015 Elsevier B.V. All rights reserved.

Please cite this article as: van den Berg, M., et al., Does energy intake influence diet selection of novel forages by horses? Livestock Science (2015), http://dx.doi.org/10.1016/j.livsci.2015.07.022i

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It has also been demonstrated that horses can adjust to decreasing energy density in plants by increasing their intake (Sneddon and Argenzio, 1998). Other equine studies have focussed on the ability to learn from the consequences of diet selection. Laut et al. (1985) offered concentrate diets of different caloric densities and showed that ponies can respond to decreasing density by increasing their intake. The ability of horses to differentiate between diets based on their flavour and nutrient content has been reported by Cairns et al. (2002). These authors showed that horses are able to select a higher energy concentrate over a lower energy one, regardless of the preferred flavour (e.g. mint or garlic), and that they can form associations between foods and their nutritional composition. However, the differentiation between these two foods of different energy densities only began after 10 meals over a 4-to 5-day period. It has been suggested that horses, due to hindgut fermentation, may have less specific association between food characteristics and post-ingestive consequences when foods are presented simultaneously (Redgate et al., 2014). Therefore, Redgate and colleagues investigated the effect of long term exposure of forages (individually) that were rich in either protein, lipids or hydrolysable carbohydrates on the choice preference of these forages in a simultaneous choice session. Redgate et al. (2014) implied that single presentations of forages modified foraging behaviour and that horses showed a greater preference for the forages rich in protein or hydrolysable carbohydrates, whereas selection for lipids remained constant throughout the testing period. The authors suggested that the horses responded to macronutrients in the diets and that a single dietary experience was adequate to facilitate memory of consequences and hence affected dietary preferences. In the studies of Cairns et al. (2002) and Redgate et al. (2014) horses received familiar concentrates or forages and horses were initially exposed to flavours or nutrients which allowed for familiarisation. However, in natural and pasture environments horses are continuously exposed to patches with a variety of plants, including familiar and novel species, which may differ in flavour, nutrients and secondary compounds throughout the year. The acceptance of such novel foods, and the neophobic and neophillic responses of horses in such situations has not been examined. It appears that horses are able to differentiate between diets and can adapt by increasing intake or changing selection, but how these choices are influenced by nutritional status of the animal warrants further research. There are a few studies that have reported effects of nutritional requirements on diet choices. Lamoot et al. (2005) demonstrated that lactating donkeys and Shetland ponies selected high quality regrowth more strongly than nonlactating females. Lactating animals have a greater energy and protein requirement, therefore it seems that these animals selected quality forages rather than increasing grazing time which allowed for greater digestible protein intake. Conversely, Celaya et al. (2011) reported that lactation state of mares did not affect diet composition in heathlands and suggest that other factors such as plant availability and forage characteristics may also play a role. To our knowledge there have been no studies that have investigated the nutritional status of horses and how this may affect diet selection and intake; in particular the proportions of familiar and novel foods selected as a result of post-ingestive consequences. Therefore, the present study aimed to examine if current levels of energy intake affects the foraging behaviour of horses and their selection of familiar versus non-familiar forages. The hypothesis was that horses would select familiar over novel forages, and that the acceptance of nutritious novel forages would be greater in horses on a lower energy intake.

2. Materials and methods 2.1. Animals and housing A total of 12 healthy horses were used for the study. The care and use of the animals followed the guidelines set by The University of New England Animal Ethics Committee, in accordance with section 25 of the Animal Research Act (1985). Horses were housed at a commercial horse facility in the New England region (NSW, Australia). The study was conducted using 12 mares from a single herd that had been managed together on the same property. The mares were between the ages of 4 and 13 years, weighing 430–500 kg (mean 7sem 461 75.9 kg) at arrival and were of Standardbred or Thoroughbred breed. Horses initially were grazing pasture (Winter) and had a Henneke's body condition score (Henneke et al., 1983) between 3 and 5 (thin to moderate). Animals received a veterinary health check and were given a broadspectrum wormer before the start of the experiment. Horses were maintained in individual yards for the duration of feeding and were housed in groups (n¼ 6) in barren paddocks when not feeding. Non-edible enrichments such as a horse ball and rubber tyres were provided between feeding times. All horses had ad libitum access to water in their paddocks. 2.2. Study design The 12 study animals were allocated to one of two dietary treatments: low energy (LE) at 80% of the recommended daily intake (RDI) and high energy (HE) at 120% of RDI in a randomised block design. Each horse was paired with another of similar weight and temperament before randomly allocating one horse from each pair to either the LE or HE diet. A behavioural test using a novel object was used to score the temperament traits of the horses (Visser et al., 2001). The total testing period lasted 8 weeks. After establishing maintenance feeding amounts (week-1), horses received their maintenance diet (energy 100% of RDI) in weeks 0, 4 and 8. In weeks 1–3, and 5–7 horses received their allocated treatment diets (LE or HE). Forage choice tests were conducted in weeks 3 and 7 (Stage 1). In week 8 individual preference of all forages used were evaluated (Stage 2). 2.3. Diets A basal ration (LE diet) was formulated to meet NRC recommendations for fibre, protein, vitamins and minerals, and 80% of the RDI of energy (Anonymous, 2007). These nutrient requirements were based on maintenance level (2% body weight (BW); average to elevated temperament) using the temperament test as reference. The basal diet comprised of oaten chaff (Avena sativa), lucerne chaff (Medicago sativa), beet pulp (Beta vulgaris) and a commercial vitamin/mineral supplement. Barley (Hordeum vulgare L.) and a vegetable oil supplement (Equi-Oil, BEC Feed Solutions) were combined with the basal diet to alter the energy density, which constituted the HE diet. The combination of oil with barley was chosen to increase energy density and to avoid reaching suggested upper limits for oil supplementation (McKenzie et al., 2002). For the maintenance diet only oil was added to increase energy levels to 100% RDI. The chemical analysis of the feed ingredients is presented in Table 1. 2.4. Preference testing Preference testing was conducted in two stages. Stage 1 compared pairs of forages, and in stage 2 horses were offered six different forages at one time. Testing for stage 1 was conducted in

Please cite this article as: van den Berg, M., et al., Does energy intake influence diet selection of novel forages by horses? Livestock Science (2015), http://dx.doi.org/10.1016/j.livsci.2015.07.022i

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Table 1 Chemical compositiona (g/kg dry matter (DM)) of the feedstuffs offered to horses (n¼ 12) during feeding experiment. Constituent

Barley steam rolled

Beet pulp flakes

Oaten chaff

Lucerne chaff Weeping willow leaf chaff

Bamboo leaf chaff

Saltbush leaf chaff

Tagasaste leaf chaff

Dry Matter Digestible Energy (MJ/ kg DM) Crude Protein Lysine Crude Fat NDF ADF NFC Starchb Calcium Phosphorus Magnesium Potassium

901 14.6

938 11.0

916 9.1

925 9.8

909 11.5

938 9.0

992 12.3

911 10.9

145 5.2 29 173 57 635 520 1.3 3.7 1.7 5.5

95 2.9 12 434 263 453 NT 10.8 0.8 2.4 4.0

91 3.3 32 546 380 247 49 4.1 2.3 1.9 13.8

175 8.9 27 459 349 273 18 14.6 2.6 2.6 20.4

93 3.2 37 324 255 477 NT 24.4 2.7 4.5 12.5

96 3.4 36 593 322 235 NT 3.9 2.2 0.7 12.3

123 4.8 25 296 179 475 NT 12.3 1.7 8.0 24.9

176 8.8 38 390 286 355 NT 4.2 2.1 2.1 13.0

a b

NDF, neutral detergent fibre; ADF, acid detergent fibre; NFC, non-fibre carbohydrates. Units are g/kg DM, unless otherwise stated. NT, not tested.

weeks 3 and 7 and stage 2 testing was conducted in week 8. 2.4.1. Forage two-choice test (Stage 1) Stage 1 testing was conducted as a split-plot design, with the 12 horses being the main-plot units. The 2 diet treatments (HE and LE) were applied at the main-plot/horse levels, with 6 replicate horses. The sub-plot levels were the 10-min two-choice tests (combined over 3 consecutive days) where the horses (one at a time) were introduced to a forage preference test (1–4). The forage preference tests were made up of pairs of feeds, with one being a familiar feed (A or B) and the other a feed that was novel to the horses (a, b, c or d). The forages offered were: Familiar forage (FF) A: oaten chaff; B: lucerne chaff. Novel forage (NF) (chaffed/wilted) a: golden bamboo (Phyllostachys aurea); b: tagasaste (Chamaecytisus palmensis); c: willow (Salix babylonica); and d: saltbush (Atriplex nummularia). The nutritional profile of the novel forages is given in Table 1. The forage preference tests were paired based on their nutritional profile (energy and crude protein): (1) oaten and bamboo (Aa); (2) lucerne and tagasaste (Bb); (3) oaten and willow (Ac); and (4) lucerne and saltbush (Bd). The horses received two forage preference tests in week 3 (with one day in between) and another two tests (consecutive) in week 7 of the experiment. Horses were allocated to the forage preference tests based on horse's body weight and temperament. The forage preference tests applied at the sub-plot level were the factorial combination of forage preference test by time applied (week 3 and 7) (Table 2). This allowed for distribution of treatments across testing days and eliminated the influences of potential weather conditions for one particular treatment. The horses received the same allocated forage treatment each day during a 3-day block. This preference testing period was selected based on previous pilot study that investigated the preference of fresh novel forages (Triebe et al., 2012). Testing was conducted between 1030 and 1500 h and horses were tested in the same order for the three testing days. The order of appearance was changed for each forage preference test and horses were allocated to a new time slot based on the previous order to reduce the influence of fasting time since the last meal. All horses received the treatment within 5 ½ h after the morning meal. A testing area (12 m  12 m) divided into 16 zones (2.5 m2) was used for the two-choice test. Each zone contained 50 g (dry chaff weight) of either a FF or NF, which was offered in feeding tubs that were placed in rubber tyres. The total amount of forages (800 g) offered in the choice test did not exceed 10% of their daily energy intake. Rubber matting 1 m  1 m was placed under the feeding tubs. There were 8 zones allocated to each forage in a

Table 2 Randomisation of preference tests through blocking procedure based on horse's body weight and temperament. The bold cells show the tests that are replicated, and the plain cells have tests in the 3-way combination of diet by forage preference by time that are not replicated. Main-plot Diet

1 2 3 4 5 6 7 8 9 10 11 12

Low Low Low Low Low Low High High High High High High

Forage preference tests (cell values) Time 1 – wk 3 (3 days)

Time 2 – wk 3 (3 days)

Time 3 – wk 7 (3 days)

Time 4 – wk 7 (3 days)

T T T T T T T T T T T T

T T T T T T T T T T T T

T T T T T T T T T T T T

T T T T T T T T T T T T

1 2 3 4 1 4 1 2 3 4 3 4

2 3 4 1 4 3 2 3 4 1 2 3

3 4 1 2 2 1 3 4 1 2 4 1

4 1 2 3 3 2 4 1 2 3 1 2

chequerboard fashion (Naujeck et al., 2005) (Fig. 1). On every testing day the forage types were randomly allocated to a new zone. There were individual yards with companion animals on both sides of the testing area. Before the start of the experiment, horses were familiarised with the test area and routine of leading them separately into the testing area. During the experiment horses were individually led into the testing area by a handler and allowed 10 min to forage the area uninhibited. 2.4.2. Forage preference test (Stage 2) At the conclusion of the second testing period, horses were returned to the maintenance diet and, following three non-test days, were individually subjected to a two-day preference test. Horses were presented with six feed buckets containing 100 g of the four NF and two FF to determine preferences, and responses (10 min per horse) recorded on video. This preference test was performed in the same testing area as the two-choice test. Forages were presented in feed buckets set in tyres and placed in a circle 2.5 m apart and randomised on both testing days. 2.5. Feeding and weight management All diets were offered twice daily between 700–930 h and 1600–1830 h. Amounts of feed offered and any refusals were weighed and recorded throughout the study. Horses consumed on

Please cite this article as: van den Berg, M., et al., Does energy intake influence diet selection of novel forages by horses? Livestock Science (2015), http://dx.doi.org/10.1016/j.livsci.2015.07.022i

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Fig. 1. Field and patch layout. A testing area (12 m  12 m) divided into 16 zones (2.5 m2). Note: There were 8 zones allocated to each forage (familiar and novel) in a chequer board fashion. On every testing day the forage types were randomly allocated to a new zone. Horses were individually led into the testing area and allowed 10 min to forage the area uninhibited, which was recorded with a video recorder and by direct observation.

average between 95% and 100% of the ration offered. The majority of the refusals occurred in the HE group. The average daily intake of the dietary chemical constituents for each of the diet groups is presented in Table 3. Horses were weighed twice in the week leading up to the experimental period and weekly during the study to monitor weight gain and loss. The difference in energy levels of the LE and HE diets was reflected in the weight gain of the horses (week 1 to 7). The average (7 sem) weight gain for the HE group was 0.37 70.03 kg/ day and the LE group remained stable at 0.04 70.01 kg/day. 2.6. Behaviours Behaviours for stage 1 and 2 were recorded for 10 min with two video recorders (Panasonic, Japan) and by a person sitting 10 m outside the testing arena (see Fig. 1). The number of visits to each zone (categorised as both front hooves being placed in a zone) and sequence to each zone/bucket were documented. In addition, the time spent foraging (labelled as standing and chewing) or moving to each zone/bucket (classified as walking towards a new zone/ bucket) was recorded. The intake of foods by each horse was determined by weighing the foods in each feeding bucket before and after each test.

2.7. Feed collection and other measurements The selection of browse: willow, bamboo, tagasaste and saltbush were chosen based on novelty and literature that described the use as an alternative/supplementary fodder in horse diets (Dann and Trimmer, 1986; Myers, 2005; Nelson et al., 1997; Triebe et al., 2012; Van den Berg, 2015). To limit the potential effect of secondary compounds on diet selection, only small NF amounts were offered in this study and plant materials were processed (wilted/dried). All novel forages were sourced from northern regions of NSW. The tree or shrub browse was stripped to leaves and twigs, dried in a climate-controlled room at 27 °C for 3–4 days and cut in pieces similar to the familiar chaff form (2–3 cm). Only the saltbush leaves had a smaller particle size of 0.5–1 cm. Feed samples (for nutrient analysis) of the basal diet (e.g. lucerne, oaten, barley and beetpulp) were taken weekly throughout the study period, and the NF samples were taken twice weekly during the testing weeks. The collected feed samples were pooled and sub-samples of 500 g were taken for chemical analysis. These ‘as fed’ samples were sent to a laboratory (Equi-analytical labs, Ithaca, NY) for analysis. Weather observations were registered daily during the testing days. The average (7 sem) minimum temperature was 2.3 71.3 °C, maximum temperature was 18.4 71.4 °C (maximum), rainfall was

Please cite this article as: van den Berg, M., et al., Does energy intake influence diet selection of novel forages by horses? Livestock Science (2015), http://dx.doi.org/10.1016/j.livsci.2015.07.022i

M. van den Berg et al. / Livestock Science ∎ (∎∎∎∎) ∎∎∎–∎∎∎ Table 3 Mean daily intake (g DM)7 sem per kg bodyweight (BW) of dietary chemical constituents of the feed offered to the low (n¼ 6) and high (n¼ 6) energy diet group during weeks 1–3 and week 5–7. Mean percentage of daily recommended nutrient intake (NRC maintenance 2% BW; average/elevated temperament) is presented in brackets where applicable. Constituent a

Lucerne (g) Oaten Beetpulp Barley Oil Dry Matter Digestible Energy (MJ)b Crude Protein Lysine Crude fat NDF ADF NFC Starch

High energy

Low energy

sem

5.88 5.82 1.03 1.91 0.58 15.01 (75%) 0.17 (120%) 1.97 (146%) 0.13 (224%) 0.94 6.60 5.02 3.93 0.26

6.05 5.99 1.07 – – 13.16 (65%) 0.12 (82%) 1.72 (127%) 0.12 (207%) 0.35 6.01 4.57 3.58 0.24

0.03 0.03 o 0.01 NA NA 0.25 o 0.01 0.02 o 0.01 o 0.01 0.11 0.08 0.07 o 0.01

NA Not applicable: no barley and oil was given to the low energy group during treatment weeks. a b

Units are grams (DM) unless otherwise stated. DM basis.

2.8 71.7 mm/day, evaporation was 3.5 70.4 mm/day, wind gust was 21 72.8 km/h (maximum 52.4 74.2 km/h) and sea level pressure was 1011 71.6 hPa. 2.8. Statistical analysis Data are presented as mean 7sem, unless stated otherwise. Forage intake, zones visited and time spent foraging and moving were analysed in GenStat version 16 (VSN International Lt, Hemel Hempstead, Hertfordshire, UK) and all data were checked for normality and transformed were necessary. The level of significance was set to 5% for all testing. Protected least significant difference (lsd) tests were used for pair-wise comparisons of treatments if the overall effect was significant. 2.8.1. Forage two-choice test (Stage 1) To determine the influence of dietary energy intake on FF and NF selection, the proportions of NF over the three testing days were examined by fitting linear mixed models and the estimation of variance components using the method of residual maximum likelihood (REML). The proportion of NF intake out of the total intake of the i-th day, j-th diet, k-th preference test and h-th horse that belongs to the b-th time of the test referred to as block with pijkh was calculated. In the model diet, forage preference test and day with a two-way and three-way interaction were included as fixed effects and horse and the horse  time block interaction as random effects. The model was;

angular (Pijkh ) = (μ + αday + αdiet + αtest + αdayxdiet + αday xtest + αdiet xtest +αday xdiet xtest + βblock | horse + βhorse + error, μ is the intercept and with αday we denote the effect of the day factor, i.e. αday×i (i = 1, 2, 3) and similarly for the diet and the test factors. With αday x diet we denote the coefficient of the day-diet interaction and βblock | horse is the random effect of the time block and βhorse is the random effect of the individual horse. Angular transformation was used to equalise residual variances of NF proportions. To investigate trends in NF intake and proportions between the two diet groups based on the nutritional profile Test 1 (oaten and

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bamboo; Aa) was paired with Test 3 (oaten and willow; Ac) and Test 2 (lucerne and Tagasaste; Bb) with Test 4 (lucerne and saltbush; Bd). Test 1 and 3 were classified as the lower energy/protein pairing and Test 2 and 4 as a higher energy/protein pairing and added to the model as an energy/protein and test term. The preference tests were nested within the energy/protein factor (low; Aa–Ac vs. high; Bd–Bd). Further analysis was conducted to determine if there were any differences in the intake (g) of the four NF choices between the two diet groups. A square root transformation was applied to normalise the residuals. The above analyses were repeated for zone count and time spent foraging and moving, but no transformations were required for these parameters. 2.8.2. Forage preference test (Stage 2) The intake of FF and NF by the horses over the two testing days were investigated by fitting linear mixed models and the estimation of variance components using the method of REML. A logarithmic transformation was applied to normalise the residuals. In the model forage choice was included as a fixed effect and test day and the test day  horse interaction as random effects. This model was repeated for zone count, time spent foraging and moving. No transformation was necessary for the zone visits but for time spent foraging and moving a square root transformation was applied.

3. Results 3.1. Forage two-choice test The proportions (percentage) of NF (high and low energy/ protein pairing) and total intake, zone count and time spent foraging or moving by the LE and HE diet groups over three testing days are given in Table 4. The average empirical intake (g) of NF by the two diet groups is depicted in Fig. 2. 3.1.1. Intake of novel forages Data on NF proportions consumed are presented as angular transformed mean values (back-transformed where applicable). The average proportion of NF intake by the two diet groups over the three testing days did not significantly differ, with both groups ingesting relatively small amounts (HE; 6.8% vs LE; 6.1%). Only a day effect was observed, with the two diet groups consuming a significantly greater proportion of NF on Day 1 (9.2 (2.5)%) compared to Day 2 (5.5 (0.9)%) and Day 3 (4.7 (0.7)%) (7 sem; 0.97%; Po 0.001). No differences between the high and low energy/protein forage pairing were observed for the diet groups and days. Comparable trends were apparent for the mean intake (g) of the NF choices and no differences between the four NF were recorded. 3.1.2. Time spent foraging and moving Similar to the trends in NF intake, both diet groups spent on average a small proportion of their time foraging on NF (HE; 9.1% vs. LE; 10.2%) over the three-testing days and there were no differences between the diet groups. The two diet groups spent a significantly greater time foraging on NF on Day 1 (12.1%) compared to Day 2 (8.9%) and 3 (8.0%) (7sem; 0.7%; P o0.001). In addition, a significant difference was observed for the forage energy/protein pairing and diet interaction (high energy/protein; 10.1 (HE) and 13.3 (LE) vs. low energy/protein; 8.1 (HE) and 7.1 (LE) (7sem; 1.0%; P¼ 0.03)). Both diet groups spent less time sampling “low energy/protein” NF. There were no differences in the proportions of time spent moving towards NF between the diet groups and days. The HE group spent on average about 47.0% and the LE group 49.5% of their time moving towards the NF over the three testing days,

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Table 4 Results of the two-choice forage tests (10 min; Stage 1); mean 7sem proportion (%) of novel forage and total intake, zone counts and time spent foraging or moving by the low (n¼ 6) and high (n ¼6) energy diet groups over three testing days. Data on novel forage proportions consumed are angular transformed mean 7 sem (back-transformed mean). Energy/Protein pairing†

High Energy Diet Group Day 1

Low Energy Diet Group

Day 2

Day 3

Day 1

Day 2

Day 3

4.0 7 1.79 (0.5)b 3707 21.4 5.7 7 1.79 (1.0)b 323 7 25.1

7.7 7 1.79 (2.0)a 390 7 13.1 10.2 7 1.79 (3.8)a 380 7 17.6

5.2 7 1.79 (0.9)b 4007 5.3 4.4 7 1.79 (0.6)b 364 7 14.5

5.7 7 1.79 (1.0)b 389 7 12.9 3.4 7 1.79 (0.4)b 3537 26.7

b) Total time spent foraging (s) and proportions (%) of time spent consuming novel forages 9.4 71.31 b 8.4 7 1.31b High Proportion % 12.4 7 1.31a Total time foraging (s) 422 7 24.8 448 729.1 452 7 22.8 Low Proportion % 11.8 7 1.31a 6.5 71.31b 6.3 7 1.31b Total time foraging (s) 486 7 17.8 475 722.9 4717 34.5 Diet: NS, Day: nnn, Energy/Protein pairing: nnn and Diet x Energy/Protein pairing: n

14.4 7 1.31a 4077 28.7 9.8 7 1.31 a 499 7 13.1

13.2 7 1.31b 4437 20.6 6.3 7 1.31b 5107 13.2

12.2 7 1.31b 4437 26.0 5.17 1.31b 4987 22.5

c) Total time spent moving (s) and proportions (%) of time spent moving towards novel forages High Proportion % 47.3 7 1.98 45.7 71.98 47.2 7 1.98 Total time moving (s) 1257 7.8 110 712.0 1087 12.4 Low Proportion % 47.17 1.98 50.1 71.98 44.6 7 1.98 Total time moving (s) 967 14 95 719.2 827 13.3 Diet: NS, Day: NS and Energy/Protein pairing: NS

49.67 1.98 1357 18.9 49.07 1.98 887 11.4

48.47 1.98 1237 13.1 50.2 7 1.98 807 11.1

50.2 7 1.98 1087 15.3 49.67 1.98 777 7.5

d) Total zone count and proportions (%) of novel forage zones visited High Proportion % 48.87 1.11 48.3 71.11 Total zone count 347 6.9 36 72.9 Low Proportion % 48.37 1.11 48.3 71.11 Total zone count 257 6.5 29 73.6 Diet: nn, Day: NS and Energy/Protein pairing: NS

49.77 1.11 367 4.4 49.67 1.11 267 3.3

48.37 1.11 447 5.5 51.0 7 1.11 307 3.3

51.2 7 1.11 417 3.8 47.9 7 1.11 287 3.0

a) Total forage intake (g) and novel forage proportions (%) consumed 5.1 71.79 (0.8)b High Proportion % 6.9 7 1.79 (1.6)a Total intake (g; as fed) 3777 8.3 377720.9 Low Proportion % 11.8 7 1.79 (4.3)a 7.3 71.79 (1.7)b Total intake (g; as fed) 3597 24.5 332 720.8 Diet: NS, Day: nnn and Energy/Protein pairing: NS

47.3 7 1.11 377 4.0 45.8 7 1.11 287 4.0

n 0.01o P o0.05. nn0.001o Po 0.01. nnnPo 0.001. NS, not significant. a,b,c: Proportions with different superscript are significantly different within diet group and Energy/ Protein pairing and between days (rows).

† “Low Energy/Protein Forages” are oaten paired with bamboo (Aa) and willow (Ac) and the “High Energy/Protein Forages” are lucerne paired with tagasaste (Bb) and saltbush (Bd).

3.1.3. Zones/buckets visited A small but significant difference was observed between the diet groups in the proportion of NF zones visited (P ¼0.009). The average percentage of NF zones visited was 47.8% for the HE group and 49.6% for the LE group (7sem; 0.47%). Similar to the time spent moving, these results suggest an almost balanced proportion for NF and FF zone counts. No variations between the testing days were recorded for either diet group and no differences between the high and low energy/protein forage pairing were apparent for the diet groups and days. 3.2. Forage preference test

Fig. 2. The mean 7sem (empirical) intake (g; as fed) of novel forage chaff by the low and high energy diet groups over three testing days.

showing an almost equal proportion of movement towards NF and FF. No differences between the high and low energy/protein forage pairing were apparent for the diet groups and days.

A summary of the mean forage intake, zone count and time spent foraging or moving by the horses (n ¼12) is given in Table 5. Data on intake are presented as logarithmical transformed and time spent foraging or moving are square-root transformed mean values (back-transformed mean). The horses consumed 99–100% of the offered familiar forages on both test days. While five out of the 12 horses increased their NF intake by consuming 50–100% of the offered willow and bamboo forage by day 2, the overall mean

Table 5 Mean 7 sem intake, zone visits and time spent foraging or moving by the horses (n¼12) over two testing days in the forage preference test (10 min; Stage 2). Data on intake are logarithmical transformed and time spent foraging or moving are square-root transformed mean 7sem (back-transformed mean). Means with different superscript are significantly different (P o0.05). Forage

Lucerne

Oaten

Willow

Bamboo

Saltbush

Tagasaste

Intake (g; as fed) Zone count Time spent foraging (s) Time spent moving (s)

4.6 7 0.31 (99.6)a 3.3 7 0.3ab 10.9 7 0.62 (119.7)a 3.8 7 0.27 (14.6)a

4.6 7 0.31 (99.2)a 3.0 7 0.3b 11.8 7 0.62 (139.3)a 3.7 7 0.27 (13.3)ab

1.7 70.31 (4.5)b 3.3 70.3ab 5.3 70.62 (27.7)b 3.9 70.27 (15.2)a

1.6 7 0.31 (3.3)b 3.6 7 0.3a 5.4 7 0.62 (28.8)b 4.0 7 0.27 (15.8)a

1.3 7 0.31 (2.7)b 3.17 0.3ab 4.5 7 0.62 (20.0)b 3.6 7 0.27 (13.2)ab

0.6 7 0.31 (0.8)c 2.5 7 0.3c 3.0 7 0.62 (9.0)c 3.2 7 0.27 (10.0)b

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intake (Day 1 and 2) showed a significantly higher intake for willow (1.7 (4.5)), bamboo (1.6 (3.3)) and saltbush (1.3 (2.7)) with tagasaste (0.6 (0.8)) being the least preferred ( 7sem; 0.31 g; P ¼0.01). Similar patterns were also observed for the time spent foraging. The mean visits to the bamboo forage zone (3.6) were significantly greater than the visits to the oaten forage (3.0), but both forages did not significantly differ from lucerne (3.3), willow (3.3) and saltbush (3.1) and the least visits were made to the tagasaste forage zone (2.5) ( 7sem; 0.3; P ¼0.001). The time spent moving towards bamboo (4.0 (15.8)), willow (3.9 (15.2)) and lucerne (3.8 (14.6)) were significantly greater than the tagasaste forage (3.2 (10.0)), but movement towards tagasaste did not differ from oaten (3.7 (13.3)) and saltbush (3.6 (13.2)) ( 7sem; 0.27 sec; P ¼0.001).

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horses showed a higher preference for the grasses with the shorter sward height and higher quality (CP: 13.5%, NDF: 55.5%) (Edouard et al., 2010). Even though the digestible DM and energy intake rates were higher for the taller swards, the authors suggest that digestible protein was the better indicator of the likely selection patterns of horses but the evidence for this is not at all clear. It is well known that horses as hindgut fermenters are far less efficient at utilising dietary nitrogen (Anonymous, 2007; Cheeke and Dierenfeld, 2010) but the implications for this on diet selection is still far from being well-defined. In our study horses in both HE and LE groups had an adequate intake of CP (HE; 146% RDI and LE; 127% RDI) and lysine (HE; 224% RDI and LE; 207% RDI) and therefore deficiency of protein should not have been a factor influencing food choice and sampling of alternative food sources. Whether nitrogen deficit would influence alternative food choices is evidently a subject for further research.

4. Discussion 4.2. Plant characteristics and pre-ingestive cues This study aimed to investigate the effect of energy intake on foraging behaviour in horses and the selection of FF and NF. It was hypothesised that horses would have a higher preference for FF, but horses on a low energy diet would show a neophilic response toward the NF and increase the intake of nutritious alternatives. The key findings of this study were (1) horses had a higher preference for FF, but there were no differences between the LE and HE diet groups in the intake of NF. (2) There was a change in intake pattern over time with a higher acceptance of NF on day 1, which declined on day 2 and 3 for both diet groups. (3) The overall preference test showed a greater acceptance of the NF willow and bamboo. This difference in the final preference test seemed to not be related to the nutritional profile (i.e. low energy/protein pairing) but may have been linked to other food characteristics. 4.1. Forage selection Horses demonstrated a greater preference for FF and tended to have a neophobic response towards the NF options. This is in agreement with the theory that animals will select initially FF and subsequently sample novel options and make accordingly diet decisions based on nutritional status and post-ingestive feedback mechanisms (Provenza et al., 1998; Wang and Provenza, 1996). However, our study was not able to confirm the second supposition that when nutritional and physiological conditions are inadequate animals would increase the intake of alternative foods, provided these novel foods are nutritious and there is a trade-off for toxicological effects (Provenza, 1995). In the present study, horses on a LE diet did not show a greater acceptance of NF, even for those forages that had a high energy and protein nutritional profile. Horses fed the LE diet in our study had an average intake of about 82% of the RDI for energy. During the two-choice tests, horses in the LE diet group were able to increase their energy intake with approximately 5% of the RDI for energy if they consumed all of the offered FF. This added energy from FF and the increased sampling of NF on Day 1 could have contributed to the limited intake of unfamiliar foods on Day 2 and 3. However, it is also possible that energy may have not been the main reason for horses to mix diets and sample NF and that other dietary requirements (e.g. protein) may have a greater influence on diet selection (Edouard et al., 2010). Studies of patch selection have shown that diet choices in horses are less responsive to energy costs and more by quality or other plant factors (Edouard et al., 2009, 2010; Fleurance et al., 2009, 2012). Edouard et al. (2009) demonstrated that horses preferred pastures of taller sward heights, when offered grasses with heights between 6 and 17 cm and of equal quality. In a second experiment, the taller sward height had matured (CP: 7% DM, NDF: 62% DM) and in contrast

In the present study horses initially sampled the NF on Day 1 but intake declined on days 2 and 3. It seems plausible that the flavour (texture, odour, smell, taste) of the NF may have played a role in the trend observed. Smell and taste appear to be important cues for herbivores to evaluate potential forages and foods because they can provide rapid and detailed information linked to the biochemical characteristics of feedstuffs. In addition, animals can use smell and taste to access memory information about foods (pre-ingestive) without actual ingestion (Pain and Revell, 2007). The novel browse forages used here, although presented in a similar conserved chaff form as lucerne and oaten, were from trees and shrubs that varied in texture and odour. The novelty of the texture may have been a reason for the lower consumption of novel foods. Naujeck et al. (2005) reported that horses preferred longer pasture sward height over short and suggested that this may have been due to the novel textures (due to mowing). However, it is more likely that the odour profile of some of the forages used in the present study may have influenced the foraging decisions by the horses. A pilot study investigated the acceptance of two fresh NF (bamboo and tagasaste) in a two-choice test and showed that horses increased the intake of bamboo and decreased the number of samplings of tagasaste over the course of 3 days (Triebe et al., 2012). When fresh leaves of tagasaste are crushed (by bites) they release a strong volatile smell and this may explain why intake (and time spent foraging) was limited after day 1 compared to bamboo. While, in the present study dried versions of the NF were used, most of the forages had a characteristic smell that was easily detected by human senses. The influence of plant odours on herbivore feeding behaviour and dietary preferences has been clearly demonstrated in sheep (Arnold et al., 1980). In addition, odour profiling has also been used to make predictions about the preferences (and links with nutritional traits) for oaten and lucerne hay by horses and dairy cattle (Pain and Revell, 2009). 4.3. Animal morpho-physiological constraints In the present study horses appeared to rely more on smell and taste (pre-ingestive) rather than the post-ingestive mechanisms when selecting forages and this was apparent with frequent scanning of the novel forages by the horses placing their nose over the buckets without ingesting the forages. For example, Tagasaste was the least preferred and fewer visits to this forage were made; even though it had a similar nutritional profile to the well-liked familiar forage, lucerne. Although it has some phenolic compounds it is considered to be a good alternative fodder (fresh, wilted and dried) for grazing animals and has been reported to be consumed by sheep, goats, cattle and even horses (Assefa et al.,

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2012; Borens and Poppi, 1990; Dann and Trimmer, 1986; Van den Berg, 2015). Horses rely on hindgut fermentation to gain energy from plant fibrous materials and therefore potentially do not have the same “feedback” systems available to ruminants. It appears that horses are less sensitive to cell wall content in forages than ruminants and this is most likely because horses do not have a reticuloomasal orifice (Cymbaluk, 1990; Dulphy et al., 1997b). The reticuloomasal orifice is highly sensitive to, and selectively retains, large forage particles for regurgitation and masticate foods until particles are small enough to move further down the digestive tract. Consequently it seems likely that organoleptic qualities of forages (e.g. taste, odour, ease of prehension, texture etc.) may be of greater importance for diet selection in horses (Doreau et al., 1990; Ralston, 1984). This may also be evident in the morphology of the nose and size of the nasal epithelium. Horses have long noses and olfactory senses are likely to be well developed and used in relation with other signals to respond to their environment (Pain et al., 2005). Ruminants and horses also differ in their ability to detoxify plant secondary compounds due to the differences in digestive anatomy. Non-ruminants (e.g. pigs, poultry and horses) are usually more susceptible to plant toxins than ruminants which have the capacity to denature toxins in the rumen (Norton, 1998). Overall, this difference may help to explain why horses in this study rejected foodstuffs based on smell without actually ingesting the foods to avoid possible malaise. 4.4. Preferences for novel forages A final preference test was conducted after the two-choice test to review any preference developed between the NF. Horses showed an increased trend in the intake of willow and bamboo forages and made significantly more visits to the bamboo zone compared to oaten, but neither differed from the lucerne, willow and saltbush zone visits. In hindsight it would have been interesting to determine if the preferences persisted over time as postingestive consequences were identified. The increased trend in sampling of NF observed when all six forages were presented may suggest that when given more choice (more than two) animals are more likely to increase intake. Diet mixing and increased plant species richness has shown to increase the intake of feed by ruminants and animals switch between preferred and less preferred species or feed stuffs (Burritt and Provenza, 2000; Wang et al., 2010). However it can also be that exposure and recognition may have allowed for greater acceptance of novel feeds. There is some indication that previous exposure to forages may increase sampling interest; Yearsley et al. (2006) showed that the rate of learning new food types (i.e. the rate of information transfer) is maximised when food types occur with a particular frequency in the diet. However, these authors suggest that during the encounters with novel foods, this “optimal-learning diet” is likely to conflict with the classical foraging theory based on nutrient or energy intake (Stephens and Krebs, 1986). Of all the NF Tagasaste was the least preferred by the horses. It remains unclear why Tagasaste was strongly disliked, as it had the best energy/protein profile. Further investigation is warranted on the role of smell and taste in diet selection by horses and if exposure (or training) will change the preferences for novel quality foods in horses. 4.5. Patch selection The design of this study also allowed us to assess the impact of energy levels on patch selection of horses, to our knowledge the first such study. While it was recorded that horses on a LE diet had a higher proportion of NF zone visits than the horses on a HE diet,

this difference was small and both averages were close to 50%. The almost equal proportion of familiar and novel was also observed in the time spent moving towards different “forage zones” which suggests that animals did not use short-term spatial memory to identify preferred (familiar) patches. Illius and Gordon (1990) observed similar patterns in ruminants and suggested that grazing animals may rely on visual or orosensory cues rather than on memory of spatial cues. When ungulates forage they select a feeding site, a patch in the site, within which numerous bites are selected. Researchers suggest that the memory necessary to remember each bite or feeding station would be excessive (Bailey et al., 1996; Senft et al., 1987). Spatial recognition seems to relate more to a larger spatial scale and temporal level in the foraging hierarchy such as feeding site (1–4 h feeding bouts), camps (1–4 weeks) and home range (1 month–2 years). Whether this applies to horses is currently unknown. While cognitive function (long and short-term memory) and leaning in horses have been assessed, there are somewhat variable results when the short-term memory in foraging tasks was tested. For example, McLean (2004) used a two-choice apparatus whereby horses were maintained immobile in a test arena and visually and aurally experienced the delivery of food into one of two feed goals. The results showed that horses were able to achieve the correct feed goal in the immediate-release trials but seem to be unsuccessful in the 10 s release trials. Conversely, other studies that used a two-choice or three-choice design suggest that horses do recall the location of a food reward (after a human or device placed the food under/in bucket) even with a delayed release of 10 s or more (Hanggi, 2010; Lovrovich et al., 2015). The discrepancy between the studies is most likely due to sample size, the designs used and/or the causal inference of the effect of a visual cue (human or device placing the food reward under/in bucket). Lovrovich et al. (2015) is the only study that used a control group that did not receive any visual (human) cues and relied on smell and random attempts (sampling) to find the correct food target. While the horses that were given human cues were able to find the food reward more accurately in the first attempts, as the trial proceeded the horses showed similar feeding strategies as the control group (switching from accuracy to speed). The results suggest that orosensory cues and trial and error or sampling have to be acknowledged as important components of foraging behaviour and highlight that experimental designs need to be carefully considered when analysing short-term memory in foraging “tasks”. Therefore it seems that further equine research is needed if we are to understand shortterm spatial memory and the role of sensory cues in different spatial scales and temporal levels of the foraging hierarchy.

5. Conclusion This study has demonstrated that differences in energy intake the weeks prior to testing did not significantly alter the proportion of FF and nutritious NF ingested by horses. While there was a greater intake of NF on the first days of testing, proportions ingested were low and after day 1 horses showed a consistent neophobic response towards the novel browse forages. Although the consumption of all FF on the first days may have added to the energy intake for the LE diet group, our results suggest that energy is not the main determinant of diet choice in horses. Future studies should investigate the effects of other plant components and nutrients on diet selection and the likelihood of diet mixing (partial preference). In addition, multiple choice and nutritional geometry models could be adopted to further examine diet selection patterns in horses and how individual animals regulate their intake of multiple nutrients to maintain target physiological states. Furthermore, hindgut fermentation may be the reason for the slow

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response to post-ingestive feedback which may be why horses rely more on the organoleptic qualities of forages to make appropriate diet choices, but further studies are needed to confirm this.

Conflict of interest Funding for this project was kindly provided by the University of New England, New South Wales, Australia. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no additional financial support for this work that could have influenced its outcome.

Acknowledgements We are grateful to Tom Armytage and Jock Weston for arranging facilities and horses. We would like to thank Carolina Zanatta, Karina McLoughlan, Caroline Triebe and Heather Salmon for their help with taking care of the horses and assistance during the feeding experiment. Special thanks go to Michael Raue for his help during the experiment and browse forage collection/processing. We also acknowledge Mark Porter for his assistance with the collecting and processing of the browse forages. In addition, we are grateful to Kerry Bell for her statistical advice. The authors also would like to thank all our sponsors for the supply of horse care and/or feed products; BEC Feed Solutions, Axon Equine Razor Wormer, OzHorseToys, Tuffys Tubs, Burton's Saddlery, Manuka Farm/Haylage, EyKamp Kikuya Company and Horses and People Magazine.

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