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Use of a maze test to assess spatial learning and memory in cattle: Can cattle traverse a complex maze? Masahiko Hirata ∗ , Chihiro Tomita, Karin Yamada Department of Animal & Grassland Sciences, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan
a r t i c l e
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Article history: Received 6 December 2015 Received in revised form 3 April 2016 Accepted 5 April 2016 Available online xxx Keywords: Cattle Maze Exploration Spatial learning Spatial memory Personality
a b s t r a c t Maze tests were conducted to assess spatial learning and memory in Japanese Black cows (Bos taurus). The test arena (14 × 14 m) was designed so that it could contain no maze or one of four different mazes of increasing complexity (L1–L4 mazes), with a food reward at the opposite end of the release point to entice test cows. Cows were individually released into the arena and tested for 5 min to measure the time to reach the food reward (completion time) and the time spent eating the reward (eating time). Experiment 1 subjected 15 cows initially to the no-maze situation, then to the L4 maze, and finally to the no-maze situation again. None of the cows were able to traverse the L4 maze although 10 cows retained the motivation to reach the reward, showing significant (P < 0.01) differences in completion time between the no-maze and the L4 maze situations. Experiment 2 tested another 14 cows in the no-maze situation and found 8 cows highly motivated for the reward. Experiment 3 subjected 10 cows which showed strong motivation for the reward in Experiments 1 and 2, initially to the no-maze situation, then to the L1, L2, L3 and L4 mazes, and to the no-maze situation again. Five cows were able to complete the L4 maze whereas all cows retained the motivation for the reward, showing a significant (P < 0.01) difference in completion time between the no-maze and the L4 maze situations. There was a strong negative correlation between the completion time and eating time throughout the three experiments (P < 0.001). The results show Japanese Black cows are able to learn to traverse a complex maze when they are provided with stepby-step learning opportunities, although this ability is limited to about 20% of animals. Once learned, they can retain the memory of the maze configuration for up to 6 weeks. Further studies are needed to determine the limitations of the memory. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Spatially heterogeneous distribution of resources (e.g., food, water and shelter) is the major characteristic of habitats and home ranges of animals (Fisher, 2000; Ganskopp and Bohnert, 2009; Moe and Wegge, 1994; Shannon et al., 2006). Animals living in such an environment are considered to have the ability to learn and remember the locations of resources in their habitats and home ranges in order to efficiently exploit the resources and successfully fit the environment. Spatial learning and memory in large mammalian herbivores has been studied in controlled experiments mainly for domestic animals such as cattle (Bos taurus) and sheep (Ovis aries). Bailey and Sims (1998) reported that cattle remembered the locations of
∗ Corresponding author. E-mail address: [email protected] (M. Hirata).
high quality food placed in an 8-arm radial maze for 30 days. Ksiksi and Laca (2002) observed that cattle learned the locations of food patches placed in an experimental arena in 3 days and remembered the food locations accurately for at least 48 days. Edwards et al. (1996) found that sheep learned the locations of food patches in an arena in 2–3 days and remembered the food locations for at least 3 days. Lee et al. (2006) reported that sheep learned to traverse a maze including two error zones to join their flock mates in 3 days and were able to perform the task when tested 6 weeks later. However, the spatial environment (structure of the maze or food arrangement in the arena) offered to animals in most previous studies differs greatly in complexity from the one which animals encounter in their habitats. For example, mazes used previously include only one or two branching points, i.e., limited number of occasions where animals need to make a decision on route choice (Bailey and Sims, 1998; Lee et al., 2006). Whether animals are able to learn to traverse a more complex maze (referred to simply as ‘complex maze’ hereafter) remains unexplored. Furthermore, if ani-
http://dx.doi.org/10.1016/j.applanim.2016.04.004 0168-1591/© 2016 Elsevier B.V. All rights reserved.
Please cite this article in press as: Hirata, M., et al., Use of a maze test to assess spatial learning and memory in cattle: Can cattle traverse a complex maze? Appl. Anim. Behav. Sci. (2016), http://dx.doi.org/10.1016/j.applanim.2016.04.004
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mals are able to do so, another critical question is how long they can retain the memory of the correct traverse route of the complex maze without reinforcement. In the study reported here, we evaluated spatial learning and memory in cattle offered a complex maze, as one step toward complexity from the simplicity in the previous studies. The ability to learn to traverse the complex maze was assessed either by offering the maze immediately after a no-maze situation or by gradually increasing the maze difficulty from the no-maze situation to the complex maze. The ability to remember the maze configuration was assessed by retesting the animals after a retention interval of 43 days. We hypothesized that cattle would be able to learn to traverse a complex maze when they are provided with step-by-step learning opportunities, and would remember the configuration of the complex maze for up to 6 weeks.
2. Materials and methods The study comprised three experiments (Fig. 1) and was conducted at the Sumiyoshi Livestock Science Station (31◦ 59 N, 131◦ 28 E), Faculty of Agriculture, University of Miyazaki, southern Kyushu, Japan. The animals used in the study were 29 Japanese Black cows from the same herd. All experimental procedures were approved by the Animal Care and Use Committee of the university (#2012–001–2 and 3).
2.1. Experimental plot The experimental plot, established outdoors on bare ground (no grazable vegetation), comprised a test arena (14 × 14 m), a standby area (1.6 × 3.5 m) and a reward area (2 × 2.5 m) attached to the opposite sides of the test arena (Fig. 2a–e). The standby area was constructed with iron posts (1.5 m high), two wire-mesh sliding gates (1.2 m high) on the 1.6 m sides and three lines (0.4, 0.8 and 1.2 m high) of barbed wire on the 3.5 m sides. The perimeter of the test arena plus the reward area was made of two lines (0.6 and 1.2 m high) of synthetic cord with interwoven metal (2.5 mm diameter) attached to fiber-reinforced plastic (FRP) poles (1.5 m high) at intervals of 2 or 2.5 m, in order to avoid visual isolation stress in test cows. A white plastic container (71 cm width × 43 cm depth × 22 cm height) containing a food reward (1 kg grain-based concentrate; Minami Nihon Kumiai Siryo Co., Ltd., Kagoshima, Japan) was fixed at the end of the reward area, and a signboard (2 m wide, 1.8 m high) showing a blue Landolt C (Fig. 2f) in the top center was set up behind the container as a potential visual cue. Because the plastic container was 22 cm high, the food reward could not be seen by the cows until they were almost directly above the container. The test arena was designed so that it could contain no maze (Fig. 2a) or one of four different mazes of increasing complexity, shown as L1–L4 mazes (Fig. 2b–e). The configurations of the L3–L1 mazes were determined by gradually simplifying the structure of the L4 maze. Maze barriers within the arena were also made of the synthetic cord and FRP poles. The concentrate used as a reward contained approximately 14% crude protein, 2.5% crude fat, 10% crude fiber and 69% total digestible nutrients. This feed was regularly used as an attractant to collect cows for health and pregnancy check as part of management, i.e., all cows were familiar with the concentrate showing a preference for it. The amount of concentrate dispensed in the container (1 kg) was determined to ensure that the concentrate would not be eaten up by a test cow within the test period of 5 min (see Section 2.2).
2.2. Experiment 1 Experiment 1 tested 15 cows 10 times on 10 separate days at intervals of 1 day for Tests 1–7, and 3 or 4 days thereafter (Fig. 1). The intervals of 3–4 days instead of 1 day after Test 7 had to be adopted to accommodate the experimenters’ commitments as university members. The test cows were selected randomly from the herd, excluding individuals with a pre-weaning calf, near to calving or with a health problem. The mean age and body weight (± SE) of the cows were 12.2 ± 0.5 years and 459 ± 12 kg, respectively. They had no exposure to the experimental plot, the plastic container for the reward or the signboard showing the Landolt C prior to the experiment. Tests 1–6 were conducted without a maze to familiarize the cows with the experimental plot, and to assess their ability to learn of the food reward and their motivation to reach and eat the reward, by providing an opportunity to associate the Landolt C with the reward. Tests 7–9 investigated the ability of cows to complete the L4 maze offered immediately after the no-maze situation. Test 10 offered the cows the no-maze condition again to confirm their ability and motivation to reach and eat the food reward. In each test, the 15 cows were individually tested in a random sequence. Each test cow was taken out of the barn where grass hay or silage was available and kept in the standby area for about 1 min, then released into the test arena and allowed to behave freely for 5 min. Immediately after this, the cow was removed from the experimental plot through the standby area and returned to the barn in exchange for the next test cow. When the cow reached and ate the food reward, the food container was refilled with 1 kg concentrate before testing the next cow. Cows in the barn were not able to see the cow in the experimental plot (no opportunity for social learning). All tests were video-recorded using a video camera (HDRCX180; Sony, Tokyo, Japan). The camera was fitted with a fisheye conversion lens (0.28×; HDP-2800ES; Raynox, Tokyo, Japan) and fixed at a 3 m high position 4.5 m behind the standby area, so as to cover the whole experimental plot. 2.3. Experiment 2 Experiment 2 tested 14 cows three times on 3 separate days at intervals of 1 day (Fig. 1). This experiment was conducted 21 weeks after Experiment 1 so as to use individuals which were not tested in Experiment 1. The mean age and body weight of the cows were 7.2 ± 0.8 years and 512 ± 14 kg, respectively. They had no exposure to the experimental plot, the plastic container for the reward or the signboard showing the Landolt C prior to the experiment. We were not able to balance the age and body weight of cows between Experiments 1 and 2 because of the limited availability of animals due to the presence of a pre-weaning calf, calving, a health problem and culling. The objectives of the three tests in this experiment were the same as those of Tests 1–6 in Experiment 1. The number of tests, i.e., three times, was determined based on the results of Experiment 1, as the minimum number required for assessing the ability and motivation of cows to reach and eat the food reward. In each test, the 14 cows were individually tested and recorded in the same way as in Experiment 1. 2.4. Experiment 3 Experiment 3 tested 10 cows 11 times on the first 6 days, and 4 cows 6 weeks later (Fig. 1). This experiment commenced 17 weeks after Experiment 2 in order that individuals which showed relatively strong ability and motivation to reach and eat the food reward in Experiments 1 and 2 could be used in Tests 1–11, i.e., minimiz-
Please cite this article in press as: Hirata, M., et al., Use of a maze test to assess spatial learning and memory in cattle: Can cattle traverse a complex maze? Appl. Anim. Behav. Sci. (2016), http://dx.doi.org/10.1016/j.applanim.2016.04.004
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Fig. 1. Timeline for the maze tests in the three experiments. Experiments 1 and 2 used different individuals (15 and 14 cows, respectively). In Experiment 3, Tests 1–11 used 10 cows selected from Experiments 1 and 2 (three and seven cows, respectively), and Test 12 used 4 cows selected from Tests 1–11 (one and three cows from Experiments 1 and 2, respectively). When two tests were allocated to a single day (Tests 1–10 in Experiment 3), one was conducted in the morning and the other in the afternoon.
Table 1 Completion time (s) of 15 cows in the 10 tests in Experiment 1. Cow
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Completion rate Difference in completion time from the previous test
Test (maze) 1 (no maze) 2 (no maze) 3 (no maze) 4 (no maze) 5 (no maze)
6 (no maze) 7 (L4 maze) 8 (L4 maze) 9 (L4 maze) 10 (no maze)
300 300 300 300 300 274 300 236 300 300 300 300 213 300 300 0.20 –
300 23 300 75 300 166 300 8 23 17 14 16 12 300 300 0.60 NS
242 30 300 300 300 19 300 19 36 300 18 300 24 300 300 0.47 *
103 300 300 300 300 15 300 13 27 62 14 28 12 300 300 0.53 NS
106 44 300 300 300 30 300 9 36 36 13 13 14 300 300 0.60 NS
71 23 300 300 300 14 300 12 30 30 13 7 19 300 300 0.60 NS
300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 0.00 **
300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 0.00 NS
300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 0.00 NS
38 25 300 15 300 12 300 11 47 27 34 20 17 300 75 0.73 **
L4 denotes level 4. Cows which did not reach the food reward within the 5-min test time were assigned a completion time of 300 s. * P < 0.05. ** P < 0.01, NS P ≥ 0.05.
ing exclusions due to the presence of a pre-weaning calf, calving or a health problem. The 4 cows used for Test 12 were the individuals that completed the L4 maze in Tests 9 and 10 (see Section 3.2) and were available at the time of the test. The mean age and body weight of the 10 cows were 9.9 ± 1.0 years and 513 ± 22 kg, respectively. They had no exposure to the experimental plot, the plastic container for the reward or the signboard showing the Landolt C for 17–18 weeks prior to the experiment. Tests 1 and 2 were conducted in the no-maze situation to confirm that the cows retained the ability and motivation to reach and eat the food reward. Tests 3–10 investigated the ability of cows to complete the 4 levels of mazes offered in order of increasing complexity (from L1 to L4). Test 11 was carried out after removal of the L4 maze to reconfirm the ability and motivation of cows to reach and eat the food reward, and Test 12 evaluated the ability of cows to
remember the configuration of the L4 maze after a retention interval of 43 days from Test 10. In each test, the cows were individually tested and recorded in the same way as in Experiments 1 and 2.
2.5. Data analysis The video records were analyzed to quantify the two behavioral measures, the time to reach the food reward (completion time, s) and the time spent eating the food (eating time, s), for individual cows in individual tests in individual experiments. Cows which did not reach the food reward within the 5-min test time were assigned a completion time of 300 s and an eating time of 0 s. For Tests 9 and 10 in Experiment 3, the video records were analyzed also to determine the trajectory of cows within the L4 maze, using a coordinate system of 7 rows × 7 columns plus the reward area (Fig. 2e).
Please cite this article in press as: Hirata, M., et al., Use of a maze test to assess spatial learning and memory in cattle: Can cattle traverse a complex maze? Appl. Anim. Behav. Sci. (2016), http://dx.doi.org/10.1016/j.applanim.2016.04.004
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Fig. 2. Layout of the experimental plot with (a) no maze, (b) level 1 maze, (c) level 2 maze, (d) level 3 maze and (e) level 4 maze, and (f) the Landolt C painted on the signboard behind the food reward. White cells numbered 1–18 and shaded cells leading to W1–W5 in the level 4 maze indicate the correct pathway to the food reward and the wrong routes to the dead end, respectively.
Because there was a strong negative correlation between the completion time and eating time in Experiments 1–3 (Spearman rank correlation (rS ) = −0.96, P < 0.001, n = 306), the completion time was selected as a performance measure of cows and subjected to the subsequent statistical analysis. Changes in the completion time in cows across the tests in individual experiments were nonparametrically evaluated using the Friedman test and the Wilcoxon signed rank sum test. The Friedman test for Experiment 3 excluded Test 12 because of the different number of test cows from the preceding tests. Fisher’s exact test was used to detect whether cows selected from Experiment 1 (with experience with the L4 maze) differed from cows selected from Experiment 2 (with no experience with the L4 maze) in the proportion of success in reaching the reward (completion rate) in Experiment 3. Differences in the animal attributes (age, liveweight and physiological state as days from the last parturition) between the successful and unsuccessful cows in the L4 maze tests (Tests 9 and 10) in Experiment 3 were evaluated using the Wilcoxon rank sum test. All statistical analyses were performed using STATISTICA (version 10; StatSoft, Tulsa, OK, USA). 3. Results 3.1. Experiments 1 and 2 In Experiment 1, only 3 cows (Cows 6, 8 and 13) out of the 15 reached and ate the food reward in Test 1 where there was no maze, showing a completion rate (CR) of 0.20 (Table 1). Under the
repeated no-maze conditions, however, the number of rewarded cows increased to seven (Cows 1, 2, 6, 8, 9, 11 and 13) in Test 2 (CR = 0.47) and remained at eight or nine thereafter in Tests 3–6 (CR = 0.53–0.60). Once learned, cows always or almost always reached and ate the food in the subsequent tests. Then, in Tests 7–9, none of the 15 cows were able to traverse the L4 maze and reach the reward (CR = 0). In the subsequent test where cows were retested under the no-maze condition (Test 10), 11 cows (Cows 1, 2, 4, 6, 8–13 and 15) reached and ate the food reward (CR = 0.73). These cows, with one exception (Cow 15), were the individuals which reached and ate the food at least once in Tests 1–6. The completion time in cows varied significantly across the 10 tests (Friedman 2 = 64.9, P < 0.001, n = 15, df = 9), showing a decrease from Test 1 to Test 2 (P < 0.05), an increase from Test 6 to Test 7 (P < 0.01), and a decrease from Test 9 to Test 10 (P < 0.01). The 5 cows (Cows 6, 8, 9, 11 and 13) which reached and ate the food reward at least 5 times out of the first six no-maze tests (Tests 1–6) were selected as candidates for Experiment 3. In Experiment 2 where cows were always tested in the no-maze situation, 5 cows (Cows 16, 18, 22, 23 and 27) out of the 14 reached and ate the food reward in Test 1 (CR = 0.36; Table 2). The number of rewarded cows increased to eight (Cows 19, 22–27 and 29) in Test 2 and remained the same in Test 3 (CR = 0.57). Once learned, cows always reached and ate the food in the subsequent test or tests, except two individuals (Cows 16 and 18). The completion time in cows varied significantly across the 3 tests (Friedman 2 = 6.5, P < 0.05, n = 14, df = 2), showing a decrease from Test 1 to Test 2 (P < 0.05). The 8 cows (Cows 19, 22–27 and 29) which reached and
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Group 1: cows selected from Experiment 1 (with experience with the level 4 maze). Group 2: cows selected from Experiment 2 (with no experience with the level 4 maze). L1, L2, L3 and L4 denote levels 1, 2, 3 and 4, respectively. Cows which did not reach the food reward within the 5-min test time were assigned a completion time of 300 s. Difference in completion time from the previous test is for data pooled over two groups. * P < 0.05. ** P < 0.01, NS P ≥ 0.05.
12 (L4 maze)
NS ** * *
NS NS NS NS NS
2
Difference in completion rate between groups Difference in completion time from the previous test
–
**
NS
53 11 15 1.00 13 15 14 16 36 38 13 1.00 NS
11 (no maze) 10 (L4 maze)
300 300 29 0.33 39 300 29 61 300 300 60 0.57 NS 300 300 80 0.33 68 300 31 102 300 300 118 0.57 NS
9 (L4 maze) 5 (L2 maze) 4 (L1 maze) 3 (L1 maze) 2 (no maze)
6 8 11 Completion rate 19 22 23 25 26 27 29 Completion rate 1
Test (maze) Cow Group
Table 3 Completion time (s) of two groups of cows in the 12 tests in Experiment 3 (10 cows in Tests 1–11 and 4 cows in Test 12).
Among the 13 candidates selected from Experiments 1 and 2, three (Cows 9, 13 and 24) had to be excluded from Experiment 3 due to calving or culling. In Experiment 3, the 3 cows derived from Experiment 1 (Cows 6, 8 and 11 as Group 1, with experience with the L4 maze) and the 7 cows derived from Experiment 2 (Cows 19, 22, 23, 25–27 and 29 as Group 2, with no experience with the L4 maze) did not differ in CR (P ≥ 0.05) in any of the 12 tests (Table 3). Across the two groups, all 10 cows reached and ate the food reward in Tests 1 and 2 where there was no maze (CR = 1; Table 3). Thereafter, the number of rewarded cows decreased to seven (Cows 6, 11, 19, 25–27 and 29) in Tests 3–4 with the L1 maze (overall CR = 0.55 (=11/20)), increased to eight (Cows 8, 11, 19, 23, 25–27 and 29) in Tests 5–6 with the L2 maze (overall CR = 0.8 (=16/20)), remained almost the same in Tests 7–8 with the L3 maze (overall CR = 0.75 (=15/20)), and decreased to five (Cows 11, 19, 23, 25 and 29) in Tests 9–10 with the L4 maze (overall CR = 0.5 (=10/20)). Then, when the maze was removed in Test 11, all 10 cows successfully reached and ate the food reward again (CR = 1). Furthermore, after a 6-week retention interval, all 4 test cows completed the L4 maze in Test 12 (CR = 1), reaching the reward as quick as in Test 10 (29–61 s in Test 10 and 26–80 s in Test 12). The completion time in cows varied significantly across the 11 tests (Friedman 2 = 72.5, P < 0.001, n = 10, df = 10; excluding Test 12 with n = 4), showing increases from Test 2 to Test 3 (P < 0.01) and from Test 8 to Test 9 (P < 0.05) and decreases from Test 9 to Test 10 (P < 0.05) and from Test 10 to Test 11 (P < 0.01). Cows differed considerably in the ability to traverse the 4 levels of mazes in Tests 3–10, despite having the ability to reach the reward in the no-maze situation (Table 3). Cow 22 was unable to traverse any of the mazes, and Cow 6 was able to complete only the L1 maze in Tests 3 and 4. Three cows (Cows 8, 26 and 27) failed to traverse the L4 maze in Tests 9 and 10 despite the completion in the L2 and L3 mazes in Tests 5–8. Cows that successfully traversed the L4 maze did not differ (P > 0.05) from unsuccessful cows in age, liveweight or physiological state. Among the 5 cows which traversed the L4 maze in Tests 9 and 10 (maximum correct cell number = 18), one (Cow 23) reached the
6 (L2 maze)
3.2. Experiment 3
300 49 37 0.67 87 300 20 21 30 47 18 0.86 NS
8 (L3 maze)
ate the food reward at least twice out of the three no-maze tests (Tests 1–3) were chosen as candidates for Experiment 3.
300 39 28 0.67 300 300 31 23 34 116 23 0.71 NS
7 (L3 maze)
Cows which did not reach the food reward within the 5-min test time were assigned a completion time of 300 s. * P < 0.05, NS P ≥ 0.05.
300 108 51 0.67 50 300 64 28 62 112 31 0.86 NS
*
300 95 52 0.67 53 300 43 42 143 68 36 0.86 NS
300 300 300 24 300 300 20 20 22 20 30 29 300 16 0.57 NS
76 300 105 0.67 216 300 300 30 300 300 160 0.43 NS
3 (no maze)
300 300 300 168 300 300 24 33 157 134 108 21 300 11 0.57
61 300 65 0.67 47 300 300 50 97 193 300 0.57 NS
2 (no maze)
216 300 253 300 300 300 157 147 300 300 300 215 300 300 0.36 –
13 12 13 1.00 26 47 17 12 14 19 14 1.00 NS
16 17 18 19 20 21 22 23 24 25 26 27 28 29 Completion rate Difference in completion time from the previous test
Test (maze) 1 (no maze)
1 (no maze)
Cow
25 12 16 1.00 20 235 19 15 15 16 18 1.00 NS
Table 2 Completion time (s) of 14 cows in the 3 tests in Experiment 2.
5
– – 26 1.00 28 – 28 80 – – – 1.00 NS
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Table 4 Maximum correct cell number reached and occurrence of wrong route selection (W1–W5) by two groups of cows in Tests 9 and 10 in Experiment 3 (level 4 maze). Group
Cow
Test
Maximum correct cell number
W1
W2
W3
W4
W5
1
6
9 10 9 10 9 10 9 10 9 10 9 10 9 10 9 10 9 10 9 10
8 10 11 11 18 18 18 18 11 8 18 18 18 18 12 11 11 11 18 18
0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1 0 1 1 0 0 1 1 1 1 0 0 0 0 0 0 1 1 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1 2 3 2 0 0 0 0 2 2 0 0 1 1 2 2 1 1 1 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
8 11 2
19 22 23 25 26 27 29
Group 1: cows selected from Experiment 1 (with experience with the level 4 maze). Group 2: cows selected from Experiment 2 (with no experience with the level 4 maze). See Fig. 2e for correct cell numbers and wrong routes (W1–W5). Maximum correct cell numbers of 18 and <18 indicate success and failure in reaching the food reward, respectively.
food reward without taking a wrong route in either test (Table 4). Two (Cows 11 and 29) reached the reward only after an error (W1 or W4) in Test 9. The other two (Cows 19 and 25) reached the reward after an error (W2 or W4) in both tests. In contrast, the 5 cows which failed to traverse the L4 maze (Cows 6, 8, 22, 26 and 27) selected a wrong route at least twice during each test and were able to follow the correct route only as far as the cell number of 8, 10, 11 or 12. Among the five wrong routes, W4 was most frequently selected (18 times), followed by W2 (7 times) and W1 (once). 4. Discussion In the current study, the ability of cows to complete the maze task and remember the maze configuration can be evaluated only when they know the presence of the food reward and have the motivation to reach and eat the reward. Performance of cows in the no-maze tests is thus a critical criterion for assessing cows’ ability. It is not surprising in Experiment 1 that cows which failed to reach the food reward throughout the initial six no-maze tests (Cows 3, 5, 7, 14 and 15) did not traverse the L4 maze thereafter (Table 1). Only cows that did not traverse the L4 maze despite the retention of the motivation to reach and eat the reward in the subsequent no-maze test (Cows 1, 2, 4, 6 and 8–13 in Experiment 1 (Table 1); Cows 6, 8, 22, 26 and 27 in Experiment 3 (Table 3)) are regarded as lacking the ability to complete the maze. 4.1. Learning of the food reward A major finding from the present study is that individual cows differed considerably in the ability to learn of the food reward in the no-maze situation in Tests 1–6 in Experiment 1 (Table 1) and in Tests 1–3 in Experiment 2 (Table 2). No cows had experience with the experimental plot including the plastic container for the reward and the signboard with the Landolt C prior to the two experiments, and they were tested individually in the tests. Hence, cows that learned of the food reward in the initial one or two tests in each experiment can be rated less subject to social isolation (i.e., less
social) and more exploratory (i.e., more curious, risk-taking and bold) than those which failed to find the reward. It has been well documented that individual animals consistently differ through time and across situations in behaviors such as aggressiveness, fearfulness, neophobia, exploration, boldness (shyness) and sociability, which is known as a behavioral syndrome, personality or temperament (Bell, 2007; Gibbons et al., 2010; Searle et al., 2010; Sih et al., 2004). Thus, the individual differences in the ability to find the food reward in the no-maze situation may be interpreted to reflect the personality of individual cows, though the current study did not evaluate the intra-animal consistency in this ability in varying situations across time (e.g., changing positions of the reward area).
4.2. Spatial learning of the L4 maze Another major finding from the current study is that some cows were able to learn to traverse the L4 maze when they were offered step-by-step learning opportunities (Tests 3–10 in Experiment 3; Table 3), whereas no cows were able to complete the L4 maze when the maze was offered immediately after the no-maze condition (Tests 7–9 in Experiment 1; Table 1). Our first hypothesis was true for 5 cows (Cows 11, 19, 23, 25 and 29) out of the 10 (Tests 9–10 in Experiment 3; Table 3). The failure in the other five (Cows 6, 8, 22, 26 and 27) was not due to the loss of the motivation to reach and eat the reward, because they were shown to retain the motivation in Test 11. When the completion rate in the L4 maze (Table 3) is applied to the number of candidates for Experiment 3 (see Section 3.1), the number of cows which have the ability to complete the L4 maze is estimated at 1.65 (=0.33 × 5) and 4.56 (=0.57 × 8) for cows selected from Experiments 1 and 2, respectively, i.e., giving a total of 6.21 cows. Thus the overall completion rate for the 29 cows used in the present study is estimated at 0.21 (=6.21/29). This estimate is much lower than the completion rate of 0.71 for barrows (Sus domesticus) subjected to a maze with five dead ends (Jansen et al., 2009) and 0.77–0.91 for sheep subjected to a maze with two error zones (Doyle et al., 2014). Variation in the success rate in a maze test can be attributed to a number of factors such as animal species, complexity of maze configuration, motivation to traverse the maze and method and frequency of training. The fact that the number of rewarded cows decreased drastically to five in the L4 maze after the relative constancy of 7–8 in the L2 and L3 mazes (Table 3) indicates the substantial difficulty of the L4 maze for cows as compared with the other mazes. Previous studies reported that the ability of sheep to learn to traverse a maze was affected by animal attributes such as sex and prenatal nutrition level (T-maze; Erhard et al., 2004), and age and experience (Y-maze; Hunter et al., 2015). However, the present study did not detect differences in age, liveweight or physiological state between successful and unsuccessful cows in the L4 maze tests (Tests 9 and 10) in Experiment 3. This may suggest the possibility of the involvement of the personality (e.g., activity, exploration and boldness) in the maze learning ability in cattle as well as the reward-learning ability in the no-maze situation. Carere and Locurto (2011) highlighted the personality as an important source of variation that may affect cognitive performance in individual animals. Sneddon (2003) investigated boldness and shyness as personality traits in hatchery-reared rainbow trout (Oncorhynchus mykiss) and found that bold fish learned a simple conditioning task more quickly than shy fish, although little is known about the linkage between personality and learning ability in cattle (Webb et al., 2015). Cow 23 who completed the L4 maze without taking a wrong route in either Test 9 or 10 (Table 4) is taken to have a marked ability of spatial learning. Cows 19 and 25 who
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traversed the L4 maze after an error in both tests are taken to have a high ability to modify their behavior to get back from the error.
differences in the spatial learning and memory are reflected in the resource exploitation and the fit to the environment as a group.
4.3. Spatial memory of the L4 maze
5. Conclusion
The other major finding from the present study is that cows were able to remember how to traverse the L4 maze after a retention interval of 6 weeks (Test 12 in Experiment 3; Table 3). The fact that cows evaluated in Test 12 reached the food reward as quickly as they did in Test 10 indicates no decay in the cows’ memory. These results support the second hypothesis. Ksiksi and Laca (2002) reported that cattle remembered the locations of food patches placed in an experimental arena for at least 48 days. Lee et al. (2006) observed that sheep retained the memory of the configuration of a relatively simple maze (with two error zones) for 6 weeks. Hunter et al. (2015) reported that sheep remembered Y-maze tasks for 22 weeks. Our study demonstrates that cattle can retain the memory of the spatial environment of an even more complex structure for at least 43 days. Further studies are warranted to determine the limitations of this ability.
Japanese Black cows are able to learn to traverse a complex maze when they are provided with step-by-step learning opportunities with increasing difficulty, although this ability is limited to some (estimated at about 20%) individuals. Once learned, cows can retain the memory of the maze configuration for up to 6 weeks. Individual cows differ in the ability of both spatial learning and memory. Further studies are needed to determine the limitations of the memory.
4.4. Effect of the Landolt C sign on cows’ motivation Motivating animals to traverse a maze is a critical part of a maze test. Previous studies used conspecifics to join (Hunter et al., 2015; Lee et al., 2006), a home pen to return to (Jansen et al., 2009) or food to eat (Erhard et al., 2004) as the reward for motivating animals. In the current study, we placed not only the food reward but also the signboard showing the Landolt C behind the reward (Fig. 2) in expectation that the sign would enhance or summon up the motivation of cows serving as the visual cue of the reward. Strictly, our study cannot prove the benefit of the Landolt C sign, because the study did not compare the cows’ performance with and without the sign. In this regard, in Experiment 3, all 10 cows successfully reached and ate the food reward in Tests 1 and 2 after the intervals of 17–18 weeks (Table 3). Furthermore, all 4 cows used in Test 12 completed the L4 maze after the interval of 43 days. These results suggest a possibility that the Landolt C made some contribution to the evocation of the motivation in cows for a period of up to 18 weeks. Use of visual cues by large mammalian herbivores to locate foods has been widely known (Edwards et al., 1997; Howery et al., 2000; Renken et al., 2008; Taylor et al., 2010), and Japanese Black cows have been reported to retain an association between a visual cue and a food reward for 1 year without reinforcement (Hirata and Takeno, 2014). It should be noted, however, that cows had not completed the L4 maze in Test 12 as quickly as in Test 10 if they had impaired or lost the memory of the maze structure, even if they had the motivation to reach the reward. 4.5. Ecological implication Our finding that only limited individuals of cattle were able to learn and remember the spatial structure of the complex maze may pose a question how individuals lacking the ability can cope with the heterogeneously distributed resources (e.g., food, water and shelter) in their habitats and home ranges. However, many large mammalian herbivores are group-living animals. Individual members of a group can use social information about the resource in their environment. For instance, individuals foraging in a group can acquire information on the location of preferred food by monitoring the foraging of others (Greenwood and Rittenhouse, 1997; Shrader et al., 2007). Thus, all members of a group may not need to have the ability to learn and remember a complex spatial structure. Further studies are necessary for investigating how the individual
Conflict of interest There are no conflicts of interest to report. Acknowledgements We thank Minamo Hamada, Fuki Hirota, Ai Matsubara, Keishi Tanaka, Moeko Uchimura, Takayoshi Saito, Yui Shiraishi, Sakura Yuda, Chie Arimoto, Tomoe Ikegami, Hiroyasu Inoue, Yuki Oshige, Rune Oda, Chihiro Shibata, Yuri Hatanaka and Naofumi Yoshida for field assistance, and Kiichi Fukuyama, Ikuo Kobayashi and Koichiro Henmi for animal management. This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS KAKENHI Grant Number 23580371; to M. Hirata). References Bailey, D.W., Sims, P.L., 1998. Association of food quality and locations by cattle. J. Range Manage. 51, 2–8. Bell, A.M., 2007. Future directions in behavioural syndromes research. Proc. R. Soc. B 274, 755–761. Carere, C., Locurto, C., 2011. Interaction between animal personality and animal cognition. Curr. Zool. 57, 491–498. Doyle, R.E., Freire, R., Cowling, A., Knott, S.A., Lee, C., 2014. Performance of sheep in a spatial maze is impeded by negative stimuli. Appl. Anim. Behav. Sci. 151, 36–42. Edwards, G.R., Newman, J.A., Parsons, A.J., Krebs, J.R., 1996. The use of spatial memory by grazing animals to locate food patches in spatially heterogeneous environments: an example with sheep. Appl. Anim. Behav. Sci. 50, 147–160. Edwards, G.R., Newman, J.A., Parsons, A.J., Krebs, J.R., 1997. Use of cues by grazing animals to locate food patches: an example with sheep. Appl. Anim. Behav. Sci. 51, 59–68. Erhard, H.W., Boissy, A., Rae, M.T., Rhind, S.M., 2004. Effects of prenatal undernutrition on emotional reactivity and cognitive flexibility in adult sheep. Behav. Brain Res. 151, 25–35. Fisher, D.O., 2000. Effects of vegetation structure, food and shelter on the home range and habitat use of an endangered wallaby. J. Appl. Ecol. 37, 660–671. Ganskopp, D.C., Bohnert, D.W., 2009. Landscape nutritional patterns and cattle distribution in rangeland pastures. Appl. Anim. Behav. Sci. 116, 110–119. Gibbons, J.M., Lawrence, A.B., Haskell, M.J., 2010. Measuring sociability in dairy cows. Appl. Anim. Behav. Sci. 122, 84–91. Greenwood, P.T., Rittenhouse, L.R., 1997. Feeding area selection: the leader–follower phenomena. Proc. West Sect. Am. Soc. Anim. Sci 48, 267–269. Hirata, M., Takeno, N., 2014. Do cattle (Bos taurus) retain an association of a visual cue with a food reward for a year? Anim. Sci. J. 85, 729–734. Howery, L.D., Bailey, D.W., Ruyle, G.B., Renken, W.J., 2000. Cattle use visual cues to track food locations. Appl. Anim. Behav. Sci. 67, 1–14. Hunter, D.S., Hazel, S.J., Kind, K.L., Liu, H., Marini, D., Owens, J.A., Pitcher, J.B., Gatford, K.L., 2015. Do I turn left or right? Effects of sex, age, experience and exit route on maze test performance in sheep. Physiol. Behav. 139, 244–253. Jansen, J., Bolhuis, J.E., Schouten, W.G.P., Spruijt, B.M., Wiegant, V.M., 2009. Spatial learning in pigs: effects of environmental enrichment and individual characteristics on behaviour and performance. Anim. Cogn. 12, 303–315. Ksiksi, T., Laca, E.A., 2002. Cattle do remember locations of preferred food over extended periods. Asian-Aust. J. Anim. Sci 15, 900–904. Lee, C., Colegate, S., Fisher, A.D., 2006. Development of a maze test and its application to assess spatial learning and memory in Merino sheep. Appl. Anim. Behav. Sci. 96, 43–51. Moe, S.R., Wegge, P., 1994. Spacing behaviour and habitat use of axis deer (Axis axis) in lowland Nepal. Can. J. Zool. 72, 1735–1744.
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Use of a maze test to assess spatial learning and memory in cattle: Can cattle traverse a complex maze? Masahiko Hirata ∗ , Chihiro Tomita, Karin Yamada Department of Animal & Grassland Sciences, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan
a r t i c l e
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Article history: Received 6 December 2015 Received in revised form 3 April 2016 Accepted 5 April 2016 Available online xxx Keywords: Cattle Maze Exploration Spatial learning Spatial memory Personality
a b s t r a c t Maze tests were conducted to assess spatial learning and memory in Japanese Black cows (Bos taurus). The test arena (14 × 14 m) was designed so that it could contain no maze or one of four different mazes of increasing complexity (L1–L4 mazes), with a food reward at the opposite end of the release point to entice test cows. Cows were individually released into the arena and tested for 5 min to measure the time to reach the food reward (completion time) and the time spent eating the reward (eating time). Experiment 1 subjected 15 cows initially to the no-maze situation, then to the L4 maze, and finally to the no-maze situation again. None of the cows were able to traverse the L4 maze although 10 cows retained the motivation to reach the reward, showing significant (P < 0.01) differences in completion time between the no-maze and the L4 maze situations. Experiment 2 tested another 14 cows in the no-maze situation and found 8 cows highly motivated for the reward. Experiment 3 subjected 10 cows which showed strong motivation for the reward in Experiments 1 and 2, initially to the no-maze situation, then to the L1, L2, L3 and L4 mazes, and to the no-maze situation again. Five cows were able to complete the L4 maze whereas all cows retained the motivation for the reward, showing a significant (P < 0.01) difference in completion time between the no-maze and the L4 maze situations. There was a strong negative correlation between the completion time and eating time throughout the three experiments (P < 0.001). The results show Japanese Black cows are able to learn to traverse a complex maze when they are provided with stepby-step learning opportunities, although this ability is limited to about 20% of animals. Once learned, they can retain the memory of the maze configuration for up to 6 weeks. Further studies are needed to determine the limitations of the memory. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Spatially heterogeneous distribution of resources (e.g., food, water and shelter) is the major characteristic of habitats and home ranges of animals (Fisher, 2000; Ganskopp and Bohnert, 2009; Moe and Wegge, 1994; Shannon et al., 2006). Animals living in such an environment are considered to have the ability to learn and remember the locations of resources in their habitats and home ranges in order to efficiently exploit the resources and successfully fit the environment. Spatial learning and memory in large mammalian herbivores has been studied in controlled experiments mainly for domestic animals such as cattle (Bos taurus) and sheep (Ovis aries). Bailey and Sims (1998) reported that cattle remembered the locations of
∗ Corresponding author. E-mail address: [email protected] (M. Hirata).
high quality food placed in an 8-arm radial maze for 30 days. Ksiksi and Laca (2002) observed that cattle learned the locations of food patches placed in an experimental arena in 3 days and remembered the food locations accurately for at least 48 days. Edwards et al. (1996) found that sheep learned the locations of food patches in an arena in 2–3 days and remembered the food locations for at least 3 days. Lee et al. (2006) reported that sheep learned to traverse a maze including two error zones to join their flock mates in 3 days and were able to perform the task when tested 6 weeks later. However, the spatial environment (structure of the maze or food arrangement in the arena) offered to animals in most previous studies differs greatly in complexity from the one which animals encounter in their habitats. For example, mazes used previously include only one or two branching points, i.e., limited number of occasions where animals need to make a decision on route choice (Bailey and Sims, 1998; Lee et al., 2006). Whether animals are able to learn to traverse a more complex maze (referred to simply as ‘complex maze’ hereafter) remains unexplored. Furthermore, if ani-
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mals are able to do so, another critical question is how long they can retain the memory of the correct traverse route of the complex maze without reinforcement. In the study reported here, we evaluated spatial learning and memory in cattle offered a complex maze, as one step toward complexity from the simplicity in the previous studies. The ability to learn to traverse the complex maze was assessed either by offering the maze immediately after a no-maze situation or by gradually increasing the maze difficulty from the no-maze situation to the complex maze. The ability to remember the maze configuration was assessed by retesting the animals after a retention interval of 43 days. We hypothesized that cattle would be able to learn to traverse a complex maze when they are provided with step-by-step learning opportunities, and would remember the configuration of the complex maze for up to 6 weeks.
2. Materials and methods The study comprised three experiments (Fig. 1) and was conducted at the Sumiyoshi Livestock Science Station (31◦ 59 N, 131◦ 28 E), Faculty of Agriculture, University of Miyazaki, southern Kyushu, Japan. The animals used in the study were 29 Japanese Black cows from the same herd. All experimental procedures were approved by the Animal Care and Use Committee of the university (#2012–001–2 and 3).
2.1. Experimental plot The experimental plot, established outdoors on bare ground (no grazable vegetation), comprised a test arena (14 × 14 m), a standby area (1.6 × 3.5 m) and a reward area (2 × 2.5 m) attached to the opposite sides of the test arena (Fig. 2a–e). The standby area was constructed with iron posts (1.5 m high), two wire-mesh sliding gates (1.2 m high) on the 1.6 m sides and three lines (0.4, 0.8 and 1.2 m high) of barbed wire on the 3.5 m sides. The perimeter of the test arena plus the reward area was made of two lines (0.6 and 1.2 m high) of synthetic cord with interwoven metal (2.5 mm diameter) attached to fiber-reinforced plastic (FRP) poles (1.5 m high) at intervals of 2 or 2.5 m, in order to avoid visual isolation stress in test cows. A white plastic container (71 cm width × 43 cm depth × 22 cm height) containing a food reward (1 kg grain-based concentrate; Minami Nihon Kumiai Siryo Co., Ltd., Kagoshima, Japan) was fixed at the end of the reward area, and a signboard (2 m wide, 1.8 m high) showing a blue Landolt C (Fig. 2f) in the top center was set up behind the container as a potential visual cue. Because the plastic container was 22 cm high, the food reward could not be seen by the cows until they were almost directly above the container. The test arena was designed so that it could contain no maze (Fig. 2a) or one of four different mazes of increasing complexity, shown as L1–L4 mazes (Fig. 2b–e). The configurations of the L3–L1 mazes were determined by gradually simplifying the structure of the L4 maze. Maze barriers within the arena were also made of the synthetic cord and FRP poles. The concentrate used as a reward contained approximately 14% crude protein, 2.5% crude fat, 10% crude fiber and 69% total digestible nutrients. This feed was regularly used as an attractant to collect cows for health and pregnancy check as part of management, i.e., all cows were familiar with the concentrate showing a preference for it. The amount of concentrate dispensed in the container (1 kg) was determined to ensure that the concentrate would not be eaten up by a test cow within the test period of 5 min (see Section 2.2).
2.2. Experiment 1 Experiment 1 tested 15 cows 10 times on 10 separate days at intervals of 1 day for Tests 1–7, and 3 or 4 days thereafter (Fig. 1). The intervals of 3–4 days instead of 1 day after Test 7 had to be adopted to accommodate the experimenters’ commitments as university members. The test cows were selected randomly from the herd, excluding individuals with a pre-weaning calf, near to calving or with a health problem. The mean age and body weight (± SE) of the cows were 12.2 ± 0.5 years and 459 ± 12 kg, respectively. They had no exposure to the experimental plot, the plastic container for the reward or the signboard showing the Landolt C prior to the experiment. Tests 1–6 were conducted without a maze to familiarize the cows with the experimental plot, and to assess their ability to learn of the food reward and their motivation to reach and eat the reward, by providing an opportunity to associate the Landolt C with the reward. Tests 7–9 investigated the ability of cows to complete the L4 maze offered immediately after the no-maze situation. Test 10 offered the cows the no-maze condition again to confirm their ability and motivation to reach and eat the food reward. In each test, the 15 cows were individually tested in a random sequence. Each test cow was taken out of the barn where grass hay or silage was available and kept in the standby area for about 1 min, then released into the test arena and allowed to behave freely for 5 min. Immediately after this, the cow was removed from the experimental plot through the standby area and returned to the barn in exchange for the next test cow. When the cow reached and ate the food reward, the food container was refilled with 1 kg concentrate before testing the next cow. Cows in the barn were not able to see the cow in the experimental plot (no opportunity for social learning). All tests were video-recorded using a video camera (HDRCX180; Sony, Tokyo, Japan). The camera was fitted with a fisheye conversion lens (0.28×; HDP-2800ES; Raynox, Tokyo, Japan) and fixed at a 3 m high position 4.5 m behind the standby area, so as to cover the whole experimental plot. 2.3. Experiment 2 Experiment 2 tested 14 cows three times on 3 separate days at intervals of 1 day (Fig. 1). This experiment was conducted 21 weeks after Experiment 1 so as to use individuals which were not tested in Experiment 1. The mean age and body weight of the cows were 7.2 ± 0.8 years and 512 ± 14 kg, respectively. They had no exposure to the experimental plot, the plastic container for the reward or the signboard showing the Landolt C prior to the experiment. We were not able to balance the age and body weight of cows between Experiments 1 and 2 because of the limited availability of animals due to the presence of a pre-weaning calf, calving, a health problem and culling. The objectives of the three tests in this experiment were the same as those of Tests 1–6 in Experiment 1. The number of tests, i.e., three times, was determined based on the results of Experiment 1, as the minimum number required for assessing the ability and motivation of cows to reach and eat the food reward. In each test, the 14 cows were individually tested and recorded in the same way as in Experiment 1. 2.4. Experiment 3 Experiment 3 tested 10 cows 11 times on the first 6 days, and 4 cows 6 weeks later (Fig. 1). This experiment commenced 17 weeks after Experiment 2 in order that individuals which showed relatively strong ability and motivation to reach and eat the food reward in Experiments 1 and 2 could be used in Tests 1–11, i.e., minimiz-
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Fig. 1. Timeline for the maze tests in the three experiments. Experiments 1 and 2 used different individuals (15 and 14 cows, respectively). In Experiment 3, Tests 1–11 used 10 cows selected from Experiments 1 and 2 (three and seven cows, respectively), and Test 12 used 4 cows selected from Tests 1–11 (one and three cows from Experiments 1 and 2, respectively). When two tests were allocated to a single day (Tests 1–10 in Experiment 3), one was conducted in the morning and the other in the afternoon.
Table 1 Completion time (s) of 15 cows in the 10 tests in Experiment 1. Cow
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Completion rate Difference in completion time from the previous test
Test (maze) 1 (no maze) 2 (no maze) 3 (no maze) 4 (no maze) 5 (no maze)
6 (no maze) 7 (L4 maze) 8 (L4 maze) 9 (L4 maze) 10 (no maze)
300 300 300 300 300 274 300 236 300 300 300 300 213 300 300 0.20 –
300 23 300 75 300 166 300 8 23 17 14 16 12 300 300 0.60 NS
242 30 300 300 300 19 300 19 36 300 18 300 24 300 300 0.47 *
103 300 300 300 300 15 300 13 27 62 14 28 12 300 300 0.53 NS
106 44 300 300 300 30 300 9 36 36 13 13 14 300 300 0.60 NS
71 23 300 300 300 14 300 12 30 30 13 7 19 300 300 0.60 NS
300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 0.00 **
300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 0.00 NS
300 300 300 300 300 300 300 300 300 300 300 300 300 300 300 0.00 NS
38 25 300 15 300 12 300 11 47 27 34 20 17 300 75 0.73 **
L4 denotes level 4. Cows which did not reach the food reward within the 5-min test time were assigned a completion time of 300 s. * P < 0.05. ** P < 0.01, NS P ≥ 0.05.
ing exclusions due to the presence of a pre-weaning calf, calving or a health problem. The 4 cows used for Test 12 were the individuals that completed the L4 maze in Tests 9 and 10 (see Section 3.2) and were available at the time of the test. The mean age and body weight of the 10 cows were 9.9 ± 1.0 years and 513 ± 22 kg, respectively. They had no exposure to the experimental plot, the plastic container for the reward or the signboard showing the Landolt C for 17–18 weeks prior to the experiment. Tests 1 and 2 were conducted in the no-maze situation to confirm that the cows retained the ability and motivation to reach and eat the food reward. Tests 3–10 investigated the ability of cows to complete the 4 levels of mazes offered in order of increasing complexity (from L1 to L4). Test 11 was carried out after removal of the L4 maze to reconfirm the ability and motivation of cows to reach and eat the food reward, and Test 12 evaluated the ability of cows to
remember the configuration of the L4 maze after a retention interval of 43 days from Test 10. In each test, the cows were individually tested and recorded in the same way as in Experiments 1 and 2.
2.5. Data analysis The video records were analyzed to quantify the two behavioral measures, the time to reach the food reward (completion time, s) and the time spent eating the food (eating time, s), for individual cows in individual tests in individual experiments. Cows which did not reach the food reward within the 5-min test time were assigned a completion time of 300 s and an eating time of 0 s. For Tests 9 and 10 in Experiment 3, the video records were analyzed also to determine the trajectory of cows within the L4 maze, using a coordinate system of 7 rows × 7 columns plus the reward area (Fig. 2e).
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Fig. 2. Layout of the experimental plot with (a) no maze, (b) level 1 maze, (c) level 2 maze, (d) level 3 maze and (e) level 4 maze, and (f) the Landolt C painted on the signboard behind the food reward. White cells numbered 1–18 and shaded cells leading to W1–W5 in the level 4 maze indicate the correct pathway to the food reward and the wrong routes to the dead end, respectively.
Because there was a strong negative correlation between the completion time and eating time in Experiments 1–3 (Spearman rank correlation (rS ) = −0.96, P < 0.001, n = 306), the completion time was selected as a performance measure of cows and subjected to the subsequent statistical analysis. Changes in the completion time in cows across the tests in individual experiments were nonparametrically evaluated using the Friedman test and the Wilcoxon signed rank sum test. The Friedman test for Experiment 3 excluded Test 12 because of the different number of test cows from the preceding tests. Fisher’s exact test was used to detect whether cows selected from Experiment 1 (with experience with the L4 maze) differed from cows selected from Experiment 2 (with no experience with the L4 maze) in the proportion of success in reaching the reward (completion rate) in Experiment 3. Differences in the animal attributes (age, liveweight and physiological state as days from the last parturition) between the successful and unsuccessful cows in the L4 maze tests (Tests 9 and 10) in Experiment 3 were evaluated using the Wilcoxon rank sum test. All statistical analyses were performed using STATISTICA (version 10; StatSoft, Tulsa, OK, USA). 3. Results 3.1. Experiments 1 and 2 In Experiment 1, only 3 cows (Cows 6, 8 and 13) out of the 15 reached and ate the food reward in Test 1 where there was no maze, showing a completion rate (CR) of 0.20 (Table 1). Under the
repeated no-maze conditions, however, the number of rewarded cows increased to seven (Cows 1, 2, 6, 8, 9, 11 and 13) in Test 2 (CR = 0.47) and remained at eight or nine thereafter in Tests 3–6 (CR = 0.53–0.60). Once learned, cows always or almost always reached and ate the food in the subsequent tests. Then, in Tests 7–9, none of the 15 cows were able to traverse the L4 maze and reach the reward (CR = 0). In the subsequent test where cows were retested under the no-maze condition (Test 10), 11 cows (Cows 1, 2, 4, 6, 8–13 and 15) reached and ate the food reward (CR = 0.73). These cows, with one exception (Cow 15), were the individuals which reached and ate the food at least once in Tests 1–6. The completion time in cows varied significantly across the 10 tests (Friedman 2 = 64.9, P < 0.001, n = 15, df = 9), showing a decrease from Test 1 to Test 2 (P < 0.05), an increase from Test 6 to Test 7 (P < 0.01), and a decrease from Test 9 to Test 10 (P < 0.01). The 5 cows (Cows 6, 8, 9, 11 and 13) which reached and ate the food reward at least 5 times out of the first six no-maze tests (Tests 1–6) were selected as candidates for Experiment 3. In Experiment 2 where cows were always tested in the no-maze situation, 5 cows (Cows 16, 18, 22, 23 and 27) out of the 14 reached and ate the food reward in Test 1 (CR = 0.36; Table 2). The number of rewarded cows increased to eight (Cows 19, 22–27 and 29) in Test 2 and remained the same in Test 3 (CR = 0.57). Once learned, cows always reached and ate the food in the subsequent test or tests, except two individuals (Cows 16 and 18). The completion time in cows varied significantly across the 3 tests (Friedman 2 = 6.5, P < 0.05, n = 14, df = 2), showing a decrease from Test 1 to Test 2 (P < 0.05). The 8 cows (Cows 19, 22–27 and 29) which reached and
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Group 1: cows selected from Experiment 1 (with experience with the level 4 maze). Group 2: cows selected from Experiment 2 (with no experience with the level 4 maze). L1, L2, L3 and L4 denote levels 1, 2, 3 and 4, respectively. Cows which did not reach the food reward within the 5-min test time were assigned a completion time of 300 s. Difference in completion time from the previous test is for data pooled over two groups. * P < 0.05. ** P < 0.01, NS P ≥ 0.05.
12 (L4 maze)
NS ** * *
NS NS NS NS NS
2
Difference in completion rate between groups Difference in completion time from the previous test
–
**
NS
53 11 15 1.00 13 15 14 16 36 38 13 1.00 NS
11 (no maze) 10 (L4 maze)
300 300 29 0.33 39 300 29 61 300 300 60 0.57 NS 300 300 80 0.33 68 300 31 102 300 300 118 0.57 NS
9 (L4 maze) 5 (L2 maze) 4 (L1 maze) 3 (L1 maze) 2 (no maze)
6 8 11 Completion rate 19 22 23 25 26 27 29 Completion rate 1
Test (maze) Cow Group
Table 3 Completion time (s) of two groups of cows in the 12 tests in Experiment 3 (10 cows in Tests 1–11 and 4 cows in Test 12).
Among the 13 candidates selected from Experiments 1 and 2, three (Cows 9, 13 and 24) had to be excluded from Experiment 3 due to calving or culling. In Experiment 3, the 3 cows derived from Experiment 1 (Cows 6, 8 and 11 as Group 1, with experience with the L4 maze) and the 7 cows derived from Experiment 2 (Cows 19, 22, 23, 25–27 and 29 as Group 2, with no experience with the L4 maze) did not differ in CR (P ≥ 0.05) in any of the 12 tests (Table 3). Across the two groups, all 10 cows reached and ate the food reward in Tests 1 and 2 where there was no maze (CR = 1; Table 3). Thereafter, the number of rewarded cows decreased to seven (Cows 6, 11, 19, 25–27 and 29) in Tests 3–4 with the L1 maze (overall CR = 0.55 (=11/20)), increased to eight (Cows 8, 11, 19, 23, 25–27 and 29) in Tests 5–6 with the L2 maze (overall CR = 0.8 (=16/20)), remained almost the same in Tests 7–8 with the L3 maze (overall CR = 0.75 (=15/20)), and decreased to five (Cows 11, 19, 23, 25 and 29) in Tests 9–10 with the L4 maze (overall CR = 0.5 (=10/20)). Then, when the maze was removed in Test 11, all 10 cows successfully reached and ate the food reward again (CR = 1). Furthermore, after a 6-week retention interval, all 4 test cows completed the L4 maze in Test 12 (CR = 1), reaching the reward as quick as in Test 10 (29–61 s in Test 10 and 26–80 s in Test 12). The completion time in cows varied significantly across the 11 tests (Friedman 2 = 72.5, P < 0.001, n = 10, df = 10; excluding Test 12 with n = 4), showing increases from Test 2 to Test 3 (P < 0.01) and from Test 8 to Test 9 (P < 0.05) and decreases from Test 9 to Test 10 (P < 0.05) and from Test 10 to Test 11 (P < 0.01). Cows differed considerably in the ability to traverse the 4 levels of mazes in Tests 3–10, despite having the ability to reach the reward in the no-maze situation (Table 3). Cow 22 was unable to traverse any of the mazes, and Cow 6 was able to complete only the L1 maze in Tests 3 and 4. Three cows (Cows 8, 26 and 27) failed to traverse the L4 maze in Tests 9 and 10 despite the completion in the L2 and L3 mazes in Tests 5–8. Cows that successfully traversed the L4 maze did not differ (P > 0.05) from unsuccessful cows in age, liveweight or physiological state. Among the 5 cows which traversed the L4 maze in Tests 9 and 10 (maximum correct cell number = 18), one (Cow 23) reached the
6 (L2 maze)
3.2. Experiment 3
300 49 37 0.67 87 300 20 21 30 47 18 0.86 NS
8 (L3 maze)
ate the food reward at least twice out of the three no-maze tests (Tests 1–3) were chosen as candidates for Experiment 3.
300 39 28 0.67 300 300 31 23 34 116 23 0.71 NS
7 (L3 maze)
Cows which did not reach the food reward within the 5-min test time were assigned a completion time of 300 s. * P < 0.05, NS P ≥ 0.05.
300 108 51 0.67 50 300 64 28 62 112 31 0.86 NS
*
300 95 52 0.67 53 300 43 42 143 68 36 0.86 NS
300 300 300 24 300 300 20 20 22 20 30 29 300 16 0.57 NS
76 300 105 0.67 216 300 300 30 300 300 160 0.43 NS
3 (no maze)
300 300 300 168 300 300 24 33 157 134 108 21 300 11 0.57
61 300 65 0.67 47 300 300 50 97 193 300 0.57 NS
2 (no maze)
216 300 253 300 300 300 157 147 300 300 300 215 300 300 0.36 –
13 12 13 1.00 26 47 17 12 14 19 14 1.00 NS
16 17 18 19 20 21 22 23 24 25 26 27 28 29 Completion rate Difference in completion time from the previous test
Test (maze) 1 (no maze)
1 (no maze)
Cow
25 12 16 1.00 20 235 19 15 15 16 18 1.00 NS
Table 2 Completion time (s) of 14 cows in the 3 tests in Experiment 2.
5
– – 26 1.00 28 – 28 80 – – – 1.00 NS
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Table 4 Maximum correct cell number reached and occurrence of wrong route selection (W1–W5) by two groups of cows in Tests 9 and 10 in Experiment 3 (level 4 maze). Group
Cow
Test
Maximum correct cell number
W1
W2
W3
W4
W5
1
6
9 10 9 10 9 10 9 10 9 10 9 10 9 10 9 10 9 10 9 10
8 10 11 11 18 18 18 18 11 8 18 18 18 18 12 11 11 11 18 18
0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1 0 1 1 0 0 1 1 1 1 0 0 0 0 0 0 1 1 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1 2 3 2 0 0 0 0 2 2 0 0 1 1 2 2 1 1 1 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
8 11 2
19 22 23 25 26 27 29
Group 1: cows selected from Experiment 1 (with experience with the level 4 maze). Group 2: cows selected from Experiment 2 (with no experience with the level 4 maze). See Fig. 2e for correct cell numbers and wrong routes (W1–W5). Maximum correct cell numbers of 18 and <18 indicate success and failure in reaching the food reward, respectively.
food reward without taking a wrong route in either test (Table 4). Two (Cows 11 and 29) reached the reward only after an error (W1 or W4) in Test 9. The other two (Cows 19 and 25) reached the reward after an error (W2 or W4) in both tests. In contrast, the 5 cows which failed to traverse the L4 maze (Cows 6, 8, 22, 26 and 27) selected a wrong route at least twice during each test and were able to follow the correct route only as far as the cell number of 8, 10, 11 or 12. Among the five wrong routes, W4 was most frequently selected (18 times), followed by W2 (7 times) and W1 (once). 4. Discussion In the current study, the ability of cows to complete the maze task and remember the maze configuration can be evaluated only when they know the presence of the food reward and have the motivation to reach and eat the reward. Performance of cows in the no-maze tests is thus a critical criterion for assessing cows’ ability. It is not surprising in Experiment 1 that cows which failed to reach the food reward throughout the initial six no-maze tests (Cows 3, 5, 7, 14 and 15) did not traverse the L4 maze thereafter (Table 1). Only cows that did not traverse the L4 maze despite the retention of the motivation to reach and eat the reward in the subsequent no-maze test (Cows 1, 2, 4, 6 and 8–13 in Experiment 1 (Table 1); Cows 6, 8, 22, 26 and 27 in Experiment 3 (Table 3)) are regarded as lacking the ability to complete the maze. 4.1. Learning of the food reward A major finding from the present study is that individual cows differed considerably in the ability to learn of the food reward in the no-maze situation in Tests 1–6 in Experiment 1 (Table 1) and in Tests 1–3 in Experiment 2 (Table 2). No cows had experience with the experimental plot including the plastic container for the reward and the signboard with the Landolt C prior to the two experiments, and they were tested individually in the tests. Hence, cows that learned of the food reward in the initial one or two tests in each experiment can be rated less subject to social isolation (i.e., less
social) and more exploratory (i.e., more curious, risk-taking and bold) than those which failed to find the reward. It has been well documented that individual animals consistently differ through time and across situations in behaviors such as aggressiveness, fearfulness, neophobia, exploration, boldness (shyness) and sociability, which is known as a behavioral syndrome, personality or temperament (Bell, 2007; Gibbons et al., 2010; Searle et al., 2010; Sih et al., 2004). Thus, the individual differences in the ability to find the food reward in the no-maze situation may be interpreted to reflect the personality of individual cows, though the current study did not evaluate the intra-animal consistency in this ability in varying situations across time (e.g., changing positions of the reward area).
4.2. Spatial learning of the L4 maze Another major finding from the current study is that some cows were able to learn to traverse the L4 maze when they were offered step-by-step learning opportunities (Tests 3–10 in Experiment 3; Table 3), whereas no cows were able to complete the L4 maze when the maze was offered immediately after the no-maze condition (Tests 7–9 in Experiment 1; Table 1). Our first hypothesis was true for 5 cows (Cows 11, 19, 23, 25 and 29) out of the 10 (Tests 9–10 in Experiment 3; Table 3). The failure in the other five (Cows 6, 8, 22, 26 and 27) was not due to the loss of the motivation to reach and eat the reward, because they were shown to retain the motivation in Test 11. When the completion rate in the L4 maze (Table 3) is applied to the number of candidates for Experiment 3 (see Section 3.1), the number of cows which have the ability to complete the L4 maze is estimated at 1.65 (=0.33 × 5) and 4.56 (=0.57 × 8) for cows selected from Experiments 1 and 2, respectively, i.e., giving a total of 6.21 cows. Thus the overall completion rate for the 29 cows used in the present study is estimated at 0.21 (=6.21/29). This estimate is much lower than the completion rate of 0.71 for barrows (Sus domesticus) subjected to a maze with five dead ends (Jansen et al., 2009) and 0.77–0.91 for sheep subjected to a maze with two error zones (Doyle et al., 2014). Variation in the success rate in a maze test can be attributed to a number of factors such as animal species, complexity of maze configuration, motivation to traverse the maze and method and frequency of training. The fact that the number of rewarded cows decreased drastically to five in the L4 maze after the relative constancy of 7–8 in the L2 and L3 mazes (Table 3) indicates the substantial difficulty of the L4 maze for cows as compared with the other mazes. Previous studies reported that the ability of sheep to learn to traverse a maze was affected by animal attributes such as sex and prenatal nutrition level (T-maze; Erhard et al., 2004), and age and experience (Y-maze; Hunter et al., 2015). However, the present study did not detect differences in age, liveweight or physiological state between successful and unsuccessful cows in the L4 maze tests (Tests 9 and 10) in Experiment 3. This may suggest the possibility of the involvement of the personality (e.g., activity, exploration and boldness) in the maze learning ability in cattle as well as the reward-learning ability in the no-maze situation. Carere and Locurto (2011) highlighted the personality as an important source of variation that may affect cognitive performance in individual animals. Sneddon (2003) investigated boldness and shyness as personality traits in hatchery-reared rainbow trout (Oncorhynchus mykiss) and found that bold fish learned a simple conditioning task more quickly than shy fish, although little is known about the linkage between personality and learning ability in cattle (Webb et al., 2015). Cow 23 who completed the L4 maze without taking a wrong route in either Test 9 or 10 (Table 4) is taken to have a marked ability of spatial learning. Cows 19 and 25 who
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traversed the L4 maze after an error in both tests are taken to have a high ability to modify their behavior to get back from the error.
differences in the spatial learning and memory are reflected in the resource exploitation and the fit to the environment as a group.
4.3. Spatial memory of the L4 maze
5. Conclusion
The other major finding from the present study is that cows were able to remember how to traverse the L4 maze after a retention interval of 6 weeks (Test 12 in Experiment 3; Table 3). The fact that cows evaluated in Test 12 reached the food reward as quickly as they did in Test 10 indicates no decay in the cows’ memory. These results support the second hypothesis. Ksiksi and Laca (2002) reported that cattle remembered the locations of food patches placed in an experimental arena for at least 48 days. Lee et al. (2006) observed that sheep retained the memory of the configuration of a relatively simple maze (with two error zones) for 6 weeks. Hunter et al. (2015) reported that sheep remembered Y-maze tasks for 22 weeks. Our study demonstrates that cattle can retain the memory of the spatial environment of an even more complex structure for at least 43 days. Further studies are warranted to determine the limitations of this ability.
Japanese Black cows are able to learn to traverse a complex maze when they are provided with step-by-step learning opportunities with increasing difficulty, although this ability is limited to some (estimated at about 20%) individuals. Once learned, cows can retain the memory of the maze configuration for up to 6 weeks. Individual cows differ in the ability of both spatial learning and memory. Further studies are needed to determine the limitations of the memory.
4.4. Effect of the Landolt C sign on cows’ motivation Motivating animals to traverse a maze is a critical part of a maze test. Previous studies used conspecifics to join (Hunter et al., 2015; Lee et al., 2006), a home pen to return to (Jansen et al., 2009) or food to eat (Erhard et al., 2004) as the reward for motivating animals. In the current study, we placed not only the food reward but also the signboard showing the Landolt C behind the reward (Fig. 2) in expectation that the sign would enhance or summon up the motivation of cows serving as the visual cue of the reward. Strictly, our study cannot prove the benefit of the Landolt C sign, because the study did not compare the cows’ performance with and without the sign. In this regard, in Experiment 3, all 10 cows successfully reached and ate the food reward in Tests 1 and 2 after the intervals of 17–18 weeks (Table 3). Furthermore, all 4 cows used in Test 12 completed the L4 maze after the interval of 43 days. These results suggest a possibility that the Landolt C made some contribution to the evocation of the motivation in cows for a period of up to 18 weeks. Use of visual cues by large mammalian herbivores to locate foods has been widely known (Edwards et al., 1997; Howery et al., 2000; Renken et al., 2008; Taylor et al., 2010), and Japanese Black cows have been reported to retain an association between a visual cue and a food reward for 1 year without reinforcement (Hirata and Takeno, 2014). It should be noted, however, that cows had not completed the L4 maze in Test 12 as quickly as in Test 10 if they had impaired or lost the memory of the maze structure, even if they had the motivation to reach the reward. 4.5. Ecological implication Our finding that only limited individuals of cattle were able to learn and remember the spatial structure of the complex maze may pose a question how individuals lacking the ability can cope with the heterogeneously distributed resources (e.g., food, water and shelter) in their habitats and home ranges. However, many large mammalian herbivores are group-living animals. Individual members of a group can use social information about the resource in their environment. For instance, individuals foraging in a group can acquire information on the location of preferred food by monitoring the foraging of others (Greenwood and Rittenhouse, 1997; Shrader et al., 2007). Thus, all members of a group may not need to have the ability to learn and remember a complex spatial structure. Further studies are necessary for investigating how the individual
Conflict of interest There are no conflicts of interest to report. Acknowledgements We thank Minamo Hamada, Fuki Hirota, Ai Matsubara, Keishi Tanaka, Moeko Uchimura, Takayoshi Saito, Yui Shiraishi, Sakura Yuda, Chie Arimoto, Tomoe Ikegami, Hiroyasu Inoue, Yuki Oshige, Rune Oda, Chihiro Shibata, Yuri Hatanaka and Naofumi Yoshida for field assistance, and Kiichi Fukuyama, Ikuo Kobayashi and Koichiro Henmi for animal management. This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS KAKENHI Grant Number 23580371; to M. Hirata). References Bailey, D.W., Sims, P.L., 1998. Association of food quality and locations by cattle. J. Range Manage. 51, 2–8. Bell, A.M., 2007. Future directions in behavioural syndromes research. Proc. R. Soc. B 274, 755–761. Carere, C., Locurto, C., 2011. Interaction between animal personality and animal cognition. Curr. Zool. 57, 491–498. Doyle, R.E., Freire, R., Cowling, A., Knott, S.A., Lee, C., 2014. Performance of sheep in a spatial maze is impeded by negative stimuli. Appl. Anim. Behav. Sci. 151, 36–42. Edwards, G.R., Newman, J.A., Parsons, A.J., Krebs, J.R., 1996. The use of spatial memory by grazing animals to locate food patches in spatially heterogeneous environments: an example with sheep. Appl. Anim. Behav. Sci. 50, 147–160. Edwards, G.R., Newman, J.A., Parsons, A.J., Krebs, J.R., 1997. Use of cues by grazing animals to locate food patches: an example with sheep. Appl. Anim. Behav. Sci. 51, 59–68. Erhard, H.W., Boissy, A., Rae, M.T., Rhind, S.M., 2004. Effects of prenatal undernutrition on emotional reactivity and cognitive flexibility in adult sheep. Behav. Brain Res. 151, 25–35. Fisher, D.O., 2000. Effects of vegetation structure, food and shelter on the home range and habitat use of an endangered wallaby. J. Appl. Ecol. 37, 660–671. Ganskopp, D.C., Bohnert, D.W., 2009. Landscape nutritional patterns and cattle distribution in rangeland pastures. Appl. Anim. Behav. Sci. 116, 110–119. Gibbons, J.M., Lawrence, A.B., Haskell, M.J., 2010. Measuring sociability in dairy cows. Appl. Anim. Behav. Sci. 122, 84–91. Greenwood, P.T., Rittenhouse, L.R., 1997. Feeding area selection: the leader–follower phenomena. Proc. West Sect. Am. Soc. Anim. Sci 48, 267–269. Hirata, M., Takeno, N., 2014. Do cattle (Bos taurus) retain an association of a visual cue with a food reward for a year? Anim. Sci. J. 85, 729–734. Howery, L.D., Bailey, D.W., Ruyle, G.B., Renken, W.J., 2000. Cattle use visual cues to track food locations. Appl. Anim. Behav. Sci. 67, 1–14. Hunter, D.S., Hazel, S.J., Kind, K.L., Liu, H., Marini, D., Owens, J.A., Pitcher, J.B., Gatford, K.L., 2015. Do I turn left or right? Effects of sex, age, experience and exit route on maze test performance in sheep. Physiol. Behav. 139, 244–253. Jansen, J., Bolhuis, J.E., Schouten, W.G.P., Spruijt, B.M., Wiegant, V.M., 2009. Spatial learning in pigs: effects of environmental enrichment and individual characteristics on behaviour and performance. Anim. Cogn. 12, 303–315. Ksiksi, T., Laca, E.A., 2002. Cattle do remember locations of preferred food over extended periods. Asian-Aust. J. Anim. Sci 15, 900–904. Lee, C., Colegate, S., Fisher, A.D., 2006. Development of a maze test and its application to assess spatial learning and memory in Merino sheep. Appl. Anim. Behav. Sci. 96, 43–51. Moe, S.R., Wegge, P., 1994. Spacing behaviour and habitat use of axis deer (Axis axis) in lowland Nepal. Can. J. Zool. 72, 1735–1744.
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