Animal Cognition

Why do rabbits have big ears? Nursing time stimulation by vocal playback of rabbit
pups
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Manuscript Number: ANCO-D-17-00086

Full Title: Why do rabbits have big ears? Nursing time stimulation by vocal playback of rabbit
pups

Article Type: Short Communication

Corresponding Author: Nilson Evilásio Souza Filho, Ph.D.

Universidade Federal de Santa Maria
santa maria, rs BRAZIL

Corresponding Author Secondary

Information:

Corresponding Author's Institution: Universidade Federal de Santa Maria

Corresponding Author's Secondary

Institution:

First Author: Nilson Evilásio Souza Filho, Ph.D.

First Author Secondary Information:

Order of Authors: Nilson Evilásio Souza Filho, Ph.D.

Ana Carolina Kohlrausch Klinger, Ms

Vinícius Mourão Alves Souza, Dr.

Geni Salete Pinto Toledo, Dra

Order of Authors Secondary Information:

Funding Information: Conselho Nacional de Desenvolvimento Ms Ana Carolina Kohlrausch Klinger

Científico e Tecnológico
Fundação de Amparo à Pesquisa do Dr. Vinícius Mourão Alves Souza
Estado de São Paulo

Abstract: The vocal behavior of rabbit pups was monitored during their first 15 days of life. It was
possible to estimate the average of pup vocalizations issued in the nest. The
vocalizations can be important in pup recognition and consequently, stimulate the doe
to nurse their offspring. We performed experiments of vocal playback of pups and
observed the behavior between the offspring and the doe during the period of lactation.
In rabbits, the vocal playback of a pup stimulates the doe to nurse the offspring.

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1 Why do rabbits have big ears?

2 Nursing time stimulation by vocal playback of rabbit pups
3
4 N.E. Souza Filho1, A.C.K. Klinger2, V.M.A. Souza3, G.S.P. Toledo2
5
6 1Engenharia Acústica, Universidade Federal de Santa Maria, Santa Maria-RS, Brazil
7 2Departamento de Zootecnia, Universidade Federal de Santa Maria, Santa Maria-RS, Brazil
8 3Instituto de Matemática e Computação, Universidade de São Paulo, São Carlos-SP, Brazil

9
10 Abstract
11
12 The vocal behavior of rabbit pups was monitored during their first 15 days of life. It was possible to
13 estimate the average of pup vocalizations issued in the nest. The vocalizations can be important in pup
14 recognition and consequently, stimulate the doe to nurse their offspring. We performed experiments of
15 vocal playback of pups and observed the behavior between the offspring and the doe during the period of
16 lactation. In rabbits, the vocal playback of a pup stimulates the doe to nurse the offspring.
17
18 Keywords: rabbit; sound pattern recognition; vocal playback; induced nursing time.
19
20 Introduction
21
22 Laparos are the pups of rabbits that accompany them from birth to weaning during the lactation period of
23 one month. They are born without hair, with eyes and ears sealed, limited motor coordination and
24 incapacity of thermoregulation (Hudson and Distel 1982). Initially, their social contact is limited to the
25 doe during lactation (Coureaud et al. 2000). It is known that the breeding phase is where the laparos are
26 more fragile, and this phase presents high rates of mortality (Hudson and Distel 1982). Rabbits present a
27 peculiar behavior and they are still less studied when compared to other animal species pups (Hudson and
28 Distel 1982). Understanding the behavior of litters assists the identification of anomalous signs, as
29 situations of stress that may contribute to the mortality rate in the breeding phase.
30
31 The means of communication in most mammals species include visual and olfactory input, body contact,
32 and pheromones in urine (Liu et al. 2013, Patris et al. 2008). Vocal communication has the special
33 importance in mother-offspring interactions in many mammals. In rabbits, pup distress call is the main
34 trigger to parental care, essential for offspring survival and development.
35
36 The communication between parent and offspring is important to adjust the care the offspring needs.
37 Signals from the young may act to indicate a pup’s immediate need, such as for thermoregulation or food
38 (Liu et al. 2013). Few studies have been conducted to investigate the communication pattern used by
39 rabbit in commercial breeding (Schuh et al. 2005). In this study we extracted the pattern of vocalizations
40 in the nest, and observe the consequences of its use during lactation period.
41
42 We perform our experiments in three stages. In stage 1, sound monitoring of four nests was carried over
43 two weeks of lactation considering typical commercial breeding conditions. Among the known methods
44 for sound pattern recognition (Souza et al. 2015, Souza Filho et al. 2014), we used, in stage 2, the
45 Spectrographic Image Template Matching (SITM) to quantifies the number of the pup calls from each
46 nest.
47
48 In stage 3, the pup call of each one of the first three nests was chosen for the vocal playback experiments
49 in the two first nests and we observed the behavior of litters through vocal communication between the
50 pups and the doe. Here, we want to know if the doe may be motivated to breastfeed the offspring with an
51 induced stimulation by vocal communication cues. We observe the conditions associated with the
52 maternal behavior of rabbits in relation to vocal communication of consanguineous and non-
53 consanguineous pups.
54
55
56 Methods
57
58 The experiments arrangements were conducted in the Cunicultura, Departamento de Zootecnia,
59 Universidade Federal de Santa Maria, Brazil. The investigation was carried out in 2016 between May and
60 June. The European rabbits (Oryctolagus cuniculus) were kept in anodized aluminum cages equipped
 61 with wooden nest boxes. The first weeks of lactation could be monitored by a recording system with
62 microphones.
63
64 Stage 1 - Nests sound monitoring: The vocalizations of the pups in four nest were continuously recorded
65 over a 24 hour period with four microphones, two measurement microphones (Superlux, ECM-999) and
66 two condenser microphones (Behringeer, C2), both pairs with a frequency range of 40 to 20000 Hz which
67 was fixed in a hole on top of the nest. The microphones were powered by a pre-amplifier audio interface
68 (Presonus Audiobox, 1818VSL) connected to a computer via USB.
69
70 Stage 2 - Identification of calls of rabbit pups: It is well established that rabbits nurse only once a day
71 (Hudson and Distel 1982, Coureaud et al. 2000, Patris et al. 2008, Schuh et al. 2005, Canali et al. 1991).
72 Furthermore, this ability is motivated by biological factors as olfactory input, body contact, pheromones
73 in urine, thermoregulation (Hudson and Distel 1982, Coureaud et al. 2000, Patris et al. 2008) and
74 especially by vocal communication. The rabbit pups usually vocalize intensely just before breastfeeding,
75 and this vocalization was recognized and quantified by SITM method (Souza Filho et al. 2014) from
76 sound files of nest monitoration.
77
78 Stage 3 - Playback Experiments: We defined the individual call vocalizations of pups as sound stimulus
79 and use the individual pup calls as the main mechanism to induce the nursing time. The sound stimulus
80 (individual call vocalizations of the pups) was recorded with a manual recorder (Tascam, Dr100 MkII) on
81 the third day of life. Recordings were sampled at 44.1 kHz with a resolution of 32 bit and were transferred
82 to a PC.
83
84 Were used different vocalizations from three nests to perform the playback experiment. Pup calls were
85 played at 45 dB, corresponding to their natural volume. Six seconds of pure tone of 440 Hz was used as a
86 control stimulus (45 dB). We standardized the amplitude of the stimuli, using the ‘normalize’ function in
87 the software Cool Edit. Playbacks were played directly from a smartphone, connected to a pre-amplifier,
88 in a rectangular speaker of 2 X 3.5 inch with 5 W and 16 Ω, camouflaged in the nest. The calls were
89 broadcasted with a peak sound pressure level (SPL) of 45 dB as measured at 1,0 m distance from the
90 loudspeaker with a sound level meter. Each female received one playback trial of pup calls.
91
92 Females distinguish between calls of their own and non-consanguineous pups. Thus, we conducted
93 playback experiments with females under two conditions. We always started with the playback of the
94 control sound. In condition 1, we tested responses of lactating females to playback of calls recorded from
95 their own pup. In condition 2, we tested responses of lactating females to playback of calls recorded from
96 non-consanguineous pup.
97
98 To analyze the behavioral response data, we consider that the occurrence of mother’s breastfeeding,
99 returning to the nest or vigilance responses were used as dichotomous dependent variables. First was
100 comparing the responses of these three variables with sound control (fixed factor with two levels). Then,
101 was compared the behavioral responses to playbacks calls of non-consanguineous and sanguineous pups.
102
103
104 Results
105
106 Were performed 360h of recordings in four nests simultaneously, 1440h of total recording. Rabbit pups
107 vocalize in the first weeks of life. The calls are uttering low whistles (duration 0.05-150 s; frequency
108 range 0.50-4 kHz) and whistles (duration 0.15-550 s; frequency range 0.50-30 kHz) and nuzzled which
109 frequently occur together in bouts. The spectrographic evaluation was conducted with 4 litters over 15
110 days. The mean litter size was (4.5 ± 0.5) pups. The quantitative analysis performed by SITM showed that
111 an average of 53 communication calls were issued in the 15-day period, or 13.3 calls/day in each litter,
112 approximately 3 calls/pup in 24 hours.
113
114 The vocalizations spectrograms used for playback are shown in Figure 1. All audios have around 7
115 seconds and the sonograms are set to show frequency ranging 0 to 5 kHz. To avoid pseudo replication
116 (Kroodsma et al. 2001), we prepared for each female a playback file with calls from a different
117 individual.
118
119 One person played the audios and another person wrote down the behavioral responses. Before playing
120 the playbacks, was assigned non-informative codes to all sound files for observer and was played them
121 for each female. With this procedure, we avoid the observer knowing which playback is from a
122 consanguineous pup. The conditions made it possible to test if the nursing time can be induced by calls
123 from consanguineous or non-consanguineous pups. The experiments were performed twice a day for six
124 days, from the fourth day of life of the pups. The behaviour of mother and offspring was observed, and it
125 was checked if the doe was induced to vigilance; return to nest, or breastfeeding. The Table 1, Fig.2 and
126 Fig.3 show the results of the playback experiments
127
128
129 Discussion
130
131 A rabbit’s ear contains an extensive network of blood vessels that provide a large surface area for heat
132 exchange, they assist their thermoregulation (Hill and Veghte 1976). Rabbits can pick up sounds from
133 considerable distances, perhaps up to 3km. Their ears can be rotated 270 degrees to help detect the source
134 of a sound, and can revolve independently to monitor different noises. In various species, offspring calls
135 (distress cries, fear screams) are emitted in situations of direct threat when young are injured or at risk of
136 being killed (Rödel et al. 2013). Scientist already conducted playback experiments with animals of a field
137 enclosure population to test whether pup distress vocalizations induce maternal behaviours indicative of
138 offspring defense (Rödel et al. 2013). But they never used calls from consanguineous pup.
139
140 We worked under commercial conditions of captivity. Four nests were monitored for 15 days. From the
141 sound recordings it was possible to estimate the average of calls issued in the nests using the SITM
142 pattern recognition. It was determined that the pups issued on average 13.3 calls/day in each litter.
143 Mothers leave their altricial young in a burrow and only visit them for a brief nursing bout of around 3
144 min once a day (Hudson and Distel 1982). We used a vocal playback of non-consanguineous and
145 consanguineous pups to stimulate the nursing time. Animals were considered to be vigilant when they
146 abruptly raised the head above shoulder height while maintaining the feeding posture, or adopted an
147 upright posture, either quadrupedal or bipedal, lifted the ears, often looked around and stopped all current
148 activities (Rödel et al. 2013). Return to nest or exploration at the burrow was defined as sniffing with
149 head down to the ground with frequent head movements to left and right, while the animal was moving
150 slowly or stopped moving forward (Rödel et al. 2013) . Breastfeeding is considered when the doe stayed
151 in a position allowing pup to suckle, as the doe's ability to actively eject milk is the primordial phase of
152 nursing time.
153
154 The graphs in Fig. 2 show that mother rabbit does not respond to any signal. With playback of 440 Hz
155 control signal, it presented the vigilance behavior in 18% of the cases, returned to the nest 9.1% and did
156 not breastfeed. While for pups calls, the doe always shows the behavior of vigilance, 80% returned to the
157 nest and breastfeeding 18% of times.
158
159 The graphs in Fig.3 confirm that the does always show the behavior of vigilance for any call, but for own
160 pup call, the does always return to the nest and 70% of the times they breastfeeding the offspring. While
161 for calls of non-consanguineous pups, such rabbits returned to nest 75% but did not breastfeed the
162 offspring. This evidence suggests the realization of new similar experiments with a larger sample, to
163 study the cognitive relation between vocal communication and rabbits maternal care.
164
165 The doe responds significantly more strongly to playback of calls from its own pups than to calls from
166 non-consanguineous pups, suggesting that rabbit mothers are able to use vocal cues in pup recognition.
167 Our playback experiments show that acoustic cues are relevant in mother-pup Oryctolagus cuniculus
168 communication. The vocalization of the pups cannot be the exclusive trigger that animates the doe to
169 nurse, but it was observed that the pup calls are the main mechanisms. This new concept of stimulation of
170 nursing time by vocal reproduction of a pup may be useful in many ways, as in control of malnutrition
171 and mortality in the breeding phase or even parental brain analysis during induced of maternal care.
172
173 Acknowledgments
174 Capes. Fapesp.
175 Prof. Lucio Strazzabosco Dorneles.
176
177 Compliance with ethical standards
178
179 Conflict of interest
180 The authors declare that they have no conflict of interest.
181
182 Ethical approval
183 All applicable international, national, and/or institutional guidelines for the care and use of animals were
184 followed. No specific permissions were required under Brazilian law for this noninvasive study.
185 References
186
187 Canali, E., Ferrante, V., Todeschini, R., Verga, M., & Carenzi, C. (1991). Rabbit nest construction and its
188 relationship with litter development. Applied Animal Behaviour Science, 31(3-4), 259-266.
189 doi:10.1016/0168-1591(91)90010-U
190
191 Coureaud, G., Schaal, B., Coudert, P., Rideaud, P., Fortun-Lamothe, L., Hudson, R., & Orgeur, P. (2000).
192 Immediate postnatal sucking in the rabbit: Its influence on pup survival and growth. Reproduction
193 Nutrition Development, 40(1), 19-32. doi:10.1051/rnd:2000117
194
195 Hill, R. W., & Veghte, J. H. (1976). Jackrabbit ears: surface temperatures and vascular responses.
196 Science, 194(4263), 436-438. doi:10.1126/science.982027
197
198 Hudson, R., & Distel, H. (1982). The pattern of behaviour of rabbit pups in the nest. Behaviour, 79(2),
199 255-271. doi:10.1163/156853982X00292
200
201 Kroodsma, D. E., Byers, B. E., Goodale, E., Johnson, S., & Liu, W. C. (2001). Pseudoreplication in
202 playback experiments, revisited a decade later. Commentaries, Animal Behaviour, 61, 1029–1033.
203 doi:10.1006/anbe.2000.1676
204
205 Liu, H. X., Lopatina, O., Higashida, C., Fujimoto, H., Akther, S., Inzhutova, A., ... & Sumi, K. (2013).
206 Displays of paternal mouse pup retrieval following communicative interaction with maternal
207 mates. Nature communications, 4, 1346. doi:10.1038/ncomms2336
208
209 Patris, B., Perrier, G., Schaal, B., & Coureaud, G. (2008). Early development of filial preferences in the
210 rabbit: implications of nursing-and pheromone-induced odour learning?. Animal Behaviour, 76(2), 305-
211 314. doi:10.1016/j.anbehav.2008.01.017
212
213 Schuh, D., Hoy, S., & Selzer, D. (2005). Vocalization of rabbit pups in the mother-young relationship.
214 In Proceedings of the 8th World Rabbit Congress, September 7-10, 2004, Pueblo, Mexico (pp. 1266-
215 1270). World Rabbit Science Association (WRSA).
216 www.cabdirect.org/cabdirect/abstract/20053160942
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218 Souza, V. M., Batista, G. E., & Souza-Filho, N. E. (2015, July). Automatic classification of drum sounds
219 with indefinite pitch. In Neural Networks (IJCNN), 2015 International Joint Conference on (pp. 1-8).
220 IEEE. doi:10.1109/IJCNN.2015.7280342
221
222 Souza Filho, N. E., Oliveira, B. C., da Silva, M. L., & Vielliard, J. (2014). Automatic classification of
223 Turdus rufiventris song notes by spectrographic image template matching. Ciência e Natura, 36, 646.
224 doi:10.5902/2179460X11303
225
226 Rödel, H. G., Landmann, C., Starkloff, A., Kunc, H. P., & Hudson, R. (2013). Absentee Mothering–Not
227 So Absent? Responses of European Rabbit (Oryctolagus cuniculus) Mothers to Pup Distress Calls.
228 Ethology, 119(11), 1024-1033. doi:10.1111/eth.12149
229
230
231
232 Figure 1 – Spectrogram of pup calls for playback experiments. (a) Call of individual pup from nest A; (b)
233 Call of individual pup from nest B; (c) Call of individual pup from nest C;
234
235 Table 1 – Behavioral responses of the doe to playback experiments, in which playbacks A, B and C were
236 set in Figure 1.
237
Playback Doe of Nest A Doe of Nest B Playback Doe of Nest A Doe of Nest B
18/06/2016; 8am: 19/06/2016; 8am: 18/06/2016; 8am: 19/06/2016; 8am:
Control Vigilance [X] Vigilance [ ] A Vigilance [X] Vigilance [X]
440 Hz Return to nest [ ] Return to nest [ ] Return to nest [X] Return to nest [X]
Breastfeeding [ ] Breastfeeding [ ] Breastfeeding [X] Breastfeeding [ ]
19/06/2016; 7pm: 19/06/2016; 7pm:
Control -------------------- Vigilance [ ] A -------------------- Vigilance [X]
440 Hz Return to nest [ ] Return to nest [X]
Breastfeeding [ ] Breastfeeding [ ]
20/06/2016; 8am: 21/06/2016; 8am: 20/06/2016; 8am: 21/06/2016; 8am:
Control Vigilance [X] Vigilance [ ] B Vigilance [X] Vigilance [X]
440 Hz Return to nest [X] Return to nest [ ] Return to nest [X] Return to nest [X]
Breastfeeding [ ] Breastfeeding [ ] Breastfeeding [ ] Breastfeeding [ ]
20/06/2016; 7pm: 21/06/2016; 5pm: 20/06/2016; 7pm: 21/06/2016; 5pm:
Control Vigilance [ ] Vigilance [ ] B Vigilance [X] Vigilance [X]
440 Hz Return to nest [ ] Return to nest [ ] Return to nest [X] Return to nest [X]
Breastfeeding [ ] Breastfeeding [ ] Breastfeeding [ ] Breastfeeding [X]
22/06/2016; 8am: 23/06/2016; 7pm: 22/06/2016; 8am: 23/06/2016; 7pm:
Control Vigilance [ ] Vigilance [ ] C Vigilance [X] Vigilance [X]
440 Hz Return to nest [ ] Return to nest [ ] Return to nest [X] Return to nest [ ]
Breastfeeding [ ] Breastfeeding [ ] Breastfeeding [ ] Breastfeeding [ ]
22/06/2016; 5pm: 24/06/2016; 8am: 22/06/2016; 5pm: 24/06/2016; 8am:
Control Vigilance [ ] Vigilance [ ] C Vigilance [X] Vigilance [X]
440 Hz Return to nest [ ] Return to nest [ ] Return to nest [X] Return to nest [ ]
Breastfeeding [ ] Breastfeeding [ ] Breastfeeding [ ] Breastfeeding [ ]
238
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284 Figure 2 – Percentage of behavioral response to playback of 440 Hz and pup call. (a) Vigilance; (b)
285 Return to nest; (c) Breastfeeding. Proportions of positive responses are given inside the bars.
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345 Figure 3 – Percentage of behavioral response of (a) Total number of playbacks; (b) Playback of non-
346 consanguineous pup; (c) Playback of consanguineous pup. Proportions of positive responses are given
347 inside the bars.
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349