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Ultrasonic vocalisations of kits during maternal kit-retrieval in farmed mink, Mustela vison
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Applied Animal Behaviour Science 114 (2008) 582–592 www.elsevier.com/locate/applanim
Ultrasonic vocalisations of kits during maternal kit-retrieval in farmed mink, Mustela vison Karin Tubbert Clausen a, Jens Malmkvist b,*, Annemarie Surlykke c a
University of Aarhus, Department of Biological Sciences, Building 1131, C.F. Moellers Alle`, DK-8000 Aarhus C, Denmark b University of Aarhus, Department of Animal Health, Welfare and Nutrition, P.O. Box 50, DK-8830 Tjele, Denmark c University of Southern Denmark, Institute of Biology, Campusvej 55, DK-5230 Odense M, Denmark Accepted 26 March 2008 Available online 12 May 2008
Abstract Mink kits are born immature and are dependent on their dams’ protection, warmth and milk for survival. We hypothesize that vocalisations by mink kits may be of importance for communication between kit and dam, increasing the individual’s chance of survival. However, very little is known about the nature and function of mink kits’ vocalisation. In the present study, we aimed to (i) describe the kit vocalisation, when it is placed away from warm nest/dam and littermates, (ii) investigate the influence of these vocalisations on maternal behaviour, in terms of dams orientation towards kit and kit-retrieval, and (iii) test whether active/ stereotyping dams differed from less active/no-stereotyping dams in their kit-retrieval response. We used 58 first parity 1-year-old female mink of two genetic lines (colour type ‘Palomino’, n = 23, and ‘Black’, n = 35), allocated into equal-sized groups of active (including stereotyping) or passive (no stereotypic behaviour observed) individuals. All recordings were made during a standardised kit-retrieval test performed 5 days after birth. Our recordings demonstrated for the first time that mink kits are able to produce complex ultrasonic vocalisation with energy up to 50 kHz. The calls consist of long trains of multiharmonic pulses with a relative long pulse duration (average 264–874 ms) and a high repetition rate (0.6–2.3 pulses/s). The pulses contained most energy between 2.5 and 34 kHz (mean of F max and F min). Delayed maternal kit-retrieval increased the number of mink vocalisations (P = 0.007) but the repetition rate, average and S.D. in duration of pulses could not, in isolation, be identified as significant parts of the calls eliciting the maternal kit-retrieval response (P > 0.1). Genetic line affected both kit vocalisations and maternal retrieval since Palomino kits had a higher variation in pulse duration than Black kits (P = 0.023), and Palomino females had an impaired kit-retrieval compared to Black females (P = 0.008). We found no significant difference in kit-retrieval behaviour between groups of females according to their activity level.
* Corresponding author. Tel.: +45 89991314; fax: +45 89991500. E-mail addresses: [email protected] (K.T. Clausen), [email protected] (J. Malmkvist). 0168-1591/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.applanim.2008.03.008
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In conclusion, mink kits do produce ultrasonic vocalisation when isolated from their mothers and nests. Genetic and other factors influenced both (i) the nature of kit vocalisations (effects of colour type and litter size), and (ii) the maternal behaviour (effects of colour type and kit gender) during a standardised kitretrieval test. # 2008 Elsevier B.V. All rights reserved. Keywords: American mink; Behaviour; Mustela vison; Parent–offspring communication; Ultrasonic vocalisation
1. Introduction Specialized parent–offspring communication has evolved in several species of animals, and may have consequences for fitness and reproductive outcome (as exemplified for some species of domestic mammals in Nowak et al., 2000). For infants that are born immature and blind, e.g. mink kits, vocalisation may be a particular important means of communication. The American mink, Mustela vison, is a seasonal breeder and in the Northern hemisphere births are concentrated during a short period, from late April to early May. The altricial mink kit is dependent on the dam’s protection and care, and the warmth in the nest for survival (Tauson et al., 2006; Malmkvist et al., 2007). We hypothesize that the vocalisations by mink kits are an important part of the communication between kit and dam, increasing the individual’s chance of survival. However, no published studies have previously focused on the nature and function of vocalisations in mink kits. Young rodents typically vocalise during isolation from their dam and/or nest (Mandelli and Sales, 2004; Ehret, 2005), and we expect the mink kits to emit calls when they are isolated from their dam, nest and littermates. During high predation risks some mammals lower their vocalisation while a stronger vocalisation more often is produced when there are no predators around. In addition, odour and tactile stimuli also affect the vocalisation of rodent pups (Hofer, 1996). Nearly all existing studies on mustelid vocalisation have focused on adults only. In these studies, reports of vocalisation never exceeded 8 kHz during recordings of Long-Tailed Weasel, Mustela frenata, (Svendsen, 1976), Least Weasel, Mustela nivalis (Huff and Price, 1968; Svendsen, 1976), Siberian Ferret, Mustela eversmanni (Farley et al., 1987) and American mink, M. vison (Gilbert, 1969). One experiment tested the vocalisation of an adult Least Weasel female and her four 3-week offspring and detected no ultrasonic vocalisation (Huff and Price, 1968). These findings may be a consequence of the limited performance of the equipment used (e.g. in Huff and Price, 1968 and Svendsen, 1976), rather than a true reflection of the mustelid vocalisations. Thus, the previous recordings may have revealed the lower harmonics of multiharmonic signals. We therefore wanted to measure vocalisations in mink with equipment sensitive to higher frequencies (up to 100 kHz). In smaller mammals, ultrasonic vocalisation around 50 kHz has been reported in rodents such as voles (Mandelli and Sales, 2004), rats (Hofer, 1996; Knutson et al., 1999; Burman et al., 2007) and mice (Holy and Guo, 2005). For 3-day-old common voles frequencies up to 35 kHz were found and for field voles a frequency range of 25–60 kHz (Mandelli and Sales, 2004). We wanted to examine if mink kits emit ultrasonic sound and if so to describe spectral and temporal aspects of their sounds, when they vocalise during isolation from the dam and nest. In addition, we investigated whether kit’s vocalisation influences the kit-retrieval by the dam. In one study, stereotyping female mink were associated with a higher fertility, as they produced larger litters and had a lower mortality rate among their kits (Jeppesen et al., 2004). In
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addition, females with low early kit mortality performed more active kit-directed behaviour than more passive females with high kit mortality (Malmkvist et al., 2007). In the present study, we therefore tested whether active and stereotyping mink differ in their kit-retrieval response compared to more passive mink dams, during a standardised test situation. 2. Animal, material and methods 2.1. Animals and housing A total of 80 mated female mink, M. vison, of Palomino and Black genetic lines in their first parity were kept in standard wire cages (W: 30 cm, H: 45 cm, L: 91 cm) connected to a straw covered wooden nest box (W: 28 cm, H: 20 cm, L: 23 cm) at the farm unit at Research Centre Foulum, University of Aarhus, Denmark. Mink were kept under conventional farming conditions with food and water ad libitum. The experimental mink were given equal and ad libitum access to straw for nest building, since a previous study has shown that access to straw in the peripartureint period may affect the maternal behaviour in mink (Malmkvist and Palme, 2008). The temperature in the house was similar to the outside temperature. An empty cage separated each female. Prior to the experiment, the mink were selected according to their level of activity: active (n = 40) and passive (n = 40); the proportion of Black (65 %) and Palomino (35 %) mink was equal in the two activity groups. This selection was based on 16 scanning observations during 4 days of mild feed restriction in January 2007, using an original population of 300 individuals. The active females were active (including stereotyping) in more than 40% of all observations, while passive females were not observed active (i.e. running, stereotyping) during any of the observations. Barren females and females without kits on the fifth day after birth were excluded from further investigation, leaving 58 female mink with young (n = 31 active, n = 27 passive; average litter size: 5.6 0.29, range 1–9). Twenty-three of these (40%) were Palomino mink and thirty-five (60 %) were Black. There were no differences in the litter size between the colour types (t-test: T56 = 0.9; P = 0.36) or between the activity groups (active vs. passive, t-test: T56 = 0.4; P = 0.71). 2.2. The kit-retrieval test The kit-retrieval test and simultaneous recording of kit vocalisation were carried out on the fifth day after birth, between 10 am and 1 pm during the period from 30th April to 14th May 2007. Two persons performed this test; one person handled the animals and registered the behaviour based on direct observations using a handheld computer while another person recorded sound. The kit-retrieval test has been developed as a measure of maternal reactivity towards a 5-day-old progeny placed outside the nest (Malmkvist and Houbak, 2000). The handler selected one kit from the litter located inside the nest box, and after restricting the dam in the nest box, placed the kit in the middle of the wire cage (a specific point 31 cm from the opening of the nest box) with its head directed towards the nest box entrance. The test started when the female regained access to the wire cage and stopped when she retrieved the kit into the nest box. The observer registered latency to touch kit and latency to retrieve kit into nest. In the case of no kit-retrieval within a test time of 240 s, the test stopped, and the observer returned the kit to the nest. To test the effect of gender we alternately tested a female and a male kit. Each dam/kit pair was tested once. We used a fixed waiting time of 20 s for the female in the nest box, before the slide gate was removed and retrieval of the kit was possible. 2.3. Recording of kit vocalisation during tests For recordings we used a 1/400 (0.64 cm) microphone (Type 4135, Bru¨el & Kjaer, Denmark), placed in a holder 74 cm above the cage floor and connected through a pre-amplifier (Bru¨el & Kjaer 2633) to an amplifier (Microphone power supply type 2804, Bru¨el & Kjaer) set at 40 dB amplification. The microphone was pointed towards the vocalising kit in the initial position. The amplifier output was digitized with a
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DAQcard (sampling rate: 200 kHz 12-bit, DAQCard-6062E, National Instruments) and stored on a laptop (HP Compaq NC 8430) controlled by BatSound Pro Software (version 3.1, Pettersson Elektronik AB, Sweden). The recordings started from the start of the kit-retrieval test and consecutive files of 60 s were stored. 2.4. Analysis of recorded vocalisations We used the Batsound Pro Software to analyze the recordings. To reduce background noise, the files were high-pass filtered 1000 Hz (Butterworth, filter order six). For the analysis, we first listened to the recordings by playing back the files through headphones at one-fourth of the recording speed. Only periods where the test-kit could be identified and isolated were used for analysing the vocalisations. The vocalisations were displayed as oscillograms for analysis of temporal parameters and as spectrograms or spectra for analysis of spectral parameters. For all kits in the test we randomly chose 10 pulses and determined the mean and standard deviation of pulse duration and the repetition rate, defined as vocal pulses per second. Vocalisations of three randomly chosen kits were analyzed in detail to describe variation and spectral characteristics of the sounds. For each of the three kits we analyzed five pulses, two from the beginning, one from the middle, and two from the end of a vocalisation. The vocalisations were displayed as spectrograms to determine number of harmonics, and frequency modulations with time. The vocalisations were also displayed as spectra and minimum frequency (F min) and maximum frequency (F max) were determined as 20 dB low frequency and 20 dB high frequency cut-off. Bandwidth (BW20 dB) was equal to the distance between F min and F max. Due to the relatively long duration and slow frequency modulation of the pulses we used a 2048 sample FFTwindow for spectral analyses giving a frequency resolution of 97.7 Hz. 2.5. Statistical analyses 2.5.1. Description of the kit vocalisations We analyzed three temporal variables of kit pulses using an ANOVA in the statistical software SAS (version 9.1, SAS Institute Inc., USA): (1) repetition rate, (2) mean pulse duration, and (3) standard deviation (S.D.) of pulse duration. The models included sex (male or female test kit), colour type (Palomino or Black), dam activity group (active or passive) as explanatory class variables, and handling time of the dam during test (21–194 s) and litter size (1–9 kits) as explanatory covariates. The model also included the interactions between sex and colour type, and between sex and activity group. The models were reduced by stepwise removal of insignificant terms (P > 0.10), starting with the interactions. The data were normally distributed. Logarithmic transformation of the response variables was used to stabilise the variance as it resulted in better residuals. The demand for dispersion and variance homogeneity was tested and the validity of the final model justified by the appearance of the residuals. 2.5.2. Factors influencing dam behaviour in the kit-retrieval test Initially, we used simple chi-square test with one degree of freedom to test whether the proportion of retrieved and non-retrieved kits differed according to dam activity group (active, passive), sex of the test kit and the colour type (Palomino, Black). The latencies for the dam to touch and to retrieve the kit were strongly correlated (r = 0.92, P < 0.001) and therefore only the latency for the dam to retrieve the kit was tested further. The latency for the dam to leave the nest and latency for kit-retrieval were also, but less strongly correlated (r = 0.52, P < 0.001). Latencies for leaving nest and for retrieving the test kit were analyzed using methods for survival analysis (Allison, 1995), considering censored data (thus including dams that did not leave the nest/retrieve within the maximum test time of 240 s as right censored observations), using the computer software SAS with models including sex, colour type, dam activity group, repetition rate, mean and S.D. of pulse duration. A probability level (P) of 0.05 was chosen as a limit for statistical significance in all tests. P-values between 0.05 and 0.1 are reported as trends in the results.
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3. Results 3.1. Description of kit vocalisation Vocalisation occurred in 30 out of the 58 performed kit-retrieval tests. Useful vocalisation data from the test kits were obtained for 70% of these (n = 21) and the three main observed variables of vocalisation are summarized in Table 1. The rest of the files were not analyzed further because of recording failures or due to background noise masking the vocalisations of the test kit. The three kits randomly chosen to illustrate the nature and variation of vocalisation between and within individuals were not subjected to statistical analyses, but the results are presented in Fig. 1. Each kit varied in pulse duration, frequency modulation rate and direction, as well as in relative energy of harmonics. There were individual characteristics making it easy to discriminate between individuals when listening to the slowed down version of the recordings. Kit I (Fig. 1A and B left panel) emitted pulses which typically started with an initial upward frequency modulation, followed by a section with more shallow or no modulation, to end with a steeper downward frequency modulation. The pulses were multiharmonic with a first harmonic (fundamental) around 2.4 kHz in the middle part of the pulse. The second harmonic had more energy than the first harmonic, and all harmonics up to number 10–12 had high energy. Which of those harmonics had maximum energy changed within and between pulses. Low and high frequency limit and bandwidth of the sounds were determined from an average spectrum of 30 pulses. At low frequencies, noise from surroundings and other nearby minks obscured the low frequency limit, so F min in the spectra was set by the high pass filter: 1 kHz, but the first harmonic of the multiharmonic pulse (ca. 2.2–2.4 kHz) was probably the lower limit of the kit’s own emission. F max was 45 kHz, and thus the bandwidth BW20 dB was approx. 42 kHz for kit I (Fig. 1B left panel). Kit II and III both made more complicated frequency modulation patterns sweeping up and down more than once during a pulse (Fig. 1B). The average spectra of 30 pulses showed that Kit II had a first harmonic around 2.4 kHz, an F max of 42 kHz, giving a BW20 dB = 40 kHz (Fig. 1B middle panel). Kit II emitted relatively low energy at midfrequencies from 10 to 20 kHz (Fig. 1C). Kit III emitted the highest frequencies of the three. Its average spectrum showed a first harmonic of approx. 2.1 kHz, an F max of 51 kHz and thus BW20 dB of 49 kHz (Fig. 1B right panel). Analysis of the calls from all 21 kits confirmed this by showing large variation within and between individual kit’s vocalisations. The vocalisations or calls consisted of a long train of pulses (as exemplified in Fig. 1A). All pulses were multiharmonic as described above for the three selected kits with a first harmonic between 2 and 3 kHz and energy up to around 50 kHz. Our data clearly demonstrate that mink kits do indeed produce vocalisations with much energy at ultrasonic frequencies. The pulse durations were between 264 and 874 ms and the repetition rate relatively high averaging 1.3 pulses per second (Table 1). However, there were considerable Table 1 Repetition rate, mean and standard deviation in pulse duration of vocalisations of 5-day-old mink kit (n = 21) during kitretrieval test, given as mean (S.E.M.) and range of the observations
Pulse repetition rate in vocalisation (1/s) Mean pulse duration (ms) S.D. pulse duration (ms) a
Mean (S.E.M.)
Range (minimum–maximum)
1.26 (0.090) 537 (38.4) 104 (11.4)a
0.59–2.31 264–874 33–235
Significant effects of colour type and litter size were found for this variable, please refer to the text.
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Fig. 1. Vocalisations from three mink kits. (A) Shows a train of pulses from Kit I with spectrograms below, illustrating the variation, but also the similarity in general structure of pulses, which were slow frequency modulated multiharmonic pulses with significant energy up to at least the 10–12th harmonic. (B) Shows in the left panel expanded spectrograms of two of the pulses (marked with asterisks) from Kit I, and for comparison two pulses from Kit II in the middle panel and Kit III in the right panel. (C) Shows average spectra of 30 pulses from each of the three kits as indicated by the colour code. Due to the frequency modulations the spectra do not show the multiharmonic structure of the pulses, but illustrate that the pulses contain much energy up to frequencies of 40–50 kHz.
variations in the vocalisation within each kit and between kits during the test. The detailed acoustic analysis not only revealed a surprisingly complex structure of the vocalisations, but also variation within and between kits of all parameters. Thus, kit vocalisations differed in the acoustic parameters: duration, number of harmonics, relative weight (energy) of harmonics and
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Fig. 2. Variation (S.D.) in pulse duration (ms) of kit vocalisation during test in relation to litter size. The variation in pulse duration fell with increasing litter size of the tested kit (P = 0.002).
modulation rate. The results also demonstrated that these parameters could change from pulse to pulse, allowing individual kits a large repertoire of possible acoustic communication. Pulse durations averaged 516 ms for Black kits and 573 ms for Palomino kits, and the overall average pulse duration was 537 ms (Table 1). The mean durations were not significantly different, but standard deviation of pulse duration was significantly affected by both the colour type of mink (F 1,18 = 6.15, P = 0.023) and the litter size (F 1,18 = 12.96, P = 0.002; Fig. 2). Palomino kits had a larger standard deviation (110 ms) of pulse duration than Black kits (100 ms). We found no significant effects of the tested factors on the repetition rate. However, the repetition rate of pulses within the test kit vocalisation may increase with increasing litter size, as reflected by a non significant trend in data (F 1,19 = 3.83, P = 0.065). 3.2. Factors influencing dam behaviour on the kit-retrieval test Simple chi-square tests showed that a higher proportion of female kits than male kits were retrieved (female: 97% vs. male: 79%, x21 ¼ 4:062, P = 0.044) and Palomino dams retrieved a lower proportion of their kits than did Black dams (73% vs. 97%, x21 ¼ 7:057, P = 0.008), whereas there were insignificant effects with regard to active/passive dams on the retrieval/nonretrieval of kit (P = 0.55). Survival analysis of latency performed on all data (i.e. including those that did not react within the test time of 240 s) showed an effect on the latency for dam to leave the nest with regard to gender. Latency for dams to leave the nest was shorter when the kit was a female (median (quartiles 25%; 75%): 8 (3; 21) s; n = 29, 0% censored) than when it was a male (16 (6; 34) s; n = 29, 20.7% censored). Hazard ratio (HR) for female/male test kit was 2.2 (x21 ¼ 7:21, P = 0.007) indicating that female kits resulted in dams leaving the nest sooner. The colour type of mink showed a significant effect on the latency for dams to leave the nest (HR for Palomino/ Black = 0.3, x21 ¼ 15:5, P < 0.001), Palomino dams leaving their nests markedly later (24 (16; 46) s, n = 23; 21.7% censored) than Black dams (7 (2; 16) s, n = 35; 2.9% censored). Palomino dams had a longer latency of kit-retrieval (68 (48; 123) s, n = 23, 26.1% censored) than Black dams (24 (17; 46) s, n = 35, 2.9% censored) (HR for Palomino/Black = 0.3,
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Fig. 3. Cumulative probability of kit-retrieval during the test time of 240 s, with upper and lower 95% confidence intervals. Black colour type: solid black line, Palomino colour type: dotted blue line. The latency of kit-retrieval was significant shorter in Black (median 24 s) than in Palomino dams (median 68 s; P < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
x21 ¼ 15:2, P < 0.001), with no significant effect of any of the other tested factors, e.g. gender of the test kit (HR female/male = 1.3, P = 0.36). Fig. 3 illustrates the cumulative probability of the kit-retrieval latency in the two colour types. In a supplemental ANOVA including the retrievers only (n = 51, 23 without and 28 with kit vocalisation), latency of maternal kit-retrieval and the number of vocalisations in mink kits were positively related (F 1,46 = 7.9, P = 0.007). However, we failed to demonstrate significant effects of any of the elements within the kit vocalisations we measured – i.e. the repetition rate, the mean and S.D. of pulse duration – on the latencies of the dam to leave the nest (P > 0.1) and to retrieve her kit (P > 0.1). 4. Discussion We found that 5-day-old American mink kits do produce ultrasonic vocalisation when isolated from their mothers and nests. To our knowledge, this study is the first to measure mink kits’ vocalisation with equipment that is sensitive to high frequencies. Our results show that mink kits produce almost continuous trains of multiharmonic ultrasonic pulses of relatively long duration and with high pulse repetition rate. Numerous studies of ultrasonic vocalisation have been performed in young rodents. Most rodents produce ultrasonic vocalisations that are relatively short (20–80 ms; Hahn et al., 1998; Mandelli and Sales, 2004), whereas mink kits produce pulses of about 10 times longer duration (mean 264–874 ms). The pulse duration in calls emitted by kits resemble that of calls of adult mink (50–100 ms; Gilbert, 1969) and other adult mustelids; the Long-Tailed Weasel (150– 350 ms; Svendsen, 1976), the Siberian Ferret (88–1300 ms; Farley et al., 1987), and the Least Weasel (130–510 ms; Huff and Price, 1968). Palomino kits produced less uniform pulses when vocalising compared to Black mink kits. This is to our knowledge the first time that fundamental differences in kit vocalisations have been demonstrated between genetic lines of mink. Ultrasonic vocalisation characteristics have also
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been found to be influenced by genotype in mice pups, Mus musculus (Hahn et al., 1997, 1998; Thornton et al., 2005). Furthermore, the dams from the two genetic mink lines differed markedly in their behaviour in the kit-retrieval test, with Palomino dams being slower and less effective. Studies have demonstrated hearing deficits developing in certain colour types of farmed mink e.g. the white ‘Hedlund’ mink (Flottorp and Foss, 1979) but we have at present no data on differences in adult hearing capacity in Black and Palomino mink. The complex pulse structure and individual characteristics in all acoustic parameters between kits would potentially allow for individual recognition by females; however this has not yet been studied in mink. In addition, the variation over time between and within pulses indicates that acoustic parameters might serve to disclose the present state of the kit. We standardised the age of testing (day 5 postpartum) but had variable litter sizes. Litter size affected the vocalisations, since kits from larger litters produced pulses with more homogeneous durations within their calls. Litter size and kit weight may be negatively correlated in the early period after parturition (Hoy et al., 1998; Malmkvist and Palme, 2008). It is currently unknown whether kit weight affects the nature of vocalisation and thereby whether the effect of litter size on pulse homogeneity partly reflects differences in body weights. In rat pups, a positive correlation between call duration and body weight has been reported during cold exposure (Tonkiss et al., 2003). Heat loss and deviation from the normal nest temperature may elicit ultrasonic vocalisations in young rodents (Hofer, 1996; Ehret, 2005). Mink is altricial (like rat and mice) in the early postnatal period and unable to thermoregulate independently until 22–29 days of age (Harjunpaa¨ and Rouvinen-Watt, 2004; Tauson et al., 2006). The heat loss during cold exposure increases linearly with decreasing body weight (tested 8–9 days postpartum in Malmkvist, 2006); presumably a consequence of the higher surface to volume ratio in the smaller kits. Other factors than heat loss may affect the level of offspring vocalisation. Mandelli and Sales (2004) suggested that the interval since infant Short-Tailed field voles had fed could influence the vocalisation. We have no observations of the timing of suckling in the mink kits but any nursing would be interrupted as the kit-retrieval test was prepared. In mice and rats vocalisations of infants play a role in retrieval, initiating maternal orientation towards the stimulus source, followed by the retrieval of a pup back to the nest (Hahn et al., 1998; Ehret, 2005). Hahn et al. (1998) further suggested that the call length of an ultrasonic vocalisation produced by an infant mouse and perceived by the mother would allow her to make a decision about whether or not to retrieve the pup. In addition, results suggest that adult laboratory rats, Rattus norvegicus, vocalise around 22 kHz as a reflection of a negative emotional state (Burman et al., 2007). In our study, delayed maternal kit-retrieval increased the occurrence of vocalisation in mink kits. This may indicate that the functional role of the vocalisation of isolated mink kits is to elicit maternal kit-retrieval. It is also evident that other factors than calls play a role, since a large proportion of the non-vocalising kits (82%) were successfully retrieved as well. Thus, the ultrasonic vocalisation is not the ultimate or only factor involved in dam decision-making during the kit-retrieval test. It may also be that offspring placed outside the nest has to ‘‘fight for priority’’ against the rest of the litter (as suggested for rodents by Ehret, 2005). Our results do not confirm such a ‘trade-off hypothesis’ affecting mink dam behaviour, as no significant effects of the number of kits in the nest box were evident for the dam to leave the nest, to touch and to retrieve the test kit. However, even though litter size was included in the statistical models, the study may not be optimal for testing this specific hypothesis since we did not use litter size as a factor in the design. We hypothesized that mink kits vocalising with a greater repetition rate would be retrieved earlier than kits that vocalised less. This hypothesis, however, was not confirmed in our study as
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the repetition rate affected neither the latency for the dam to leave the nest nor latency for kitretrieval. Maternal attention towards the kit away from the nest depended on the gender of the test kit: dams left their nests earlier when the test kit was female. Sex-biased difference in maternal care has not previously been studied in farmed mink. In other species, sex difference in maternal care has been reported, but often in favour of male offspring. Ferret mothers, Mustela putorious furo, directed more anogenital licking (i.e. not other behaviours and not licking on other body parts) towards male kits after a period of maternal separation, e.g. 6 days postpartum, but not consistently throughout the postpartum period (day 3–35; Baum et al., 1996). Rat mothers may also direct more of this behaviour to males than females during the immediate postpartum period (Moore and Morelli, 1979) and may retrieve males before females at a certain postnatal age (Deviterne and Desor, 1990). At present, the finding of preferential maternal approach towards female over male kits in mink stands alone. Active dams have been associated with a higher fertility, a lower mortality rate of kits, and a larger litter size (Jeppesen et al., 2004), which could be indicators linked to maternal abilities. Using groups of highly active or passive females (based upon observations in the premating period), we were unable to establish any significant relation between the activity level of mink and their reaction in the kit-retrieval test. However, due to the low number of animals used, we suggest that aspects of the link between maternal activity and ability affecting the offspring should be studied further. 5. Conclusion Mink kits do produce ultrasonic vocalisation when isolated from their mothers and nests. The calls consist of almost continuous trains of multiharmonic ultrasonic pulses of relatively long duration and a high rate of pulse repetition. Genetic and other factors influenced both (i) the nature of kit vocalisations (effects of colour type and litter size), and (ii) the maternal behaviour (effects of colour type and kit gender) during a standardised kit-retrieval test. Acknowledgements We thank Birthe Houbak, Aarhus University, Department of Animal Health, Welfare and Nutrition for participation in the data collection, Peter Teglberg Madsen and Frants Havmand Jensen, Aarhus University, Department of Biology for their help with programs, Birte Lindstrøm Nielsen, Janne Winther Christensen, Aarhus University, Department of Animal Health, Welfare and Nutrition, Søren Foged, and Jill Tubbert for manuscript improvements. This study received funding from Danish Fur Breeders Association. References Allison, P.D., 1995. Survival Analysis using the SAS System: A Practical Guide. SAS Institute, Cary, North Carolina, USA. Baum, M.J., Bressler, S.C., Daum, M.C., Veiga, C.A., McNamee, C.S., 1996. Ferret mothers provide more anogenital licking to male offspring: possible contribution to psychosexual differentiation. Physiol. Behav. 60, 353–359. Burman, O.H.P., Ilyat, A., Jones, G., Mendl, M., 2007. Ultrasonic vocalizations as indicators of welfare for laboratory rats (Rattus norvegicus). Appl. Anim. Behav. Sci. 104, 116–129. Deviterne, D., Desor, D., 1990. Selective pup retrieving by mother rats: sex and early developmental characteristics as discrimination factors. Dev. Psychobiol. 23, 361–368.
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Ehret, G., 2005. Infant rodent ultrasounds – a gate to the understanding of sound communication. Behav. Genet. 35, 19– 29. Farley, S.D., Lehner, P.N., Clark, T., Trost, C., 1987. Vocalizations of the Siberian Ferret (Mustela eversmanni) and comparisons with other mustelids. J. Mamm. 68, 413–416. Flottorp, G., Foss, I., 1979. Development of hearing in hereditarily deaf white mink (Hedlund) and normal mink (standard) and the subsequent deterioration of the auditory response in hedlund mink. Acta Otolaryngol. 87, 16–27. Gilbert, F.F., 1969. Analysis of basic vocalizations of the ranch mink. J. Mamm. 50, 625–627. Hahn, M.E., Hewitt, J.K., Schanz, N., Weinreb, L., Henry, A., 1997. Genetic and developmental influences on infant mouse ultrasonic calling. I. A diallel analysis of the calls of 3-day olds. Behav. Genet. 27, 133–143. Hahn, M.E., Karkowski, L., Weinreb, L., Henry, A., Schanz, N., Hahn, E.M., 1998. Genetic and developmental influences on infant mouse ultrasonic calling. II. Developmental patterns in the calls of mice 2–12 days of age. Behav. Genet. 28, 315–325. Harjunpaa¨, S., Rouvinen-Watt, K., 2004. The development of homeothermy in mink (Mustela vison). Comp. Biochem. Physiol. A 137, 339–348. Hofer, M.A., 1996. Multiple regulators of ultrasonic vocalization in the infant rat. Psychoneuroendocrinology 21, 203– 217. Holy, T.E., Guo, Z., 2005. Ultrasonic songs of male mice. PLoS Biol. 3, 1–10. Hoy, S., Mengs, U., Wenzel, U.D., 1998. Influence of birth weight on live weight development up to weaning in farm mink (Mustela vison). Archiv fu¨r Tierzucht 41, 497–504 (in German, with English abstract). Huff, J.N., Price, E.O., 1968. Vocalization of the Least Weasel, Mustela nivalis. J. Mamm. 49, 548–550. Jeppesen, L.L., Heller, K.E., Bildsøe, M., 2004. Stereotypies in female farm mink (Mustela vison) may be genetically transmitted and associated with higher fertility due to effects on body weight. Appl. Anim. Behav. Sci. 86, 137–143. Knutson, B., Burgdorf, J., Panksepp, J., 1999. High-frequency ultrasonic vocalizations index conditioned pharmacological reward in rats. Physiol. Behav. 66, 639–643. Malmkvist, J., 2006. Sma˚ mink – store udfordinger. Nyt om den tidlige hvalpedødelighed. (Small mink – big challenges. News about the early kit mortality). Report in Animal Husbandry no. 2 (in Danish with abstract in English), September 2006, Danish Institute of Agricultural Sciences, pp. 6–11. Malmkvist, J., Houbak, B., 2000. Measuring maternal care: kit retrieval test. In: Proceedings 7th International Scientific Congress in Fur Animal Production. Scientifur 24, 159–161. Malmkvist, J., Palme, R., 2008. Periparturient nest building: Implications for parturition, kit survival, maternal stress and behaviour in farmed mink (Mustela vison). Appl. Anim. Behav. Sci. 114, 270–283. Malmkvist, J., Gade, M., Damm, B.I., 2007. Parturient behaviour in farmed mink (Mustela vison) in relation to early kit mortality. Appl. Anim. Behav. Sci. 107, 120–132. Mandelli, M.-J., Sales, G., 2004. Ultrasonic vocalizations of infant Short-Tailed Field Voles, Microtus agrestis. J. Mamm. 85, 282–289. Moore, C.L., Morelli, G.A., 1979. Mother rats interact differently with male and female offspring. J. Comp. Physiol. 93, 677–684. Nowak, R., Porter, R.H., Le´vy, F., Orgeur, P., Schaal, B., 2000. Role of mother–young interactions in the survival of offspring in domestic mammals. Rev. Reprod. 5, 153–163. Svendsen, G.E., 1976. Vocalizations of the Long-Tailed Weasel (Mustela frenata). J. Mamm. 57, 398–399. Tauson, A.-H., Chwalibog, A., Tygesen, M.P., 2006. Late development of homeothermy in mink (Mustela vison) kits – a strategy for maximum survival rate. J. Anim. Physiol. 90, 38–45. Thornton, L.M., Hahn, M.E., Schanz, N., 2005. Genetic and developmental influences on infant mouse ultrasonic calling. III. Patterns of inheritance in the calls of mice 3–9 days of age. Behav. Genet. 35, 73–83. Tonkiss, J., Bonnie, K.E., Hudson, J.L., Shultz, P.L., Duran, P., Galler, J.R., 2003. Ultrasonic call characteristics of rat pups are altered following prenatal malnutrition. Dev. Psychobiol. 43, 90–101.
Applied Animal Behaviour Science 114 (2008) 582–592 www.elsevier.com/locate/applanim
Ultrasonic vocalisations of kits during maternal kit-retrieval in farmed mink, Mustela vison Karin Tubbert Clausen a, Jens Malmkvist b,*, Annemarie Surlykke c a
University of Aarhus, Department of Biological Sciences, Building 1131, C.F. Moellers Alle`, DK-8000 Aarhus C, Denmark b University of Aarhus, Department of Animal Health, Welfare and Nutrition, P.O. Box 50, DK-8830 Tjele, Denmark c University of Southern Denmark, Institute of Biology, Campusvej 55, DK-5230 Odense M, Denmark Accepted 26 March 2008 Available online 12 May 2008
Abstract Mink kits are born immature and are dependent on their dams’ protection, warmth and milk for survival. We hypothesize that vocalisations by mink kits may be of importance for communication between kit and dam, increasing the individual’s chance of survival. However, very little is known about the nature and function of mink kits’ vocalisation. In the present study, we aimed to (i) describe the kit vocalisation, when it is placed away from warm nest/dam and littermates, (ii) investigate the influence of these vocalisations on maternal behaviour, in terms of dams orientation towards kit and kit-retrieval, and (iii) test whether active/ stereotyping dams differed from less active/no-stereotyping dams in their kit-retrieval response. We used 58 first parity 1-year-old female mink of two genetic lines (colour type ‘Palomino’, n = 23, and ‘Black’, n = 35), allocated into equal-sized groups of active (including stereotyping) or passive (no stereotypic behaviour observed) individuals. All recordings were made during a standardised kit-retrieval test performed 5 days after birth. Our recordings demonstrated for the first time that mink kits are able to produce complex ultrasonic vocalisation with energy up to 50 kHz. The calls consist of long trains of multiharmonic pulses with a relative long pulse duration (average 264–874 ms) and a high repetition rate (0.6–2.3 pulses/s). The pulses contained most energy between 2.5 and 34 kHz (mean of F max and F min). Delayed maternal kit-retrieval increased the number of mink vocalisations (P = 0.007) but the repetition rate, average and S.D. in duration of pulses could not, in isolation, be identified as significant parts of the calls eliciting the maternal kit-retrieval response (P > 0.1). Genetic line affected both kit vocalisations and maternal retrieval since Palomino kits had a higher variation in pulse duration than Black kits (P = 0.023), and Palomino females had an impaired kit-retrieval compared to Black females (P = 0.008). We found no significant difference in kit-retrieval behaviour between groups of females according to their activity level.
* Corresponding author. Tel.: +45 89991314; fax: +45 89991500. E-mail addresses: [email protected] (K.T. Clausen), [email protected] (J. Malmkvist). 0168-1591/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.applanim.2008.03.008
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In conclusion, mink kits do produce ultrasonic vocalisation when isolated from their mothers and nests. Genetic and other factors influenced both (i) the nature of kit vocalisations (effects of colour type and litter size), and (ii) the maternal behaviour (effects of colour type and kit gender) during a standardised kitretrieval test. # 2008 Elsevier B.V. All rights reserved. Keywords: American mink; Behaviour; Mustela vison; Parent–offspring communication; Ultrasonic vocalisation
1. Introduction Specialized parent–offspring communication has evolved in several species of animals, and may have consequences for fitness and reproductive outcome (as exemplified for some species of domestic mammals in Nowak et al., 2000). For infants that are born immature and blind, e.g. mink kits, vocalisation may be a particular important means of communication. The American mink, Mustela vison, is a seasonal breeder and in the Northern hemisphere births are concentrated during a short period, from late April to early May. The altricial mink kit is dependent on the dam’s protection and care, and the warmth in the nest for survival (Tauson et al., 2006; Malmkvist et al., 2007). We hypothesize that the vocalisations by mink kits are an important part of the communication between kit and dam, increasing the individual’s chance of survival. However, no published studies have previously focused on the nature and function of vocalisations in mink kits. Young rodents typically vocalise during isolation from their dam and/or nest (Mandelli and Sales, 2004; Ehret, 2005), and we expect the mink kits to emit calls when they are isolated from their dam, nest and littermates. During high predation risks some mammals lower their vocalisation while a stronger vocalisation more often is produced when there are no predators around. In addition, odour and tactile stimuli also affect the vocalisation of rodent pups (Hofer, 1996). Nearly all existing studies on mustelid vocalisation have focused on adults only. In these studies, reports of vocalisation never exceeded 8 kHz during recordings of Long-Tailed Weasel, Mustela frenata, (Svendsen, 1976), Least Weasel, Mustela nivalis (Huff and Price, 1968; Svendsen, 1976), Siberian Ferret, Mustela eversmanni (Farley et al., 1987) and American mink, M. vison (Gilbert, 1969). One experiment tested the vocalisation of an adult Least Weasel female and her four 3-week offspring and detected no ultrasonic vocalisation (Huff and Price, 1968). These findings may be a consequence of the limited performance of the equipment used (e.g. in Huff and Price, 1968 and Svendsen, 1976), rather than a true reflection of the mustelid vocalisations. Thus, the previous recordings may have revealed the lower harmonics of multiharmonic signals. We therefore wanted to measure vocalisations in mink with equipment sensitive to higher frequencies (up to 100 kHz). In smaller mammals, ultrasonic vocalisation around 50 kHz has been reported in rodents such as voles (Mandelli and Sales, 2004), rats (Hofer, 1996; Knutson et al., 1999; Burman et al., 2007) and mice (Holy and Guo, 2005). For 3-day-old common voles frequencies up to 35 kHz were found and for field voles a frequency range of 25–60 kHz (Mandelli and Sales, 2004). We wanted to examine if mink kits emit ultrasonic sound and if so to describe spectral and temporal aspects of their sounds, when they vocalise during isolation from the dam and nest. In addition, we investigated whether kit’s vocalisation influences the kit-retrieval by the dam. In one study, stereotyping female mink were associated with a higher fertility, as they produced larger litters and had a lower mortality rate among their kits (Jeppesen et al., 2004). In
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addition, females with low early kit mortality performed more active kit-directed behaviour than more passive females with high kit mortality (Malmkvist et al., 2007). In the present study, we therefore tested whether active and stereotyping mink differ in their kit-retrieval response compared to more passive mink dams, during a standardised test situation. 2. Animal, material and methods 2.1. Animals and housing A total of 80 mated female mink, M. vison, of Palomino and Black genetic lines in their first parity were kept in standard wire cages (W: 30 cm, H: 45 cm, L: 91 cm) connected to a straw covered wooden nest box (W: 28 cm, H: 20 cm, L: 23 cm) at the farm unit at Research Centre Foulum, University of Aarhus, Denmark. Mink were kept under conventional farming conditions with food and water ad libitum. The experimental mink were given equal and ad libitum access to straw for nest building, since a previous study has shown that access to straw in the peripartureint period may affect the maternal behaviour in mink (Malmkvist and Palme, 2008). The temperature in the house was similar to the outside temperature. An empty cage separated each female. Prior to the experiment, the mink were selected according to their level of activity: active (n = 40) and passive (n = 40); the proportion of Black (65 %) and Palomino (35 %) mink was equal in the two activity groups. This selection was based on 16 scanning observations during 4 days of mild feed restriction in January 2007, using an original population of 300 individuals. The active females were active (including stereotyping) in more than 40% of all observations, while passive females were not observed active (i.e. running, stereotyping) during any of the observations. Barren females and females without kits on the fifth day after birth were excluded from further investigation, leaving 58 female mink with young (n = 31 active, n = 27 passive; average litter size: 5.6 0.29, range 1–9). Twenty-three of these (40%) were Palomino mink and thirty-five (60 %) were Black. There were no differences in the litter size between the colour types (t-test: T56 = 0.9; P = 0.36) or between the activity groups (active vs. passive, t-test: T56 = 0.4; P = 0.71). 2.2. The kit-retrieval test The kit-retrieval test and simultaneous recording of kit vocalisation were carried out on the fifth day after birth, between 10 am and 1 pm during the period from 30th April to 14th May 2007. Two persons performed this test; one person handled the animals and registered the behaviour based on direct observations using a handheld computer while another person recorded sound. The kit-retrieval test has been developed as a measure of maternal reactivity towards a 5-day-old progeny placed outside the nest (Malmkvist and Houbak, 2000). The handler selected one kit from the litter located inside the nest box, and after restricting the dam in the nest box, placed the kit in the middle of the wire cage (a specific point 31 cm from the opening of the nest box) with its head directed towards the nest box entrance. The test started when the female regained access to the wire cage and stopped when she retrieved the kit into the nest box. The observer registered latency to touch kit and latency to retrieve kit into nest. In the case of no kit-retrieval within a test time of 240 s, the test stopped, and the observer returned the kit to the nest. To test the effect of gender we alternately tested a female and a male kit. Each dam/kit pair was tested once. We used a fixed waiting time of 20 s for the female in the nest box, before the slide gate was removed and retrieval of the kit was possible. 2.3. Recording of kit vocalisation during tests For recordings we used a 1/400 (0.64 cm) microphone (Type 4135, Bru¨el & Kjaer, Denmark), placed in a holder 74 cm above the cage floor and connected through a pre-amplifier (Bru¨el & Kjaer 2633) to an amplifier (Microphone power supply type 2804, Bru¨el & Kjaer) set at 40 dB amplification. The microphone was pointed towards the vocalising kit in the initial position. The amplifier output was digitized with a
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DAQcard (sampling rate: 200 kHz 12-bit, DAQCard-6062E, National Instruments) and stored on a laptop (HP Compaq NC 8430) controlled by BatSound Pro Software (version 3.1, Pettersson Elektronik AB, Sweden). The recordings started from the start of the kit-retrieval test and consecutive files of 60 s were stored. 2.4. Analysis of recorded vocalisations We used the Batsound Pro Software to analyze the recordings. To reduce background noise, the files were high-pass filtered 1000 Hz (Butterworth, filter order six). For the analysis, we first listened to the recordings by playing back the files through headphones at one-fourth of the recording speed. Only periods where the test-kit could be identified and isolated were used for analysing the vocalisations. The vocalisations were displayed as oscillograms for analysis of temporal parameters and as spectrograms or spectra for analysis of spectral parameters. For all kits in the test we randomly chose 10 pulses and determined the mean and standard deviation of pulse duration and the repetition rate, defined as vocal pulses per second. Vocalisations of three randomly chosen kits were analyzed in detail to describe variation and spectral characteristics of the sounds. For each of the three kits we analyzed five pulses, two from the beginning, one from the middle, and two from the end of a vocalisation. The vocalisations were displayed as spectrograms to determine number of harmonics, and frequency modulations with time. The vocalisations were also displayed as spectra and minimum frequency (F min) and maximum frequency (F max) were determined as 20 dB low frequency and 20 dB high frequency cut-off. Bandwidth (BW20 dB) was equal to the distance between F min and F max. Due to the relatively long duration and slow frequency modulation of the pulses we used a 2048 sample FFTwindow for spectral analyses giving a frequency resolution of 97.7 Hz. 2.5. Statistical analyses 2.5.1. Description of the kit vocalisations We analyzed three temporal variables of kit pulses using an ANOVA in the statistical software SAS (version 9.1, SAS Institute Inc., USA): (1) repetition rate, (2) mean pulse duration, and (3) standard deviation (S.D.) of pulse duration. The models included sex (male or female test kit), colour type (Palomino or Black), dam activity group (active or passive) as explanatory class variables, and handling time of the dam during test (21–194 s) and litter size (1–9 kits) as explanatory covariates. The model also included the interactions between sex and colour type, and between sex and activity group. The models were reduced by stepwise removal of insignificant terms (P > 0.10), starting with the interactions. The data were normally distributed. Logarithmic transformation of the response variables was used to stabilise the variance as it resulted in better residuals. The demand for dispersion and variance homogeneity was tested and the validity of the final model justified by the appearance of the residuals. 2.5.2. Factors influencing dam behaviour in the kit-retrieval test Initially, we used simple chi-square test with one degree of freedom to test whether the proportion of retrieved and non-retrieved kits differed according to dam activity group (active, passive), sex of the test kit and the colour type (Palomino, Black). The latencies for the dam to touch and to retrieve the kit were strongly correlated (r = 0.92, P < 0.001) and therefore only the latency for the dam to retrieve the kit was tested further. The latency for the dam to leave the nest and latency for kit-retrieval were also, but less strongly correlated (r = 0.52, P < 0.001). Latencies for leaving nest and for retrieving the test kit were analyzed using methods for survival analysis (Allison, 1995), considering censored data (thus including dams that did not leave the nest/retrieve within the maximum test time of 240 s as right censored observations), using the computer software SAS with models including sex, colour type, dam activity group, repetition rate, mean and S.D. of pulse duration. A probability level (P) of 0.05 was chosen as a limit for statistical significance in all tests. P-values between 0.05 and 0.1 are reported as trends in the results.
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3. Results 3.1. Description of kit vocalisation Vocalisation occurred in 30 out of the 58 performed kit-retrieval tests. Useful vocalisation data from the test kits were obtained for 70% of these (n = 21) and the three main observed variables of vocalisation are summarized in Table 1. The rest of the files were not analyzed further because of recording failures or due to background noise masking the vocalisations of the test kit. The three kits randomly chosen to illustrate the nature and variation of vocalisation between and within individuals were not subjected to statistical analyses, but the results are presented in Fig. 1. Each kit varied in pulse duration, frequency modulation rate and direction, as well as in relative energy of harmonics. There were individual characteristics making it easy to discriminate between individuals when listening to the slowed down version of the recordings. Kit I (Fig. 1A and B left panel) emitted pulses which typically started with an initial upward frequency modulation, followed by a section with more shallow or no modulation, to end with a steeper downward frequency modulation. The pulses were multiharmonic with a first harmonic (fundamental) around 2.4 kHz in the middle part of the pulse. The second harmonic had more energy than the first harmonic, and all harmonics up to number 10–12 had high energy. Which of those harmonics had maximum energy changed within and between pulses. Low and high frequency limit and bandwidth of the sounds were determined from an average spectrum of 30 pulses. At low frequencies, noise from surroundings and other nearby minks obscured the low frequency limit, so F min in the spectra was set by the high pass filter: 1 kHz, but the first harmonic of the multiharmonic pulse (ca. 2.2–2.4 kHz) was probably the lower limit of the kit’s own emission. F max was 45 kHz, and thus the bandwidth BW20 dB was approx. 42 kHz for kit I (Fig. 1B left panel). Kit II and III both made more complicated frequency modulation patterns sweeping up and down more than once during a pulse (Fig. 1B). The average spectra of 30 pulses showed that Kit II had a first harmonic around 2.4 kHz, an F max of 42 kHz, giving a BW20 dB = 40 kHz (Fig. 1B middle panel). Kit II emitted relatively low energy at midfrequencies from 10 to 20 kHz (Fig. 1C). Kit III emitted the highest frequencies of the three. Its average spectrum showed a first harmonic of approx. 2.1 kHz, an F max of 51 kHz and thus BW20 dB of 49 kHz (Fig. 1B right panel). Analysis of the calls from all 21 kits confirmed this by showing large variation within and between individual kit’s vocalisations. The vocalisations or calls consisted of a long train of pulses (as exemplified in Fig. 1A). All pulses were multiharmonic as described above for the three selected kits with a first harmonic between 2 and 3 kHz and energy up to around 50 kHz. Our data clearly demonstrate that mink kits do indeed produce vocalisations with much energy at ultrasonic frequencies. The pulse durations were between 264 and 874 ms and the repetition rate relatively high averaging 1.3 pulses per second (Table 1). However, there were considerable Table 1 Repetition rate, mean and standard deviation in pulse duration of vocalisations of 5-day-old mink kit (n = 21) during kitretrieval test, given as mean (S.E.M.) and range of the observations
Pulse repetition rate in vocalisation (1/s) Mean pulse duration (ms) S.D. pulse duration (ms) a
Mean (S.E.M.)
Range (minimum–maximum)
1.26 (0.090) 537 (38.4) 104 (11.4)a
0.59–2.31 264–874 33–235
Significant effects of colour type and litter size were found for this variable, please refer to the text.
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Fig. 1. Vocalisations from three mink kits. (A) Shows a train of pulses from Kit I with spectrograms below, illustrating the variation, but also the similarity in general structure of pulses, which were slow frequency modulated multiharmonic pulses with significant energy up to at least the 10–12th harmonic. (B) Shows in the left panel expanded spectrograms of two of the pulses (marked with asterisks) from Kit I, and for comparison two pulses from Kit II in the middle panel and Kit III in the right panel. (C) Shows average spectra of 30 pulses from each of the three kits as indicated by the colour code. Due to the frequency modulations the spectra do not show the multiharmonic structure of the pulses, but illustrate that the pulses contain much energy up to frequencies of 40–50 kHz.
variations in the vocalisation within each kit and between kits during the test. The detailed acoustic analysis not only revealed a surprisingly complex structure of the vocalisations, but also variation within and between kits of all parameters. Thus, kit vocalisations differed in the acoustic parameters: duration, number of harmonics, relative weight (energy) of harmonics and
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Fig. 2. Variation (S.D.) in pulse duration (ms) of kit vocalisation during test in relation to litter size. The variation in pulse duration fell with increasing litter size of the tested kit (P = 0.002).
modulation rate. The results also demonstrated that these parameters could change from pulse to pulse, allowing individual kits a large repertoire of possible acoustic communication. Pulse durations averaged 516 ms for Black kits and 573 ms for Palomino kits, and the overall average pulse duration was 537 ms (Table 1). The mean durations were not significantly different, but standard deviation of pulse duration was significantly affected by both the colour type of mink (F 1,18 = 6.15, P = 0.023) and the litter size (F 1,18 = 12.96, P = 0.002; Fig. 2). Palomino kits had a larger standard deviation (110 ms) of pulse duration than Black kits (100 ms). We found no significant effects of the tested factors on the repetition rate. However, the repetition rate of pulses within the test kit vocalisation may increase with increasing litter size, as reflected by a non significant trend in data (F 1,19 = 3.83, P = 0.065). 3.2. Factors influencing dam behaviour on the kit-retrieval test Simple chi-square tests showed that a higher proportion of female kits than male kits were retrieved (female: 97% vs. male: 79%, x21 ¼ 4:062, P = 0.044) and Palomino dams retrieved a lower proportion of their kits than did Black dams (73% vs. 97%, x21 ¼ 7:057, P = 0.008), whereas there were insignificant effects with regard to active/passive dams on the retrieval/nonretrieval of kit (P = 0.55). Survival analysis of latency performed on all data (i.e. including those that did not react within the test time of 240 s) showed an effect on the latency for dam to leave the nest with regard to gender. Latency for dams to leave the nest was shorter when the kit was a female (median (quartiles 25%; 75%): 8 (3; 21) s; n = 29, 0% censored) than when it was a male (16 (6; 34) s; n = 29, 20.7% censored). Hazard ratio (HR) for female/male test kit was 2.2 (x21 ¼ 7:21, P = 0.007) indicating that female kits resulted in dams leaving the nest sooner. The colour type of mink showed a significant effect on the latency for dams to leave the nest (HR for Palomino/ Black = 0.3, x21 ¼ 15:5, P < 0.001), Palomino dams leaving their nests markedly later (24 (16; 46) s, n = 23; 21.7% censored) than Black dams (7 (2; 16) s, n = 35; 2.9% censored). Palomino dams had a longer latency of kit-retrieval (68 (48; 123) s, n = 23, 26.1% censored) than Black dams (24 (17; 46) s, n = 35, 2.9% censored) (HR for Palomino/Black = 0.3,
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Fig. 3. Cumulative probability of kit-retrieval during the test time of 240 s, with upper and lower 95% confidence intervals. Black colour type: solid black line, Palomino colour type: dotted blue line. The latency of kit-retrieval was significant shorter in Black (median 24 s) than in Palomino dams (median 68 s; P < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
x21 ¼ 15:2, P < 0.001), with no significant effect of any of the other tested factors, e.g. gender of the test kit (HR female/male = 1.3, P = 0.36). Fig. 3 illustrates the cumulative probability of the kit-retrieval latency in the two colour types. In a supplemental ANOVA including the retrievers only (n = 51, 23 without and 28 with kit vocalisation), latency of maternal kit-retrieval and the number of vocalisations in mink kits were positively related (F 1,46 = 7.9, P = 0.007). However, we failed to demonstrate significant effects of any of the elements within the kit vocalisations we measured – i.e. the repetition rate, the mean and S.D. of pulse duration – on the latencies of the dam to leave the nest (P > 0.1) and to retrieve her kit (P > 0.1). 4. Discussion We found that 5-day-old American mink kits do produce ultrasonic vocalisation when isolated from their mothers and nests. To our knowledge, this study is the first to measure mink kits’ vocalisation with equipment that is sensitive to high frequencies. Our results show that mink kits produce almost continuous trains of multiharmonic ultrasonic pulses of relatively long duration and with high pulse repetition rate. Numerous studies of ultrasonic vocalisation have been performed in young rodents. Most rodents produce ultrasonic vocalisations that are relatively short (20–80 ms; Hahn et al., 1998; Mandelli and Sales, 2004), whereas mink kits produce pulses of about 10 times longer duration (mean 264–874 ms). The pulse duration in calls emitted by kits resemble that of calls of adult mink (50–100 ms; Gilbert, 1969) and other adult mustelids; the Long-Tailed Weasel (150– 350 ms; Svendsen, 1976), the Siberian Ferret (88–1300 ms; Farley et al., 1987), and the Least Weasel (130–510 ms; Huff and Price, 1968). Palomino kits produced less uniform pulses when vocalising compared to Black mink kits. This is to our knowledge the first time that fundamental differences in kit vocalisations have been demonstrated between genetic lines of mink. Ultrasonic vocalisation characteristics have also
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been found to be influenced by genotype in mice pups, Mus musculus (Hahn et al., 1997, 1998; Thornton et al., 2005). Furthermore, the dams from the two genetic mink lines differed markedly in their behaviour in the kit-retrieval test, with Palomino dams being slower and less effective. Studies have demonstrated hearing deficits developing in certain colour types of farmed mink e.g. the white ‘Hedlund’ mink (Flottorp and Foss, 1979) but we have at present no data on differences in adult hearing capacity in Black and Palomino mink. The complex pulse structure and individual characteristics in all acoustic parameters between kits would potentially allow for individual recognition by females; however this has not yet been studied in mink. In addition, the variation over time between and within pulses indicates that acoustic parameters might serve to disclose the present state of the kit. We standardised the age of testing (day 5 postpartum) but had variable litter sizes. Litter size affected the vocalisations, since kits from larger litters produced pulses with more homogeneous durations within their calls. Litter size and kit weight may be negatively correlated in the early period after parturition (Hoy et al., 1998; Malmkvist and Palme, 2008). It is currently unknown whether kit weight affects the nature of vocalisation and thereby whether the effect of litter size on pulse homogeneity partly reflects differences in body weights. In rat pups, a positive correlation between call duration and body weight has been reported during cold exposure (Tonkiss et al., 2003). Heat loss and deviation from the normal nest temperature may elicit ultrasonic vocalisations in young rodents (Hofer, 1996; Ehret, 2005). Mink is altricial (like rat and mice) in the early postnatal period and unable to thermoregulate independently until 22–29 days of age (Harjunpaa¨ and Rouvinen-Watt, 2004; Tauson et al., 2006). The heat loss during cold exposure increases linearly with decreasing body weight (tested 8–9 days postpartum in Malmkvist, 2006); presumably a consequence of the higher surface to volume ratio in the smaller kits. Other factors than heat loss may affect the level of offspring vocalisation. Mandelli and Sales (2004) suggested that the interval since infant Short-Tailed field voles had fed could influence the vocalisation. We have no observations of the timing of suckling in the mink kits but any nursing would be interrupted as the kit-retrieval test was prepared. In mice and rats vocalisations of infants play a role in retrieval, initiating maternal orientation towards the stimulus source, followed by the retrieval of a pup back to the nest (Hahn et al., 1998; Ehret, 2005). Hahn et al. (1998) further suggested that the call length of an ultrasonic vocalisation produced by an infant mouse and perceived by the mother would allow her to make a decision about whether or not to retrieve the pup. In addition, results suggest that adult laboratory rats, Rattus norvegicus, vocalise around 22 kHz as a reflection of a negative emotional state (Burman et al., 2007). In our study, delayed maternal kit-retrieval increased the occurrence of vocalisation in mink kits. This may indicate that the functional role of the vocalisation of isolated mink kits is to elicit maternal kit-retrieval. It is also evident that other factors than calls play a role, since a large proportion of the non-vocalising kits (82%) were successfully retrieved as well. Thus, the ultrasonic vocalisation is not the ultimate or only factor involved in dam decision-making during the kit-retrieval test. It may also be that offspring placed outside the nest has to ‘‘fight for priority’’ against the rest of the litter (as suggested for rodents by Ehret, 2005). Our results do not confirm such a ‘trade-off hypothesis’ affecting mink dam behaviour, as no significant effects of the number of kits in the nest box were evident for the dam to leave the nest, to touch and to retrieve the test kit. However, even though litter size was included in the statistical models, the study may not be optimal for testing this specific hypothesis since we did not use litter size as a factor in the design. We hypothesized that mink kits vocalising with a greater repetition rate would be retrieved earlier than kits that vocalised less. This hypothesis, however, was not confirmed in our study as
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the repetition rate affected neither the latency for the dam to leave the nest nor latency for kitretrieval. Maternal attention towards the kit away from the nest depended on the gender of the test kit: dams left their nests earlier when the test kit was female. Sex-biased difference in maternal care has not previously been studied in farmed mink. In other species, sex difference in maternal care has been reported, but often in favour of male offspring. Ferret mothers, Mustela putorious furo, directed more anogenital licking (i.e. not other behaviours and not licking on other body parts) towards male kits after a period of maternal separation, e.g. 6 days postpartum, but not consistently throughout the postpartum period (day 3–35; Baum et al., 1996). Rat mothers may also direct more of this behaviour to males than females during the immediate postpartum period (Moore and Morelli, 1979) and may retrieve males before females at a certain postnatal age (Deviterne and Desor, 1990). At present, the finding of preferential maternal approach towards female over male kits in mink stands alone. Active dams have been associated with a higher fertility, a lower mortality rate of kits, and a larger litter size (Jeppesen et al., 2004), which could be indicators linked to maternal abilities. Using groups of highly active or passive females (based upon observations in the premating period), we were unable to establish any significant relation between the activity level of mink and their reaction in the kit-retrieval test. However, due to the low number of animals used, we suggest that aspects of the link between maternal activity and ability affecting the offspring should be studied further. 5. Conclusion Mink kits do produce ultrasonic vocalisation when isolated from their mothers and nests. The calls consist of almost continuous trains of multiharmonic ultrasonic pulses of relatively long duration and a high rate of pulse repetition. Genetic and other factors influenced both (i) the nature of kit vocalisations (effects of colour type and litter size), and (ii) the maternal behaviour (effects of colour type and kit gender) during a standardised kit-retrieval test. Acknowledgements We thank Birthe Houbak, Aarhus University, Department of Animal Health, Welfare and Nutrition for participation in the data collection, Peter Teglberg Madsen and Frants Havmand Jensen, Aarhus University, Department of Biology for their help with programs, Birte Lindstrøm Nielsen, Janne Winther Christensen, Aarhus University, Department of Animal Health, Welfare and Nutrition, Søren Foged, and Jill Tubbert for manuscript improvements. This study received funding from Danish Fur Breeders Association. References Allison, P.D., 1995. Survival Analysis using the SAS System: A Practical Guide. SAS Institute, Cary, North Carolina, USA. Baum, M.J., Bressler, S.C., Daum, M.C., Veiga, C.A., McNamee, C.S., 1996. Ferret mothers provide more anogenital licking to male offspring: possible contribution to psychosexual differentiation. Physiol. Behav. 60, 353–359. Burman, O.H.P., Ilyat, A., Jones, G., Mendl, M., 2007. Ultrasonic vocalizations as indicators of welfare for laboratory rats (Rattus norvegicus). Appl. Anim. Behav. Sci. 104, 116–129. Deviterne, D., Desor, D., 1990. Selective pup retrieving by mother rats: sex and early developmental characteristics as discrimination factors. Dev. Psychobiol. 23, 361–368.
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