Reprod Dom Anim 50, 321–326 (2015); doi: 10.1111/rda.12494 ISSN 0936–6768
Vocalizations During Electroejaculation in Anaesthetized Adult and Young Pampas Deer (Ozotoceros bezoarticus) Males F Fumagalli1, JP Damian2 and R Ungerfeld3 1 Clınica Semiol ogica, Facultad de Veterinaria, Universidad de la Repu´blica, Montevideo, Uruguay; 2Departamento de Biologıa Molecular y Celular, Facultad de Veterinaria, Universidad de la Rep ublica, Montevideo, Uruguay; 3Departamento de Fisiologıa, Facultad de Veterinaria, Universidad de la Rep ublica, Montevideo, Uruguay
Contents The aim of this study was to characterize the vocalizations produced during electroejaculation under general anaesthesia in pampas deer males and to determine whether the characteristics of those vocalizations differ in adult and young pampas deer males. Electroejaculation was applied to 13 adults (AM) and 13 young (YM) males under general anaesthesia. Vocalizations were digitally recorded, and the number and duration of vocalizations, the latency in relation to each voltage, the total time vocalizing, and the structure of the fundamental frequency (F0) [initial frequency (Fstart), maximal frequency (Fmax), minimal frequency (Fmin) and final frequency (Fend)] were analysed. No male vocalized with 0 V; the number of animals that vocalized increased at 2 and 3 V and increased again at 4, 5 and 6 V (p < 0.05). The latency time from the beginning of each series (each voltage) decreased until 4 V (p < 0.01). The number of vocalizations/voltage increased from 4 V (p < 0.05). The length of each vocalization and the total time during which animals vocalized were greater in YM than AM (p = 0.02 and p = 0.01, respectively). Similarly, the fundamental frequencies were higher in YM than AM (p ≤ 0.05). Overall, we concluded that the vocalizations emitted during electroejaculation in pampas deer under general anaesthesia are related to the voltage applied during the process. Young males vocalize more time, probably due to a greater sensibility to the electric stimulation. The differences in the characteristics of the vocalizations between adult and young males may be related to the anatomic differences in the neck of adult or young males.
Introduction The pampas deer (Ozotoceros bezoarticus, Linnaeus, 1758) used to be a widespread species originally distributed in the open grasslands of South America (Jackson and Langguth 1987). However, habitat fragmentation, agriculture development, competition with farmed animals and unregulated hunting led to the decrease in size and distribution of the species (Demarıa et al. 2003). This species is listed in Appendix I (see supporting information) of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES 2014). In Uruguay, there are two wild populations, and there is also a third semicaptive group (Ungerfeld et al. 2008). This third population is allocated at the Estaci on de Crıa de Fauna Aut octona Cerro Pan de Az ucar (ECFA) (Maldonado, Uruguay; 34°30 S, 54°00 W) and consists of approximately 80 individuals. The cryopreservation of gametes and the development of gene banks are useful techniques to improve conservation © 2015 Blackwell Verlag GmbH
status of endangered species (Garde et al. 2006). For this purpose, techniques for semen collection should be adequately adapted for each species. As artificial vagina can only be used in tame and trained individuals, electroejaculation (EE) is the most widely used technique for semen collection in wild animals. In deer, due to nervous temperament and thus, risks for operators, EE is conventionally used under general anaesthesia (Umapathy et al. 2007; Martınez et al. 2008; Martınez-Pastor et al. 2009). Electroejaculation produces some undesirable physiological responses. For example, in anaesthetized pampas deer, we previously observed increases in heart and pulse rates, as well as in creatine kinase, aspartate aminotransferase and alkaline phosphatase concentrations, and a decrease in rectal temperature (Fumagalli et al. 2012). Responses to EE may also differ in relation to males’ age, as adult males had a greater increase in heart rate, but lower increases in alkaline phosphatase and creatine kinase concentrations. These negative responses have been associated with the occurrence of vocalizations in bulls (Falk et al. 2001; Whitlock et al. 2012) and rams (Damian and Ungerfeld 2011), which are considered as reliable indicators of pain (Damian and Ungerfeld 2010). Although we did not record vocalizations in anaesthetized domestic (rams, bucks) or wild (antelopes, muflon, dama deer) ruminants, strikingly we have previously observed that pampas deer males vocalize during EE even while they are maintained under general anaesthesia. At least under several other contexts, the increases in cortisol secretion, and the number, duration and characteristics (high-frequency) of vocalizations are reliable indicators for pain (Prunier et al. 2013). Considering all this information, a first aim of this study was to characterize the vocalizations produced during EE under general anaesthesia in pampas deer males. Another objective was to determine whether the characteristics of the vocalizations, as well as the cortisol and creatine kinase serum concentration, differ in adults and young pampas deer males during EE under general anaesthesia.
Materials and Methods Animals and facilities The study was conducted from September to March (spring to autumn), at the ECFA, with 26 males. From
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these, 13 were adult [group AM; 4–7 year old, 29.8 3.0 kg (mean SEM)] and 13 young (group YM; 1.5–2 year-old, 25.6 1.6 kg) males. All these males were anaesthetized and electroejaculated at least once before this study. Both groups were housed in different paddocks (0.5 ha), with free access to water, native pastures, trees and shrubs. Animals also received approximately 600 g of dairy cow ration/deer daily. The study was approved by the Comisi on Honoraria de Experimentaci on Animal (CHEA), from the Universidad de la Rep ublica, Uruguay. Capture and anaesthesia The animals were captured and managed as described by Fumagalli et al. (2012). Briefly, they were captured with anaesthetic darts fired from a blowpipe (Telinject USA, Inc. Soledad Canyon Road Agua Dulce, Newhall, CA, USA) containing 2 mg/kg of xylazine (Sedomin 10%, Laboratorio K€ onig, Buenos Aires, Argentina), 1.6 mg/kg of ketamine (Vetanarcol 5%, Laboratorio K€ onig S.A.) and 0.013 mg/kg of atropine (Sulfate de Atropine 1& ION, Laboratorio ION, Montevideo, Uruguay). After the animals were anaesthetized (induction time), they were placed on a stretcher, transported, weighed and placed in right lateral position on a padded table. An intravenous catheter was placed in the cephalic vein, and sodium chloride 0.9% was administered to have a continuous via open. Electroejaculation Electroejaculation was carried out using a rectal probe (300 mm length, 19 mm width, with 30 mm longitudinal electrodes; Model 303, P-T Electronics, Boring Oregon, USA). The EE was performed according to Beracochea et al. (2014), with series of 10 periods (4–5 s) of voltage stimulus with the same voltage, with rest intervals of 2–3 sec, increasing 1V after each 10 pulses series, beginning with 1V until ejaculation (maximum = 7 V). Registration of vocalizations Vocalizations were recorded using a microphone and mp4 equipment (Insignia NS-4V24, China). The distance between the microphone and the head of the recorded animal was 150 cm. The sound measurements and spectral analysis were performed with WaveSurfer 1.8.5 acquisition software (open software, KTH Royal Institute of Technology, Stockholm, Sweden, 2005). Average and total time duration of vocalizations, latency (time from first electric pulse of each series to first vocalization emitted in that series), and the initial (Fstart), final (Fend), minimal (Fmin) and maximal (Fmax) frequencies (Hz) of the fundamental frequency (F0) were calculated.
F Fumagalli, JP Dami an and R Ungerfeld
Cortisol and creatine kinase serum determination Two blood samples were collected immediately before and immediately after EE. Samples were allowed to clot at room temperature, centrifuged at 1080 9 g for 20 min, and the serum was stored at 20°C. Creatinine kinase (CK) serum concentrations were measured in the Laboratorio de Analisis Clınicos (Facultad de Veterinaria, Montevideo, Uruguay), using a clinical analyser (Metrolab 1600 DR; Metrolab, Buenos Aires, Argentina). Serum cortisol concentrations were measured in the Laboratorio de Tecnicas Nucleares (Facultad de Veterinaria) by radioimmunoassay using a solid phase kit (DPC; Siemens, Los Angeles, CA, USA). The analytical sensitivity of the assay for cortisol was 2.0 nmol/l. The intra-assay and interassay coefficients of variation were both below 12.5%. Recovery of the animals After the procedure finished, the animals were taken back to their respective paddocks. Anaesthesia was reversed with a jugular vein injection of 0.26 mg⁄kg of yohimbine hydrochloride 1% (Reverze, Laboratorio Vetcross, Montevideo, Uruguay), and the time from administration to recovery of the animals was recorded (recovery time). Data analysis The induction and the recovery time, and the number of series in which AM and YM began to ejaculate was compared with ANOVA. Separated linear regressions were performed for AM and YM between the total number of pulses and the total number of vocalizations. The differences between categories in the number of vocalizations/voltage, the total time vocalizing, length time of each vocalization, the Fstart, Fend, Fmax and Fmin, during EE were also compared by ANOVA. The number of AM and YM that vocalized in each voltage was compared with the Fishers’ exact probability test. The influence of voltage on the latency to the first vocalization was compared with ANOVA for repeated measures, considering the category of the male (AM vs YM) and the voltage as fixed effects, and the male as a random effect into each experimental group. Concentrations of cortisol and creatine kinase serum were compared by ANOVA for repeated measures. The model included the effect of category (AM vs YM), time (before and after EE), as well as their interaction. Data are expressed as mean SEM.
Results The induction time was 12.0 0.6 and 14.0 10.0 min, and the recovery time was 2.6 2.4 and 2.3 1.8 min in AM and YM, respectively. Semen was collected from all animals: ejaculation began at 3.6 0.7 and 3.5 0.5 volts in adults and yearlings (ns), respectively.
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Electroejaculation and Vocalizations
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Number of vocalizations
16
b
12 a
8 4 0 1
2
3 4 Voltages (V)
5
6
Fig. 1. Number of vocalizations emitted in each voltage used during electroejaculation in adults (black column n = 13) and young (grey column n = 13) anaesthetized pampas deer males. Values are expressed as mean SEM. a vs b: p < 0.05
75 Total number of vocalizaƟons
Vocalizations There were no vocalizations recorded before or after EE in any animal. Only one AM male did not vocalize during EE. The number of animals that vocalized with each voltage did not differ in AM and YM males, so it is presented pooled in Table 1. No male vocalized with 0 V, the number of animals that vocalized increased at 2 and 3 V, and increased again at 4, 5 and 6 V (Table 1). The latency from the beginning of each pulse of the series to the first vocalization in that series was not affected by the group, and there was no interaction between voltage and group, so it is presented pooled despite the group in Table 1. This latency time was shorter with greater voltages (p < 0.0001) (Table 1). The number of vocalizations/voltage increased from 4 V (p < 0.05), without differences between AM or YM (Fig. 1). The total number of vocalizations was positively related with the total number of pulses in AM (R2 = 0.55; p = 0.014) and tended to do so in YM (R2 = 0.28; p = 0.09) (Fig. 2). The total number of vocalizations was similar in AM and YM males, but the length of each vocalization and the total time during which animals vocalized were greater in YM than AM (p = 0.02 and p = 0.01, respectively; Table 2). Similarly, the fundamental frequencies: Fmax, Fend, Fmin were higher in YM than AM (p = 0.04, p = 0.05 and p = 0.05, respectively), and the Fstart tended to be higher in YM than AM (p = 0.06) (Table 2). The sonogram of one vocalization from an AM and one from a YM are included as examples in Fig. 3, and the recordings are included as supplementary files. In these sonograms, it can be observed the difference between AM (bass sound) and YM (high-pitched sound).
50
25
0 30
40
50
60
70
80
Total number of pulses
Fig. 2. Relationship between the total number of pulses during electroejaculation and the total number of vocalizations in adult () and young (○) pampas deer males. Adult males: R2 = 0.55; p = 0.014 young males: R2 = 0.28; p = 0.09
Cortisol and creatine kinase serum The cortisol and creatine kinase serum concentrations increased after the EE. Both, cortisol and creatine kinase concentrations increased after the procedure (p = 0.006 and p = 0.01, respectively), with no effect of group or interaction between group and time in the serum concentrations of cortisol and creatine kinase (Fig. 4a and b, respectively).
Table 2. Number of vocalizations, length of each vocalization, total time vocalizing during electroejaculation and fundamental frequencies of adult (AM) and young (YM) pampas deer males during electroejaculation under general anaesthesia. Values are expressed as mean SEM
Table 1. Number of animal that vocalized and the latency time (min) for voltage (1V–6V) during electroejaculation under general anaesthesia. Values are expressed as mean SEM
N Number of vocalizations Length time of vocalization (s) Total time of vocalization (s) Fstart (Hz) Fmax (Hz) Fend (Hz) Fmin (Hz)
Voltage 1 2 3 4 5 6
Total 0/26 4/26 10/26 17/26 18/26 17/26
(0%)a (15%)b (39%)b (65%)c (69%)c (65%)c
Latency (min) 3.0 2.0 1.1 0.4 0.08 0.03
0.3w 0.3x 0.2y 0.2z 0.05z 0.01z
Different letters within columns indicate significant differences (a vs b vs c: p < 0.05; w vs x vs y vs z: p < 0.01).
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AM 13 11 1.4 12 286 314 243 222
2 0.2 2 51 59 51 43
YM 13 10 2.0 20 405 484 377 331
2 0.2 2 50 67 59 47
p
ns 0.02 0.01 0.06 0.04 0.05 0.05
Fundamental frequencies: Fstart (initial frequency), Fmax (maximum frequency), Fend (final frequency), Fmin (minimum frequency), NS (not significant).
Discussion According to our knowledge, this is the first description of the vocalizations emitted in animals during EE under general anaesthesia. Although the animals were anaes-
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F Fumagalli, JP Dami an and R Ungerfeld
(a)
(b)
Fig. 3. Spectrograms of the vocalizations of adult (a) and young (b) pampas deer males recorded during the electroejaculation under general anaesthesia. While adult males show bass sounds, young males show high-pitched sounds
thetized, the anaesthetic combination used in this work (xylazine, ketamine and atropine) does not affect the ability to vocalize (McKelvey and Hollingshead 2003). Although the causes of vocalizations cannot be determined in this study, it is clear that its expression is directly related with the electric pulses. This may be a consequence of a lower effect of the anaesthesia combination in this species, but this is not reflected in other physiological parameters (Fumagalli et al. 2012). Other possible explanation is a greater sensitivity of pampas deer to EE, determining that even anaesthetized they elicit a clear response. An alternative strategy to determine this would be to test other anaesthetic preparations or analgesics, such as titelamine or zolazepam, as both may provide better analgesia than the combination used in this study (Santiago-Moreno et al. 2011). Other combinations, such as the method previously used plus inhalation of isoflurane anaesthesia (dos Santos et al. 2010), or the addition of analgesic agents as fentanyl citrate (Asher et al. 2011) should also be tested. The use of alternative techniques, such as the use of transrectal ultrasound-guided massage of the accessory sex glands (TUMASG), a technique reported to be effective in aoudad (Ammotragus lervia sahariensis)
(Santiago-Moreno et al. 2013) should be tested. Unfortunately, due to the low number of pampas deer available for research, advancement in the development of effective alternative techniques is slow in the species. As the number of vocalizations emitted increased with the increase in voltage, and at the same time the latency to the first vocalization decreased, it seems that the voltage used is directly related to these responses. However, we cannot discard that the increased response observed with higher voltage can be a consequence of the accumulated process, as the animals received lower voltage before. In any case, considering that vocalizations seem to be reliable indicators of pain produced by EE in other species (Damian and Ungerfeld 2010), it would be important to study whether shorter protocols, even beginning with greater voltage, are as effective as this one. It is interesting to note that acoustic communication seems not to be as important in the pampas deer as in other deer species, probably because it lives in open grasslands, with easy visual communication. Vocalizations in the same population of pampas deer have been previously studied (see review: Ungerfeld et al. 2008), but after several trials, we have only recorded vocalizations for offspring calling by the dams (Olazabal et al. © 2015 Blackwell Verlag GmbH
Electroejaculation and Vocalizations 70
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**
(a)
CorƟsol (nmol/l)
60 50 40 30
20 10 0
BEE 400
(b)
AEE
**
CK (UI/l)
300
200
100
explain this difference. The neck perimeter is a secondary sexual character of the deer, directly influenced by the anabolic action of testosterone before the rut (Handelsman 2006). As in the pampas deer, both the testosterone concentrations and the neck size are greater in adult than in young animals during the rut (Ungerfeld et al. 2011), the larynx is probably larger in AR than YR, provoking differences in the resonance of the vocalizations. Overall, we concluded that the vocalizations emitted during EE in pampas deer under general anaesthesia are related to the voltage applied during the process. Young males vocalize for a greater time length, probably due to a greater sensitivity to the electric stimulation. The differences in the characteristics of the vocalizations may be related to the anatomic differences in the neck of adult or young males. Given changes in stress related physiology demonstrate herein along with the recognition that the vocalization during EE is related to pain in other species, it seems important to develop alternative techniques to EE with less negative effects on welfare when applied to wild animals.
0
BEE
AEE
Fig. 4. a) Cortisol and b) CK concentrations before (BEE) and after (AEE) electroejaculation in adult (black bars) and young (white bars) pampas deer males. **p < 0.01
2013), and in one occasion during courtship (MoralesPi~ neyrua and Ungerfeld 2012). Therefore, this reinforces the hypothesis that these vocalizations observed out from a social context are probably related to pain caused by EE. It is interesting that YM had longer vocalizations and a greater total time vocalizing than AM males. As all animals were electroejaculated in the past, we can affirm that this difference was not related to the previous experience of EE. This may be at least partially explained by the anatomical differences between the two age groups: as YM were smaller in size and body weight, they may have a smaller pelvic and rectal region. As this is the region where the electrical stimulation was applied, a shorter distance from the electric stimulation to the nerve pathways may explain the greater sensitivity of YM males. The YM vocalized with a higher fundamental frequency during EE than AM. In agreement with our results, Reby et al. (2005) observed that the frequencies with which red deer males emit spontaneous roars (vocalizations during the breeding season) were greater in young than adult males. This difference may also be explained by the anatomical differences between categories. Reby et al. (2005) proposed that the differences in the anatomy of the neck region, and especially of the larynx, which is the phonation organ in deer may
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Acknowledgements Authors are grateful to Tabare Gonzalez-Sierra, who was the Director of the ECFA; Matıas Villagran, Solana Gonzalez, Florencia Beracochea, Lorena Lacuesta, Julia Giriboni helped in data collection; Johnny Brioso, Ricardo Sorello, and Edgardo Barrios, workers from the ECFA, for their help with animal management. Financial support: CSIC (Universidad de la Rep ublica, Montevideo, Uruguay), Intendencia Departamental de Maldonado (Uruguay), and Agencia Nacional de Investigaci on e Innovaci on (ANII).
Conflict of interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the article.
Author contributions Fernando Fumagalli recorded the data, made the initial analysis of the data and drafted the first version of the manuscript. Juan Pablo Damian helped with data recording and analysis, especially with the sonogram analysis, and participated in writing the manuscript. Rodolfo Ungerfeld directed and coordinated the study and the analysis of data, coordinated the manuscript preparation writing several parts of the article including the submitted version.
Supporting Information Additional Supporting Information may be found in the online version of this article: Audio S1 Audio of vocalization of an adult deer during electroejaculation. Audio S2 Audio of vocalization of a young deer during electroejaculation.
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Submitted: 20 Nov 2014; Accepted: 13 Jan 2015 Author’s address (for correspondence): R Ungerfeld, Departamento de Fisiologıa, Facultad de Veterinaria, Universidad de la Rep ublica, Lasplaces 1620, 11600 Montevideo, Uruguay. E-mail: rungerfeld@gmail. com
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Vocalizations During Electroejaculation in Anaesthetized Adult and Young Pampas Deer (Ozotoceros bezoarticus) Males F Fumagalli1, JP Damian2 and R Ungerfeld3 1 Clınica Semiol ogica, Facultad de Veterinaria, Universidad de la Repu´blica, Montevideo, Uruguay; 2Departamento de Biologıa Molecular y Celular, Facultad de Veterinaria, Universidad de la Rep ublica, Montevideo, Uruguay; 3Departamento de Fisiologıa, Facultad de Veterinaria, Universidad de la Rep ublica, Montevideo, Uruguay
Contents The aim of this study was to characterize the vocalizations produced during electroejaculation under general anaesthesia in pampas deer males and to determine whether the characteristics of those vocalizations differ in adult and young pampas deer males. Electroejaculation was applied to 13 adults (AM) and 13 young (YM) males under general anaesthesia. Vocalizations were digitally recorded, and the number and duration of vocalizations, the latency in relation to each voltage, the total time vocalizing, and the structure of the fundamental frequency (F0) [initial frequency (Fstart), maximal frequency (Fmax), minimal frequency (Fmin) and final frequency (Fend)] were analysed. No male vocalized with 0 V; the number of animals that vocalized increased at 2 and 3 V and increased again at 4, 5 and 6 V (p < 0.05). The latency time from the beginning of each series (each voltage) decreased until 4 V (p < 0.01). The number of vocalizations/voltage increased from 4 V (p < 0.05). The length of each vocalization and the total time during which animals vocalized were greater in YM than AM (p = 0.02 and p = 0.01, respectively). Similarly, the fundamental frequencies were higher in YM than AM (p ≤ 0.05). Overall, we concluded that the vocalizations emitted during electroejaculation in pampas deer under general anaesthesia are related to the voltage applied during the process. Young males vocalize more time, probably due to a greater sensibility to the electric stimulation. The differences in the characteristics of the vocalizations between adult and young males may be related to the anatomic differences in the neck of adult or young males.
Introduction The pampas deer (Ozotoceros bezoarticus, Linnaeus, 1758) used to be a widespread species originally distributed in the open grasslands of South America (Jackson and Langguth 1987). However, habitat fragmentation, agriculture development, competition with farmed animals and unregulated hunting led to the decrease in size and distribution of the species (Demarıa et al. 2003). This species is listed in Appendix I (see supporting information) of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES 2014). In Uruguay, there are two wild populations, and there is also a third semicaptive group (Ungerfeld et al. 2008). This third population is allocated at the Estaci on de Crıa de Fauna Aut octona Cerro Pan de Az ucar (ECFA) (Maldonado, Uruguay; 34°30 S, 54°00 W) and consists of approximately 80 individuals. The cryopreservation of gametes and the development of gene banks are useful techniques to improve conservation © 2015 Blackwell Verlag GmbH
status of endangered species (Garde et al. 2006). For this purpose, techniques for semen collection should be adequately adapted for each species. As artificial vagina can only be used in tame and trained individuals, electroejaculation (EE) is the most widely used technique for semen collection in wild animals. In deer, due to nervous temperament and thus, risks for operators, EE is conventionally used under general anaesthesia (Umapathy et al. 2007; Martınez et al. 2008; Martınez-Pastor et al. 2009). Electroejaculation produces some undesirable physiological responses. For example, in anaesthetized pampas deer, we previously observed increases in heart and pulse rates, as well as in creatine kinase, aspartate aminotransferase and alkaline phosphatase concentrations, and a decrease in rectal temperature (Fumagalli et al. 2012). Responses to EE may also differ in relation to males’ age, as adult males had a greater increase in heart rate, but lower increases in alkaline phosphatase and creatine kinase concentrations. These negative responses have been associated with the occurrence of vocalizations in bulls (Falk et al. 2001; Whitlock et al. 2012) and rams (Damian and Ungerfeld 2011), which are considered as reliable indicators of pain (Damian and Ungerfeld 2010). Although we did not record vocalizations in anaesthetized domestic (rams, bucks) or wild (antelopes, muflon, dama deer) ruminants, strikingly we have previously observed that pampas deer males vocalize during EE even while they are maintained under general anaesthesia. At least under several other contexts, the increases in cortisol secretion, and the number, duration and characteristics (high-frequency) of vocalizations are reliable indicators for pain (Prunier et al. 2013). Considering all this information, a first aim of this study was to characterize the vocalizations produced during EE under general anaesthesia in pampas deer males. Another objective was to determine whether the characteristics of the vocalizations, as well as the cortisol and creatine kinase serum concentration, differ in adults and young pampas deer males during EE under general anaesthesia.
Materials and Methods Animals and facilities The study was conducted from September to March (spring to autumn), at the ECFA, with 26 males. From
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these, 13 were adult [group AM; 4–7 year old, 29.8 3.0 kg (mean SEM)] and 13 young (group YM; 1.5–2 year-old, 25.6 1.6 kg) males. All these males were anaesthetized and electroejaculated at least once before this study. Both groups were housed in different paddocks (0.5 ha), with free access to water, native pastures, trees and shrubs. Animals also received approximately 600 g of dairy cow ration/deer daily. The study was approved by the Comisi on Honoraria de Experimentaci on Animal (CHEA), from the Universidad de la Rep ublica, Uruguay. Capture and anaesthesia The animals were captured and managed as described by Fumagalli et al. (2012). Briefly, they were captured with anaesthetic darts fired from a blowpipe (Telinject USA, Inc. Soledad Canyon Road Agua Dulce, Newhall, CA, USA) containing 2 mg/kg of xylazine (Sedomin 10%, Laboratorio K€ onig, Buenos Aires, Argentina), 1.6 mg/kg of ketamine (Vetanarcol 5%, Laboratorio K€ onig S.A.) and 0.013 mg/kg of atropine (Sulfate de Atropine 1& ION, Laboratorio ION, Montevideo, Uruguay). After the animals were anaesthetized (induction time), they were placed on a stretcher, transported, weighed and placed in right lateral position on a padded table. An intravenous catheter was placed in the cephalic vein, and sodium chloride 0.9% was administered to have a continuous via open. Electroejaculation Electroejaculation was carried out using a rectal probe (300 mm length, 19 mm width, with 30 mm longitudinal electrodes; Model 303, P-T Electronics, Boring Oregon, USA). The EE was performed according to Beracochea et al. (2014), with series of 10 periods (4–5 s) of voltage stimulus with the same voltage, with rest intervals of 2–3 sec, increasing 1V after each 10 pulses series, beginning with 1V until ejaculation (maximum = 7 V). Registration of vocalizations Vocalizations were recorded using a microphone and mp4 equipment (Insignia NS-4V24, China). The distance between the microphone and the head of the recorded animal was 150 cm. The sound measurements and spectral analysis were performed with WaveSurfer 1.8.5 acquisition software (open software, KTH Royal Institute of Technology, Stockholm, Sweden, 2005). Average and total time duration of vocalizations, latency (time from first electric pulse of each series to first vocalization emitted in that series), and the initial (Fstart), final (Fend), minimal (Fmin) and maximal (Fmax) frequencies (Hz) of the fundamental frequency (F0) were calculated.
F Fumagalli, JP Dami an and R Ungerfeld
Cortisol and creatine kinase serum determination Two blood samples were collected immediately before and immediately after EE. Samples were allowed to clot at room temperature, centrifuged at 1080 9 g for 20 min, and the serum was stored at 20°C. Creatinine kinase (CK) serum concentrations were measured in the Laboratorio de Analisis Clınicos (Facultad de Veterinaria, Montevideo, Uruguay), using a clinical analyser (Metrolab 1600 DR; Metrolab, Buenos Aires, Argentina). Serum cortisol concentrations were measured in the Laboratorio de Tecnicas Nucleares (Facultad de Veterinaria) by radioimmunoassay using a solid phase kit (DPC; Siemens, Los Angeles, CA, USA). The analytical sensitivity of the assay for cortisol was 2.0 nmol/l. The intra-assay and interassay coefficients of variation were both below 12.5%. Recovery of the animals After the procedure finished, the animals were taken back to their respective paddocks. Anaesthesia was reversed with a jugular vein injection of 0.26 mg⁄kg of yohimbine hydrochloride 1% (Reverze, Laboratorio Vetcross, Montevideo, Uruguay), and the time from administration to recovery of the animals was recorded (recovery time). Data analysis The induction and the recovery time, and the number of series in which AM and YM began to ejaculate was compared with ANOVA. Separated linear regressions were performed for AM and YM between the total number of pulses and the total number of vocalizations. The differences between categories in the number of vocalizations/voltage, the total time vocalizing, length time of each vocalization, the Fstart, Fend, Fmax and Fmin, during EE were also compared by ANOVA. The number of AM and YM that vocalized in each voltage was compared with the Fishers’ exact probability test. The influence of voltage on the latency to the first vocalization was compared with ANOVA for repeated measures, considering the category of the male (AM vs YM) and the voltage as fixed effects, and the male as a random effect into each experimental group. Concentrations of cortisol and creatine kinase serum were compared by ANOVA for repeated measures. The model included the effect of category (AM vs YM), time (before and after EE), as well as their interaction. Data are expressed as mean SEM.
Results The induction time was 12.0 0.6 and 14.0 10.0 min, and the recovery time was 2.6 2.4 and 2.3 1.8 min in AM and YM, respectively. Semen was collected from all animals: ejaculation began at 3.6 0.7 and 3.5 0.5 volts in adults and yearlings (ns), respectively.
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Electroejaculation and Vocalizations
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Number of vocalizations
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b
12 a
8 4 0 1
2
3 4 Voltages (V)
5
6
Fig. 1. Number of vocalizations emitted in each voltage used during electroejaculation in adults (black column n = 13) and young (grey column n = 13) anaesthetized pampas deer males. Values are expressed as mean SEM. a vs b: p < 0.05
75 Total number of vocalizaƟons
Vocalizations There were no vocalizations recorded before or after EE in any animal. Only one AM male did not vocalize during EE. The number of animals that vocalized with each voltage did not differ in AM and YM males, so it is presented pooled in Table 1. No male vocalized with 0 V, the number of animals that vocalized increased at 2 and 3 V, and increased again at 4, 5 and 6 V (Table 1). The latency from the beginning of each pulse of the series to the first vocalization in that series was not affected by the group, and there was no interaction between voltage and group, so it is presented pooled despite the group in Table 1. This latency time was shorter with greater voltages (p < 0.0001) (Table 1). The number of vocalizations/voltage increased from 4 V (p < 0.05), without differences between AM or YM (Fig. 1). The total number of vocalizations was positively related with the total number of pulses in AM (R2 = 0.55; p = 0.014) and tended to do so in YM (R2 = 0.28; p = 0.09) (Fig. 2). The total number of vocalizations was similar in AM and YM males, but the length of each vocalization and the total time during which animals vocalized were greater in YM than AM (p = 0.02 and p = 0.01, respectively; Table 2). Similarly, the fundamental frequencies: Fmax, Fend, Fmin were higher in YM than AM (p = 0.04, p = 0.05 and p = 0.05, respectively), and the Fstart tended to be higher in YM than AM (p = 0.06) (Table 2). The sonogram of one vocalization from an AM and one from a YM are included as examples in Fig. 3, and the recordings are included as supplementary files. In these sonograms, it can be observed the difference between AM (bass sound) and YM (high-pitched sound).
50
25
0 30
40
50
60
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Total number of pulses
Fig. 2. Relationship between the total number of pulses during electroejaculation and the total number of vocalizations in adult () and young (○) pampas deer males. Adult males: R2 = 0.55; p = 0.014 young males: R2 = 0.28; p = 0.09
Cortisol and creatine kinase serum The cortisol and creatine kinase serum concentrations increased after the EE. Both, cortisol and creatine kinase concentrations increased after the procedure (p = 0.006 and p = 0.01, respectively), with no effect of group or interaction between group and time in the serum concentrations of cortisol and creatine kinase (Fig. 4a and b, respectively).
Table 2. Number of vocalizations, length of each vocalization, total time vocalizing during electroejaculation and fundamental frequencies of adult (AM) and young (YM) pampas deer males during electroejaculation under general anaesthesia. Values are expressed as mean SEM
Table 1. Number of animal that vocalized and the latency time (min) for voltage (1V–6V) during electroejaculation under general anaesthesia. Values are expressed as mean SEM
N Number of vocalizations Length time of vocalization (s) Total time of vocalization (s) Fstart (Hz) Fmax (Hz) Fend (Hz) Fmin (Hz)
Voltage 1 2 3 4 5 6
Total 0/26 4/26 10/26 17/26 18/26 17/26
(0%)a (15%)b (39%)b (65%)c (69%)c (65%)c
Latency (min) 3.0 2.0 1.1 0.4 0.08 0.03
0.3w 0.3x 0.2y 0.2z 0.05z 0.01z
Different letters within columns indicate significant differences (a vs b vs c: p < 0.05; w vs x vs y vs z: p < 0.01).
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AM 13 11 1.4 12 286 314 243 222
2 0.2 2 51 59 51 43
YM 13 10 2.0 20 405 484 377 331
2 0.2 2 50 67 59 47
p
ns 0.02 0.01 0.06 0.04 0.05 0.05
Fundamental frequencies: Fstart (initial frequency), Fmax (maximum frequency), Fend (final frequency), Fmin (minimum frequency), NS (not significant).
Discussion According to our knowledge, this is the first description of the vocalizations emitted in animals during EE under general anaesthesia. Although the animals were anaes-
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F Fumagalli, JP Dami an and R Ungerfeld
(a)
(b)
Fig. 3. Spectrograms of the vocalizations of adult (a) and young (b) pampas deer males recorded during the electroejaculation under general anaesthesia. While adult males show bass sounds, young males show high-pitched sounds
thetized, the anaesthetic combination used in this work (xylazine, ketamine and atropine) does not affect the ability to vocalize (McKelvey and Hollingshead 2003). Although the causes of vocalizations cannot be determined in this study, it is clear that its expression is directly related with the electric pulses. This may be a consequence of a lower effect of the anaesthesia combination in this species, but this is not reflected in other physiological parameters (Fumagalli et al. 2012). Other possible explanation is a greater sensitivity of pampas deer to EE, determining that even anaesthetized they elicit a clear response. An alternative strategy to determine this would be to test other anaesthetic preparations or analgesics, such as titelamine or zolazepam, as both may provide better analgesia than the combination used in this study (Santiago-Moreno et al. 2011). Other combinations, such as the method previously used plus inhalation of isoflurane anaesthesia (dos Santos et al. 2010), or the addition of analgesic agents as fentanyl citrate (Asher et al. 2011) should also be tested. The use of alternative techniques, such as the use of transrectal ultrasound-guided massage of the accessory sex glands (TUMASG), a technique reported to be effective in aoudad (Ammotragus lervia sahariensis)
(Santiago-Moreno et al. 2013) should be tested. Unfortunately, due to the low number of pampas deer available for research, advancement in the development of effective alternative techniques is slow in the species. As the number of vocalizations emitted increased with the increase in voltage, and at the same time the latency to the first vocalization decreased, it seems that the voltage used is directly related to these responses. However, we cannot discard that the increased response observed with higher voltage can be a consequence of the accumulated process, as the animals received lower voltage before. In any case, considering that vocalizations seem to be reliable indicators of pain produced by EE in other species (Damian and Ungerfeld 2010), it would be important to study whether shorter protocols, even beginning with greater voltage, are as effective as this one. It is interesting to note that acoustic communication seems not to be as important in the pampas deer as in other deer species, probably because it lives in open grasslands, with easy visual communication. Vocalizations in the same population of pampas deer have been previously studied (see review: Ungerfeld et al. 2008), but after several trials, we have only recorded vocalizations for offspring calling by the dams (Olazabal et al. © 2015 Blackwell Verlag GmbH
Electroejaculation and Vocalizations 70
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**
(a)
CorƟsol (nmol/l)
60 50 40 30
20 10 0
BEE 400
(b)
AEE
**
CK (UI/l)
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100
explain this difference. The neck perimeter is a secondary sexual character of the deer, directly influenced by the anabolic action of testosterone before the rut (Handelsman 2006). As in the pampas deer, both the testosterone concentrations and the neck size are greater in adult than in young animals during the rut (Ungerfeld et al. 2011), the larynx is probably larger in AR than YR, provoking differences in the resonance of the vocalizations. Overall, we concluded that the vocalizations emitted during EE in pampas deer under general anaesthesia are related to the voltage applied during the process. Young males vocalize for a greater time length, probably due to a greater sensitivity to the electric stimulation. The differences in the characteristics of the vocalizations may be related to the anatomic differences in the neck of adult or young males. Given changes in stress related physiology demonstrate herein along with the recognition that the vocalization during EE is related to pain in other species, it seems important to develop alternative techniques to EE with less negative effects on welfare when applied to wild animals.
0
BEE
AEE
Fig. 4. a) Cortisol and b) CK concentrations before (BEE) and after (AEE) electroejaculation in adult (black bars) and young (white bars) pampas deer males. **p < 0.01
2013), and in one occasion during courtship (MoralesPi~ neyrua and Ungerfeld 2012). Therefore, this reinforces the hypothesis that these vocalizations observed out from a social context are probably related to pain caused by EE. It is interesting that YM had longer vocalizations and a greater total time vocalizing than AM males. As all animals were electroejaculated in the past, we can affirm that this difference was not related to the previous experience of EE. This may be at least partially explained by the anatomical differences between the two age groups: as YM were smaller in size and body weight, they may have a smaller pelvic and rectal region. As this is the region where the electrical stimulation was applied, a shorter distance from the electric stimulation to the nerve pathways may explain the greater sensitivity of YM males. The YM vocalized with a higher fundamental frequency during EE than AM. In agreement with our results, Reby et al. (2005) observed that the frequencies with which red deer males emit spontaneous roars (vocalizations during the breeding season) were greater in young than adult males. This difference may also be explained by the anatomical differences between categories. Reby et al. (2005) proposed that the differences in the anatomy of the neck region, and especially of the larynx, which is the phonation organ in deer may
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Acknowledgements Authors are grateful to Tabare Gonzalez-Sierra, who was the Director of the ECFA; Matıas Villagran, Solana Gonzalez, Florencia Beracochea, Lorena Lacuesta, Julia Giriboni helped in data collection; Johnny Brioso, Ricardo Sorello, and Edgardo Barrios, workers from the ECFA, for their help with animal management. Financial support: CSIC (Universidad de la Rep ublica, Montevideo, Uruguay), Intendencia Departamental de Maldonado (Uruguay), and Agencia Nacional de Investigaci on e Innovaci on (ANII).
Conflict of interest None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the article.
Author contributions Fernando Fumagalli recorded the data, made the initial analysis of the data and drafted the first version of the manuscript. Juan Pablo Damian helped with data recording and analysis, especially with the sonogram analysis, and participated in writing the manuscript. Rodolfo Ungerfeld directed and coordinated the study and the analysis of data, coordinated the manuscript preparation writing several parts of the article including the submitted version.
Supporting Information Additional Supporting Information may be found in the online version of this article: Audio S1 Audio of vocalization of an adult deer during electroejaculation. Audio S2 Audio of vocalization of a young deer during electroejaculation.
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Submitted: 20 Nov 2014; Accepted: 13 Jan 2015 Author’s address (for correspondence): R Ungerfeld, Departamento de Fisiologıa, Facultad de Veterinaria, Universidad de la Rep ublica, Lasplaces 1620, 11600 Montevideo, Uruguay. E-mail: rungerfeld@gmail. com
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