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Behavioural study of a progeny of a bull clone
Livestock Science 162 (2014) 209–213
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Livestock Science journal homepage: www.elsevier.com/locate/livsci
Short communication
Behavioural study of a progeny of a bull clone C. Federici a,1, M. Speroni a,n, M. Capelletti a,1, F. Abeni a,1, G. Pirlo a,1, R. Aleandri b,2 a Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per le produzioni foraggere e lattiero-casearie, sede di Cremona, via Porcellasco 7, 26100 Cremona, Italy b Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Direzione Dipartimento Biologia e Produzioni Animali, via Nazionale 82, 00184 Roma, Italy
a r t i c l e i n f o
abstract
Article history: Received 6 March 2012 Received in revised form 2 January 2014 Accepted 17 January 2014
Behaviour of progeny of a bull clone was compared to that of offspring from the donor bull. A total of fifteen Friesian female calves were used. Seven of them were progeny of a bull clone (CLp) and eight were offspring from its donor bull (DONp). During the collection of blood samples for hemocromocytometric analysis, behavioural response of calves to restraint was examined. Calves were individually restrained for blood sampling at 24–48 h, and at 1, 2, 3, 4, 8, 12, 16, 20, 24 weeks of age. Number of struggling movements (STR) and vocalisations (VO) occurring within the first minute of blood sampling were recorded by direct observation. The calves were also monitored at 2nd, 3rd, 4th, and between 5th and 8th week of age for lying, standing, nutritive and non-nutritive oral, sniffing, self-grooming behaviours and vocalisation. Each calf was observed in a morning and an afternoon time band, each lasting 3 h. No difference was found between CLp and DONp on the overall mean for STR and VO at restraint. The study of general behaviour did not reveal difference between CLp and DONp for the average proportion of any of the considered traits. Our results suggest that the adaptive ability of progeny of cloned bull to challenges and conditions imposed by a farm environment are comparable to what is normally observed in dairy calves reared under similar conditions. & 2014 Elsevier B.V. All rights reserved.
Keywords: Behaviour Cloning Progeny Dairy cattle
1. Introduction Cloning livestock by somatic cell nuclear transfer implies reprogramming a somatic cell into a totipotent embryonic cell. An incomplete nuclear reprogramming due to epigenetic dysregulation is considered to be the main reason of abnormalities and impaired health observed in livestock clones in peri-natal period (EFSA, 2012). These aspects have
n Correspondence to: Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per le produzioni foraggere e lattierocasearie, sede di Cremona, via Porcellasco 7, 26100 Cremona, Italy. Tel.: þ 39 0372 433029; fax: þ39 0372 435056. E-mail addresses: [email protected], [email protected] (M. Speroni). 1 Tel.: þ39 0372 433029; fax: þ39 0372 435056. 2 Tel.: þ39 06 47836277; fax: þ 39 06 47836210.
1871-1413/$ - see front matter & 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.livsci.2014.01.018
raised ethical and scientific concerns about the welfare of both clones and their progeny, due the possibility that epigenetic abnormalities observed in cloned subjects could be transmitted to the offspring. Several studies were carried out on the progeny of cattle clones. Results of these studies showed that the offspring of clones displayed haematological and biochemical profile (Abeni et al., 2012), as well as growth and reproductive parameters (Abeni et al., 2012; Heyman et al., 2004; Kasai et al., 2007; Panarace et al., 2007; Watanabe and Nagai, 2008) comparable with healthy, non-clone animals, suggesting that epigenetic possible alterations present in clones were reset in the germ line of clone. This was further supported by Couldrey et al. (2011) who analysed the sperm DNA methylation patterns of Friesian bull clones. Few studies were carried to investigate the effects of cloning on behaviour of cattle; these studies show that
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bovine clones exhibit less playing behaviour, higher levels of curiosity, more grooming activities (Savage et al., 2003) and more exploratory behaviour than conventional controls (Coulon et al., 2007). No difference was found between clones and conventional heifers for cognitive capacities and kin discrimination (Coulon et al., 2010). To our knowledge no specific study on behaviour of progeny of bull clones has been done. In farm animals, behaviour is an important and early indicator of proper maturation of physiological systems underlying the vital functions and ability to cope with the environment. Restraint, even in absence of painful stimuli, has been shown to be a powerful stressor in several species; in cattle, behavioural responses to restraint can be used as indicators of coping ability and fearfulness, which are traits mediated by underlying biological functions. Also the behavioural repertoire, the rhythms of rest and activity, the development of behaviour with age are potential indicators of the level at which the farm animals are adapted to their environment. Calves need to spend a lot of time resting and sleeping; Hänninen et al. (2005) found a positive correlation between the total duration of rest and the growth rate in calves; sleep is essential for regulation of several hormones and brain development in growing animals. The aim of the present study was to assess whether there was a difference between progeny of a bull clone and offspring of the corresponding donor bull in (a) the level of excitability to routine handling, measured by the behavioural response, and (b) the general behaviour expression, as indicators of adaptive ability to farm environment. 2. Materials and methods 2.1. Animals and management The study was carried out at the experimental dairy farm of CRA-FLC, located in Cremona (Italy). A total of fifteen Holstein female calves were used. The calves were born at different times from Holstein recipient heifers implanted with embryos obtained by IVF with frozen semen of a bull clone (n ¼7; CLp) or its nuclear transfer donor bull (n ¼8; DONp, as control); oocytes for in vitro production of embryos were collected from six Holstein donor cows. Deliveries were concentrated between November and January; median of birth dates were December 13th and December 23rd for CLp and DONp, respectively; standard deviation of birth dates was higher in CLp (42 days) than in DONp (21 days) due to one CLp born on September. The mean of birth weight were 38.4 kg (SD ¼8.2 kg; range¼26.0–52.0) and 42.7 kg (SD ¼8.9 kg; range ¼27.0–54.0) for CLp and DONp, respectively. Calves were separated from their respective surrogate mothers immediately after birth and moved to a calf barn, where they were housed in individual straw-littered cages. Cages (182 90 cm2) had solid sides, with an opening in the front to allow the calf access to bucket of milk and feeder. A heat lamp was directed onto the cage for the first 48 h of life. Calves were fed colostrum by bottle for the first two day postpartum. Starting from 3rd day of age, calves were fed
reconstituted milk, twice daily (06:00–15:00), by nippled milk bucket (2 L/meal, on average). At approximately one week of age, the milk diet was supplemented with calfstarter and ad libitum roughage in the form of hay, distributed daily, immediately after the morning milk meal. At 8 weeks of age, calves were transferred outdoors into group pens (6.15 3.33 m2; to a maximum of 4 calves/pen). The calves were weaned in the successive 2 weeks, by a progressive decrease in milk supply and after the assessment of an adequate solid feed intake (approximately above 700–800 g/day of DM). All calves were healthy at birth, but during the first moth of life three DONp died for neonatal diarrhoea. 2.2. Data collection 2.2.1. Behaviour at restraint Behavioural data were collected during sample session for the study of haematological profile and clinical chemistry. According to that protocol, each calf was subjected to manual restraint for sampling blood at jugular vein at 24–48 h and 1, 2, 3, 4, 8, 12, 16, 20, 24 weeks of age. Blood sampling sessions were performed between 06:00 and 07:00, before milk distribution. On each occasion, an operator entered the cage and manually restrained the calf firmly against a wall of the cage until the end of blood sample collection, while another operator outside the cage kept the calf's head. Blood sampling started immediately after immobilisation of the calf. Blood was drawn from external jugular vein using a 20 G needle connected to vacuum tube. From the insertion to removal of the needle passed at least 1 min per calf because of the need to collect from 3 to 4 samples. This was obtained by changing vacuum tubes, without removal of the needle from jugular vein. At the end of blood sampling the calf was released. In calves raised into group pens, restraint consisted of head immobilisation by the headlocks; a rope was also placed around the muzzle. Blood sampling procedure is described above. In cattle, it was already shown that acute restraint per se elicits a variety of physiological and behavioural responses, where struggling is the most common behavioural expression. Struggling movement was defined as vigorous trunk movements in attempting to free oneself from the squeeze by operator or head immobilisation by headlocks. Number of struggling movements (STR) and vocalisations (VO) occurring within the first minute of blood sampling were recorded by direct observation. Struggling movements were considered as a distinct event when a single or a series of trunk movements were interrupted by a period of body immobility of at least 10 s. 2.2.2. Behaviour over the pre-weaning period Each calf was observed during four daily sessions at 2nd, 3rd, 4th, and between the 5th and 8th week of age; on the basis of preliminary observations of conventional calves reared on the same experimental farm, a morning (MTB) and an afternoon time band (ATB) were chosen for observations. Morning time band started 2 h after milk meal distribution and lasted 3 consecutive hours; observation during this time band aimed to compare the two groups mainly for resting behaviour; in ATB, calves were observed 1 h before,
C. Federici et al. / Livestock Science 162 (2014) 209–213
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Table 1 Catalogue of behaviours selected for observation of calves. Behaviour/posture
Description
Lying inactive with neck relaxed
Lying on the sternum or on-side, neck relaxed, head on the ground, or turned back on the neck, or front legs, or flank, without any obvious activity Lying on the sternum, head lifted up, with opened or closed eyes, without any obvious activity Standing, head turned towards any of solid wall of the cage, without any obvious activity Standing, head turned towards or outside the open side of the cage, without any obvious activity Eating solid food from the feeder Chewing rhythmically while not eating, in standing or lying position Sucking at some parts of the cage or at an object Licking some part of cage Self-licking or scratching or rubbing herself against some parts of cage Sniffing walls or litter of the cage All types of vocalisations Swinging the tongue outside of the mouth from one side to the other, or contorts and rolls it inside the mouth, or stretches it out
Lying inactive with head up Standing inactive Standing inactive at the feeder Eating Chewing Non-nutritive sucking Licking Self-Grooming Sniffing Vocalizing Tongue-playing
at the hour and 1 h after milk meal, to emphasise behaviours normally associated with milk distribution. Individual calf behaviour was recorded by a video camera provided with internal hard disk, mounted on a tripod and placed in front of the cage. The videos were successively transferred to a personal computer and the behavioural data sampled from the video by instantaneous sampling at 3 min intervals. The description of recorded behaviours is given in Table 1. Each occurrence was counted; VO occurrences were expressed as the total number per hour; the occurrences of other behaviours were expressed as percentage of total number of scans per hour.
2.3.2. Behaviour over the pre-weaning period Data from only 13 calves (7 CLp; 6 DONp) were used for the analysis, because, due to technical faults, 2 DONp had more than one incomplete daily observation. Percentages data, with exception for VO, were arcsine transformed. Transformed data were analysed by mixed models for repeated measures: sire (CLp or DONp), time band (MTB or ATB), hour within time band (1, 2, 3 within MTB or ATB), age (2nd, 3rd, 4th, 5th–8th week) and their interactions were considered as fixed factors and calf as random factor. Back-transformed values were used in tables. 3. Results
2.3. Statistical analysis 3.1. Behaviour at restraint Data were analysed using MIXED procedure of SAS/ STAT software in the SAS system for Windows, Release 9.2 (SAS Institute, Inc. 2008). 2.3.1. Behaviour at restraint Data from only 14 calves (6 CLp; 8 DONp) were analysed; the CLp that was born on September was excluded from analysis due its late inclusion in the monitoring protocol. Counts of STR and VO were square root transformed before statistical analysis. A mixed model for repeated measures was used to analyse the fixed effect of the bull (CLp or DONp), age (0, 1, 2, 3, 4, 8, 12, 16, 20, 24 weeks), birth weight (o40 kg, Z40 kg or r50 kg, 450 kg), level of assistance at birth (unassisted; assisted by one person, assisted by more than one person, caesarean section) and bull by age interaction. Calf was considered as random factor. Statistical significance was set at Po0.05. Literature reports that production of embryos by in vitro procedure and embryo transfer techniques can result in the birth of larger offspring with higher incidence of dystocia, perinatal loss and anomalies, compared to AI (Galli et al., 2003); for this reason the level of assistance at birth and birth weight were introduced in the model to test their effect and exclude any influence on calf reactivity. No effect of birth weight, level of assistance at birth and bull by age interaction were found on STR or VO; thus the final model included only bull and age as fixed effects and calf as random effect.
All calves struggled and vocalized when restrained for sampling, but no difference was found between CLp and DONp on the overall mean for STR and VO. A reduction (P ¼0.0008) of the number of vocalisations with age was observed, but this effect did not differ between CLp and DONp. 3.2. Behaviour over the pre-weaning period No difference between CLp and DONp was found for the average proportion of time spent in any of the considered behaviours. An effect of the time band, the hour within the time band, and the age on most of the behaviours was found, but these effects did not differ between CLp and DONp, with exception for licking. There was no case of tongue rolling in any of the calves monitored. For licking, a sire by age interaction was found; in particular, CLp showed at 2 and 3 weeks of age a higher proportion of licking than DONp, while at 4 weeks of age this behaviour resulted more expressed in DONp than in CLp. Development over time of the other behaviours was the same for both groups. As calves were growing, eating solid food increased, whereas chewing activity decreased. A clear decrease was observed also for vocalisation, as well as for sniffing (Table 2).
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Table 2 Least square means (99% CI) of behaviour (LMS (99% CI)) of the calves at different age. Behaviour
Age (weeks) 2nd
Lying inactive with neck relaxed (%) Lying inactive with head up (%) Standing inactive (%) Standing inactive at feeder (%) Eating (%) Chewing (%) Non-nutritive sucking (%) Licking (%) Self grooming (%) Sniffing (%)
17.43 8.38 2.25 7.04 0.54 12.35 0.07 0.22 1.23 2.19
3rd (12.03–23.81) (5.14–12.42) (1.48–3.18) (4.58–10.03)a (0.06–1.54)c (8.10–17.49)a (0.01–0.16) (0.08–0.42) (0.63–2.03)ab (1.41–3.13)a
14.20 7.16 1.93 4.16 4.48 10.21 0.06 0.12 0.68 1.08
(9.33–20.08) (4.17–10.95) (1.22–2.82) (2.31–6.55)a (2.59–6.89)b (6.35–14.99)ab (0.01–0.15) (0.02–0.29) (0.26–1.31)b (0.55–1.79)ab
4th
5th–8th
13.55 (8.71–19.43) 8.27 (4.96–12.41) 1.02 (0.51–1.70) 5.49 (3.29–8.25)a 6.47 (4.32–9.70)ab 4.92 (2.34–8.46)b 0.03 (0.00–0.11) 0.14 (0.03–0.32) 0.99 (0.45–1.75)b 0.90 (0.42–1.56)b
14.05 13.21 1.15 1.47 10.22 5.74 0.08 0.14 2.24 0.54
(9.21–19.90) (8.99–18.22) (0.62–1.84) (0.48–2.99)b (7.25–13.70)a (2.97–9.43)b (0.00–0.07) (0.03–0.32) (1.39–3.29)a (0.20–1.04)b
Means with different letters on same row differ significantly (P o0.05).
Table 3 Least square means (99% CI) of behaviour performed by calves at different hours of the day, within MTB and ATB. Behaviour
Hours within time bands H1(MTB)
H2(MTB)
H3(MTB)
H1(ATB)
H2(ATB)
H3(ATB)
9.11 (4.90–14.60)c 2.14 (0.42–5.19)c 5.32 (2.26–9.68)c Lying inactive 34.26 (25.68–43.98 )a 34.93 (26.20–44.89)a 20.17 (13.64–27.98)b with neck relaxed (%) Lying inactive 17.73 (12.37–24.03)a 12.26 (7.85–17.65)ab 8.96 (5.24–13.68)ab 11.62 (7.29–16.95)ab 2.01 (0.49–4.56)c 6.68 (3.52–10.85)b with head up (%) 0.66 (0.05–1.00)d Standing 0.71 (0.07–2.03)d 3.55 (1.69–6.10)c 4.87 (2.62–7.81)bc 9.0 (5.79–12.93)ab 13.12 (9.21–17.73)a inactive at feeder (%) Standing 0.55 (0.17–1.14) d 0.66 (0.21–1.24)cd 1.73 (0.96–2.71)abc 1.31 (0.66–2.19)bcd 2.75 (1.74–3.99)ab 3.28 (2.18–4.61)a inactive (%) Eating (%) 1.32 (0.34–2.95)b 1.73 (0.55–3.56)b 2.52 (1.02–4.67)b 9.55 (6.31–13.46)a 15.24 (11.02–20.14)a 3.7 (1.81–6.27)b Chewing (%) 2.63 (0.76–5.61)b 4.15 (1.64–7.79)b 6.85 (3.45–11.41)b 7.91 (4.19–12.80)b 18.3 (12.29–25.51)a 13.12 (8.16–19.24)ab Non-nutritive 0.00 (0.0294–0.021) 0.00 (0.018–0.035) 0.00 (0.0296–0.023) 0.02 (0.001–0.087) 0.73 (0.4769–1.04) 0.10 (0.024–0.231) sucking (%) 0.13 (0.02–0.24)bc 0.05 (0.00–0.16)c 0.53 (0.27–0.32)a 0.42 (0.19–0.19)ab 0.04 (0.00–0.89)c Licking (%) 0.04 (0.00–0.42)c Self grooming 1.46 (0.72–2.46)abc 0.8 (0.28–1.59)bc 2.42 (1.41–3.69)a 0.71 (0.23–1.48)bc 0.51 (0.12–1.18)c 2.01 (1.10–3.19)ab (%) Sniffing (%) 0.85 (0.33–1.60) 0.78 (0.28–1.52) 1.31 (0.62–2.24) 1.41 (0.69–2.38) 1.86 (1.01–2.97) 0.64 (0.201.34) Means with different letters on same row differ significantly (P o0.05).
Distribution of the activities within MTB and ATB was similar in CLp and DONp; in MTB, there was no difference between CLp and DONp in proportion of time spent lying inactive with neck relaxed or head up. In ATB, they spent the same time in oral activities. Eating, as well as chewing, reached the maximal values around milk-meal time; licking and vocalizing peaked some time before milk-meal distribution. Non-nutritive sucking was observed immediately after calves emptied the milk bucket. The time spent by calves in standing inactive reached maximal values some time after milk-meal distribution (Table 3). 4. Discussion In our study, progeny of bull clone and its donor responded in the same way to handling. Both groups struggled and vocalized when restrained for blood sampling; magnitude of behavioural response was similar for the two groups. Unlike the struggling response, which remained unchanged over
time, vocalisations decreased as calves were growing, in both progeny of bull clone and control. Overall, these data suggest that all calves reacted with the same emotional level to handling. Thus, our data do not seem to confirm the finding, based on physiological data, of a lower excitability of progeny of a bull clone compared to conventional control during manipulation in the chute reported by Ortegon et al. (2007). General behaviour of progeny from bull clone was similar to that of the progeny of its donor. Behaviours associated with resting represented the more expressed behaviour for both groups and peaked at some distance from the morning meal. It is commonly reported that healthy young calves rest for much of the day and that most of resting occurred at night time or after the morning meal (Hänninen et al., 2005). We did not observe oral stereotypies in any of the monitored calves. Other oral non-nutritive behaviours, such as sucking and licking oriented to structures of the
C. Federici et al. / Livestock Science 162 (2014) 209–213
cage also were at very low levels. Licking is reported as common in pre-weaning calves, more expressed in those housed individually, evocated by a certain level of frustration and or boredom (Kopp et al., 1986). In our study, the early access to solid food as well as to the straw of litter, may have led the calves to satisfy their need for oral manipulation by chewing activity, helping to keep low levels of licking and to prevent the development of oral stereotypies. We observed calves sucking some parts of the cage immediately after the milk bucket was emptied. This is a common behaviour in calves, elicited by ingestion of milk (De Passillé, 2001). In both groups, a high alertness due to expectation for milk meal, was expressed by a peak in licking and vocalisation some time before milk distribution and roused by the presence of caretakers preparing milk substitute. Development of behaviour over age was the same for progeny of bull clone and control. A progressive increase with age was found for eating solid food, according to findings by other authors for calves raised under similar conditions (Hepola et al., 2006). Chewing decreased as calves aged; an increased chewing efficiency, as well as a reduction of the need of oral manipulation, could explain this finding. Other oral non-nutritive behaviours also remained at very low levels over the experimental period in all calves so that it is not easy to provide a biological explanation for different patterns of licking activity that was lower in DONp than in CLp were at 2 and 3 weeks of age, whether at 4th week was lower in CLp than in DONp. In both groups, inactivity in the standing position facing the feeder, as well as sniffing the walls or the ground of the cage decreased with age. It is likely that the increase with age in time spent eating solid food reduced time dedicated by calves to other behaviours. For both groups, the highest number of vocalisations was recorded at the beginning of the observational period. Probably, the increasing intake of solid food covered the nutritional requirements of calves and reduced the level of hunger at the time of the milk-meal. 5. Conclusion In this study, the progeny of cloned bull showed similar reactivity to its counterpart when faced with the same aversive experience of handling. Afterward, offspring of bull clone showed behavioural repertoire, behavioural rhythms and development with age comparable to that commonly reported for preweaning calves raised under similar conditions. These results suggest that adaptive ability of progeny of cloned bull to challenges and conditions imposed by a farm environment was normal. Conflict of interest statement There is no conflict of interest.
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Acknowledgements This work was funded by the Italian Ministry of Instruction, University and Research (MIUR), as a part of the project TECLA (Tecniche di clonazione bovina e sicurezza alimentare del latte). References Abeni, F., Federici, C., Speroni, M., Petrera, F., Pisacane, V., Terzano, G.M., Capelletti, M., Pirlo, G., Aleandri, R., 2012. Body growth, hematological profile, and clinical biochemistry of heifer calves sired by a bull or its clone. Theriogenology 78, 542–559. Couldrey, C., Wells, D.N., Lee, R.S.F., 2011. DNA methylation patterns are appropriately established in the sperm of bulls generated by somatic cell nuclear transfer. Cell. Reprogr. 13, 171–177. Coulon, M., Baudoin, C., Abdi, H., Heyman, Y., Deputte, B.L., 2010. Social behaviour and kin discrimination in a mixed group of cloned and non cloned heifers (Bos taurus). Theriogenology 74, 1596–1603. Coulon, M., Baudoin, C., Depaulis-Carre, M., Heyman, Y., Renard, J.P., Richard, C., Deputte, B.L., 2007. Dairy cattle exploratory and social behaviours: is there an effect of cloning? Theriogenology 68, 1097–1103. De Passillé, A.M., 2001. Sucking motivation and related problems in calves. Appl. Anim. Behav. Sci. 72, 175–187. EFSA (European Food Safety Authority), 2012. Update on the state of play of animal health and welfare and environmental impact of animals derived from SCNT cloning and their offspring, and food safety of products obtained from those animals. EFSA J. 10 (7, 2794), 1–42. Galli, C., Duchi, R., Crotti, P., Turini, N., Ponderato, N., Colleoni, S., Lagutina, I., Lazzari, G., 2003. Bovine embryo technologies. Theriogenology 59, 559–616. Hänninen, L., de Passillé, A.M., Rushen, J., 2005. The effect of flooring type and social grouping on the rest and growth of dairy calves. Appl. Anim. Behav. Sci. 91, 193–204. Heyman, Y., Richard, C., Rodriguez-Martinez, H., Lazzari, G., ChavattePalmer, P., Vignon, X., Galli, C., 2004. Zootechnical performance of cloned cattle and offspring: preliminary results. Cloning Stem Cells 6, 111–120. Hepola, H., Hanninen, L., Pursinianen, P., Tuure, V.M., Syrjala-Qvist, L., Pyykkonen, M., Salonienmi, H., 2006. Feed intake and oral behaviour of dairy calves housed individually or in groups in warm or cold buildings. Livest. Sci. 105, 94–104. Kasai, K., Sano, F., Miyashita, N., Watanabe, S., Nagai, T., 2007. Comparison of the growth performances of offspring produced by a pair of cloned cattle and their nuclear donor animals. J. Reprod. Dev. 53, 135–142. Kopp, M.B., Friend, T.H., Dellmeier, G.R., 1986. Effect of feeding method on nutritive oral activities in Holstein calves. J. Dairy Sci. 69, 3094–3099. Ortegon, H., Betts, D.H., Lin, L., Coppola, G., Perrault, S.D., Blondin, P.W., King, A., 2007. Genomic stability and physiological assessments of live offspring sired by a bull clone, Starbuck II. Theriogenology 67, 116–126. Panarace, M., Aguero, J.I., Garrote, M., Jauregui, G., Segovia, A., Cane', L., Gutierrez, J., Marfil, M., Rigali, F., Pugliese, M., Young, S., Lagioia, J., Garnil, C.D., Forte Pontes, J.E., Ereno Junio, J.C., Mower, S., Medina, M., 2007. How healthy are clones and their progeny: 5 years of field experience. Theriogenology 67, 142–151. Savage, A.F., Maull, J., Tian, X.C., Taneja, M., Katz, L., Darre, M., Yang, X., 2003. Behavioural observations of adolescent Holstein heifers cloned from adult somatic cells. Theriogenology 60, 1097–1110. Watanabe, S., Nagai, T., 2008. Health status and productive performance of somatic cell cloned cattle and their offspring produced in Japan. J. Reprod. Dev. 54, 6–17.
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Livestock Science journal homepage: www.elsevier.com/locate/livsci
Short communication
Behavioural study of a progeny of a bull clone C. Federici a,1, M. Speroni a,n, M. Capelletti a,1, F. Abeni a,1, G. Pirlo a,1, R. Aleandri b,2 a Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per le produzioni foraggere e lattiero-casearie, sede di Cremona, via Porcellasco 7, 26100 Cremona, Italy b Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Direzione Dipartimento Biologia e Produzioni Animali, via Nazionale 82, 00184 Roma, Italy
a r t i c l e i n f o
abstract
Article history: Received 6 March 2012 Received in revised form 2 January 2014 Accepted 17 January 2014
Behaviour of progeny of a bull clone was compared to that of offspring from the donor bull. A total of fifteen Friesian female calves were used. Seven of them were progeny of a bull clone (CLp) and eight were offspring from its donor bull (DONp). During the collection of blood samples for hemocromocytometric analysis, behavioural response of calves to restraint was examined. Calves were individually restrained for blood sampling at 24–48 h, and at 1, 2, 3, 4, 8, 12, 16, 20, 24 weeks of age. Number of struggling movements (STR) and vocalisations (VO) occurring within the first minute of blood sampling were recorded by direct observation. The calves were also monitored at 2nd, 3rd, 4th, and between 5th and 8th week of age for lying, standing, nutritive and non-nutritive oral, sniffing, self-grooming behaviours and vocalisation. Each calf was observed in a morning and an afternoon time band, each lasting 3 h. No difference was found between CLp and DONp on the overall mean for STR and VO at restraint. The study of general behaviour did not reveal difference between CLp and DONp for the average proportion of any of the considered traits. Our results suggest that the adaptive ability of progeny of cloned bull to challenges and conditions imposed by a farm environment are comparable to what is normally observed in dairy calves reared under similar conditions. & 2014 Elsevier B.V. All rights reserved.
Keywords: Behaviour Cloning Progeny Dairy cattle
1. Introduction Cloning livestock by somatic cell nuclear transfer implies reprogramming a somatic cell into a totipotent embryonic cell. An incomplete nuclear reprogramming due to epigenetic dysregulation is considered to be the main reason of abnormalities and impaired health observed in livestock clones in peri-natal period (EFSA, 2012). These aspects have
n Correspondence to: Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per le produzioni foraggere e lattierocasearie, sede di Cremona, via Porcellasco 7, 26100 Cremona, Italy. Tel.: þ 39 0372 433029; fax: þ39 0372 435056. E-mail addresses: [email protected], [email protected] (M. Speroni). 1 Tel.: þ39 0372 433029; fax: þ39 0372 435056. 2 Tel.: þ39 06 47836277; fax: þ 39 06 47836210.
1871-1413/$ - see front matter & 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.livsci.2014.01.018
raised ethical and scientific concerns about the welfare of both clones and their progeny, due the possibility that epigenetic abnormalities observed in cloned subjects could be transmitted to the offspring. Several studies were carried out on the progeny of cattle clones. Results of these studies showed that the offspring of clones displayed haematological and biochemical profile (Abeni et al., 2012), as well as growth and reproductive parameters (Abeni et al., 2012; Heyman et al., 2004; Kasai et al., 2007; Panarace et al., 2007; Watanabe and Nagai, 2008) comparable with healthy, non-clone animals, suggesting that epigenetic possible alterations present in clones were reset in the germ line of clone. This was further supported by Couldrey et al. (2011) who analysed the sperm DNA methylation patterns of Friesian bull clones. Few studies were carried to investigate the effects of cloning on behaviour of cattle; these studies show that
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bovine clones exhibit less playing behaviour, higher levels of curiosity, more grooming activities (Savage et al., 2003) and more exploratory behaviour than conventional controls (Coulon et al., 2007). No difference was found between clones and conventional heifers for cognitive capacities and kin discrimination (Coulon et al., 2010). To our knowledge no specific study on behaviour of progeny of bull clones has been done. In farm animals, behaviour is an important and early indicator of proper maturation of physiological systems underlying the vital functions and ability to cope with the environment. Restraint, even in absence of painful stimuli, has been shown to be a powerful stressor in several species; in cattle, behavioural responses to restraint can be used as indicators of coping ability and fearfulness, which are traits mediated by underlying biological functions. Also the behavioural repertoire, the rhythms of rest and activity, the development of behaviour with age are potential indicators of the level at which the farm animals are adapted to their environment. Calves need to spend a lot of time resting and sleeping; Hänninen et al. (2005) found a positive correlation between the total duration of rest and the growth rate in calves; sleep is essential for regulation of several hormones and brain development in growing animals. The aim of the present study was to assess whether there was a difference between progeny of a bull clone and offspring of the corresponding donor bull in (a) the level of excitability to routine handling, measured by the behavioural response, and (b) the general behaviour expression, as indicators of adaptive ability to farm environment. 2. Materials and methods 2.1. Animals and management The study was carried out at the experimental dairy farm of CRA-FLC, located in Cremona (Italy). A total of fifteen Holstein female calves were used. The calves were born at different times from Holstein recipient heifers implanted with embryos obtained by IVF with frozen semen of a bull clone (n ¼7; CLp) or its nuclear transfer donor bull (n ¼8; DONp, as control); oocytes for in vitro production of embryos were collected from six Holstein donor cows. Deliveries were concentrated between November and January; median of birth dates were December 13th and December 23rd for CLp and DONp, respectively; standard deviation of birth dates was higher in CLp (42 days) than in DONp (21 days) due to one CLp born on September. The mean of birth weight were 38.4 kg (SD ¼8.2 kg; range¼26.0–52.0) and 42.7 kg (SD ¼8.9 kg; range ¼27.0–54.0) for CLp and DONp, respectively. Calves were separated from their respective surrogate mothers immediately after birth and moved to a calf barn, where they were housed in individual straw-littered cages. Cages (182 90 cm2) had solid sides, with an opening in the front to allow the calf access to bucket of milk and feeder. A heat lamp was directed onto the cage for the first 48 h of life. Calves were fed colostrum by bottle for the first two day postpartum. Starting from 3rd day of age, calves were fed
reconstituted milk, twice daily (06:00–15:00), by nippled milk bucket (2 L/meal, on average). At approximately one week of age, the milk diet was supplemented with calfstarter and ad libitum roughage in the form of hay, distributed daily, immediately after the morning milk meal. At 8 weeks of age, calves were transferred outdoors into group pens (6.15 3.33 m2; to a maximum of 4 calves/pen). The calves were weaned in the successive 2 weeks, by a progressive decrease in milk supply and after the assessment of an adequate solid feed intake (approximately above 700–800 g/day of DM). All calves were healthy at birth, but during the first moth of life three DONp died for neonatal diarrhoea. 2.2. Data collection 2.2.1. Behaviour at restraint Behavioural data were collected during sample session for the study of haematological profile and clinical chemistry. According to that protocol, each calf was subjected to manual restraint for sampling blood at jugular vein at 24–48 h and 1, 2, 3, 4, 8, 12, 16, 20, 24 weeks of age. Blood sampling sessions were performed between 06:00 and 07:00, before milk distribution. On each occasion, an operator entered the cage and manually restrained the calf firmly against a wall of the cage until the end of blood sample collection, while another operator outside the cage kept the calf's head. Blood sampling started immediately after immobilisation of the calf. Blood was drawn from external jugular vein using a 20 G needle connected to vacuum tube. From the insertion to removal of the needle passed at least 1 min per calf because of the need to collect from 3 to 4 samples. This was obtained by changing vacuum tubes, without removal of the needle from jugular vein. At the end of blood sampling the calf was released. In calves raised into group pens, restraint consisted of head immobilisation by the headlocks; a rope was also placed around the muzzle. Blood sampling procedure is described above. In cattle, it was already shown that acute restraint per se elicits a variety of physiological and behavioural responses, where struggling is the most common behavioural expression. Struggling movement was defined as vigorous trunk movements in attempting to free oneself from the squeeze by operator or head immobilisation by headlocks. Number of struggling movements (STR) and vocalisations (VO) occurring within the first minute of blood sampling were recorded by direct observation. Struggling movements were considered as a distinct event when a single or a series of trunk movements were interrupted by a period of body immobility of at least 10 s. 2.2.2. Behaviour over the pre-weaning period Each calf was observed during four daily sessions at 2nd, 3rd, 4th, and between the 5th and 8th week of age; on the basis of preliminary observations of conventional calves reared on the same experimental farm, a morning (MTB) and an afternoon time band (ATB) were chosen for observations. Morning time band started 2 h after milk meal distribution and lasted 3 consecutive hours; observation during this time band aimed to compare the two groups mainly for resting behaviour; in ATB, calves were observed 1 h before,
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Table 1 Catalogue of behaviours selected for observation of calves. Behaviour/posture
Description
Lying inactive with neck relaxed
Lying on the sternum or on-side, neck relaxed, head on the ground, or turned back on the neck, or front legs, or flank, without any obvious activity Lying on the sternum, head lifted up, with opened or closed eyes, without any obvious activity Standing, head turned towards any of solid wall of the cage, without any obvious activity Standing, head turned towards or outside the open side of the cage, without any obvious activity Eating solid food from the feeder Chewing rhythmically while not eating, in standing or lying position Sucking at some parts of the cage or at an object Licking some part of cage Self-licking or scratching or rubbing herself against some parts of cage Sniffing walls or litter of the cage All types of vocalisations Swinging the tongue outside of the mouth from one side to the other, or contorts and rolls it inside the mouth, or stretches it out
Lying inactive with head up Standing inactive Standing inactive at the feeder Eating Chewing Non-nutritive sucking Licking Self-Grooming Sniffing Vocalizing Tongue-playing
at the hour and 1 h after milk meal, to emphasise behaviours normally associated with milk distribution. Individual calf behaviour was recorded by a video camera provided with internal hard disk, mounted on a tripod and placed in front of the cage. The videos were successively transferred to a personal computer and the behavioural data sampled from the video by instantaneous sampling at 3 min intervals. The description of recorded behaviours is given in Table 1. Each occurrence was counted; VO occurrences were expressed as the total number per hour; the occurrences of other behaviours were expressed as percentage of total number of scans per hour.
2.3.2. Behaviour over the pre-weaning period Data from only 13 calves (7 CLp; 6 DONp) were used for the analysis, because, due to technical faults, 2 DONp had more than one incomplete daily observation. Percentages data, with exception for VO, were arcsine transformed. Transformed data were analysed by mixed models for repeated measures: sire (CLp or DONp), time band (MTB or ATB), hour within time band (1, 2, 3 within MTB or ATB), age (2nd, 3rd, 4th, 5th–8th week) and their interactions were considered as fixed factors and calf as random factor. Back-transformed values were used in tables. 3. Results
2.3. Statistical analysis 3.1. Behaviour at restraint Data were analysed using MIXED procedure of SAS/ STAT software in the SAS system for Windows, Release 9.2 (SAS Institute, Inc. 2008). 2.3.1. Behaviour at restraint Data from only 14 calves (6 CLp; 8 DONp) were analysed; the CLp that was born on September was excluded from analysis due its late inclusion in the monitoring protocol. Counts of STR and VO were square root transformed before statistical analysis. A mixed model for repeated measures was used to analyse the fixed effect of the bull (CLp or DONp), age (0, 1, 2, 3, 4, 8, 12, 16, 20, 24 weeks), birth weight (o40 kg, Z40 kg or r50 kg, 450 kg), level of assistance at birth (unassisted; assisted by one person, assisted by more than one person, caesarean section) and bull by age interaction. Calf was considered as random factor. Statistical significance was set at Po0.05. Literature reports that production of embryos by in vitro procedure and embryo transfer techniques can result in the birth of larger offspring with higher incidence of dystocia, perinatal loss and anomalies, compared to AI (Galli et al., 2003); for this reason the level of assistance at birth and birth weight were introduced in the model to test their effect and exclude any influence on calf reactivity. No effect of birth weight, level of assistance at birth and bull by age interaction were found on STR or VO; thus the final model included only bull and age as fixed effects and calf as random effect.
All calves struggled and vocalized when restrained for sampling, but no difference was found between CLp and DONp on the overall mean for STR and VO. A reduction (P ¼0.0008) of the number of vocalisations with age was observed, but this effect did not differ between CLp and DONp. 3.2. Behaviour over the pre-weaning period No difference between CLp and DONp was found for the average proportion of time spent in any of the considered behaviours. An effect of the time band, the hour within the time band, and the age on most of the behaviours was found, but these effects did not differ between CLp and DONp, with exception for licking. There was no case of tongue rolling in any of the calves monitored. For licking, a sire by age interaction was found; in particular, CLp showed at 2 and 3 weeks of age a higher proportion of licking than DONp, while at 4 weeks of age this behaviour resulted more expressed in DONp than in CLp. Development over time of the other behaviours was the same for both groups. As calves were growing, eating solid food increased, whereas chewing activity decreased. A clear decrease was observed also for vocalisation, as well as for sniffing (Table 2).
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Table 2 Least square means (99% CI) of behaviour (LMS (99% CI)) of the calves at different age. Behaviour
Age (weeks) 2nd
Lying inactive with neck relaxed (%) Lying inactive with head up (%) Standing inactive (%) Standing inactive at feeder (%) Eating (%) Chewing (%) Non-nutritive sucking (%) Licking (%) Self grooming (%) Sniffing (%)
17.43 8.38 2.25 7.04 0.54 12.35 0.07 0.22 1.23 2.19
3rd (12.03–23.81) (5.14–12.42) (1.48–3.18) (4.58–10.03)a (0.06–1.54)c (8.10–17.49)a (0.01–0.16) (0.08–0.42) (0.63–2.03)ab (1.41–3.13)a
14.20 7.16 1.93 4.16 4.48 10.21 0.06 0.12 0.68 1.08
(9.33–20.08) (4.17–10.95) (1.22–2.82) (2.31–6.55)a (2.59–6.89)b (6.35–14.99)ab (0.01–0.15) (0.02–0.29) (0.26–1.31)b (0.55–1.79)ab
4th
5th–8th
13.55 (8.71–19.43) 8.27 (4.96–12.41) 1.02 (0.51–1.70) 5.49 (3.29–8.25)a 6.47 (4.32–9.70)ab 4.92 (2.34–8.46)b 0.03 (0.00–0.11) 0.14 (0.03–0.32) 0.99 (0.45–1.75)b 0.90 (0.42–1.56)b
14.05 13.21 1.15 1.47 10.22 5.74 0.08 0.14 2.24 0.54
(9.21–19.90) (8.99–18.22) (0.62–1.84) (0.48–2.99)b (7.25–13.70)a (2.97–9.43)b (0.00–0.07) (0.03–0.32) (1.39–3.29)a (0.20–1.04)b
Means with different letters on same row differ significantly (P o0.05).
Table 3 Least square means (99% CI) of behaviour performed by calves at different hours of the day, within MTB and ATB. Behaviour
Hours within time bands H1(MTB)
H2(MTB)
H3(MTB)
H1(ATB)
H2(ATB)
H3(ATB)
9.11 (4.90–14.60)c 2.14 (0.42–5.19)c 5.32 (2.26–9.68)c Lying inactive 34.26 (25.68–43.98 )a 34.93 (26.20–44.89)a 20.17 (13.64–27.98)b with neck relaxed (%) Lying inactive 17.73 (12.37–24.03)a 12.26 (7.85–17.65)ab 8.96 (5.24–13.68)ab 11.62 (7.29–16.95)ab 2.01 (0.49–4.56)c 6.68 (3.52–10.85)b with head up (%) 0.66 (0.05–1.00)d Standing 0.71 (0.07–2.03)d 3.55 (1.69–6.10)c 4.87 (2.62–7.81)bc 9.0 (5.79–12.93)ab 13.12 (9.21–17.73)a inactive at feeder (%) Standing 0.55 (0.17–1.14) d 0.66 (0.21–1.24)cd 1.73 (0.96–2.71)abc 1.31 (0.66–2.19)bcd 2.75 (1.74–3.99)ab 3.28 (2.18–4.61)a inactive (%) Eating (%) 1.32 (0.34–2.95)b 1.73 (0.55–3.56)b 2.52 (1.02–4.67)b 9.55 (6.31–13.46)a 15.24 (11.02–20.14)a 3.7 (1.81–6.27)b Chewing (%) 2.63 (0.76–5.61)b 4.15 (1.64–7.79)b 6.85 (3.45–11.41)b 7.91 (4.19–12.80)b 18.3 (12.29–25.51)a 13.12 (8.16–19.24)ab Non-nutritive 0.00 (0.0294–0.021) 0.00 (0.018–0.035) 0.00 (0.0296–0.023) 0.02 (0.001–0.087) 0.73 (0.4769–1.04) 0.10 (0.024–0.231) sucking (%) 0.13 (0.02–0.24)bc 0.05 (0.00–0.16)c 0.53 (0.27–0.32)a 0.42 (0.19–0.19)ab 0.04 (0.00–0.89)c Licking (%) 0.04 (0.00–0.42)c Self grooming 1.46 (0.72–2.46)abc 0.8 (0.28–1.59)bc 2.42 (1.41–3.69)a 0.71 (0.23–1.48)bc 0.51 (0.12–1.18)c 2.01 (1.10–3.19)ab (%) Sniffing (%) 0.85 (0.33–1.60) 0.78 (0.28–1.52) 1.31 (0.62–2.24) 1.41 (0.69–2.38) 1.86 (1.01–2.97) 0.64 (0.201.34) Means with different letters on same row differ significantly (P o0.05).
Distribution of the activities within MTB and ATB was similar in CLp and DONp; in MTB, there was no difference between CLp and DONp in proportion of time spent lying inactive with neck relaxed or head up. In ATB, they spent the same time in oral activities. Eating, as well as chewing, reached the maximal values around milk-meal time; licking and vocalizing peaked some time before milk-meal distribution. Non-nutritive sucking was observed immediately after calves emptied the milk bucket. The time spent by calves in standing inactive reached maximal values some time after milk-meal distribution (Table 3). 4. Discussion In our study, progeny of bull clone and its donor responded in the same way to handling. Both groups struggled and vocalized when restrained for blood sampling; magnitude of behavioural response was similar for the two groups. Unlike the struggling response, which remained unchanged over
time, vocalisations decreased as calves were growing, in both progeny of bull clone and control. Overall, these data suggest that all calves reacted with the same emotional level to handling. Thus, our data do not seem to confirm the finding, based on physiological data, of a lower excitability of progeny of a bull clone compared to conventional control during manipulation in the chute reported by Ortegon et al. (2007). General behaviour of progeny from bull clone was similar to that of the progeny of its donor. Behaviours associated with resting represented the more expressed behaviour for both groups and peaked at some distance from the morning meal. It is commonly reported that healthy young calves rest for much of the day and that most of resting occurred at night time or after the morning meal (Hänninen et al., 2005). We did not observe oral stereotypies in any of the monitored calves. Other oral non-nutritive behaviours, such as sucking and licking oriented to structures of the
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cage also were at very low levels. Licking is reported as common in pre-weaning calves, more expressed in those housed individually, evocated by a certain level of frustration and or boredom (Kopp et al., 1986). In our study, the early access to solid food as well as to the straw of litter, may have led the calves to satisfy their need for oral manipulation by chewing activity, helping to keep low levels of licking and to prevent the development of oral stereotypies. We observed calves sucking some parts of the cage immediately after the milk bucket was emptied. This is a common behaviour in calves, elicited by ingestion of milk (De Passillé, 2001). In both groups, a high alertness due to expectation for milk meal, was expressed by a peak in licking and vocalisation some time before milk distribution and roused by the presence of caretakers preparing milk substitute. Development of behaviour over age was the same for progeny of bull clone and control. A progressive increase with age was found for eating solid food, according to findings by other authors for calves raised under similar conditions (Hepola et al., 2006). Chewing decreased as calves aged; an increased chewing efficiency, as well as a reduction of the need of oral manipulation, could explain this finding. Other oral non-nutritive behaviours also remained at very low levels over the experimental period in all calves so that it is not easy to provide a biological explanation for different patterns of licking activity that was lower in DONp than in CLp were at 2 and 3 weeks of age, whether at 4th week was lower in CLp than in DONp. In both groups, inactivity in the standing position facing the feeder, as well as sniffing the walls or the ground of the cage decreased with age. It is likely that the increase with age in time spent eating solid food reduced time dedicated by calves to other behaviours. For both groups, the highest number of vocalisations was recorded at the beginning of the observational period. Probably, the increasing intake of solid food covered the nutritional requirements of calves and reduced the level of hunger at the time of the milk-meal. 5. Conclusion In this study, the progeny of cloned bull showed similar reactivity to its counterpart when faced with the same aversive experience of handling. Afterward, offspring of bull clone showed behavioural repertoire, behavioural rhythms and development with age comparable to that commonly reported for preweaning calves raised under similar conditions. These results suggest that adaptive ability of progeny of cloned bull to challenges and conditions imposed by a farm environment was normal. Conflict of interest statement There is no conflict of interest.
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