子牛における隔離および輸送に対する生理および行動反応
誌名
誌名 Animal behaviour and management ISSN
ISSN 18802133
著者 著者
小木野, 瑞奈 山崎, 淳 松浦, 晶央 入交, 眞巳 甫立, 孝一 巻/号
巻/号 47巻1号
掲載ページ
掲載ページ p. 12-20 発行年月
発行年月 2011年3月
農林水産省 農林水産技術会議事務局筑波産学連携支援センター
Tsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research Council Secretariat
一 OriginalArticle ‑
Physiological and behavioral responses to social isolation and transportation in calves
Mizuna OGINO， Atusi YAMAZAKI， Akihiro MATSUURA， Mami IRIMAJIRI， Koichi HODATE*
Department of Animal Science， School of Veterinary Medicine， Kitasato University， Towada‑shi， Japαn
* Conesponding audlor. E‑mail address:[email protected]‑u.acj.p
Abstract
The aim of iliis study was to investigate the influence of social isolation (Experiment 1) andむansportatlOn (Experiment 2) on behaviors， salivary and plasm註levelsof clu"omogranin A (CgA) and cortisol (CORT)， plasma levels ofprolactin (PRL)， and heart rate variability (HRV) in calves. In Experiment 1， isolation did not increase the concentrations of salivruy CORT， plasma CORT， or Cg A，although it increased the concentration of plasma PRL and heart rate in ilie case of註letreated calves (Pく 0.05).Furthermore， the behavioral states of dle isolated calves highIighted ili吋 discomfort.In Experiment 2， the plasma CORT and PRL levels and low企equency/highfrequency ratio were significantly increased inunediately after ilie calves were loaded into the transportation包uck(P < 0.05). The results obtained in iliis study suggest役latisolation under出epresent condition was not sufficiently severe to activate the hypothalamic‑pituitary柵adrenal(HP A) axis or the sympathetic nervous system in calves. However， the transportation r・esultedin changes in CORT and HRV parameters involved in the acti
、
rationof ilie HP A a幻sand iliesympathetic nervous system but not in the CgA levels in calves.
Key words: calves， chromogranin A， cortisol， heart rate variabilityラprolactin
Animal Behaviour and Management， 47 (1)・ 12‑20，2011 (Received 25 Jun 2010; Accepted fo1' publication 22 Feb 2011)
Introduction
The capacity to cope Witll enviromnental chaIlenges has an important influence on tlle welfare of individuals (Broom， 1988). On a conunon fann， ca抗le are kept in complex enviromnents and are exposed to potentiaIly stressful chaIlenges such as novel situationsう social stimuli， physical restraints， and transportation For animal welfare， it is necessary to assess ilie responses to these stimuli企omdiverse viewpoints such as neurophysiology， physiology， and behavioral indices.
Over the past decade， neurophysiological indices such as heart rate variability (HRV) has been increasingly used as a pru"ameter in frum animal research to assess changes in sympailiovagal balance related to diseases， psychological and environmental stressors， or individual chru"acteristics such as temperament and coping strategies (von Borell et aヲ.l 2007). The HRV pru羽netersreflect activation of the autonomlc ner吋vous system; ilie power in the high‑fr・equency(HF) band represents vagal activity and that in ilie low働fi"equency(LF) band is associated with sympailietic or sympathetic plus vagal activity. Therefore， tlle LFIHF ratio is considered a measure of sympathovagal balance and has been used as an index
of sympailietic nervous activity、
Several physiological and b巴havioralchanges have been reported in ruminants living under sむessful conditions Cortisol (CORT) has been used as a stress marlωr in vru'ious animals， including ruminants because it is ilie main active hOlmone of ilie hypothalamic欄pituitary楠adr・巴nal(HPA) axis (Morrr泌氏 et a ，.l 2007). Moreover， studies have shown iliat prolactin (PRL) release in cows is affected by several stress stimuli (Johke， 1970; Raud et a ，.1 1971). Behavioral responses to stressお1conditions have been previously reported in cattle， such as ilie numberof attempts to lie down， lying time， rumination tim，巴 vocalizationラandlocomotion (Lidfors， 1989; Haley et a ，.l2000; PhiIlips， 2002; MruIteuffel et a ，.l2004; Van Reenen et a ，.l2005)
Clu'omogranin A (CgA) is an acidic protein coreleased wiili catecholamine by exocytosis from ilie
呂drenalmedulla組 dsympathetic nerve endings (Smitll
& Kirslmer， 1967; Smith & Wirホler，1967; O'Conner・
et aヲ.l1984).Aliliough s巴cretionsof CgA in ruminants have not been previously reported， CgA serves as a valuable indicator of sympailioadrenal activity (Taupenot et a ，.l 2003)， and CgA responses reflect activation of ilie sympailioadrenal‑medullaη(SAM) axis under st:ress conditions. The plasma CgA level was
O G部
o
，Y品1:AZAKI，MATSUURA， 1RIMAJIRI AND HODATEreported to be a useful index for evaluation of acute sなessresponse in dogs (Akiyoshi et a ，.l2005). CgA is produced by not only the adrenal medulla but also the submandibular gland in humans (Saruta et a ，.l2005). In addition， CgA is reportedly secreted into the saliva by stimulation of the isolated and pe出 sed rat submandibular gland with noradrenalin and acetylcholine (Kanno et a ，.l 1999). ln other words， salivary CgA level is also increased by activation ofthe sympathetic nervous system as well as plasma CgA. Thus， it has been reported that plasma and saIivary CgA is a sensitive and reIiable index for evaluating stress in humans (Nakane et a ，.l1998，2002).
In this s加dy，we exposed calves to sむ‑essful conditions such as social isolation and tr釦sportation， which are characterized by changes in salivary and plasma CgA and CORT levels， plasma PRL levels， behavior， and HRV parameters. A study had shown that cattle in socially isolated siぬationsand/or in unfamiliar sunoundings have increased CORT levels， HRs， and vocalizations (Boissy and Le Neindr久 1997).Fぽther， another study had shown thatラincattle， transportation is a weII剛knownn巴gativestress factor・asevidenced through mortality rate and changes in HR， adrenal activity， and enzyme activity as wel1 as immunological e紅白ts，carcass quaIity， and behavioral responses (Trunkfield & Broom， 1990). 1n ruminants， whether・
salivary CgA is secreted from the submandibular gland and whether saIivary and plasma CgA can respond to stressful conditions has not yet been investigated. Therefore， we prelirninarily meaSill・ed the CgA concentrations in saliva obtained from the parotid and submandibular glands of calves. We then measured the neurophysiological， physiological， and behavioral indices as previously noted in calves during conditions of social isolation and transportation.
Materials and Methods
The research protocols in this study w巴reapproved by the Animal Experiment and Care Comrnittee of Kitasato University， Towada， Japan.
Animals and experime出alprocedures
Preliminary test: Detection of salivary CgA levels in calves
明re assessed the CgA levels in the saliva obtained 企omthe parotid and submandibular glands of calves. The test was pelformed in 4 male Holstein calves (mean土SD;age， 7.3土0.1months; body weight [BW]， 218土 4.0 kg) ill1der・ xylazine‑induced anesthesia Carmulas were inserted into the ducts of the parotid and submandibular glands， and saliva from these glands was simultaneously collected. The saliva samples wer・e stored at ‑30oC until they were assayed.
ExpeI加lent1: Effect of social isolation
Four male Holstein calves (age， 4.1土0.1months; B W，122ま4.2kg) were reared in the s創nepen (home pen) and fとda pel1eted diet twice daily (at 09.00 and 15.30 hours); water and hay were available ad libitum. Two randomly selected experimental animals were fitted with a catheter into the jugular vein 1 day before the experimental day. On the experimental day， 2 calves were connected to portable electrocardiographs (Digital Holter Recorder FM・180; Fukuda Denshi， To勾0，Japan) at 11.00 hours. At 12.00 hoursラ1ofthe 2 s巴lected calves was moved to a stall， a noveI environment that was located 30 m from the home pen， wher・easthe other calf was kept as a control in the home pen with the other 2 calves. The isolation treatment continued until 24 h after the calf was transferred to the stall. This experimental process was repeatedly perfonned for the 4 calves. An intervaI of at least 1 day was maintained between each experimental day.
Blood samples were collected via the indwelling jugular catheters inωbes containing heparin sodium or EDTA at ‑10， 0 Gust after relocation from the home pen)， 10，20， and 30 min， and at 1，2，3，6， 12， and 24 h. Saliva from the mouth was simuItaneously collected with the blood samples by using Salivette sampling devices (Sarstedt 1nc.， Rommelsdorf， Gennany)， including a small cotton swab. All blood and saliva samples were stored on ice. Ai記rcentrifugation (3000 rpmラ40C，30 min)， the plasma and saliva samples were stored at ‑30oC until they were assayed. The electrocardiogram (ECG) was monitored throughout the experimental period.百lebehavior of individual calves was continuously recorded during the experiment by using a digital video camera.
Experiment 2: Effect of transportation
Before transportation， 4 male Holstein calves (age， 2.4土 0.1months) were reared in pens that housed 2 calves each， and the calves were fed a pel1eted diet daily; water was provided ad libitmn. On the day of the experiment， 3 of the 4 calves in the pen were connected to portable electrocardiographs at 14.00 hours; after 10 min， ECG monitoring was initiated. Thereafter， all the calves were moved approximately 40 m 企omせlepen， loaded into the truck， and transported for 4 h. 1n the
仕uck，all calves were individually tied to the truck with a rope. The dimensions ofthe tmck were 6.1 m x 2.0 m x 3.2 m (length x width x height). The joumey started at 16.00 hours and terrnInated at 20.00 hours. After the transportation， the calves were moved to a new pen， which was different 合om the location before the transportation and was unfamiliar surroundings for calves. Then， the transpOlted calves were fed a pel1eted diet twice daily (at 09.00 and 15.30 hours); water and hay were available ad libitum.
Blood and saliva samples were obtained during 6 different stages: in the pen， before設letransportation as a basal condition (14.00 hours); immediately after moving and loading也ecalves into the位uck，i.e.，
RESPONSES TO STRESS IN CAL VES immediately before theむansportation(16.00 hours);
hal向aythrough the transportation (18.00 hours); at the end of transportation， while the atumals were in the truck (20.00 hours); and at 6 and 24 h a会erthe end of the transportation， when the anim註Iswere in the new pen (at 02.00 and 20.00 hours). Jugular venous blood samples were drawn by venipuncture and collected in tubes containing heparin sodium or EDT A. Saliva satnpling was perfonned before blood satnpling in order to eliminate the influence of the sむ・巴ssassociated with venipunctur・e.All blood and saliva samples were stored on ice until the end of the experiment. After the experiment， the plasma and saliva samples were centri白ged(3000 rpm， 40C， 30 min) and stored at
‑300C until they were assayed. The ECG was monitor吋 th1'oughoutthe transportation period. Measurements
Hormone assays
Salivary and plasma CgA concenむatlOns were analyzed using the YK070 Human CgA ELISA kit (Yanaihara Institute， ShizuokaラJapan).The int1'a‑and inte1'‑assay coe伍cientsof variation (CV s) for・註leCgA assay we1'e 2.5% and 9.3%， respectively. The CORT levels in the saliva and plasma were detennined using the Cortisol Expr巴ssEIA kit (Cayman Chemical， A1m Arbor， MI， USA) and Enzaplate cortisol (Siemens Medical Solutions Diagnostics， すo防0， Japan)， respectively. The intra‑assay CV s for salivary atld plasma CORT were 3.8% and 0.2%， 1'espectively. The inte1'‑assay CVs for salivary atld plasma CORT were 5.5% and 11.1%， respectively. The salivary CgA and CORT concentrations were cOITected for・totalp1'otein concenむヨtions(mg/mL)， which were detennined using a BCA Protein Assay Kit (Pie1'ce， Rockfo1'd， IL， USA).
Plasma PRL concentration was detennined by radioimmunoassay according to the methods described by Johke (1969). The intra柵assayCV fo1' PRL was 0.7%.
HRVanalysis
The ECG was analyzed using an ECG analyzer (SCM・5000system; Fukuda Denshi， To材0，Japan) to obtain the R‑R intervals創ld was automatically subjected to power岨spectmm analysis， which was perfonned using an analysis tool (MemCalc/CHIRAM;
GMS， Tokyo， Japan). The HR， HF， LF， and 印 刷F ratio we1'e calculated from the tachograms by setting the limits of the LF band to 0.04 Hz (lower limit) and 0.3 Hz (upper linut) and those ofthe HF band to 0.3 Hz (lower limit) and 0.8 Hz (upper limit) for the calves (Mohr et a ，.l 2002). The calculation was perfonned using the tachogratn data obtained for 10 consecutive minutes at each sampling interva. 1
Behavioral analysis
The behavior of the calves was continuously
1'ecorded企om…10min to 24 h after isolation by using
a digital video catnera (Experiment 1). In preparation of behavioral observation， all behaviors that calves displayed during the experiment were categorized as access to hay and water， contact with humansラ elimination of urine and stool， head‑up pos旬re， investigating behavior， kicking， moving， mbbing， self‑grooming， social behavior， vocalizationラ attempts at lying down， atld ml1Unating・We observed the f‑iequencies of these behaviors， and the counts per minute of thes巴behaviorswere analyzed， except for mminating. The percentages of time spent standing or lying and f Wninating during出e24 h after the isolation were calculated.
Statistical analysis
In Exper泊lent1， the effects of time and treatm巴nts on the mean concentrations of CgA， CORT， and PRL;
the mean value of the HRV parameters; and continuous behavioral data were analyzed using repe昌ted‑measures two哨 ay analysis of variance (ANOVA). If the interaction between time and treatments was significant， calves in the control group were compared with those in the isolation treatment group with regard to the behavior and CgA， CORT， PRL， and HRV values昌t each sampling interval by using paired トtests.The total number and percent difference in behavior beれNeenthe con位。1and isolation仕eatmentgroups were statistically compared using paired トtests. In Experiment 2ラ the mean concentrations of CgA， CORT，註ndPRL and the m巴an value of the HRV pat'anleters beれNeen the satnpling times were compat'ed using repeated‑measures one‑way ANOVA. Subsequentlyヲ a post‑hoc test with Tukey's multiple comparison test was performed
Results Preliminary test
The concentration of CgA in the submandibular・
saliva was 429.49土 258.21 pmoνmL (minimum 100.85 pmol/mL， maximum: 731.47 pmol/mL)， whereas the cOITesponding value in the parotid saliva was below the detection linut (<0.14 pmol/mL). Experiment 1
Plasma CgA， salivary CgA， and CORT levels wer・e not significantly altered after social isolation. The plasma CgA concentration at ‑10 noo (basal condition) in control calves was l.28土0.62pmol/mL and that in isolated calves was l.45土0.90pmol/mL. The salivaty CgA concentration at ‑10 min in control calves was 28.66土 47.61pmol/mg protein and that in isolated calves was 5.42土 2.48pmol/mg protein. The plasma CORT concentration at ‑10 min in contr叫 calveswas 10.00土7.93ng/mL and that in isolated calves w昌s6.54
土3.05ng/mL. The salivary CORT concentraむonat‑lO min in control calves was 0.72土 0.85nダmgprotein and that in isolated calves was 0.39土 0.30ng/mg
OG取0，YAMAZAKl， MATSUURA， IRIMAJIRIAND HODATE
Table 1. Percentage of time spent standing or Iying and ruminating and occurrence of behaviors from ‑10 min to 24 h after isolation.
B~havior C Standing
{号、。ftimc)
0.029
0.029
ど0.001 0.002 0.002
319.25土172.57
119.75土73.59 品7.48土3.06 32.52土3.06 17.33土3.34
609.75土147.20
74.25土72.63 61.37士8.44
33.33土5.09 38.63土8.44 Lying (% oftime)
Accessing water (number) Ruminating (%oftime) Accc5sing hay
(numberl
protein. On the other hand， the plasma PRL levels increased at 10 min (Fig. 1) and HR increased at 0， 10， 20， and 30 min and at 1 and 2 h after isolation (Fig. 2) in the calves subjected to this treatment compared with the co汀espondinglevels in the control calves (P <
0.05). The HF and LFIHF components were not altered by social isolation. The HF at ‑10 min in contr叫calves was 15.32土 12.88ms2. The HF at ‑10 min in isolated calves was 6.43土0.62ms2. The LF圧iFvalues at ‑10 min in control and isolated calves were 10.41土8.77 and 13.32土11.12，respectively
Table 1 shows the behavioral data recorded for the control and isolated calves during the experiment. The time spent lying and rurninating and the frequ巴ncyof accessing hay were less， but the frequency of accessing water， elimination of urine and stool， movement，
0.243 32.75土20.07
16.75土15.06 Comacl with human
(number)
0.013
0.122
0.063
0.129 0.016 39.25士8.77
134，75士112.79
695.25土436.55
2072.25土1835.10 884.00土315.05 13.75土9.60
7.00士12目68
149.50士59.50
183.75土148.72 66.50士34.11 Elimination ofurine
and 51∞1 (numberl HcadぺJpposlllrc
(nllmber) lnvestigating behavior (nllmber)
Kicking (number) Moving (number) 140 牢
{
活
120~I! : :f 100
} 岬
・ 開嗣 制ヤ 究
開
。
p : ;
d
=
匂今 明
間仏 80
40 20
0.113 0.517
0.041 23.50土27.26 433.00土610.82
0.00土0.00
1387，50土801.95 41.75士17.69
96.25土49.20
10.00土10.83 230.25土8287 RlIbbing (numbcr)
Social behavior (nllmber) ScIιgroommg
(nllmber)
Vocalizalion (number) よ4
11I
Timl'AftぞrIsolation
Fig. 1 Concentration of plasma prolactin (PRし)in the isolated calves (慰)and control calves (0). Mean + SD (n
=
4).安Significantdifference between the control and isolated groups (Repeated‑measures two‑way ANOVA， Fロ 3.50，P < 0.05; Paired t‑test， P <
0.05)
iよ 6 3
‑
ラ30 1 1Il1ll 20
。
10Auempt al Iying do¥Vn (numberJ
t :
P value obtained by paired t‑tes. t
1[: The statistical test for the number of social behaviors was not peげormed，because the counts were 0 in the case of the isolated calves.
0.043 58.25土7.85
28.75土10.05
帽3・Contt01
j
叫 山1ヰ:
bh・・
b侃
•
vocalization， and the number of attempts to lie down were higher in the isolated calves than in the control calves (Pく 0.05).The total number of investigating behaviors tended to increase in the treated calves (P <
0.1). Because the plasma levels of PRL were significant1y altered at 10 min and HR was significant1y altered from 0 to 2 h after isolation in the treated calves， the number of behaviors per minute during this time was analyzed in detai .1The behavioral data during 4 periods (‑10三Plく
o
min; 0三 回 <20 min; 20 min ~ P3く 1h; and 1 ~ P4く 2h after isolation) were obtained， and the effects of time and trea加lents on the number of behaviors were statistically assessed. The treated calves tended to move frequently from 0 min to 2 h a食erisolation (Pく 1.Jhl
Timl' Aftl'l' Isolatioll
Fig. 2 Changes in the heart rate in the isolated calves (塑)and control calves (0). Mean + SD (n
=
4).*Significant difference between the control and isolated groups (Repeatedmeasures two例way ANOVA， F
=
2.93， P < 0.05; Paired ιtest， P <0.05).
;h4 」 ν ム ]
::t::
。
12 3 .ヲ30
llltll より
。
10RESPONSES TO SτRESS IN CALVES
CgA is secreted into the saliva from the submandibular gland in rats and humans (K叙moet a ，.1
1999; Saruta et a ，.12005). In the present preliminary study， CgA was highly secreted in the submandibular saliva but was not detected in the parotid saliva. This result indicates that in calves， salivruy CgA is mainly secreted from the submandibular gland， similar to that observed ill rats and humans
In the isolated calves， the HR， and the indices of sympathetic nervous activity increased after the isolationラおrther，vocalization was more frequently observed than in th巴 con位01 calves. This result is consistent with that of the study by Boissy and Le Neindre (1997) Because increases in HR reflect locomotor activity when isolated cows are allowed to move freely (Hopster & Blockhuis， 1994)， the incr・ease in HR at 0 min may be attributable to the movement of the experimental calves from the home pen to the individual stall. In addition， the significant increase in HIミpersistedfor 2 h after isolation of the calvesヲbut the fi'equency of movement also continuously increased until at least 2 h after the isolation (Fig・3a). Furthermore， previous studies have shown that assessment of HR V parameters may be mor・euseful than simple HR analysis to assess the level of physiological， psychological， and envirorunental stress in an organism， for exrunpl巴 in catt1e and horses (Bachmann et aラ.12003;Hagen et a ，.12005); tller巴fore， the unaltered LF/HF ratio and tlle HF component in the preser託 study may indicate that isolation did not sufficient1y affect animals psychologにally. These evidences suggest that the significant increase in HR was caused by increase of movement rather than psychological stress in isolated calves.
1n addition to the increase in the frequ巴ncyof vocalization， behavioral changes wer巴observed，such as increases in the frequencies of movement and the number of attempts to lie down， and decreases in the time spent lying down and IUminating. These results suggest that unfanlIliar sUIToundings without companions caused discomfort to the isol註tedcalves. The same suggestIons resu¥ted from several behavioral data in previous studies (Lidforsラ1989;Haley et a ，.1 2000; Phillips， 2002; Manteuffel et a ，.l 2004). Van Reen巴net al. (2005) indicated that in case of calves subjected to open field or novel object tests， locomotion may reflect the characteristics of activity or coping style
loaded into the truck (Pく0.05)
Discussion
v
‑C‑Control
欄後‑Isolatioll
{日)
‑ nv
︐a宰事︑
2 2
~ 1.8 g i 6 と ユ i。ヰ
さ
， 1 2 g i
~ 0.8 ー争 0.6
~. 0.4
1
語 0.2
~ 0 記
言 0.6
匂竺
s
0ぅo
、
，
:: 0.4
。
303 供ν
。... 'o 0.1
".
. . . .
急，
tZ 0:::
p‑+ P('rIQ(¥
Fig. 3 Frequency of moving (a) and vocalization (b) in the isolated calves (磁)and control (0) calves. ‑10 :::; P1 < 0 min: 0 :::; P2 < 20 min: 20 min話P3< 1 h; and 1 :::; P4 < 2 h after isolation. Mean + SD (n
=
4).t
Significant di汗erencebetween the control and isolated groups (Repeated‑measures two需way ANOVA， F=
3.57， P < 0.05; Pairedt ‑ t e s t
， P <0.1 ).
P3 P2
o Pl
0.1) (Fig. 3a). Vocalization was observed in the isolated calves but not in tlle control calves (counts， 0) after isolation (Fig. 3b).
Experiment 2
Table 2 shows the changes in the plasma and in the salivruy CgA， CORT， plasma PRL and HRV par創ueters before， during， and after住ansportatlOn. Compared with the basal levels， the plasma CORT levels were significantly increased during loading of the calves into the transportation t印 ck and at th巴
halfway point of transportation (P < 0.05). The CORT levels in the saliva at the end of transportation were th巴
highest ruuong tllOse at all other stages (P < 0.05). The plasma PRL levels were si伊ificantlyhigher when tlle calves were loaded into the truck as compared to the basallevels (P < 0.05). The CgA levels in the plasma and saliva were not altered at any stage of transportation as compared to the basallevels.
The HR was not altered throughout th巴expenment. The LF/HF ratio was elevated when the calves were
OG応10，Y AMAZAKI， MATSUURA， lRIMAJIRI AND HODA:τE
Table2. Changes in the plasma and salivary chromogranin A (CgA)， cortisol (CORT)， plasma prolactin (PRし)and heart rate variability (HRV) parameters before， during， and after transportation (Mean土SEM，
n
口4).Stages 01' trnnsportation
before transpoれation transportation after transportation Variables basal aJler loading into
hall日ay end 6 hours 24hours thc truck
P/osllla
CgA (pmol/mL) 1.19AB土0.19 1.06B土0.13 1.48'¥日土0.28 2.50AB土0.60 2.63.¥B土0.71 2.84A土0.95 CORT (nglmL) 3.8511士0.64 9.06"士1.22 9.57.¥土1.49 6.79AB土0.80 5.40"1l士1.55 5.91AIl土 1.22 PRL(nglml.) 95.261lC、土56.51 214.01'¥土42.15 59.871lC 土9.54 29.72正 土4.88 78.5SBC'土31.77 122.031l土36.27 So/ivo
仁gA(pmol!mg protcin)t 13.00土2.67 11.48 :l: 1.05 12.79士1.32 19.30土9.57 11.72土1.85 9.16土1.56 CORT (ng/mg protcin)t 0.30B土0.17 0.931l土0.19 0.56B土0.08 2.29"土0.54 0.49B土0.20 0.37B土0.06 HRV parameler唱
HR (bcats/min) 93.52土3.63 97.64土3.93 89.39土2.71 86.03士3.76 86.92土4.10 104.84土8.30 LF(ms‑) 276.57土116.11 189.56土17.39 233.32土72.72 366.89土159.86 103.70土47.10 209.87土40.77 日F(ms2) 80.06土18.56 20.48士5.10 63.60士11.98 73.56土18.59 89.85士44.88 62.21土6.97 LF/HF 3.79B土1.46 10.2柱、土1.58 3ρg白土1.14 4.58AB土0.96 2.83B土 1.26 3.63B土 1.01
t :
The concentrations of salivary CgA and CORT were corrected for saliva total protein concentration (mg/mL).τHRV parameters of 3 calves.
Different letters indicate significant differences within the row (Repeated‑measures one‑way ANOVA， all P
< 0.03; Tukey's multiple comparison test， all P < 0.05).
surroundings and increased locomotion and time speぽ
in interaction with the stimulus in the novel object test (Van Reenen et a ，.12009). In the present study， salivary and plasm昌 CORTresponses were not altered in the isolated calves， but the frequency of locomotion and vocalization were high; these effects are identical to those of an anxiolytic drug. Thus， the ph)引 ologicaland behavioral reactions of the isolated calves in our study were thought to represent the motivation to be in close proximity to the companions rather than to represent fearfulness. This might account fOlせle unvaried response of plasma and salivary CgA in isolated calves
ln this study， the salivruy and plasma CgA levels were not altered after social isolation. ln humans， salivary CgA is a more sensitive marker of psychological stress than of physical stress (Nakane et a ，.11998). Furthelmore， the isolation stimuli under the present condition might not be sufficiently strong to increase the plasma and salivary CgA levels in calves. Because the individual stalls were not located far from the home pen， and because the isolated calves could perceive voices of their peers and other cattle，出ey may not have felt fearfu .1
ln the present study， the plasma PRL levels considerably increased at 10 min after・isolationof the calves. ln other words， physical stimuli that occulTed secondary to movement of the calves could have had a significant effi巴ct on the increase in PRL release observed in our study. The peripheral blood
concentration of PRL increased in response to many different s回 ssorsin both rodents and humans (τomer
&
Neumann， 2002). Physical 路 esses reportedly affected plasma PRL concentrations in cows (Johke，1970; Raud et a ，.11971)， whereas arecent study on heifers revealed that the plasma PRL concentration was not cOITelated with the principal component score associated with fearfulness in a novel environment (Yayou et al.， 2010).
ln cattle， transportation is a well‑known negative stress factor， as evidenced by the mortality rate; changes in HR， adrenal activity， and en勾Tmeact1vlty; immunological effects; carcass quality; and behavioral responses (Trunkfield & Broom， 1990). During transportation， the plasma and salivary CORT levels in the experimental calves were significantly increased， suggesting that the calves were in a highly stressed. ln addition， the calves exhibited the highest LF注IFratio immediately after也巴ywere moved and loaded into the truck， and this finding indicates that sympathetic nervous activation was accelerated at that time Physical stimuli， locomotion， and loading of animals may have increased the PRL levels， similar to也e increase in PRL levels observed after isolation of dle calves in Experiment 1. However， an increase in the plasma or salivary CgA levels during the experiment was not observed. The reason is not clear presently， but there is a possibility that negative feedback by catestatin (Mahata et a ，.11997; Rao et a ，.12007)， a
RESPONSES TO STRESS IN CALVES fi‑agment of Cg A，might explain why CgA levels in the
present study did not increase when the sympathetic nervous activation was accelerated by enviromnental stimulation during transportation. To clarifシthis， further experiments involving frequent sampling intervals are necessruy.
In summary， the results of the present study indicate that salivary CgA is secreted fi‑om the submandibular gland in calves and that isolated calves expressed their discomfort by behavioral changes; however， these changes may not involve feru布lness.ln addition， physical stimuli involved in isolation prutly changed their neurophysiological and physiological states; however， the isolation under the present condition was not sufficiently strong to activate both the HP A axis and the sympathetic nervous system in calves. However， the transportation resulted in changes in CORT and HRV parameters involved in the activation ofthe HPA axis and the sympathetic nervous system but not in the CgA levels in calves
Acknowledgements
The autllors ru'e grateful to Yuko Amanoヲ Akiko Ishizawa， TakrulOri Ogawa， Saori Fukuhar丸 NozomI Morikawa， Takahruu Yamashinぇand Dr¥Hide戸lki T北ahashifor assisting in the care of the animals ruld for providing technical help. The authors ru'e also grateお1to Shoji Kubota ruld the staff of the field science center， Kitasato University， for providing facilities ruld technical help.
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