Onderzoeksstageverslag

Annemarie Kemmink Studentnummer: 0461318 Begeleider C. . !estermann

Aero"i# glu#ose meta"olism during e$er#ise in %arm"lood &orses A. Kemmink' C. . !estermann(' ).*. van der Kolk
+e,artment o- ./uine S#ien#es' edi#ine Se#tion' 0a#ult1 o- 2eterinar1 edi#ine'

3tre#&t 3niversit1' 4.O. Bo$ 80.156' 3508 7+ 3tre#&t' t&e 8et&erlands' 0a$ 931:30: 653;<<0' .mail C. .!estermann=uu.nl
*

Corresponding author

ABSTRACT The aim of the research presented here is to quantify glucose metabolism during moderate exercise in warmblood horses employing the hyperglycemic clamp technique. Se en healthy warmblood mares! all in diestrus! with an a erage age and weight of "".# $ %.& years and '#( $ &) *g! were fasted for "% hours. These horses were sub+ected to a hyperglycemic clamp in fi e different inter als of wor*, At rest " -until steady state plasma glucose concentrations were reached with a minimum of .) minutes/! wal* -") minutes! ".' m0s/! trot -%) minutes! &.& m0s/! wal* -") minutes! ".' m0s/! and finally rest % -same conditions as first rest/. The a erage amount of glucose metaboli1ed -expressed in 2, The amount of glucose ta*en up per *g bodyweight per minute/ was calculated and analy1ed by a one3way A456A statistical test with post hoc Bonferroni ad+ustments. 23 alues were obser ed of "'.) $ %." at rest - 1/! %'." $ #.% at wal* -6/! .7.& $ (." at trot -3/! ...) $ ".." at wal* -4/! and "8.7 $ &.# 9mol0*g B:0min at rest -5/. The amount of glucose metaboli1ed differed significantly between 1 and 3 -p ; ).)))/! 1 and 4 -p ; ).))%/! 3 and 5 -p ; ).))"/! and 4 and 5 -p ; ).)%%/. <n conclusion! glucose metabolism more than doubled in trot compared to rest and shows a lag phase during reco ery. These results are compatible with pre ious wor* using radio3labeling.

<4TR5=>CT<54 Blood glucose is an important source of energy consumed by muscles. 5ther sources are muscle glycogen and fatty acids. Research of glucose metabolism during exercise has been performed in different species! howe er! limited *nowledge is a ailable of glucose metabolism of horses in exercise -Coggan "(("/. ?uantification of endogenous glucose production and glucose upta*e in dogs and humans during exercise re eals a tight regulation of production and upta*e -Berger et al. "((&@ Aen*ins et al. "(8'/. <n horses! howe er! there appears to be a mismatch in this balance, B en during moderate exercise plasma glucose concentrations can rise -Carris et al. "(('/. Dnowledge of exercise physiology of horses depends hea ily on the precise quantification of their glucose metabolism. Currently! horses are in ol ed in ery different *inds of exercise li*e dressage! show3+umping! and endurance. Eigh3performance is often demanded of the muscles of these equine athletes. <mpro ement of muscle strength! endurance or both would not only be of great interest for researchers! but also for trainers of horses. Curthermore! better refined ad ices for food composition and supplements could be pro ided. Feor et al. were the first to report material about glucose metabolism in horses in different circumstances -Feor et al. %)))a@ Feor et al. %)))b! c/. They utili1ed radio3acti e glucose in order to trace the upta*e. The hyperglycemic clamp technique is an alternati e non3isotope based method to quantify the glucose metabolism of horses. A critical assumption made in this technique! which was alidated by Ri+nen and an der Dol*! is that during hyperglycemia after the appropriate corrections the glucose metabolism is equal to the glucose infusion rate -Ri+nen and an der Dol* %))./. This is due to the complete suppression of endogenous glucose production. The aim of this research is to quantify the amount of glucose metaboli1ed during exercise in horses by means of the hyperglycemic clamp technique. The following hypotheses are tested in the present study, Flucose metabolism increases during exercise and is ele ated during the reco ery phase in comparison to the pre3exercise phase.

2ATBR<AGS A4= 2BTE5=S Animals H This study was appro ed by the Committee on Animal :elfare of the Caculty of 6eterinary 2edicine! >ni ersity of >trecht. Se en =utch warmblood mares! aged 8 to "& years -A6F ""$%/ were included in this study! all in diestrus at the time of the experiment. The horses were used to frequent handling and tredmill exercise. Bodyweight ranged from '%% to #&8 *g -A6F '#($&) *g/. The horses were housed on pasture! but during the experiment they were *ept in indi idual stables for . days. Cood was withheld for "% hours prior to the experiment to rule out glucose upta*e from the small intestines. :ater was supplied ad libitum. After the experiment the horses were fed hay and pellets! monitored for one night and sent bac* to pasture.

*1,ergl1#emi# #lam, te#&ni/ue H To get familiar with the method! a pilot of the experiment was performed two months prior to the definite experiments. ?uantification of glucose metabolism using this technique is described by Ri+nen and an der Dol* -Ri+nen and an der Dol* %))./.5n the day before each experiment! two ". cm 2<GA I catheters were inserted in both +ugular eins and co ered with 6etrapI and GeucoplastI during nighttime. 5n the day of the experiment the horses were weighed to calculate the priming dose of glucose. The horses were positioned on the tredmilla. An BCF de iceb was installed and turned on. Eeartrates were monitored during the whole experiment. Two bloodsamples were ta*en for determination of basal plasma glucose3concentration. Eereafter! the bodyweight3dependent3 priming dose -BJ=/ of glucose -as a ')K solution/ was gi en <6 within ") minutes. The priming dose aried from "%' to "'# ml -A6F ".#.($(.7 ml/. <t was calculated by the following equation, BJ= -ml/ ; )!%& x B: . After this! the continuous infusion started at a bodyweight3dependend rate -A6F )..%"$).).' ml0h0*gB:/ with a calibrated infusion pumpc. B ery ") minutes a enous bloodsample -heparini1ed/ was ta*en for immediate determination of glucose concentration in an automated analy1er d. Blood glucose concentrations were considered hyperglycemic at L ") m2. A steady concentration was defined as a plasma glucose concentration within narrow ranges -$ ).%/ for at least .) minutes -& samples/ and no earlier than () minutes after the start of the clamp. Also! sodium!

potassium! chloride! lactate and pE were measured in enous blood. =uring steady state plasma glucose concentrations! additional enous blood samples -heparin acuettes/ were ta*en for determination of plasma insulin3concentration. These samples were centrifuged for ' minutes at #))) M g. Jlasma was separated and stored at 3%)NC until analysis of insulin could be performed. <nsulin concentration was measured with a radioimmunoassay *it e alidated for samples of horses - an der Dolk et al. "(('/. <f this steady state -Orest "P/ continued for L.) minutes! the exercise procedure was started. This comprised ") minutes of wal*ing -Owal* "P/! %) minutes of trotting -OtrotP/ and another ") minutes of wal*ing -Owal* %P/. Tredmill speeds ranged from ".& to ".7 m0s during wal* and ..( to &.# m0s during trot. The chosen speed depended on si1e and stride length of each indi idual horse. =uring the exercise procedure we ad+usted the infusion rate of glucose! in order to achie e steady concentrations of plasma glucose -Q") m2/. =uring exercise! bloodsamples were ta*en e ery ' minutes. After the protocol the horses rested again and the experiment ended when concentrations of blood glucose were steady for L.) minutes -at Orest %P/. At the end of each experiment! urine samples were collected to calculate the loss of glucose in urine.

Cal#ulations H <n this study! we assumed that endogenous production of glucose during the experiment is totally suppressed due to the hyperglycemia. Therefore! the amount of glucose infused -<4C/ equals the amount of glucose metaboli1ed -2/. The <4C -')K glucose3solution@ mmol0*gB:0min/ was calculated by use of the following equation, <4C;-mG0h M 8..../0-B: M "8)/ where B: stands for bodyweight in *ilograms. This method needs two correction factors. =uring hyperglycemia! glucose is lost in urine and therefore a correction is made for the amount of glucose loss in urine ->C/. The >C was calculated by the following equation, >C;%(..70-B: M "8)/. =uring the hyperglycemic clamp test! glucose3concentrations are not maintained perfectly constant. Therefore a correction must be made! called the space correction. <t ad+usts for the amount of glucose! which has been added or remo ed from the glucose space. The formula is based on human alues! with the assumption that ).)%8 mmol of glucose is remo ed from each deciliter of glucose space in a %)3minute period -=eCron1o et al. "(7(/. The glucose space in G is gi en by )."( M B:. The SC was calculated by use of the following equation,

SC;--F"3F%/ M -)."( M B:/ 0 ") M B: which is reduced to the following, SC;-F"3F%/ M ).)"( The 2 -mmol0*g0min/ was calculated by use of the following equation, 2;<4C3>C3SC

The insulin #on#entration > ?,mol@AB during the steady state in the experiment is a measure of bRtacell response to hyperglycemia. The 20< ratio is an index of tissue3sensiti ity to insulin! because it reflects the quantity of glucose metaboli1ed per unit of insulin.

Assa1s and data anal1sis H all 2 alues were calculated and a one way A456A with post hoc Bonferroni statistical test was performed with SJSS f. =ifferences were considered significant at alues of pS).)'.

RBS>GTS
At first we determined the basal plasma glucose concentration after "% hours of withholding food. The mean basal plasma glucose concentrations ranged from &.8 to '.' m2 -A6F '."$).%7 m2/.

The mean time to reach a steady blood glucose concentration within hyperglycemic ranges -L") m2/ was ""#$%" min -Orest "P/. At rest "! the a erage blood glucose concentration was "..%($"."& m2. Cor wal* "! trot! wal* % and rest % these alues were ".."%$".%" m2@ "%.''$".%' m2@ "".8"$".)( m2 and ""..&$"."( m2 respecti ely. The infusion rates ranged from ).%8 ml0h0*g B: to )..8 ml0h0*g B: -A6F )..%$).). ml0h0*g B:/ during different gaits. <n table "! all 2 -metabolism/ alues are shown. <n table % ranges and a erage 2 alues are shown. >rine glucose3concentrations ranged from 8." to #7.( m2 -A6F %&.% $ %%.. m2/. 2 alues were "'.)$%." 9mol0*g B:0min@ %'."$#.%) 9mol0*g B:0min@ .7.&$(."' 9mol0*g B:0min@ ...)$"..)( 9mol0*g B:0min@ "8.7$""..' 9mol0*g B:0min at rest "! wal* "! trot! wal* % and at rest % respecti ely.

<n figure "! a erage glucose metabolism alues -2/ with standard de iations are shown. <n figure %! the a erage 2 alues were tested with an one way A456A and post hoc Bonferroni statistical test. Significant differences -pS).)'/ were calculated for rest " s. trot and wal* %@ trot s. rest % and wal* % s. rest % and ice ersa. <n figure . is isible that lactate blood concentrations were far below the aerobic lactate threshold concentration of & m2. The speed of the tredmill was not the same for each horse! but the heart rates were rather similar as can be seen in figure &. <n figure '! mean pE alues are shown. Significant differences -pS).)'/ were calculated between rest " s. trot and wal* %@ wal* " s. trot@ trot s. rest % and ice ersa. Cigure # shows increasing plasma potassium concentrations during exercise. 2aximal alues are reached during trot and lower alues are determined at rest % than at rest ". Significant

differences -pS).)'/ were calculated between rest " s. rest % and trot@ wal* " s. trot and rest %@ trot s. rest % and wal* %@ wal* % s. rest % and ice ersa. Sodium and chloride were also determined and figures 7 and 8 show that plasma sodium and chloride concentrations remain constant throughout the experiment.

=<SC>SS<54
A quantitati e measurement of glucose metabolism in horses during rest and different grades of exercise has been described.

Flucose as a substrate for ATJ production comes from arious sources, Flucose can be ta*en up in the small intestine! it can be produced by the li er in gluconeogenesis! or by glycogenolysis in the li er and muscles. 2uscle glycogen is the most important resource for glucose in the exercise phase. A relati ely small amount of glucose is withdrawn from the blood -Einchcliff D.:. %))&/. <n a state of hyperglycemia! more glucose is ta*en from the blood circulation! but the absolute amount of muscle glycogen consumed remains constant -Feor et al. %)))c/. The contribution of plasma glucose to total carbohydrate oxidation -plasma glucose! muscle glycogen and lactate/ is approximately %#K during induced hyperglycemia and "7K without exogenous glucose infusion -Feor et al. %)))c/.

The calculated glucose metabolism -2/ alues re eal interesting differences between all gaits. The glucose metabolism during trot more than doubles compared to the rest " phase. Apparently! muscles ta*e up a large amount of glucose from the hyperglycemic blood circulation during exercise. <n comparison the results of Feor et al are, Flucose upta*e alues -Rd/ were 8..$).( 9mol0*g B:0min at rest! %(."$.." 9mol0*g B:0min at &' minutes of exercise -.&$"K of 65% max/ and &).&$%.( 9mol0*g B:0min between #) and () minutes of exercise -Feor et al. %)))c/. These results are in good agreement with our findings.

Feor et al -Feor et al. %)))c/ compared glucose upta*e rates between a group with glucose infusion and a control group during exercise. They assumed that there would be a large mismatch between hepatic glucose production -EFJ/ and glucose upta*e -Rd/ in the control group! i.e. a hyperglycemia would occur during exercise. <n contrast! they found a close match between EFJ and Rd during low3intensity exercise. <n the other group! EFJ was only partially suppressed during the whole exercise procedure. Eowe er! EFJ increased mostly after &' minutes of exercise. The assumption of EFJ suppression by exogenous glucose

infusion can be defended! because measurements were done during %) minutes of low3 intensity exercise. There must be an explanation for the -small/ mismatch in EFJ and Rd in horses. Apparently! EFJ is not only influenced by metabolic feedbac* but also by a feed forward regulation. <n the horse! it is possibly due to higher sympathoadrenal acti ation during low intensity exercise! compared to humans and dogs. Another important finding is that the glucose upta*e is enhanced by hyperglycemia. <n a state of -induced/ hyperglycemia! muscles rely on glucose more than with normal blood glucose concentrations -Feor et al. %)))a/. =uring hyperglycemia! also hyperinsulinemia occurs and this facilitates glucose upta*e in the muscles and the whole body.

An important difference between the method used by Feor et al and the hyperglycemic clamp technique is that with the current method glucose metabolism is quantified by achie ing steady concentrations of blood glucose abo e ") m2 at a certain infusion rate -Feor et al. %)))c/.

Aager et al determined a decrease in blood glucose concentration on the onset of exercise! followed by an increase after "' minutes -Fraaf3Roelfsema %))7/. This increase is probably due to enhanced glycogenolysis after "' minutes of exercise.

:e obser ed a lag phase in glucose metabolism during wal* % and rest %. =uring the hyperglycemic clamp technique! we expect plasma insulin le els to be ele ated. According to Aager et al and Creestone et al! plasma insulin is low during exercise and a hyperinsulinemia rebound occurs after exercise -Creestone et al. "(("@ Fraaf3Roelfsema %))7/. This rebound can be caused by withdrawal of sympathetic inhibition of the pancreatic beta3cells or by a rise of endogenous opioid peptides that enhance insulin release -Creestone et al. "((%/. =uring hyperglycemia and exercise! plasma insulin is possibly less ele ated than during rest! and increases again in rest %. This can explain the ele ated glucose metabolism, insulin3mediated glycogenogenesis and glucose upta*e in the muscle by FG>T &.

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Jlasma catecholamine le els are decreased during reco ery and cause lower rates of lipolysis and 4BCAPs in the blood circulation. Flucose is therefore the most important substrate for energy repletion in the muscle cell -Eyyppa et al. "((7/. These mechanisms explain the increased metabolism of glucose during reco ery.

Feor et al found that muscle glycogen consumption is not affected by plasma glucose concentrations! therefore we considered muscle biopsies unnecessary -Feor et al. %)))c/.

Jlasma lactate concentrations are low during all gaits and ne er exceeded concentrations Q " m2 -figure ./. The threshold le el of lactate is & m2! because it is exported out of the cell until this alue is reached. After exceeding & m2! lactate rises exponentially in the cytoplasm. The alues for lactate might be inaccurate! as these ha e been determined in whole blood samples. <n the horse! RBCs function as Olactate sin*sP. >p to ')K of the lactate may be in RBC -Joso A.R. "(('@ 6aih*onen G.D. "(((/. Garge differences of this characteristic ha e been reported interindi idually -6aih*onen G.D. "(((@ 6aih*onen G.D. "((8/. Eowe er! plasma lactate concentrations are sufficiently low for aerobic exercise.

<nstead of collecting all urine during the experiment! we estimated the a erage glucose concentration in urine ->C/ from the measurement of one sample collected at the end of the experiment. This approach can be +ustified by the fact that the >Cs do not influence the 2 alues to a great extent. Because our clamptime is longer than % hours -.3' h/! the total amount of urine is different from the assumption of the formula to calculate the >C. The increased production due to glucosuria should also be considered. >rine glucose concentrations were ery different for each horse.

The formula used for the space correction is made for blood sample inter als of ") minutes. =uring exercise! blood samples were ta*en with a ' minute inter al.

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The amount of effort deli ered by the horses has been monitored by an BCF de ice. Comparable heart rates indicate that All horses exercised at approximately the same le el -figure &/.

Jlasma potassium increases during exercise and decreases in the reco ery phase. This is due to two opposite factors, -i/ Release of potassium from contacting muscle cells and -ii/ increased upta*e of potassium by inacti e muscle cells -Cleroux et al. "(87/. =uring moderate exercise! release o errules upta*e and plasma potassium rises.

The pE has been determined to monitor the acid0base balance. A erage pE alues are presented in figure '. The pE shows a significant ele ation during exercise. This can be explained by the release of potassium to the blood circulation with constant plasma sodium concentrations. Eydrogen ions exchange for potassium and E T rises in the muscle cells. <n the blood circulation the pE decreases due to lower E T concentrations.

<n conclusion! glucose metabolism more than doubles during trot compared to rest ". Curthermore! metabolism during wal* % is significantly higher than during rest " and rest %. There is no significant difference between rest " s. rest % and wal* " s. wal* %.

Curther recommendations for research are quantification of glucose metabolism during longer moderate exercise or during high intensity and anaerobic exercise.

0ootnotes
a

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AJJB4=<M

Table ", mean metabolism alues of all horses based on . measurements -Umol0*gB:0min/ Rest " "8.7$.." "..($..( "..8$%.7 "&.8$%.& "%.'$".# "#.8$..% "&.8$..% :al* " %'.7$).) "(."$..8 %).($".) %%.&$).) %".)$).) .).7$".) .#."$7.# Trot .&.'$).) ...&$).) ...7$..8 %'.8$&.8 ....$..8 '".7$)." &8."$'.& :al* % %7.($""." %&.8$".( %..($).) "(.&$".) .%.#$".) ').($".) '".#$".( Rest % "8."$'.& "#.)$..( "7."$%.& "&.8$..% "'..$).( %7.7$&.7 %%.)$%.&

Caldo Jrincess 4octurne 4anda 5pium Truffel Rolindra

Table %, Ranges and a erage 2 alues -Umol0*g0min/ Rest " "%.' "8.7 "'.) %." :al* " "(." .#." %'." #.% Trot %'.8 '".7 .7.& (." :al* % "(.& '".# ...) ".." Rest % "&.8 %7.7 "8.7 &.#

2inimum 2aximum A erage S=

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ean meta"olism %it& S+

') &' glu#ose meta"olism ?Cmol@kg@minB &) .' .) %' %) "' ") ' ) " % . gait & '

Cigure ", Flucose metabolism alues with standard de iations

eta"olism means
') &' &) .' .) %' %) "' ") ' )

glu#ose meta"olism ?Cmol@kgB!@minB

*W

X V XW

*V

"

.
gait

&

'

"3& V p;)!))% &3' X p;)!)%% .3' W p;)!))" "3. * p;)!))) Cigure %, 2ean metabolism profile

14

ean ,lasma la#tate #on#entrations

).( ).8 ).7 ).#

la#tate ?m B

).' ).& ).. ).% )." ) " % . & '

gait

Cigure ., 2ean plasma lactate concentrations

ean &eart rate values

"&) "%) ")) *0 ?$@minB 8) #) &) %) ) " % . gait & '

Cigure &, 2ean heart rates

15

ean ,* values
7.&8 7.&7 7.&# 7.&' ,* 7.&& 7.&. 7.&% 7.&" 7.& 7..( " % . gait & ' VX W X

VW

"3& V p ; ).)))

"3& X p ; ).).(

%3. W p ; ).)).

.3' * p ; ).)))

Cigure ', 2ean pE alues

ean ,lasma ,otassium #on#entrations

..( ..7 WV

,lasma ,otassium ?m B

..' ... W

X V
.." %.( %.7 %.' "

Z YX

&

'

gait
"3. V p ; ).))) .3& N p ; ).))) "3' X p ; ).)&8 .3' Y p ; ).))) %3. W p ; ).))" &3' Z p ; ).))# %3' * p ; ).))"

Cigure #, 2ean plasma potassium concentrations

16

ean ,lasma sodium #on#entrations

"&. "&% "&" "&) ".( ".8 ".7 ".# ".' ".& " % . & '

sodium ?m

gait

Cigure 7, 2ean plasma sodium concentrations

ean ,lasma #&loride #on#entrations

")' ")& "). ")% C&loride ?m B ")" ")) (( (8 (7 (# (' " % . gait & '

Cigure 8, 2ean plasma chloride concentrations

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RBCBRB4CBS

Berger, C.M., Sharis, P.J., Bracy, D.P., Lacy, D.B. an !asser"an, D.#. $1994% Sensi&i'i&y () e*ercise+in ,ce increase in he-a&ic g.,c(se -r( ,c&i(n &( g.,c(se s,--.y an e"an . The American journal of physiology 267, /411+ 421. C.er(,*, J., Pe&ers(n, M. an Leenen, 0.#. $1987% /*ercise+in ,ce hy-er1a.ae"ia2 e))ec&s () 3e&a+a ren(ce-&(r 3.(c1er 's i,re&ic. British journal of clinical pharmacology 24, 225+229. C(ggan, 4.5. $1991% P.as"a g.,c(se "e&a3(.is" ,ring e*ercise in h,"ans. Sports medicine (Auckland, N.Z 11, 102+124. De0r(n6(, 5.4., 7(3in, J.D. an 4n res, 5. $1979% 8.,c(se c.a"- &echni9,e2 a "e&h( )(r 9,an&i)ying ins,.in secre&i(n an resis&ance. The American journal of physiology 237, /214+223. 0arris, J.!., #inchc.i)), :.!., Mc:ee'er, :.#. an La"3, D.5. $1995% 8.,c(se in),si(n increases "a*i"a. ,ra&i(n () -r(.(nge &rea "i.. e*ercise in S&an ar 3re h(rses. E uine !eterinairy "ournal Supplements 18, 357+361. 0rees&(ne, J.0., Bea .e, 5., Sh(e"a1er, :., Bessin, 5.7., !(.)shei"er, :.J. an Ch,rch, C. $1992% ;"-r('e ins,.in sensi&i'i&y in hy-erins,.inae"ic -(nies &hr(,gh -hysica. c(n i&i(ning an c(n&r(..e )ee in&a1e. E uine !et " 24, 187+190. 0rees&(ne, J.0., !(.)shei"er, :.J., :a"er.ing, S.8., Ch,rch, 8., #a"ra, J. an Bag<e.., C. $1991% /*ercise in ,ce h(r"(na. an "e&a3(.ic changes in 7h(r(,gh3re h(rses2 e))ec&s () c(n i&i(ning an ace-r("a6ine. E uine !et " 23, 219+223. 8e(r, 5.J., #inchc.i)), :.!., McC,&che(n, L.J. an Sa"s, 5.4. $2000a% /-ine-hrine inhi3i&s e*(gen(,s g.,c(se ,&i.i6a&i(n in e*ercising h(rses. " Appl #hysiol 88, 1777+1790. 8e(r, 5.J., #inchc.i)), :.!. an Sa"s, 5.4. $20003% 3e&a+a renergic 3.(c1a e a,g"en&s g.,c(se ,&i.i6a&i(n in h(rses ,ring gra e e*ercise. " Appl #hysiol 89, 1086+1098. 8e(r, 5.J., #inchc.i)), :.!. an Sa"s, 5.4. $2000c% 8.,c(se in),si(n a&&en,a&es en (gen(,s g.,c(se -r( ,c&i(n an enhances g.,c(se ,se () h(rses ,ring e*ercise. " Appl #hysiol 88, 1765+1776. 8raa)+5(e.)se"a, /. $2007% Endocrinological and $eha%ioural adaptations to e&perimentally induced physical stress in horses, =&rech& =ni'ersi&y, =&rech&. #inchc.i)) :.!., 8., 5.J. $2004% 'ntegrati%e physiology of e&ercise, Sa,n ers, / in3,rgh. - 6.

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#yy--a, S., 5asanen, L.4. an P(s(, 4.5. $1997% 5esyn&hesis () g.yc(gen in s1e.e&a. ",sc.e )r(" s&an ar 3re &r(&&ers a)&er re-ea&e 3(,&s () e*ercise. American journal of %eterinary research 58, 162+166. Jen1ins, 4.B., Chish(.", D.J., Ja"es, D./., #(, :.>. an :raegen, /.!. $1985% /*ercise+in ,ce he-a&ic g.,c(se (,&-,& is -recise.y sensi&i'e &( &he ra&e () sys&e"ic g.,c(se s,--.y. (eta$olism) clinical and e&perimental 34, 431+436. P(s( 4.5., L.#.J., 5asanen L.4. $1995% Dis&ri3,&i(n () .ac&a&e 3e&<een re 3.(( ce..s an -.as"a a)&er e*ercise. E uine !eterinary "ournal Supplements 18. 5i?nen, :./. an 'an er :(.1, J.#. $2003% De&er"ina&i(n () re)erence range 'a.,es in ica&i'e () g.,c(se "e&a3(.is" an ins,.in resis&ance 3y ,se () g.,c(se c.a"- &echni9,es in h(rses an -(nies. American journal of %eterinary research 64, 1260+1264. @aih1(nen L.:., #.S., P(s( 4.5. $1999% 0ac&(rs a))ec&ing acc,",.a&i(n () .ac&a&e in re 3.(( ce..s. E uine !eterinary "ournal Supplements 30. @aih1(nen L.:., P.4.5. $1998% ;n&erin i'i ,a. i))erences in &(&a. an carrier "e ia&e .ac&a&e in).,* in&( re 3.(( ce..s. Am " #hysiol *eg 'ntegr +omp #hysiol 274. 'an er :(.1, J.#., !ensing, 7., :a.s3ee1, #.C. an Bre,1in1, #.J. $1995% La3(ra&(ry iagn(sis () e9,ine -i&,i&ary -ars in&er"e ia a en("a. ,omestic animal endocrinology 12, 35+39.

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