J Physiol 565.3 (2005) pp 1019–1030 1019

Development of cardiovascular function in the horse fetus

Dino A. Giussani, Alison J. Forhead and Abigail L. Fowden

Department of Physiology, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK

In mammals, the mechanisms regulating an increase in fetal arterial blood pressure withadvancing gestational age remain unidentified. In all species studied to date, the prepartumincrease in fetal plasma cortisol has an important role in the maturation of physiological systemsessential for neonatal survival. In the horse, the prepartum elevation in fetal cortisol and arterialblood pressure are delayed relative to other species. Hence, the mechanisms governing theontogenic increase in arterial blood pressure in the horse fetus may mature much closer to termthan in other fetal animals. In the chronically instrumented pony mare and fetus, this studyinvestigated how changes in fetal peripheral vascular resistance, in plasma concentrations ofnoradrenaline, adrenaline and vasopressin, and in the maternal-to-fetal plasma concentrationgradient of oxygen and glucose relate to the ontogenic changes in fetal arterial blood pressureand fetal plasma cortisol concentration as term approaches. The data show that, towards termin the horse fetus, the increase in arterial blood pressure occurs together with reductions inmetatarsal vascular resistance, elevations in plasma concentrations of cortisol, vasopressin,adrenaline and noradrenaline, and falls in the fetal : maternal ratio of blood Pa,O2

and glucoseconcentration. Correlation analysis revealed that arterial blood pressure was positively relatedwith plasma concentrations of vasopressin and noradrenaline, but not adrenaline in the fetus,and inversely related to the fetal : maternal ratio of blood Pa,O2

, but not glucose, concentration.This suggests that increasing vasopressinergic and noradrenergic influences as well as changesin oxygen availability to the fetus and uteroplacental tissues may contribute to the ontogenicincrease in fetal arterial blood pressure towards term in the horse.

(Received 2 November 2004; accepted after revision 23 March 2005; first published online 24 March 2005)Corresponding author D. A. Giussani: Department of Physiology, University of Cambridge, Cambridge CB2 3EG, UK.Email: dag26@cam.ac.uk

Towards term there are maturational changes in a numberof physiological systems in the fetus, which ensure survivalboth in utero and at birth. For instance, arterial bloodpressure increases in the fetus with advancing gestationalage in a number of species, including the horse (Reeveset al. 1972; Boddy et al. 1974; Dawes et al. 1980;Macdonald et al. 1983; Kitanaka et al. 1989; Forheadet al. 2000a). This increases the perfusion pressure ofthe fetal vascular tree, and maintains placental bloodflow and the delivery of oxygen and nutrients to thefetal tissues as their demands for nutrients rise withincreased growth in late gestation. However, the cardio-vascular causes of this ontogenic increase in fetal arterialblood pressure remain unclear. Increased cardiac outputdoes not appear to be a contributing factor in fetalsheep, as the combined ventricular output expressedper kilogram of body weight does not increase towardsterm (Rudolph & Heymann, 1970). Even less is knownabout the contributions made by developmental changesin peripheral vascular resistance, as there have been nomeasurements of total peripheral vascular resistance in the

fetus with increasing gestational age towards term in anyspecies.

Like many of the maturational changes essential forneonatal survival (Liggins, 1994; Fowden et al. 1998),the ontogenic rise in fetal arterial blood pressure alsoappears to be glucocorticoid dependent. First, a closetemporal relationship exists between elevations in arterialblood pressure and plasma cortisol in the fetus close toterm in a number of species (Macdonald et al. 1983;Forhead et al. 2000a,b). Second, exogenous treatment offetal sheep with synthetic (Derks et al. 1997; Fletcheret al. 2002) or natural (Wood et al. 1987) glucocorticoidselevates fetal arterial blood pressure. Third, developmentalincreases in fetal arterial blood pressure can beprevented by fetal bilateral adrenalectomy and restored inadrenalectomised fetuses by cortisol replacement (Unnoet al. 1999). In the horse, fetal plasma cortisol risesmuch closer to term than in other species (Fowden& Silver, 1995). Glucocorticoid-dependent maturationof the fetal cardiovascular system may therefore occurcomparatively late in gestation in equids compared with

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1020 D. A. Giussani and others J Physiol 565.3

Table 1. Details of individual animals

Gestational age (days) at Foal

Proportion Physiological WeightMare of gestation Surgery recordings Delivery Outcome Sex (kg)

1 0.9 328 333–336 337 LB Male 162 0.9 328 332–339 341 LB Unrecorded 19.43 0.9 327 328 329 LB Unrecorded Unrecorded4 0.9 327 328–333 334 LB Unrecorded Unrecorded5 0.9 323 327–329 330 LB Male 23

6 0.9 321 325–326 327 LB Female 237 0.9 315 318–323 329 LB Male 208 0.9 304 306–312 313 LB Male 239 0.9 302 305–323 324 LB Unrecorded 1510 0.9 299 305–327 328 LB Female 15.8

11 0.9 289 292–310 311 LB Female Unrecorded12 0.9 286 288–294 296 SB Female 2013 0.9 285 289–294 295 SB Male 19.714 0.9 281 284–296 297 SB Female 1615 0.9 275 283 287 LB Unrecorded 15

16 0.9 260 265–269 283 SB Female 10.417 0.6 219 224–228 337 LB Female 2318 0.6 216 218–223 224 SB Male 3.219 0.6 210 213–218 227 SB Unrecorded 7.520 0.6 195 196–213 215 SB Unrecorded 7.3

21 0.6 190 194–198 335 LB Female 2022 0.6 180 185–191 192 SB Male 523 0.6 179 181–189 190 SB Female 3.324 0.6 150 153–168 191 SB Unrecorded 425 0.6 143 148–157 163 SB Male 4.5

The proportion of gestation encompasses the time between surgery and the end of the study for all individual equinepreparation. LB, live-born; SB, still-born.

other species. It has been reported that fetal arterialblood pressure increases late in gestation in the horsein association with the delayed surge in fetal circulatingglucocorticoid concentration (Forhead et al. 2000a).Furthermore, the prepartum increase in plasma cortisolcoincides with maturational changes in the activityof the renin–angiotensin system in the horse fetus,suggesting that maturing pressor actions of angiotensin IImay be responsible, at least in part, for the ontogenicincrease in fetal arterial blood pressure in this species(Forhead et al. 2000a). However, the relationships betweenplasma cortisol concentrations, arterial blood pressure andchanges in plasma concentrations of other vasoconstrictoragents, such as catecholamines and arginine vasopressin,in the horse fetus remain unknown.

The aim of the present study was to identify, inthe chronically instrumented horse fetus, mechanismscontributing to the ontogenic increase in arterial bloodpressure as the fetus approaches term, by comparingcardiovascular function between 0.6 and 0.9 of gestation.The objectives were to determine how changes in fetal peri-pheral vascular resistance, and in plasma concentrations

of noradrenaline, adrenaline and vasopressin, relate tochanges in fetal arterial blood pressure and plasma cortisolconcentration with advancing gestational age.

Methods

Animals

Welsh Pony mares (n = 25) carrying fetuses of predictedgestational ages between 0.6 and 0.9 of gestation were used(Table 1). The ponies were housed in individual stables andwere fed 500 g concentrates (Horse Stud Mix; Moulton’sFeed Supplies, Lincolnshire, UK) twice a day with accessto hay and water ad libitum. On the day preceding surgery,mares were moved into an indoor horsebox within themain animal facility. Food, but not water, was withdrawn18 h prior to surgery, and the cyclooxygenase inhibitor,meclofenamic acid (2 mg kg−1; Arquel, Pharmacia andUpjohn, Sussex, UK) was given orally the night beforesurgery, and again the morning after surgery to reduce end-ogenous prostaglandin production associated with fastingand surgery in this species (Silver et al. 1979).

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Surgical preparation

All procedures were performed under the UK Animals(Scientific Procedures) Act 1986. Gestational age was, first,estimated by ultrasound scan and measurement of aorticdiameter and/or heart rate, and then confirmed at delivery.Surgery was performed between day 143 and 328 of pre-dicted gestation (term is approx 330–350 days, Table 1),using techniques previously described (Taylor et al. 2001).In brief, on the morning of surgery, mares were pre-medicated with 20 µg kg−1 acepromazine (ACP; C-Vet,Leyland, UK), 20 µg kg−1 butorphanol (Torbugesic; FortDodge, Southampton, UK) and 10 µg kg−1 detomidine(Domosedan; Pfizer, Sandwich, UK) via a jugular catheter(Vygon intraflon 2 14G). Thirty minutes later, anaesthesiawas induced with a further 10 µg kg−1 detomidine i.v.,followed by 2 mg kg−1 ketamine i.v. (Vetalar; Pharmaciaand Upjohn, Corby, UK). The trachea was intubatedwith a cuffed tube (Portex Ltd.; Hythe, Kent, UK)as soon as the mare became recumbent, and 100%oxygen was supplied via a to-and-fro rebreathing system(Fluotec; Ohmeda, Hatfield, UK). General anaesthesia wasmaintained in the recumbent position with i.v. infusionof propofol (Rapinovert; Schering Plough, Harefield, UK)at 200 µg kg−1 min−1 for the first 60 min, and then at areduced rate of 180 and 130 µg kg−1 min−1 as requiredfor the remaining period of surgery. After 10 min ofpropofol infusion, a ketamine infusion was commencedvia a separate jugular catheter at 40 µg kg−1 min−1 for60 min, and then reduced to 20–30 µg kg−1 min−1 asrequired (Baker et al. 1999; Taylor et al. 2001). Maternalarterial blood pressure via the facial artery was recordedthroughout anaesthesia.

Under general anaesthesia, catheters were inserted intothe maternal dorsal aorta via the circumflex artery, amain uterine vein via a small branch and the umbilicalvein, and into the fetal dorsal aorta and caudal vena cavavia peripheral hind limb vessels. A further catheter wassecured to the fetal hind limb to measure amniotic fluidpressure. In 13 of the pregnancies, an ultrasonic flow trans-ducer (2RS or 3RS Transonic Systems Inc., NY, USA) wasimplanted around one of the fetal metatarsal arteries, tomeasure peripheral blood flow. The fetal membranes weretied tightly and the uterine incision was closed in layers.Following fetal i.v. administration of ampicillin (25 mg (kgestimated bodyweight)−1; Penbritin; Beecham AnimalHealth, Brentford) and gentamycin (5 mg (kg estimatedbodyweight)−1; Frangen-100; Biovet Ltd, Mullingar), allcatheters were filled with heparinised saline (50 i.u.heparin ml−1 in 0.9% NaCl), plugged with brass pins, andwere then exteriorised with the flow probe lead througha key hole incision in the maternal flank. The maternalabdominal and skin incisions were closed. Fetal catheterswere maintained patent via a slow infusion of heparinisedsaline (100 i.u. heparin ml−1) via battery-operated mini

pumps (MS16A syringe driver, Graseby Ltd, Watford, UK).Pumps, catheters and cables were kept in a plastic pouchsutured onto the maternal flank. Ampicillin (1 g, i.v.) wasadministered to the mare at surgery and for a further3 days.

Experimental protocol

Commencing 5 days after surgery, cardiovascular,metabolic and endocrine measurements were made everyother day at 10 : 00 for the duration of the experimentalperiod, at gestational ages ranging from 148 to 341 days.On each measurement day, fetal arterial blood pressure,fetal heart rate and mean metatarsal blood flow wererecorded continually at 1 s intervals for 2 h using acomputerised Data Acquisition System. Fetal arterialblood pressure was corrected for amniotic pressure andfetal heart rate was triggered via a tachometer from eitherthe arterial blood pressure or the metatarsal blood flowpulsatility. Whenever possible, simultaneous samples ofmaternal arterial, uterine venous, umbilical venous andfetal arterial blood (5 ml each) were taken. Values for pH(pHa), and partial pressures of oxygen and carbon dioxide(PO2 and PCO2 ), were obtained using a blood gas analyser(ABL 5; Radiometer, Copenhagen, Denmark), correctedto 39.5◦C for fetal blood and 38◦C for maternal blood.Blood haemoglobin concentration [Hb] and percentagesaturation of haemoglobin with oxygen (Sat.Hb) weredetermined using a haemoximeter (OSM2; Radiometer).Blood glucose and lactate concentrations were determinedwith an automated analyser (Yellow Springs 2300 Stat PlusGlucose/Lactate Analyser; YSI Ltd, Farnborough, UK).The remainder of the maternal and fetal blood (∼4 ml)was transferred into appropriately treated chilled tubes,for measurement of plasma levels of cortisol, vasopressinand catecholamines. All tubes were centrifuged (4 min,4000 r.p.m. at 4◦C), and plasma aliquots transferredto PVC tubes, which were maintained at −70◦C untilbiochemical analysis.

Biochemical analyses

Plasma concentrations of cortisol were measured byradioimmunoassay validated for equine plasma (RIA;Rossdale et al. 1982; Silver & Fowden, 1984). Theminimum detectable quantity of cortisol in the assaywas 1 ng ml−1. The intra-assay coefficient of variationwas 2.7% for a mean value of 29.5 ng ml−1. The inter-assay coefficients of variation for two plasma pools (meanconcentrations: 10.7 and 29.5 ng ml−1) were 9.4 and 7.8%,respectively. The cross-reactivities of the antiserum at 50%binding with other cortisol-related compounds were: 0.5%cortisone; 2.3% corticosterone; 0.3% progesterone; 4.6%deoxycortisol.

Plasma arginine vasopressin (AVP) concentrations weremeasured using a commercially available double-antibody

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1022 D. A. Giussani and others J Physiol 565.3

RIA kit (Nichols Institute Diagnostics ltd, Saffron Walden,UK) validated for equine plasma. The lower limit ofdetection of the assay was 1.3 pg ml−1. The intra-assaycoefficients of variation for four control plasma pools(mean concentrations: 3.2, 9.9, 12.2 and 28.9 pg ml−1)were 10.0, 6.7, 3.7 and 4.6%, respectively. The inter-assay coefficients of variation for two plasma samples(2.71 and 5.55 pg ml−1 AVP) were 4.1 and 9.8%,respectively. The anti-AVP antiserum was highly specificfor [Arg8]vasopressin, with cross-reactivities of <0.05%against oxytocin, [Lys8]vasopressin and [Arg8]vasotocin.

Plasma noradrenaline and adrenaline concentrationswere measured by high pressure liquid chromatographywith electrochemical detection (Silver & Fowden, 1995).Samples were prepared by absorption of 250 µl of plasmaonto acid-washed alumina, and 20 µl aliquots of the100 µl perchloric acid eluates were injected onto thecolumn. Dihydroxybenzylamine was added as the inter-nal standard to each plasma sample before absorption.Recovery ranged from 63 to 97%, and all catecholaminevalues were corrected for their respective recovery. Theinterassay coefficients of variation for noradrenaline andadrenaline were 6.2% and 7.3%, respectively, and theminimum detectable dose was 10 pg ml−1.

Calculations

Fetal arterial blood pressure was corrected for amnioticpressure, which was used as zero reference. Fetal peri-pheral vascular resistance was calculated by dividingsupra-amniotic mean arterial blood pressure bymean metatarsal blood flow. The fetal rate pressureproduct, an index of cardiac work, was calculatedby multiplying fetal arterial blood pressure by fetalheart rate. Arterial blood oxygen content (Ca,O2

),oxygen capacity (O2,cap), fetal peripheral arterial oxygendelivery (O2,del), fetal peripheral arterial glucose delivery(Glucosedel), maternal oxygen extraction (O2,maternal ext),fetal oxygen extraction (O2,fetal ext), maternal glucoseextraction (Glucosematernal ext), and fetal glucose extraction(Glucosefetal ext) were calculated using eqns (1)–(8),respectively:

Ca,O2(mmol l−1)

= [Hb](g dl−1) × Sat.Hb (%)/100 × 0.62 (1)

O2,cap (mmol.l−1) = [Hb](g dl−1) × 0.62 (2)

O2,del (µmol.min−1)

= Ca,O2 (µmol ml−1) × tarsal blood flow (ml min−1) (3)

Glucosedel (µmol.min−1)

= [Glucose](µmol ml−1) × tarsal blood flow (ml min−1)(4)

O2,maternal ext(%)

= (MA Ca,O2− MV Ca,O2

/MA Ca,O2) × 100 (5)

O2,fetal ext (%)

= (FUV Ca,O2− FA Ca,O2

/FUV Ca,O2 ) × 100 (6)

Glucose,maternal ext (%)

= (MA[glucose] − MV [glucose]/MA [glucose]) × 100(7)

Glucosefetal ext (%) =(FUV[glucose] − FA [glucose]/FUV [glucose]) × 100

(8)

where one molecule of haemoglobin (MW = 64 450)binds four molecules of oxygen. The contribution ofoxygen dissolved in plasma was regarded as beingnegligible. MA, maternal descending aorta; MV, uterinevein; FA, fetal descending aorta; FUV, umbilical vein.

Data and statistical analyses

Recordings of arterial blood pressure and heart rate wereobtained from 17 of the 25 horse fetuses. In 2 of the13 fetuses, the implanted tarsal flow probe developed anacoustic error, leaving 11 of 25 horse fetuses with successfulperipheral blood flow recording. Measurements of bloodgases, metabolic status and plasma cortisol concentrationswere made in all pregnancies. Simultaneous blood sampleswere taken from the maternal descending aorta, uterinevein, umbilical vein and fetal descending aorta in 18 of the25 pregnancies. Measurements of plasma catecholamineand vasopressin concentrations were made in 14 fetuses.

Variables are presented either as individual dailyvalues with respect to gestational age (mean ± s.e.m.)for pregnancies <250 days (range: 148–228 days;193.8 ± 2.7 days; 0.6 of gestation; n = 9), or forpregnancies >250 days (range: 265–341 days;309.6 ± 2.4 days; 0.9 of gestation; n = 16). All datawere assessed for normality of distribution usingthe Kolmogorov–Smirnov test. Data were normallydistributed and were assessed for significance usingparametric statistical tests. Significant differences betweenvariables at 0.6 and 0.9 of gestation were determined bythe Student’s t test for unpaired data. Pearson productmoment and partial correlation analyses were used todetermine the relationships between cardiovascular,metabolic and endocrine variables with gestational ageor with fetal plasma cortisol levels (Jandel SigmaStat2.0 and Statview 4.02). Significance was accepted whenP < 0.05.

Results

Table 1 describes details of the animals used, withinformation on the number of days with successful physio-logical recordings, the fate of the animals, and, whenrecorded, the sex and weight of the foal at delivery.

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J Physiol 565.3 Fetal equine cardiovascular function 1023

Cardiovascular function

Horse fetuses at 0.9 of gestation had significantlygreater arterial blood pressure, rate–pressure product andperipheral blood flow, but significantly lower heart rateand peripheral vascular resistance, than horse fetuses at0.6 of gestation (P < 0.05; Fig. 1).

Correlation of fetal cardiovascular variables withgestational age and with the prevailing fetal plasma cortisolconcentration showed significant positive relationshipsbetween fetal arterial blood pressure, rate–pressure

Figure 1. Cardiovascular function in the horse fetusValues are either mean ± S.E.M. at 0.6 (193.8 ± 2.7 days, range: 148–228 days; n = 9) and 0.9 (309.6 ± 2.4 days;range: 283–341 days, n = 16) of gestation, or values from individual horse fetuses in relation to gestational agefor arterial blood pressure, heart rate, the rate–pressure product, peripheral blood flow and peripheral vascularresistance. r, Pearson product moment correlation coefficient; n, number of observations; ∗P < 0.05, 0.6 vs 0.9gestation.

product and peripheral blood flow, and both gestationalage and fetal plasma cortisol (Figs 1 and 2), andshowed significant negative relationships between peri-pheral vascular resistance and both gestational ageand plasma cortisol (Figs 1 and 2), and a significantnegative relationship between heart rate and gestationalage (Fig. 1). Partial correlation analyses further revealedthat gestational age and fetal plasma cortisol equallydetermined fetal arterial blood pressure and fetalperipheral blood flow (arterial blood pressure × gestation:

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1024 D. A. Giussani and others J Physiol 565.3

0.78, n = 28, P < 0.01; arterial blood pressure × cortisol:0.65, n = 28, P < 0.01; peripheral blood flow × gestation:0.79, n = 51, P < 0.01; peripheral blood flow × cortisol:0.78, n = 44, P < 0.01). However, gestational agewas a greater determinant than plasma cortisol offetal peripheral vascular resistance (peripheral vascularresistance × gestation: −0.76, n = 19, P < 0.01; peri-pheral vascular resistance × cortisol: −0.07, n = 19,P = NS). Conversely, plasma cortisol concentration betterdetermined rate–pressure product than gestational age(rate–pressure product × cortisol: 0.57, n = 18, P < 0.05;rate–pressure product × gestation: 0.46, n = 18, P = NS).

Hormone concentrations

Horse fetuses at 0.9 of gestation had significantlygreater plasma concentrations of vasopressin, adrenaline,noradrenaline and cortisol than horse fetuses at 0.6of gestation (Fig. 3). Fetal plasma concentrations of allhormones were low for most of the gestational periodstudied. However, at ∼290 days of gestation, abruptelevations in the plasma concentrations of vasopressin,

Figure 2. Relationship between cardiovascular function and plasma cortisol in the horse fetusAssociation between plasma cortisol concentration and the values of arterial blood pressure, the rate–pressureproduct, peripheral blood flow and peripheral vascular resistance in all individual horse fetuses studied. r, Pearsonproduct moment correlation coefficient; n, number of observations.

adrenaline and noradrenaline occurred, in close temporalassociation with the prepartum surge in fetal plasmacortisol (Fig. 3).

Correlation of the plasma concentration of vaso-pressin, adrenaline and noradrenaline with gestationalage in individual horse fetuses showed significantpositive relationships between all hormones andgestational age (Fig. 3). However, significant positiverelationships were only seen between fetal plasmavasopressin and noradrenaline with cortisol (Fig. 4),but not between adrenaline and cortisol (0.36, n = 14,P = NS). Partial correlation analysis revealed thatcortisol was a greater determinant than gestationalage of vasopressin and noradrenaline concentrationsin fetal plasma (cortisol × vasopressin: 0.90, n = 27,P < 0.01; cortisol × noradrenaline: 0.90, n = 14,P < 0.01; gestation × vasopressin: 0.09, n = 27, P = NS;gestation × noradrenaline: 0.29, n = 14, P = NS).Significant positive relationships were obtained betweenfetal plasma vasopressin and noradrenaline with fetalarterial blood pressure (Fig. 4), but not between adrenalineand fetal arterial blood pressure (0.34, n = 11, P = NS).

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Maternal plasma concentrations of vasopressin,noradrenaline, adrenaline and cortisol were similar at 0.6and 0.9 of gestation (Table 2). No significant relationshipswere observed between any of the maternal hormoneconcentrations and gestational age.

Arterial blood gas and metabolic status

Maternal basal blood gas and metabolic status did notchange significantly from 0.6 to 0.9 of gestation (Table 2).In contrast, horse fetuses at 0.6 of gestation had higherbasal values for Pa,O2

and blood glucose concentrationthan horse fetuses at 0.9 of gestation (P < 0.05;Table 3). Therefore, a decrease in the fetal : maternalratio of Pa,O2

(0.26 ± 0.01–0.24 ± 0.01) and blood glucoseconcentration (0.5 ± 0–0.36 ± 0.01) occurred from 0.6 to

Figure 3. Hormone concentrations in the horse fetusValues are either mean ± S.E.M. at 0.6 (193.8 ± 2.7 days, range: 148–228 days; n = 9) and 0.9 (309.6 ± 2.4 days;range: 283–341 days, n = 16) of gestation, or values from individual horse fetuses in relation to advancinggestational age for fetal plasma concentrations of vasopressin, adrenaline, noradrenaline and cortisol. r, Pearsonproduct moment correlation coefficient; n, number of observations. ∗P < 0.05, 0.6 vs 0.9 gestation.

0.9 of gestation (P < 0.05). Fetuses at 0.9 of gestationhad significantly greater concentrations of haemoglobinthan those at 0.6 of gestation. Consequently, despite thedifference in Pa,O2

, basal oxygen content and oxygencapacity in the equine fetus were greater at 0.9 than at 0.6 ofgestation (P < 0.05; Table 2). Greater values for peripheralblood flow contributed to greater delivery of oxygen andglucose to the peripheral circulation in horse fetuses at 0.9than at 0.6 of gestation (Table 3).

When individual values for blood gas and metabolicstatus were correlated with gestational age or with theprevailing fetal plasma cortisol concentration in all horsepregnancies studied, significant negative relationshipswere observed between gestational age and the followingfactors: fetal Pa,O2

, fetal blood glucose concentrationand the fetal: maternal ratio of Pa,O2

and blood

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1026 D. A. Giussani and others J Physiol 565.3

glucose (P < 0.05; Table 4). Conversely, there weresignificant positive relationships between gestational ageand haemoglobin concentration, oxygen content andoxygen capacity in fetal blood (P < 0.05; Table 4). Partialcorrelation analysis revealed that delivery of oxygen andglucose to the fetal peripheral vasculature was equallyrelated both to gestational age and to fetal plasma cortisol(Table 4).

When individual values for blood gas and metabolicstatus were correlated with fetal arterial blood pressure,neither fetal Pa,O2

nor blood glucose were significantlyrelated to blood pressure, although the correlation of fetalPa,O2

with fetal arterial blood pressure just fell outsidesignificance (Pa,O2

× fetal arterial blood pressure: −0.40,n = 25, P = 0.06; glucose × fetal arterial blood pressure:−0.03, n = 16, P = 0.91). When the fetal : maternal ratioof Pa,O2

and blood glucose concentration were correlatedto fetal arterial blood pressure, negative relationshipswere obtained, which reached significance only between

Figure 4. Relationships between hormone concentrations and either plasma cortisol or arterial bloodpressure in the horse fetusAssociation between plasma vasopressin and noradrenaline concentrations with either the prevailing plasma cortisolconcentration or the arterial blood pressure in all individual horse fetuses studied. r, Pearson product momentcorrelation coefficient; n, number of observations.

the fetal : maternal ratio of Pa,O2and fetal arterial blood

pressure (−0.68, n = 18, P < 0.01), but not between thefetal : maternal ratio of glucose and fetal arterial bloodpressure (−0.41, n = 16, P = NS).

Discussion

The data in this study show that arterial blood pressure,rate–pressure product, peripheral blood flow, and plasmaconcentrations of noradrenaline, adrenaline, vasopressinand cortisol increased with advancing gestational age upto term in the equine fetus. In contrast, heart rate, peri-pheral vascular resistance and the fetal : maternal ratio ofPa,O2

and blood glucose concentration decreased towardsterm. While most cardiovascular and endocrine changes inthe horse fetus showed significant associations with bothgestational age and fetal plasma cortisol concentrations,partial correlation analyses revealed that gestational ageand fetal plasma cortisol contributed equally to the

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Table 2. Maternal arterial blood gases, metabolic status andendocrinology at two different proportions of gestation

0.6 0.9

pHa 7.43 ± 0 7.42 ± 0Pa,CO2 (mmHg) 38.8 ± 0.8 38.5 ± 0.4Pa,O2 (mmHg) 108 ± 2 109 ± 2Sat.Hb (%) 93.4 ± 0.7 95.0 ± 0.3[Hb] (g dl−1) 11.3 ± 0.4 10.9 ± 0.3Ca,O2 (mmol l−1) 6.3 ± 0.3 6.3 ± 0.2O2 cap (mmol l−1) 6.8 ± 0.3 6.8 ± 0.2[Glucose] (mM) 5.0 ± 0.3 5.0 ± 0.2[Lactate] (mM) 0.7 ± 0.1 0.7 ± 0.1Oxygen extraction (%) 14.6 ± 2.7 17.0 ± 1.6Glucose extraction (%) 7.1 ± 2.2 5.9 ± 1.1Plasma vasopressin (pg ml−1) 1.2 ± 0.1 1.6 ± 0.2Plasma noradrenaline (pg ml−1) 222.8 ± 58.6 158.3 ± 22.1Plasma adrenaline (pg ml−1) 91.8 ± 23.5 100.6 ± 8.6Plasma cortisol (ng ml−1) 42.4 ± 3.3 50.6 ± 4.6

Values are mean ± S.E.M. at 0.6 (193.8 ± 2.7 days, range:148–228 days; n = 9) or 0.9 (309.6 ± 2.4 days; range:283–341 days, n = 16) of gestation. There were no significantdifferences between the two age groups. Terms used areexplained in Table 3.

ontogenic increase in fetal arterial blood pressure. Incontrast, gestational age is a greater determinant thancortisol of fetal heart rate and peripheral vascularresistance, while cortisol is a greater determinant thangestational age of the rate–pressure product, and plasmaconcentrations of noradrenaline and vasopressin. FetalPa,O2

and blood glucose, and the fetal : maternal ratioof Pa,O2

and blood glucose concentration, were onlyrelated to advancing gestational age and not to cortisol.Arterial blood pressure was positively related with plasmaconcentrations of vasopressin and noradrenaline, butnot adrenaline in the fetus, and inversely related to thefetal : maternal ratio of blood Pa,O2

, but not glucose,concentration.

In the simplest terms, mean arterial blood pressure inthe fetus is determined by the combined ventricular outputand the total peripheral vascular resistance. Therefore,ontogenic increases in arterial blood pressure in thefetus could be accounted for by ontogenic increases ineither or both of these variables. The most comprehensivestudy of changes in regional blood flow during fetaldevelopment is that of Rudolph & Heymann (1970) whomeasured the combined ventricular output and organblood flow distribution in fetal sheep from 60 days ofgestation to term. They reported that the combinedventricular output of the sheep fetus increased withadvancing gestational age up to term in absolute terms,but not when expressed per kg fetal body weight. Theseobservations suggest that increases in ventricular outputtowards term occur in parallel with the growing volumeof the fetal vasculature and, hence, are unlikely to makea major contribution to the ontogenic increase in fetal

Table 3. Fetal arterial blood gas and metabolic status at twodifferent proportions of gestation

0.6 0.9

pHa 7.35 ± 0.01 7.35 ± 0Pa,CO2 (mmHg) 55.2 ± 1 55.2 ± 0.6Pa,O2 (mmHg) 28 ± 1 25 ± 0∗

Sat.Hb (%) 53.5 ± 1.2 50.3 ± 1.3[Hb] (g dl−1) 9.3 ± 0.3 13.0 ± 0.6∗

Ca,O2 (mmol.l−1) 3.1 ± 0.1 3.9 ± 0.1∗

O2 cap (mmol.l−1) 5.7 ± 0.2 8.1 ± 0.3∗

[Glucose] (mM) 2.4 ± 0.1 1.9 ± 0.1∗

[Lactate] (mM) 1.1 ± 0.2 1.2 ± 0.1Peripheral O2 del (µmol min−1) 25.1 ± 3.3 146.7 ± 11.0∗

Peripheral glucosedel(µmol min−1) 15.8 ± 1.5 71.7 ± 9.4∗

Oxygen extraction (%) 35.8 ± 2.2 33.3 ± 1.5Glucose extraction (%) 15.9 ± 2.5 14.9 ± 1.7

Values are mean ± S.E.M. at 0.6 (193.8 ± 2.7 days, range:148–228 days; n = 9) and 0.9 (309.6 ± 2.4 days; range:283–341 days, n = 16) of gestation. pHa, arterial pH; Pa,CO2 ,arterial CO2 partial pressure; Pa,O2 , arterial O2 partial pressure;Sat.Hb, percentage saturation of haemoglobin; [Hb], bloodhaemoglobin concentration; Ca,O2 , oxygen content in thearterial circulation; O2 cap, oxygen carrying capacity in thearterial circulation; [X], arterial concentration of substance; del,delivery. ∗Significant differences (P < 0.05): Student’s t test forunpaired data.

blood pressure. Little is known about changes in totalperipheral vascular resistance towards term in fetal sheep,although the resistance of several vascular beds is knownto be responsive to adverse intrauterine conditions duringlate gestation (e.g. Cohn et al. 1974; Rudolph, 1984;Jensen & Berger, 1991; Giussani et al. 1993). In thepresent study, there was a pronounced fall in the vascularresistance of the metatarsal circulation of the fetal horseduring late gestation, which was due to a greater ontogenicincrease in hind limb blood flow than fetal arterial bloodpressure. Since resistance of the metatarsal circulation wasmore closely related to gestational age than fetal plasmacortisol, the ontogenic fall in peripheral vascular resistanceis more likely to reflect angiogenesis and an increasein the cross-sectional area of the vascular beds in thegrowing fetus than maturational changes in vasoreactivityof the peripheral vasculature. Indeed, when peripheralblood flow was expressed per kg body weight, there wasno change in metatarsal blood flow with increasing agein the fetal horse (data not shown). These observationssuggest that the ontogenic increase in fetal arterial bloodpressure in the horse is not due to changes in total peri-pheral vascular resistance, although vascular resistance inthe metatarsal circulation may not be the best index ofoverall changes in total peripheral vascular resistance inthis species. In the absence of direct changes in eitherfetal cardiac output or peripheral vascular resistance, analternative explanation for the ontogenic rise in fetalblood pressure is that placental vascular resistance rises

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1028 D. A. Giussani and others J Physiol 565.3

Table 4. Correlation statistical analysis

Gestation Fetal cortisol

r n P r n P

Fetal Pa,O2 (mmHg) −0.38 125 <0.05 −0.23 53 NSFetal [Hb] (g dl−1) 0.40 117 <0.0001 0.05 44 NSFetal Ca,O2 (mmol.l−1) 0.43 113 <0.0001 −0.26 44 NSFetal O2 cap (mmol.l−1) 0.40 113 <0.0001 0.05 44 NSFetal [Glucose] (mM) −0.44 108 <0.0001 −0.18 47 NSFetal peripheral O2 del (µmol min−1) 0.90 38 <0.001 0.63 33 <0.001Fetal peripheral glucosedel (µmol min−1) 0.70 40 <0.001 0.68 35 <0.001FA : MA [Glucose] −0.43 74 <0.0001 −0.22 60 NSFA : MA [Pa,O2] −0.24 80 <0.05 −0.18 64 NS

The table shows significant changes in maternal (MA) and fetal (FA) arterial blood gas and metabolic status inequine pregnancy in relation to gestational age and the fetal plasma cortisol concentration. r, Pearson productmoment correlation coefficient; n, number of observations, NS not significant. Terms used are explained inTable 3.

relative to the total vascular resistance of the fetus, eitherby a greater rise in placental resistance or a greater fallin fetal total vascular resistance as term approaches. Inthis context, it has been reported that weight-normalisedumbilico-placental blood flow decreases with advancinggestation in the horse (Fowden et al. 2000a) and in thesheep (Hedriana et al. 1995), but changes little in thehuman (Sutton et al. 1990). However, since a similarprogression of mean arterial blood pressure, as seen in thefetus, also occurs after birth (Louey et al. 2000), a role forchanges in placental vascular resistance in contributing toprogressive changes in arterial blood pressure during thefetal period is not supported.

In the present study, there was a fall in the heartrate of the horse fetus with advancing gestational age, incommon with other species (Reeves et al. 1972; Boddyet al. 1974; Dawes, 1980; Macdonald et al. 1983; Kitanakaet al. 1989; Forhead et al. 2000a). Several studies haveshown that rate–pressure product is a useful marker ofcardiac work, correlating well with changes in myocardialoxygen consumption (Jorgensen et al. 1973; Nelson et al.1974). The increasing metabolic demand of the equinefetal heart with advancing gestation may reflect not onlychanges in cardiac growth and stroke volume (Machidaet al. 1988), but also in increased cardiac work secondaryto the imposed changes in cardiac afterload as a result ofthe ontogenic increase in fetal arterial blood pressure.

Previous studies in horses and other species haveshown that developmental changes in fetal arterial bloodpressure correlate strongly with ontogenic changes inthe renin–angiotensin system and adrenocortical cortisolsecretion (Forhead et al. 2000a,b). In the present study,abrupt elevations in the fetal concentrations of plasmavasopressin, adrenaline and noradrenaline occurred inclose temporal association with the prepartum surgein plasma cortisol in the horse fetus. While significantcorrelations were observed between gestational age orfetal plasma cortisol concentration and fetal vaso-

pressin, adrenaline and noradrenaline, only fetal plasmaconcentrations of noradrenaline and vasopressin werecorrelated with fetal plasma cortisol and fetal arterial bloodpressure. These results suggest that ontogenic changesin fetal plasma noradrenaline and vasopressin, but notadrenaline, are also involved in mediating the ontogenicincrease in fetal arterial blood pressure in the horse,presumably by constricting circulations other than thoserepresented by the metatarsal vascular bed. Whether theseontogenic changes in plasma noradrenaline and vaso-pressin are matched by similar changes in the density of therespective receptor population with advancing gestationalage remains unknown in the fetal horse. In fetal rats,expression of vasopressin V1 receptors is developmentallyregulated (Ostrowski et al. 1993) and, in fetal baboons,the maximum vasoconstrictor response to noradrenalineof small branches of the femoral artery also increases withadvancing gestastional age (Anwar et al. 2001). However,in vivo treatment of immature fetal sheep with cortisoldid not enhance their pressor response to exogenousnoradrenaline (Tangalakis et al. 1992), and in vitrovasoconstrictor responses to exogenous noradrenaline ofperipheral vessels obtained from immature sheep fetusestreated with betamethasone were also not enhanced(Anwar et al. 1999).

In the present study, a fall in basal values of Pa,O2and

blood glucose concentration occurred with advancinggestational age in fetal but not maternal blood.Consequently, there were decreases in the fetal: maternalratios of Pa,O2

and blood glucose concentration between0.6 and 0.9 of gestation, consistent with previousobservations in pregnant rabbits (Gilbert et al. 1984),sheep (Hay, 1995) and horses (Fowden et al. 2000a,b).The falls in fetal Pa,O2

and blood glucose concentration,together with changes in the rates of oxygen and glucoseconsumption by the placenta, help to maintain thenet maternal-to-fetal plasma concentration gradients forglucose molecules needed to meet the increasing nutrient

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J Physiol 565.3 Fetal equine cardiovascular function 1029

demands of the growing fetus (Meschia et al. 1980; Bellet al. 1987; Molina et al. 1991; Fowden et al. 2000a,b).The current finding that the fetal: maternal ratios ofPa,O2

and blood glucose concentration were significantlyrelated to gestational age but not to fetal plasmacortisol concentrations supports the suggestion thatthese metabolic changes reflect fetal growth rather thanfetal maturation in the horse. The relative fetal hypo-xaemia and hypoglycaemia observed at 0.9 of gestationmay contribute, in part, to the ontogenic increase infetal arterial blood pressure, by triggering cardiovascularchemoreflex and neuroendocrine mechanisms that ensuredelivery of sufficient oxygen and glucose to the fetaltarget organs to meet the increasing demands for growth(see Giussani et al. 1994 for review). This suggestion issupported by the inverse relationship observed betweenfetal blood pressure and the fetal : maternal ratios of Pa,O2

and blood glucose concentration, although only therelationship of fetal : maternal Pa,O2

reached statisticalsignificance. It is of interest that umbilical venous andarterial PO2 also fall progressively with advancing gestationin the human fetus (Soothill et al. 1986).

In conclusion, the present data show that the increasein arterial blood pressure towards term in the horsefetus occurs in association with ontogenic reductionsin metatarsal vascular resistance, elevations in plasmaconcentrations of vasopressin and noradrenaline, and adecrease in the fetal : maternal ratio of Pa,O2

. This suggeststhat increasing vasopressinergic and noradrenergicinfluences as well as changes in oxygen uteroplacentaland fetal metabolism may contribute to the mechanismsthat drive fetal arterial blood pressure up with advancinggestational age in equine pregnancy.

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Acknowledgements

This work was funded by The Horserace Betting Levy Board. Theauthors would like to thank Mr Paul Hughes, Dr Polly Taylorand Mrs Sue Nicholls for their excellent assistance. Dr DinoA. Giussani is a Fellow of The Lister Institute for PreventiveMedicine, UK.

C© The Physiological Society 2005