Publisher: CSIRO; Journal: RD:Reproduction, Fertility and Development Article Type: Research Paper; Volume: ; Issue: ; Article ID: RD17298

DOI: 10.1071/RD17298; TOC Head:

Doppler ultrasound can be used to monitor umbilical arterial blood flow in lightly sedated pigs at multiple gestational ages

Claire StenhouseA, Peter TennantA, W. Colin DuncanB and Cheryl J. AshworthB,C

A Developmental Biology Division, The Roslin Institute and Royal (Dick) School of Veterinary

Studies, University of Edinburgh, Easter Bush Campus, Edinburgh, EH25 9RG, U.K.

BCentre for Reproductive Health, The Queen’s Medical Research Institute, University of Edinburgh,

Edinburgh, EH16 4TJ, U.K.

CCorresponding author. Email: cheryl.ashworth@roslin.ed.ac.uk

Doppler ultrasound was performed under moderate sedation (ketamine and azaperone) for 30 min to monitor

umbilical arterial (UA) blood flow in one uterine horn of Large White × Landrace gilts (n = 23) at Gestational

Days (GD) 30, 45, 60 and 90. Gilts were scanned before euthanasia they were killed to examine relationships

between litter size, sex ratio and five UA parameters. In gilts where in which scans were obtained from all

fetuses in the scanned horn, relationships between fetal weight and sex were examined. A subset of gilts were

was sedated, scanned and recovered (SSR) earlier in gestation (GD30 or GD45) to assess the effectsinfluence of

sedation on later fetal development by comparisong with control litters (no previous sedation). Temporal

changes were observed in all UA parameters (PP ≤ 0.001). At GD60 and GD90, fetal heart rate decreased

with increasing duration of sedation (PP ≤ 0.001). Sex ratio and fetal weight affected UA blood flow, whereas

litter size and fetal sex did not. SSR at GD30 and GD45 was associated with decreased fetal weight at GD60

(PP ≤ 0.001) and GD90 (P = 0.06) respectively, compared with controls. These results suggest maternal

sedation during gestation affects fetal development, which should be investigated further. Measuring UA blood

flow in growth- restricted porcine fetuses throughout gestation may be feasible.

Additional keywords: gestation, gilt, Doppler ultrasound, sedation., gestation, umbilical arterial blood flow,

pig

C. Stenhouse et al.

Doppler ultrasound in sedated pregnant gilts

Growth- restriction in humans can be identified by monitoring umbilical blood flow by ultrasound. This study

presented presents novel relationships between umbilical blood flow in the pig with and both foetal weight and

the sex ratio of the litter, and umbilical blood flow in the pig. In additionAdditionally, maternal sedation early in

pregnancy decreased foetal weight in late pregnancy. It is hoped this method of Doppler ultrasound under light

sedation could be usedutilised further to improve the our understanding of the mechanisms governing foetal

growth in the pig.

Introduction

Increased litter size has been a focus of recent pig genetic selection programs as an effective means

to increase the profitability of the pig industry (Bee, 2007). This has increased the within-litter

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Publisher: CSIRO; Journal: RD:Reproduction, Fertility and Development Article Type: Research Paper; Volume: ; Issue: ; Article ID: RD17298

DOI: 10.1071/RD17298; TOC Head: variation in birthweight observed, with many litters containing an intrauterine growth- restricted

(IUGR) piglet (Ashworth et al. 2001; Wu et al. 2006; Bee, 2007; Vallet et al. 2014; Yuan et al. 2015).

The decrease in birthweight observed in the IUGR piglet has severe consequences for postnatal

development, with several disadvantageous phenotypes observed, including impaired adaptation to

extra-uterine life, a permanently reduced growth rate, altered organ growth and development,

decreased reproductive performance and increased disease susceptibility (Wu et al. 2006).

It is essential that the umbilical cord and placenta function efficiently throughout gestation to

support the growing fetus, with inadequate placental function being associated with adverse

pregnancy outcomes, including pre-eclampsia and IUGR (Baschat, 2003). It is hypothesised that due

to the placental insufficiency associated with IUGR, the fetus re-directs available blood flow to its

most vital organs, namely the brain and heart, to spare their growth at the expense of the other organs

(Giussani, 2011). In human clinical practice, non-invasive Doppler ultrasound is heavily widely

usedutilised to identify and monitor umbilical arterial (UA) blood flow in IUGR pregnancies,

ensuring that clinical interventions occur at the optimal time (Detti et al. 2002). The waveforms

produced by Doppler ultrasound can be used to calculate several parameters, examples of which

includeincluding: peak systolic velocity (PSV); ), end diastolic velocity (EDV), the; A/B ratio (or

PSV to EDV (S/D) ratio); ), resistance index (RI); and the pulsatility index (PI;) (Thompson et al.

1988; Berkley et al. 2012). These parameters fall into two categories: angle dependent and angle-

independent. Velocity measurements are categorised as angle-dependent measurements, whereby to

obtain a truly accurate measurement of the velocity present, the angle of the ultrasound beam needs to

be as close to zero 0° degrees as possible (McDicken, 1991; Detti et al. 2002). As the angle increases,

the accuracy of determining the velocity decreases. Considering this, angle- independent parameters,

such as the A/B ratio, RI and PI, are commonly used in clinical practice (Trudinger et al. 1985; Detti

et al. 2002; Berkley et al. 2012). These parameters provide an indication of vascular resistance,

which, in turn, provides information regarding the arterial blood flow and placental vascular

resistance.

In large animals, Doppler ultrasound has been usedutilised to investigate uterine, umbilical and

fetal vessels in cattle (Bollwein et al. 2002; Panarace et al. 2006; Silva and Ginther, 2010), goats

(Serin et al. 2010; Elmetwally et al. 2016), horses (Bollwein et al. 2000; , Bollwein et al. 2004) and

sheep (Elmetwally et al. 2016). There have also been limited investigations into gestational changes

in UA blood flow in the pig (Brüssow et al. 2012; Harris et al. 2013). It has been suggested that an

invasive laparoscopic approach is the most effective method to monitor UA blood flow in the pig

(Brüssow et al. 2012),; although the effectinfluence of general anaesthesia is unknown. In addition,

the relationship between UA blood flow and fetal growth and development in the pig is poorly

understood.

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Publisher: CSIRO; Journal: RD:Reproduction, Fertility and Development Article Type: Research Paper; Volume: ; Issue: ; Article ID: RD17298

DOI: 10.1071/RD17298; TOC Head: The aim of the presentis study aimed was to relate several parameters (, including litter size, fetal

weight and sex, and the sex ratio of the litter) to UA blood flow parameters throughout gestation in

the pig, in a non-invasive manner, under light sedation. In addition, this protocol was usedutilised to

monitor gestational changes within the same pregnancy, with an additional objective of investigating

the effectinfluence of light sedation in early gestation on later fetal development.

Materials and methods

All procedures were performed with approval from The Roslin Institute (University of Edinburgh)

Animal Welfare and Ethical Review Board and in accordance with the UK Animals (Scientific

Procedures) Act, 1986.

Experimental animals

AltogetherIn all, 23 Large White × Landrace gilts (age 11–14 months; mean weight at slaughter

death 188.76 kg) were included in this study. All gilts were observed daily for signs of oestrus and

were housed 6–8six to eight animals per pen, unless otherwise stated otherwise. Oestrous cyclicity

and ovarian function were controlled in accordance with routine normal practice at The Roslin

Institute Large Animal Unit. In a subset of the gilts (n = 5), oestrus was synchronised by daily feeding

of 20 mg altrenogest (Regumate;, Hoechst Roussel Vet Ltd) for 18 days followed by injection of

pregnant mare’s serum gonadotrophin Pregnant Mare’s Serum Gonadotrophin (PMSG) and human

chorionic gonadotrophin (hCG), as described by Lillico et al. (2013). All gilts were inseminated twice

daily for the duration of oestrus with semen from one of two sires (Large White). The first day of

insemination was assigned as Gestational Day (GD) 0. The average ovulation rate (determined by the

number of corpora lutea present) and percentage prenatal survival (determined by as the number of

live fetuses present divided by the ovulation rate multiplied by 100) was 20.5% and 66.75%

respectively.

Doppler ultrasound procedure

Gilts were sedated by intramuscular injection with 1 mg/kg ketamine (Henry Schein Animal

Health) and 2 mg/kg azaperone (Henry Schein Animal Health). Once the animals were sedated (~10–

15 min after drug administration), they animals were positioned in lateral recumbency on their left

side. The presumed right uterine horn was scanned by a single operator from the cervix towards the

ovary using a Philips iU22 colour Doppler ultrasound machine, with an L9–-3 transducer probe

(Philips Healthcare). The scanning procedure lasted for approximately ~30 min. UA traces were

obtained from the fetuses and the following parameters were measured: fetal heart rate (FHR; beats

per minute (bpm)),; peak systolic velocity (PSV; cm/s);, end diastolic velocity (EDV; cm/s);,

Resistance Index (RI) and the A/B ratio (PSV : : EDV). The number of fetuses and waveform data

collected are summarised in Table 1.

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Publisher: CSIRO; Journal: RD:Reproduction, Fertility and Development Article Type: Research Paper; Volume: ; Issue: ; Article ID: RD17298

DOI: 10.1071/RD17298; TOC Head: Experiment 1: temporal changes in UA blood flow

Pregnant gilts were used in this study at gestational day (GD) 30 (range GD= 29–30; n = 3); ),

GD45 (range = GD43–46; n = 6), GD; 60 (range GD= 60–61; n = 5) and GD90 (range GD= 89–92; n

= 9). Following completion of the scanning procedure, the gilts were euthanised killed with 0.4 mL

kg–1 of 20% w/v sodium pentobarbitone 20% w/v (Henry Schein Animal Health), injected at a dose of

0.4 ml/kg by intravenously injection via a cannula inserted in the ear vein.

A subset of the pregnant gilts which were sedated and scanned at GD60 (n = 4) and GD90 (n = 3)

were sedated, scanned and allowed to regain consciousness (recovered; SSR) at GD30 (range GD=

29–33; n = 4) and GD45 (range = GD43–44; n = 3) respectively. The sedation and scanning procedure

was performed as described above. Following this, sedated gilts were hoisted onto a surgical trolley

and transferred to a recovery pen. Once the animals had fully recovered, they were transferred to a

shed where they were housed individually, in close proximity to other pregnant gilts, for the

remainder of the experiment. These animals were euthanised killed immediately after the second

scanning occasionprocedure. To assess temporal changes in UA blood flow, the SSR traces at GD30

and GD45 were combined with those of the euthanised killed animals (GD30, n = 7 litters; GD45, n =

9 litters; GD60, n = 5 litters; GD90, n = 9 litters).

Experiment 2: effectInfluence of litter size and sex reatio of the litter on UA blood flow

Following confirmation of death in euthanised animals, the reproductive tract was revealed by a

mid-ventral incision revealed the reproductive tract. The tract was lifted from the body cavity and

placed in on a dissecting tray. Both uterine horns were dissected, from the ovaries towards the cervix,

allowing determination of whether traces were obtained from all fetuses in the right uterine horn. All

fetuses were identified as ‘live’ or ‘dead’ based on their morphology at the time of dissection. Fetuses

They were weighed and, at GD45, GD60 and GD90, fetal sex was determined morphologically. DNA

was isolated from the GD30 fetuses using the DNeasy Blood and Tissue DNA extraction kit (Qiagen),

and polymerase chain reaction (PCR)PCR was performed for the SRY region of the Y chromosome

(forward primer, 5-GAAAGCGGACGATTACAGCC GAAAGCGGACGATTACAGCC-–3;

reverse primer, 5-TTGCGACGAGGTCGGTATTT-3). Primers for the homologous zfx/zfy regions

of the sex chromosomes served as a positive control (forward primer 5-

ATAATCACATGGAGAGCCACAAGCT-3; reverse primer 5-

GCACTTCTTTGGTATCTGAGAAAGT-3) ; (Oliver et al. 2011). For the reaction, 7μL nuclease-

free water, 5 μL of 10 × PCR Buffer, 1 μL dNTP mix (10 mM), 1 μL FastStart Taq (all solutions from

the Roche FastStart Taq kit;, Sigma Aldrich) and 5 μL of both forward and reverse primers (5 μM

stock) were added to 1 µg of sample DNA. A no- template control and postnatal porcine testicular and

ovarian DNA were run as controls. The PCR conditions had an initial step of 95°C for 4 min 30 s,

which was followed by 30 cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 30 s, with a final

extension of at 72°C for 4 min 30 s and an indefinite hold at 4°C. Samples were processed in 96-well

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Publisher: CSIRO; Journal: RD:Reproduction, Fertility and Development Article Type: Research Paper; Volume: ; Issue: ; Article ID: RD17298

DOI: 10.1071/RD17298; TOC Head: microplates (NUNC, ; Thermofisher) on a 96-well thermocycler (Biometra). The PCR products were

then analysed on a 1.2% (wt/vol) agarose 1 × SybrSafe (Thermofisher) gel with 1 × Tris/–acetic

acid–/EDTA buffer. This allowed the results to be related to the number of live fetuses and the sex

ratio of the litter at all GD.

Experiment 3: effectsinfluence of fetal size and sex on UA blood flow

As described in Experiment 2, fetal weight and sex was recorded for all fetuses in all of thekilled

euthanised litters at all GD investigated. In a subset of the euthanisedkilled GD90 litters (n = 4), UA

waveforms were obtained from all fetuses in the right uterine horn, which allowed relation of the

parameters obtained to be related to fetal size and sex.

Experiment 4: effectInfluence of sedation

The difference in time since the first fetal trace measurement to each subsequent trace measured

was calculated within gilt, to determine whetherif the fetuses were affected by maternal sedation over

the scanning period. The mean FHR for each fetus was then related to the time since the first fetal

trace measurement. To determine whetherif the sedation in early gestation for the SSR procedure

affectedinfluenced fetal development later in gestation, fetal weight, the number of live fetuses in the

uterus and the sex ratio of the litters were compared in the SSR gilts were compared with GD-

matched control gilts that had not been subjected to the SSR procedure (n = 8 and 4 control and SSR

GD60 litters respectively; n = 6 and 3 control and SSR GD90 litters respectively).

Statistical analysis

All statistical analyses were performed using Minitab 17 or GENSTAT 13.1 (VSN International Ltd).

The mean value for each parameter was calculated for each fetus and normality was assessed. Outliers

were tested for using a Grubbs outlier test and were excluded systematically, with normality within

each group being reassessed following each exclusion. Log-transformed data were used for PSV at

GD45, GD60 and GD90, and as well as for EDV at GD45 and GD90, to improve the distribution of

the data. Non-parametric tests were used whenever the data were not found to have be not normallya

normal distributed ion after transformation.

Temporal changes in FHR were analysed by one-way analysis of variance (ANOVA) followed by

Tukey analysis. The gilt term was blocked to account for the common maternal environment. For the

remainder of the parameters, where the data did not have a normal distribution with the four GD

combined, a Kruskal–Wallis analysis test was performed used to investigate temporal changes. Linear

regressions were performed within GD for all parameters against the number of live fetuses and

percentage of males in the litter (sex ratio). Two-sample t-tests were performed to determine

whetherif fetal sex affectedinfluenced any of the parameters investigated. A restricted maximum

likelihood (REML) variance component analysis was performed with gilt fitted as a random effect to

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Publisher: CSIRO; Journal: RD:Reproduction, Fertility and Development Article Type: Research Paper; Volume: ; Issue: ; Article ID: RD17298

DOI: 10.1071/RD17298; TOC Head: identify the effectsinfluence of fetal weight on FHR, PSV, EDV, A/B ratio and RI. The difference in

time from the first fetal trace measurement to each subsequent trace measured was calculated within

gilt. The results were grouped by GD and gilt was fitted as a random effect in an REML variance

component analysis, to account for the variation attributed to gilt within GD. Similarly, two-way

ANOVA (GD × treatment (SSR or control)) with a block for gilt was performed to compare the

number of live fetuses, the percentage of males in the litter and the s.d. in fetal weight in SSR and

control litters. The weight of the SSR and control fetuses were compared within GD (Mann–Whitney

at GD60; ANOVA with gilt block at GD90) and fetal weight was then compared within sex (two-way

ANOVA (sex × treatment) with gilt block). In all cases, two-sided PP values of ≤ 0.05 were was

considered to be statistically significant. Data are presented as the mean ± s.e.m.

Results

Experiment 1: temporal changes in FHR, PSV, EDV, RI and A/B ratio were observed

UA waveforms were obtained at all GD studied (Fig. 1). Temporal changes were observed in all

parameters investigated, with a decrease in FHR, A/B ratio and RI (all FPr/P ≤ 0.001), and an

increase in PSV and EDV (all P ≤ 0.001; Fig. 2) with advancing gestation. FHR gradually

decreased gradually with advancing gestation however, but the largest differences in the other

parameters occurred between GD60 and GD90; , reflecting the presence of diastolic blood flow at

GD90 but not at earlier GD.

Experiment 2: effects of lLitter sex ratio but and not litter size influences on UA blood flow

The number of fetuses in the litter or right uterine horn did not affectinfluence any of the

parameters investigated within GD (Table 2). A negative relationship between the percentage of

males in the litter and EDV at GD30 (P = 0.077) and GD60 (PP ≤ 0.05) was observed. In

additionAdditionally, a positive relationship was observed at GD30 between the percentage of males

in the litter with and both the A/B ratio (P ≤ 0.05) and RI (P ≤ 0.05) was observed at GD30.

Experiment 3: effects of fFetal weight but not fetaland sex influences on UA blood flow

In four of the GD90 litters, UA waveforms were obtained from all fetuses in the right uterine horn,

allowing relationships between UA parameters and both fetal size and sex to be explored. FHR was

did not differ significantly between not statistically different between male (n = 16) and female (n =

10) fetuses (mean ± s.e.M; 135.0 ± 3.0 bpm. n = 16) and females (132.9 ± 3.2 b.p.m. respectively; n =

10) (Fig. 3Aa). Similarly, no differences were observed between male (n = 16) and female (n = 9)

fetusesalterations in PSV (69.29 ± 6.54 and 81.45 ± 7.88 cm s–1 respectively) or EDV was observed

between males (69.29 ± 6.54 cm/s and( 15.03 ± 2.24 cm/s respectively. n = 16) and females (81.45 ±

7.88 and 16.54 ± 1.88 cm s–1 respectively. n = 9) (; Fig. 3Bb and, Cc). Sex did not affectinfluence the

A/B ratio (males: 5.62 ± 0.62 , n = 16. females:and 5.12 ± 0.45 in male (n = 16) and female (n = 9)

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Publisher: CSIRO; Journal: RD:Reproduction, Fertility and Development Article Type: Research Paper; Volume: ; Issue: ; Article ID: RD17298

DOI: 10.1071/RD17298; TOC Head: fetuses respectively, n = 9) or RI (males: 0.79 ± 0.02, n = 16. and females: 0.79 ± 0.02 respectively; )

(Fig. 3Dd and, Ee).

Fetal weight was negatively related to FHR (P = 0.089), A/B ratio (PP ≤ 0.05) and RI (P ≤P

0.05).

Experiment 4: effects of sSedation influences on FHR and later fetal development

A significant decrease in FHR over the sedation period was observed at GD60 and GD90 (P ≤ P

0.001), but not at GD30 or GD45 (Fig. 4). The mean decrease in FHR ± s.e.M. at GD30, GD45,

GD60 and GD90 was 8.7 ± 4.6 bpm, 5.3 ± 6.2 bpm, 18.4 ± 5.7 bpm and 20.6 ± 10.3 b.p.m.

respectively. The SSR process at GD30 and GD45 did not affectinfluence the number of live fetuses

or the sex ratio of the litter at GD60 and GD90 , when compared with control fetuses whose dams had

not previously been sedated previously. Intriguingly, fetal weight was significantly lower in GD60

SSR fetuses compared with control GD60 fetuses (P ≤ P 0.001; Fig. 5Aa and, Cc). A similar trend

was observed at GD90 (P = 0.061; Fig. 5Bb and, Dd). Analysis of the s.d. in fetal weight within the

litter revealed a lower s.d. in SSR litters when compared with controls at GD90, but not at GD60 (P ≤

P 0.05; Fig. 5Ee).

Discussion

The broad objective of the presentthis study was to determine whetherif non-invasive Doppler

ultrasound could be used to monitor UA blood flow in pregnant gilts under light sedation. To our

knowledge, this is the first report of the use of Doppler ultrasound at this early stage of gestation in

the pig. This method was successfully used to obtain traces at all GD analysed, and highlighted novel

temporal changes in all parameters investigated. The length of the sedation period affectedinfluenced

FHR at GD60 and GD90, but not earlier in gestation, suggesting that fetuses in late gestation may be

more affected by maternal sedation, albeit with modest numbers. Pregnant gilts were subjected to an

SSR procedure to allow measurements on two GD within the same litter. Interestingly, the most

striking finding from the presentthis study was that approximately ~30 min of light sedation in early

gestation decreased fetal weight in later gestation, highlighting surprising potential long-term

programming effects thatwhich warrant further investigation.

Similar temporal changes in UA blood flow parameters have been reported in sheep and goats

(Serin et al. 2010; Carr et al. 2012). The two published studies describing pig UA blood flow

parameters (Brüssow et al. 2012; Harris et al. 2013) both reported a decrease in FHR as gestation

proceedsproceeded, but only Harris et al. (2013) observed temporal changes in RI and PI between

GD39 and GD81. Changes in FHR observed in both studies follow a similar pattern to those seen in

the presentcurrent study however, but not all parameters studied exhibited the same temporal trends in

the three studies. The three experiments were performed under different ing sedation conditions, with

Harris et al. (2013) using non-sedated gilts, ‘rubbing’ the udder of the gilts to produce a ‘calming’

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Publisher: CSIRO; Journal: RD:Reproduction, Fertility and Development Article Type: Research Paper; Volume: ; Issue: ; Article ID: RD17298

DOI: 10.1071/RD17298; TOC Head: effect in the animals, whereaswhilst Brüssow et al. (2012) performed the procedure by scanning the

exposed uterus directly while the pig was under general anaesthesia. Comparison of the mean FHR

values reported in these studies with the values obtained in the presentcurrent experiment suggests

that increasing the level of sedation decreases the mean FHR observed at all stages of gestation

studied.

The sex ratio of pig litters is an area of significant interest to the pig industry, with gilts from

female-biased litters having an increased number of teats, a higher fertility rate and an increased

conception rate on their first breeding attempt than gilts from male-biased litters (Drickamer et al.

1997; Górecki, 2003). It has also been proposed that male piglets are disadvantaged, with male piglets

being crushed by the sow more than females, more males dying from disease-related causes than

females, and males having impaired thermoregulation compared with females (Baxter et al. 2012). In

the presentcurrent study, the percentage of males in the litter affectedinfluenced EDV, the A/B ratio

and RI, highlighting that the sex ratio of the litter could affectinfluence UA blood flow. Recent studies

have revealed that sexual dimorphism exists in human placentas (Adibi et al. 2017; for a review,

seereviewed by Kalisch-Smith et al. 2017; Adibi et al. 2017). In additionAdditionally, it has been

shown that male babies have increased perinatal mortality and morbidity when human pregnancy is

complicated by pre-eclampsia and IUGR (Challis et al. 2013; Muralimanoharan et al. 2013).

Similarly, fetal sex has been shown to affectinfluence the expression of placental genes and the

inflammatory response in humans, which is thought to be attributed to sexual dimorphism in placental

function (Ghidini and Salafia, 2005; Challis et al. 2013). It has also been shown in the pig that

placental and endometrial gene expression and vascularity are associated with both fetal size and sex

(C. Stenhouse, C. O. Hogg and C. J. Ashworth, unpubl. obs.ished). Widnes et al. (2017) reported that

in humans, UA PI is increased in females compared with males, although, these authors y did not

suggest that fetal sex affectedinfluenced any of the other parameters investigated. Therefore, if

differences are observed in placental and endometrial tissue supplying fetuses of different sex, this

may indicate that the uterine environment of a male-biased litter would be very different to that of a

female-biased litter, which may explain the relationships between the sex ratio of the litter and the UA

blood flow parameters measured in the presentthis study.

Surprisingly, light sedation in early gestation decreased fetal weight in later gestation. This,

alongside the decrease in FHR over the sedation period, suggests that sedation during gestation could

have significant effects on fetal development. The mechanisms behind this decrease in fetal weight

warrant much further investigation. It has previously been suggested that azaperone has a vasodilatory

effect, and can therefore result in a decrease in blood pressure and body temperature (Van Woerkens

et al. 1990; Hall and Clarke, 1991; Brodbelt and Taylor, 1999; Hodgkinson, 2007). Although care was

taken to ensure that the gilts were kept warm throughout the process, temperature and blood pressure

were not monitored in the presentcurrent study. In addition, there is some evidence suggesting that

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Publisher: CSIRO; Journal: RD:Reproduction, Fertility and Development Article Type: Research Paper; Volume: ; Issue: ; Article ID: RD17298

DOI: 10.1071/RD17298; TOC Head: azaperone can promote activation of the hypothalamic–us pituitary– adrenal cortex (HPA) axis. Daş

et al. (2016) investigated the effects of azaperone- and azaperone plus ketamine- induced general

anaesthesia on plasma cortisol levels in pigs. Administration of azaperone led to a prolonged and

significant increase in plasma cortisol within 45 min of drug administration for the remaining 255 min

of the experiment, and this effect was increased further with the addition of ketamine (Daş et al.

2016). This may indicate that cortisol mediates the observed physiological effects of azaperone,

suggesting that azaperone may promote an increased stress response in pigs.

Considering this information, it could be hypothesised that sedation with azaperone and ketamine

promoted a decrease in body temperature and blood pressure, activating the maternal HPA axis and

increasing cortisol production by the adrenal cortex. Although cortisol is metabolised by 11β-

hydroxysteroid dehydrogenase 2 into inactive corticosterone at the placenta from GD24 in the pig

(Klemcke, 2003), not all of the available cortisol will be converted into inactive corticosterone,

leading to an increase in the levels of maternal cortisol crossing the placenta to affect the fetus. This

would ultimately act as a stressful environment for the fetus, leading to the observed decreased fetal

weight in late gestation, and potentially having effects postnatally. Interestingly, it has previously

been suggested that there is an inverse relationship between fetal plasma cortisol and fetal size

(Klemcke and Christenson, 1997), indicating that lighter fetuses may have a higher baseline stress

response. To test the proposed mechanism of action in the future, it would be useful to monitor

maternal rectal and surface temperature, blood pressure and salivary cortisol at regular intervals

throughout the Doppler process.

In conclusion, we have demonstrated that Doppler ultrasound can be used to non-invasively

monitor UA blood flow in the pregnant pig from GD30. Our results have indicated, for the first time,

that fetal weight and the sex ratio of the litter could affectinfluence UA blood flow in the pig.

Intriguingly, sedation with azaperone and ketamine had a significant effect on fetal development; the

mechanisms and long-term effects of this warrant much further investigation and could have a clinical

relevance. It is hoped that further optimisation of this techniqueDoppler ultrasound under light

sedation would allow evaluation of UA blood flow parameters in relation to fetal growth and

development, especially in IUGR fetuses.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

The authorsWe thank the staff of The Roslin Institute Large Animal Unit for skilled assistance,

Professor Eddie Clutton for helpful discussions and advice and Dr Darren Shaw for statistical advice.

The Roslin Institute receives Institute Strategic Grant funding from the Biotechnology and Biological

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DOI: 10.1071/RD17298; TOC Head: Sciences Research Council (BBSRC) (BBS/E/D/30002276). C. Stenhouse CS is in receipt of a

studentship from the University of Edinburgh.

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DOI: 10.1071/RD17298; TOC Head: doi:10.1631/jzus.B1500010 </jrn></jrn>

Manuscript received 28 July 2017, accepted 28 March 2018

Fig. 1. Representative umbilical arterial waveforms produced at Gestational Day (GD) 30, GD45, GD60 and

GD90. Pregnant gilts were sedated and non-invasive Doppler ultrasound was performed on umbilical arteries.

Representative Doppler ultrasound images were obtained using the Philips iU22 colour Doppler ultrasound

machine at GD30 (top left), 45 (top right), 60 (bottom left) and 90 (bottom right). Abbreviations: GD =

Gestational Day.

Fig. 2. Temporal changes were observed in umbilical arterial blood flow parameters. Non-invasive Doppler

ultrasound was performed on sedated pregnant gilts at Gestational Day (GD) 30, GD45, GD60 and GD90.

GD30, 45, 60 and 90. Mean values for each parameter were calculated for each image to give a mean value per

fetus. These values were then used to calculate the mean within each GD. Data show the mean ± s.e.m. (a(A)

Fetal heart rate (FHR) decreased with advancing gestation (ANOVA with block for gilt;. dDifferent ing letters

signify indicate significant differences in mean values between groups determined by Tukey analysis). (b(B)

Peak systolic velocity ( PSV) and (c(C) end diastolic velocity (EDV) increased with advancing gestation

(Kruskal–Wallis). (d(D) The A/B ratio (PSV : EDV) and (Ee) resistance index (RI) decreased with advancing

gestation (Kruskal–Wallis). * PP ≤ 0.05,. ***P ≤ 0.001. Error bars represent s.e.m. Abbreviations: FHR =

Fetal Heart Rate; bpm = beats per minute; PSV = Peak Systolic Velocity; EDV = end diastolic velocity; A/B

Ratio = Ratio of PSV to EDV; RI = resistance index; GD = Gestational Day; s.e.m. = Standard Error of the

Mean.

Fig. 3. Fetal sex did not influence affect (a) fetal heart rate (FHR)FHR, (b) peak systolic velocity (PSV), (c)

end diastolic velocity (EDV), (d) the A/B ratio (PSV : EDV) or (e) the resistance index PSV, EDV, RI or A/B

Ratio at Gestational Day GD90. Mean values for each parameter were calculated for each image to give a mean

value per fetus. These values were used to calculate the mean within each sex. Data show the mean ± s.e.m.(A)

FHR, (B) PSV, (C) EDV, (D) A/B ratio, and (E) RI were not influenced by the sex of the fetus. Error bars

represent s.e.m. Abbreviations: FHR = Fetal Heart Rate; bpm = beats per minute; PSV = Peak Systolic Velocity;

EDV = End Diastolic Velocity; A/B Ratio = Ratio of SV to EDV; RI = Resistance Index; GD = Gestational

Day; s.e.m. = Standard Error of the Mean.

Fig. 4. Mean fetal heart rate (FHR) over the sedation period within gestational day (GD):GD. A:(a) GD30, B:

(b) GD45, (C:c) GD60, and D:(d) GD90. Pregnant gilts were sedated and non-invasive Doppler ultrasound was

performed on umbilical arteries. To determine whetherif the sedation process had an effect on fetal heart

rateFHR, time since the first fetal trace was measured and plotted against FHR. The results were grouped by

GD, as illustratedshown, and gilt was fitted as a random effect in a restricted maximum likelihood

(REML)REML variance component analysis to account for the variation attributed to gilt within GD. A: GD30,

B: GD45, C: GD60, and D: GD90. Each symbol represents a different gilt. Abbreviations used: GD =

Gestational Day; FHR = Fetal Heart Rate; bpm = beats per minute; h = hours; min = minutes.

Fig. 5. (a–d) Fetal weight and (e) the s.d. in of fetal weight in litters thatwhich had been sedated in early

gestation and control litters. Non-invasive Doppler ultrasound was performed on sedated pregnant gilts at

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DOI: 10.1071/RD17298; TOC Head: Gestational Day (GD) GD30 and GD45. Following this, the gilts were allowed to recover (sedated, scanned and

recovered; = SSR) before undergoing the procedure for a second time in late gestation (GD60 or GD90) as a

terminal procedure. The weight of the control (black) and SSR (grey) fetuses were was compared within GD

(Mann–Whitney at GD60; ANOVA with gilt block at GD90) and the fetal weight was then compared within sex

(two-way ANOVA (sex × treatment) with gilt block). In all cases, P values of ≤ 0.05 were was considered to

be statistically significant. Control litters were identified as those thatwhich had not been subjected to the SSR

procedure. (a, b(A) Fetal weight at GD60 (a) . (B) Fetal weight at and GD90 (b). (Cc, d) Fetal weight at GD60

within sex(c) and . (D) Fetal weight at GD90 (d) within sex. (E) s.d. in fetal weightData are the mean ± s.e.m. (.

n = 8 and 4 control and SSR GD60 litters respectively;. n = 6 and 3 control and SSR GD90 litters respectively).

Mean values presented and the error bars represent s.e.m. *PP ≤ 0.05, ** P ≤ P 0.01, *** P ≤ P 0.001.

Abbreviations used: SSR = Sedate, Scan, Recovery; GD = Gestational Day; s.d. = Standard Deviation; s.e.m. =

Standard Error of the Mean.

Table 1. Measurements of umbilical arterial each parameters per gestational day

Unless indicated otherwise, data show mean values, with the range in parentheses. Abbreviations: GD

= Gestational Day; FHR, = fetal heart rate; bpm = beats per minute; PSV, = peak systolic velocity;

EDV, = end diastolic velocity; A/B ratio, = Ratio of PSV :  to EDV ratio; RI, = resistance index

GD Parameter (units) Number ofNo. fetuses

Number ofNo. litters

Number ofNo. waveforms per /Ffetus (mean;

range)

Number ofNo. fetuses per

/litter (mean; range)

GD30 FHR (b.p.m.) 18 7 5.78 (1–165.78; 1–16)

2.57 (1–42.57; 1–4)

30 PSV (cm /s–1 ) 18 7 6.06 (1–176.06; 1–17)

2.57 (1–42.57; 1–4)

30 EDV (cm s–1 /s) 15 7 6.06 (1–176.06; 1–17)

2.14 (1–42.14; 1–4)

30 A/B ratio 18 7 6.06 (1–176.06; 1–17)

2.57 (1–42.57; 1–4)

30 RI 17 7 6.06 (1–176.06; 1–17)

2.43 (1–42.43; 1–4)

GD45 FHR (b.p.m.)FHR (bpm)

39 9 7.15 (2–157.15; 2–15)

4.33 (2–74.33; 2–7)

45 PSV (cm s–1 )PSV (cm/s)

39 9 7.69 (2–167.69; 2–16)

4.33 (2–74.33; 2–7)

45 EDV (cm s– 1 )EDV (cm/s)

35 9 7.69 (2–167.69; 2–16)

3.89 (1–73.89; 1–7)

45 A/B ratioA/B Ratio

39 9 7.69 (2–167.69; 2–16)

4.33 (2–74.33; 2–7)

45 RIRI 37 9 7.69 (2–167.69; 2–16)

4.11 (1–74.11; 1–7)

GD 60 FHR (b.p.m.)FHR (bpm)

29 5 8.07 (1–228.07; 1–22)

6.00 (5–76.00; 5–7)

60 PSV (cm s–1 )PSV (cm/s)

31 5 8.29 (3–228.29; 3–22)

6.20 (5–76.20; 5–7)

60 EDV (cm s– 1 )EDV (cm/s)

29 5 8.29 (3–228.29; 3–22)

5.80 (4–75.80; 4–7)

60 A/B ratioA/B Ratio

30 5 8.29 (3–228.29; 3–22)

6.00 (4–76.00; 4–7)

60 RIRI 30 5 8.29 (3–228.29; 3–22)

6.00 (5–76.00; 5–7)

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Publisher: CSIRO; Journal: RD:Reproduction, Fertility and Development Article Type: Research Paper; Volume: ; Issue: ; Article ID: RD17298

DOI: 10.1071/RD17298; TOC Head: GD90 FHR (b.p.m.)FHR

(bpm)45 9 6.73 (1–196.73;

1–19)6.00 (3–86.00;

3–8)90 PSV (cm s–1 )PSV

(cm/s)48 7 7.11 (1–237.11;

1–23)7.00 (5–

107.00; 5–10)90 EDV (cm s–

1 )EDV (cm/s)50 7 7.11 (1–237.11;

1–23)7.00 (5–

107.00; 5–10)90 A/B ratioA/B

Ratio55 9 7.11 (1–237.11;

1–23)6.11 (3–

106.11; 3–10)90 RIRI 55 9 7.11 (1–237.11;

1–23)6.11 (3–

106.11; 3–10)

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Publisher: CSIRO; Journal: RD:Reproduction, Fertility and Development Article Type: Research Paper; Volume: ; Issue: ; Article ID: RD17298

DOI: 10.1071/RD17298; TOC Head: Table 2. Summary of linear regressions performed to investigate the relationship between total number of fetuses, total number of fetuses in the

right uterine horn and percentage of males in the litter

Abbreviations: FHR = Fetal Heart Rate; PSV = Peak Systolic Velocity; EDV = End Diastolic Velocity; A/B Ratio = Ratio of PSV to EDV; RI = Resistance

Index. P -values of ≤ 0.1 are indicated in bolded. GD, gestation day; FHR, fetal heart rate; PSV, peak systolic velocity; EDV, end diastolic velocity; A/B

ratio, PSV : EDV ratio; RI, resistance index; RSq, R-squared.xxx

Gestational Day

Parameter Total Number ofno. fetuses in uterus

Total Number ofno. fetuses in right uterine horn Percentage of% Males in the litter

RSq (%)

PP- Vvalue

Number ofNo.

oObservations

RSq (%) P-valueP Value

No. observationsNumber

of Observations

RSq (%) P-valueP Value

No. observationsNumber

of ObservationsGD30 FHR 0 0.542 3 12.3 0.461 3 0 0.767 330 PSV 0 0.787 3 0 0.868 3 0 0.095 330 EDV 0 0.614 3 0 0.696 3 97.1 0.077 330 A/B ratio 0 0.653 3 0 0.734 3 99.3 0.038 330 RI 0 0.640 3 0 0.721 3 98.7 0.051 3GD45 FHR 30.1 0.151 6 0 0.574 6 0 0.896 645 PSV 0 0.939 6 0 0.848 6 0 0.896 645 EDV 0 0.436 6 0 0.665 6 0 0.479 645 A/B ratio 0 0.640 6 0 0.844 6 0 0.630 645 RI 18.6 0.217 6 0 0.426 6 0 0.726 6GD60 FHR 11 0.309 5 11.8 0.304 5 0 0.485 560 PSV 0 0.474 5 0 0.909 5 0 0.449 560 EDV 0 0.683 5 0 0.975 5 82.4 0.021 560 A/B ratio 0.2 0.390 5 0 0.826 5 0.6 0.386 560 RI 0 0.774 5 0 0.628 5 14.9 0.283 5GD90 FHR 4 0.286 9 0 0.572 9 0 0.923 990 PSV 0 0.603 9 0 0.587 9 22.7 0.110 990 EDV 0 0.558 9 0 0.507 9 25.2 0.096 990 A/B ratio 0 0.927 9 0 0.616 9 9.2 0.221 990 RI 6.5 0.253 9 7.4 0.241 9 0 0.663 9Combined FHR 4.2 0.176 23 1.6 0.258 23 1.6 0.257 23Combined PSV 0.5 0.302 23 0.1 0.325 23 9.1 0.088 23Combined EDV 2.41 0.227 23 2.1 0.239 23 11.9 0.059 23Combined A/B ratio 2.41 0.230 23 0 0.363 23 0 0.796 23Combined RI 12.8 0.052 23 10.6 0.071 23 1.7 0.254 23

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Publisher: CSIRO; Journal: RD:Reproduction, Fertility and Development Article Type: Research Paper; Volume: ; Issue: ; Article ID: RD17298

DOI: 10.1071/RD17298; TOC Head:

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