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: [email protected]
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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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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