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Background: Huntington’s disease (HD) is a fatal neurodegenerative autosomal dominant disorder with prevalence of 1 : 20000 that has no effective treatment to date. Translatability of candidate therapeutics could be enhanced by additional testing in large animal models because of similarities in brain anatomy, size, and immunophysiology. These features enable realistic pre-clinical studies of biodistribution, efficacy, and toxicity. Objective and Methods: Here we non-invasively characterized alterations in brain white matter microstructure, neurochemistry, neurological status, and mutant Huntingtin protein (mHTT) levels in cerebrospinal fluid (CSF) of aged OVT73 HD sheep. Results: Similar to HD patients, CSF mHTT differentiates HD from normal sheep. Our results are indicative of a decline in neurological status, and alterations in brain white matter diffusion and spectroscopy metric that are more severe in aged female HD sheep. Longitudinal analysis of aged female HD sheep suggests that the decline is detectable over the course of a year. In line with reports of HD human studies, white matter alterations in corpus callosum correlates with a decline in gait of HD sheep. Moreover, alterations in the occipital cortex white matter correlates with a decline in clinical rating score. In addition, the marker of energy metabolism in striatum of aged HD sheep, shows a correlation with decline of clinical rating score and eye coordination. Conclusion: This data suggests that OVT73 HD sheep can serve as a pre-manifest large animal model of HD providing a platform for pre-clinical testing of HD therapeutics and non-invasive tracking of the efficacy of the therapy.

Gene therapy continues to be a promising contender for the treatment of cystic fibrosis (CF) airway disease. We have previously demonstrated that airway conditioning with lysophosphatidylcholine (L...

Gene therapy continues to be a promising contender for the treatment of cystic fibrosis (CF) airway disease. We have previously demonstrated that airway conditioning with lysophosphatidylcholine (LPC) followed by delivery of a HIV-1-based lentiviral (LV) vector functionally corrects the CF transmembrane conductance regulator (CFTR) defect in the nasal airways of CF mice. In our earlier pilot study we showed that our technique can transduce marmoset lungs acutely; this study extends that work to examine gene expression in this nonhuman primate (NHP) 1 month after gene vector treatment. A mixture of three separate HIV-1 vesicular stomatitis virus G (VSV-G)-pseudotyped LV vectors containing the

Preclinical imaging studies of fetal hemodynamics require anesthesia to immobilize the animal. This may induce cardiovascular depression and confound measures under investigation. We compared the impact of four anesthetic regimes upon maternal and fetal blood gas and hemodynamics during baseline periods of normoxia, and in response to an acute hypoxic challenge in pregnant sheep. Merino ewes were surgically prepared with maternal and fetal vascular catheters and a fetal femoral artery flow probe at 105–109 days gestation. At 110–120 days gestation, ewes were anesthetized with either isoflurane (1.6%), isoflurane (0.8%) plus ketamine (3.6 mg·kg−1·h−1), ketamine (12.6 mg·kg−1·h−1) plus midazolam (0.78 mg·kg−1·h−1), propofol (30 mg·kg−1·h−1), or remained conscious. Following 60 min of baseline recording, nitrogen was administered directly into the maternal trachea to displace oxygen and induce maternal and thus fetal hypoxemia. During normoxia, maternal PaO2 was ~30 mmHg lower in anesthetized ewes compared to conscious controls, regardless of the type of anesthesia (p .05), but heart rate was 32 ± 8 bpm lower in fetuses from ewes administered isoflurane (p = .044). During maternal hypoxia, fetal MAP increased, and peripheral blood flow decreased in all fetuses except those administered propofol (p, Preclinical imaging studies of fetal haemodynamics require anaesthesia to immobilise the animal. In the presence of isoflurane, isoflurane/ketamine and ketamine/midazolam, but not propofol anaesthesia, maternal and fetal hypoxaemia increased fetal mean arterial pressure and decreased peripheral blood flow. Acute fetal hypoxaemia induced fetal tachycardia regardless of anaesthetic type. These results have implications for the design and interpretation of preclinical imaging studies where anaesthesia is required.

Pre-existing neutralizing antibody (NAb) against adeno-associated virus (AAV) commonly found in primates is a major host barrier that can severely compromise in vivo gene transfer by AAV vectors. To achieve proof-of-concept success in clinical development of recombinant AAV (rAAV)-based in vivo gene therapy, it is crucial to consider the potential interference of NAb and to enroll serologically compatible study subjects. In this study, we report a large AAV NAb dataset comprising multiple large animal species and AAV serotypes and compare two NAb assays based on in vitro or in vivo transduction inhibition, respectively. Together with previously published AAV seroepidemiology studies, these data can serve as a reference for selecting suitable serotypes, study subjects of large animal species, and potentially human patients for rAAV treatment. In addition, we modeled the intrathalamus rAAV9 delivery in the presence of circulating anti-AAV9 NAb generated by either pre-immunization or passive transfer of NAb-positive large animal serum to mice. The data showed that circulating NAb may not be the sole determinant to inhibit brain transduction. Other aspects of pre-existing AAV immunity following natural infection or rAAV administration may be further studied to establish a more accurate inclusion criterion for clinical studies employing intraparenchymal rAAV9 injections. Keywords: adeno-associated virus, CNS gene transfer, gene therapy, large animals, neutralizing antibody

The primary metabolic pathway required to produce ATP differs as a result of tissue type, developmental stage and substrate availability.We utilized molecular and histological techniques to define the metabolic status in foetal and adult, adipose and skeletal muscle tissues. Redox ratios of these tissues were also determined optically by two-photon microscopy.Adult perirenal adipose tissue had a higher optical redox ratio than fetal perirenal adipose tissue, which aligned with glycolysis being used for ATP production;whereas adult skeletal muscle had a lower optical redox ratio than fetal skeletal muscle, which aligned with oxygen demanding oxidative phosphorylation activity being utilized for ATP production. We have compared traditional molecular and microscopy techniques of metabolic tissue characterization with optical redox ratios to provide a more comprehensive report on the dynamics of tissue metabolism. Refereed/Peer-reviewed

Sepsis remains a significant health burden and a major clinical need exists for therapeutics to dampen the excessive and uncontrolled immune activation. Nuclear protein high mobility group box protein 1 (HMGB1) is released following cell death and is a late mediator in sepsis pathogenesis. While approaches targeting HMGB1 have demonstrated reduced mortality in pre-clinical models of sepsis, the impact of HMGB1 blockade on the complex septic inflammatory milieu and the development of subsequent immunosuppression remain enigmatic. Analysis of plasma samples obtained from septic shock patients established an association between increased HMGB1 and non-survival, higher APACHE II scores, and increased pro-inflammatory cytokine responses. Pre-clinically, administration of neutralising ovine anti-HMGB1 polyclonal antibodies improved survival in murine endotoxaemia and caecal ligation and puncture-induced sepsis models, and altered early cytokine profiles to one which corresponded to patterns observed in the surviving patient cohort. Additionally, anti-HMGB1 treated murine sepsis survivors were significantly more resistant to secondary bacterial infection and exhibited altered innate immune cell phenotypes and cytokine responses. These findings demonstrate that anti-HMGB1 antibodies alter inflammation in murine sepsis models and reduce sepsis mortality without potentiating immunosuppression.

This study investigates the effect of contaminant zinc (Zn) on lead (Pb) bioavailability and bioaccessibility in six contrasting soils spiked with 1500 mg Pb/kg and aged 12 months under laboratory conditions. Zn was added to the soils (7500 mgZn/kg soil) and aged for a further two weeks. In vivo studies were conducted using juvenile swine as a surrogate model for young children. Two compartment pharmacokinetic models were used to analyze the biological response produced by Pb oral solution and spiked soils. Absolute and relative bioavailability of Pb in soils (oral dose of 100 μ g Pb/kg body weight/day) were estimated by comparing them with intravenously administered soluble Pb salt (25 μ g Pb/kg/day) and orally administered the same Pb salt [Pb acetate =(CH3COO)2Pb⋅3H2O] administered to 3 juvenile pigs per treatment. Lead bioaccessibility was calculated using the in vitro RBALP (i.e. relative bioaccessibility leaching procedure) method. The in vitro results of RBALP were compared to in vivo relative Pb bioavailability to ascertain whether the changes in bioaccessibility correlated with the in vivo data. Although the in vivo Pb relative bioavailability (RB) in all soils except in MLA (Mount Lofty Acidic) revealed an increase (18%–159%) in the presence of Zn, the in vitro RBALP bioaccessibility results indicated otherwise (1%–38% decrease). In vivo RB of Pb in MLA declined by 37% in the presence of Zn. However, the RBALP in vitro bioaccessible Pb did not correlate with the relative bioavailabilities of Pb in the juvenile swine dosing experiment. Caution is therefore needed when predicting Pb bioavailability/bioaccessibility in the presence of metal mixtures. The literature contains much information on the correlation of metal and metalloid bioaccessibility with their bioavailability. There is, however, a paucity of studies investigating the effects of other metals on Pb and their IVIVC (in vitro and in vivo correlations). The current study addresses this knowledge gap by assessing in vivoand in vitro bioavailability of Pb in the presence of Zn.

Sepsis is a highly complex and often fatal syndrome which varies widely in its clinical manifestations, and therapies that target the underlying uncontrolled immune status in sepsis are needed. The failure of preclinical approaches to provide significant sepsis survival benefit in the clinic is often attributed to inappropriate animal disease models. It has been demonstrated that high mobility group box protein 1 (HMGB1) blockade can reduce inflammation, mortality and morbidity in experimental sepsis without promoting immunosuppression. Within this study, we explored the use of ovine anti-HMGB1 antibodies in a model of ovine septic shock incorporating intensive care supports (OSSICS). Results: Septic sheep exhibited elevated levels of HMGB1 within 12 h after the induction of sepsis. In this study, sepsis was induced in six anaesthetized adult Border Leicester × Merino ewes via intravenous instillation of E. coli and sheep monitored according to intensive care unit standard protocols for 26 h, with the requirement for noradrenaline as the primary endpoint. Septic sheep exhibited a hyperdynamic circulation, renal dysfunction, deranged coagulation profile and severe metabolic acidosis. Sheep were assigned a severity of illness score, which increased over time. While a therapeutic effect of intravenous anti-HMGB1 antibody could not be observed in this model due to limited animal numbers, a reduced bacterial dose induced a septic syndrome of much lower severity. With modifications including a reduced bacterial dose, a longer timeframe and broad spectrum antibiotics, the OSSICS model may become a robust tool for preclinical assessment of sepsis therapeutics.

Global gene delivery to the CNS has therapeutic importance for the treatment of neurological disorders that affect the entire CNS. Due to direct contact with the CNS, cerebrospinal fluid (CSF) is an attractive route for CNS gene delivery. A safe and effective route to achieve global gene distribution in the CNS is needed, and administration of genes through the cisterna magna (CM) via a suboccipital puncture results in broad distribution in the brain and spinal cord. However, translation of this technique to clinical practice is challenging due to the risk of serious and potentially fatal complications in patients. Herein, we report development of a gene therapy delivery method to the CM through adaptation of an intravascular microcatheter, which can be safely navigated intrathecally under fluoroscopic guidance. We examined the safety, reproducibility, and distribution/transduction of this method in sheep using a self-complementary adeno-associated virus 9 (scAAV9)-GFP vector. This technique was used to treat two Tay-Sachs disease patients (30 months old and 7 months old) with AAV gene therapy. No adverse effects were observed during infusion or post-treatment. This delivery technique is a safe and minimally invasive alternative to direct infusion into the CM, achieving broad distribution of AAV gene transfer to the CNS.

Background: There are many uncertainties concerning variations in benzo[a]pyrene (B[a]P) soil guidelines protecting human health based on carcinogenic data obtained in animal studies. Although swine is recognised as being much more representative of the human child in terms of body size, gut physiology and genetic profile the rat/mice model is commonly used in practice. Objectives: We compare B[a]P bioavailability using a rat model to that estimated in a swine model, to investigate the correlation between these two animal models. This may help reduce uncertainty in applying bioavailability to human health risk assessment. Methods: Twelve spiked soil samples and a spiked silica sand (reference material) were dosed to rats in parallel with a swine study. B[a]P bioavailability was estimated by the area under the plasma B[a]P concentration-time curve (AUC) and faecal excretion as well in the rats. Direct comparison between the two animal models was made for: firstly, relative bioavailability (RB) using AUC assay; and secondly, the two assays in the rat model. Results: Both AUC and faecal excretion assays showed linear dose-response for the reference material. However, absolute bioavailability was significantly higher when using faecal excretion assay (p

Huntington's disease (HD) is a fatal neurodegenerative disease caused by a genetic expansion of the CAG repeat region in the huntingtin (HTT) gene. Studies in HD mouse models have shown that artificial miRNAs can reduce mutant HTT, but evidence for their effectiveness and safety in larger animals is lacking. HD transgenic sheep express the full-length human HTT with 73 CAG repeats. AAV9 was used to deliver unilaterally to HD sheep striatum an artificial miRNA targeting exon 48 of the human HTT mRNA under control of two alternative promoters: U6 or CβA. The treatment reduced human mutant (m) HTT mRNA and protein 50-80% in the striatum at 1 and 6 months post injection. Silencing was detectable in both the caudate and putamen. Levels of endogenous sheep HTT protein were not affected. There was no significant loss of neurons labeled by DARPP32 or NeuN at 6 months after treatment, and Iba1-positive microglia were detected at control levels. It is concluded that safe and effective silencing of human mHTT protein can be achieved and sustained in a large-animal brain by direct delivery of an AAV carrying an artificial miRNA.

The heart has high metabolic demand to maintain function. The primary source of energy supply to support correct contractile muscle function differs between a fetus and an adult. In fetal life, ATP is primarily generated by glycolysis and lactate oxidation, whereas following birth, there is a shift towards a reliance on mitochondrial metabolism and fatty acid oxidation. This change in metabolic status is an adaptation to different fuel availability, oxygenation and growth patterns. In this study, we have employed 2-photon excitation fluorescence microscopy to define the relationship between two critical metabolic cofactors nicotinamide adenine dinucleotide(P)H and flavin adenine dinucleotide, effectively utilizing a redox ratio to differentiate between the metabolic status in fetal (proliferative) and adult (quiescent/hypertrophic) hearts. Two-photon imaging was also used to visually confirm the known increase in collagen deposition in the adult heart. The changes observed were consistent with a hypertrophic growth profile and greater availability of fatty acids in the adult heart, compared to the proliferative fetal heart. Two-photon excitation fluorescence microscopy is therefore a convenient imaging technology that enables the monitoring of striated muscle architecture and the metabolic status of heart tissue. This imaging technology can potentially be employed to visualize cardiac and other muscle pathologies. Refereed/Peer-reviewed

Soil plays a significant role in controlling the potential bioavailability of contaminants in the environment. In this study, eleven soils were used to investigate the relationship between soil properties and relative bioavailability (RB) of lead (Pb). To minimise the effect of source of Pb on in vivo bioavailability, uncontaminated study soils were spiked with 1500 mg Pb/kg soil and aged for 10-12 months prior to investigating the relationships between soil properties and in vivo RB of Pb using swine model. The biological responses to oral administration of Pb in aqueous phase or as spiked soils were compared by applying a two-compartment pharmacokinetic model to blood Pb concentration. The study revealed that RB of Pb from aged soils ranged from 30±9% to 83±7%. The very different RB of Pb in these soils was attributed to variations in the soils' physico-chemical properties. This was established using sorption studies showing: firstly, Freundlich partition coefficients that ranged from 21 to 234; and secondly, a strongly significant (R(2)=0.94, P

Background Multidrug resistance-associated protein 1 (MRP1) overexpression plays a major role in chemoresistance in glioblastoma multiforme (GBM) contributing to its notorious deadly nature. Although MRP1-siRNA transfection to GBM in vitro has been shown to sensitise the cells to drug, MRP1 silencing in vivo and the phenotypic influence on the tumour and normal tissues upon MRP1 down-regulation have not been established. Here, porous silicon nanoparticles (pSiNPs) that enable high-capacity loading and delivery of siRNA are applied in vitro and in vivo. Result We established pSiNPs with polyethyleneimine (PEI) capping that enables high-capacity loading of siRNA (92 µg of siRNA/mg PEI-pSiNPs), and optimised release profile (70% released between 24 and 48 h). These pSiNPs are biocompatible, and demonstrate cellular uptake and effective knockdown of MRP1 expression in GBM by 30%. Also, siRNA delivery was found to significantly reduce GBM proliferation as an associated effect. This effect is likely mediated by the attenuation of MRP1 transmembrane transport, followed by cell cycle arrest. MRP1 silencing in GBM tumour using MRP1-siRNA loaded pSiNPs was demonstrated in mice (82% reduction at the protein level 48 h post-injection), and it also produced antiproliferative effect in GBM by reducing the population of proliferative cells. These results indicate that in vitro observations are translatable in vivo. No histopathological signs of acute damage were observed in other MRP1-expressing organs despite collateral downregulations. Conclusions This study proposes the potential of efficient MRP1-siRNA delivery by using PEI-capped pSiNPs in achieving a dual therapeutic role of directly attenuating the growth of GBM while sensitising residual tumour cells to the effects of chemotherapy post-resection. Electronic supplementary material The online version of this article (10.1186/s12951-018-0365-y) contains supplementary material, which is available to authorized users.

It is unclear how septic shock causes acute kidney injury (AKI) and whether this is associated with histological change.We aimed to determine the nature and extent of changes in renal structure and function over time in an ovine model of septic shock.Fifteen sheep were instrumented with a renal artery flow probe and renal vein cannula. Ten were given intravenous Escherichia coli to induce septic shock, and five acted as controls. Animals were mechanically ventilated for 48 hours, while receiving protocol-guided parenteral fluids and a norepinephrine infusion to maintain mean arterial pressure. Renal biopsies were taken every 24 hours or whenever animals were oliguric for 2 hours. A renal pathologist, blinded to tissue source, systematically quantified histological appearance by light and electron microscopy for 31 prespecified structural changes.Sheep given E. coli developed septic shock, oliguria, increased serum creatinine, and reduced creatinine clearance (AKI), but there were no changes over time in renal blood flow between groups (P 0.30) or over time within groups (P 0.50). Renal oxygen consumption increased only in nonseptic animals (P = 0.01), but there was no between-group difference in renal lactate flux (P 0.50). There was little structural disturbance in all biopsies and, although some cellular appearances changed over time, the only difference between septic and nonseptic animals was mesangial expansion on electron microscopy.In an intensive care-supported model of gram-negative septic shock, early AKI was not associated with changes in renal blood flow, oxygen delivery, or histological appearance. Other mechanisms must contribute to septic AKI.

Gaucher disease arises from mutations in the beta-glucocerebrosidase gene which encodes an enzyme required for the lysosomal catabolism of glucosylceramide. We have identified a naturally occurring mutation in the beta-glucocerebrosidase gene in sheep that leads to Gaucher disease with acute neurological symptoms. Here we have examined the clinical phenotype at birth and subsequently quantified lipids in Gaucher lamb brain, in order to characterise the disorder. Enzyme activity assessments showed that a reduction in beta-glucocerebrosidase activity to 1-5% of wild-type occurs consistently across newborn Gaucher lamb brain regions. We analyzed glucosylceramide, glucosylsphingosine, bis(monoacylglycero)phosphate and ganglioside profiles in brain, liver, and spleen, and observed 30- to 130-fold higher glucosylceramide, and 500- to 2000-fold higher glucosylsphingosine concentrations in Gaucher diseased lambs compared to wild-type. Significant increases of bis(monoacylglycero)phosphate and gangliosides [GM1, GM2, GM3] concentrations were also detected in the brain. As these glycosphingolipids are involved in many cellular events, an imbalance or disruption of the cell membrane lipid homeostasis would be expected to impair normal neuronal function. To our knowledge, this is the first detailed analysis of glycosphingolipids in various brain regions in a large animal model of neuronal disease, which permits the mechanistic investigation of lipid deregulation and their contribution to neurodegenerative process. Refereed/Peer-reviewed

Study Overview The incidence of adverse pregnancy outcomes is higher in pregnancies where the fetus is male. Sex specific differences in feto-placental perfusion indices identified by Doppler assessment have recently been associated with placental insufficiency and fetal growth restriction. This study aims to investigate sex specific differences in placental perfusion and to correlate these changes with fetal growth. It represents the largest comprehensive study under field conditions of uterine hemodynamics in a monotocous species, with a similar long gestation period to the human. Primiparous 14mo heifers in Australia (n=360) and UK (n=180) were either individually or group fed, respectively, diets with differing protein content (18, 14, 10 or 7% crude protein (CP)) from 60d prior to 98 days post conception (dpc). Fetuses and placentae were excised at 98dpc (n = 48). Fetal development an median uterine artery blood flow were assessed monthly from 36dpc until term using B-mode and Doppler ultrasonography. MUA blood flow to the male feto-placental unit increased in early pregnancy associated with increased fetal growth. Protein restriction before and shortly after conception (-60d up to 23dpc) increased MUA diameter and indices of velocity during late pregnancy, reduced fetal heart weight in the female fetus and increased heart rate at birth, but decreased systolic blood pressure at six months of age. Conclusion and Significance Sex specific differences both in feto-placental Doppler perfusion indices and response of these indices to dietary perturbations were observed. Further, maternal diet affected development of fetal cardiovascular system associated with altered fetal haemodynamics in utero, with such effects having a sex bias. The results from this study provide further insight into the gender specific circulatory differences present in the fetal period and developing cardiovascular system.

Advanced biosensors in future medicine hinge on the evolvement of biomaterials. Porous silicon (pSi), a generally biodegradable and biocompatible material that can be fabricated to include environment-responsive optical characteristics, is an excellent candidate for in vivo biosensors. However, the feasibility of using this material as a subcutaneously implanted optical biosensor has never been demonstrated. Here, we investigated the stability and biocompatibility of a thermally-hydrocarbonised (THC) pSi optical rugate filter, and demonstrated its optical functionality in vitro and in vivo. We first compared pSi films with different surface chemistries and observed that the material was cytotoxic despite the outstanding stability of the THC pSi films. We then showed that the cytotoxicity correlates with reactive oxygen species levels, which could be mitigated by pre-incubation of THC pSi (PITHC pSi). PITHC pSi facilitates normal cellular phenotypes and is biocompatible in vivo. Importantly, the material also possesses optical properties capable of responding to microenvironmental changes that are readable non-invasively in cell culture and subcutaneous settings. Collectively, we demonstrate, for the first time, that PITHC pSi rugate filters are both biocompatible and optically functional for lab-on-a-chip and subcutaneous biosensing scenarios. We believe that this study will deepen our understanding of cell-pSi interactions and foster the development of implantable biosensors. Refereed/Peer-reviewed

Background The safety and efficiency of gene therapies for cystic fibrosis (CF) need to be assessed in pre-clinical models. Using the normal ferret, this study sought to determine whether ferret airway epithelia could be transduced with a lysophosphatidylcholine (LPC) pre-treatment followed by a VSV-G pseudotyped HIV-1 based lentiviral (LV) vector, in preparation for future studies in CF ferrets. Methods Six normal ferrets (7 -8 weeks old) were treated with a 150 μL LPC pre-treatment, followed one hour later by a 500 μL LV vector dose containing the LacZ transgene. LacZ gene expression in the conducting airways and lung was assessed by X-gal staining after 7 days. The presence of transduction in the lung, as well as off-target transduction in the liver, spleen and gonads, were assessed by qPCR. The levels of LV vector p24 protein bio-distribution in blood sera were assessed by ELISA at 0, 1, 3, 5 and 7 days. Results The dosing protocol was well tolerated. LacZ gene expression was observed en face in the trachea of all animals. Histology showed that ciliated and basal cells were transduced in the trachea, with rare LacZ transduced single cells noted in lung. p24 levels was not detectable in the sera of 5 of the 6 animals. The LacZ gene was not detected in the lung tissue and no off-target transduction was detected by qPCR. Conclusions This study shows that ferret airway epithelia are transducible using our unique two-step pre-treatment and LV vector dosing protocol. We have identified a number of unusual anatomical factors that are likely to influence the level of transduction that can be achieved in ferret airways. The ability to transduce ferret airway epithelium is a promising step towards therapeutic LV-CFTR testing in a CF ferret model.

Study Overview: The incidence of adverse pregnancy outcomes is higher in pregnancies where the fetus is male. Sex specific differences in feto-placental perfusion indices identified by Doppler assessment have recently been associated with placental insufficiency and fetal growth restriction. This study aims to investigate sex specific differences in placental perfusion and to correlate these changes with fetal growth. It represents the largest comprehensive study under field conditions of uterine hemodynamics in a monotocous species, with a similar long gestation period to the human. Primiparous 14mo heifers in Australia (n=360) and UK (n=180) were either individually or group fed, respectively, diets with differing protein content (18, 14, 10 or 7% crude protein (CP)) from 60d prior to 98 days post conception (dpc). Fetuses and placentae were excised at 98dpc (n = 48). Fetal development an median uterine artery blood flow were assessed monthly from 36dpc until term using B-mode and Doppler ultrasonography. MUA blood flow to the male feto-placental unit increased in early pregnancy associated with increased fetal growth. Protein restriction before and shortly after conception (-60d up to 23dpc) increased MUA diameter and indices of velocity during late pregnancy, reduced fetal heart weight in the female fetus and increased heart rate at birth, but decreased systolic blood pressure at six months of age. Conclusion and Significance: Sex specific differences both in feto-placental Doppler perfusion indices and response of these indices to dietary perturbations were observed. Further, maternal diet affected development of fetal cardiovascular system associated with altered fetal haemodynamics in utero, with such effects having a sex bias. The results from this study provide further insight into the gender specific circulatory differences present in the fetal period and developing cardiovascular system.