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Contents An Oldenburg colt with wry nose was autopsied after having lived for only 30 min. It presented cyanotic oral mucosae, underdeveloped eyes and a right-sided temporal osseous mass. The applicable nomenclature for the defects is discussed, and the potential etiopathogenesis is explored by describing the normal embryonic development of the affected body parts. [ABSTRACT FROM AUTHOR]

Asphyxiated neonates often undergo therapeutic hypothermia (TH) to reduce morbidity and mortality. As perinatal asphyxia and TH impact neonatal physiology, this could also influence enzyme functionality. Therefore, this study aimed to unravel the impact of age, hypothermia and hypoxia on porcine hepatic cytochrome P450 (CYP) gene expression, protein abundance and activity. Hepatic CYP expression, protein abundance and activity were assessed in naive adult and neonatal Göttingen minipigs, alongside those from an (non-survival) in vivo study, where four conditions-control (C), therapeutic hypothermia (TH), hypoxia (H), hypoxia and TH (H + TH)-were examined. Naive neonatal Göttingen minipigs exhibited 75% lower general CYP activity and different gene expression patterns than adults. In vitro hypothermia (33°C) decreased general CYP activity in adult liver microsomes by 36%. Gene expression was not different between TH and C while hypoxia up-regulated several genes (i.e., CYP3A29 [expression ratio; E R  = 5.1472] and CYP2C33 [E R  = 3.2292] in the H group and CYP2C33 [E R  = 2.4914] and CYP2C42 [E R  = 4.0197] in the H + TH group). The medical treatment and the interventions over 24 h, along with hypoxia and TH, affected the protein abundance. These data on CYP expression, abundance and activity in young animals can be valuable in building physiologically-based pharmacokinetic models for neonatal drug dose predictions., (© 2024 The Author(s). Basic & Clinical Pharmacology & Toxicology published by John Wiley & Sons Ltd on behalf of Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

The baubellum (os clitoridis) is a bone found in the clitoris of many female eutherian mammals and is homologous to the baculum in males. In contrast to the baculum, the baubellum has received very little attention regarding its morphological or interspecific diversity, or on hypotheses for its function. The presence of the baubellum in bears (Ursidae) has only been established and mentioned in the literature for the Ursus genus, and not for the other genera of bears. Moreover, no scaled photographs are available for baubella of this clade, and the sizes reported vary between sources. We hereby present and describe the baubellum of a spectacled bear (Tremarctos ornatus), providing a detailed account of baubella in a basal ursid species. The baubellum of Tremarctos is slightly bowed dorsally, with two small prominences at the distal apex. The length of the Tremarctos baubellum in this study is comparable to that of Ursus americanus (American black bear). We infer the specific shape, with longitudinal ridges, of the baubellum in Tremarctos could indicate a discrete function during copulation or sexual arousal. However, future studies, especially regarding the associated soft tissues, will be required to confirm whether this is indeed the case. Our study expands the understanding of baubella within Ursidae, providing new data (including a three-dimensional model) that can be used to further explore the morphological diversity and function of this enigmatic extra-skeletal bone., (© 2024 Anatomical Society.)

Novel antimicrobial strategies are necessary to tackle using antibiotics during the suckling and weaning period of piglets, often characterized by E. coli-induced diarrhea. In the last decades, acetate, propionate, and butyrate, all short-chain fatty acids (SCFAs), have been proposed as an alternative to antibiotics. SCFAs are instrumental in promoting the proliferation of enterocytes, preserving intestinal integrity, and modulating the microbial community by suppressing the growth of pathogenic bacteria in pigs. The effect of individual SCFAs (proprionate, acetate and butyrate) on the regenerative capacity of intestinal cells was investigated via an optimized wound-healing assay in IPEC-J2 cells, a porcine jejunal epithelial cell line. IPEC-J2 cells proved a good model as they express the free fatty acid receptor 2 (FFAR2), an important SCFA receptor with a high affinity for proprionate. Our study demonstrated that propionate (p = 0.005) and acetate (p = 0.037) were more effective in closing the wound than butyrate (p = 0.190). This holds promise in using SCFA's per os as an alternative to antibiotics., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Van Bockstal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

This observational study explored the early-life challenges of intra-uterine growth restricted (IUGR), low birth body weight (LBW), and normal birth body weight (NBW) piglets. The aim was to understand the impact of birth weight and intra-uterine growth restriction phenotype on neonatal survival and behavior. Based on weight and phenotype, piglets were classified as IUGR ( n = 32), LBW ( n = 34), and NBW ( n = 29) immediately after birth. The piglets were litter- and sex-matched. Vitality scores were assigned based on motor activity and breathing and complemented with an assessment of umbilical cord condition, rectal temperature, crown-rump length (CRL), time to reach the udder, time to suckle, colostrum intake, and weight gain over 24 h. Beyond the lower birth weight, reduced CRL, and higher mortality rate, IUGR piglets faced several other challenges compared with LBW and NBW piglets. Growth-impaired piglets often struggled to engage in early feeding behaviors and displayed consistently lower rectal temperatures at 1, 3 and 24 h after birth. IUGR piglets showed inadequate colostrum intake and weight loss, which were also observed for LBW counterparts. In contrast, no significant differences were observed in vitality scores and umbilical cord conditions across the groups. In conclusion, our findings underscore the impact of intra-uterine growth restriction on neonatal piglets, emphasizing the need for specialized care strategies to improve survival and health outcomes in IUGR.

Asphyxiated neonates often undergo therapeutic hypothermia (TH) to reduce morbidity and mortality. Since both perinatal asphyxia (PA) and TH influence physiology, altered pharmacokinetics (PK) and pharmacodynamics (PD) are expected. Given that TH is the standard of care for PA with moderate to severe hypoxic-ischemic encephalopathy, disentangling the effect of PA versus TH on PK/PD is not possible in clinical settings. However, animal models can provide insights into this matter. The (neonatal) Göttingen Minipig, the recommended strain for nonclinical drug development, was selected as translational model. Four drugs-midazolam (MDZ), fentanyl (FNT), phenobarbital (PHB), and topiramate (TPM)-were intravenously administered under four conditions: control (C), therapeutic hypothermia (TH), hypoxia (H), and hypoxia plus TH (H+TH). Each group included six healthy male neonatal Göttingen Minipigs anesthetized for 24 hours. Blood samples were drawn at 0 (predose) and 0.5, 2, 2.5, 3, 4, 4.5, 6, 8, 12, and 24 hours post drug administration. Drug plasma concentrations were determined using validated bioanalytical assays. The PK parameters were estimated through compartmental and noncompartmental PK analysis. The study showed a statistically significant decrease in FNT clearance (CL; 66% decrease), with an approximately threefold longer half-life (t 1/2 ) in the TH group. The H+TH group showed a 17% reduction in FNT CL, with a 62% longer t 1/2 compared with the C group; however, it was not statistically significant. Although not statistically significant, trends toward lower CL and longer t 1/2 were observed in the TH and H+TH groups for MDZ and PHB. Additionally, TPM demonstrated a 28% decrease in CL in the H group compared with controls. SIGNIFICANCE STATEMENT: The overarching goal of this study using the neonatal Göttingen Minipig model was to disentangle the effects of systemic hypoxia and TH on PK using four model drugs. Such insights can subsequently be used to inform and develop a physiologically based pharmacokinetic model, which is useful for drug exposure prediction in human neonates., (Copyright © 2024 by The Author(s).)

Zebrafish embryo-based assays are a promising alternative for animal testing to screen new compounds for developmental toxicity. However, recent studies in zebrafish embryos showed an immature intrinsic cytochrome P450 (CYP)-mediated biotransformation capacity, as most CYPs were only active at the end of the organogenesis period. Data on other phase I enzymes involved in the biotransformation of xenobiotics in zebrafish embryos is limited. This information is pivotal for proteratogens needing bioactivation to exert their teratogenic potential. Therefore, this study aimed to investigate whether carbamazepine (CBZ) and levetiracetam (LTC), two anti-epileptic drugs that require bioactivation to exert their teratogenic potential, are biotransformed into non-CYP mediated metabolites in the zebrafish embryo and whether one or more of these metabolites cause developmental toxicity in this species. In the first step, zebrafish embryos were exposed to LTC and CBZ and their non-CYP mediated human metabolites, etiracetam carboxylic acid (ECA) and 9-acridine carboxaldehyde (9ACA), acridine (AI), and acridone (AO), respectively, from 5.25 to 120 hpf and morphologically evaluated. Next, the uptake of all compounds and the formation of the metabolites were assessed using LC-MS methods. As LTC and ECA were, respectively, poorly or not taken up by zebrafish larvae during the exposure experiments, we could not determine if LTC and ECA are teratogenic. However, biotransformation of LTC into ECA was observed at 24 hpf and 120 hpf, which indicates that the special type of B-esterase is already active at 24 hpf. CBZ and its three metabolites were teratogenic, as a significant increase in malformed embryos was observed for all of them. All three metabolites were more potent teratogens than CBZ, with AI being the most potent, followed by 9ACA and AO. The myeloperoxidase (MPO) homologue is already active at 24 hpf, as CBZ was biotransformed into 9ACA and AO in 24 hpf zebrafish embryos, and into 9ACA in 120 hpf larvae. Moreover, 9ACA was also found to be biotransformed into AI and AO, and AI into AO. As such, one or more of these metabolites probably contribute to the teratogenic effects observed in zebrafish larvae after exposure to CBZ., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)

Identifying the potential presence of stress at the pig farm is fundamental since it affects pig welfare. As a result, a reliable and straightforward tool to monitor stress could record the welfare status of the animals. Although numerous methods to assess the welfare of pigs have been developed in the past, no gold standard has been established yet. Recently, the value of saliva as a tool to identify chronic stress in piglets was explored, as it can be collected fast and non-invasively. Since the protein composition, i.e., the proteome of porcine saliva, responds to stress, the affected proteins could be used as salivary stress biomarkers. The present review first defines stress and its relationship with welfare. Next, the porcine gland-specific salivary proteome is characterized. Finally, six potential salivary biomarkers for stress are proposed, i.e., odorant-binding protein, vomeromodulin-like protein, chitinase, lipocalin-1, long palate lung and nasal epithelium protein, and alpha-2-HS-glycoprotein.

Intrauterine growth restriction (IUGR) is frequently observed in pig production, especially when using highly prolific sows. IUGR piglets are born with low body weight and shape indicative of differences in organ growth. Insufficient uteroplacental nutrient transfer to the fetuses is the leading cause of growth restriction in the pig. Supplementing the sow's gestation diet with arginine and/or glutamine improves placenta growth and functionality and consequently is able to reduce IUGR incidence. IUGR piglets are at higher risk of dying preweaning and face higher morbidity than their normal-weight littermates. A high level of surveillance during farrowing and individual nutrient supplementation can reduce the mortality rates. Still, these do not reverse the long-term consequences of IUGR, which are induced by persistent structural deficits in different organs. Dietary interventions peri-weaning can optimize performance but these are less effective in combating the metabolic changes that occurred in IUGR, which affect reproductive performance later in life. IUGR piglets share many similarities with IUGR infants, such as a poorer outcome of males. Using the IUGR piglet as an animal model to further explore the structural and molecular basis of the long-term consequences of IUGR and the potential sex bias could aid in fully understanding the impact of prenatal undernutrition and finding solutions for both species and sexes., (© 2022 The Authors. Molecular Reproduction and Development published by Wiley Periodicals LLC.)

Monitoring chronic stress in pigs is not only essential in view of animal welfare but is also important for the farmer, given that stress influences the zootechnical performance of the pigs and increases their susceptibility to infectious diseases. To investigate the use of saliva as a non-invasive, objective chronic stress monitoring tool, twenty-four 4-day-old piglets were transferred to artificial brooders. At the age of 7 days, they were assigned to either the control or the stressed group and reared for three weeks. Piglets in the stressed group were exposed to overcrowding, absence of cage enrichment, and frequent mixing of animals between pens. Shotgun analysis using an isobaric labelling method (iTRAQ) for tandem mass spectrometry performed on saliva samples taken after three weeks of chronic stress identified 392 proteins, of which 20 proteins displayed significantly altered concentrations. From these 20 proteins, eight were selected for further validation using parallel reaction monitoring (PRM). For this validation, saliva samples that were taken one week after the start of the experiment and samples that were taken at the end of the experiment were analysed to verify the profile over time. We wanted to investigate whether the candidate biomarkers responded fast or rather slowly to the onset of chronic exposure to multiple stressors. Furthermore, this validation could indicate whether age influenced the baseline concentrations of these salivary proteins, both in healthy and stressed animals. This targeted PRM analysis confirmed that alpha-2-HS-glycoprotein was upregulated in the stressed group after one and three weeks, while odorant-binding protein, chitinase, long palate lung and nasal epithelium protein 5, lipocalin-1, and vomeromodulin-like protein were present in lower concentrations in the saliva of the stressed pigs, albeit only after three weeks. These results indicate that the porcine salivary proteome is altered by chronic exposure to multiple stressors. The affected proteins could be used as salivary biomarkers to identify welfare problems at the farm and facilitate research to optimise rearing conditions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Prims et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Animal models provide useful information on mechanisms in human disease conditions, but also on exploring (patho)physiological factors affecting pharmacokinetics, safety, and efficacy of drugs in development. Also, in pediatric patients, nonclinical data can be critical for better understanding the disease conditions and developing new drug therapies in this age category. For perinatal asphyxia (PA), a condition defined by oxygen deprivation in the perinatal period and possibly resulting in hypoxic ischemic encephalopathy (HIE) or even death, therapeutic hypothermia (TH) together with symptomatic drug therapy, is the standard approach to reduce death and permanent brain damage in these patients. The impact of the systemic hypoxia during PA and/or TH on drug disposition is largely unknown and an animal model can provide useful information on these covariates that cannot be assessed separately in patients. The conventional pig is proven to be a good translational model for PA, but pharmaceutical companies do not use it to develop new drug therapies. As the Göttingen Minipig is the commonly used pig strain in nonclinical drug development, the aim of this project was to develop this animal model for dose precision in PA. This experiment consisted of the instrumentation of 24 healthy male Göttingen Minipigs, within 24 h of partus, weighing approximately 600 g, to allow the mechanical ventilation and the multiple vascular catheters inserted for maintenance infusion, drug administration and blood sampling. After premedication and induction of anesthesia, an experimental protocol of hypoxia was performed, by decreasing the inspiratory oxygen fraction (FiO 2 ) at 15%, using nitrogen gas. Blood gas analysis was used as an essential tool to evaluate oxygenation and to determine the duration of the systemic hypoxic insult to approximately 1 h. The human clinical situation was mimicked for the first 24 h after birth in case of PA, by administering four compounds (midazolam, phenobarbital, topiramate and fentanyl), frequently used in a neonatal intensive care unit (NICU). This project aimed to develop the first neonatal Göttingen Minipig model for dose precision in PA, allowing to separately study the effect of systemic hypoxia versus TH on drug disposition. Furthermore, this study showed that several techniques that were thought to be challenging or even impossible in these very small animals, such as endotracheal intubation and catheterization of several veins, are feasible by trained personnel. This is relevant information for laboratories using the neonatal Göttingen Minipig for other disease conditions or drug safety testing., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Stroe, Van Bockstal, Valenzuela, Ayuso, Leys, Annaert, Carpentier, Smits, Allegaert, Zeltner, Mulder, Van Ginneken and Van Cruchten.)

Despite considerable progress in understanding drug metabolism in the human pediatric population, data remains scarce in preterm neonates. Improving our knowledge of the ADME properties in this vulnerable age group is of utmost importance to avoid suboptimal dosing, which may lead to adverse drug reactions. The juvenile (mini)pig is a representative model for hepatic drug metabolism in human neonates and infants, especially phase I reactions. However, the effect of prematurity on the onset of hepatic phase I and phase II enzyme activity has yet to be investigated in this animal model. Therefore, the aim of this study was to assess the ontogeny of CYP3A and UGT enzyme activity in the liver of preterm (gestational day 105-107) and term-born (gestational day 115-117) domestic piglets. In addition, the ontogeny pattern between the preterm and term group was compared to examine whether postconceptional or postnatal age affects the onset of enzyme activity. The following age groups were included: preterm postnatal day (PND) 0 ( n = 10), PND 5 ( n = 10), PND 11 ( n = 8), PND 26 ( n = 10) and term PND 0 ( n = 10), PND 5 ( n = 10), PND 11 ( n = 8), PND 19 ( n = 18) and PND 26 ( n = 10). Liver microsomes were extracted, and the metabolism of CYP3A and UGT-specific substrates assessed enzyme activity. Preterm CYP3A activity was only detectable at PND 26, whereas term CYP3A activity showed a gradual postnatal increase from PND 11 onwards. UGT activity gradually increased between PND 0 and PND 26 in preterm and term-born piglets, albeit, being systematically lower in the preterm group. Thus, postconceptional age is suggested as the main driver affecting porcine CYP3A and UGT enzyme ontogeny. These data are a valuable step forward in the characterization of the preterm piglet as a translational model for hepatic drug metabolism in the preterm human neonate., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Buyssens, Valenzuela, Prims, Ayuso, Thymann, Van Ginneken and Van Cruchten.)

Antisense oligonucleotide (ASO) is a therapeutic modality that enables selective modulation of undruggable protein targets. However, dose- and sequence-dependent platelet count reductions have been reported in nonclinical studies and clinical trials. The adult Göttingen minipig is an acknowledged nonclinical model for ASO safety testing, and the juvenile Göttingen minipig has been recently proposed for the safety testing of pediatric medicines. This study assessed the effects of various ASO sequences and modifications on Göttingen minipig platelets using in vitro platelet activation and aggregometry assays. The underlying mechanism was investigated further to characterize this animal model for ASO safety testing. In addition, the protein abundance of glycoprotein VI (GPVI) and platelet factor 4 (PF4) was investigated in the adult and juvenile minipigs. Our data on direct platelet activation and aggregation by ASOs in adult minipigs are remarkably comparable to human data. Additionally, PS ASOs bind to platelet collagen receptor GPVI and directly activate minipig platelets in vitro, mirroring the findings in human blood samples. This further corroborates the use of the Göttingen minipig for ASO safety testing. Moreover, the differential abundance of GPVI and PF4 in minipigs provides insight into the influence of ontogeny in potential ASO-induced thrombocytopenia in pediatric patients.

Locomotor kinematics have been challenging inputs for automated diagnostic screening of livestock. Locomotion is a highly variable behavior, and influenced by subject characteristics (e.g., body mass, size, age, disease). We assemble a set of methods from different scientific disciplines, composing an automatic, high through-put workflow which can disentangle behavioral complexity and generate precise individual indicators of non-normal behavior for application in diagnostics and research. For this study, piglets ( Sus domesticus ) were filmed from lateral perspective during their first 10 h of life, an age at which maturation is quick and body mass and size have major consequences for survival. We then apply deep learning methods for point digitization, calculate joint angle profiles, and apply information-preserving transformations to retrieve a multivariate kinematic data set. We train probabilistic models to infer subject characteristics from kinematics. Model accuracy was validated for strides from piglets of normal birth weight (i.e., the category it was trained on), but the models infer the body mass and size of low birth weight (LBW) piglets (which were left out of training, out-of-sample inference) to be "normal." The age of some (but not all) low birth weight individuals was underestimated, indicating developmental delay. Such individuals could be identified automatically, inspected, and treated accordingly. This workflow has potential for automatic, precise screening in livestock management., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mielke, Van Ginneken and Aerts.)

Introducing hyperprolific sows has led to proportionally more (very) low birth weight ((V)LBW) piglets, accompanied by higher mortality. To improve the survival of (V)LBW piglets, drenching a dense milk replacer (DMR) could be applied. A first experiment evaluated the effect of drenching DMR (1 or 3 doses within 24 h after birth) to LBW ((mean litter birth weight - 1*SD) and weighing between 1 kg and 750 g) and VLBW piglets ((mean litter birth weight - 1.5*SD) and weighing less than 750 g). On days 1, 2, 3, 9, and two days post-weaning, body weight, growth, skin lesions, and mortality were monitored. No effect of DMR was observed on any of the parameters. In a second experiment, LBW piglets were supplemented with DMR (similarly to experiment 1) at two farms differing in the level of perinatal care. The same parameters were evaluated, and again none were affected by drenching DMR. Overall survival of the LBW piglets was significantly higher at the farm with high perinatal care. It can be concluded that good perinatal management is more effective in enhancing the survival of LBW piglets than drenching.

This experiment evaluated the impacts of essential oils (EO) and protease (PRO), independently or in combination, on growth performance, antioxidation, inflammation and intestinal function of weaned pigs. One hundred and sixty weaned pigs (21 d of age, BW of 6.74 ± 0.20 kg) were randomly divided into 4 treatments with 8 replicate pens of 5 pigs per pen. Dietary treatments included the following: 1) control diet (CON), 2) CON with 300 mg/kg essential oils (EO), 3) CON with 500 mg/kg protease (PRO), 4) CON with 300 mg/kg essential oil and 500 mg/kg protease (EO + PRO). On d 8, one pig from each pen was selected for sampling. The remaining pigs were fed for an additional week and growth performance was monitored during this period. Dietary treatments had no marked effects ( P  > 0.05) on the growth performance of pigs. However, pigs receiving EO diet had higher ( P  < 0.05) serum glutathione peroxidase (GSH-Px) activity, and tended to decrease ( P  = 0.063) serum concentration of tumor necrosis factor-α (TNF-α). In addition, pigs receiving EO diet had higher ( P  < 0.05) abundances of phylum Actinobacteria, and genera Bifidobacterium , and lower ( P  < 0.05) phylum Bacteroidetes and genera Alloprevotella in colonic digesta. Pigs receiving PRO diet decreased ( P  < 0.05) the serum concentration of malondialdehyde (MDA) and diamine oxidase activity, increased ( P  < 0.05) the villus height and the ratio of villus height to crypt depth in duodenum, increased sucrase activity in jejunal mucosa, and also increased the abundance of phylum Actinobacteria in colonic digesta. Furthermore, the synergistic effects of EO and PRO was observed ( P  < 0.05) for pigs with decreasing serum TNF-α concentration and increasing serum GSH-Px activity. Collectively, the results indicated that dietary supplementation of EO and PRO had no significant effects on growth performance of weaned pigs. EO diet appeared to improve antioxidant activity and intestinal microbiota, while PRO diet improved intestinal morphology and digestive enzyme activity, and there was a synergistic effect of EO and PRO on reducing inflammatory parameters in weaned pigs., Competing Interests: We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper., (© 2022 Chinese Association of Animal Science and Veterinary Medicine. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd.)

The introduction of hyperprolific sows has resulted in more low birth weight (LBW) piglets, accompanied by higher mortality. A possible strategy to enhance the resilience and survival of LBW piglets is oral supplementation (drenching) of bioactive substances. This study evaluated the supplementation of bovine colostrum, short-chain fructo-oligosaccharides (scFOS) or quercetin that were dissolved separately in a milk replacer. The study was divided into two sub-experiments. First, the milk replacer was compared with a sham drenched group. Secondly, each dissolved compound was compared with the milk replacer. The LBW piglets, defined as weighing between (mean litter birth weight -1*SD) and (mean litter birth weight -2.5*SD), were randomly allocated to the different treatments and drenched once a day for seven days. On day 1, 3, 9, 24 and 38, piglets were weighed and scored for skin lesions. Blood samples were collected on day 9 and 38 and analyzed to determine glucose, non-esterified fatty acids, urea, immunoglobulin G, insulin-like growth factor 1, and a standard blood panel test. There was no difference between sham drenched piglets and piglets that were drenched with milk replacer regarding any of the parameters. No effect was observed between the milk replacer group and any of the bioactive compounds either, except a higher mortality within the scFOS group. In conclusion, this study showed that drenching the evaluated bioactive compounds, in the used dosages, did not improve LBW piglets' resilience or survival and more research is required to determine the effect of scFOS on small piglets.

Dimethyl sulfoxide (DMSO) is a popular solvent for developmental toxicity testing of chemicals and pharmaceuticals in zebrafish embryos. In general, it is recommended to keep the final DMSO concentration as low as possible for zebrafish embryos, preferably not exceeding 100 μL/L (0.01%). However, higher concentrations of DMSO are often required to dissolve compounds in an aqueous medium. The aim of this study was to determine the highest concentration of DMSO that can be safely used in our standardized Zebrafish Embryo Developmental Toxicity Assay (ZEDTA). In the first part of this study, zebrafish embryos were exposed to different concentrations (0-2%) of DMSO. No increase in lethality or malformations was observed when using DMSO concentrations up to 1%. In a follow-up experiment, we assessed whether compounds that cause no developmental toxicity in the ZEDTA remain negative when dissolved in 1% DMSO, as false positive results due to physiological disturbances by DMSO should be avoided. To this end, zebrafish embryos were exposed to ascorbic acid and hydrochlorothiazide dissolved in 1% DMSO. Negative control groups were also included. No significant increase in malformations or lethality was observed in any of the groups. In conclusion, DMSO concentrations up to 1% can be safely used to dissolve compounds in the ZEDTA., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hoyberghs, Bars, Ayuso, Van Ginneken, Foubert and Van Cruchten.)

The zebrafish ( Danio rerio ) embryo is gaining interest as a bridging tool between in-vitro and in-vivo developmental toxicity studies. However, cytochrome P450 (CYP)-mediated drug metabolism in this model is still under debate. Therefore, we investigated the potential of zebrafish embryos and larvae to bioactivate two known anti-epileptics, carbamazepine (CBZ) and phenytoin (PHE), to carbamazepine-10,11-epoxide (E-CBZ) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH), respectively. First, zebrafish were exposed to CBZ, PHE, E-CBZ and HPPH from 5¼- to 120-h post fertilization (hpf) and morphologically evaluated. Second, the formations of E-CBZ and HPPH were assessed in culture medium and in whole-embryo extracts at different time points by targeted LC-MS. Finally, E-CBZ and HPPH formation was also assessed in adult zebrafish liver microsomes and compared with those of human, rat, and rabbit. The present study showed teratogenic effects for CBZ and PHE, but not for E-CBZ and HPPH. No HPPH was detected during organogenesis and E-CBZ was only formed at the end of organogenesis. E-CBZ and HPPH formation was also very low-to-negligible in adult zebrafish compared with the mammalian species. As such, other metabolic pathways than those of mammals are involved in the bioactivation of CBZ and PHE, or, these anti-epileptics are teratogens and do not require bioactivation in the zebrafish.

Background: Preterm infants frequently show neuromotor dysfunctions, but it is not clear how reduced gestational age at birth may induce developmental coordination disorders. Advancing postnatal age, not only post-conceptional age, may determine neuromuscular development, and early interventions in preterm newborns may improve their later motor skills. An animal model of preterm birth that allows early postnatal detection of movement patterns may help to investigate this hypothesis. Methods: Using pigs as a model for moderately preterm infants, preterm (106-day gestation, equivalent to 90% of normal gestation time; n = 38) and term (115-day gestation, equivalent to 99% of normal gestation time; n = 20) individuals were delivered by cesarean section and artificially reared until postnatal day 19 (preweaning period). The neuromotor skills of piglets were documented using spatiotemporal gait analyses on video recordings of locomotion at self-selected speed at postnatal age 3, 4, 5, 8, and 18 days. Results were controlled for effects of body weight and sex. Results: Both preterm and term piglets reached mature neuromotor skills and performance between postnatal days 3-5. However, preterm pigs took shorter steps at a higher frequency, than term piglets, irrespective of their body size. Within preterm pigs, males and low birth weight individuals took the shortest steps, and with the highest frequency. Conclusion: Postnatal development of motor skills and gait characteristics in pigs delivered in late gestation may show similarity to the compromised development of gait pattern in preterm infants. Relative to term pigs, the postnatal delay in gait development in preterm pigs was only few days, that is, much shorter than the 10-day reduction in gestation length. This indicates rapid postnatal adaptation of gait pattern after reduced gestational age at birth. Early-life physical training and medical interventions may support both short- and long-term gait development after preterm birth in both pigs and infants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Vanden Hole, Ayuso, Aerts, Van Cruchten, Thymann, Sangild and Van Ginneken.)

The adult Göttingen Minipig is an acknowledged model for safety assessment of antisense oligonucleotide (ASO) drugs developed for adult indications. To assess whether the juvenile Göttingen Minipig is also a suitable nonclinical model for pediatric safety assessment of ASOs, we performed an 8-week repeat-dose toxicity study in different age groups of minipigs ranging from 1 to 50 days of age. The animals received a weekly dose of a phosphorothioated locked-nucleic-acid-based ASO that was assessed previously for toxicity in adult minipigs. The endpoints included toxicokinetic parameters, in-life monitoring, clinical pathology, and histopathology. Additionally, the ontogeny of key nucleases involved in ASO metabolism and pharmacologic activity was investigated using quantitative polymerase chain reaction and nuclease activity assays. Similar clinical chemistry and toxicity findings were observed; however, differences in plasma and tissue exposures as well as pharmacologic activity were seen in the juvenile minipigs when compared with the adult data. The ontogeny study revealed a differential nuclease expression and activity, which could affect the metabolic pathway and pharmacologic effect of ASOs in different tissues and age groups. These data indicate that the juvenile Göttingen Minipig is a promising nonclinical model for safety assessment of ASOs intended to treat disease in the human pediatric population.

The Göttingen Minipig is gaining ground as nonrodent species in safety testing of drugs for pediatric indications. Due to developmental changes in pharmacokinetics and pharmacodynamics, physiologically based pharmacokinetic (PBPK) models are built to better predict drug exposure in children and to aid species selection for nonclinical safety studies. These PBPK models require high quality physiological and ADME data such as protein abundance of drug metabolizing enzymes. These data are available for man and rat, but scarce for the Göttingen Minipig. The aim of this study was to assess hepatic cytochrome P450 (CYP) protein abundance in the developing Göttingen Minipig by using mass spectrometry. In addition, sex-related differences in CYP protein abundance and correlation of CYP enzyme activity with CYP protein abundance were assessed. The following age groups were included: gestational day (GD) 84-86 ( n = 8), GD 108 ( n = 6), postnatal day (PND) 1 ( n = 8), PND 3 ( n = 8), PND 7 ( n = 8), PND 28 ( n = 8) and adult ( n = 8). Liver microsomes were extracted and protein abundance was compared to that in adult animals. Next, the CYP protein abundance was correlated to CYP enzyme activity in the same biological samples. In general, CYP protein abundance gradually increased during development. However, we observed a stable protein expression over time for CYP4A24 and CYP20A1 and for CYP51A1, a high protein expression during the fetal stages was followed by a decrease during the first month of life and an increase toward adulthood. Sex-related differences were observed for CYP4V2&#95;2a and CYP20A1 at PND 1 with highest expression in females for both isoforms. In the adult samples, sex-related differences were detected for CYP1A1, CYP1A2, CYP2A19, CYP2E1&#95;2, CYP3A22, CYP4V2&#95;2a and CYP4V2&#95;2b with higher values in female compared to male Göttingen Minipigs. The correlation analysis between CYP protein abundance and CYP enzyme activity showed that CYP3A22 protein abundance correlated clearly with the metabolism of midazolam at PND 7. These data are remarkably comparable to human data and provide a valuable step forward in the construction of a neonatal and juvenile Göttingen Minipig PBPK model., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Buyssens, De Clerck, Schelstraete, Dhaenens, Deforce, Ayuso, Van Ginneken and Van Cruchten.)

Intestinal development is compromised in low birth weight (LBW) pigs, negatively impacting their growth, health, and resilience. We investigated the molecular mechanisms of the altered intestinal maturation observed in neonatal and juvenile LBW female piglets by comparing the changes in intestinal morphology, gene expression, and methylation in LBW versus normal birth weight (NBW) female piglets. A total of 16 LBW/NBW sibling pairs were sacrificed at 0 hours, 8 hours, 10 days, and 8 weeks of age. The gastrointestinal tract was weighed, measured, and the small intestine was sampled for histomorphology, gene expression, and methylation analyses. Impaired intestinal development, with shorter villi and shallower crypts, was observed in LBW female piglets. The expression of intestinal development markers (ALPI and OLFM) rapidly peaked after birth in NBW but not in LBW female piglets. The lower expression of genes involved in nutrient digestion (ANPEP and SI) and barrier function (OCLN and CLDN4) in LBW, together with their delayed development of intestinal villi and crypts could help to explain the compromised health and growth potential of LBW female piglets. The changes in methylation observed in LBW in key regulators of intestinal development (OLFM4 and FZD5) suggest long-term effects of BW on intestinal gene expression, development, and function. Accordingly, experimental demethylation induced in IPEC-J2 cells led to increased expression of intestinal genes (MGA, DPP4, and GLUT2). Overall, we have identified the alterations in transcription or epigenetic marking at a number of genes critical to intestinal development, which may contribute to both the short- and long-term failure of LBW female piglets to thrive., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

The increase in litter sizes in recent years has resulted in more low birth weight (LBW) piglets, accompanied by a higher mortality. A potential intervention to overcome this is drenching bioactive substances. However, if the act of drenching provokes additional stress in LBW piglets, it might counteract the supplement's effect and be detrimental for the piglet's survival. To study the effect of the drenching act, piglets from 67 sows were weighed within 4 h after birth. The mean litter birth weight (MLBW) and standard deviation (SD) were calculated. LBW piglets ( n = 76) were defined as weighing between (MLBW-1*SD) and (MLBW-2.5*SD). They were randomly allocated to two treatments: "sham" (conducting the act of drenching by inserting an empty 2.5 mL syringe in the mouth during 20 s, once a day, d1 till d7; n = 37) or "no treatment" (no handling; n = 39). On day 1, 3, 9, 24 and 38, piglets were weighed and scored for skin lesions. Blood samples were collected on day 9 and 38 and analyzed to determine glucose, non-esterified fatty acids (NEFA), urea, immunoglobulin G (IgG), insulin-like growth factor 1 (IGF-1) and a standard blood panel test. There was no difference between sham drenched and untreated piglets regarding any of the parameters. In conclusion, this study showed that drenching does not impose a significant risk to LBW piglets and can be applied safely during the first 7 days after birth.

Pharmacotherapy in pediatric patients is challenging in view of the maturation of organ systems and processes that affect pharmacokinetics and pharmacodynamics. Especially for the youngest age groups and for pediatric-only indications, neonatal and juvenile animal models can be useful to assess drug safety and to better understand the mechanisms of diseases or conditions. In this respect, the use of neonatal and juvenile pigs in the field of pediatric drug discovery and development is promising, although still limited at this point. This review summarizes the comparative postnatal development of pigs and humans and discusses the advantages of the juvenile pig in view of developmental pharmacology, pediatric diseases, drug discovery and drug safety testing. Furthermore, limitations and unexplored aspects of this large animal model are covered. At this point in time, the potential of the neonatal and juvenile pig as nonclinical safety models for pediatric drug development is underexplored.

Transient receptor potential (TRP) channels contribute to sensory transduction in the body, agonized by a variety of stimuli, such as phytochemicals, and they are predominantly distributed in afferent neurons. Evidence indicates their expression in non-neuronal cells, demonstrating their ability to modulate gastrointestinal function. Targeting TRP channels could potentially be used to regulate gastrointestinal secretion and motility, yet their expression in the pig is unknown. This study investigated TRPA1 and TRPV1 expression in different gut locations of piglets of varying age. Colocalization with enteroendocrine cells was established by immunohistochemistry. Both channels were expressed in the gut mucosa. TRPV1 mRNA abundance increased gradually in the stomach and small intestine with age, most notably in the distal small intestine. In contrast, TRPA1 exhibited sustained expression across ages and locations, with the exception of higher expression in the pylorus at weaning. Immunohistochemistry confirmed the endocrine nature of both channels, showing the highest frequency of colocalization in enteroendocrine cells for TRPA1. Specific co-localization on GLP-1 immunoreactive cells indicated their possible role in GLP-1 release and the concomitant intestinal feedback mechanism. Our results indicate that TRPA1 and TRPV1 could play a role in gut enteroendocrine activity. Moreover, age and location in the gut significantly affected gene expression.

Digitization of video recordings often requires the laborious procedure of manually clicking points of interest on individual video frames. Here, we present progressive tracking, a procedure that facilitates manual digitization of markerless videos. In contrast to existing software, it allows the user to follow points of interest with a cursor in the progressing video, without the need to click. To compare the performance of progressive tracking with the conventional frame-wise tracking, we quantified speed and accuracy of both methods, testing two different input devices (mouse and stylus pen). We show that progressive tracking can be twice as fast as frame-wise tracking while maintaining accuracy, given that playback speed is controlled. Using a stylus pen can increase frame-wise tracking speed. The complementary application of the progressive and frame-wise mode is exemplified on a realistic video recording. This study reveals that progressive tracking can vastly facilitate video analysis in experimental research., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Several pharmaceutical and chemical companies are using the zebrafish embryo as an alternative to animal testing for early detection of developmental toxicants. Unfortunately, the protocol of this zebrafish embryo assay varies between labs, resulting in discordant data for identical compounds. The assay also has some limitations, such as low biotransformation capacity and fewer morphological endpoints in comparison with the in vivo mammalian developmental toxicity studies. Consequently, there is a need to standardize and further optimize the assay for developmental toxicity testing. We developed a Zebrafish Embryo Developmental Toxicity Assay (ZEDTA) that can be extended with a metabolic activation system and/or skeletal staining to increase its sensitivity. As such, the ZEDTA can be used as a modular system depending on the compound of interest.•Our protocol is customized with a metabolic activation system for test compounds, using human liver microsomes. This system ensures exposure of zebrafish embryos to metabolites that are relevant for human risk and safety assessment. As human liver microsomes are toxic for the zebrafish embryo, we developed a preincubation system with an ultracentrifugation and subsequent dilution step.•Additionally, we developed a skeletal staining protocol that can be added to the ZEDTA modular system. Our live Alizarin Red staining method detects several bone structures in 5-day old zebrafish larvae in a consistent manner., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Author(s). Published by Elsevier B.V.)

The feed industry continuously seeks new molecules with antioxidant capacity since oxidative stress plays a key role in intestinal health. To improve screening of new antioxidants, this study aims to set up an assay to assess oxidative stress in the porcine small intestinal epithelial cell line IPEC-J2 using plate-reader-based analysis of fluorescence. Two oxidants, H 2 O 2 and menadione, were tested at 1, 2 and 3 mM and 100, 200 and 300 µM, respectively. Trolox (2 mM) was used as the reference antioxidant and the probe CM-H2DCFDA was used to indicate intracellular oxidative stress. Cell culture, reactive oxygen species (ROS) production and assessment conditions were optimized to detect a significant ROS accumulation that could be counteracted by pre-incubation with trolox. Menadione (200 µM) reproducibly increased ROS levels, H 2 O 2 failed to do so. Trolox significantly decreased intracellular ROS levels in menadione (200 µM)-exposed cells in a consistent way. The system was further used to screen different concentrations of the commercially available antioxidant ELIFE ® . Concentrations between 100 and 200 ppm protected best against intracellular ROS accumulation. In conclusion, the combination of CM-H2DCFDA fluorescence analysis by a plate-reader, trolox as a reference antioxidant and 200 µM of menadione as a stressor agent, provides a replicable and reliable medium-throughput setup for the evaluation of intracellular oxidative stress in IPEC-J2 cells.

Blood supply to enterocytes dictates intestinal health and nutrient absorption. These two aspects are impaired in low birthweight (LBW) piglets, but whether the perfusion to intestinal tissues is implicated as well is still unknown. Thus, structural changes in the microvasculature of LBW and normal birthweight (NBW) piglets were investigated during early postnatal development. Additionally, the presence of endothelial nitric oxide synthase (eNOS) in the intestinal mucosa was assessed given its important role to assure perfusion. A total of 22 pigs (11 LBW and 11 NBW) were sacrificed at days 0, 3, 8 and 19 of life. Body weight and intestinal length were recorded and a piece of the small intestine was sampled for immunohistochemical analysis of von Willebrand Factor (vWF, an endothelial cell marker) and eNOS. LBW piglets had a relatively (to body weight) longer intestine than their NBW counterparts. Age did not affect microvasculature, which was more abundant (85% larger vWF-positive area) in NBW than LBW pigs. However, an interaction age*BW was observed for eNOS-IR, showing that eNOS presence peaked in NBW piglets on the first day of life and subsequently decreased. This pattern was not observed in LBW piglets. The less abundant intestinal endothelial mass and the different pattern of eNOS expression observed in LBW piglets suggests microcirculation as a contributing factor in the impaired digestive functioning and gut health of LBW pigs. However, revealing whether the origin of this alteration is prenatal or postnatal, for example due to a lower milk intake, needs further study., (© 2019 Wiley-VCH GmbH.)