Welfare Assessment in Pigs Using the Salivary Proteome.

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.

Drug Disposition in Neonatal Göttingen Minipigs: Exploring Effects of Perinatal Asphyxia and Therapeutic Hypothermia.

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 (t1/2) in the TH group. The H+TH group showed a 17% reduction in FNT CL, with a 62% longer t1/2 compared with the C group; however, it was not statistically significant. Although not statistically significant, trends toward lower CL and longer t1/2 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.

First description of the baubellum in the spectacled bear Tremarctos ornatus (Mammalia: Ursidae).

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.

Early Development of Locomotion in the Term Piglet Model: Does Size Matter?

Intrauterine undernutrition in humans typically results in low birth weight ([small for gestational age] SGA) and delayed postnatal neuromotor maturation. Since SGA and intrauterine growth retardation are also common in domestic pigs, piglets are premised as models to study delayed motor development. Applied to the locomotor paradigm, however, questions emerge: (i) how to map the developmental time scale of the precocial model onto the altricial target species and (ii) how to distinguish size from maturation effects? Gait data were collected at self-selected voluntary walking speed during early development (0-96 hours postpartum; pp) for SGA- and normal ([appropriate for gestational age] AGA) piglets. Dimensionless spatiotemporal gait characteristics (according to dynamic similarity) become invariant already after 4 hours pp, suggesting rapid postnatal neuromotor maturation. Moreover, dimensionless gait data are largely identical for SGA- and AGA-siblings, indicating that primarily size effects explain absolute locomotor differences. This is further supported by (i) normalized force-generating capacity of limb muscles, (ii) joint kinematics (<10 hours pp), and (iii) normalized ground reaction forces (<5 days pp) being indifferent between SGA- and AGA- piglets. Furthermore, predictive modeling based on limb joint kinematics is unable to discern the majority of SGA- from AGA-piglets (<10 hours pp). All this leads to the conclusion that, although smaller than the AGA piglets in absolute terms, SGA-piglets mature (neuromechanically speaking) just like, and equally fast as their AGA littermates. Yet, it remains a fact that early SGA piglets are reported to be less mobile, less vital, and less competitive than their AGA siblings (even often die before day 3 pp). This conspicuous difference likely results from the energy level (blood glucose and glycogen) and its mobilization being considerably different between the piglet categories during early development.

Ontogeny of CYP3A and UGT activity in preterm piglets: a translational model for drug metabolism in preterm newborns.

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.

Chronic exposure to multiple stressors alters the salivary proteome of piglets.

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.

Development of a neonatal Göttingen Minipig model for dose precision in perinatal asphyxia: technical opportunities, challenges, and potential further steps.

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 (FiO2) 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.

Platelet Activation by Antisense Oligonucleotides (ASOs) in the Göttingen Minipig, including an Evaluation of Glycoprotein VI (GPVI) and Platelet Factor 4 (PF4) Ontogeny.

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.

A workflow for automatic, high precision livestock diagnostic screening of locomotor kinematics.

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.

Preweaning performance in intrauterine growth-restricted piglets: Characteristics and interventions.

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.

Effects of dietary supplementation with essential oils and protease on growth performance, antioxidation, inflammation and intestinal function of weaned pigs.

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.

The Effect of Drenching (Very) Low Birth Weight Piglets with a Dense, Concentrated Milk Replacer at Farms with Differing Farrowing Management.

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.

Developmental Toxicity and Biotransformation of Two Anti-Epileptics in Zebrafish Embryos and Early Larvae.

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 5»- 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.

Preterm Birth Affects Early Motor Development in Pigs.

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.

Safety Testing of an Antisense Oligonucleotide Intended for Pediatric Indications in the Juvenile Göttingen Minipig, including an Evaluation of the Ontogeny of Key Nucleases.

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.

Hepatic Cytochrome P450 Abundance and Activity in the Developing and Adult Göttingen Minipig: Pivotal Data for PBPK Modeling.

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_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_2, CYP3A22, CYP4V2_2a and CYP4V2_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.

Low birth weight female piglets show altered intestinal development, gene expression, and epigenetic changes at key developmental loci.

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.

Handling Associated with Drenching Does Not Impact Survival and General Health of Low Birth Weight Piglets.

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.

DMSO Concentrations up to 1% are Safe to be Used in the Zebrafish Embryo Developmental Toxicity Assay.

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.

Drenching Bovine Colostrum, Quercetin or Fructo-Oligosaccharides Has No Effect on Health or Survival of Low Birth Weight Piglets.

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.