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.

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.

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.

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.

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.

Glucose and glycogen levels in piglets that differ in birth weight and vitality.

In the pig, intrauterine crowding can greatly affect postnatal characteristics, among which birth weight and locomotion. In a previous study, we discovered that piglets with a low birth weight/low vitality (L piglets) have a reduced motor performance compared to piglets with a normal birth weight/normal vitality (N piglets). A possible explanation is that L piglets lack the energy to increase their motor performance to the level of that of N piglets. Blood glucose levels (GLU) and glycogen concentrations in skeletal muscle of the front (GLYFRONT) and hind leg (GLYHIND) and the liver (GLYLIVER) at birth and during the first 96 h postpartum were compared between L and N piglets. GLU at birth was the same for both groups. After birth, GLU immediately increased in N piglets, whereas it only increased after 8 h in L piglets. L piglets showed a lower GLYHIND at birth and did not use this glycogen during the first 8 h postpartum, while N piglets showed a gradual depletion. GLYLIVER at birth was 50% lower for L piglets and was unused during the studied period while N piglets consumed half of their GLYLIVER during the first 8 h. Based on these results, it is possible that lower glycogen concentrations at birth, the delayed increase in GLU and the lower use of glycogen during the first 8 h after birth negatively affect motor performance in L piglets. However, based on this study, it is unclear whether the low mobilization of glycogen by L piglets is a consequence, rather than a cause of their lower motor performance.

Does intrauterine crowding affect the force generating capacity and muscle composition of the piglet front limb?

In the pig, intrauterine competition (IUC) greatly affects postnatal traits, such as birth weight, but also locomotor capacities. In a previous study, our group discovered a lower motor performance in piglets with a low birth weight and low vitality (L piglets), compared to piglets with a normal birth weight and normal vitality (N piglets). In order to explain the force deficit causing this reduced motor performance, in a subsequent study, we investigated whether this deficit in L piglets was caused by a lower force generating capacity (FGC) of the extensors of the hind limb and/or a lower number of type II (fast-twitch) fibers in m. vastus lateralis. L piglets had a lower absolute FGC, but surprisingly, a higher relative FGC (to birth weight) in the hind limb, compared to N piglets. In addition, we found no differences in fiber composition of m. vastus lateralis. In the present study, we assessed whether this higher relative FGC is a common feature for front and hind limb locomotor muscles of L piglets. To that end, the physiological cross-sectional area of the main extensor muscles of the front limb was calculated from their volume and fiber length, in order to calculate both the absolute and the relative FGC. By immunohistochemical staining of m. triceps brachii caput longum, the percentage of type II (fast-contracting) fibers could be determined. Similar to the results of the hind limb, we found a smaller absolute FGC, but a larger relative FGC in the front limb of L piglets, compared to N piglets. In addition, m. triceps brachii caput longum did not have a different muscle fiber composition in L and N piglets. As such, we can conclude that IUC affects the locomotor muscles in the front and hind limb in a similar way and that the observed force deficit in L piglets cannot be explained by a different force generating capacity or a lower percentage of type II muscle fibers.

On the characterisation of the porcine gland-specific salivary proteome.

To expand the knowledge on the porcine salivary proteome, secretions from the three major salivary glands were collected from anaesthetised piglets. Pilocarpine and isoproterenol were simultaneously injected intraperitoneally to increase the volume and protein concentration of the saliva, respectively. The protein composition and relative protein-specific abundance of saliva secreted by the parotid gland and by the mandibular and monostomatic sublingual gland, were determined using iTRAQ. When combining two detection methods, MALDI-TOF/TOF MS and Q-Exactive orbitrap MS/MS, a total of 122 porcine salivary proteins and 6 mammalian salivary proteins with a predicted porcine homolog were identified. Only a quantitative and not a qualitative difference was observed between both ductal secretions. The 128 proteins were detected in both secretions, however, at different levels. Twenty-four proteins (20 porcine and 4 mammalian with a predicted porcine homolog) were predominantly secreted by the parotid gland, such as carbonic anhydrase VI and alpha-amylase. Twenty-nine proteins (all porcine) were predominantly secreted by the mandibular and sublingual glands, for example salivary lipocalin and submaxillary apomucin protein. Data are available via ProteomeXchange with identifier PXD008853. SIGNIFICANCE: In humans, more than 3000 salivary proteins have been identified. To our knowledge, previous studies on porcine saliva only identified a total of 34 proteins. This research increased the total number of identified proteins in porcine saliva to 143. This insight into the porcine salivary proteome will facilitate the search for potential biomarkers that may help in the early detection of pathologies and follow-up of animal welfare. Moreover, it can also endorse the value of a porcine animal model and contribute to a better understanding of the animal's physiology. Additionally, this was the first study to collect and analyse gland specific saliva of pigs. The obtained relative-quantitative knowledge of the identified proteins is valuable when comparing data of stimulated (chewing on a device) vs. unstimulated (passive) saliva collection in the future, since salivary stimulation changes the relative contribution of the major salivary glands to the whole saliva in the oral cavity. For example, carbonic anhydrase VI, which is present in higher concentrations in parotid saliva, has a higher concentration in stimulated whole saliva because of the larger contribution of the parotid gland after stimulation by chewing.

How does intrauterine crowding affect locomotor performance in newborn pigs? A study of force generating capacity and muscle composition of the hind limb.

Intrauterine crowding (IUC) considerably influences postnatal traits in a polytocous species such as the pig. Previously, our group described how IUC affects locomotion during the piglet's first days of life (until 96 h after birth). We noted a reduced motor performance in piglets with a low birth weight and low vitality (L piglets), compared to piglets with a normal birth weight and normal vitality (N piglets), indicating L piglets are unable to produce enough force. Our current study investigates whether this observed force deficit in L piglets is caused by a reduced force generating capacity in the muscles and/or a lower percentage of type II (fast-contracting) fibers. Volume and fiber length of the main extensor muscles of the hind limb were used to estimate the physiological cross-sectional area (PSCA) and hence calculate the maximal isometric force generating capacity (Fiso-max) of the hind limb. To check for developmental differences between the muscles of L and N piglets, Fiso-max was normalized to body weight (BW), thus yielding a dimensionless variable F'iso-max. To check for differences in muscle composition, m. vastus lateralis was stained immunohistochemically in order to determine the percentage of type II fibers through image analysis. Our results indicate that L piglets have a reduced absolute force generating capacity due to a lesser muscle mass, compared to N piglets. However, when normalized to BW L piglets actually show a larger force generating capacity, suggesting their muscles are more voluminous, given their body mass, than those of N piglets. However, no differences between L and N piglets were detected with regard to muscle composition of the m. vastus lateralis. Based on our data, we can say that neither normalized force generating capacity, nor muscle composition (of the m. vastus lateralis) can explain the observed force deficit in L piglets and as such the effect of IUC on locomotor performance.

The porcine tonsils and Peyer's patches: A stereological morphometric analysis in conventionally and artificially reared piglets.

Selection for prolificacy in modern pig farming has resulted in increasing litter sizes. Since rearing large litters is challenging, artificial rearing of piglets with a milk replacer is an alternative strategy. It is hypothesized that the development of the piglets' mucosa-associated lymphoid tissues (MALT) is affected by these artificial conditions. Therefore, the stereologically estimated volumes of the tonsil of the soft palate, and the lingual, nasopharyngeal and paraepiglottic tonsils, as well as the jejunal and ileal Peyer's patches were statistically compared at day 21 postpartum between six conventionally reared piglets and six piglets that were artificially reared from day 7 onwards. In addition, six 7-day-old sow-fed piglets were examined to evaluate the effect of age. All tonsils and Peyer's patches significantly increased in volume with age. The rearing strategy had no significant effect on the volumes of the tonsil of the soft palate and the lingual tonsil. The former tonsil was by far the largest with a mean volume of 967.2 ± 122.4 mm3 and 822.3 ± 125.4 mm3 in the conventionally and artificially reared piglets, respectively. The lingual tonsil only measured 9.4 ± 6.4 mm3 and 6.3 ± 2.6 mm3 in conventionally and artificially reared groups, respectively. In contrast, the rearing strategy did affect the volumes of the nasopharyngeal and paraepiglottic tonsils, which had a mean volume of 137.1 ± 32.4 mm3 and 84.4 ± 26.9 mm3, and 30.7 ± 7.8 mm3 and 20.0 ± 3.9 mm3 in conventionally and artificially reared piglets, respectively. The rearing strategy did not affect the development of the Peyer's patches. At day 21, the jejunal Peyer's patches of the conventionally and artificially reared piglets presented a volume of 1.6 ± 0.4 cm3 and 1.3 ± 0.2 cm3, respectively. The volumes of the ileal Peyer's patch amounted to 15.1 ± 3.0 cm³ in conventionally reared piglets and 12.0 ± 2.6 cm³ in artificially reared piglets at day 21. The results showed that artificial rearing hampers the morphological development of the tonsils that are exposed to inhaled antigens, but the voluminous lymphoid tissues that sample oral antigens are not influenced. Since it is unlikely that the observed differences in both tonsils are due to the milk replacer, artificial rearing could be a valuable alternative for raising large litters. In addition, the presence of developing MALT in piglets allows for investigating the value of nasal and oral vaccination in this species for human or veterinary purposes.

Does intrauterine crowding affect locomotor development? A comparative study of motor performance, neuromotor maturation and gait variability among piglets that differ in birth weight and vitality.

In polytocous species, such as pigs, the growth of an individual fetus is affected by competition from its littermates and the sow. This intrauterine competition greatly influences postnatal traits such as birth weight and vitality (physical strength). A lowered vitality is most often observed among low birth weight piglets. Since it has been argued that locomotion might be key to unraveling vitality-related differences, we compared gait development in piglets with a low birth weight and low vitality (L piglets) with piglets with a normal birth weight and normal vitality (N piglets) by means of spatio-temporal gait analysis during locomotion at self-selected speed. Video recordings of L and N piglets walking along a corridor at ten time points (between birth and 96 h after birth) were made and the footfalls were digitized. Hence, self-selected speed, spatio-temporal characteristics and gait symmetry were analyzed to compare motor performance, neuromotor maturation (motor task, interlimb and intralimb coordination) and gait variability for L and N piglets. The analysis included both absolute and normalized data (according to the dynamic similarity concept), to distinguish neuromotor maturation from effects caused by growth. Results indicate that intrauterine crowding affects locomotion, mainly by impairing growth in utero, with a lowered motor performance during the first 96 h of age as a consequence. A difference in neuromotor skills was also visible, though only for swing and stance duration, implying a difference in neuromotor development in utero. However, further maturation during the first days after birth does not seem to be affected by intrauterine crowding. We can therefore conclude that L piglets might be considered a smaller and fictitious younger version of N piglets.

How innate is locomotion in precocial animals? A study on the early development of spatio-temporal gait variables and gait symmetry in piglets.

Locomotion is one of the most important ecological functions in animals. Precocial animals, such as pigs, are capable of independent locomotion shortly after birth. This raises the question whether coordinated movement patterns and the underlying muscular control in these animals is fully innate or whether there still exists a rapid maturation. We addressed this question by studying gait development in neonatal pigs through the analysis of spatio-temporal gait characteristics during locomotion at self-selected speed. To this end, we made video recordings of piglets walking along a corridor at several time points (from 0 h to 96 h). After digitization of the footfalls, we analysed self-selected speed and spatio-temporal characteristics (e.g. stride and step lengths, stride frequency and duty factor) to study dynamic similarity, intralimb coordination and interlimb coordination. To assess the variability of the gait pattern, left-right asymmetry was studied. To distinguish neuromotor maturation from effects caused by growth, both absolute and normalized data (according to the dynamic similarity concept) were included in the analysis. All normalized spatio-temporal variables reached stable values within 4 h of birth, with most of them showing little change after the age of 2 h. Most asymmetry indices showed stable values, hovering around 10%, within 8 h of birth. These results indicate that coordinated movement patterns are not entirely innate, but that a rapid neuromotor maturation, potentially also the result of the rearrangement or recombination of existing motor modules, takes place in these precocial animals.

Artificial rearing influences the morphology, permeability and redox state of the gastrointestinal tract of low and normal birth weight piglets.

BACKGROUND: In this study the physiological implications of artificial rearing were investigated. Low (LBW) and normal birth weight (NBW) piglets were compared as they might react differently to stressors caused by artificial rearing. In total, 42 pairs of LBW and NBW piglets from 16 litters suckled the sow until d19 of age or were artificially reared starting at d3 until d19 of age. Blood and tissue samples that were collected after euthanasia at 0, 3, 5, 8 and 19 d of age. Histology, ELISA, and Ussing chamber analysis were used to study proximal and distal small intestine histo-morphology, proliferation, apoptosis, tight junction protein expression, and permeability. Furthermore, small intestine, liver and systemic redox parameters (GSH, GSSG, GSH-Px and MDA) were investigated using HPLC. RESULTS: LBW and NBW artificially reared piglets weighed respectively 40 and 33% more than LBW and NBW sow-reared piglets at d19 (P < 0.01). Transferring piglets to a nursery at d3 resulted in villus atrophy, increased intestinal FD-4 and HRP permeability and elevated GSSG/GSH ratio in the distal small intestine at d5 (P < 0.05). GSH concentrations in the proximal small intestine remained stable, while they decreased in the liver (P < 0.05). From d5 until d19, villus width and crypt depth increased, whereas PCNA, caspase-3, occludin and claudin-3 protein expressions were reduced. GSH, GSSG and permeability recovered in artificially reared piglets (P < 0.05). CONCLUSION: The results suggest that artificial rearing altered the morphology, permeability and redox state without compromising piglet performance. The observed effects were not depending on birth weight.

In Vitro Investigation of Six Antioxidants for Pig Diets.

Oxidative stress in the small intestinal epithelium can lead to barrier malfunction. In this study, the effect of rosmarinic acid (RA), quercetin (Que), gallic acid (GA), lipoic acid (LA), ethoxyquin (ETQ) and Se-methionine (SeMet) pre-treatments using 2 mM Trolox as a control on the viability and the generation of intracellular reactive oxygen species (iROS) of oxidatively (H2O2) stressed intestinal porcine epithelial cells (IPEC-J2) was investigated. A neutral red assay showed that RA (50-400 µM), Que (12.5-200 µM), GA (50-400 µM), ETQ (6.25-100 µM), and SeMet (125-1000 µM) pre-treatments but not LA significantly increased the viability of H2O2-stressed IPEC-J2 cells (p < 0.05). A 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) fluorescent probe showed that RA (100-600 µM), Que (25-800 µM), ETQ (3.125-100 µM) and SeMet (500-2000 µM) pre-treatments significantly reduced iROS in IPEC-J2 monolayers (p < 0.05). Moreover, RA and Que were most effective in reducing iROS. Therefore, the effects of RA and Que on barrier functioning in vitro were examined. RA and Que pre-treatments significantly decreased fluorescein isothiocyanate (FITC)-conjugated dextran-4 (4 kDa) permeability and transepithelial electrical resistance (TEER) of an IPEC-J2 cell monolayer (p < 0.05). These in vitro results of RA and Que hold promise for their use as antioxidants in pig feed.