Rapid Postnatal Adaptation of Neurodevelopment in Pigs Born Late Preterm.

Preterm birth interrupts intrauterine brain growth and maturation and may induce a delay in postnatal neurodevelopment. Such developmental delays can result from the reduced fetal age at birth, together with the clinical compli-cations of preterm birth (e.g., hypoxia, ischemia, and inflammation). We hypothesized that late preterm birth, inducing only mild clinical complications, has minimal effects on brain-related outcomes such as motor function and behavior. Using the pig as a model for late preterm infants, piglets were cesarean delivered preterm (90%, 106 days gestation) or at full term, reared by identical procedures, and euthanized for tissue collection at birth or after 11 days (e.g., term-corrected age for preterm pigs). Clinical variables and both structural and functional brain endpoints were assessed. The preterm pigs were slow to get on their feet, gained less weight (-30%), and had a higher cerebral hydration level and blood-to-cerebrospinal fluid permeability than the term pigs. At term-corrected age (11 days), the absolute weight of the brain and the weights of its regions were similar between 11-day-old preterm and newborn term pigs, and both were lower than in 11-day-old term pigs. Postnatally, physical activity and movements in an open field were similar, except that preterm pigs showed a reduced normalized stride length and increased normalized maximum stride height. Perinatal brain growth is closely associated with advancing postconceptional age in pigs, and late preterm birth is initially associated with impaired brain growth and physical activity. Postnatally, neuromuscular functions mature rapidly and become similar to those in term pigs, even before term-corrected age. Neuromuscular functions and behavior may show rapid postnatal adaptation to late preterm birth in both pigs and infants.

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.

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.

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.

Short-chain fructo-oligosaccharides supplementation to suckling piglets: Assessment of pre- and post-weaning performance and gut health.

Farmers face difficulties in redeeming their investment in larger litter sizes since this comes with larger litter heterogenicity, lower litter resilience and risk of higher mortality. Dietary oligosaccharides, given to the sow, proved beneficial for the offspring's performance. However, giving oligosaccharides to the suckling piglet is poorly explored. Therefore, this field trial studied the effect of dietary short-chain fructo-oligosaccharides (scFOS; 1g/day; drenched) supplementation to low (LBW, lower quartile), normal (NBW, two intermediate quartiles) and high (HBW, upper quartile) birth weight piglets from birth until 7 or 21 days of age. Performance parameters, gut microbiome and short-chain fatty acids profile of feces and digesta were assessed at birth (d 0), d 7, weaning (d 21.5) and 2 weeks post-weaning (d 36.5). Additional parameters reflecting gut health (intestinal integrity and morphology, mucosal immune system) were analysed at d 36.5. Most parameters changed with age or differed with the piglet's birth weight. Drenching with scFOS increased body weight by 1 kg in NBW suckling piglets and reduced the post-weaning mortality rate by a 100%. No clear difference in the IgG level, the microbiota composition and fermentative activity between the treatment groups was observed. Additionnally, intestinal integrity, determined by measuring intestinal permeability and regenerative capacity, was similar between the treatment groups. Also, intestinal architecture (villus lenght, crypt depth) was not affected by scFOS supplementation. The density of intra-epithelial lymphocytes and the expression profiles (real-time qPCR) for immune system-related genes (IL-10, IL-1ß, IL-6, TNFα and IFNγ) were used to assess mucosal immunity. Only IFNγ expression, was upregulated in piglets that received scFOS for 7 days. The improved body weight and the reduced post-weaning mortality seen in piglets supplemented with scFOS support the view that scFOS positively impact piglet's health and resilience. However, the modes of action for these effects are not yet fully elucidated and its potential to improve other performance parameters needs further investigation.

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.

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.

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.

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.