World Package 3 (WP3)

WP3.1: COL feeding following preterm or term birth

The preterm pig born at 90% gestation is the only animal model that shows the characteristics of VLBW infants born <32 wks (65-80% gestation) with immature organs, including lungs, liver, gut, and brain.

To validate the immaturity in COL-fed preterm pigs, relative to term pigs, we need to compare development of multiple organs between term and preterm pigs at birth and term-corrected age (+11 d) to maximize translational value of results to preterm infants.

Methods: Preterm and term pigs are delivered by caesarean section at day 106 or 116 of gestation (one litter each). Half of preterm and term pigs are euthanized right after delivery for tissue collection (day 1). The remaining pigs are nourished by parenteral nutrition and enteral nutrition using infant-relevant COL volumes, followed by transition to mature milk. Parameters of gut, immunity, brain and other organs are investigated at birth and +11 d.

Expected results: All organs in preterm pigs are expected to be immature relative to term pigs, not only at the same postnatal age, but also the same post-conceptional age. COL-fed preterm pigs are expected to show gradual organ development after birth without signs of intestinal and immune diseases, but they will not reach the same degree of maturity after 11 postnatal days, relative to term pigs of the same post-conceptional age. The postnatal organ developmental trajectory in preterm pigs is predicted to be more than just an effect of advancing age, as it is affected by both prematurity and environmental factors (e.g. feeding, colonization, oxygen, cardiovascular).

WP3.2: COL feeding of preterm pigs with infections

Background: Many preterm infants suffer from systemic infections due to immature gut and immune systems at birth. Excessive antibiotics use is required to combat these infections in infants and pigs and this may increase antimicrobial resistance. It is important to know if COL improves infection resistance (e.g. less sepsis-induced brain damage). With existing competence in conducting fetal studies and our initial success in doing neonatal infection studies, we develop new animal models of fetal/neonatal infection to mimic infant conditions.

1. Fetal infection model. Few days prior to preterm birth, fetal pigs receive an intra-amniotic challenge with bacteria/bacterial components or saline control. Following preterm birth at day 106 of gestation, half of piglets from each group are euthanized for tissue collection and the remaining pigs are fed infant formula until euthanasia on day 5 (n = 10-15 pigs in each sub-group) to study postnatal effects of fetal infection.

2. Neonatal infection model. Shortly after preterm birth (day 106), pigs are systemically challenged with S. epidermidis (a common pathogen identified in septic infants) or saline, followed by formula feeding until euthanasia on day 5 (n = 10-15 pigs each group). Extra funding is sought to be able to run the experiment more long-term (19 days) with cognition endpoints. For both two models, temporal blood samples are analyzed to follow the kinetics of immune responses and pathogen clearance following infection while at euthanasia, gut, blood, brain and other tissues are collected to analyze the effects of infection.

3. Testing COL effects. The fetal and neonatal infection models are subjected to COL feeding to test the efficacy of COL relative to infant formula.

Expected results: We expect that the two infection model experiments trigger profound gut and systemic effects in preterm pigs and COL feeding can attenuate detrimental effects of prenatal/neonatal infection, relative to formula feeding.

WP3.3: COL combined with formula feeding

Background: During the first weeks after birth, the timing, feeding advancement rate and milk composition (MM, DM, IF) are highly variable for preterm infants. If MM is available, then this is the first choice, MM may be supplemented or even replaced, by DM or IF, if MM is insufficient or not available. In WP4.1, we test if COL is an optimal diet in the first 1-2 weeks after birth, before transition to MM or IF. To support infant studies, it is important to know if early/slow COL feeding (minimal enteral nutrition, MEN) protects against the possible adverse effects of later IF feeding (feeding intolerance, maldigestion, gut atrophy, NEC, sepsis).

Methods: In feeding regimens relevant for preterm infants, preterm pigs (11 days preterm) are fed total parenteral nutrition (TPN) or TPN supplemented with COL or IF for 5 days (0-64 mL/kg/d). Over the next 5-6 days all piglets are slowly transitioned to full IF feeding (64-120 mL/kg/d). Following tissue collection on day 11, standard gut and immunity endpoints are investigated.

Expected results: That early/slow COL feeding for 5 days is superior to induce gut maturation and resistance against the adverse effects of later IF feeding, relative to early TPN or TPN+IF feeding.

WP3.4: COL used as a fortifier to donor milk

Background: From 1-2 weeks after preterm birth, MM and DM contain inadequate nutrients (protein, minerals) to support optimal growth of preterm infants. Adverse effects are reported after adding fortifiers based on concentrated IF or DM. For WP4.2 infant studies, it is important to document that IF-based nutrient fortifiers to DM are detrimental also in preterm pigs, relative to fortification with COL as a novel, competitive fortification product.

Methods: Preterm pigs are delivered by caesarean section and provided parenteral nutrition support together with increasing volumes of enteral nutrition during the first 2-5 days. After this the pigs are fed total enteral nutrition with either an unfortified base diet (i.e. bovine milk or human donor milk), or the base diet fortified with COL or an IF-based fortifier (eg. Similac, FM85, Enfamil products). Body weight accretion is studied either 5 days, 11 days or up to day 19 (in separate experiments). Tissues and blood are collected at euthanasia for gut, immunity and brain analyses, as in WP 3.1.

Expected results: As a primary outcome we expect to see higher growth velocity in pigs supplemented with COL relative to IF and an unfortified base diet. Second, we expect to see improvements in markers of innate immunity and cognitive function.