EXPLORING ENVIRONMENTAL AND NUTRITIONAL FACTORS THAT INFLUENCE REGIONAL PARACELLULAR PERMEABILITY OF THE GASTROINTESTINAL TRACT IN LACTATING DAIRY CATTLE

This dissertation aimed to (1) develop and validate an in vivo dual-marker technique to distinguish paracellular permeability of the total gastrointestinal tract (GIT) from the post-ruminal regions during a 60% feed restriction (FR) or 100% ad-libitum intake (AL), and (2) apply this technique to evaluate interactions between dietary cation-anion difference (DCAD) and dietary buffer during heat stress (HS), as well as the effects of a probiotic-postbiotic blend (PB) during mammary inflammation in lactating dairy cows. In Chapter 3, six ruminally cannulated Holstein cows were assigned to a crossover design and fed either 100% (AL) or 40% of ad libitum feed intake (FR) for 5 d (CHAL). To assess total tract and post-ruminal paracellular permeability, equimolar doses of Cr-EDTA and Co-EDTA were bolus dosed into the rumen and abomasum (0.369 mmol/kg BW), respectively, on d 3 of CHAL. Urine was collected for 96 h, and 15 blood samples were collected over 64 h. Plasma Cr area under the curve (AUC) was 34% greater and Co AUC tended to be greater in FR than AL. Urinary Cr recovery was unaffected, while Co recovery was 36% greater in FR cows. Expressing urinary Co recovery as a percentage of Cr recovery provided an estimate of the proportion of total tract paracellular permeability occurring post-ruminally; although not statistically different, Co represented 61% and 69% of urinary Cr for AL and FR, respectively. These results suggest greater susceptibility of post-ruminal regions to increased paracellular permeability. Plasma AUC for both Cr and Co were strongly correlated with urinary marker recovery (r = 0.76 and 0.87, respectively), and blood sampling at hour 2, 8, 20, 40, and 48 relative to marker infusions accurately predicted total AUC within 1.9% (Cr) and 6.2% (Co) of the full 15-blood sample AUC model. The validated dual-marker technique provides a novel, minimally invasive tool for assessing regional GIT paracellular permeability in adult ruminants. In Chapter 4, 16 cows were enrolled in a replicated 4 × 4 Latin square and fed diets with low (LD; 17.5 mEq/100g DM) or high DCAD (HD; 39.6 mEq/100g DM) concentrations, adjusted using NH₄Cl and Na-acetate. Within each DCAD concentration, cows received diets with low (LB; 0% CaMg(CO₃)₂) or high buffer (HB; 1% CaMg(CO₃)₂) concentrations during mild heat stress (THI ≥ 68 for 15 h/d with night cooling). Feeding HB reduced urinary Co excretion by 27% and tended to reduce Cr excretion, with no effects of DCAD. Urinary Co accounted for 72% of the urinary Cr recovery, indicating that post-ruminal permeability accounted predominantly for total tract paracellular permeability during mild HS. Feeding HD increased blood HCO3-, urine volume (+4 kg/d), and milk fat yield (+36.5 g/d). Increasing ruminal Na+ supply did not affect in vivo short chain fatty acid (SCFA) disappearance from the isolated reticulo-rumen. Chapter 5 utilized Ussing chambers to further investigate the effects of high (140 mM) and low (10 mM) Na+ under high (7.4) or low (6.2) pH conditions on ex vivo apical SCFA uptake and barrier function of isolated ruminal epithelial tissue. There were no effects of Na+ on transporter-mediated and unmediated acetate and butyrate uptake, nor mucosal-to-serosal flux of 3H-mannitol. However, high Na+ stabilized tissue conductance across flux periods. These findings support the lack of a DCAD effect observed in Chapter 4 on SCFA absorption and regional paracellular permeability in vivo. Chapter 6 was a pilot study evaluating the dose-response to an intramammary (IMM) lipopolysaccharide (LPS) challenge; while Chapters 7 and 8 evaluated the effects of dietary PB provision during an IMM LPS infusion on regional GIT paracellular permeability, systemic inflammation, milk performance, and circulating neutrophil mRNA transcript abundance. In Chapter 6, the highest dose of LPS (200 µg/rear quarter) appeared to elicit the greatest reductions in dry matter intake (DMI) and milk yield with possible parity effects. In Chapter 7 and 8, exposure to IMM LPS (IML; 200 µg/rear quarter) increased somatic cell score and markers of systemic inflammation for up to 12 d, and reduced DMI and milk yield for 4 d, with milk fat yield suppressed for 13 d compared to cows that did not receive an IMM infusion (CON). Interestingly, on d 1 post-challenge, Cr and Co AUC were reduced by 24% and 28%, respectively, for IML compared to CON. By d 6, Co AUC remained 33% lower for IML but was no longer different by d 11. Feeding PB reduced circulating serum amyloid A and neutrophil abundance and tended to accelerate body temperature recovery but did not accelerate recovery of DMI or milk production. These studies demonstrate that FR and HS increase GIT paracellular permeability, with greater impacts observed for post-ruminal regions; whereas, IMM inflammation reduced total GIT and post-ruminal paracellular permeability despite systemic inflammation and transient hypophagia. During HS, dietary CaMg(CO₃)₂) mitigated an increase in post-ruminal paracellular permeability, whereas PB provision during IMM inflammation did not modulate GIT paracellular permeability responses. Collectively, these findings suggest that GIT paracellular permeability is stressor-dependent and may be selectively modulated through targeted dietary interventions.