Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
Dairy Science
Non Technical Summary
Heat stress (HS) affects US dairy herds of all sizes and in every region of the country costing US agriculture $1.7 billion annually. In dairy cows, HS reduces milk production efficiency by affecting milk yield and milk composition. Much of the loss in milk production is due to reduced feed intake. However, reduced feed intake only explains about 50% of the observed decrease in milk production suggesting that HS itself affects milk production. We hypothesize that HS causes leaky gut in high-producing dairy cows resulting in immune system activation thereby partitioning nutrients away from milk production. Furthering the explanation of decreased milk production during HS, we hypothesize that amino acids (AA) supplied by the diet and body stores do not keep pace with demand for milk production and the immune response to leaky gut. Using a pair-feeding model to adjust for feed intake, high-producing dairy cows will be used in experiments to address two Specific Aims: 1) Determine how HS affects gut integrity, immune system function, and whole body AA utilization. 2) Demonstrate that supplementation of specific AA can amend negative effects of HS and improve whole body AA efficiency by balancing the effects of body AA release, gut integrity and function, immune system requirements, and milk production requirements. This project will improve US agriculture through the enhancement of AA utilization for milk production in high-producing dairy cattle under HS conditions, aligning well with the Animal Nutrition, Growth and Lactation (A1231) program area.
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
0%
Goals / Objectives
Using a pair-feeding model to adjust for feed intake, high-producing dairy cows will be used in experiments to address two Specific Aims: 1) Determine how HS affects gut integrity, immune system function, and whole body AA utilization. 2) Demonstrate that supplementation of specific AA can amend negative effects of HS and improve whole body AA efficiency by balancing the effects of body AA release, gut integrity and function, immune system requirements, and milk production requirements.
Project Methods
Specific Aim 1 Methods Overview. Work for this Aim will center on one in vivo experiment designed to test the following hypotheses, phrased as questions: 1a) Does HS directly cause leaky gut and activate the immune system in lactating cows? 1b) Is the supply of AA from the diet and from endogenous proteolysis significantly altered by HS, leading to either inadequate or of an improper balance to support demands for both milk production and an immune response? 1c) To what extent does HS alter enterocyte fuel substrate use and mitochondrial function? Sixteen healthy lactating Holstein cows will be used in a pair-feeding experiment, as described by Gao et al. (2017). Cows will be randomly allocated to one of two treatments with two periods per treatment. After 14 d of acclimation (period 1), cows on the heat stress treatment (HS) will be exposed to cyclical heat stress for 4 d whereas cows on the pair-fed thermal neutral treatment (PFTN) will not be exposed to HS and intake will be matched to HS. A common diet will be fed to all cows twice daily; intake will be measured. Cow vital signs will be recorded 3x/d throughout the experiment. To assess gut integrity, each cow will be orally drenched with inert markers: chromium-EDTA, D-mannitol, and sucralose on a single day per period. Hourly subsamples of total volume of urine collected within 24 h of dosing will be used to assess percent recovery of each marker. On d 7 of period 1, two jugular catheters will be placed in each cow; catheters will be maintained until the end of the experiment and used for labelled AA infusion (below) and blood sampling for measurement of acute phase proteins. Histology and immunohistochemistry will be used to assess intestinal integrity at the end of period 2. Cows will be milked twice daily throughout both periods; milk yield and composition will be measured at each milking. Amino acid entry from the diet (EAA), de novo synthesis (non-essential AA), release from whole body protein, incorporation into body protein, and clearance will be assessed over a 24 h period at the end of both periods. We will use the basic methodology of Estes et al. (2018), wherein each cow will receive a constant infusion of 0.5 g of a 13C-AA mixture. Blood samples will be assessed for AA enrichment (Estes et al., 2018). A 3-pool model (mirrored for isotope movement) will be fitted to the resulting data to derive rates of whole body AA incorporation into and release from protein, AA entry rates (absorption for EAA and absorption plus synthesis for NEAA), and AA irreversible loss rates. At the end of period 2, all cows will be slaughtered. To quantify HS effects on skeletal muscle metabolism, semitendinosus muscle biopsies (excisional or surgical) will be obtained from all cows on the final day of each period and analyzed according to methods of Xie et al. (2016). Jejunal mucosal scrapings from all HS and PFTN animals will be obtained at slaughter. These enterocyte-rich scrapings will be used in substrate oxidation and metabolic flexibility assays (Zhao et al., 2018, Frisard et al., 2010, Yang et al., 2014, Tarpey et al., 2017).Evaluation, Specific Aim 1:Aim 1 will use a randomized complete block design with 16 cows allotted to one of two treatments (HS or PFTN) and one of four (1, 2, 3, 4) groups. Each cow will experience 2 periods. In period 1, no treatments will be assigned and therefore data for period 1 will be considered baseline data. Data pertaining to period 2 will be adjusted with the baseline data for period 1. Cow within treatment and group will be considered the random term used to test for differences. Data will be reported as least square means and considered significant if P ≤ 0.05.Expected Outputs, Specific Aim 1:Compared to PFTN, HS will exhibit changes to vital signs (e.g, increased respiration, increased rectal temperature). Also, relative to our original questions, the following results are expected:1a) Does HS directly cause leaky gut and activate the immune system in lactating cows?We expect increased urine excretion of all 3 orally dosed inert markers in HS compared to PFTN.Visual evidence for reduced gut integrity in HS compared to PFTN by histological assessment.Increased circulating blood leukocytes and blood acute phase proteins in HS compared to PFTN, indicative of an inflammatory immune response.Increased irreversible loss of EAA during HS due to immune requirements.1b) Is the supply of AA from the diet and from endogenous proteolysis either inadequate or of an improper balance to support demand for both milk production and an immune response in HS cows?Despite similar dry matter intake, HS cows are expected to show ~50% further reduction in milk yield compared to PFTN.Altered blood plasma free AA concentrations and net supply in HS compared to PFTNThe same or reduced rates of absorbed AA due to gut integrity problems, and increased rates of irreversible loss of one or more AA.Increased whole body proteolysis.1c) To what extent does extent HS alter enterocyte fuel substrate use and mitochondrial function?Enterocytes exposed to HS are expected to switch their major oxidative fuel source away from the preferred substrate glutamine, perhaps to glucose.The molecular basis for apparently altered fuel substrate metabolism in HS will be revealed through our mitochondrial assays.Specific Aim 2 Methods Overview. Work for this Aim will center on one in vivo experiment designed to test the following hypothesis, phrased as a question:2) Can increasing the AA status of HS cows be a way to support milk production, obtain less protein mobilization, improve gut integrity, and avoid of immune system activation?The animal experiment for this Aim will be similar to the methods reported for Aim 1. Notable updates here include:Doubling number of total cows used (32 used in Aim 2; 16 used in Aim 1) to account for 4 treatments:PFTN-0; pair-fed thermoneutral, minus EAA+Gln (n = 8).PFTN-AA; pair-fed thermoneutral, plus EAA+Gln (n = 8).HS-0; heat stress, minus EAA+Gln (n = 8).HS-AA; heat stress, plus EAA+Gln (n = 8).No stable isotope infusions.No slaughter in this experiment.No assays of enterocyte fuel substrate preference or mitochondrial function in this experiment.The crystalline AA in the treatment mixture will contain a blend of all EAA and the NEAA Gln (included for its purported role in intestinal function; Harmon, 1986; Kessel et al., 2007; Oba et al., 2004; Okine et al., 1995; El-Kadi et al., 2009; Zuhl et al., 2015). The supplemented amount of each EAA will be determined based on Aim 1 results using the model of the effects of individual AA and energy supply on milk protein production developed by co-investigator Hanigan as described in Aim 1. Cows assigned to AA treatments will be infused continuously via a jugular catheter with a sterile AA mixture designed to correct the apparent imbalance or deficiency in EAA supply caused by the HS and the Gln. The same blood, feces, and urine sampling scheme used in Aim 1 will be repeated here.Expected Outputs, Specific Aim 2:Compared to PFTN, HS will exhibit changes to vital signs (e.g, increased respiration, increased rectal temperature). Also, going along with our originally phrased question for this Aim, we expect the following:Lower evidence of leaky gut and immune system activation in HS+AA when compared to HS-0.Plasma AA concentrations for the HS animals that more closely match the PFTN-0.Improved AA utilization and improved N balance.Higher milk yield, milk lactose, and milk protein in HS+AA when compared to HS-0, that is similar to PFTN-0.Improved N balance, maintained gut integrity and function (and thus no inflammatory immune response), and improved milk yield and protein yield in HS-AA compared to HS-0.Evaluation, Specific Aim 2:Data analysis will be as in Aim 1, with statistical models updated to include the fixed effect of AA supplementation.