Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Animal Science
Non Technical Summary
The objective of our proposed work is to study the impact of citrulline supplementation to pregnant ewes on the growth of brown adipose tissue in her fetus. Because brown adipose tissue is responsible for 50% of the heat produced by the lamb at the time of birth our work will have a direct and beneficial effect on lamb survival, where death loss is the greatest on the first day of life, with cold and cold related starvation being the primary non-predator related cause of death.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
0%
Goals / Objectives
Our primary objective is to determine if maternal citrulline supplementation during the latter third of pregnancy will increase fetal brown adipose tissue development in the lamb. This objective is part of our long term goal to reduce perinatal death loss in ruminant livestock species.
Project Methods
Ewes Pregnancy will be confirmed via transabdominal ultrasound on d 28 of gestation and pregnant ewes will be weighed and individually housed in outdoor covered pens on a concrete floor with unrestricted access to drinking water. Ewes will be fed twice daily at 0700 and 1700 with the daily allotment of feed set to meet 100% NRC requirements. On d 35 of pregnancy fetal number (single or twin) will be determined by transabdominal ultrasound and for the remainder of the study ewes will be fed based on NRC dietary requirements for her individual fetal number. Ewes will be weighed weekly before feeding and feed amounts adjusted accordingly. Thus, the overall experimental design is a 2 x 2 factorial with diet and amino acid supplement being the independent variables to be tested. This period of amino acid supplementation was chosen because this is the period of rapid proliferation of brown adipose tissue in the fetal lamb and we have previously shown that maternal i.v. infusion of arginine during this time increases neonatal thermogenesis in response to cold (McKnight et al., 2020) At 2 h of age the lamb will have its rectal temperature recorded and be humanely euthanized. Fetal parameters of growth will be assessed and organs and tissues dissected, weighed, and preserved for future molecular biology analyses. The proposed study has been approved by the Texas A&M University Institutional Animal Care and Use Committee. Amino acids and polyamines will be quantified using fluorometric HPLC methods after a derivatization reaction with o-phthaldialdehyde (Wu et al., 1997). The integration of chromatographic peaks will be performed using Millenium-32 Software (Waters, Milford, MA).Ammonia will be measured using enzymatic methods as previously described (Lassala et al., 2010b). Urea will be analyzed using a colorimetric method involving urease, phenol, and hypochlorite (Fu et al., 2005). Insulin concentrations in plasma will be measured by EIA (catalog# 80-INSOV-E01, ALPCO Diagnostics, Salem, NH) according to manufacturer's recommendations (Satterfield et al., 2011). Plasma glucose concentrations will be analyzed in duplicate using enzymatic methods as previously described (Wu, 1995; Wu et al., 1995; Fu et al., 2005). NEFAs will be measured using a NEFA-HR Assay (Wako Diagnostics, Mountain View, CA, USA) according to manufacturer's instructions.Histological analyses: Histological analyses of BAT will be conducted using methods previously described (Gray et al., 2000). Briefly, embedded tissues will be sectioned (5 µm), deparaffinized, and hydrated prior to staining with Hematoxylin Stain, Gill's Formulation #3 (Fisher Scientific, New Jersey, NJ). Destaining will be performed using a 1.0% acid ethanol solution. Slides will be developed in a 3% ammonium hydroxide solution, counterstained with 1% Eosin Y (Statlab Medical Products, Lewisville, TX), and destained in 95% ethanol followed by dehydration through a series of alcohol and xylene.Immunohistochemistry: Immunocytochemical localization of immunoreactive von Willebrand's factor (VWF), voltage dependent anionic channel 1 (VDAC1), UCP1, and Ki-67 proteins in BAT will be performed as described previously (Taylor et al., 2000; Gray et al., 2004). VDAC1, VWF, and UCP1 proteins will be detected using primary rabbit monoclonal antibodies at final concentrations of 1:500, 1:500, 1:1000, respectively and a Vectastain ABC anti-rabbit kit (Vector Laboratories, Inc., Burlingame, CA). Ki-67 protein will be detected using a mouse monoclonal antibody at a final concentration of 1:500 and a Vectastain ABC anti-mouse kit (Vector Laboratories, Inc., Burlingame, CA). Negative controls for respective antibodies will include substitution of the primary antibody with non-immune rabbit or mouse IgG (Sigma). Immunoreactive protein will be visualized using diaminobenzidine tetrahydrochloride (Sigma) as the chromagen. Sections will then be dehydrated and coverslips affixed with Permount (Fisher).Photomicroscopy: Photomicrographs will be taken using a Nikon Eclipse E1000 photomicroscope (Nikon Instruments, Melville, NY). Digital images will be captured using a Nikon DXM 1200 digital camera and assembled using Adobe Photoshop 7.0 (Adobe Systems, Seattle, WA). Statistical analyses: Data will be subjected to ANOVA, using the General Linear Model procedures of the Statistical Analysis System (SAS, 2002). In addition to the main effects of diet and amino acid supplementation, fetal sex, fetal number and all interactions will also be considered in the statistical model where appropriate. Differences between means will be determined using the Student-Newman-Keuls multiple comparison test. Probability values ≤ 0.05 will be taken to indicate statistical significance.