Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
Animal Sciences
Non Technical Summary
Brangus cattle are found throughout the Southeastern US and are abundant in Florida due to their environmental thermotolerance. While the animals more easily survive the harsh temperatures, they grow slower and produce a carcass that is often less desirable than their Angus contemporaries. To improve upon these parameters, a better understanding of the inherent differences in the musculature of Angus and Brangus cattle is needed. The goal of this project is to examine muscle growth of the two breeds during early and late pregnancy. Specifically, we propose to measure the numbers of immature muscle cells and their ability to respond to endogenous growth factors as well as the numbers and sizes of muscle fibers at two developmental time points. Conventional cellular and molecular biology approaches will be used to measure breed differences in prenatal muscle formation. We predict that the muscles of Brangus animals grow slower due to a reduction in the numbers of muscle cells formed in utero and a blunted response to growth factors. By establishing the developmental timeframe of muscle formation, strategies to improve the growth potential of this valuable commodity can be designed.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Goals / Objectives
Brangus calves have smaller birth weights, grow at slower rates and produce carcasses that are lighter muscled at slaughter than their Angus counterparts. A long term goal of the research group is to identify genetic, dietary and management strategies that improve growth rates and muscle deposition in Brangus calves. Postnatal muscle accretion is a result of increased muscle fiber size or hypertrophy; there is no evidence for postnatal muscle fiber hyperplasia. Fiber hypertrophy occurs when protein synthesis exceeds degradation. To meet the protein synthesis demands, satellite cells are added to the growing fiber. Satellite cells are the resident stem cells of skeletal muscle. These entities typically reside in a quiescent state immediately adjacent to the muscle fiber. During periods of growth or repair, satellite cells become mitotically active, proliferate and fuse with the existing muscle fiber. Satellite cells are essential to postnatal muscle growth. Mice genetically ablated for Pax7, the lineage marker of satellite cells, exhibit a significant reduction in muscle fiber size, are deficient in repair activity and suffer early neonatal death. Moreover, satellite cell activation and proliferative responses to growth factors is blunted in aged animals. Parabiosis studies in mice demonstrate that exposure of old animals to a young systemic environment restores the muscle regenerative capabilities that are mediated by satellite cells. The positive effectors found in the circulation likely include Notch ligands. However, it is possible that young animals also contain reduced amounts of the negative myogenic factor, myostatin. Based on these observations, the goal of this proposal is to determine if the differences between Angus and Brangus mature muscle size is due to inherent disparities in satellite cell numbers and myogenic activities. Preliminary data indicates that Brangus newborns exhibit a tendency toward lower levels of Pax7 mRNA content, an estimate of satellite cell numbers, than Angus calves at birth. The first objective will measure the numbers of satellite cells, migratory myoblasts, primary and secondary muscle fibers in Angus and Brangus embryos (gestational day 42) and fetuses (gestational day 180). The second objective will establish primary clonal cultures of embryonic and fetal muscle cells and measure their ability to respond to myostatin and Notch invoked signals. Experimental outcomes will provide the foundation for a graduate student thesis and the results will be disseminated to cattle producers at the UF Beef Cattlemen's Shortcourse, an annual extension activity.
Project Methods
Angus and Brangus dams will be superovulated and inseminated with a single sire of the appropriate breed. Embryos will be flushed, cryopreserved and stored frozen in liquid nitrogen. Holstein recipient cows will synchronized and implanted with the aforementioned embryos. Pregnancies will be confirmed by ultrasonography at day 28 of gestation. At gestational day 42, one half of the Angus and Brangus recipients will be slaughtered and the embryos will be recovered. Gross morphology, crown-rump length, limb length and organ weights will be recorded. The limbs will be microdissected free from the trunk and embedded in cryopreservative media. Frozen tissue cryosections will be collected and analyzed by histology, immunocytochemistry and in situ hybridization for numbers of migratory myoblasts, muscle fibers and formation of skeletal elements. Fetal and secondary myogenesis will be evaluated on Angus and Brangus pregnancies terminated on gestational day 180. Clonal cultures will be established from myogenic cells isolated from the right hindlimb musculature. Cells will be fixed with paraformaldehyde after 1, 2 and 10 days in culture and immunostained for expression of Pax7, Myf5 and myosin heavy chain. Cell morphometrics will be measured with NIS Elements and ImageJ computer software. Individual muscles of the left hindlimb will be cryopreserved. Tissue sections will be analyzed by immunocytochemistry for muscle fiber numbers and size, and myoblast cell identity and numbers. Morphometrics will be measured as described above. The proliferative and myogenic capabilities of fetal Angus and Brangus myoblasts will be measured in mass cultures of gestational day 180 cell isolates. In brief, myogenic cells will be treated with hepatocyte growth factor for 48 hours prior to fixation and immunocytochemical detection of muscle lineage and proliferation markers. Myogenic activities of established fetal myoblast cells will be evaluated following 48 hours of treatment with myostatin. Three different myostatin proteins will be tested; myostatin F94L, a point mutant found in Limousin, myostatin E226X a variant found in MaineAnjou cattle and myostatin Q204X typically found in Charlais cattle. Cells will be treated for 48 hours in fusion-competent media with the respective myostatins proteins, followed by fixation and immunocytochemical detection of myosin heavy chain and MyoD. We predict that the muscle groups of Brangus animals will contain fewer myoblasts, satellite cells and muscle fibers during embyrogenesis and fetal development than Angus contemporaries. In addition, we predict that myogenic cells isolated from Brangus animals will activate and proliferate at lower rates in response to HGF. Importantly, the Brangus myogenic cells are expected to demonstrate a heightened response to myostatin by comparison to Angus myoblasts.