Source: AGRICULTURAL RESEARCH SERVICE submitted to
IN VIVO MANIPULATION OF MAMMARY STEM CELLS TO IMPROVE MILK PRODUCTION EFFICIENCY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0213058
Grant No.
2008-35206-18825
Project No.
MDR-2007-04259
Proposal No.
2007-04259
Multistate No.
(N/A)
Program Code
42.0
Project Start Date
Feb 15, 2008
Project End Date
Feb 14, 2013
Grant Year
2008
Project Director
Capuco, A. V.
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
RM 331, BLDG 003, BARC-W
BELTSVILLE,MD 20705-2351
Performing Department
(N/A)
Non Technical Summary
Efficiency of milk production can be enhanced by decreasing the length of nonlactating periods, or increasing the benefit of these periods to a cow's lifetime production. Two such periods are the time from birth to first lactation, and the nonlactating period between successive lactations (dry period). We propose a unique approach to increase efficiency of milk production by increasing the number of mammary stems. Cell growth and replacement are functions of mammary stem cells. We have established procedures to identify mammary stem cells and to augment their number in vivo by simple infusion of xanthosine (ribonucleoside, a metabolic precursor for DNA and RNA), into the mammary gland. We hypothesize that increasing the mammary stem cell population will enhance the production and turnover of mammary secretory cells. This should permit rapid rearing of heifers (young female cows) and a shortened dry period, without the deleterious effects on milk production that typically occur under these management conditions. We propose three specific aims to determine effects of xanthosine or inosine (another ribonucleoside) when these are administered: (1) to heifers reared at different rates of gain before puberty (2) to heifers during first pregnancy, when most mammary development occurs, and (3) to cows during an abbreviated dry period, when growth and cell replacement must occur. In addition to production parameters, we will evaluate effects of treatment on mammary gene expression, using state-of-the-art molecular approaches. The potential to increase production efficiency by modulating adult stem cell function is exciting, with implications to other production systems and biological processes.
Animal Health Component
(N/A)
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3043410104020%
3053410102030%
3053410103030%
3053450102020%
Knowledge Area
305 - Animal Physiological Processes; 304 - Animal Genome;

Subject Of Investigation
3450 - Milk; 3410 - Dairy cattle, live animal;

Field Of Science
1020 - Physiology; 1030 - Cellular biology; 1040 - Molecular biology;
Goals / Objectives
The objectives of this project are as follows: 1) Determine if treatment of mammary glands of prepubertal heifers with xanthosine or inosine will increase the number of mammary stem cells and progenitor cells, causing a permanent increase in future milk yield. 2) Determine if treatment of mammary glands of primigravid heifers with xanthosine or inosine will increase the number of mammary stem cells and progenitor cells, causing a permanent increase in future milk yield. 3) Determine if treatment of mammary glands of multiparous cows during the dry period with inosine or xanthosine will enhance mammary cell turnover and increase milk production in the subsequent lactation. Results of the research will be published in peer-reviewed scientific journals.
Project Methods
Our broad objective is to develop strategies that promote cell proliferation and the replacement of senescent cells in the bovine mammary gland, in order to increase lactation productivity and efficiency. We previously demonstrated a noninvasive and novel method to expand the putative mammary stem cell population by infusion of xanthosine into the bovine mammary gland, a procedure that was the first non-transgenic, in vivo method to expand a somatic stem cell population. We will test the ability of intramammary infusion of xanthosine or inosine to enhance mammary cell proliferation and milk production. Experiments are designed as half-udder studies in which one side of the udder provides control glands and the other side provides treated glands. This design will increase our ability to detect treatment effects with fewer animals. We will evaluate the impact of treatment during the prepubertal period, first gestation and the dry period, on milk yield and composition (udder-halves). Additionally, mammary tissue obtained from control and treated mammary glands of prepubertal heifers and multiparous cows during the dry period will be evaluated for parameters of cell kinetics and gene expression. Efficacy of the treatment to stimulate expansion of the mammary stem cell population in prepbuertal heifers will be assessed based on quantitative immunohistochemical analysis of the number of cells that retain bromodeoxyuridine for an extended period after initial in vivo labeling (immortal DNA strand). Epithelial cells, isolated by laser microdissection of cryosections, will also be assessed for telomerase activity and extracted RNA subjected to microarray analysis. Furthermore, ability of xanthosine treatment of the mammary glands of prepubertal heifers to increase subsequent milk production will be tested in heifers reared at normal or accelerated rates of gain. Mammary tissues from cows treated with xanthosine or inosine during an abbreviated dry period will be assessed for parameters of cell kinetics and expression of genes of interest (by quantitative RT-PCR). These tissue analyses will provide a means to evaluate the nature and efficacy of treatments and to assess the relationship between the presence or absence of treatment-induced changes in cell populations and milk production. The latter information will be important for designing future experiments that may be more efficient or effective. Achievement of our specific aims will increase efficiency of lactation in accordance with two definitions of efficiency: 1) By decreasing the length of non-revenue-generating periods, lifetime milk production efficiency will be increased. This has been a goal of heifer rearing programs for many years and is behind the current reevaluation of shortened dry periods. 2) By increasing the number of highly secretory mammary epithelial cells, milk production will be increased disproportionately to nutrient intake, increasing efficiency of lactation. Achievement of these goals will have significant impact on future research and application by the dairy industry.

Progress 02/15/08 to 02/14/09

Outputs
OUTPUTS: Two animal experiments were initiated during the first year of this project. The first experiment was designed to confirm and extend our previous finding that xanthosine infusion into the mammary gland of prepubertal Holstein heifers increased the number of putative stem cells, assessed by retention of BrdU-labeled DNA for an extended period (~40 d). Five Holstein heifer calves (~3 mo of age) were administered xanthosine into two ipsilateral mammary glands for five consecutive days. Another five calves were administered inosine. BrdU was administered after each nucleoside treatment. Heifers were sacrificed 45 d after the last treatment and tissues collected. We are in final stages of assessing the percentage of label retaining epithelial cells (LREC) in each quarter to determine efficacy of each nucleoside. A second experiment was initiated to test the hypothesis that stimulated expansion of the mammary stem cell population via xanthosine treatment of prepubertal heifers will produce a cascade of progenitor and epithelial cell proliferation, promote entry into lactation with an increased number of functional secretory cells, and support increased milk yield. This will be assessed in heifers reared at moderate (650 g/d) and accelerated (1000 g/d) rates of daily gain prior to puberty. At 2 mo of age, 26 Holstein heifers were randomly assigned to one of the two dietary groups fed to achieve the targeted rates of gain. At 3 mo of age, each heifer had two quarters within the udder treated with xanthosine while contralateral quarters serve as controls. After heifers reach puberty they will be housed and managed in accordance with the BARC herd's standard rearing and breeding system. Onset of puberty is being monitored by blood progesterone screening of heifers. Targeted rates of gain are being achieved and heifers are beginning to reach puberty. Following parturition, cows will be milked twice daily and milk yield recorded through lactation. One d/wk week cows will be milked with a quarter-milker and milk weights recorded. Milk component analysis and somatic cell count will be assessed. We anticipate the first cow entering lactation in early 2010. To achieve the goals of this project a postdoctoral scientist was hired in May 2008. Her background is well suited for this work, and her skills are being expanded to include microarray analysis. PARTICIPANTS: The PI has overseen all aspects of the current study. He was responsible for administering all treatments in the first experiment and for harvesting tissue. The PI established protocols for the second study, he administered most of the treatments. However, after appropriate training, he was assisted by a postdoctoral scientist who had been hired. The PI is assisting with blood sampling, progesterone RIA and oversees all aspects of data collection and interpretation. A postdoctoral scientist was hired after completion of tissue processing from the experiment. However, she has performed all immunohistochemical procedures, quantitation of LREC, and is performing statistical analyses on the data generated from that experiment. For the second experiment, the postdoctoral scientist assisted with treatment administration, maintains the animal growth database and is tracking data from progesterone RIA to determine onset of puberty. She is responsible for scheduling bleeding and RIA. She is currently being trained in laser microdissection of tissue sections and microarray analysis for future applications. The support scientist in my laboratory has provided technical assistance with all phases of this project. She prepares solutions, does all ordering, coordinates animal procedures and assists or performs analyses as required. She will perform the telomerase assay. TARGET AUDIENCES: The immediate target audience for this research is the scientific community. Data will provide general information pertaining to mammary stem cells and regulation of mammary growth. Should treatments provide a production benefit, the ultimate target will be the dairy industry. As an outreach effort, the PI and postdoctoral scientist on this project have mentored a local high school student on a project to evaluate expression of cytokines in bovine mammary tissue. PROJECT MODIFICATIONS: Thus far, there have been no project modifications. Should nucleoside treatment prove to be ineffective for stimulating proliferation of putative mammary stem cells, adjustments may be necessary this year. We will consider microarray analysis of tissues from the current study and the previous study that demonstrated an effect of xanthosine administration and we will consider further in vitro studies to evaluate the impact of nucleosides on stem cell expansion, so that we can determine if there may be an underlying methodological complication with in vivo studies.

Impacts
Our first experiment was designed to confirm our previous finding that xanthosine infusion into the mammary gland of prepubertal Holstein heifers increased the number of putative stem cells, assessed by retention of BrdU-labeled DNA for an extended period (~40 d). Initial analysis of data from that study suggest that the increase in label retaining epithelial cells LREC that was previously observed persists as a trend in the current study. These histological data are being more thoroughly analyzed and the impact of treatment on telomerase activity within the mammary tissue will be assessed. Funding from a related project will be used to evaluate the impact of xanthosine and inosine on mammary cells in vitro to provide additional information that may be valuable for determining an appropriate course of action for successive studies.

Publications

  • No publications reported this period