Source: UNIV OF IDAHO submitted to
EFFECT OF HIGH UREA AND LOW PH ON UTERINE RESPONSE TO INTERFERON TAU
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0214653
Grant No.
2008-35203-04505
Project No.
IDA00805-CG
Proposal No.
2008-00571
Multistate No.
(N/A)
Program Code
41.0
Project Start Date
Aug 1, 2008
Project End Date
Jul 31, 2012
Grant Year
2013
Project Director
Ahmadzadeh, A.
Recipient Organization
UNIV OF IDAHO
(N/A)
MOSCOW,ID 83843
Performing Department
ANIMAL & VETERINARY SCIENCE
Non Technical Summary
Reproductive performance of dairy cattle, an important contributor to profitability, has declined steadily over the last few decades. To increase milk yield and profitability, dairy farmers commonly feed diets that contain high levels of protein. However, feeding high dietary protein results in elevated urea and acidic pH in the uterus, which can be detrimental to fertility. Elevated urea concentration and reduced uterine pH, may alter uterine secretions and affect fertility by impairing sperm, ova, or the early embryo. During maternal recognition of pregnancy, the uterine endometrium responds to the embryonic signal interferon-tau (IFN tau), and becomes highly secretory, producing a variety of proteins (histotroph), which are essential for embryo survival and attachment. In cattle, uterine glands secrete proteins including ISG15 and Mx1 in response to IFN tau. Because these two proteins are upregulated during early pregnancy and they are IFN tau-stimulated proteins; they may be critical to the function and establishment of the uterine microenvironment during early pregnancy. The effect of high concentrations of urea and acidic pH on secretion of the uterine epithelium is unknown. If high urea concentrations and (or) low pH affect the uterine environment, it is possible that uterine gland secretion may be reduced or abrogated, even in the presence of IFN tau. Therefore, alteration in the uterine environment and interruption of communication between the embryo and the dam, may explain in part, the associations between high dietary protein and decreased fertility in dairy cows. A long term goal of our research is to elucidate the mechanism by which high dietary protein intake affects dairy cow fertility, specifically through the investigation of the interaction of the conceptus and the uterine environment. The specific aims of this seed grant proposal is to examine the effect of urea, pH, and high dietary protein on IFN tau-induced protein expression in bovine endometrial cells and the uterus of lactating cows. The findings will be presented in the regional and national scientific meetings. The proposed experiments will provide the foundation for future investigations into the role of urea and pH in uterine secretion, specifically aimed at achieving our long term goals, i.e., to 1) elucidate the mechanism by which high dietary protein affects the interaction of the conceptus and the uterine environment, and 2) improve the reproductive efficiency of dairy cattle through the greater understanding of the interaction between nutrition and reproduction. Impact: Conception rates have been reported to be decreased in cows with high blood urea. With growing evidence that dairy profitability increases as pregnancy rate (PR) increases, it is financially important to the US dairy industry to further investigate the effect of high dietary protein intake and fertility. Adjusting the dietary protein and lowering the level of BUN, especially during breeding and early embryo development, might improve PR in dairy cattle. Each 1% increase in PR results in the return of approximately $US 12 to 15 per cow per year regardless of herd size.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3013410102025%
3013410103025%
3053410102025%
3053410103025%
Goals / Objectives
The effect of high concentrations of urea and acidic pH on secretion of the uterine epithelium is unknown. If high urea concentrations and (or) low pH affect the uterine environment, it is possible that uterine gland secretion may be reduced or abrogated, even in the presence of interferon tau (IFN tau). Hypothesis: High urea concentration and low uterine pH, associated with high protein diets, affect uterine endometrial function. Specific Aims:1) To determine the effect of urea and pH on protein expression (ISG15, Mx1) and secretion in uterine epithelial cells in response to IFN using bovine endometrial cells (BEND cells). 2) To determine the effect of high dietary protein on endometrial expression of ISG 15 and Mx1 in response to IFN tau in lactating cows. Outputs: The results of these experiment will be presented at the regional and national scientific meetings. Also, the results will be published in the scientific journals. Through this proposal a graduate student will be trained and several undergraduate students will be trained in the area of basic and applied reproductive physiology.
Project Methods
Specific aim 1: Bovine endometrial cells (BEND) will be plated ans exposed to various urea concentration or pH levels. After 24 h of incubation, cells will be challenged with 0 or 10,000 U/mL of recombinant IFN tau Cells and media from all flasks will be harvested simultaneously 48 h after initial treatment and further processed for ISG15 and Mx1 mRNA. The experiment then will be repeated for isolation of protein to analyze ISG15 and Mx1 by Western Blot analysis. Specific Aim 2: To determine the effect of high dietary protein on endometrial expression of ISG15 and Mx1 mRNA and proteins in response to IFN tau in cows, eight non-pregnant lactating Holstein cows will be used. Cows will be randomly assigned to one of two diets containing 15 or 20% CP in dry matter basis designed to result in either moderate blood urea or high blood urea classifications. Estrus will be synchronized and on day 15 of the estrous cycle Uterine endometrial tissues from all cows will be obtained. A portion will be used for extraction of mRNA for ISG15 and Mx1. The other portion will be used to measure tissue protein response to IFN tau. Statistical Analyses: The in vitro experiment will be conducted as a completely randomized block design with three replicates. Repeated measure analysis will be utilized to analyze dependent variables (separately for each experiment). The statistical model will include the factors dose (IFN taue, 2 levels), treatment (urea, 4 levels; or DMD, 4 levels), and the repeated effect of time. For the in vivo experiment, the mixed procedure of SAS will be utilized to analyze dependent variables the statistical model included dietary treatment (high or low protein; 2 levels), dose (IFN tau, 2 levels), and their interaction as sources of variation. Outputs: This proposal addresses the question: Does urea and (or) pH affect the ability of the bovine uterus to respond to IFN tau? We expect that in vitro uterine cell secretion, in response to IFN tau will be decreased in a high urea and acidic pH environment. These findings will increase our knowledge about possible causative mechanisms between elevated dietary protein and decreased fertility in cattle.

Progress 08/01/08 to 07/31/12

Outputs
OUTPUTS: A graduate student was trained on this project for the partial fulfillment of the requirements of Masters Degree. She earned her M.S. degree. Another graduate student has been trained and has worked on specific Aim 2 of this project. She will complete her M.S. degree within a year. The project indicated in the Specific Aims 1 ( i.e. determining the effect of urea and pH on protein expression (ISG15, Mx1) and secretion in uterine epithelial cells [BEND cells] in response to IFN tau ) has been completed. Also, project indicated in the revised Specific Aims 2 i.e. Characterize the response of the endometrium (ISG15 and Mx1 protein) to IFN-tau following exposure of BEND cells to urea and acidic pH and in the presence and absence of progesterone has been completed. The results related to effect of urea on uterine response was presented at the 2011 Annual Meeting of American Society of Animal Science was held in New Orleans, LA. The results related to effect of pH on uterine response was presented at the 2012 Annual Meeting of American Society of Animal Science was held in Phoenix, AZ. The findings related to specific Aim 2 will be presented at the 2013 Annual Meeting of American Society of Animal Science (Indianapolis, IN). The findings of experiment two is novel and for the first time, it is shown that Acidic pH abrogated bovine endometrial cells Mx1 and ISG15 production in response to IFN-tau in vitro. The benefit of feeding high dietary protein to maximize milk production can be offset by its potentially harmful effect on reproduction of dairy cattle. Without understanding the site and mechanism of action of high protein diets on fertility, it will be very difficult to develop strategies on diet and management of cows for optimum milk production and fertility. The findings of these studies help 1) to elucidate the mechanism by which high dietary protein affects dairy cow pregnancy, and 2) improve the reproductive efficiency of dairy cattle through the greater understanding of the interaction between nutrition and reproduction. PARTICIPANTS: The PI (Amin Ahmadzadeh) and the CO-PI's Drs. Tracy Davis (faculty and reproductive physiologist) and K. C. Carnahan (Support Scientist and reproductive physiologist) were fully involved with project. Moreover, two graduate students (Chloe Autran and Jen Spencer) worked and received training and two undergraduate students assisted with the project. We have collaborated with Dr. Troy Ott (Penn State University) and K. Austin (University of Wyoming). The individual assisted us with laboratory procedures and provided us with Mx-1 and ISG-15 antibodies. TARGET AUDIENCES: Dairy veterinarians, nutritionist, dairy reproduction specialists, dairy producers will gain more knowledge in the area nutrition -reproduction interaction which can help dairy producers to improve the reproductive efficiency of dairy cattle. The above information has been disseminated in the classroom (Dairy Cattle Mgt. course). Moreover, the information has been presented in a workshop/ experiential learning settings for the allied industry (i.e. Select Sires). Lastly, the findings of our experiments have presented t in outreach and extension setting to the producers and stakeholders (Idaho Dairymen association). PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Effect of Urea on Uterine Response to Interferon-tau (IFN tau): High uterine urea concentrations can reduce fertility in dairy cows. The objective was to determine the effects of urea on two IFN tau stimulated proteins, ISG15 and Mx1, of the bovine endometrial cells (BEND) in response to IFN-tau). BEND cells were grown (24 hr) and treated with media containing 0, 5, 7.5, or 10 mM urea and were challenged with 0 or 10,000 UI of IFN-tau. Once harvested, BEND cells were analyzed for protein content. IFN-tau increased (P < 0.01) Mx1 and ISG15 production regardless of treatment. There was no effect of any urea treatment or urea by IFN-tau interaction on Mx1 and ISG15 production after 24 (P = 0.9) or 36 h (P = 0.4) of culture.These results show that there is no disruption of IFN tau-stimulated Mx1 or ISG15 production, when BEND cells were exposed to various concentrations of urea in vitro. Effect of Urea on Uterine Response to IFN tau in progesterone-treated environment: We have shown that there is no disruption of IFN tau-stimulated Mx1 or ISG15 production, when uterine cells were exposed to urea in vitro. However, it has been shown that uterine cells IFN-tau induction of Mx1 gene expression be altered by progesterone. The objective was to characterize the protein response of the endometrium (ISG15 and Mx1 protein) to IFN-tau following exposure of BEND cells to urea in the presence of progesterone (P4). BEND cells were grown in P4 containing media, treated with media containing 0, 5, 7.5, or 10 mM urea, and were challenged with 0 or 10,000 UI of IFN-tau. IFN-tau increased (P < 0.01) Mx1 and ISG15 production regardless of treatment.. There was no effect of any urea treatment or urea by IFN-tau interaction on Mx1 and ISG15 production after 24 hr of P4-containing culture. These results show that there is no disruption of IFN tau-stimulated Mx1 or ISG15 production even when BEND cells were exposed to various concentrations of urea in presence of P4. Effect of acidic pH on Uterine Response to IFN tau : Low uterine pH associated with high blood urea and dietary protein, can reduce fertility in dairy cows. The objective was to determine the effects of acidic pH on protein expression of the BEND cells in response to IFN-tau. Dimethadione (DMD), a weak non-metabolizable acid, was used to decrease culture medium pH. BEND cells were cultured in media containing 0 (pH= 7.3), 10 (pH=7.2), 15 (pH=7.0), or 20 (pH=6.8) mM DMD and subsequently challenged with 0 or 10,000 IFN-tau and were incubated for 24 h. DMD decreased (P < 0.01) the pH of the culture media. There was effect of DMD (P < 0.01) and DMD by IFN-tau interaction (P < 0.01) on both Mx1 and ISG15. The 15 and 20mM DMD reduced (P < 0.01) IFN-tau-induced Mx1 expression, whereas 20 mM DMD reduced (P < 0.01) ISG15 expression in response to IFN-tau indicating that acid pH abrogated BEND cell Mx1 and ISG15 production in response to IFN-tau. These results show that acidic pH disrupted IFN tau-stimulated Mx1 or ISG15 production, and it may indicate that high dietary protein may compromise fertility by inducing lower pH in uterine secretions altering proteins essential for pregnancy.

Publications

  • Ahmadzadeh, A. T. Davis, K. Carnahan, and C. Autran, 2012. Effect of acidic pH on uterine response to interferon-τ. J. Dairy Sci. 95(Suppl. 2):733.
  • Ahmadzadeh, A., T. L. Davis, and K. G. Carnahan. 2011. Effect of urea on interferon-tau response in the bovine endometrium. J. Dairy Sci. 94 (Suppl. 1):486.
  • Ahmadzadeh, A. T. Davis, K. Carnahan, and C. Autran, 2012. Effect of acidic pH on uterine response to interferon-τ. In Proc.. 16th Roy Wallace Memorial Symposium on Bovine Reproduction. Columbus, OH


Progress 08/01/10 to 07/31/11

Outputs
OUTPUTS: A graduate student was trained and on this project for the partial fulfillment of the requirements of Masters Degree. Approximately 90% of project indicated in the Specific Aims 1 ( i.e. determining the effect of urea and pH on protein expression (ISG15, Mx1) and secretion in uterine epithelial cells [BEND cells] in response to IFN tau ) has been completed. The results related to effect of urea on uterine response was presented at the 2011 Annual Meeting of American Society of Animal Science was held in New Orleans, LA. These findings have increased our knowledge about possible causative mechanisms between elevated dietary protein and decreased fertility in cattle. PARTICIPANTS: Currently, the PI (Amin Ahmadzadeh)and thh CO-PI's Drs. Tracy Davis (faculty and reproductive physiologist) and K.C. Carnahan (Support Scientist and reproductive physiologist) are fully involved with the project. Moreover, one student (Jenifer Spencer) is working on this project. TARGET AUDIENCES: Dairy veterinarians, nutritionist, faculty, dairy reproduction specialists, dairy producers will gain more knowledge in the area nutrition-reproduction interaction which can help dairy producers to improve the reproductive efficiency of dairy cattle. PROJECT MODIFICATIONS: The Specific Aims 2) of the proposal was to determine the effect of high dietary protein on endometrial expression of ISG 15 and Mx1 in response to IFN-tau in lactating cows (in vivo). However, as indicated in the progress report, the results show that there is no disruption of IFN-tau-stimulated Mx1 or ISG15 production, when BEND cells were exposed to various concentrations of urea in vitro. Because the in vivo experiments do not appear to show any effect, it would not be logical to conduct the extensive experiment on the whole animal (Specific Aim 2). The goal of the research is to elucidate the mechanism by which high dietary protein affects the interaction of the conceptus and the uterine environment. Therefore, the PI is requesting a change in the direction of the project by continuing with in vitro experiments in a different setting but with the same objective stated in Specific Aim 1. As proposed under the "Future Research" in the original proposal, we would like to change the Specific Aim 2 to the following objectives (Aim 3) Aim 3) Characterize the response of the endometrium (ISG15 and Mx1 protein) to IFN-tau following exposure of BEND cells to urea and acidic pH and in the presence and absence of progesterone. A previous in vivo study, using ovine cells, showed the induction of Mx1 gene expression by IFN-tau may be altered by progesterone.

Impacts
High urea concentrations, associated with high dietary protein, can reduce fertility in dairy cows. The objective was to determine the direct effects of urea on protein expression of the endometrial cells of the bovine uteri in response to interferon-tau (IF-tau).Using bovine endometrial (BEND) cells as a model, the experiment was designed to determine the effects of urea on the production of two IFN tau stimulated proteins, ISG15 and Mx1. Bovine endometrial cells were grown to 80% confluency and treated with media containing 0, 5, 7.5, or 10 mM urea for 24 h. Subsequently, BEND cells were challenged with 0 or 10,000 UI of recombinant IFN-tau and cells were incubated for an additional 24 or 36 h. Cells were in culture for the same period of time regardless of treatment and then harvested. Once harvested, BEND cells were lysed and the lysate was analyzed for protein content. The protein was separated by SDS-page and subjected to western blot analysis and immunoblotting to assess the production of Mx1 and ISG15. Based on optical density, IFN-tau increased (P < 0.01) Mx1 and ISG15 production regardless of treatment after 24 and 36 h. There was no effect of any urea treatment or urea by IFN-tau interaction on Mx1 and ISG15 production after 24 (P = 0.9) or 36 h (P = 0.4) of culture. Moreover, there was no effect of either 24 or 36 h or time by urea treatment interaction on the production of Mx1 and ISG15, in response to IFN-tau. These results show that there is no disruption of IFN tau-stimulated Mx1 or ISG15 production, when BEND cells were exposed to various concentrations of urea in vitro. Perhaps, in cows, the negative effect of urea on reproduction is mediated through a different system other than the uterus.

Publications

  • A. Ahmadzadeh, T. L. Davis, and K. G. Carnahan. 2011. Effect of urea on interferon-tau response in the bovine endometrium. J. Dairy Sci. 94 (Suppl. 1):486.


Progress 08/01/09 to 07/31/10

Outputs
OUTPUTS: Reproductive performance of dairy cattle, an important contributor to profitability, has declined steadily over the last few decades. To increase milk yield and profitability, dairy farmers commonly feed diets that contain high levels of protein. However, feeding high dietary protein results in elevated urea and acidic pH in the uterus, which can be detrimental to fertility. Elevated urea concentration and reduced uterine pH, may alter uterine secretions and affect fertility by impairing sperm, ova, or the early embryo. If high urea concentrations and (or) low pH affect the uterine environment, it is possible that uterine gland secretion may be reduced or abrogated, even in the presence of IFN&#61556;. Therefore, alteration in the uterine environment and interruption of communication between the embryo and the dam, may explain in part, the associations between high dietary protein and decreased fertility in dairy cows. The objective was to determine the direct effects of urea on protein expression of the endometrial cells of the bovine uteri in response to interferon-tau (IF-tau).Using bovine endometrial (BEND) cells as a model, the experiment was designed to determine the effects of urea on the production of two IFN tau stimulated proteins, ISG15 and Mx1. Bovine endometrial cells were grown to 80% confluency and treated with media containing 0, 5, 7.5, or 10 mM urea for 24 h. Subsequently, BEND cells were challenged with 0 or 10,000 UI of recombinant IFN-tau and cells were incubated for an additional 24 or 36 h. Cells were in culture for the same period of time regardless of treatment and then harvested. Once harvested, BEND cells were lysed and the lysate was analyzed for protein content. The protein was separated by SDS-page and subjected to western blot analysis and immunoblotting to assess the production of Mx1 and ISG15. The results of this study will be presented at the national meeting of American Society of Animal Science and American Dairy Science Association (New Orleans, LA). PARTICIPANTS: Dr. Tracy Davis (Univ. of Idaho Faculty) and Dr. Carnahan (Support Scientist) were involved in this project. An undergraduate student and a graduate student received partial training through this research. TARGET AUDIENCES: Through the national presentation, faculty from other universities, as well other scientists from industry become familiar with different aspects of interaction between dietary protein and uterine environment. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Based on optical density, IFN-tau increased (P < 0.01) Mx1 and ISG15 production regardless of treatment after 24 and 36 h. There was no effect of any urea treatment or urea by IFN-tau interaction on Mx1 and ISG15 production after 24 (P = 0.9) or 36 h (P = 0.4) of culture. Moreover, there was no effect of either 24 or 36 h or time by urea treatment interaction on the production of Mx1 and ISG15, in response to IFN-tau. These results show that there is no disruption of IFN tau-stimulated Mx1 or ISG15 production, when BEND cells were exposed to various concentrations of urea in vitro. The findings of the current research will enable us to further investigate the relationship between the conceptus and the specific uterine environment by characterize the response of the endometrium (ISG15 and Mx1 proteinz) to IFN tau following exposure of BEND cells to urea and acidic pH and in the presence and absence of progesterone.

Publications

  • No publications reported this period


Progress 08/01/08 to 07/31/09

Outputs
OUTPUTS: A graduate student is being trained and currently working on this project for the partial fulfillment of the requirements of Masters Degree. Approximately 80% of project indicated in the Specific Aims 1 ( i.e. determining the effect of urea and pH on protein expression (ISG15, Mx1) and secretion in uterine epithelial cells [BEND cells] in response to IFN tau ) has been completed. The results related to this aim will be presented at the 2010 Annual Meeting of American Society of Animal Science which will be held in Denver, Colorado. These findings will increase our knowledge about possible causative mechanisms between elevated dietary protein and decreased fertility in cattle. PARTICIPANTS: Currently the PI (Amin Ahmadzadeh) and the CO-PI's Drs. Tracy Davis (faculty and reproductive physiologist) and K. C. Carnahan (Support Scientist and reproductive physiologist) are fully involved with project. Moreover, a graduate student (Kaleigh Perry) is working in this project and earning her master's degree. TARGET AUDIENCES: Dairy veterinarians, nutritionist, dairy reproduction specialists, dairy producers will gain more knowledge in the area nutrition -reproduction interaction which can help dairy producers to improve the reproductive efficiency of dairy cattle. PROJECT MODIFICATIONS: The only change that occurred is that the effect of treatment was tested only at 24 and 36 hours and after BEND cells were challenged with IFN tau. The initial experiment was suppose to test the effect after, 8, 16, 24, 32, 40, or 48 h of culture. This change was made due to the new findings during the preliminary experiment, in which it was fund that the maximal IFN-induced expression of ISG 15 and Mx occurred at 24 hours.

Impacts
Bovine endometrial cells (BEND) were successfully cultured with media containing 0, 5, 7.5, or 10 mM urea for 24 h. After 24 h of incubation, cells were challenged with 0 or 10,000 U/mL of recombinant IFN&#61556;. Cells were harvested simultaneously 48 or 60 h after initial treatment and analyzed for ISG15 and Mx1 by Western Blot analysis.&#61472; To examine the effect of pH on ISG15 and Mx1 expression in response to IFN tau&#61484; BEND cells were exposed to 0, 10, 15 or 20 mM of DMD inorganic acid (media pH 7.3, 7.1, 6.9, and 6.8, respectively) at 0 h. After 24 h of incubation, cells were challenged with 0 or 10,000 U/mL of recombinant IFN tau. Cells were harvested simultaneously at 48 or 60 h after initial treatment and analyzed for ISG15 and Mx1 by Western Blot analysis. There was not sufficient evidence indicating that urea in any concentration affected BEND cells response with IFN-induced ISG15 or Mx1. In other words mean ISG 15 or Mx1 was not different among treatment at 24 or 36 after IFN tau challenge. These results may indicated that the observed effect of elevated uterine urea on dairy cows fertility is not mediated through its effect on uterine response to embryo signaling, i.e. IFN tau. Currently Western Blot analysis is being conducted to examine the effect of pH on protein expression (ISG15, Mx1) and secretion in response to IFN tau. More experiment will be conducted to determine the effect of high dietary protein on endometrial expression of ISG 15 and Mx1 in response to IFN&#61556; in lactating cows". The experiments outlined in this proposal will provide the foundation for future investigations into the role of urea and pH in uterine secretion, specifically aimed at achieving our long term goals, i.e., to 1) elucidate the mechanism by which high dietary protein affects the interaction of the conceptus and the uterine environment, and 2) improve the reproductive efficiency of dairy cattle through the greater understanding of the interaction between nutrition and reproduction.

Publications

  • No publications reported this period