Source: University of Northern Colorado submitted to
INFLUENCE OF FISH OIL ON CORPUS LUTEUM FUNCTION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1024909
Grant No.
2021-67016-33839
Cumulative Award Amt.
$500,000.00
Proposal No.
2020-02778
Multistate No.
(N/A)
Project Start Date
Jul 1, 2021
Project End Date
Jun 30, 2026
Grant Year
2021
Program Code
[A1211]- Animal Health and Production and Animal Products: Animal Reproduction
Project Director
Burns, P.
Recipient Organization
University of Northern Colorado
501 20th St.
Greeley,CO 80639
Performing Department
(N/A)
Non Technical Summary
Early pregnancy failures continue to be a major problem in the beef and dairy industries resulting in millions of dollars lost in meat and milk production. The most significant window of pregnancy loss occurs around day 14 - 18 after mating when slow developing embryos fail to prevent onset of the next estrous cycle. Immature embryos are unable to robustly communicate their presence, and thus prostaglandin F2a escapes the pregnant uterus and destroys the corpus luteum (luteolysis) and its essential progesterone support to the nascent pregnancy. Data show that supplementing dietary omega-3 fatty acids from fish oil extends the pregnancy recognition window by de-sensitizing the corpus luteum to uterine prostaglandin F2a, but the underlying mechanisms are still largely unknown. A mature corpus luteum exposed to prostaglandin F2a experiences a rapid (within 6 hours) reduction in blood flow and diminished progesterone production due to downregulation of critical progesterone synthesis genes. It is hypothesized that dietary omega-3 fatty acids from fish oil will prevent prostaglandin F2a-induced reductions in luteal blood flow and progesterone production. The objectives of the current study will be to deliver a natural dose of prostaglandin F2a to cattle synchronized to day 14-18 of the estrous cycle and determine whether dietary omega-3 fatty acids preserve luteal: 1) blood flow, 2) progesterone production, and 3) abundance of key progesterone synthesis proteins. It is anticipated that dietary omega-3 fatty acids from fish oil will reduce sensitivity of the corpus luteum to a natural prostaglandin F2a challenge, preserving luteal blood flow and sustaining progesterone secretion. Outcomes from these studies would suggest that the reproductive efficiency of cattle operations could be improved through development of novel, cost-effective omega-3 fatty acid supplementation strategies that reduce luteal prostaglandin F2a sensitivity and maintain progesterone support for the embryo during the critical window that a pregnancy is trying to establish.
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
3013310102025%
3013310103025%
3013310104025%
3013310101025%
Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3310 - Beef cattle, live animal;

Field Of Science
1040 - Molecular biology; 1030 - Cellular biology; 1010 - Nutrition and metabolism; 1020 - Physiology;
Goals / Objectives
Our overarching goal is to reduce early embryonic mortality and improve profitability in farm animals through supplemental feeding strategies. Dietary supplementation of fish oil is luteal protective to intrauterine infusions of prostaglandin F2a. However, there is currently a knowledge gap in our understanding of how fish products affect corpus luteum function in ruminants, specifically luteal sensitivity to prostaglandin F2a. The goal of this project is to bridge this gap of knowledge. The specific objectives of this project will be to 1) determine luteal blood flow following intrauterine infusion of prostaglandin F2a and 2) determine the expression of key genes and abundance of critical proteins that regulate progesterone biosynthesis following prostaglandin F2a treatment.
Project Methods
Beef cows will be individually fed diets that consist of 95% mixed hay and will be randomly allotted to a 5% vegetable or fish oil supplement group. Rations will be delivered daily at a dry matter intake equivalent to 2.0% body weight for 60 days. Supplements will be formulated to be isonitrogenous and isocaloric. Body weights will be determined weekly and diets adjusted as needed to maintain desired supplementation at 5% dry matter intake. Weekly blood samples will be collected to monitor plasma omega-3 fatty acid composition. Estrous cycles will be synchronized using two injections of prostaglandin F2a at 14-day intervals. Data collection will commence between day 10 to 12 following synchronized estrus, which will be approximately day 60 of the supplementation period. Cows will be administered two intrauterine infusions of 0.25 mL saline or 0.5 mg prostaglandin F2a diluted in 0.25 mL saline at 12-h intervals. Jugular blood samples will be collected immediately before infusion and at 3-h intervals for the first 24 h. After which, samples will be collected at 6-h intervals until ovariectomy to determine serum concentrations of progesterone. Ovariectomies will be performed at 30 or 48 h of the experimental period. In experiment 1, color-Doppler ultrasonography will be used to estimate luteal blood flow following prostaglandin F2a treatment. The percentage of luteal area with color-Doppler signals of blood flow will be estimated at the first intrauterine infusion (0 h) and at second intrauterine infusion (12 h). Relative peak systolic velocity and time-averaged maximum velocity at each time point will be estimated. Cows infused with two doses of prostaglandin F2a will be grouped as having a regressed or functional corpus luteum. Cows with regressed corpus luteum will have serum progesterone below 1 ng/mL at the time of ovariectomy. The effects of dietary supplementation (vegetable oil or fish oil), treatment (saline, prostaglandin F2a regressed corpus luteum, or prostaglandin F2a functional corpus luteum), and time on changes in luteal blood flow will be analyzed using the mixed models procedure of SAS with repeated measures. The statistical model will include dietary supplementation, treatment, time and all possible interactions as sources of variation. Animal within dietary supplementation will be considered as a random variable in the statistical model. If main effects or interactions are significant (P < 0.05), means will be separated using Tukey's. The corpus luteum will be cut into four equal sections at the time of ovariectomy and snap frozen in liquid nitrogen. In experiment 2a, one section of tissue will be used to determine fold change in expression of gene that regulate progesterone biosynthesis (steroidogenic acute regulatory protein, P450 side chain cleavage enzyme, 3-beta hydroxysteroid dehydrogenase, and low-density lipoprotein receptor) following prostaglandin treatment using qPCR. In experiment 2b, another section of tissue will be used to determine changes in abundance of protein for these genes using western blotting. Prostaglandin F2a treated animals will be sorted into regressed or functional CL as described above. The effects of dietary supplementation (vegetable oil or fish oil), treatment (saline, prostaglandin F2a regressed CL and prostaglandin F2a functional CL), and time on fold-change in mRNA or protein abundance (increase or decrease) of steroidogenic acute regulatory protein, P450 side-chain cleavage enzyme, 3-beta hydroxysteroid dehydrogenase and low-density lipoprotein receptor will be analyzed using the mixed models procedure of SAS. The statistical model will include dietary supplementation, treatment, time, and all possible interactions as sources of variation. Animal within dietary supplementation will be considered as a random variable in the statistical model. If main effects or interactions are significant (P < 0.05), means will be separated using Tukey's.

Progress 07/01/23 to 06/30/24

Outputs
Target Audience:The primary target audience is research scientists and graduate/undergraduate students in reproductive biology, beef/dairy extension specialists, large animal veterinarians and eventually beef/dairy producers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three Master candidates (Tyra Goodluck, Rebecca Phelps, and Amanda Hibbard) are currently working on the project. Six undergraduate students have assisted with both collection of field data and bench laboratory analysis. Two undergraduate students were accepted into the Rocky Mountain Alliance for Minority Participation (RM-AMP) program where they actively participated in data collection and analysis. Following completion of the program, these students presented their research at our department's weekly seminar. Furthermore, Chole Seto will be presenting findings on luteal cortisol at the Annual Biomedical Research Conference for Minoritized Scientists conference and Denisse Reyna will be presenting luteal blood flow data at the Society for Advancement of Chicanos/Hispanics & Native Americans in Science in November. These experiences provide students with unique opportunities that prepare them for a career in reproductive biology. How have the results been disseminated to communities of interest?Preliminary findings have been presented at local, regional, and international scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?Preliminary data have shown differential expression of genes associated with fibroblast in tissue collected from fish meal supplemented cows treated with PGF. We will continue investigating expression of genes as well as conduct western blot analysis luteal tissue during the next reporting period. We will continue to mine the proteomic data set for unique proteins that may be expressed in response to dietary fish meal supplementation.

Impacts
What was accomplished under these goals? We have currently collected data on 57 animals. Regardless of dietary supplementation (corn gluten meal vs fish meal), all cows (100%) receiving intrauterine infusion of saline retained a functional corpus luteum (CL) at the time of ovariectomy. Twenty percent more cows supplemented with fish meal retained a functional CL following a prostaglandin challenge as compared to cows receiving the control supplement. We have initiated a proteomic analysis on luteal tissue collected from 6 corn gluten meal and 7 fish meal supplemented cows that received intrauterine infusion of saline. 5,694 proteins were identified, and 240 proteins were differently expressed between supplement groups. Pathway analysis conducted using ShinyGO 0.80 revealed an enrichment in pathways associated with angiogenesis, vasculature development, and blood vessel morphogenesis in luteal tissue obtained from cows supplemented with fish meal. However, luteal blood flow as determined by color flow Doppler ultrasonography did not differ between supplement groups in cows infused with saline (P>0.05). We did observe an increase in blood flow following PGF infusion with a more robust response after the second infusion of PGF (P<0.05). We are in the process of looking at differences in cows that had a regressed or functional CL following infusion and if dietary supplementation affected this response. We are also exploring the influence of low dose uterine infusions of PGF2a on luteal fibroblasts. S100A4 and fibroblast activation protein-a (FAP) are two known genes expressed by luteal fibroblasts during CL regression. S1004A is a calcium binding protein which regulates the assembly and degradation of cytoskeletal proteins leading to remodeling of the extracellular matrix. Likewise, FAP, a serine protease, regulates extracellular matrix remodeling as well as angiogenesis and epithelial-to-mesenchymal transition. PGF2a induced a robust increase in expression of both genes in luteal tissue collected from cows that had a regressed CL following infusion (P<0.05). However, there was a modest increase in tissue obtained from cows with a functional CL, but did not differ from saline treated cows (P>0.05). Furthermore, dietary supplementation did not influence expression following PGF2a treatment (P>0.05). Clearly, there is a greater proportion of cows that retain a functional CL following PGF2a infusions, but the mechanisms remain elusive.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Does the Supplementation of Fishmeal Alter the Cellular Composition of the Corpus Luteum Following Low Dose Prostaglandin F2a? Amanda Hibbard, Anika Shelrud, James Haughian, Jason Bruemmer, Patrick Burns. 2024. Rocky Mountain Reproductive Sciences Symposium. May 2024, Fort Collins, CO.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Intrauterine Infusions of Prostaglandin F2a Increases Luteal Blood Flow. Rebecca Phelps, Denisse Rodriguez Reyna, Makayla Luke, Nia Price, Tyra Goodluck, Amanda Hibbard, James Haughian, Jason Bruemmer, and Patrick Burns. 2024. Rocky Mountain Reproductive Sciences Symposium. May 2024, Fort Collins, CO.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Developing a protocol to quantify lipid droplets in the bovine luteal cell. Frank Gase, Grace Barton, Patrick Burns. Rocky Mountain Reproductive Sciences Symposium. May 2024, Fort Collins, CO.


Progress 07/01/22 to 06/30/23

Outputs
Target Audience:The primary target audience is research scientists and graduate/undergraduate students in reproductive biology, beef/dairy extension specialists, large animal veterinarians and eventually beef/dairy producers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Anika Shelrud completed her Master's Thesis in December 2022 and received the Dean's Citation for her work. Two current Master candidates (Tyra Goodluck and Amanda Hibbard) are working on the project and findings were presented at the 56th annual SSR meeting in Ottawa, Ontario. Elevenundergraduate students have assisted with collection of field data and bench laboratory analysis. Students presented their findings at local and regional scientific meetings. These experiences provide students with unique opportunities that prepare them for a career in reproductive biology. How have the results been disseminated to communities of interest?Preliminary findings have been presented at local, regional, and international scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?We anticipate collecting data on an additional twenty animals during the next reporting period. This will increase sample size and allow us to test the main effects of supplementation and time of ovariectomy on response variables. We will begin conducting RT-qPCR and western blotting analysis to determine expression of genes that are involved with regulated cell death pathways (apoptosis and necroptosis) in luteal tissue collected from cows that retained a functional or regressed CL following PGF2a infusions.

Impacts
What was accomplished under these goals? Studies are ongoing and we have collected data on 50 animals. Regardless of dietary supplementation (corn gluten meal vs fish meal), all cows (100%) that receivedintrauterine infusion of saline retained a functional CL at the time of ovariectomy. However, dietary supplementation affected percentage of cows retaining a functional CL following intrauterine infusion of PGF2a (P <0.05). Seventy-eight percent of the cows receiving fish meal supplementation retained a functional CL following PGF2a infusion, while only 50% retained functional CL following PGF in corn gluten meal supplemented animals - a 50% increase in retained functional CL for the fish meal supplemented group. We have analyzed two key genes that regulate progesterone biosynthesis (STARD1 and CYP11A). Steady-state mRNA expression for STARD1 decreased in luteal tissue collected from cows that received PGF2a at the time of ovariectomy as compared to cows administered saline (P < 0.05). Numerically, steady-state mRNA levels for STARD1 were greater in luteal tissue collected from cows with a functional CL that received PGF2a as compared to tissue collected from cows that had a regressed CL but did not differ statistically. StAR protein did not differ between saline treated cows and cows that retained a functional CL following PGF2a infusions but was decreased in tissue collected from cows that had a regressed CL (P<0.05). Steady-state mRNA expression for CYP11A1 did not differ between saline treated cows and those cows that retained a functional CL following PGF2a infusions but was decreased in tissue collected from cows that had a regressed CL (P < 0.05). However, P450scc protein did not differ among treatment groups. It appears that cows that retain a functional CL following PGF2a infusions maintain StAR protein to support progesterone production. Supplementation does not appear to affect expression of these genes following PGF2a infusions. Clearly, there is a greater proportion of cows that retain a functional CL following PGF2a infusions, but the mechanism remains elusive.

Publications

  • Type: Other Status: Published Year Published: 2023 Citation: Mario Cavazos, Patrick Burns, James Haughian, Anika Shelrud, Jason Bruemmer. 2023. Is progesterone output by the corpus luteum affected by omega 3 fatty acids? Rocky Mountain Reproductive Sciences Symposium. May 2023, Fort Collins, CO.
  • Type: Other Status: Accepted Year Published: 2023 Citation: Tyra Goodluck, Patrick Burns, James Haughian Anika Shelrud, Jason Bruemmer. 2023. Plasma from fish meal supplemented cows improves mitochondria morphology, but not during acute hypoxia. Rocky Mountain Reproductive Sciences Symposium. May 2023, Fort Collins, CO.
  • Type: Other Status: Accepted Year Published: 2023 Citation: Amanda Hibbard, Risa Canton, Anika Shelrud, Tyra Goodluck, Mario Cavazos, Patrick Burns, James Haughian, Jason Bruemmer. 2023. Mechanisms of luteal protection with fish meal supplementation in bovine. Rocky Mountain Reproductive Sciences Symposium. May 2023, Fort Collins, CO.
  • Type: Other Status: Accepted Year Published: 2023 Citation: Amanda Hibbard, Tyra Goodluck, Mario Cavazos, Anika Shelrud, Patrick Burns, James Haughian, Jason Bruemmer. 2023. Luteal protective mechanisms of fish meal supplementation in bovine. Society for the Study of Reproduction. July 2023, Ottawa, Ontario.
  • Type: Other Status: Accepted Year Published: 2023 Citation: Tyra Goodluck, Patrick D. Burns, Anika Shelrud. 2023. Does plasma from fish meal supplemented cows protect luteal cells undergoing acute hypoxia? Society for the Study of Reproduction. July 2023, Ottawa, Ontario.
  • Type: Other Status: Accepted Year Published: 2023 Citation: Patrick Burns. 2023. Omega-3 fatty acids as a nutraceutical for improving corpus luteum function. Regis University. February 2023, Denver, CO.
  • Type: Theses/Dissertations Status: Accepted Year Published: 2022 Citation: Anika Shelrud Mechanisms Behind the Luteoprotective Effects of Fish Meal Supplementation in Bovine: Cytokine Sensitivity and Luteal Blood Supplementation in Bovine: Cytokine Sensitivity and Luteal Blood FlowFlow


Progress 07/01/21 to 06/30/22

Outputs
Target Audience:The primary target audience is research scientists and graduate/undergraduate students in reproductive biology, beef/dairy extension specialists, large animal veterinarians and eventually beef/dairy producers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One M.S. student (Anika Shelrud) is currently working on the project, and she presented preliminary outcomes at the 55th annual SSR meeting in Spokane, WA. Thirteen undergraduate students have assisted with collection of field data and bench laboratory analysis. Students presented their findings at local and regional scientific meetings. One group of students received 1st place honors for their poster presentation at the University of Northern Colorado Academic Excellence Week. This experience provides students with unique opportunities that prepares them for a career in reproductive biology. How have the results been disseminated to communities of interest?Preliminary findings have been presented at local, regional, and international scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?We anticipate collecting data on an additional twenty animals during the next reporting period. This will increase sample size and allow us to test the main effects of supplementation and time of ovariectomy on response variables. We will begin conducting western blotting analysis to determine abundance of proteins in luteal tissue collected from cows that retained a functional or regressed CL following PGF treatment.

Impacts
What was accomplished under these goals? Studies are ongoing and we have collected data on 37 animals. All control cows (100%) receiving saline infusions retained a functional CL at the time of ovariectomy. Cows receiving fish meal supplementation and intrauterine PGF infusion retained a functional CL 64% of the time, while only 44% retained functional CL following PGF in corn gluten meal supplemented animals - a 48% relative increase in retained functional CL for the fish meal supplemented group. Analysis is ongoing for effects of supplementation (corn gluten meal vs. fish meal) or time of ovariectomy (30 vs. 48 h) on other proposed response variables: serum progesterone, luteal mass, steady-state mRNA, or luteal blood flow. Results of these analyses will continue as we work to reach sample sizes with sufficient statistical power. Though as indicated above, we have looked preliminarily at differences in CL function between saline vs. PGF infusion, and fish meal vs. corn gluten meal diets at the time of ovariectomy and see trends toward improvements with fish meal supplementation. Serum progesterone was greater than 1 ng/mL for control cows treated with saline throughout the experimental period. Likewise, serum progesterone remained greater than 1 ng/mL in cows that retained a functional CL following two intrauterine doses of PGF. In this group, progesterone declined initially, but later rebounded between 24 to 30 hours of the study. In cows that had a regressed CL at the time of ovariectomy, progesterone had declined below 1 ng/mL (the threshold defining luteal regression) within 12 to 24 hours following PGF administration. There were minimal changes in luteal diameter (mm) and mass (g) for cows treated with saline or cows administered PGF that retained a functional CL at the time of ovariectomy. Both the CL diameter and mass were decreased in cows treated with PGF that had a regressed CL. Luteal steady-state mRNA expression for key genes that regulate progesterone biosynthesis (STARD1, CPY11A and LDLR) was decreased in tissue for cows that received PGF at the time of ovariectomy as compared to control cows given saline. Numerically, steady-state mRNA levels for these genes was greater in luteal tissues collected from cows with a functional CL that received PGF as compared to tissue collected from cows that had a regressed CL, but did not differ statistically. Thus, there appears to be no apparent rebound in gene expression for key genes that regulate progesterone biosynthesis in tissue collected from cows administered PGF that retain a functional CL. Luteal blood flow measured by color Doppler ultrasonography appears to be greater for cows supplemented with fish meal prior to infusion of either saline or PGF. We are in the process of analyzing luteal blood flow across the treatment groups. A preliminary proteomic analysis has been conducted on luteal tissue collected from saline treated cows to determine if dietary supplementation influences protein abundance. Eight proteins were differentially expressed using a false discovery rate of P < 0.05. GO terms revealed these proteins are associated with metabolism and protein translation.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Anika Shelrud, Winford Rule, Mario Cavazos James Haughian, Jason Bruemmer, Patrick Burns. 2002. Effect of Dietary Fish Meal Supplementation on Bovine Corpus Luteum Blood Flow and Serum Progesterone. Society for Study of Reproduction. July 2022, Spokane, WA.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Winford Rule, Aubrey Chacon, Mario Cavazos, Jason Bruemmer, James Haughian, Anika Shelrud, Patrick Burns. 2022. Effect of Fish Meal Supplementation on Bovine Corpus Luteum Blood Flow and Serum Progesterone. Rocky Mountain Reproductive Sciences Symposium. April 2022, Fort Collins, CO.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Alexander Mazza, Madeline Sanders, Amanda Hibbard, Jason Bruemmer, James Haughian, Anika Shelrud, Patrick Burns. 2002. The Effects of Fish Meal Supplementation on Bovine Corpus Luteum Function. Rocky Mountain Reproductive Sciences Symposium. April 2022, Fort Collins, CO.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Ethan Shepherd, Madison Gremillion, Samantha Marez, Jason Bruemmer, James Haughian, Anika Shelrud, Patrick Burns. 2002. The Effects of Omega-3 Fatty Acids on Steroidogenic Gene Expression on the Bovine Corpus Luteum Following Prostaglandin Infusion. Rocky Mountain Reproductive Sciences Symposium. April 2022, Fort Collins, CO.