Source: UNIVERSITY OF TENNESSEE submitted to NRP
FUNCTIONAL IMPORTANCE OF HIGHER ESTRUS-ASSOCIATED BODY TEMPERATURES (HEAT) TO MAXIMIZE CATTLE FERTILITY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1027936
Grant No.
2022-67015-36374
Cumulative Award Amt.
$650,000.00
Proposal No.
2021-06848
Multistate No.
(N/A)
Project Start Date
Feb 1, 2022
Project End Date
Jan 31, 2026
Grant Year
2022
Program Code
[A1211]- Animal Health and Production and Animal Products: Animal Reproduction
Recipient Organization
UNIVERSITY OF TENNESSEE
2621 MORGAN CIR
KNOXVILLE,TN 37996-4540
Performing Department
Animal Science
Non Technical Summary
Reproductive failure weighs heavily on the minds of cattle producers. Estimated percentage of females failing to become pregnant after a single insemination ranges from 35 to 40% in beef and may be as high as 50 to 70+% in dairy cattle. Failures of this magnitude are not sustainable and are incredibly costly to the US cattle industry. While basis for failure is multi-factorial, heightened levels of estrus activity matter when pregnancy is a desired outcome after breeding. Activity is a major driver of elevated body temperature in females exhibiting estrus. The central hypothesis to be tested in this proposal is Higher Estrus-Associated body Temperatures (HEAT) is functionally important to maximize pregnancy outcomes through direct effects on ovulatory follicle components, especially the oocyte and its surrounding cumulus cells. To test hypothesis, two specific aims will be pursued: 1) determine the extent to which an acute episode of elevated body temperature in estrual females directly impacts ovulatory follicle components, and 2) identify cumulus-oocyte factors promoting meiotic maturation when appropriately activated by HEAT. This novel research utilizes original and complementary basic and applied studies. If confirmed, outcomes would provide invaluable insight regarding inexpensive approaches to improving fertility in cattle. Answers to this interesting question could lead to significant gains in the management of reproduction to improve pregnancy rates.
Animal Health Component
75%
Research Effort Categories
Basic
25%
Applied
75%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3013310102070%
3013410102030%
Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3310 - Beef cattle, live animal; 3410 - Dairy cattle, live animal;

Field Of Science
1020 - Physiology;
Goals / Objectives
An overarching goal of our research is to understand how higher estrus-associated body temperatures (HEAT) are functionally important to maximize cattle fertility. To this end, two objectives with the specific aim of 1) determining extent to which elevated body temperature in cows exhibiting estrus is functionally important to maximize pregnancy outcomes through direct effects on ovulatory follicle components, and 2) identifying factors promoting meiotic maturation in the cumulus-oocyte complex when appropriately activated by an acute physiologically-relevant elevated temperature will be pursued.
Project Methods
SPECIFIC AIM ONE: Determine if acute elevations in body temperature due to heightened levels of estrus activity are functionally important to maximize pregnancy outcomes.Study ONE: Determine impact of HEAT to alter follicular fluid components that may potentiate ovulation and promote meiotic progression of the oocyte. Estrus will be pharmacologically induced in cyclic females. Once a female stands to be mounted by another, it will be randomly allocated to one of two treatments: 1) Restricted activity or 2) Higher activity. Restricted activity females will be moved to a nearby pen to minimize activity-induced increases in body temperature. Higher activity heifers will be moved to a pen with others exhibiting estrus to promote higher vaginal temperatures. Follicular fluid, the cumulus-oocyte complex, and other ovulatory follicle contents (granulosa cells) will be removed by ultrasound-guided vaginal aspiration. Activity and vaginal temperature will be monitored "continuously" via sensor technologies. A portion of follicular fluid samples will be subjected to iTRAQ-based proteomics; other portions will be analyzed for progesterone, estradiol and other protein components (e.g., cytokine profiles). In vivo derived oocytes will be processed for RNA sequencing. Granulosa cell RNA may be isolated to validate reported outcomes and to possibly examine other transcripts in follow on efforts outside scope of this study. DATA WILL BE ANALYZED as described previously by our laboratory and collaborators (cow blood/follicular fluid components and follicle assessment variables/proteome & transcriptome analyses). RESULTS WILL BE USED to document the extent to which estrus activity drives elevated body temperature during the follicular phase of the estrous cycle. If follicular-based consequences are noted, we would then better know the amenability of activity to induce functional changes related to temperature during this critical time period. Validation of candidate proteins and transcripts, biological processes and/or networks will depend on the outcome of this aim. We are excited about the many and varied options that will be available to perform follow-on studies from the incredible base of foundational knowledge to be gained.STUDY TWO: Determine if elevating body temperature through increased walking activity at or around the time of a pharmacologically-induced LH surge improves pregnancy outcomes of beef cows submitted to a fixed time AI. Healthy multiparous beef cows will be randomly allocated to one of two treatments: Control or Increased Activity. Cows allocated to Control group will be moved directly to working facility on horseback in the usual manner and sorted from their calf immediately before breeding limiting the time away from their calves. Cows allocated to the Increased Activity group will be walked for an additional amount of time-distance to elevate body temperature while being moved by the same set of individuals to the working facility on horseback or motorized vehicle in a low stress manner. A pedometer may be placed on horse and/or people walking along with cattle as they make their way to the working facility to record distance and time related to cattle movement. Rectal temperature, chute exit velocity, blood sample (progesterone and estradiol) and ovarian ultrasonography/location (right/left ovary) and mapping of the largest follicle and any significant subordinate follicle will be performed when CIDR is removed and immediately before FTAI. On farm ambient temperature will be recorded at the exact time AI is performed per each animal which will allow for appropriate covariate adjustment during data analysis. Artificial insemination will be performed by same technician(s) while taking effort to ensure even distribution per each treatment to reduce inseminator bias on pregnancy outcome. Seven days after FTAI, a blood sample will be taken for progesterone analysis. Corpus luteum will be sized by ultrasonography. Pregnancy status will be determined 30 to 35 days, 60 to 90 days, and confirmed at calving. At a single location, a subset of females per each treatment (n=20) will be monitored to assess changes in activity (via Hobo G Pendant accelerometers) and vaginal temperatures where a thermometer-based data logger is affixed to a blank CIDR (no hormone) to record temperature every one minute. Devices for this subset of animals will be applied/inserted at the time of CIDR removal. DATA WILL BE ANALYZED using a mixed multivariable logistic regression model to determine if treatment-induced changes in rectal temperature is associated with probability for pregnancy. RESULTS WILL BE USED to determine functional significance, if any, of elevating body temperatures at or around the time of a pharmacologically-induced LH surge. Depending on the extent to which elevated body temperature may be influential on pregnancy outcomes, obtained results will provide insight about possible opportunities to alter management practices to further promote cow fertility submitted to a fixed time AI.SPECIFIC AIM TWO: Identify cumulus-oocyte factors promoting meiotic maturation when appropriately activated by an acute physiologically-relevant elevated temperature: Cumulus-oocyte complexes will be cultured in the maturation medium at 38.5°C or 41°C up to 12 h followed by 38.5°C. Subsets will be evaluated for FYN, miR-125a-3p, and CAV1 RNA (Fig. 13) by ddPCR. FYN protein will be evaluated using a fluorescent bead-based immunoassay at different time during maturation. DATA WILL BE ANALYZED as a randomized block design, blocking on replicate, using generalized linear mixed models (SAS 9.4). Fixed effects in model will be temperature, time, and respective interaction. When relevant, nonlinear regression may be useful to further characterize potential treatment differences. RESULTS WILL BE USED to provide new and novel information regarding presence and abundance of FYN and other variables of interest during bovine oocyte maturation while also charactering impact of heat for different durations. Although beyond scope of this initial study, once new knowledge is gained regarding each of the cumulus-oocyte factors to be evaluated throughout maturation in the bovine, use of chemical inhibitors or enhanced/inhibition through microinjection to assess functional importance of FYN and other components to impact GV-breakdown and/or continued meiotic progression will be possible.

Progress 02/01/24 to 01/31/25

Outputs
Target Audience:Target audiences reached by our efforts include livestock producers attending a UT Institute of Agriculture beef and forage field day and others in attendance at different seminars. We discussed past and current research data highlighting how elevated body temperature during estrus is impactful on components in the preovulatory follicle, especially the oocyte resident within. We also highlighted instances where higher estrous associated temperatures may be a 'friend' to cattle fertility. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students, along with others (undergraduate, graduate and technical staff) have been trained or are in the process of being trained to work with beef cows/heifers as important to conduct animal studies. Undergraduate and graduate students working with other faculty but interested in helping us also learned various different procedural aspects important for beginning cow based studies. The PI's graduate students participated in a University sponsored precision beef field day. Graduate students who had or was in the process of completing thesis, learned to compose a manuscript to withstand peer-review (one published in 2024 is now at Mississippi State pursuing PhD degree; the other graduate student will submit publication in 2025). This latter graduate student and their associated learning while pursuing MS degree, prepared them well to begin a PhD program, spring 2025. How have the results been disseminated to communities of interest?Results have been shared with beef producers attending beef field day in different years. Results have and will continue to be presented to peers and others in attendance at professional meetings (e.g., society for study of reproduction and international embryo transfer society meetings). What do you plan to do during the next reporting period to accomplish the goals?Continue with data collection, analysis and summarization of different study outcomes.

Impacts
What was accomplished under these goals? Toward determining extent to which elevated body temperature in cows exhibiting estrus is functionally important to maximize pregnancy outcomes through direct effects on ovulatory follicle components, studies remain ongoing; data collection continues. We are excited about other data that will be summarized in an upcoming publication that will highlight a positive relationship of higher estrous associated temperature and engagement in estrous active behaviors with advanced preovulatory follicle progression. Regarding specific aim 2 aimed at identifying factors promoting meiotic maturation in the cumulus-oocyte complex when appropriately activated by an acute physiologically-relevant elevated temperature, data have been collected. A manuscript was published January, 2025 highlighting direct effects of an exposure to a physiologically relevant elevated temperature for as few as 2 to 4 hours impacted transcripts with gene products related to progesterone production and signaling, germinal vesicle breakdown, oocyte meiotic progression, transcriptional activity and/or alternative splicing, cell cycle, cumulus expansion, and/or ovulation. Use of multidimensional scaling plots to 'visualize' samples highlighted that oocytes exposed to an acute elevation in temperature are more advanced at the molecular level during the initial stages of maturation. These described efforts represent important steps toward providing novel insight into the dynamic physiology of the COC in the estuarial female bovid, during HEAT and after body temperature returns to baseline.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Mills, M.D., A.B. Pollock, I.E. Batey, M.A. ONeil, F.N. Schrick, R.R. Payton, S.E. Moorey, P. Fioravanti, W. Hipsher, S.M. Zoca and J.L. Edwards. 2024. Magnitude and persistence of higher estrus associated temperatures in beef heifers and suckled cows. Journal of Animal Science, 102. Skae079 Open Access https://doi.org/10.1093/jas/skae079
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Klabnik, J.L., J.E. Beever, R.R. Payton, K.H. Lamour, F.N. Schrick and J.L. Edwards. 2025. A step toward understanding direct impacts of a higher estrus associated temperature (HEAT): Transcript level changes in cumulus-oocyte complexes directly exposed to acute elevated temperature. Animals 15(4), 517 https://doi.org/10.3390/ani15040517
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Fioravanti, P., S.E. Moorey, I. Batey, M. Mills, K. Hill, A. Pollock, R.R. Payton, E. Hessock, F.N. Schrick, S.R. Campagna, M.A. ONeil, and J.L. Edwards. 2024. Impact of Higher Estrus-Associated Temperatures (H.E.A.T.) and Mounting Activity on the Periovulatory Follicular Fluid Metabolome in Beef Heifers. Abstract-Poster presentation at International Embryo Technology Society Meetings, Ft. Worth, TX, January 18-22, 2025.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Klabnik, J.L., J.E. Beever, R.R. Payton, K.H. Lamour, F. N. Schrick, J.L. Edwards. 2024. A step toward understanding direct impacts of a higher estrus associated temperature (HEAT): Transcript level changes in cumulus-oocyte complexes directly exposed to acute elevated temperature. Abstract-Poster presentation at International Embryo Technology Society Meetings, Ft. Worth, TX, January 18-22, 2025.


Progress 02/01/23 to 01/31/24

Outputs
Target Audience:Target audiences reached by our efforts include livestock producers attending a UT Institute of Agriculture beef and forage field day. We discussed past and current research data highlighting how elevated body temperature may be FOE and a friend to cattle fertility. Functional importance of higher estrus-associated body temperatures (HEAT) to maximize cattle fertility was also shared with other scientists in attendance at the USDA-NIFA AFRI Animal Reproduction Annual Project Director Meeting, summer 2023 via poster and co-PI S. Mooreyand participants of the Virginia Tech-Multistate ReproductiveBiology Club (attendees are scientifics, professional staff, graduate students and other). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students, along with others (undergraduate, graduate and technical staff) have beentrained or are in the process of being trained to work with beef cows/heifers as important to conduct animal studies.Undergraduate and graduate students working with other faculty but interested in helping us also learned various differentprocedural aspects important for beginning cow based studies. The PI's graduate students participated in a University sponsored precision beef field day. One wasable to attend theSociety for Study of Reproduction meetings, held in Canada this past year. Two graduate students completed thesis, learned to compose a manuscript to withstand peer-review. One of our graduate student's manuscript was published in 2023; associated learning prepared her well to begin a PhD program. The other graduate student leaned to compose a manuscript for submission for peer review. This associated learning prepared her well to begin a PhD program at Mississippi State. How have the results been disseminated to communities of interest?Results have been shared with beef producers attending beef field day in each of two different years. What do you plan to do during the next reporting period to accomplish the goals?Continue with data collection, analysis and summarization of different study outcomes.

Impacts
What was accomplished under these goals? Towards determining extent to which elevated body temperature in cows exhibiting estrus is functionally important to maximize pregnancy outcomes through direct effects on ovulatory follicle components, studies are ongoing; data collection continues. We are beginning to make progress with publishing some of our findings and are excited about data that will be summarized in upcoming publications. Regarding specific aim 2 aimed at identifying factors promoting meiotic maturation in the cumulus-oocyte complex when appropriately activated by an acute physiologically-relevant elevated temperature, data have been collected. A manuscript is in the final stages of preparation to highlight research outcomes for this objective.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Pollock, A.B., S.E. Moorey, E.A. Hessock, J.L. Klabnik, R.R. Payton, F.N. Schrick, S.R. Campagna, and J.L. Edwards. 2023. Relationship between higher estrus-associated temperatures (HEAT) and the bovine preovulatory follicular fluid metabolome. Frontiers in Animal Science, Volume 4; https://doi.org/10.3389/fanim.2023.1241033
  • Type: Journal Articles Status: Under Review Year Published: 2024 Citation: 1) Mills, M.D., A.B. Pollock, I.E. Batey, M.A. ONeil, F.N. Schrick, R.R. Payton, S.E. Moorey, P. Fioravanti, W. Hipsher, S.M. Zoca and J.L. Edwards. 2023. Magnitude and persistence of higher estrus associated temperatures in beef heifers and suckled cows.
  • Type: Theses/Dissertations Status: Published Year Published: 2023 Citation: Abigayle Pollock, M.S. August 2023. Thesis title Relationship between higher estrus associated temperatures (HEAT) on the bovine preovulatory follicular fluid metabolome
  • Type: Theses/Dissertations Status: Published Year Published: 2023 Citation: Megan Mills, M.S. December 2023. Thesis title Higher estrus associated temperatures and associated factors in beef heifers and suckled cows
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Pollock, A.B., S.E. Moorey, E.A. Hessock, J.L. Klabnik, R.R. Payton, F.N. Schrick, and J.L. Edwards. 2023. Relationship of higher estrus-associated temperatures (H.E.A.T.) on the bovine periovulatory follicular fluid metabolome. Abstract-poster presentation SSR meetings, Ottawa, Canada. July 11-14, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Pollock, A.B., E.A. Hessock, S.E. Moorey, L.G. Schneider, Y. Zhao, X. Yang and J.L. Edwards. 2022. Positive relationship of estrus activity and body temperature changes in cattle. Abstract-poster presentation SSR meetings, July 26-29, 2023 Spokane, Washington.


Progress 02/01/22 to 01/31/23

Outputs
Target Audience:Target audiences reached by our efforts during this reporting period include livestock producers attending a UT Institute of Agriculture beef and forage field day. We discussed past and current research data highlighting how elevated body temperature and be FOE and friend to cattle fertility. Functional importance of higher estrus-associated body temperatures (HEAT) to maximize cattle fertility was also shared with other scientists in attendance at the USDA-NIFA AFRI Animal Reproduction Annual Project Director Meeting, summer 2022 and participants of the Virginia Tech-Multistate Reproductive Biology Club Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A PhD student in the final stages of their program acquired important training related to the collection and examination of transcript abundance in cumulus oocyte complexes which is an important part of one of our aims. Two other graduate students have been hired. These individuals, along with others (undergraduate, graduate and technical staff) have been trained or are in the process of being trained to work with beef cows/heifers as important to conduct animal studies. Undergraduate and graduate students working with other faculty but interested in helping us also learned various different procedural aspects important for beginning cow based studies. The PI's graduate students participated in a University sponsored beef and forage field day and were able to attend the Society for Study of Reproduction meetings, held in Spokane Washington. Athough much of their efforts focused on presenting preliminary data appearing in funded proposal, insight acquired by discussing some aspects of proposed or ongoing studies was helpful for ongoing and/or future studies. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Continue with data collection, analysis and summarization of different study outcomes.

Impacts
What was accomplished under these goals? Estimated percentage of females failing to become pregnant after a single insemination ranges from 35 to 40% in beef and may be as high as 50 to 70+% in dairy cattle. Failures of this magnitude are not sustainable and are incredibly costly to the cattle industry. While basis for failure is multi-factorial, heightened levels of estrus activity matter when pregnancy is a desired outcome after breeding. Activity is a major driver of elevated body temperature in females exhibiting estrus. We are seeking to determine if Higher Estrus-Associated body Temperatures (HEAT) are functionally important to maximize pregnancy outcomes through direct effects on ovulatory follicle components with special emphasis on the oocyte and its surrounding cumulus cells. This novel research utilizes original and complementary basic and applied studies. If confirmed, outcomes could provide invaluable insight regarding inexpensive approaches to improving fertility in cattle. Answers to this interesting question could lead to significant gains in the management of reproduction to improve pregnancy rates. Towards determining extent to which elevated body temperature in cows exhibiting estrus is functionally important to maximize pregnancy outcomes through direct effects on ovulatory follicle components, studies have been initiated and data collection has begun. Regarding specific aim 2 aimed at identifying factors promoting meiotic maturation in the cumulus-oocyte complex when appropriately activated by an acute physiologically-relevant elevated temperature, a preliminary study was conducted by my PhD student to determine the extent to which an elevated temperature observed in females exhibiting a heightened level of estrus activity impacts abundance of certain transcripts others have associated with promoting oocyte maturation.

Publications

  • Type: Theses/Dissertations Status: Other Year Published: 2022 Citation: Jessica Bradford Klabnik, Ph.D. December 2022. Dissertation title: Elevations in body temperature: Impacts on the transcriptome of the periovulatory follicle components with direct effects on the maturing cumulus-oocyte complex Embargoed until Publication of relevant chapter in refereed journal article.