Progress 01/01/21 to 12/19/23
Outputs
Target Audience:The target audience for the proposed work primarily includes scientists with interests in mitochondrial biology, lactation biology, and stress physiology, and undergraduate and graduate students in animal science and dairy science. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Over the course of the full performance period of this grant, a total of 2 graduate students, 20 undergraduates, and 2 research technicians were trained in cattle handling and on-farm data collection, and in various basic and advanced laboratory techniques. Students improved their technical proficiency, laboratory skills, scientific skill set, critical thinking and troubleshooting skills, and advanced their education in animal science and cellular/molecular biology through the process. Additionally, the lead graduate student presented results of the study at the American Dairy Science Association (ADSA) meeting in 2022 and in 2023 and participated in the Master's student poster competition at ADSA in 2022. The meeting in 2022 was the first professional conference attended by the student. Attending the conference increased their confidence in science communication, broadened their scope of knowledge, and expanded their professional network. The graduate student also presented their research on the podcast, "The Dairy Signal" organized by Professional Dairy Producers of Wisconsin (PDPW) and was invited to give two additional presentations to animal science groups in Chile and Puerto Rico. Additionally, the PI was invited to give presentations in Uruguay and at ADSA in Ottawa, which increased visibility of the study and enabled the establishment of relationships with potential future collaborators and prospective graduate students. How have the results been disseminated to communities of interest?Research results have been disseminated to target audiences through a number of presentations at professional conferences and universities around the world, through incorporation into undergraduate and graduate courses at the University of Idaho, and through the podcast, "The Dairy Signal" organized by the Professional Dairy Producers of Wisconsin (PDPW). The topic (heat stress and mitochondrial metabolism) were covered in depth across two review papers by my lab group. An empirical paper is forthcoming. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
For objective 1, we successfully completed two animal trials with a total of 24 heat-stressed and pair-fed thermoneutral dairy cows and collected data including production metrics (i.e. milk yield), cow respiration rates, body temperatures, body weights, body condition scores, feed intake, ambient temperature, and ambient relative humidity. We also collected biological samples including blood, mammary gland and liver biopsies, and milk samples for analysis of milk components. All laboratory assays proposed for the collected samples have been completed which included, mitochondrial oxygen consumption (i.e. mitochondrial respiration), RT-PCR (to quantify expression of mitochondrial and nuclear genes), mitochondrial reactive oxygen species production, activity of antioxidant proteins, oxidative damage, blood metabolites (for glucose, non-esterified fatty acids, and beta-hydroxybutyrate) and blood cortisol concentration. Additionally, assays to quantify activity of enzyme complexes in the electron transport chain and western blots (to quantify abundance of mitochondrial and nuclear proteins) were completed this year. The projected was largely managed by a graduate student with PI oversight and guidance and carried out with a team of graduate and undergraduate students. Over the course of the project, two research technicians were also heavily involved by training students and running assays. The graduate student finished her degree program and the results from this project culminated in a Master's thesis. We are currently in the process of completing an empirical manuscript for this objective, which will be submitted for publication. While working on the project, the lead graduate student also wrote a literature review on the mitochondrial response to heat stress, which was published in the Journal of Dairy Science earlier this year. Additionally, the project galvanized a review paper by the PI focused on mitochondrial bioenergetics and metabolism during lactation and with heat stress, which was accepted and is currently in press in the Journal of Dairy Science Communications. Summary statistics and results for objective 1: For this objective, we assigned multiparous Holstein dairy cows to one of two treatment groups in mid-lactation (approximately 100 d in milk): 1) heat stress (HS) or 2) thermoneutral (TN) and housed both groups in a naturally ventilated barn where the temperature-humidity index was below the heat stress threshold for dairy cattle. The trial consisted of two experimental periods (P1 and P2) separated by a 3 d thermoneutral period. During P1, cows in both treatments were maintained under thermoneutral conditions and fed ad libitum for 7 d. For the duration of P2 (10 d), HS cows wore an electric heat blanket. Daily reduction in feed intake for HS cows was calculated as a percent of average daily intake during P1. During P2, TN cows remained under TN conditions but were pair-fed to match the reduction in HS cows' feed intake. This design allowed us to assess the direct effects of heat stress on mitochondrial function. We have completed statistical analyses for all data generated thus far except for protein expression, which is currently underway. Our heat stressed cows had higher body temperatures and slightly elevated respiration rates throughout the trial relative to the thermoneutral cows, indicating that the heat group was indeed heat stressed. In addition, the milk yield dropped by approximately 1.5 kg/cow/day after the heat blankets were applied to the heat stressed group. However, the heat stress appeared to be mild; for example, typical respiration rate for moderately heat stressed cows is above 66 breaths per minute and our heat stressed cows had an average respiration rate of 59 breaths per minute. As in other heat stress studies, non-esterified fatty acid concentrations in circulation decreased in our heat stressed group during P2 compared to P1. In our study heat stress also affected mammary gland ultrastructure; there were fewer mitochondria in the mammary glands of heat stressed cows relative to thermoneutral cows. However, we did not detect significant differences in any other variables measured including gene expression, mitochondrial respiration, oxidant production, or oxidative damage in liver or in mammary tissue. Thus, it does not appear that mild heat stress during lactation has much direct effect on hepatic or mammary mitochondrial function. Key outcomes for objective 1: Through this project, we generated valuable information regarding mitochondrial function during heat stress. We did not detect many differences in mitochondrial function, suggesting that mild, acute, heat stress has little effect on cellular function. However, given that our control group was pair-fed to match the reduction in feed intake in the heat stress group, the lack of mitochondrial differences between treatment groups could indicate that a decrease in dry matter intake is a major factor precipitating impacts of heat stress at the subcellular level. The results from this project will serve as the basis for future grant proposals to assess mitochondrial function relative to dry matter intake in heat stress and thermoneutral conditions. This project enabled hands-on experiential learning opportunities for a number of graduate and undergraduate students and provided practical on-farm experiences where students were able to apply information learned in the classroom. In addition, this project culminated in several presentations, publications, and a Master's thesis. For objective 2, in summer 2022, we attempted to isolate and grow primary bovine mammary epithelial cells to perform an in vitro study to assess the role of hormones in mitochondrial dysfunction associated with heat stress. However, the cells were not viable. We tried again in spring 2023, but again were unsuccessful in isolating viable cells due to issues with the collagenase enzyme used. We recently procured frozen cells from another group and purchased all supplies necessary to complete the in vitro work in early 2024. Data management was carried out according to the data management plan. All biological samples are stored frozen at -80 or -20?C in the PI lab. All electronic data are saved in the raw format and as excel or word files. All written data are stored in notebooks in the PI lab and electronic copies made by scanning. All electronic copies are backed up on an external hard drive and on the cloud. Data will be uploaded into the Data Repository managed by the Research Computing and Data Services group at the University of Idaho upon publication of the empirical work.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2023
Citation:
Skibiel, A. L. In press. Invited review: Hepatic mitochondrial bioenergetics and metabolism across lactation and in response to heat stress in dairy cows. Submitted to Journal of Dairy Science Communications.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Marquez-Acevedo, A.S., W. R. Hood, R. J. Collier, A. L. Skibiel. 2023. Graduate student literature review: Mitochondrial response to heat stress and its implications on dairy cattle bioenergetics, metabolism, and production. Journal of Dairy Science 106: 7295-7309.
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
Marquez-Acevedo, A. S., R. J. Collier, A. L. Skibiel. 2023. Direct effects of heat stress on mitochondrial structure and energy metabolism in lactating dairy cows. Journal of Dairy Science 106 (Suppl. 1): 422.
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
Skibiel, A. L., M. Zachut, G. E. Dahl, A. N. Kavazis, W. R. Hood. 2023. Mitochondrial bioenergetics of extramammary tissues during lactation and in response to heat stress. Journal of Dairy Science 106 (Suppl. 1): 21.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Skibiel, A. L., M. Zachut, G. E. Dahl, A. N. Kavazis, W. R. Hood. Mitochondrial bioenergetics of extramammary tissues during lactation and in response to heat stress. American Dairy Science Association Annual Meeting, Joint Breeding and Genetics and Lactation Biology Symposium: Genomics and Phenomics of Lactation, Ottawa, Canada, June 26, 2023.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Marquez-Acevedo, A. S., R. J. Collier, A. L. Skibiel. Direct effects of heat stress on mitochondrial structure and energy metabolism in lactating dairy cows. Poster presentation, American Dairy Science Association Annual Meeting, Ottawa, Canada, June 25-28, 2023.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Marquez-Acevedo, A. S. and A. L. Skibiel. Efectos metabolicos del estres por calor en la vaca lechera Holstein. Department of Industria Pecuaria, Universidad National de Colombia, Simposio Metabolismo De Energ�a, Bogota, Colombia, March 14, 2023.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Skibiel, A. L. Energy metabolism of dairy cows across lactation and in response to environmental stress. Facultad de Agronom�a, Universidad de la Republica, Montevideo, Uruguay, March 7, 2023.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Marquez-Acevedo, A. S. and A. L. Skibiel. Efectos metabolicos del estres por calor en la vaca lechera Holstein. Department of Industria Pecuaria, University of Puerto Rico at Mayag�ez, Departmental Symposium on Energy Metabolism, virtual presentation, December 6, 2022.
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2023
Citation:
Marquez-Acevedo, A. S. 2023. Direct effects of heat stress on hepatic and mammary mitochondrial function of lactating dairy cattle. Masters thesis, University of Idaho. University of Idaho Theses and Dissertations Digital Collection. https://www.lib.uidaho.edu/digital/etd/
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Progress 01/01/22 to 12/31/22
Outputs
Target Audience:The target audience for the proposed work primarily includes scientists with interests in mitochondrial biology, lactation biology, and stress physiology, and undergraduate and graduate students in animal science and dairy science. Changes/Problems:Since we needed to complete 2 animal trials to achieve the necessary sample size for aim 1 and given grant reviewer comments on the initial proposal regarding the aim 2 approach, we plan to do an in vitro study for aim 2 instead of the original in vivo study. The in vitro approach will allow us to experimentally manipulate incubation temperature and the hormonal milieu of the growing cells in tandem to investigate hormonal regulation of mitochondrial performance under heat stress and thermoneutral conditions in primary bovine mammary epithelial cells. In summer 2022, we attempted to isolate and grow primary bovine mammary epithelial cells, but the cells were not viable. We will either try to isolate cells again in spring 2023, making adjustments based on identified problems this year, or collaborate with a group who already has frozen cells that could be used in the experiment. What opportunities for training and professional development has the project provided?Through the second year of this grant, I have trained 2 graduate students and 13 undergraduates in cattle handling and on-farm data collection and in lab techniques, such as RT-PCR, respiration assays, blood metabolite analysis, and ELISA, among others. These students have improved their technical proficiency, laboratory skills, scientific skill set, critical thinking and troubleshooting skills, and animal science education through the process. Additionally, the graduate students presented at their first professional meeting, American Dairy Science Association meeting where they competed in the graduate studentpresentation competition. The graduate students also presented their research on the podcast, "The Dairy Signal" organized by Professional Dairy Producers of Wisconsin (PDPW). How have the results been disseminated to communities of interest?Thus far we have disseminated results to a small group of animal scientists at the University of Idaho and we've also presented at the annual American Dairy Science Association meeting. Results have also been disseminated to the targeted audience by participating in the podcast, "The Dairy Signal" organized by Professional Dairy Producers of Wisconsin (PDPW). What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period we will finish the remaining laboratory assays (western blot and enzyme complex assays) from the animal trials. Furthermore, we will complete objective 2, finish data analysis, and disseminate results through conferences and publications. One publication will be submitted this year and the second publication stemming from this research will be submitted in 2023.
Impacts
What was accomplished under these goals?
In the second year of funding for this work, we have successfully completed an additional animal trial with approximately 10 heat-stressed and pair-fed thermoneutral dairy cows and procured biological samples, such as blood, mammary gland and liver biopsies, and milk samples. Completing a second trial allowed us to achieve the sample size needed for statistical power. We have completed the majority of the laboratory assays for these samples, but still have enzyme complex assays and western blots to complete. The PI had oversight of the animal trial and benchwork. The work was largely managed by a graduate student and carried out with a team of graduate and undergraduate students. A research technician was also heavily involved in this project by training students and running assays. The heat blanket method of inducing heat stress induced a mild heat stress response (elevated body temperature and only slightly elevated respiration rate). Although milk yield decreased in the heat group after the heat blankets were applied relative to baseline obtained prior to fitting the cows with blankets, there did not appear to many differences in mitochondrial gene expression or respiration, oxidant production, or oxidative damage between the heat-stressed and pair-fed thermoneutral group. The specific objectives of the project are to 1) define patterns of tissue-specific mitochondrial function in heat-stressed lactating cows and 2) to identify the endocrine signaling mechanisms contributing to heat stress-induced mitochondrial dysfunction. Activities completed and data collected for objective 1: 1. Completed an animal trial with approximately 10 heat-stressed and pair-fed thermoneutral dairy cows 2. Collected biological samples - blood, milk, mammary tissue, and liver tissue 3. Collected other physiological, health, production, and environmental data on farm, including cattle respiration rates, body temperatures, body weights, body condition scores, feed intake, milk yield, ambient temperature, and ambient relative humidity 4. Measured mitochondrial respiration in harvested tissues 5. Quantified mitochondrial reactive oxygen species production 6. Completed assays to quantify blood metabolites, circulating hormone concentrations, antioxidant activity, and oxidative damage to protein and lipids. 7. Quantified expression of nuclear and mitochondrial genes that modulate mitochondrial function Summary statistics and results for objective 1: We have completed statistical analyses for all data generated thus far. Our heat stressed cows had higher body temperatures and slightly elevated respiration rates throughout the trial relative to the thermoneutral cows, indicating that the heat group was indeed heat stressed, although the heat stress was mild. Both groups were housed in the same barn where the temperature-humidity index was below the heat stress threshold for dairy cattle. This confirms success of the experimental treatments applied. While we found some numerical differences for particular variables between treatment groups, such as between mitochondrial respiration and gene expression, we did not detect differences in mRNA expression, mitochondrial respiration, oxidant production, or oxidative damage in liver or in mammary tissue. Key outcomes for objective 1: Thus far we have generated valuable information regarding mitochondrial function during heat stress. We did not detect many differences in mitochondrial function, suggesting that mild, acute, heat stress has little effect on cellular function. Alternatively, given that our control group was pair-fed the same percentage reduction in feed intake as the heat stress group, the lack of mitochondrial differences between treatment groups could indicate that a decrease in dry matter intake is a major factor precipitating impacts of heat stress at the subcellular level. This information will serve as the basis for future grant proposals to assess mitochondrial function relative to dry matter intake in heat stress and thermoneutral conditions and mild versus moderate or extreme heat stress. This project has also enabled hands-on experiential learning opportunities for a number of graduate and undergraduate students and provided practical on-farm experiences where students have been able to apply information learned in the classroom. Activities completed and data collected for objective 2: In summer 2022, we attempted to isolate and grow primary bovine mammary epithelial cells to perform an in vitro study to assess the role of hormones in mitochondrial dysfunction associated with heat stress. However, the cells were not viable. We will either try to isolate cells again in spring 2023, making adjustments based on identified problems this year or collaborate with a group who already has frozen cells that could be used in the experiment.
Publications
- Type:
Other
Status:
Published
Year Published:
2022
Citation:
Marquez-Acevedo, A. S., P. Villamediana, C. C. Josefson, R. J. Collier, A. L. Skibiel. 2022. Direct effects of heat stress on hepatic mitochondrial function in lactating dairy cows. Journal of Dairy Science 105 (Suppl. 1): 354.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Marquez-Acevedo, A. S. and A. L. Skibiel. Effects of heat stress on hepatic and mammary epithelial cells and the impact of wildfire-smoke inhalation on dairy calf health. Professional Dairy Producers (PDPW), The Dairy Signal podcast, Rising Stars: Graduate Research Showcase, sponsored by Balchem, July 27, 2022.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Marquez-Acevedo, A. S., P. Villamediana, C. C. Josefson, R. J. Collier, A. L. Skibiel. Direct effects of heat stress on hepatic mitochondrial function in lactating dairy cows. Poster presentation, Production Division Poster MS competition, American Dairy Science Association Annual Meeting, Kansas City MO, June 19-22, 2022.
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Progress 01/01/21 to 12/31/21
Outputs
Target Audience:The target audience for the proposed work primarily includes scientists with interests in mitochondrial biology, lactation biology, and stress physiology, and undergraduate and graduate students. Efforts for this reporting period included discussion of the project in formal classroom instruction, specifically courses in lactation physiology and environmental physiology. Preliminary results of the project were also disseminated to an audience of graduate students and faculty scientists in a departmental seminar. Changes/Problems:This year we completed an animal trial for objective 1, however to due limitations on animal numbers at the university dairy farm and the loss of two animals from our trial due to health issues unrelated to the project, we do not yet have the sample size needed for statistical power as indicated in our power analysis. For this reason, we will repeat this trial with additional animals in spring 2022. What opportunities for training and professional development has the project provided?Through the first year of this grant, I have trained 3 graduate students and 7 undergraduates in cattle handling and on-farm data collection and trained a graduate student and 3 undergraduates in lab techniques, such as RT-PCR, respiration assays, blood metabolite analysis, and ELISA, among others. These students have improved their technical proficiency, laboratory skills, scientific prowess, and animal science education through the process. During the first year of the project, the PI and graduate students have attended conferences, including ADSA and ICFAE-BOLFA. How have the results been disseminated to communities of interest?Thus far we have disseminated preliminary results to a small group of animal scientists at the University of Idaho. After completion of the second animal trial in 2022, results will be presented at a professional conference and submitted for publication. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period we will finish the remaining laboratory assays (adenine nucleotide quantification and analysis of protein expression) from our first animal trial. We will complete a second animal trial for objective 1 to improve our sample size. Furthermore, we will complete objective 2, finish data analysis, and disseminate results through conferences and publication.
Impacts
What was accomplished under these goals?
High ambient temperature and humidity, resulting in heat stress in cattle, is a billion-dollar problem in the dairy industry. Heat stressed cows produce less milk, and are therefore less profitable, and heat stressed cows are more susceptible to disease, thereby increasing treatment costs and culling rates on farm. As global temperatures continue to rise, it is paramount that we develop a solid understanding of the physiological changes occurring in heat stressed cows that underlie these adverse outcomes. Ultimately, this knowledge can be leveraged to devise novel methods to mitigate heat impacts on cattle. In this work, we proposed to focus on mitochondrial structural and functional aberrations as a consequence of heat stress that may contribute to poor performance in heat stressed cows as mitochondria produce needed energy and nutrient precursors for milk synthesis and they are sensitive to fluctuations in temperature. In the first year of funding for this work, we have successfully completed an animal trial with roughly 20 heat-stressed and pair-fed thermoneutral dairy cows and procured biological samples, such as blood, mammary gland and liver biopsies, and milk samples. We have completed the majority of the laboratory assays for these samples. The PI had oversight of the animal trial and benchwork. The work was largely managed by a graduate student and carried out with a team of graduate and undergraduate students. A research technician was also heavily involved in this project by training students and running assays. Thus far our data suggest that heat stress may impair milk production by inducing mitochondrial damage in liver and mammary tissue resulting in lower expression of nuclear genes involved in mitochondrial function and greater oxidative stress. However, caution must be exercised at this point in the project because while there are some numerical differences between treatment groups, we currently lack the statistical power to detect significant differences and the trial must be repeated to increase sample size. This will be accomplished in spring 2022. If these trends hold up with a larger sample size, it would suggest that mitochondria may be a viable target to improve milk production under heat stress conditions. The specific objectives of the project are to 1) define patterns of tissue-specific mitochondrial function in heat-stressed lactating cows and 2) to identify the endocrine signaling mechanisms contributing to heat stress-induced mitochondrial dysfunction. Activities completed and data collected for objective 1: 1. Completed an animal trial with approximately 20 heat-stressed and pair-fed thermoneutral dairy cows 2. Collected biological samples - blood, milk, mammary tissue, and liver tissue 3. Collected other physiological, health, production, and environmental data on farm, including cattle respiration rates, body temperatures, body weights, body condition scores, feed intake, milk yield, ambient temperature, and ambient relative humidity 4. Measured mitochondrial respiration in harvested tissues 5. Quantified mitochondrial reactive oxygen species production 6. Completed assays to quantify blood metabolites, circulating hormone concentrations, antioxidant activity, and oxidative damage to protein and lipids. 7. Finished transmission electron microscopy on mammary and liver tissue 8. Quantified expression of nuclear and mitochondrial genes that modulate mitochondrial function Summary statistics and results for objective 1: We have completed statistical analyses for all data generated thus far. Our heat stressed cows had higher body temperatures and respiration rates throughout the trial relative to the thermoneutral cows. Both groups were housed in the same barn where the temperature-humidity index was below the heat stress threshold for dairy cattle. This confirms success of the experimental treatments applied. While we found some numerical differences for particular variables between treatment groups, such as between mitochondrial respiration and gene expression, we do not yet have enough statistical power to make any robust conclusions. We are limited in animal number on the UI Dairy Farm and two animals were removed from our trial due to health issues unrelated to the project, so we are shy of our target sample size for statistical analysis. For this reason, we will repeat this trial again in spring 2022 to increase sample size. Key outcomes for objective 1: Thus far we have generated valuable information regarding mitochondrial function during heat stress. Furthermore, this project has enabled hands-on experiential learning opportunities for a number of graduate and undergraduate students and provided practical on-farm experiences where students have been able to apply information learned in the classroom. Activities completed and data collected for objective 2: Progress has not yet been made on objective 2, however we plan to complete this work by summer 2022.
Publications
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