Source: UNIV OF WISCONSIN submitted to
UNRAVELLING CHANGES IN MAMMARY GLAND DEVELOPMENT DUE TO IN UTERO HEAT STRESS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1026119
Grant No.
2019-67015-34538
Project No.
WIS04020
Proposal No.
2020-11480
Multistate No.
(N/A)
Program Code
A1231
Project Start Date
Nov 15, 2020
Project End Date
Nov 14, 2023
Grant Year
2023
Project Director
Laporta Sanchis, J.
Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
Animal and Dairy Sciences
Non Technical Summary
Global climate change has a massive impact on agricultural operations. In the United States alone, milk production losses associated with heat stress during lactation cost the dairy industry up to $1.5 billion annually. While most dairy producers around the globe understand the value of cooling lactating cows to limit the very visible drop in milk production, many still do not cool dry cows. The dry period is a non-lactating period between lactations and is concurrent with the last trimester of gestation in dairy cattle. Exposure of the pregnant dam to heat stress during the dry period significantly reduces her milk production by about 5 kg/d in the following lactation, but also leads to transgenerational effects on her offspring. For example, female calves born to heat stressed dams will produce ~3.5 kg/d less milk during their first and second lactations and are more likely to be culled from the herd relative to their unexposed heard mates. Our hypothesis is that if an unborn dairy calf experiences suboptimal thermal conditions in utero, they will experience abnormal growth and development of their mammary glands leading to impaired milk production in adulthood. Specific objectives are to: 1) determine how in utero exposure to heat stress impacts mammary gland growth and development at birth, weaning and around puberty, 2) determine how intrauterine heat stress impacts growth of the mammary gland in response to hormones during gestation, and 3) to assess the response of mammary tissue later in life to thermal conditions that are similar to or different from those experienced during fetal development. We expect to identify the mammary histological and molecular changes initiated by exposure to heat stress in utero that deviate from the normal developmental trajectory of the mammary gland. These changes might explain the impaired mammary gland function and milk yield of those animals later in life when they begin lactating. This proposal will contribute to fundamental concepts of fetal programming in mammary gland development and function in dairy ruminants, enhance the use of dry cow cooling as a management strategy to increase milk yield across multiple generations, and characterize critical windows of mammary development that can be targeted for interventions to alleviate the negative effects of in utero heat stress on future lactation performance.
Animal Health Component
100%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3063499104050%
3053410102050%
Knowledge Area
305 - Animal Physiological Processes; 306 - Environmental Stress in Animals;

Subject Of Investigation
3499 - Dairy cattle, general/other; 3410 - Dairy cattle, live animal;

Field Of Science
1040 - Molecular biology; 1020 - Physiology;
Goals / Objectives
The broad objective of this proposal is to unveil the effects of exposing the developing fetus to hyperthymic intrauterine environment on mammary gland development, including mammary morphology, molecular pathways, and cellular events occurring at key ontogenic stages such as birth, pre-weaning, pubertal, and gestation stages. We predict that exposure to heat stress in utero will induce a cascade of developmental alterations in the mammary gland, which will be evident at key stages of mammary ontogeny. To test this, we will assess mammary architecture and growth at gross and ultrastructural scales. Furthermore, we will investigate the underlying molecular and cellular mechanisms mediating effects of in utero heat stress on mammary growth and development.The proposed project will incorporate both in vivo and in vitro experiments to address the following objectives: 1) determine how in utero exposure to heat stress impacts mammary gland growth and development at birth, weaning and around puberty, 2) determine how intrauterine exposure to heat stress impacts growth of the mammary gland in response to mammogenic hormones during gestation, 3) assess the response of mammary tissue later in life to thermal conditions that are similar or different from those experienced during fetal development.
Project Methods
Holstein dairy heifers were gestated under two intrauterine thermic conditions: (1) in utero cooled (IUCL, n=20) and (2) in utero heat stress (IUHS, n=20) during the last 55 days of gestation. Forty heifers were relocated from the University of Florida to the University of Wisconsin-Madison (December 2020, at ~ ten weeks of age) to continue a longitudinal study. The animal portion and tissue collection for objective one of the original proposal was completed before relocating the heifers. Weaning to pubertal age. Upon arrival to the University of Wisconsin, all heifers will be under the same nutritional management and environmental conditions at the UW-Madison Arlington Research Station through their first lactation to ensure the effects observed are a result of the prenatal rather than the postnatal environment. From weaning to puberty (~2 - 12 months of age), noninvasive mammary gland measures, body weights, hip height, withers height, and rectal temperature will be recorded monthly. Blood samples will be collected monthly for hormone analyses from 6 months of age weekly until the onset of puberty to characterize the profile of circulating mammogenic hormones (e.g., estrogen, progesterone, cortisol, and prolactin) at the onset of puberty. Additionally, follicular development will be monitored weekly by ultrasound to track follicular development. One mammary biopsy will be performed at puberty. Mammary tissue will be stored at -80C for future laboratory assessment of gene and protein expression.Breeding. Heifers will be bread according to the University of Wisconsin Dairy Facility standard operating procedures. Sexed semen from two Holstein bulls of similar genetic merit will be used to inseminate all heifers balanced by treatment artificially. Pregnancies will be confirmed by ultrasound at 30 and 60 days post artificial insemination.Gestation. Pregnant heifers will remain in the herd, managed under identical conditions. From pregnancy confirmation to calving (approx. from 13 to 24 months of age), growth and noninvasive mammary gland measures be recorded. Pregnant heifers will be subjected to two mammary gland biopsies during gestation (~150 and 260 days) from alternate rear quarters. Additionally, three blood samples will be collected from tail vessels and processed for hormone analyses (e.g., estrogen, progesterone, cortisol, and prolactin) 5 days prior to biopsy and 5 days post-biopsy. Harvested mammary tissue from in utero cooled and in utero heat-stressed pregnant animals will be prepared and cultured in a humidified chamber with 5% CO2 at 38.5 °C or 40.5 °C (TN vs. TS, respectively) for 24 h in media 199 with 1 ng/mL bovine insulin, and 6.5 ng/mL hydrocortisone. After culture, explants in each treatment will be stored in RNA later, liquid nitrogen, and 4% neutral formalin for gene expression, immunoblot, and histological analyses, respectively.Downstream laboratory analyses are summarized below.DNA, protein, and fat content. For all developmental stages, DNA content of the mammary tissue will be measured to serve as an estimate of the total number of cells.Histological analyses. For all developmental stages mammary tissue will be sectioned (5 µm) and stained with hematoxylin & eosin, Masson's trichrome and red oil to quantify lipid storage in adipocytes and relevant mammary characteristics. (e.g. ductal area, number and length of ductal branches, and connective and adipose tissue area). Cell proliferation and apoptosis will be assessed through the Ki67 assay and Tdt dUTP nick end labeling assay, respectively.Gene and protein expression. For all developmental stages after mammary tissue homogenization, RNA and protein will be isolated using commercial kits. Expression of genes and proteins of the major mammary gland developmental and signaling pathways will be evaluated by qRT-PCR and immunoblotting. Target genes will include those involved in mammary gland development (e.g., branching morphogenesis, bud formation and elongation, cell growth, differentiation, survival, and metabolism. qRT-PCR data will be analyzed using the 2-ΔΔCt method.Methylation analysis. We will use reduced representation bisulfite sequencing (RRBS) approach to assess IUHT and IUCL mammary tissue's methylation patterns for all four developmental stages. Briefly, purified DNA libraries will be sequenced using the Illumina HiSeq 4000 system to generate 50 bp single-end reads. Read quality control, genome alignment, differential gene expression analysis, and pathway analyses (KEGG, GO, and GSEA) will be performed.

Progress 11/15/20 to 11/14/23

Outputs
Target Audience:The specific target audience for the research outcomes of the project will be scientific colleagues in the animal and dairy science field, particularly tomammary gland and lactation biologists, including faculty, industry scientists, graduate students, and post-doctoral trainees in the field. The outcomes of this project will be disseminated to the allied dairy industry professionals and Extension specialists to help prevent the negative consequences of heat stress at a practical level. Changes/Problems:Despite minor changes in the proposed studies, including the relocation of the principal investigator, the proposal Aims were executed and completed as planned. This report excludes ongoing laboratory work from puberty and gestation mammary biopsies and the summary of the granddaughter's (F2) growth trajectory and mammary histology. Other ongoing work includes the evaluation of the gastrointestinal tract and ovaries of the F1heifers. USDA funding will be acknowledged at the time of publication. What opportunities for training and professional development has the project provided?Two Ph.D. students have been significantly involved in all aspects of this grant. One graduated in April 2022, while the other doctoral student worked on this project with independent funding from a College Fellowship. Additionally, at least six undergraduate students assisted in various aspects of this grant and gained valuable hands-on training on the farm and in the laboratory. Furthermore, three interns and/or hourly students have been temporarily hired by this grant to help with different aspects, including animal husbandry, sample collection, and laboratory work. Finally, this grant partially supported three Postdocs over four years. How have the results been disseminated to communities of interest?The dissemination of results to the scientific community has been primarily through peer-reviewed publications. So far, 11 such papers have been published, with two manuscripts under review and two more in preparation. Additionally, the project's findings have been disseminated through local seminar series at the University of Florida and the University of Wisconsin, where Dr. Laporta and the graduate students have presented. Dr. Laporta has also presented this work at Animal and Dairy Science Annual Conferences and at various in-person and virtual meetings, webinars, and workshops across the United States throughout the grant duration (2019-2023). These presentations include the Florida Ruminant Nutrition Conference, Southwest Nutrition Conference, Tristate Nutrition Conference, Four-State Dairy Nutrition and Management Conference, American Society of Animal Science Cell Biology Symposium, and two Professional Dairy Producers of Wisconsin Dairy Signal Webinars. Dr. Dahl (Co-PI) has disseminated this work via various outlets, including webinars and workshops across the United States. Dr. Laporta has also presented the project's findings at international conferences like the 2023 European Association of Animal Production Annual Meeting (EAAP). One publication (Laporta J., Ferreira F.C., Ouellet V., Dado-Senn B., Almeida A.K., DeVries A., Dahl G.E. 2020.Late-gestation heat stress impairs daughter's and granddaughters' lifetime performance. Journal of Dairy Science. 103(8):7555-7568) was featured as Editor's Choice in the Journal of Dairy Science in 2020 and selected as one of the 100 most cited papers in 2022 and 2023. Lastly, popular press articles and extension reports have been written to summarize the practical findings of this grant for the dairy community. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have successfully completed all proposed objectives in both phases of the USDA-NIFA grant. A detailed summary for each phase 1 and 2is outlined below. Phase 1: Generation of F1 daughters.Environmental treatments (HS: heat stress,CL: active cooling with fans and soakers) were applied to 80 pregnant Holstein cows (dams) during the last 60 days of gestation for the generation of 40 in-utero HS and 40 in-utero CL (IU-HSandIU-CL; F1 = daughters, respectively) in summer-fall 2020. (1A)Birth to weaning developmental stage and euthanasia.A subset of thirty-two F1 female daughters was euthanized at two time points (day 0 = birth and day 63 = weaning) to harvest mammary gland tissue (i.e., mammary parenchyma and fat pad) for histological and molecular purposes. *Note: other tissues collected for follow-up collaborative studies include the gastrointestinal tract (ileum, jejunum, and duodenum), ovaries, adrenal glands, and skin tissue. Summary of results:At birth (day 0), the heifers born to HS cows were smaller, shorter, and had reduced apparent IgG absorption efficiency than those born to CL dams. The reduced body weight and stature measures persisted in IU-HS heifers until day 63 of age (post-weaning) with a 7% lower average daily gain, reduced starter consumption, and lower hematocrit percentages relative to IU-CL heifers. Additionally, IU-HS heifers had reduced gross thymus, spleen, thyroid gland, and heart weight at birth and larger adrenal glands and kidneys but smaller ovaries relative to their live body weight at day 63. The mammary gland of IU-HS heifers was smaller and shorter in length relative to IU-CL heifers at day 0 and day 63, even after adjusted for body weight. When evaluating mammary microstructure at birth, IU-HS heifers had fewer mammary gland parenchyma epithelial ductal structures. The IU-HS heifers' mammary gland parenchyma luminal and total ductal structure cross-sectional areas were reduced at day 0 and day 63. The adipocytes within the mammary fat pad of IU-HS heifers were smaller at birth, but differences were not detected at day 63. The IU-HS heifers had diminished mammary gland parenchyma total, stromal, and epithelial cellular proliferation at both time points.In summary, prenatal hyperthermia disrupts early-life mammary development in dairy calves with potential long-term consequences on milk production. (1B) Weaning to Puberty developmental stage.The remaining subset of F1 daughters was transported to the University of Wisconsin. Blood samples were collected to detect Progesterone as an indicator of puberty attainment. Once all F1 heifers (N=40) were confirmed cycling, an estrous synchronization protocol was applied to perform mammary gland biopsies at the time of high systemic estrogen concentrations of the cycle (at approximately 12 months of age). In addition, mammary gland biopsies of F1's were collected for histological and molecular analyses. All F1 Heifers were artificially inseminated in tandem as they reached 14 months of age and the desired height characteristics from November-February (2021). Four high genetic merit bulls were used to minimize inbreeding. Thirty-two F1 heifers were successfully pregnant (carrying female fetuses;F2= granddaughter generation). Mammary gland biopsies were performed at 6 and 9 months of gestation. In addition, non-invasive mammary measures (udder depth, width, teat length, and distance) were collected bi-monthly, and three-dimensional images were taken to track udder volume during pregnancy. Summary of results:Body weight, hip height, and front and rear teat length were measured at 120, 240, 300, and 360 d of age, and MG length at 360 d of age. After puberty attainment, [progesterone (P4) concentrations > 1 ng/mL] at 310 ± 15 d of age, heifers were assigned to an estrus synchronizing protocol: d-9: GnRH+CIDR, d-2: PGF2α, d-1: PGF2α+CIDR removal. On d0 (~24 hr pre-estrus) and d12, blood was collected to capture expected high estradiol (E2) and low P4 and low E2 and high P4, respectively. On d0 and d12, mammary gland tissue biopsies were performed on a subset of heifers for histological analysis to measure alveolar and ductal areas. Compared with IU-HS, IU-CL heifers had greater body weight and hip height at 120 and 240 days of age. The front and rear teats were greater in IU-CL heifers at d120, 240, and 360 days of age. Mammary gland length at 360 d was greater in IU-CL heifers relative to IU-HS heifers. On day 0 of the cycle, P4 concentrations were similar between treatments. However, E2 concentrations were greater in IU-CL heifers relative to IU-HS. On d12 of the cycle, P4 and E2 concentrations were similar between treatments. Alveolar and ductal inner lumen and outer area were similar between treatments. After the F1 heifers became pregnant, the growth trajectory and gestation length were similar between the IU-CL and IU-HS groups.Results from pregnancy mammary growth are being processed and analyzed.Prenatal heat abatement increased mammary gland teat length and gland size during development leading to puberty and increased E2 near estrus without major microstructural differences in mammary tissue. Phase 2: Generation of F2 granddaughters.Calving of the F2 granddaughter generation, whose F1 mothers were IU-CL or IU-HS (CL-F2andHT-F2heifers), started in September 2022 and continued until December 2022. Non-invasive measures, including body weight, hip height, withers height, chest girth, and head circumference, were measured at birth, d7, d21, d35, d49, and d70. Mammary gland teat length and left-right and front-rear teat distances were measured at d21, d35, d49, and d70. Average daily gain was calculated for the pre-weaned period (0-49d). At d70, mammary ultrasounds were performed on the rear left and right quarters to quantify mammary parenchyma and fat pad areas, and a mammary biopsy was performed to quantify epithelial structures, estrogen receptor (ERα), and cell turnover (cell proliferation/death ratio). A mammary gland biopsy was performed at weaning (70 days of age, approx. 1 week after weaning). Summary of results:F2's heifer growth (birth to d49 was similar between CL-F2 and HT-F2 for all parameters evaluated. The CLF2 heifers had shorter TD than HTF2 from birth to day 49. On day 70, CL-F2 tended to have a greater left mammary parenchyma area than HT-F2, and the average mammary parenchyma area (right and left quarters) was greater in CL-F2 than HT-F2. Although the left fat pad was smaller in HT-F2, the average left and right fat pad was not different. The lumenal and non-lumenal epithelial structures in the parenchyma of HT-F2 were significantly smaller than that of CL-F2. In addition, HT-F2 had a reduced percentage of proliferating cells in the epithelial cell and stromal compartments and a greater percentage of cells undergoing apoptosis, particularly in the stroma compartment. The percent of mammary epithelial cells expressing ERα was significantly reduced in HT-F2 heifers.In summary, although HT-F2 heifers grow at an identical rate to CL-F2 during the pre-weaning phase, their mammary glands have less parenchyma area with fewer epithelial-ductal structures characterized by reduced cell turnover and lower estrogen receptor expression.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Davidson B.D., E.T. Gonzales, G.L. Mast, J. Laporta (2023) Late-gestation heat stress in Holstein dams programs in utero development of daughter's germline, triggering skin and hair morphology adaptations of granddaughters Journal of Dairy Science Communications Published Online August 18, 2023. https://doi.org/10.3168/jdsc.2023-0400
  • Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Guadagnin A.R., Pe�agaricano F., Dahl G.E., Laporta J. Effects of maternal exposure to heat stress on offsprings adrenal gland histomorphology and transcriptome. In review at Scientific Reports
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Dado-Senn B., Field S.L., Davidson B.D., Dahl G.E., Laporta J. 2022. In utero hyperthermia in late-gestation derails dairy calf early-life mammary development. Journal of Animal Science, 100 (10), skac186.
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Davidson B., Sarlo K., Dahl G.E., Mateescu R., Laporta J. 2022. Effect of in utero exposure to hyperthermia on postnatal hair length, skin morphology, and thermoregulatory responses. Journal of Dairy Science, 105(11):8898-8910.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Laporta J., Dado-Senn B., Skibiel A.L. 2021. Late gestation hyperthermia: epigenetic programming of daughter's mammary development and function. Domestic Animal Endocrinology. Special Edition: Recent Contributions from Women Scientists to Animal Endocrinology: Pregnancy and Lactation https://pubmed.ncbi.nlm.nih.gov/34600221/
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Laporta J. 2021. ADSA Foundation Scholar Award: Early-life exposure to hyperthermia: productive and physiological outcomes, cost, and opportunities. Journal of Dairy Science, 104(11):11337-11347 https://pubmed.ncbi.nlm.nih.gov/34419283/
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Dado-Senn B., Field S. L., Davidson B. D., Casarotto L. T., Marrero M. G., Ouellet V., Cunha F., Sacher M. A., Rice C. L., Maunsell F. P., Dahl G. E., Laporta J. 2021. Late-gestation in utero heat stress limits dairy heifer early-life growth and organ development. Frontiers in Animal Science, Animal Physiology and Management. https://doi.org/10.3389/fanim.2021.750390
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Davidson, B.D., Dado-Senn B., Ouellet V., Dahl G.E., Laporta J. 2021. Effect of late-gestation heat stress in nulliparous heifers on postnatal growth, passive transfer of immunoglobulin G, and thermoregulation of their calves. JDS Communications, 2(3):165-169
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Davidson B.D., Rosa Padilla N., Fabris T.F., Trevisan L., Ouellet V. Toledo I.T., Dahl G.E., Laporta J. 2020. Late-gestation heat stress abatement in dairy heifers promotes thermoregulation and improves productivity. Journal of Dairy Science.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Ouellet V. Laporta J., Dahl G.E. 2020. Late gestation heat stress in dairy cows: effects on dam and daughter. Theriogenology.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Laporta J., Ferreira F.C., Ouellet V., Dado-Senn B., Almeida A.K., DeVries A., Dahl G.E. 2020. Late-gestation heat stress impairs daughters and granddaughters lifetime performance. Journal of Dairy Science. 103(8):7555-7568. doi: 10.3168/jds.2020-18154.


Progress 11/15/21 to 11/14/22

Outputs
Target Audience:Specific target audience for the research outcomes of the project will be scientific colleagues in the animal and dairy science field, particularly to mammary gland and lactation biologists including faculty, industry scientists, graduate students, and post-doctoral trainees in the field. The outcomes of this project will be disseminated to the allied dairy industry professionals and Extension specialists as well to help prevent the negative consequences of heat stress at a practical level. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two Ph.D. students have been integrally involved in all aspects of the project. One PhD student graduated (April 2022) and one continue to work for this project (with independent funding). Four undergraduate students assisted in this project for this reporting period. An intern was hired by this grant to assist with sample collection. One Postdoc was hired (April 2022) to continue the farm and laboratory work, and data analysis for final publications. How have the results been disseminated to communities of interest?The dissemination of results to the scientific community has been through scientific peer-reviewed publications, local seminar series (University of Wisconsin Department seminars by Dr. Laporta and graduate students), Animal and Dairy Science annual conferences (by graduate students), and in person and virtual presentations all across the country by Dr. Laporta, including the Florida Ruminant Nutrition Conference, Southwest Nutrition Conference, Tristate Nutrition Conference, Four-State Dairy Nutrition And Management Conference, and American Society of Animal Science Cell Biology Symposium, Professional dairy Producers of Wisconsin Dairy Signal Webinar. What do you plan to do during the next reporting period to accomplish the goals?The next reporting period will include pubertal and gestational mammary gland growth results (including teat length, distance, udder depth and length, and 3D imaging) and body growth (body weight, hip height and withers height). These data are currently being summarized and analyzed. The report will also include the histology results of mammary biopsies performed at puberty, mid- and late-gestation periods, and a summary of the granddaughter's (F2) growth trajectory.

Impacts
What was accomplished under these goals? Completed phases of this project (2019, 2020, and 2021 previous reporting) include generation of the daughter and mammary tissue collection. Briefly, environmental treatments (HS: heat stress, CL: active cooling with fans and soakers) were applied to 80 pregnant Holstein dams during the last 60 days of gestation for the generation of in utero HS and in utero CL, IUHS, and IUCL; F1 daughters, respectively). A subsetof F1 female claves (N=16) was euthanized at two time points (day 0 = birth, and day 63 = weaning) to harvest mammary gland tissue (i.e., mammary parenchyma and fat pad) for histological and molecular purposes record organ and carcass weights. Significant milestones were achieved during the reporting period:October 2021-October 2022 include: Mammary gland tissue analysis: birth and weaning stage. Mammary gland parenchyma and fat pad tissues harvested from birth and weaning euthanasia (d0 and d63) were analyzed and published (see publications section). Briefly, we quantified gross mammary morphology, mammary fat pad and parenchyma composition, tissue microstructure, and cellular proliferation. Relative to in utero cooled heifers, the whole mammary gland and the fat pad of in utero heat-stressed heifers were lighter at both d0 and d63. In addition, the mammary parenchyma tissue portion of the udder was smaller at birth, with stunted ductal development and significantly reduced cellular proliferation, both at birth and after weaning. Mammary gland biopsies: pubertal stage. Blood samples were collected to detect Progesterone as an indicator of puberty attainment. Once all F1 heifers (N=40) were confirmed cycling, an estrous synchronization protocol was applied to perform mammary gland biopsies at the time of high systemic estrogen concentrations of the cycle (at approximately 12 months of age). In addition, mammary gland biopsies of F1's were collected for histological and molecular analyses. Breeding/Artificial insemination. F1 Heifers were artificially inseminated in tandem as they reached 14 months of age and the desired height characteristics from November-February. Four high genetic merit bulls were used to minimize inbreeding. Mammary gland biopsies: pregnancy stage. Sixteen in utero cooled and twelve in utero heat stress F1 heifers were successfully pregnant and carrying female fetuses (N=30). Mammary gland biopsies were performed at 6 and 9 months of gestation. In addition, non-invasive mammary measures (udder depth, width, teat length, and distance) were collected bi-monthly, and three-dimensional images were taken to track udder volume during pregnancy. Currently ongoing. Calving started in September 2022 and will continue until December 2022.The daughters born to the F1 dams (F2, granddaughters; second generation) will be weighed at birth, and non-invasive measures, body, and mammary measures will be recorded to track their growth through the pre-weaning phase.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Davidson BD, Beard AD, Garcia-Guerra A, Dahl GE, Wiltbank MC Laporta J. Prenatal hyperthermia affects ovarian morphology, histology, and Anti-Mullerian hormone concentrations of pre-weaned heifers. 2022 American Dairy Science Association Annual Meeting (Abstr.).
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Casarotto LT, Liu L, Penagaricano F, Laporta J, Dahl GE. 2022. Late gestation heat stress alters placental DNA methylation in cows. 2022 American Dairy Science Association Annual Meeting (Abstr.).
  • Type: Journal Articles Status: Accepted Year Published: 2022 Citation: Laporta J, Lemley C, Chavatte-Palmer P. 2022. Editorial: The Influences of Early Life Experiences on Future Health and Productivity. Frontiers in Animal Science. https://doi.org/10.3389/fanim.2022.962580
  • Type: Journal Articles Status: Accepted Year Published: 2022 Citation: Dado-Senn B, Field SL, Davidson BD, Dahl GE, Laporta J. 2022. In utero hyperthermia in late-gestation derails dairy calf early-life mammary development. J. Anim. Sci. 100(10):skac186.
  • Type: Journal Articles Status: Accepted Year Published: 2022 Citation: Davidson B, Sarlo K, Dahl GE, Mateescu R, Laporta J. 2022. Effect of in utero exposure to hyperthermia on postnatal hair length, skin morphology, and thermoregulatory responses. J Dairy Sci. 105(11):8898-8910.


Progress 11/15/20 to 11/14/21

Outputs
Target Audience:Specific target audience for the research outcomes of the project will be scientific colleagues in the animal and dairy science field, particularly to mammary gland and lactation biologists including faculty, industry scientists, graduate students, and post-doctoral trainees in the field. The outcomes of this project will be disseminated to the allied dairy industry professionals and Extension specialists as well to help prevent the negative consequences of heat stress at a practical level. Changes/Problems:The principal investigator has changed her primary Institution from the University of Florida to the University of Wisconsin-Madison. The grant transitioned with the principal investigator and the remaining objectives will be fulfilled at UW-Madison.The present report summarizes the accomplishments of the first year at the University of Wisconsin-Madison (October 15, 2020 to October 15, 2021). We were able to maintain the animal portion of this project ongoing although laboratory work was delayedduring the pandemic. Originally a third euthanasia at puberty (10-12 months of age) was proposed to dissect the parenchyma from fat pad within the mammary glands. Instead, mammary biopsies will be collected (for histological and molecular purposes) and indirect measures of mammary growth will be implemented (including ultrasounds and 3D imaging). What opportunities for training and professional development has the project provided?Two Ph.D. students (with independent funding for her education in graduate school) have been integrally involved in all aspects of the project. These students transitioned form the University of Florida to UW-Madison and continue to work for this project. Two undergraduate students assisted in this project. One of them obtained internal scholarship, and the second one was paid by this grant. All students were actively involved in the project. How have the results been disseminated to communities of interest?The dissemination of results to the scientific community has been through scientific peer-reviewed publications (seeprevious sections for more details), local seminar series (University of Wisconsin Department seminars by Dr. Laporta and graduate students), domestic conferences (4abstracts presented by graduate studentsat the American Dairy science Associationannual meeting andthe American Dairy science Association41 Discovery conference) and in person and virtualpresentations by Dr. Laporta (including:DSM Nutrition sponsored Webinar,Dairy Cattle Welfare Council (DCWC) Webinar Series,The Dairy Signal, PDPW Rising Stars,American Dairy science Association41 Discovery conference, Cornell Nutrition Conference BalchemPre-symposium, Biologu of Lactation of Farm Animals Symposium). What do you plan to do during the next reporting period to accomplish the goals?The next reporting period will include the breeding results and gestational mammary gland growth measures (including teat length, distance, udder depth and length, and 3D imaging) and body measurements (body weight, hip height and chest girth). Mammary biopsies will be performed at mid- and late-gestation periods. The next reporting period will also include results on mammary gland histology and measures for birth and weaning euthanasia including laboratory (molecular) work currently ongoing.

Impacts
What was accomplished under these goals? Completed phases of this award include: Maternal treatments:application of environmental treatments (HS: heat stress and CL: active cooling with fans and soakers) to 80 pregnant Holstein dams during late gestation (~ 60 last days of their pregnancy). These dams gave birth to 72 female calves (heifers) that were either in utero cooled (IUCL) or in utero heat stress (IUHS) Birth euthanasia (day 0):a subset (N=16) were authanized at birth to assess organ and carcass weights, focusing on mammary gland growth characterization and tissue collection (i.e., mammary parenchyma and fat pad) for histological and molecular purposes.Organs and tissues harvested included: thyroid gland, adrenal gland, untrimmed (i.e., skin and hair remaining) and trimmed (i.e., skin and hair removed) mammary gland (MG), GI tract (stomach plus duodenum, jejunum, and ileum), ovaries, mesenteric lymph node, thymus, spleen, kidney, liver, pancreas, lungs, heart, and semitendinosus skeletal muscles. Heifer raising:the remaining heifers were raised at the University of Florida until weaning (9 weeks). Data collection included weekly thermoregulatory and growth parameters, as well as grain intake and blood collections. Weaning Euthanasia (d 63 of age):a second subset (N=16) was euthanaized to assess organ and carcass weights, focusing on mammary gland growth characterization and tissue collection (i.e., mammary parenchyma and fat pad) for histological and molecular purposes. The same organs and tissues were harvested herein. Heifer relocation and subsequent measurements:the remaining heifers (N=40) were transported to the University of Wisconsin-Madison to continue the longitudinal study. Upon arrival heifers were quarantined and tested as per UW RARC recommendations before they were introduced to the UW herd. Once admitted to the heard, monthly non-invasive measurement were collected (i.e, growth measurements and mammary growth parameters). In addition, blood samples were collected weekly from 6 to 12 months of age for the detection of Progesterone as an indicator of puberty attainment. Once all heifers were confirmed cycling, a estrous synchronization protocol was applied to perform mammary gland biopsies at two stages of the estrous cycle (i.e., high and low estrogen concentrations of the cycle) coinciding with 12 months of age. Mammary gland biopsies were collected at both stages for histological and molecular analyses. Breeding:Four high genetic merit bulls have been selected specifically to avoid inbreeding. Heifers will be artificially inseminated in November-December 2021. Brief summary of growth measurements and organ weights for the birth and weaning follows.Calves born under intrauterine hyperthermia had smaller mammary glands on a per kg body weight basis compared with in utero cooled heifers at birth. These heifers also tended to have reduced thymus and thyroid gland gross organ weights and had reduced spleen and heart gross organ weights at birth relative to in utero cooled heifers, but these effects were negated when assessed on a per kg body weight basis. There were no other differences in organ, blood, or carcass weights at birth.Heifers that were not euthanized at birth were followed until weaning to assess growth and feed intake. Heifers exposed to in utero heat stress weighed less than in utero cooled counterparts from 0 to 63 days of age. Further, these heifers had a reduced ADG relative to in utero cooled heifers during this time-period (0.71 vs. 0.76 ± 0.05 kg/day). Similar to body weight over time, heifers under in utero heat stress had reduced hip height, withers height, body length, chest girth, waist girth, and head circumference compared to in utero cooled heifers. Pre-weaning calf milk replacer intake did not differ between treatments and remained near the provided amount of 0.87 kg milk replacer per day until weaning. However, in utero heat-stressed heifers consumed less starter grain concentrate relative to in utero cooled heifers, particularly from 42 to 63 days of age. On a component basis, these heifers had reduced grain and total dry matter, metabolizable energy, crude protein, and crude fat intake.Finally, a euthanasia occurred after weaning at 63 days of age. Heifers gestated under in utero heat stress had larger adrenal glands and kidneys at 63 days and smaller ovaries relative to in utero cooled heifers when adjusted for body weight. Consistent with birth outcomes, in utero heat-stressed heifers had smaller mammary glands after weaning, adjusted for body weight.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Laporta J, Dado-Senn B, Skibiel AL. 2021. Late gestation hyperthermia: epigenetic programming of daughter's mammary development and function. Domestic Animal Endocrinology. Special Edition: Recent Contributions from Women Scientists to Animal Endocrinology: Pregnancy and Lactation https://pubmed.ncbi.nlm.nih.gov/34600221/
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2021 Citation: Laporta J. 2021. ADSA Foundation Scholar Award: Early-life exposure to hyperthermia: productive and physiological outcomes, cost, and opportunities. Journal of Dairy Science. https://pubmed.ncbi.nlm.nih.gov/34419283/
  • Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Dado-Senn B, Field S. L., Davidson B. D., Casarotto L. T., Marrero M. G., Ouellet V., Cunha F., Sacher M. A., Rice C. L., Maunsell F. P., Dahl G. E., Laporta J. Late-gestation in utero heat stress limits dairy heifer early-life growth and organ development. Frontiers in Animal Science.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Late-gestation in utero heat stress limits dairy heifer early-life growth and organ development. B. Dado-Senn, S. L. Field, B. D. Davidson, L. T. Casarotto, M. G. Marrero, V. Ouellet, F. Cunha, M. A. Sacher, C. L. Rice, F. P. Maunsell, G. E. Dahl, and J. Laporta. 41st ADSA DISCOVER Conference: Health Management of Calves - From Intrauterine Life to Successful Weaning.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Impacts of in utero heat stress on the gastrointestinal morphology of dairy calves. B. D. Davidson, S. L. Field, B.Dado-Senn, M. A. Steele, G. E. Dahl, and J. Laporta. ADSA 2021 Virtual Annual Meeting
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Late-gestation heat stress alters placental morphology in multiparous cows. L. T. Casarotto, V. Ouellet, B. Dado-Senn, J. Laporta, and G. E. Dahl. ADSA 2021 Virtual Annual Meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: In utero effects of hyperthermia on hair length and skin morphology. B. D. Davidson, K. M. Sarlo Davila, R. G. Mateescu, G. E. Dahl, and J. Laporta. ADSA 2021 Virtual Annual Meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: In utero hyperthermia impacts early-life dairy calf mammary development. B. Dado-Senn, S. L. Field, B. D. Davidson, A. J. Geiger, G. E. Dahl, and J. Laporta. ADSA 2021 Virtual Annual Meeting.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Davidson, B.D., Dado-Senn B., Ouellet V., Dahl G.E., Laporta J. 2021. Effect of late-gestation heat stress in nulliparous heifers on postnatal growth, passive transfer of immunoglobulin G, and thermoregulation of their calves. JDS Communications. 2(3):165-169