Progress report for GNE22-286
This project aims to identify associations between DNA methylation and disease status and how these associations may influence performance in dairy cattle. Disease is costly to the dairy industry and negatively impacts animal welfare. Mastitis alone is estimated to cost the U.S. industry $2 billion annually. Genetic selection for disease resistance is possible but the trait is largely impacted by environmental factors that modulate gene expression through epigenetic mechanisms such as DNA methylation. This project will consist of a whole-herd retrospective analysis of the Cornell Veterinary School Teaching Dairy (VTD) and DNA methylation analysis on a subset of animals. Performance outcomes of offspring such as production, fertility, health, and longevity will be compared based on their dam’s history of disease using electronic records of the farm. Regions of differential methylation between dam health groups, in offspring pre- and post-disease, and in offspring with differing dam disease history will be identified to characterize a possible biologic explanation for any observed associations in health and performance. This project is a pilot project for a more extensive study of epigenetic associations with disease in dairy cattle. Understanding the lasting and transgenerational effects of disease will help producers make educated management decisions. Characterizing DNA methylation as a source of phenotypic variation will also help improve genetic prediction models. These two factors will increase farmer profitability with improved selection and management of dairy cattle.
This project will consist of two methodologies: a retrospective whole-herd record analysis and DNA methylation sequencing on a subset of animals. First, an analysis of the Cornell Veterinary School Teaching Dairy (VTD) historical health and performance records will be conducted to validate the results of the Carvalho study in this herd i.e., calculate associations between maternal disease status and offspring disease risk and performance. Second, genome-wide DNA methylation analyses of a subset of animals will be conducted to investigate epigenetic mechanisms underlaying the relationships observed in the first analysis. Differences in DNA methylation between sick and healthy animals pre- and post-disease event, whether these are inherited by offspring, and whether these methylation differences have effects on health, growth, production, fertility, and longevity will be investigated.
Specific objectives within the study to meet our goal of improved understanding of the relationship between DNA methylation, disease, and phenotypic variation are as follows.
- Objective 1: Investigate associations between dam history of disease and offspring’s susceptibility to disease, production, fertility, longevity, and genetic merit using a retrospective study design of the VTD
- Objective 2: Characterize associations between DNA methylation and disease status
- Objective 3: Characterize DNA methylation inheritance with a focus on dam disease status and the association with offspring disease susceptibility
- Objective 4: Identify differentially methylated regions with putative biological functions relevant to health, growth, production, fertility, and longevity relationships observed
The purpose of this project is to identify associations between DNA methylation and disease status and how these associations may influence performance. Disease negatively impacts animal welfare and reduces farmer profitability through treatment costs, lost production, and involuntary culling. Genetic evaluations for many diseases are available in dairy cattle allowing producers to select for disease resistance and improve the overall profitability of their animals. However, the direct genetic effect on disease susceptibility is complicated by large environmental and management effects. Epigenetics are a collection of biologic mechanisms used to modulate gene expression in response to environmental changes. A specific example of this is DNA methylation which is a modification of DNA that regulates a gene’s expression. In livestock, biological and environmental factors such as nutrition, stress, and disease have been shown to cause changes in DNA methylation patterns. Furthermore, individual differences in DNA methylation patterns have been associated with phenotypic variation in milk production and immunity. There is even growing evidence that epigenetic marks can be inherited or programmed in-utero based on a dam’s environment effecting the expression of an offspring’s genes throughout their lifetime. We will examine how historic health events in a dam may influence the inheritance of DNA methylation patterns to their offspring and how offspring’s health events may change their own methylation patterns and affect their performance.
Recent studies supporting these concepts include results showing cows at lower risk for clinical disease if their dam’s experienced clinical disease before or during gestation (Carvalho et al., 2020). Additionally, studies have found that dams under metabolic stress have calves with DNA methylation patterns supporting better energy efficiency than those born to non-stressed mothers (Chaput & Sirard, 2020; Zhang et al., 2021). This suggests the dam and offspring undergo epigenetic reprogramming to better deal with maternal stressors and disease.
This project will consist of two methodologies: a retrospective whole-herd record analysis and DNA methylation sequencing on a subset of animals. First, an analysis of the Cornell Veterinary School Teaching Dairy (VTD) historical health and performance records will be conducted to validate the results of the Carvalho study in this herd i.e., calculate associations between maternal disease status and offspring disease risk and performance. Second, genome-wide DNA methylation analyses of a subset of animals will be conducted to investigate epigenetic mechanisms underlying the relationships observed in the first analysis. Differences in DNA methylation between sick and healthy animals pre- and post-disease event, whether these are inherited by offspring, and if these methylation differences have effects on health, growth, production, fertility, and longevity will be investigated. This project is intended as a preliminary investigation informing the direction of a larger study of epigenetic inheritance and epigenetic variation in dairy cattle. The goal is to improve the scientific community’s understanding of phenotypic variation and improve genetic prediction models. Improved genetic selection resources and a better understanding of the lasting effects of disease will support farmer profitability in the future.
Animals and Performance Data: The proposed project will use Holstein at the VTD which houses 175 lactating/dry cows, births 110 female calves, and raises 80 heifers annually. While this is a teaching dairy, it is operated as a commercial dairy herd that markets milk through a cooperative. Cows average 85 lbs/day and are housed in a free-stall facility. Electronic records of birth, breed, production, breeding, health, and culling are regularly maintained in DairyComp305 and will be used in this study to classify disease status and performance outcomes. The VTD has a 12% incidence of metritis, 6% retained placenta, 13% ketosis, 1% milk fever, 2% displaced abomasum, and 27% incidence of mastitis. All animals in the herd are genotyped and have genomic evaluations available that will be used to compare genetic merit. As part of this study, additional performance data that is not routinely collected by the farm will be recorded with help from our research staff moving forward. This includes monthly weight collections and weekly body condition scores as heifers and calfhood disease incidence such as pneumonia and scours. Data will be collected by farm and research staff as appropriate, and procedures will follow IACUC protocols.
Cohort Definitions and Study Design: This project relies on two methodologies: a retrospective whole-herd record analysis and DNA methylation sequencing on a subset of animals. In both methods, the study subjects will be assigned to two cohorts based on their dam’s incidence of disease in the lactation preceding their birth. Any animal whose dam had at least one recorded incidence of metritis, retained placenta, ketosis, milk fever, displaced abomasum, or mastitis will be assigned to the ‘diseased dam’ cohort and any animal whose dam has no record of any of the previously mentioned or other diseases will be assigned to the ‘healthy dam’ cohort. Due to cohort definitions, only animals born to multiparous dams will be included in the analysis. For the first methodology, differences in first lactation performance and health will be analyzed between cohorts utilizing historic records (Obj. 1). For the second methodology, a subset of the herd will be enrolled in two cohorts (6 offspring and their respective dams each) and differences in DNA methylation will be analyzed between cohorts (Obj. 2-4). The study design for the second methodology is illustrated in the attached figure.
Biological Samples and Epigenetic Data: Biological samples will only be utilized for the second methodology of the study. As part of our larger investigation, all female calves born at the VTD and their dams will undergo routine blood sample collections within 24 hours of parturition starting in May 2022. DNA will be extracted from these samples and frozen. This will create a library of samples for the larger project and allow enrollment into this project based on both dam and offspring disease status. For this project, 12 study subjects and their dams (n=12 offspring; 12 dams) will be used. Enrollment will be dependent on both the dam’s history of disease and the offspring’s disease incidence within the first 4 weeks of their 1st lactation. We will have 3 subjects from ‘diseased dams’ who also had their own disease event, 3 from ‘diseased dams’ without their own disease event, 3 from ‘healthy dams’ with their own disease event, and 3 from ‘healthy dams’ without disease. This sampling strategy is illustrated in the submitted figure. For the enrolled animals, an additional blood sample will be taken 4 weeks into their 1st lactation (n = 12 offspring) following IACUC protocols. The genome wide methylation profile will be analyzed via reduced representation bisulfite sequencing (RRBS). RRBS is cost effective as only fragments pertaining to CpG-rich regions of the DNA (1-5% of genome, mostly promoter regions) are sequenced. Hyper and hypo methylated regions and differentially methylated regions (DMR) between groups will be identified. Gene ontology and KEGG pathway enrichment analysis will be performed to functionally characterize the differentially methylated regions and genes in terms of what is known about the biological function of genes.
Objective 1: Investigate associations between dam history of disease and offspring’s susceptibility to disease, production, fertility, longevity, and genetic merit using a retrospective study design of the VTD.
Performance Outcomes: For this objective, the whole herd will be split into ‘diseased dam’ and ‘healthy dam’ cohorts to analyze differences in 1st lactation health and performance of their respective offspring. The presence or absence of any clinical disease and the number of disease events in 1st lactation will be used as health outcomes in the analysis. Total 1st lactation milk, fat, and protein production, heifer and 1st lactation insemination and pregnancy rates, and culling rate and reasons will be used as performance outcomes in the analysis. Additionally, the difference between genomic predicted production and realized first lactation production will be used as an outcome to be compared between cohorts in order to assess if maternal disease status may contribute to phenotypic variability from genomic predictions.
Statistical Analysis: Analyses similar to the Carvalho study will be used to analyze differences in offspring health and performance between cohorts using R version 4.1.1 (Carvalho et al., 2020). ANOVA will be used for continuous outcomes, logistic regression will be used for binary outcomes, and Cox proportional hazard regression will be used for intervals to events. Data such as season, age, and parity of the data will be collected and assessed as covariates in the model.
Objective 2: Characterize associations between DNA methylation and disease status.
Dam Disease Comparison: Differences in DNA methylation will be identified between dams with and without a history of clinical disease based on the samples collected at calving. Differentially methylated regions (DMR) between the diseased and non-diseased dams could stem from two possible options. One, these DMRs could indicate differences existing before the disease incident that contributed to either their susceptibility or resistance to disease or the DMRs could be differences the animals acquired in response to the disease.
Pre- and Post- Offspring Disease Comparison: In order to determine the source of the DMR found between dams, methylation patterns for individual offspring at birth will be compared to their methylation patterns 4 weeks into their first lactation and to their dams. The peak incidences of all the diseases of interest are within the first 4 weeks of lactation. Therefore, changes in methylation between birth and lactation present only in offspring that had their own disease event, not in offspring that did not have disease, could be considered changes in methylation as a response to disease. In contrast, DNA methylation patterns of offspring at birth in relation to whether they end up having a disease event in their first year of life, suggests DNA methylation provides either resistance or susceptibility to the disease. These DMRs identified in the offspring at birth or as a response to disease will be compared to the patterns found in the dams.
Objective 3: Characterize DNA methylation inheritance with a focus on dam disease status and the association with offspring disease susceptibility.
Offspring Cohort Comparison: DMR in offspring at birth will be identified between cohorts of dam disease status. DMR identified here would represent differences in methylation in offspring due to maternal disease status near gestation. The diseases studied in this project are most common in early lactation meaning they occur before or around the time of insemination. The DMR between the two cohorts of offspring would have either been the result of in-utero programming of the offspring’s methylation pattern due to its own exposure to the disease or inherited from the dam based on her methylation pattern. The analysis for objective 1 investigated the source of the methylation patterns of the dam.
Inheritance Comparison: The DMR found between the two cohorts of offspring will be compared to the DMR between sick and healthy dams. If similar regions are found, this supports that either offspring and dam experienced the same re-programming event as a response to their exposure to the disease or the dam passed her methylation pattern on to the offspring. Without more frequent sampling, it will be hard to determine which scenario is the case, but we will know the outcome relationship between offspring methylation and disease.
Objective 4: Identify differentially methylated regions with putative biological functions relevant to health, growth, production, fertility, and longevity relationships observed
Performance Analysis: A similar performance outcome analysis (excluding disease incidence) as conducted for objective 1, will be conducted for the 12 methylation study subjects. While the power of this analysis will be much lower than the whole-herd analysis, it will be conducted to assess whether the direction of trends is the same in the subset of animals as in the whole-herd. Heifer average daily gain and calfhood disease incidence will be included as additional performance outcomes. This is not something that is routinely collected by the farm but will be collected for the study subjects of this project. Previous studies that investigated the impact of maternal disease status on offspring performance, proposed poor heifer growth as a possible reason for the increased cull rate of heifers born to diseased dams but did not collect the data to investigate that claim (Carvalho et al., 2020).
Functional Analysis: Genes near the identified DMRs in Objectives 2-4 will be identified. RRBS focuses on methylation patterns near promoters which are regions that impact gene expression. Genes found near DMR could indicate differentially expressed genes between groups. Gene ontology and KEGG pathway enrichment analysis will be conducted to functionally categorize the nearby genes. Gene ontology and KEGG pathway analyses use databases having genes cross-referenced with functional research to provide annotation as to what the biological function is of a gene. This should reveal genes that have biological functions that support the phenotypic relationships between maternal disease status and offspring health and performance previously identified (in the whole herd and subset).
Methods adjustments (January 2023): Since beginning the study, a few adjustments have been made to the protocol. Health evaluations for calves have been implemented as a way of monitoring calf health between birth and weaning. These are conducted three times a week and include rectal temperatures and eye, ear, nose, cough, fecal, and body condition scores. An additional blood sample timepoint was added at 48 hours after birth. This sample is used for a serum total protein estimation using a digital refractometer. Serum total protein is a good estimator of serum IgG levels which are indicative of the success of passive transfer of immunity through colostrum. As failure of passive transfer could be a confounder for future growth, disease susceptibility and productive outcome, we can now control for this in future models. This change was implemented on 7/1/22. The first 4 calves enrolled in the study do not have serum total protein readings. Due to logistical concerns with the scale, bodyweights are now being estimated using a weigh tape once a week during the time from birth to weaning. This change was implemented on 9/21/22. The 16 calves born since implementation and all future calves will have complete pre-weaning growth information. Enrollment of bull calves has stopped as of 9/14/22. An updated version of the study design schematic has been added as figure 2.
Biological Samples and Epigenetic Data Progress (January 2023): As of January 6, 2023, 39 Holstein heifer and 5 bull calves have been successfully enrolled in the study marked by successful blood collection from both calf and dam within 24 hours of birth. Of the calves enrolled, 6 heifer calves and all bulls were sold and are no longer part of the study. One heifer calf was euthanized. Of the 32 calves that have remained in the herd, 21 were their dam’s first calf, 10 were born to multiparous dam’s with no history of transition disease in the previous lactation, and 1 was born to a dam previously treated for hypocalcemia. They have been sired by 8 different sires. 7 calves have been treated for scours and 1 was treated for a navel infection. 14 have been successfully weaned and are being transferred to the heifer raiser. The remaining heifer calves are still at the farm and are being monitored for growth and disease events until weaning.
We do have concerns about the lack of calves being born to dams with a history of transition disease. Historically, this has not been a problem, but recently breeding strategies of the VTD herd have placed more emphasis on sourcing replacement animals from primiparous animals. Most heifers are bred with sexed semen; whereas, most multiparous animals are bred with conventional dairy semen or beef semen. Furthermore, the likelihood that an animal is bred with beef semen likely increases if she is a “problem cow” that has a history of disease events. The targeted use of sexed semen and beef semen is a progressive breeding strategy allowing herds to maximize genetic progress and gain a higher sale price for calves not intended as replacements. For this study, it is a problem because there are a small proportion of Holstein heifer calves that could qualify for objectives 2-4. We are considering partnering with a larger commercial herd for sourcing the biological samples for this part of the project.
Tentatively, enrollment of all Holstein heifer calves born at the VTD will continue until June 2023. All calves enrolled will be followed to their first calving (beginning spring of 2024). At 4 weeks into their lactations, we will choose 12 heifers based on their own and their dam’s disease status (as previously described in the proposal). An additional blood sample at 4 weeks into the lactation will be collected from these animals. Birth and lactation blood samples of the 12 selected dams and calves will be analyzed by RRBS DNA methylation sequencing. The sequences will be used for Objectives 2-4.
Objective 1 Progress (January 2023): The dam disease status for all current cows in the herd is being extracted from the herd’s archived DairyComp records. To date, all current first and second lactation cows have been classified as either born to a primiparous dam (excluded from study), born to a dam with no record of clinical disease in the lactation coinciding with their gestation (“healthy dam” cohort), or born to a dam with recorded clinical disease in the lactation coinciding with their gestation (“diseased dam” cohort). Dam disease events have included mastitis, metritis, retained placenta, ketosis, displaced abomasum, and hypocalcemia and have not yet been filtered by timing in lactation. First lactation production and disease events have also been extracted for all current first and second lactation animals in the herd. Third and greater lactation animals currently in the herd will be classified by dam disease status and their archived first lactation records will be extracted in the coming months. Statistical analysis of this data will then be conducted to investigate the relationship between dam disease history and offspring performance.
Objective 2 – 4 Progress (Jan 2023 Report): DMR analysis will commence once study subjects have been selected and RRBS sequences have been received (estimated summer 2024).
Education & Outreach Activities and Participation Summary
The main deliverable of this project is to improve our knowledge of the inheritance of DNA methylation and its relationship to health and performance in dairy cattle. This knowledge will help refine genetic selection models for dairy cattle by explaining some of the phenotypic variation due to the environment which is not captured in current prediction models. This will benefit farmers in the future by improving the accuracy of selection as we understand how epigenetics effect phenotypic variation. Within the timeline of this project, dissemination of results to the agricultural and scientific community is more realistic. I will assist in dissemination of results through Dr. Huson’s annual station reports for multistate committee SCC084: Selection and mating strategies to improve dairy cattle performance, efficiency, and longevity, a committee of academic and government researchers who work with industry and advise the Council on Dairy Cattle Breeding of new opportunities to improve genetic selection. Additionally, research results are commonly shared at PRO-DAIRY webinars or events given by Dr. Huson including The Genetic Management of Heifer Reproduction and Implementing Practical Genetics for the Commercial Dairy. These are annual events and webinar series attended by producers, extension agents, and industry representatives. As a way of reaching current undergraduate and graduate students, results of this project will be included in Dr. Huson’s undergraduate Applied Dairy Cattle Genetics course, her annual lecture on large animal genetics to Cornell’s College of Veterinary Medicine students, and my presentations at Animal Science Graduate Student seminar. Publications of this preliminary project may be possible and multiple publications from the future larger study will be submitted to peer-reviewed journals to disseminate results to the broader scientific community. I also plan on attending and can present results at conferences including American Dairy Science Association, American Animal Science Association, International Society for Animal Genetics, and Plant and Animal Genome Conference as applicable.
Outreach Progress (January 2023):
In October 2022, I attended the annual meeting of the multistate research coordinating committee SCC84: Selection and mating strategies to improve dairy cattle performance, efficiency, and longevity at Michigan State University. I presented a brief description of the study design for this project as part of our institution’s station report. I also presented a more detailed description of the study design, sample collection methodology, and background research on the topic of epigenetic inheritance in the Graduate Student Research Updates Seminar of the Animal Science Department at Cornell.