EMBRYO MORTALITY ENTAILS A MASSIVE IMMUNE RESPONSE IN DAIRY COWS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1023070
• Grant No.: 2020-67034-31730
• Cumulative Award Amt.: $120,000.00
• Proposal No.: 2019-07133
• Project Start Date: Jul 1, 2020
• Project End Date: Jun 30, 2023
• Grant Year: 2020
• Program Code: [A7101]- AFRI Predoctoral Fellowships

Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523

Performing Department
Biomedical Sciences

Non Technical Summary
Although many predisposing factors for the poor fertility in Holstein-Friesian cows have been identified, the biological mechanisms underlying early embryo mortality are not completely understood. Early embryo mortality pregnancies contribute to subfertility and annual loss of $1.6 billion in the dairy cattle industry. Previously we identified genes in tissue (endometrium, corpus luteum) responses to early embryo mortality via Ingenuity Pathways Analysis software. We will be validating these genes using mRNA and protein assays and determine which genes contribute to normal pregnancies compared to early embryo mortality pregnancies. The identification of mutations in candidate genes that are associated with fertility traits will also be studied. Use of CLC Genomics Workbench software will identify these mutations which will be validated in a different group of cows (n=500) to this study. Dairies will be selected by having healthy cows and records for fertility traits in their DairyCOMP software. A genotype to phenotype association study can then be conducted to determine the direction (positive or negative) relationship of a mutation to fertility with health traits. Mutations that are positive and could be used as blood markers to improve in the selection of embryo survival and cow health. Longer term goals are tied with the genotype to phenotype study associations with early embryo mortality pregnancies providing a basis for the use of a single nucleotide polymorphism chip. Producers have the option to use this state-of-the-art tool that has blood markers that are positively associated with embryo survival and cow health. This could then potentially lead to selection of reproductively efficient dairy cows and mitigate the annual loss of 1.6 billion.

Animal Health Component

25%

Research Effort Categories

Basic

75%

Applied

25%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
315	3410	1081	100%

Knowledge Area
315 - Animal Welfare/Well-Being and Protection;

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1081 - Breeding;

Keywords

conceptus

bovine

pregnancy

rna-seq

Goals / Objectives
Although general predisposing factors for poor fertility in the modern Holstein cow have been identified, the biological mechanisms underlying early embryo mortality in ruminants are not completely understood. Further improvements in reproduction of ruminants require a better understanding of establishment of pregnancy such that strategies can be devised to mitigate the incidence of early embryo mortality. Early embryo mortality pregnancies in dairy cows cause an annual economic and productivity loss of $1.6 billion-dollars in the USA. This economic loss is mainly due to inefficient management of nonpregnant cows with pregnancy failure caused by early embryo mortality. Clarifying why early embryo mortality occurs may lead to identification of genetic biomarkers in cows that can be used to identify which animals are prone to have a pregnancy associated with early embryo mortality to facilitate selecting against them. Currently, studies are limiting that have explored the relationship between conceptus signalling and immune responses, conceptus morphology (normal or early embryo mortality) and maternal responses/environment (immune system) to pregnancy. Through discovery of single nucleotide polymorphisms that are related to a normal or early embryo mortality pregnancy, producers can select cows that are reproductively efficient and eliminate those that are not.Aim 1. Determine if early embryo mortality and impaired interferon tau production from the conceptus causes luteolytic endometrial signaling and activated inflammatory and luteolytic responses in the corpus luteum. Aim 2. Identify single nucleotide polymorphism markers in candidate genes that can predict normal compared to early embryo mortality pregnancies.

Project Methods
Animal Model and Preliminary Data:Twenty-two healthy lactating multiparous Holstein cows were selected for this study. After voluntary waiting period (60 days after postpartum), cows were randomly sorted into pregnant and non-pregnant (NP) groups. All cows were enrolled in a synchronization program that consisted of an intramuscular (IM) injection of Gonadotropin Releasing Hormone (GnRH) followed by an IM injection of Prostaglandin F2 alpha (PGF2a) seven days later, and a final GnRH IM injection was administered 56 hours later. Cows from pregnant group were artificially inseminated 16 hours after the final IM injection of GnRH with semen from the same high-fertility sire. NP cows (n=7) were not exposed to semen. On day 16, all cows had ultrasounds of their ovaries to determine presence of the corpus luteum. The following tissues were then collected: uterine flushings (UF), conceptuses, endometrium, and corpus luteum (CL). Pregnant cows were further sorted on day 16 based on conceptus morphology: Normal (N): translucent and elongated (n=9); early embryo mortality (EM): pink, red, opaque and/or non-elongated (n=6). RNA was isolated from tissues by using Trizol and any remaining DNA was removed by using RNase-free DNase and RNAeasy Mini Elute Cleanup kit. RNA was also quantified using a NanoDrop 2000. All samples were sent to Zoetis for Library preparation and subjected to RNA-Sequencing. Raw data were received mapped to the new Bos Taurus assembly (ARS-UCD 1.2). A DeSEQ2 analysis was performed in R and was then submitted into Ingenuity Pathway Analysis software to identify key biological pathways. Parameters for this particular analysis was for genes to have a fold change (1.5) and P-values of 0.05. Aim 1. Determine if early EM and impaired interferon tau (IFNT) production from the conceptus causes luteolytic endometrial signaling and activated inflammatory and luteolytic responses in the CL.Approach: Total RNA from endometrium and CL will be isolated using the Trizol method previously mentioned in our animal model. Single stranded cDNA will be synthesized using an iScript cDNA synthesis kit. Primers will be designed using Primer-Blast from NCBI with optimized temperatures of 61°C melting point and lacking self-annealing. Amplicons will range from 80-120 bp and be sequenced to confirm the identity of the target genes. RT-qPCR reactions will be performed using iQ SYBR green Supermix, RNase-free water, primer sets and amplified on a CFX96. Primers will then be validated for the detection of genes impacting ISGs and E2 action in the endometrium and inflammatory responses in the corpus luteum. Each reaction will be assessed for quality by melting curve analysis. Western blot procedures and validation of antibodies will also be performed. Quantification of proteins will be performed using a Pierce BCA Protein Asssay kit, loaded into a 12% SDS-PAGE gel and electrophoresed. Proteins will be transferred to a nitrocellulose blotting membrane and incubated in 5% non-fat dry milk in Tris-buffered saline with Tween 20 at a 7.5 pH. Use of a Chemi Doc will be used to take a picture of the western blot and be quantified by using Image Lab, an optical densitometry software.Pitfalls: Based on our previous work, we do not expect any problems validating new RT-qPCR primers to study our proposed bovine gene targets. A recognized concern is availability of suitable primary antibodies with species cross-reactivity to study protein amounts in bovine tissues using WB approaches. For this reason, we initially propose to study 5 targets with the expectation that at least 1- 2 will be successful. Alternative proteomic approaches are also a possibility, and our laboratory has experience with these approaches. Preliminary data: Conceptuses undergoing EM, displayed key biological pathways with an adaptive T helper (Th) immune responses. This was surprising at the level of the conceptus. These EM conceptuses appear to also have impaired release of IFNT, which leads to impaired activation of interferon-stimulated genes (ISGs), in the face of upregulation of genes facilitating estradiol (E2) mediated luteolytic signals from the endometrium. The induction of ISGs in the CL of EM pregnancies is diminished, while hypercytokinemia and calcium responses increase to presumably facilitate luteolysis.Aim 2. Identify SNP markers in candidate genes that can predict normal compared to EM pregnancies. Approach. SNP Discovery: Th1 pathway and IFNT genes (conceptus), and ISG gene (endometrium, CL) SNPs will be prioritized based on their association with fertility traits, developing fetus, and pregnancy in Holstein cows. CLC Genomics workbench will identify mutations that are distinct between, or concordant among EM and normal pregnancies in RNA-Seq data from all tissues. Potential functionality of identified SNPs will be determined by using the Variant Effect Predictor tool in Ensembl. SNP Validation: A genotype to phenotype association study will be executed. SNPs discovered will be validated on a separate Holstein cow population (n=500). DNA extraction from white blood cells of cows from cooperating dairies that record fertility data (phenotype) within DairyCOMP 305 software. Pitfalls: Detection of a large number of SNPs are expected to exist between Holstein cows and the bovine reference genome sequence (Hereford; ARS-UCD 1.2), rather than between N vs EM (Holstein) pregnancies. Therefore, SNPs that are found between N vs EM will be specific to Holstein cows. Also, many SNPs are expected to be found within introns because Holstein have large haplotype blocks (one of two or more DNA strand sequences are found at the same place on a chromosome and are inherited together from a single parent). The statistical analysis may need to be altered to haplotype to phenotype for specific areas of the sequence being studied. Another challenge is the selection of genes in an inflammation and/or immune pathway to associate with EM.Efforts: Science-based knowledge will be delivered throughARBL and Animal Science Department Seminars (faculty, MS/PhD students): Presentation of 50-minutes will provide a variety of opinions for a stronger project and potential collaborators. An example of more public delivery is the CSU Speaks (General Audience) venue: Use of non-jargon language and receiving input from a non-scientific crowd. Evaluation: Progress by the candidate will be evaluated in weekly chalk talks (Dr. Hansen and Dr. Thomas), and laboratory meetings with a knowledgeable group (faculty and DVMs), which provides the opportunity to present research and understand appropriate knowledge for the dissemination of results.Bi-Annual Meetings (PD's committee): Assessment of PD's advancement in research, course work, and knowledge. A written evaluation sheet provides feedback to PD on what needs to be focused/worked on more. Progress will also be evaluated through annual progress report/assessment of PhD Student (GS and BMS department). This progress report includes a presentation and written evaluation sheet to assure completion of requirements: literature competency, interpreting results, preliminary exams and dissertation progress.

Progress 07/01/20 to 06/30/23

Outputs
Target Audience:This research targeted, during the 2021-2022 reporting period, researchers at different career levels (graduate students-MS or PhD, Post-Docs, faculty), veterinarians, industry and extension focused on the topic of animal genetics, bioinformatics and (or) reproduction through: • Rocky Mountain Reproductive Science Symposium Spring 2022 in Fort Collins, Colorado: 10-minutes in person oral presentation with 2-minutes of questions and answers. • American Dairy Science Association-Summer 2022 in Kansas City, Missouri: 12-minutes in person oral presentation with 3-minutes of questions and answers. • National Association of Animal Breeders-Fall 2022 in Milwaukee, Wisconsin: 2-minute flash talk and poster presentation. •Society for the Study of Reproduction-Summer 2023 in Ottawa, Canada: poster presentation. •American Society of Animal Science-Summer 2023 in Albuquerque, New Mexico: 12-minutes in person oral presentation with 3-minutes of questions and answers. Changes/Problems:It was proposed in our 2020-2021 report that the objective aim 1 was revised from being to determine if early embryo mortality (EM) and impaired interferon tau production from the conceptus causes luteolytic endometrial signaling and activated inflammatory and luteolytic responses in the corpus luteum (CL) to further test the hypothesis that EM conceptuses with an impaired interferon production have adaptive immune responses and causes luteolytic endometrial signaling. The previous approach to aim 1 was to perform real time-quantitative polymerase chain reactions (RT-qPCR) on genes impacting interferon stimulated genes and estradiol action in the endometrium (Endo) and inflammatory responses in the CL. Western blot (WB) procedures were proposed also for endometrium and CL tissues. These were revised to for 2020-2021 to be the following: use proteomic (global mass spectroscopy) analysis of normal conceptus and endometrium tissues of cows in estrous cycle (EC), EM and N pregnancies. It was reasoned that the limited tissues would be best studied for global proteomics and would provide more extensive information rather than simply focusing on the estradiol-associated responses. Unfortunately, due to time constraints, we re-propose revisingthe approach of aim 1 for 2021-2022 by conducting two experiments. The first experiment that was conducted for aim 1 entailed performing RT-qPCR on the reproductive tissues (endometrium, corpus luteum and peripheral blood mononuclear cells) of all pregnancy states (embryo mortality pregnancy, non-pregnant and normal pregnancy) to determine the levels of ISG15 mRNA. The second experiment conducted was using western blots on the endometrium of all pregnancy states (embryo mortality pregnancy, non-pregnant and normal pregnancy) to determine the levels of ISG15 protein. Both experiments measure the levels of ISG15 on day 16 of pregnancy (embryo mortality and normal) or non-pregnancy at different time points of the central dogma (after transcription and translation has occurred). The data has aided us in reinforcing the previous results from our RNA-Seq data in which demonstrate that there are no changes in ISG15 mRNA levels in embryo mortality corpus luteum and peripheral blood mononuclear cells due to collection on day 16, which may be prior to a IFNT peripheral response and luteolysis. The statistical analysis wasconducted using a one-way ANOVA analysis in SAS software. Pitfalls for this new approach were not anticipated due to both ISG15 primers and antibodies havingbeen previously validated in our research laboratory. What opportunities for training and professional development has the project provided?Current opportunities of training and professional development for the 2021-2022 reporting period have centered on the process of conducting western blots, real time-quantitative polymerase chain reaction, pipeline for single nucleotide polymorphisms in candidate genes and constructing manuscripts for both aims 1 and 2 of the grant. How have the results been disseminated to communities of interest?Results have been disseminated at the following platforms: • Rocky Mountain Reproductive Science Symposium-Spring 2022 in Fort Collins, Colorado: 10-minutes in person oral presentation with 2-minutes of questions and answers. • American Dairy Science Association-Summer 2022 in Kansas City, Missouri: 12-minutes in person oral presentation with 3-minutes of questions and answers. • National Association of Animal Breeders-Fall 2022 in Milwaukee, Wisconsin: 2-minute flash talk and poster presentation. •Society for the Study of Reproduction-Summer 2023 in Ottawa, Canada: poster presentation. •American Society of Animal Science-Summer 2023 in Albuquerque, New Mexico: 12-minutes in person oral presentation with 3-minutes of questions and answers. What do you plan to do during the next reporting period to accomplish the goals?This is a final report for this project.Two journal papers are pending publication.

Impacts
What was accomplished under these goals? For the 2021-2022 reporting period, research efforts have been directed towards conducting two additional experiments for aim 1 and the completion of a quality control pipeline for the 69 single nucleotide polymorphisms (SNP) in candidate genes from aim 2 of the grant. The first experiment that was conducted for aim 1 entailed performing real time-quantitative polymerase chain reaction on the tissues (endometrium, corpus luteum and peripheral blood mononuclear cells) of all pregnancy states (embryo mortality pregnancy, non-pregnant and normal pregnancy) to determine the ISG15 mRNA present. The second experiment for aim 1 was conducted using western blots on the endometrium tissues of all pregnancy states (embryo mortality pregnancy, non-pregnant and normal pregnancy) to determine the ISG15 protein present. The quality control pipeline for aim 2 was based on the RNA sequencing of conceptuses (normal and embryo mortality) in Holstein-Friesian (n=15) cows from aim1 and were used to conduct the SNP discovery phase. Selection for the specific SNPs within genes were first based on the following steps: (1) Related or associated to reproductive/pregnancy/fertility traits within previous studies, (2) Differentially expressed genes within previous RNA Seq transcriptome data that had log2 fold change significant (adjusted p<0.05), (3) Sorting identified SNPs as diagnostics or non-diagnostic, (4) Evaluating in which region and type of differentially expressed SNP, (5) Asses the prediction value of the sorting intolerant from tolerant tool (SIFT) analyses when an amino acid substitution occurred and if it could affect protein function , (6) Confirm if the loci of the SNP was in proximity to other associated SNP associated to reproductive/fertility traits in Cattle Quantitative Trait Loci (Cattle QTL) database. Validation of candidate SNPs and genotype to phenotype analysis were conducted in a different cohort of Holstein-Friesian cows (n=500) by collecting blood samples to be genotyped via a genotyping assay panel and collecting cow farms records. Further filtering of candidate SNPs, after having received all samples (n=500) genotyped, started by estimating genotypic and allelic frequencies to verify which SNP were monomorphic across all animals. We then verified which SNPs were in minor allele frequency (MAF; >10%). The candidate SNPs that were not in the MFA and/or were monomorphic were eliminated from the study. The remaining candidate SNPs were evaluated using pLink software using a quality control pipeline that consisted of five steps. The first step was to remove any SNP that 20% or higher genotypes missing in the data. The second step was to remove individual animals that were not genotyped for 10% or higher of the candidate SNPs. The third step was to remove any candidate SNPs that were not in Hardy-Weinberg Equilibrium by the metric of 1e-15 (i.e., SNP that were above 1e-15). The fourth was then to evaluate the remaining candidate SNPs for linkage disequilibrium via r2 and d'. The final step was to identify tag SNPs within the candidate SNPs. If there were more than one tag SNP for a group of SNPs, the tag SNP was selected based on r2 and d' having the highest values (i.e., strongest relationship to the group of SNPs). An additional number of animals were removed from the study due to missing AI information. Furthermore, some of the reproductive (breeding date, calving of cow and dystocia score) and production traits (predicted 1st lactation milk production at 305 DIM) were also removed from the study due to missing values. By having removed these animals and traits from the study, the n of observations used for the statistical models of the study were n=466. Continuous numeric trait models were analyzed using GLM-one way ANOVA in SAS. While binary models were evaluated using logistic regression in SAS. Statistically significant models were further analyzedusing a means separation tests within LSMEANS from the mixed procedure and included the Bonferroni adjustment for p-values to minimize false discovery error. A total of sixty-nine candidate SNPs were initially discovered but only twenty-three passed the quality control pipeline in pLink software. All candidate SNPs were found to explain a higher amount of the r2 variation of each of the models and were in close proximity to SNP that were associated with quantitative trait loci of fertility traits. Results of both aim 1 and 2 of the grant have been revised and organized to be published as journal articles with the added experiments (western blots and real time-quantitative polymerase chain reaction) and procedures for quality control of the initial single nucleotide polymorphisms in 69 candidate genes for fall of 2022. Submission of aim 1 manuscript was completed in May of 2023 and aim 2 manuscript is set to be submitted in late July of 2023.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Liebig BE, Bishop JV, McSweeney KD, Van Campen H, Gonzalez-Berrios CL, Hansen TR, Thomas MG. Direct genomic value daughter pregnancy rate and services per conception are associated with characteristics of day 16 conceptuses and hormone signaling for maternal recognition of pregnancy in lactating Holstein cows. Published on Applied Animal Science.
• Type: Other Status: Awaiting Publication Year Published: 2023 Citation: Gonzalez-Berrios CL, Pierce CF, Bishop JV, Van Campen H, Pinedo P, Hansen TR, Thomas MG. Identification of 8 candidate gene variants associated with early embryo mortality in Holstein-Friesian Cows. Pending publication and submission in late July, 2023.
• Type: Other Status: Submitted Year Published: 2023 Citation: Gonzalez-Berrios CL, Pierce CF, Bishop JV, Van Campen H, Pinedo P, Hansen TR, Thomas MG. Gene variants in BOLA-DMB, DECR1, FASN and SREBF1 associated with conceptus death on day 16 of pregnancy in Holstein cows. American Society of Animal Science-Canadian Society of Animal Science-Western Section American Society of Animal Science, 2023 Annual Meeting: Abstract #135. Albuquerque, NM. NIFA award acknowledged.
• Type: Other Status: Submitted Year Published: 2023 Citation: Gonzalez-Berrios CL, Pierce CF, Bishop JV, Van Campen H, Pinedo P, Hansen TR, Thomas MG. Association of DSC2, SREBF1, UBD and UMPS gene variants with early conceptus death in Holstein cows. Society for the Study of Reproduction, 2023 Annual Meeting: Abstract Accepted. Ottawa, CA. NIFA award acknowledged.
• Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Gonzalez-Berrios CL, Sinedino LDP, Georges HM, Bishop JV, Van Campen H, Thomas MG, Hansen TR. Embryo mortality is associated with a massive T-helper transcriptome response and impaired interferon-tau release and action in lactating dairy cows. Pending publication and submission in late May, 2023.
• Type: Other Status: Submitted Year Published: 2022 Citation: Gonzalez-Berrios CL, Bishop JV, Van Campen H, Thomas MG, Hansen TR. Impaired IFNT production and action during embryo mortality in lactating Holstein-Friesian cows. American Dairy Science Association, 2022 Annual Meeting: Abstract #86960. Kansas City, MO. NIFA award acknowledged.
• Type: Other Status: Other Year Published: 2022 Citation: Gonzalez-Berrios CL, Bishop JV, Van Campen H, Thomas MG, Hansen TR. Elucidating the mechanism of early embryo mortality pregnancies in lactating Holstein-Friesian cows. National Association of Animal Breeders, 2022. Milwaukee, WI. NIFA award acknowledged.
• Type: Other Status: Submitted Year Published: 2022 Citation: Gonzalez-Berrios CL, Bishop JV, Van Campen H, Hansen TR, Thomas MG. Impaired production and action of IFNT during early embryo mortality pregnancies in lactating Holstein-Friesian cows. Rocky Mountain Reproductive Science Symposium, 2022 Annual Conference. Fort Collins, CO. NIFA award acknowledged.
• Type: Theses/Dissertations Status: Accepted Year Published: 2022 Citation: Gonzalez-Berrios, C.L. MECHANISMS AND ASSOCIATED BIOMARKERS OF EARLY EMBRYO MORTALITY IN HOLSTEIN-FRIESIAN COWS. 2022. Colorado State University.

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

Outputs
Target Audience:This research targeted, during the 2021-2022 reporting period, researchers at different career levels (graduate students-MS or PhD, Post-Docs, faculty), veterinarians, industry and extension focused on the topic of animal genetics, bioinformatics and (or) reproduction through: • Rocky Mountain Reproductive Science Symposium Spring 2022 in Fort Collins, Colorado: 10-minutes in person oral presentation with 2-minutes of questions and answers. • American Dairy Science Association- Summer 2022 in Kansas City, Missouri: 12-minutes in person oral presentation with 3-minutes of questions and answers. • National Association of Animal Breeders-Fall 2022 in Milwaukee, Wisconsin: 2-minute flash talk and poster presentation. Changes/Problems:It was proposed in our 2020-2021 report that the objective aim 1 was revised from being to determine if early embryo mortality (EM) and impaired interferon tau production from the conceptus causes luteolytic endometrial signaling and activated inflammatory and luteolytic responses in the corpus luteum (CL) to further test the hypothesis that EM conceptuses with an impaired interferon production have adaptive immune responses and causes luteolytic endometrial signaling. The previous approach to aim 1 was to perform real time-quantitative polymerase chain reactions (RT-qPCR) on genes impacting interferon stimulated genes and estradiol action in the endometrium (Endo) and inflammatory responses in the CL. Western blot (WB) procedures were proposed also for endometrium and CL tissues. These were revised to for 2020-2021 to be the following: use proteomic (global mass spectroscopy) analysis of normal conceptus and endometrium tissues of cows in estrous cycle (EC), EM and N pregnancies. It was reasoned that the limited tissues would be best studied for global proteomics and would provide more extensive information rather than simply focusing on the estradiol-associated responses. Unfortunately, due to time constraints, we re-propose to revise the approach of aim 1 for 2021-2022 by conducting two experiments. The first experiment that was conducted for aim 1 entailed performing RT-qPCR on the reproductive tissues (endometrium, corpus luteum and peripheral blood mononuclear cells) of all pregnancy states (embryo mortality pregnancy, non-pregnant and normal pregnancy) to determine the levels of ISG15 mRNA. The second experiment conducted was using western blots on the endometrium of all pregnancy states (embryo mortality pregnancy, non-pregnant and normal pregnancy) to determine the levels of ISG15 protein. Both experiments measure the levels of ISG15 on day 16 of pregnancy (embryo mortality and normal) or non-pregnancy at different time points of the central dogma (after transcription and translation has occurred). The data has aided us in reinforcing the previous results from our RNA-Seq data in which demonstrate that there are no changes in ISG15 mRNA levels in embryo mortality corpus luteum and peripheral blood mononuclear cells due to collection on day 16, which may be prior to a IFNT peripheral response and luteolysis. The statistical analysis were conducted using a one-way ANOVA analysis in SAS software. Pitfalls for this new approach were not anticipated due to both ISG15 primers and antibodies have been previously validated in our research laboratory. What opportunities for training and professional development has the project provided?Current opportunities of training and professional development for the 2021-2022 reporting period have centered on the process of conducting western blots, real time-quantitative polymerase chain reaction, pipeline and validation for single nucleotide polymorphisms in candidate genes and constructing manuscripts for both aims 1 and 2 of the grant. How have the results been disseminated to communities of interest?Results have been disseminated at the following platforms: • Rocky Mountain Reproductive Science Symposium-Spring 2022 in Fort Collins, Colorado: 10-minutes in person oral presentation with 2-minutes of questions and answers. • American Dairy Science Association-Summer 2022 in Kansas City, Missouri: 12-minutes in person oral presentation with 3-minutes of questions and answers. • National Association of Animal Breeders-Fall 2022 in Milwaukee, Wisconsin: 2-minute flash talk and poster presentation. What do you plan to do during the next reporting period to accomplish the goals?For the next reporting period (2022-2023), both manuscripts of aims 1 and 2 will have been submitted for journal publication.

Impacts
What was accomplished under these goals? For the 2021-2022 reporting period, research efforts have been directed towards conducting two additional experiments for aim 1 and the completion of a quality control pipeline for the 69 single nucleotide polymorphisms (SNP) in candidate genes from aim 2 of the grant. The first experiment that was conducted for aim 1 entailed performing real time-quantitative polymerase chain reaction on the tissues (endometrium, corpus luteum and peripheral blood mononuclear cells) of all pregnancy states (embryo mortality pregnancy, non-pregnant and normal pregnancy) to determine the ISG15 mRNA present. The second experiment for aim 1 was conducted using western blots on the endometrium tissues of all pregnancy states (embryo mortality pregnancy, non-pregnant and normal pregnancy) to determine the ISG15 protein present. The quality control pipeline for aim 2 was based on the RNA sequencing of conceptuses (normal and embryo mortality) in Holstein-Friesian (n=15) cows from aim1 and were used to conduct the SNP discovery phase. Selection for the specific SNPs within genes were first based on the following steps: (1) Related or associated to reproductive/pregnancy/fertility traits within previous studies, (2) Differentially expressed genes within previous RNA Seq transcriptome data that had log2 fold change significant (adjusted p<0.05), (3) Sorting identified SNPs as diagnostics or non-diagnostic, (4) Evaluating in which region and type of differentially expressed SNP, (5) Asses the prediction value of the sorting intolerant from tolerant tool (SIFT) analyses when an amino acid substitution occurred and if it could affect protein function , (6) Confirm if the loci of the SNP was in proximity to other associated SNP associated to reproductive/fertility traits in Cattle Quantitative Trait Loci (Cattle QTL) database. Validation of candidate SNPs and genotype to phenotype analysis were conducted in a different cohort of Holstein-Friesian cows (n=500) by collecting blood samples to be genotyped via a genotyping assay panel and collecting cow farms records. Further filtering of candidate SNPs, after having received all samples (n=500) genotyped, started by estimating genotypic and allelic frequencies to verify which SNP were monomorphic across all animals. We then verified which SNPs were in minor allele frequency (MAF; >10%). The candidate SNPs that were not in the MFA and/or were monomorphic were eliminated from the study. The remaining candidate SNPs were evaluated using pLink software using a quality control pipeline that consisted of five steps. The first step was to remove any SNP that 20% or higher genotypes missing in the data. The second step was to remove individual animals that were not genotyped for 10% or higher of the candidate SNPs. The third step was to remove any candidate SNPs that were not in Hardy-Weinberg Equilibrium by the metric of 1e-15 (i.e., SNP that were above 1e-15). The fourth was then to evaluate the remaining candidate SNPs for linkage disequilibrium via r2 and d'. The final step was to identify tag SNPs within the candidate SNPs. If there were more than one tag SNP for a group of SNPs, the tag SNP was selected based on r2 and d' having the highest values (i.e. strongest relationship to the group of SNPs). An additional number of animals were removed from the study due to missing AI information. Furthermore, some of the reproductive (breeding date, calving of cow and dystocia score) and production traits (predicted 1st lactation milk production at 305 DIM) were also removed from the study due to missing values. By having removed these animals and traits from the study, the n of observations used for the statistical models of the study were n=466. Continuous numeric trait models were analyzed using GLM-one way ANOVA in SAS. While binary models were evaluated using logistic regression in SAS. Statistically significant models were further analyzes using a means separation tests within LSMEANS from the mixed procedure and included the Bonferroni adjustment for p-values to minimize false discovery error. A total of sixty-nine candidate SNPs were initially discovered but only twenty-three passed the quality control pipeline in pLink software. All candidate SNPs were found explain a higher amount of the r2 variation of each of the models and were in close proximity to SNP that were associated with quantitative trait loci of fertility traits. Results of both aim 1 and 2 of the grant have been revised and organized to be published as journal articles with the added experiments (western blots and real time-quantitative polymerase chain reaction) and procedures for quality control of the single nucleotide polymorphisms in 69 candidate genes for fall of 2022.

Publications

• Type: Other Status: Other Year Published: 2022 Citation: Gonzalez-Berrios CL, Bishop JV, Van Campen H, Thomas MG, Hansen TR. Impaired IFNT production and action during embryo mortality in lactating Holstein-Friesian cows. American Dairy Science Association, 2022 Annual Meeting: Abstract #86960. Kansas City, MO.
• Type: Other Status: Other Year Published: 2022 Citation: Gonzalez-Berrios CL, Bishop JV, Van Campen H, Thomas MG, Hansen TR. Elucidating the mechanism of early embryo mortality pregnancies in lactating Holstein-Friesian cows. National Association of Animal Breeders, 2022. Milwaukee, WI. NIFA award acknowledged.
• Type: Other Status: Other Year Published: 2022 Citation: Gonzalez-Berrios CL, Bishop JV, Van Campen H, Thomas MG, Hansen TR. Impaired production and action of IFNT during early embryo mortality pregnancies in lactating Holstein-Friesian cows. Rocky Mountain Reproductive Science Symposium, 2022 Annual Conference. Fort Collins, CO.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Liebig BE, Bishop JV, McSweeney KD, Van Campen H, Gonzalez-Berrios CL, Hansen TR, Thomas MG. Direct genomic value daughter pregnancy rate and services per conception are associated with characteristics of day 16 conceptuses and hormone signaling for maternal recognition of pregnancy in lactating Holstein cows. Published on Applied Animal Science.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2022 Citation: Gonzalez-Berrios CL, Pierce CF, Bishop JV, Van Campen H, Piendo P, Hansen TR, Thomas MG. Identification of candidate SNPs associated to fertility traits in Holstein- Friesian Cows undergoing Early Embryo Mortality. Pending publication and submission in late June, 2022.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2022 Citation: Gonzalez-Berrios CL, Sinedino LDP, Georges HM, Bishop JV, Van Campen H, Thomas MG, Hansen TR. A transcriptome response of reproductive tissues to pregnancies with early embryo mortality in Holstein-Friesian cows. Pending publication and submission in early June, 2022.

Progress 07/01/20 to 06/30/21

Outputs
Target Audience:This research targeted, during the 2020-2021 reporting period, researchers at different career levels (graduate students-MS or PhD, Post-Docs, faculty), veterinarians, industry and extension focused on the topic of animal genetics, bioinformatics and (or) reproduction through: Rocky Mountain Reproductive Science Symposium Spring 2021 in Fort Collins, Colorado: 5-minutes virtual poster presentation with 5-minutes Q&A. Animal Reproduction and Biotechnology Laboratory Department Fall 2021 Seminar: oral presentation of 50-minutes with Q&A. American Society of Animal Sciences-Western Section Fall 2021 in Fort Collins, Colorado: poster presentation of 1 hour. Changes/Problems:It was proposed in Aim 1 of this grant to determine if early embryo mortality (EM) and impaired interferon tau production from the conceptus causes luteolytic endometrial signaling and activated inflammatory and luteolytic responses in the corpus luteum (CL). The approach for this aim was to perform Real Time-quantitative Polymerase Chain Reactions (RT-qPCR) on genes impacting interferon stimulated genes and estradiol action in the endometrium (Endo) and inflammatory responses in the CL. Western blot (WB) procedures were proposed also for endometrium and CL tissues. Unfortunately, the size and number of tissues (endometrium and CL) available for both the validation and reactions for RT-qPCR and WB may not be sufficient. Herein, we propose to revise the specific objective and approach of Aim 1. Our new objective will be to further test the hypothesis that EM conceptuses with an impaired interferon production have adaptive immune responses and causes luteolytic endometrial signaling. The approach for this new aim will be to use proteomic (global mass spectroscopy) analysis of normal conceptus and Endo tissues of cows in estrous cycle (EC), EM and N pregnancies which have not yet been analyzed. It is reasoned that the limited tissues could best be studied for global proteomics and would provide more extensive information rather than simply focusing on the estradiol-associated responses. A one-way ANOVA will be performed between all three endometrium samples (EC vs EM vs N) and between N conceptus and all three endometrium samples (EC vs EM vs N). While T-tests will be performed on the following comparisons: N conceptus vs N Endo; N conceptus vs EM Endo; EM Endo vs N Endo; EM Endo vs EC Endo; EC Endo vs N Endo. The results will be further analyzed with the criteria that proteins must have a fold change of <+1.5-> and P value of <0.05 when submitted into Ingenuity Pathway Analysis (IPA) bioinformatics software. This software will aid us in identifying key biological pathways within each comparison (N conceptus vs N Endo; N conceptus vs EM Endo; EM Endo vs N Endo; EM Endo vs EC Endo; EC Endo vs N Endo). Pitfalls for this new aim are that IPA may be unable to detect key biological pathways and therefore, a list of differentially expressed genes will be generated from IPA and submitted to STRING database to evaluate any potential protein-protein interactions. What opportunities for training and professional development has the project provided?Current opportunities of training and professional development for the 2020-2021 reporting period have centered on the process of conducting SNP Discovery, creating a custom SNP panel and validating SNPs using a different cohort of cows (n=500). How have the results been disseminated to communities of interest?Results have been disseminated at the folowing platforms: Rocky Mountain Reproductive Science Symposium Spring 2021 in Fort Collins, Colorado: 5-minutes virtual poster presentation with 5-minutes Q&A. Animal Reproduction and Biotechnology Laboratory Department Fall 2021 Seminar: oral presentation of 50-minutes with Q&A. American Society of Animal Sciences-Western Section Fall 2021 in Fort Collins, Colorado: poster presentation of 1 hour. What do you plan to do during the next reporting period to accomplish the goals?For the next reporting period (2021-2022), we have proposed a revised specific objective and approach for aim 1 (Please see changes/problems section for more detail). We plan to accomplish these new goals by using Ingenuity Pathway Analysis (IPA) bioinformatics software to identifying key biological pathways. If no key biological pathways are detected by IPA, a list of differentially expressed genes will be generated from IPA and submitted to STRING database to evaluate any potential protein-protein interactions. We also will conduct statistical analysis using a one-way ANOVA and T-tests.

Impacts
What was accomplished under these goals? For the 2020-2021 reporting period, research efforts have been directed towards the completion of aim 2 of grant, which is to identify SNP markers in candidate genes that can predict normal compared to EM pregnancies. Identification of 69 SNP candidates was achieved by using the QIAGEN CLC Genomics Workbench and previous single-end read sequences from the initial preliminary project of 24 Holstein-Friesian cows. A custom Agena Plex panel was then designed with Neogen to validate the 69 SNP candidates in a different cohort of Holstein-Friesian cows (n=500). Blood samples from these cows were then taken for DNA isolation and genotype for the 69 SNP candidates associated with early embryo mortality pregnancies, health traits and (or) reproductive traits. Results will be analyzed and published as a journal article. Publication on the preliminary data for this grant with also be published with the results of serum/plasma samples submitted for estradiol and progesterone radioimmunoassays.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Liebig BE, Bishop JV, McSweeney KD, Van Campen H, Gonzalez-Berrios CL, Hansen TR, Thomas MG. Direct genomic value daughter pregnancy rate and services per conception are associated with characteristics of day 16 conceptuses and hormone signaling for maternal recognition of pregnancy in lactating Holstein cows.Provisionally Accepted on Applied Animal Science.
• Type: Other Status: Other Year Published: 2021 Citation: Gonzalez-Berrios.Early Embryo Mortality: The Hibbie Jibbies of the Dairy Industry. Animal Reproduction and Biotechnology Laboratory Spring Seminar 2021. Fort Collins, CO.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Gonzalez-Berrios CL, Bishop JV, Van Campen H, Thomas MG, Hansen TR. Analysis of the interferon-tau genes: Bovine Genome Assembly UCD1.2vsUMD3.1. American Society of Animal Science-Western Section Annual Conference 2021. Fort Collins, CO. Poster Presentation.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2021 Citation: Sinedino LDP, Liebig BE, Bishop JV, McSweeney KD, Hess AM, Van Campen H, Gonzalez-Berrios CL, Thomas MG, Hansen TR. Transcriptomic analyses of conceptus and endometrium during pregnancy failure at the preimplantation stage reveals massive inflammatory responses and lack of maternal recognition of pregnancy in Holstein cows. Awaiting Submission on Journal of Animal Reproduction and Genetics.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2021 Citation: Gonzalez-Berrios CL, Sinedino LDP, Georges HM, Bishop JV, Van Campen H, Thomas MG, Hansen TR. 2021. A transcriptome response of reproductive tissues to early embryo mortality pregnancies in Holstein-Friesian cows. Awaiting Submission in the Journal of Animal Reproduction and Genetics.