Source: UNIVERSITY OF MISSOURI submitted to
PHYSIOLOGICAL AND GENETIC INSIGHTS INTO PREGNANCY LOSS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1017921
Grant No.
2019-67015-28998
Cumulative Award Amt.
$1,625,000.00
Proposal No.
2018-08537
Multistate No.
(N/A)
Project Start Date
Jan 1, 2019
Project End Date
Dec 31, 2025
Grant Year
2019
Program Code
[A1241]- Dual use of animals for dual benefit
Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211
Performing Department
Office of Sponsored Programs
Non Technical Summary
Infertility and subfertility are major problems in domestic animals and humans. Those fertility problems involve pregnancy loss due to embryo mortality, which is particularly prevalent during the first two months of pregnancy establishment. Many cases of infertility and pregnancy loss cannot be ascribed to a specific genetic or physiological cause. In cattle and humans, fertility issues and pregnancy loss is due to paternal, maternal and/or embryonic factors. However, our knowledge of the underlying complex biological pathways and genes governing establishment of pregnancy and paternal influences on embryo mortality and pregnancy loss remains very limited in agricultural animals and humans. Our central hypothesis is that the paternal genome has a strong influence on embryo survival and mortality during the establishment of pregnancy and thus pregnancy success and loss. The goal of this proposal is to test this hypothesis using an innovative systems biology approach and to understand the genetics of embryo mortality and pregnancy loss with a focus on pregnancy establishment in dairy cattle. To achieve this goal, Holstein bulls will be identified that possess intrinsic differences in pregnancy loss during the first two months after breeding based on early (< 32 days of gestation) and late embryonic mortality (> 32 days). These phenotyped bulls will be used to uncover the biological and genetic mechanisms governing paternal effects on embryo mortality and develop genetic selection tools to improve pregnancy rates in cattle. Specific aims are to: (1) identify sires with increased embryo mortality. This aim will identify and phenotype subfertile sires who create embryos that have a higher prevalence of early or late mortality in the first two months of pregnancy; (2) investigate biological mechanisms governing early embryo mortality. This aim will test the hypotheses that subfertile sires have defects in genes governing embryo development and/or conceptus implantation and placentation that impact early embryo mortality and establishment of pregnancy; and (3) determine genetic mechanisms underlying sire effects on embryo survival and mortality. This aim will test the hypothesis that genetic loci and genomic rearrangements associated with subfertile sire effects on early and late embryo mortality can be identified using cutting-edge genomic analyses. Completion of the research is expected to fill a substantial gap in our existing knowledge by providing novel insights into bull fertility and pregnancy loss. The systems biology approach utilizing functional genomics and discovery-based methods is innovative. Expected translational outcomes of this research are genetic markers that can be used as diagnostic tools to identify, select and create sires with increased fertility, which will increase pregnancy rates, production efficiency, profitability and sustainability of cattle enterprises. Translation of the identified markers (genes in which disruptive human variants are likely to cause embryo loss) to humans is expected to provide novel insights into idiopathic male fertility that can be used to diagnose, treat and prevent fertility problems and enhance pregnancy outcomes in natural and assisted reproduction.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3013499102050%
3013499105030%
3033499108020%
Knowledge Area
303 - Genetic Improvement of Animals; 301 - Reproductive Performance of Animals;

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

Field Of Science
1080 - Genetics; 1020 - Physiology; 1050 - Developmental biology;
Goals / Objectives
The major goals of this proposal are to determine the physiological and genetic mechanisms mediating effects of the sire on pregnancy establishment and embryo mortality using a systems biology approach. To test the central hypothesis and accomplish our goals, we propose three specific objectives:Objective 1: Identify sires with increased embryo mortality. This objective will identify subfertile Holstein bulls who sire pregnancies with a higher prevalence of early or late mortality. The approach is to analyze reproduction records of many heifer raising facilities and dairies to identify candidate sires for early (SFEarly) or late (SFLate) embryo mortality and then ensure their phenotype using a controlled fertility field trial. Objective 2: Investigate physiological mechanisms regulating early embryo loss. This objective will test the hypotheses that SFEarly sires have defects in genes controlling embryo development and/or conceptus implantation and/or placentation that underlie increased early embryonic mortality.Objective 3: Determine genetic mechanisms underlying sire influences on embryo survival and pregnancy establishment. This objective will test the hypothesis that genetic loci and genomic rearrangements associated with embryo survival and mortality and pregnancy success can be identified using cutting-edge genomic analyses of fertile and subfertile sires identified and phenotyped in Objective 1.Completion of the research is expected to fill a substantial gap in our existing knowledge by providing novel insights into the physiology and genetics of bull fertility and pregnancy loss. The systems biology approach utilizing functional genomics and discovery-based methods is innovative. Expected outcomes of this research are genetic markers that can be used as diagnostic tools to identify, select and create bulls with increased fertility, which will increase pregnancy rates, production efficiency, profitability and sustainability of cattle enterprises.
Project Methods
The physiological and genetic mechanisms mediating effects of the sire on pregnancy establishment and embryo mortality will be studied using a systems biology approach. The experiments will be statistically analyzed and the results used to test the scientific hypotheses. The experimental results and findings will be published in scientific journals. The project will be evaluated by timely completion of the goals and objectives and the publication of findings in scientific journals. Measurable indicators of success will include presentation of abstracts at national and international meetings as well as scientific publications. In this way, the project will impact the larger scientific community and be transmitted to stakeholders.

Progress 01/01/24 to 12/31/24

Outputs
Target Audience:Target audience include scientists, educators, and producers. Efforts included presentation of seminars to deliver science based knowledge to stakeholders and experiential learning opportunities. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?University of Missouri: Three graduate students have completed their degrees including Kelsey Lockhart (MS, 2021), Gessica Franco (PhD, 2021), and Lindsey Fallon (MS, 2022). Jessica Drum was a postdoctoral research fellow on this project from 2021-2022, and she joined South Dakota State University as an Assistant Professor and Extension Beef Reproductive Physiology Specialist in 2023. Kimberly Davenport was a Postdoctoral Research Fellow on this project from 2021-2023, and she joined Washington State University as an Assistant Professor of Functional Genomics in 2023. Currently, two PhD students (Makenzie Lowke and Abdallah Abdelhady) are working with the PD at the University of Missouri on the project. University of Wisconsin-Madison: One MS and one PhD student have been working on projects related to this grant. Texas A&M University: One MS and one PhD student have been working on projects related to this grant. In addition, state extension and dairy specialists have been trained to consider using sire in their producer evaluations and genetics programs. How have the results been disseminated to communities of interest?The results have been published in peer-reviewed scientific journals and also presented at regional, national and international scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?Objective 2. Investigate physiological mechanisms regulating early embryo loss. An in vitro culture system has been developed to determine the effects of sire phenotype from Objectives 1 and 2 on trophoblast differentiation. This system involves deriving trophoblast stem cells from bovine embryos and differentiating them into the complex cell types found in the bovine placenta. Single cell sequencing (Multiome ATAC-seq and RNA-seq) as well as non-radioactive in situ hybridization analyses are being used to uncover gene regulatory networks governing trophoblast differentiation. We will continue using this approach to complete Objective 2. Objective 3: Determine Genetic Mechanisms Underlying Sire Influences on Embryo Survival and Pregnancy Establishment. We are currently finishing projects that genome-wide association study (GWAS) as well as copy number variation (CNV) analysis using the whole genome sequences of phenotyped sires from Objective 1. The plan is to continue this approach to complete Objective 3.

Impacts
What was accomplished under these goals? Objective 1. Identify sires with increased embryo mortality. The objective is to identify subfertile Holstein bulls who sire pregnancies with a higher prevalence of early (< day 32) or late (> day 32) embryo mortality. The approach is to analyze reproduction records of many heifer raising facilities and dairies to identify candidate sires for early (SFEarly) or late (SFLate) embryo mortality and then ensure their phenotype using a controlled fertility field trial. Comprehensive analyses identified a set of 10 high fertility and 40 candidate SF bulls and concluded low correlations with sire conception rate (SCR). This objective has been completed, and the results published. Objective 2. Investigate physiological mechanisms regulating early embryo loss. This objective will test the hypotheses that SFEarly sires have defects in genes controlling embryo development and/or conceptus implantation and/or placentation that underlie increased early embryonic mortality. A total of 65 sires were tested in vitro, and sires with poor in vitro embryo production were also poor in vivo. Sires with divergent fertility were used to produce embryos under stress and normal conditions as a measure of adaptation in the field. Further, sires with low potential for trophectoderm growth had higher pregnancy loss and lower secretion of pregnancy associated glycoproteins (PAGs) compared to those from high fertility sires and revealed a need to further understand placentation and differentiation of cell populations in the placenta. Using a single-cell transcriptomic approach, different trophoblast cell types were identified in the bovine placenta. Further, the trophoblast differentiation pathway was investigated in conceptuses from early and mid-pregnancy. This foundational information is useful to discover essential biological pathways underpinning the development and function of the bovine placenta in normal conditions and potential candidates of genes involved in pregnancy loss. This objective has not been completed. However, some of the results were published with one manuscript recently submitted for publication. Objective 3: Determine genetic mechanisms underlying sire influences on embryo survival and pregnancy establishment. This objective is to test the hypothesis that genetic loci and genomic rearrangements associated with embryo survival and mortality and pregnancy success can be identified using cutting-edge genomic analyses of fertile and subfertile sires identified and phenotyped in Objective 1. A total of 80 bulls with in vitro phenotypes were sequenced and the dataset has been enriched with sequences from the 1000 bull genome project, and other sequences gathered in collaboration with colleagues at the University of Missouri for more than 200 sequenced bulls. This dataset was used to identify genetic commonalities (SNPs) between high and low performing sires for either preimplantation development or early placentation. This objective has been completed, and the results published with one manuscript being prepared for publication.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2024 Citation: Biase FH, Moorey SE, Schnuelle JG, Rodning S, Ortega MS, Spencer TE. Altered microRNA composition in the uterine lumen fluid in cattle (Bos taurus) pregnancies initiated by artificial insemination or transfer of an in vitro produced embryo. J Anim Sci Biotechnol. 2024 Sep 13;15(1):130. doi: 10.1186/s40104-024-01083-8. PMID: 39267128; PMCID: PMC11397056.
  • Type: Journal Articles Status: Accepted Year Published: 2024 Citation: Davenport KM, O'Neil EV, Ortega MS, Patterson A, Kelleher AM, Warren WC, Spencer TE. Single-cell insights into development of the bovine placenta . Biol Reprod. 2024 Jan 13;110(1):169-184. doi: 10.1093/biolre/ioad123. PMID: 37707543.
  • Type: Journal Articles Status: Accepted Year Published: 2024 Citation: Fallon L, Lockhart KN, Spencer TE, Ortega MS. High and low performing sires differ in their contributions to early embryonic stress in the bovine. Reprod Fertil Dev. 2024 Sep;36:RD24049. doi: 10.1071/RD24049. PMID: 39265060.
  • Type: Journal Articles Status: Accepted Year Published: 2024 Citation: Kelson VC, Kiser JN, Davenport KM, Suarez EM, Murdoch BM, Neibergs HL. Identifying Regions of the Genome Associated with Conception Rate to the First Service in Holstein Heifers Bred by Artificial Insemination and as Embryo Transfer Recipients. Genes (Basel). 2024 Jun 11;15(6):765. doi: 10.3390/genes15060765. PMID: 38927701; PMCID: PMC11202900.
  • Type: Journal Articles Status: Submitted Year Published: 2024 Citation: Davenport KM, Lowke MT, Ortega MS, Kelleher AM, Warren WC, Spencer TE. Single cell multiome analysis of the bovine placenta identifies gene regulatory networks in trophoblast differentiation. Biol Reprod. 2024 submitted.


Progress 01/01/23 to 12/31/23

Outputs
Target Audience:Target audience include scientists, educators, and producers. Efforts included presentation of seminars to deliver science based knowledge to stakeholders and experiential learning opportunities. Changes/Problems:The COVID-19 pandemic delayed our ability to complete project objectives, particularly during 2020 and 2021, due to stay-at-home restrictions and supply chain disruptions, and negative impacts on data generation from advanced technology core facilities used in the projects. In addition, recruitment of personnel to work on the project was challenging during those two years. Recently, one of the co-Investigators (Dr. Sofia Ortega) moved from the University of Missouri to the University of Wisconsin-Madison in 2023, which delayed completion of some project objectives. In addition, Dr. Kimberly Davenportwas a Postdoctoral Research Fellow on this project from 2021-2023, and she moved to Washington State University as an Assistant Professor of Functional Genomics in 2023. However, two PhD students (Makenzie Lowke and Abdallah Abdelhady) are currently working with the PD at the University of Missouri to complete the projects. What opportunities for training and professional development has the project provided?University of Missouri: Three graduate students have completed their degrees including Kelsey Lockhart (MS, 2021), Gessica Franco (PhD, 2021), and Lindsey Fallon (MS, 2022). Jessica Drum was a postdoctoral research fellow on this project from 2021-2022, and she joined South Dakota State University as an Assistant Professor and Extension Beef Reproductive Physiology Specialist in 2023. Kimberly Davenport was a Postdoctoral Research Fellow on this project from 2021-2023, and she joined Washington State University as an Assistant Professor of Functional Genomics in 2023. Currently, two PhD students (Makenzie Lowke and Abdallah Abdelhady) are working with the PD at the University of Missouri on the project. University of Wisconsin-Madison:One MS and one PhD student have been working on projects related to this grant. Texas A&M University: One MS and one PhD student have been working on projects related to this grant. In addition, state extension and dairy specialists have been trained to consider using sire in their producer evaluations and genetics programs. How have the results been disseminated to communities of interest?The results have been published in peer-reviewed scientific journals and also presented at regional, national and international scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?Objective 2. Investigate physiological mechanisms regulating early embryo loss We are currently using an in vitro culture system to determine the effects of sire phenotype from Objectives 1 and 2 on trophoblast differentiation. This effort involves deriving trophoblast stem cells from bovine embryos and differentiating them into the placenta. Expensive single cell sequencing (ATAC-seq and RNA-seq Multiome) as well as non-radioactive in situ hybridization analyses are being used to map genes potentially regulating trophoblast differentiation. The plan is to continue using this approach to complete Objective 2. Objective 3: Determine Genetic Mechanisms Underlying Sire Influences on Embryo Survival and Pregnancy Establishment We are currently finishing projects that genome-wide association study (GWAS) as well as copy number variation (CNV) analysis using the whole genome sequences of phenotype sires from Objective 1. The plan is to continue this approach to complete Objective 3 in the NCE period by genotyping bulls with a GeneSeek custom Illumina platform.

Impacts
What was accomplished under these goals? Objective 1. Identify sires with increased embryo mortality. The objective is to identify subfertile Holstein bulls who sire pregnancies with a higher prevalence of early (< day 32) or late (> day 32) embryo mortality. The approach is to analyze reproduction records of many heifer raising facilities and dairies to identify candidate sires for early (SFEarly) or late (SFLate) embryo mortality and then ensure their phenotype using a controlled fertility field trial. Data from commercial farms was retrieved, and reproduction records comprising both embryo transfer and artificial inseminations from over 150 bulls have been analyzed to identify candidate SFEarly and SFLate bulls. From these analyses, a set of 10 high fertility and 40 candidate SF bulls were identified. Analyses concluded low correlations with sire conception rate and no relationship between SFEarly and SFLate, reinforcing the need for these types of analysis. High and low pregnancy loss sires have been screened from the original data set and compared to 10 dairies in the Texas, New Mexico, and Kansas. These data provided a refined list of sires that were used for repeatability studies in pregnancy loss. There have also been specific paternal and maternal uni-parental embryos produced and transferred. This objective has been completed. The results have been published. Objective 2. Investigate physiological mechanisms regulating early embryo loss. This objective will test the hypotheses that SFEarly sires have defects in genes controlling embryo development and/or conceptus implantation and/or placentation that underlie increased early embryonic mortality. Previously, 65 bulls were phenotyped, and specific tests related to early embryo stress have been used to phenotype 20 additional sires for this project. A total of 65 sires have been tested in vitro, and sires with poor in vitro embryo production were also found to be of poor performance in vivo. These results support the use of an in vitro method as a tool to identify sires with reduced probability to establish pregnancy. To understand non-genetic male influences on early embryonic development, a project evaluating sperm morphology, protein aggregation, reactive oxygen species, and DNA damage by microscopy and flow cytometry was completed. In addition, sires of divergent fertility were used to produce embryos under stress and normal conditions as a measure of adaptation in the field. The results of these experiments have been published. Previous work from this project indicated that sires with low potential for trophectoderm growth had higher pregnancy loss and lower secretion of pregnancy associated glycoproteins compared to those from high fertility sires and revealed a need to further understand placentation and differentiation of cell populations in the placenta. Using a single-cell transcriptomic approach, different trophoblast cell types were identified in the bovine placenta. Further, the trophoblast differentiation pathway was investigated in conceptuses from early and mid-pregnancy. Candidate transcription factors associated with trophectoderm differentiation were also identified. This foundational information is useful to discover essential biological pathways underpinning the development and function of the bovine placenta in normal conditions and potential candidates of genes involved in pregnancy loss. The results have been recently published, and one manuscript is being prepared for publication.Further,we have developedin vitro culture system to determine the effects of sire phenotype from Objectives 1 and 2 on trophoblast differentiation. This effort involves deriving trophoblast stem cells from bovine embryos and differentiating them into the placenta. Expensive single cell sequencing (ATAC-seq and RNA-seq Multiome) as well as non-radioactive in situ hybridization analyses are being used to map genes potentially regulating trophoblast differentiation. Objective 3: Determine genetic mechanisms underlying sire influences on embryo survival and pregnancy establishment. This aim will test the hypothesis that genetic loci and genomic rearrangements associated with embryo survival and mortality and pregnancy success can be identified using cutting-edge genomic analyses of fertile and subfertile sires identified and phenotyped in Aim 1. A total of 80 bulls with in vitro phenotypes have been sequenced and the dataset has been enriched with sequences from the 1000 bull genome project, and other sequences gathered in collaboration with colleagues at the University of Missouri for more than 200 sequenced bulls. This represents a powerful dataset to identify genetic commonalities between high and low performing sires for either preimplantation development or early placentation. The first round of analysis yielded 8 single nucleotide polymorphisms (SNP) associated with early embryonic cleavage, 4 SNP associated with formation of a blastocyst, and 4 SNP with the development from the 4-cell to the blastocyst stage. This objective has been completed. The results have been published, and a new manuscript is being prepared for publication.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Biase FH, Moorey SE, Schnuelle JG, Rodning S, Ortega MS, Spencer TE. Extensive rewiring of the gene regulatory interactions between in vitro-produced conceptuses and endometrium during attachment. PNAS Nexus. 2023 Sep 2;2(9):pgad284. doi: 10.1093/pnasnexus/pgad284. PMID: 37711857; PMCID: PMC10498941
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Davenport KM, Ortega MS, Liu H, O'Neil EV, Kelleher AM, Warren WC, Spencer TE. Single-nuclei RNA sequencing (snRNA-seq) uncovers trophoblast cell types and lineages in the mature bovine placenta. Proc Natl Acad Sci U S A. 2023 Mar 21;120(12):e2221526120. doi: 10.1073/pnas.2221526120. Epub 2023 Mar 13. PMID: 36913592; PMCID: PMC10041116
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Fallon L, Diaz-Miranda E, Hamilton L, Sutovsky P, Zigo M, Spencer TE, Ortega MS. The development of new biomarkers of spermatozoa quality in cattle. Front Vet Sci. 2023 Oct 12;10:1258295. doi: 10.3389/fvets.2023.1258295. PMID: 37901101; PMCID: PMC10601460


Progress 01/01/22 to 12/31/22

Outputs
Target Audience:Target audience include scientists, educators, and producers. Efforts included presentation of seminars to deliver science based knowledge to stakeholders and experiential learning opportunities. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?At the University of Missour, three graduate students have completed their degrees including Kelsey Lockhart (MS-2021), Gessica Franco (Ph.D.-2021), and Lindsey Fallon (MS-2022). Jessica Drum completed her postdoctoral training in 2022, and Kimberly Davenport continues to work on the project. At Texas A&M University,one MS student and one PhD student have been working on projects related to this grant. In addition, state extension and dairy specialists have been trained to consider using sire in their producer evaluations and genetics programs. How have the results been disseminated to communities of interest?The results have been published in peer-reviewed scientific journals and also presented at regional, national and international scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?For Aim 2, single cell analysis of the developing bovine conceptus between days 17 and 50 will be completed and submitted for publication. For Aim 3, candidate markers of early embryonic development and pregnancy loss will be validated using control and selected sires and apopulation of genotyped females.Efforts will be done to complete the publication of all advances in the project.

Impacts
What was accomplished under these goals? Aim 1. Identify sires with increased embryo mortality. This aim will identify subfertile Holstein bulls who sire pregnancies with a higher prevalence of early (< day 32) or late (> day 32) embryo mortality. The approach is to analyze reproduction records of many heifer raising facilities and dairies to identify candidate sires for early (SFEarly) or late (SFLate) embryo mortality and then ensure their phenotype using a controlled fertility field trial. Data from commercial farms was retrieved, and above 40,000 reproduction records comprising both embryo transfer and artificial inseminations from a total of 145 bulls have been analyzed to identify candidate SFEarly and SFLate bulls. From these analyses, a set of 10 high fertility and 40 candidate SF bulls were identified. Analysis conclude low correlations with sire conception rate and no relationship between SFEarly and SFLate, reinforcing the need for these types of analysis. High and low pregnancy loss sires have been screened from the original data set and compared to 10 dairies in the Texas, New Mexico, and Kansas. These data provide a refined list of sires that are now being used for repeatability studies in pregnancy loss. There have also been specific paternal and maternal uni-parental embryos produced and transferred. This experiment is currently being analyzed. Aim 2. Investigate physiological mechanisms regulating early embryo loss. This aim will test the hypotheses that SFEarly sires have defects in genes controlling embryo development and/or conceptus implantation and/or placentation that underlie increased early embryonic mortality. Previously, 65 bulls were phenotyped, and specific tests related to early embryo stress have been used to phenotype 20 additional sires for this project. A total of 65 sires have been tested in vitro, and sires with poor in vitro embryo production were also found to be of poor performance in vivo. These results support the use of an in vitro method as a tool to identify sires with reduced probability to establish pregnancy. To understand non-genetic male influences on early embryonic development, a project evaluating sperm morphology, protein aggregation, reactive oxygen species, and DNA damage by microscopy and flow cytometry was completed. In addition, sires of divergent fertility were used to produce embryos under stress and normal conditions as a measure of adaptation in the field. The results of these experiments are being prepared for publication. Previous work from this project indicated that sires with low potential for trophectoderm growth had higher pregnancy loss and lower secretion of pregnancy associated glycoproteins compared to those from high fertility sires and revealed a need to further understand placentation and differentiation of cell populations in the placenta. Using a single-cell transcriptomic approach, different trophoblast cell types were identified in the bovine placenta. Further, the trophoblast differentiation pathway was investigated in conceptuses from early and mid-pregancy. Candidate transcription factors associated with trophectoderm differentiation were also identified. This foundational information is useful to discover essential biological pathways underpinning the development and function of the bovine placenta in normal conditions and potential candidates of genes involved in pregnancy loss. AIM 3: Determine Genetic Mechanisms Underlying Sire Influences on Embryo Survival and Pregnancy Establishment. This aim will test the hypothesis that genetic loci and genomic rearrangements associated with embryo survival and mortality and pregnancy success can be identified using cutting-edge genomic analyses of fertile and subfertile sires identified and phenotyped in Aim 1. A total of 80 bulls with in vitro phenotypes have been sequenced and the dataset has been enriched with sequences from the 1000 bull genome project, and other sequences gathered in collaboration with colleagues at the University of Missouri for more than 200 sequenced bulls. This represents a powerful dataset to identify genetic commonalities between high and low performing sires for either preimplantation development or early placentation. The first round of analysis yielded 8 single nucleotide polymorphisms (SNP) associated with early embryonic cleavage, 4 SNP associated with formation of a blastocyst, and 4 SNP with the development from the 4-cell to the blastocyst stage.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: O'Callaghan E, S�nchez JM, Rabaglino MB, McDonald M, Liu H, Spencer TE, Fair S, Kenny DA, Lonergan P. Influence of sire fertility status on conceptus-induced transcriptomic response of the bovine endometrium. Front Cell Dev Biol. 2022 Aug 22;10:950443. doi: 10.3389/fcell.2022.950443. PMID: 36072344; PMCID: PMC9442353.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Simintiras CA, Drum JN, Liu H, Sofia Ortega M, Spencer TE. Uterine lumen fluid is metabolically semi-autonomous. Commun Biol. 2022 Mar 1;5(1):191. doi: 10.1038/s42003-022-03134-0. PMID: 35233029; PMCID: PMC8888695.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Spencer TE, Wells KD, Lee K, Telugu BP, Hansen PJ, Bartol FF, Blomberg L, Schook LB, Dawson H, Lunney JK, Driver JP, Davis TA, Donovan SM, Dilger RN, Saif LJ, Moeser A, McGill JL, Smith G, Ireland JJ. Future of biomedical, agricultural, and biological systems research using domesticated animals. Biol Reprod. 2022 Apr 26;106(4):629-638. doi: 10.1093/biolre/ioac019. PMID: 35094055; PMCID: PMC9189970.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Ortega MS, Bickhart DM, Lockhart KN, Null DJ, Hutchison JL, McClure JC, Cole JB. Truncation of IFT80 causes early embryonic loss in Holstein cattle associated with Holstein haplotype 2. J Dairy Sci. 2022 Nov;105(11):9001-9011. doi: 10.3168/jds.2022-21853. Epub 2022 Sep 7. PMID: 36085107.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Stoecklein KS, Ortega MS, Spate LD, Murphy CN, Prather RS. Improved cryopreservation of in vitro produced bovine embryos using FGF2, LIF, and IGF1. PLoS One. 2021 Feb 3;16(2):e0243727. doi: 10.1371/journal.pone.0243727. PMID: 33534866; PMCID: PMC7857633.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Davenport KM, Ortega MS, Johnson GA, Seo H, Spencer TE. Review: Implantation and placentation in ruminants. Animals. 2023. accepted.
  • Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Davenport KM, Ortega MS, Liu H, O'Neil EV, Kelleher AM, Warren WC, Spencer TE. Single nuclei RNA sequencing (snRNA-seq) uncovers trophoblast cell types and lineages in the mature bovine placenta. Proc Nat Acad Sci USA. 2023. accepted.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: 1. Drum JN, Madureira G, Macedo MC, Rosa C, Seneda M, Campos D, Wiltbank MC, Sartori R, Ortega MS (2022) Sexual dimorphism of in vitro derived conceptus at day 32 of pregnancy. Frontiers in animal sciences. doi: 10.3389/fanim.2022.807217
  • Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Lockhart KN, Drum JN, Balboula AZ, Spinka C, Spencer TE, Ortega MS. (2023) Paternal effects on pre-implantation embryo development in the bovine. Molecular Reproduction and Development. Submitted
  • Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Lockhart J, Rizo J, Balboula A, Spinka C, Spencer TE, Ortega MS. Sire conception rate and its relationship with preimplantation embryonic development. J Dairy Sci. 2023. under review.
  • Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Ortega MS, Rizo JA, Drum JN, O'Neil EV, Pohler KY, Kerns K, Schmelze A, Green J, Spencer TE. Development of an improved in vitro model of bovine trophectoderm differentiation. Frontiers in Animal Science. 2023. accepted.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Odile Polanco, Gabriela Dalmaso de Melo, Sofia Ortega, Ramiro Oliveira Filho, Gessica Franco-Johannsen, Amanda Bega, Ronaldo Cerri, Ky Pohler, Smith B. D., Poliakiwski B., Polanco O., Singleton S., de Melo G. D., Muntari M., Oliveira Filho R. V., Pohler K. G. (2022) Decisive points for pregnancy losses in beef cattle. Reproduction, Fertility and Development 35, 70-83. https://doi.org/10.1071/RD22206
  • Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Rizo JA, Drum JN, Lockhart KN, Pohler KG, Spencer TE, and Ortega MS. (2023) Sire Influences on Trophectoderm Development in Cattle. Biology of Reproduction. submitted.


Progress 01/01/21 to 12/31/21

Outputs
Target Audience:Target audience include scientists, educators, and producers. Efforts included presentation of seminars to deliver science based knowledge to stakeholders and experiential learning opportunities. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?At the University of Missouri, two graduate students completed their degrees with this project: Kelsey Lockhart (MS) and Gessica Franco (Ph.D.). Ongoing graduate students are Jason Rizo (Ph.D.) and Lindsey Fallon (MS). Jessica Drum (postdoctoral fellow) continues to work and Kimberly Davenport (postdoctoral fellow) joined research efforts related to the project in 2021. At Texas A&M University,multiple graduate students and undergraduates have helped on this specific project, providing opportunities for training future scientist as well as providing them professional development. How have the results been disseminated to communities of interest?The results have been published in peer-reviewed scientific journals and also presented at regional, national and international scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?For the next reporting period, SFEarly bull evaluation on preimplantation development and in vitro embryo development is expected to be completed and published. Evaluation of SFEarly bulls for trophectoderm development and in vivo validation of the results is expected to be completed and published. Bulls from Aim 1 with SFLate will also be tested in smaller populations of controlled animals to demonstrate repeatability and collect additional tissue. For Aim 3, genetic associations between candidate markers/structural variants is expected to be completed in the upcoming reporting period. In addition, a candidate marker list will be generated to start validation step of this project.

Impacts
What was accomplished under these goals? Aim 1. Identify sires with increased embryo mortality. This aim will identify subfertile Holstein bulls who sire pregnancies with a higher prevalence of early (< day 32) or late (> day 32) embryo mortality. The approach is to analyze reproduction records of many heifer raising facilities and dairies to identify candidate sires for early (SFEarly) or late (SFLate) embryo mortality and then ensure their phenotype using a controlled fertility field trial. Data from commercial farms was retrieved, and above 40,000 reproduction records comprising both embryo transfer and artificial inseminations from a total of 145 bulls have been analyzed to identify candidate SFEarly and SFLate bulls. From these analyses, a set of 10 high fertility and 40 candidate SF bulls were identified. Analysis conclude low correlations with sire conception rate and no relationship between SFEarly and SFLate, reinforcing the need for these types of analysis. Data has been prepared for publication and is undergoing review. Aim 2. Investigate physiological mechanisms regulating early embryo loss. This aim will test the hypotheses that SFEarly sires have defects in genes controlling embryo development and/or conceptus implantation and/or placentation that underlie increased early embryonic mortality. Previously, 65 bulls were phenotyped, and specific tests related to early embryo stress have been used to phenotype 20 additional sires for this project. The results on the sire influence during preimplantation embryonic development, were validated in vivo, a manuscript reporting these results is in preparation. To further understand non-genetic male influences on early embryonic development, a project evaluating sperm morphology, protein aggregation, reactive oxygen species, and DNA damage is ongoing. Previously, a trophectoderm cell system was developed to study early placentation. The 22 most extreme sires based in embryonic phenotype and pregnancy outcomes have been tested in this model and results indicate clear influences of sire in trophectoderm (placenta cells) proliferation, and ability to secrete pregnancy associated glycoproteins. Sires with clear segregration in trophectoderm were also tested in vivo using artificial insemination. Sires with low potential for trophectoderm growth had higher pregnancy loss and lower secretion of pregnancy associated glycoproteins in those animals that remained pregnant. These results corroborate the clear influence of sire on early placentation and how useful is the in vitro model to study and phenotype sires with increase risk of pregnancy loss. Manuscripts are being prepared for publication. AIM 3: Determine Genetic Mechanisms Underlying Sire Influences on Embryo Survival and Pregnancy Establishment. This aim will test the hypothesis that genetic loci and genomic rearrangements associated with embryo survival and mortality and pregnancy success can be identified using cutting-edge genomic analyses of fertile and subfertile sires identified and phenotyped in Aim 1. A total of 80 bulls with in vitro phenotypes have been sequenced and the dataset has been enriched with sequences from the 1000 bull genome project, and other sequences gathered in collaboration with colleagues at the University of Missouri for more than 200 sequenced bulls. This represents a powerful dataset to identify genetic commonalities between high and low performing sires for either preimplantation development or early placentation. Preliminary results from structural variant analyses, revealed novel deletions in genes related with centromere proteins in the sperm and genes related to spermatogenesis. These are major first steps towards elucidating genetic variants associated with bull fertility. A manuscript is being prepared for publication.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Drum JN, Madureira G, Macedo MC, Rosa C, Seneda M, Campos D, Wiltbank MC, Sartori R, Ortega MS (2022) Male embryos produced in vitro deviate from their in vivo counterparts in placental gene expression on day 32 of pregnancy. Frontiers in Animal Sciences https://doi.org/10.3389/fanim.2022.807217
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Simintiras CA, Drum JN, Liu H, Sofia Ortega M, Spencer TE. Uterine lumen fluid is metabolically semi-autonomous. Commun Biol. 2022 Mar 1;5(1):191. doi: 10.1038/s42003-022-03134-0. PMID: 35233029; PMCID: PMC8888695.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2022 Citation: Ortega MS, Rizo JA, Drum JN, O'Neil EV, Pohler K, Kerns K, Schmelze A, Green J, Spencer TE. Development of an improved in vitro model of bovine trophectoderm differentiation. Frontiers in Animal Sciences 2022; in press
  • Type: Book Chapters Status: Published Year Published: 2021 Citation: Geisert RD, Spencer TE. Mammalian Placentation: A Tribute to E.C. Amoroso's Contributions to Placenta Development. Adv Anat Embryol Cell Biol. 2021;234:1-6. doi: 10.1007/978-3-030-77360-1_1. PMID: 34694474.


Progress 01/01/20 to 12/31/20

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Opportunities for training was primarily via Kelsey Clark, Jason Rizo, and Gessica Franco who are pursuing degees in Animal Sciences, and conducting experiments related to the project. In addition, Jessica Drum a postdoctoral associate joined research efforts related to the project. How have the results been disseminated to communities of interest?The results have been published in peer-reviewed scientific journals and also presented at regional, national and international scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?For the next reporting period, SFEarly bull evaluation on preimplantation development and in vitro embryo development is expected to be completed and published. Evaluation of SFEarly bulls for trophectoderm development and in vivo validation of the results is expected to be completed and published. Genetic associations between candidate markers/structural variants is expected to be completed and published.

Impacts
What was accomplished under these goals? Aim 1. Identify sires with increased embryo mortality. This aim will identify subfertile Holstein bulls who sire pregnancies with a higher prevalence of early (< day 32) or late (> day 32) embryo mortality. The approach is to analyze reproduction records of many heifer raising facilities and dairies to identify candidate sires for early (SFEarly) or late (SFLate) embryo mortality and then ensure their phenotype using a controlled fertility field trial. Above 40,000 reproduction records comprising both embryo transfer and artificial inseminations from a total of 145 bulls were analyzed to identify candidate SFEarly and SFLate bulls. Results indicate that sire fertility classification was no indicative of pregnancy establishment under artificial insemination or embryo transfer. A manuscript has been submitted for publication to the Journal of Dairy Science and it is under peer review. Aim 2. Investigate physiological mechanisms regulating early embryo loss. This aim will test the hypotheses that SFEarly sires have defects in genes controlling embryo development and/or conceptus implantation and/or placentation that underlie increased early embryonic mortality. A total of 65 Bulls were tested to identify potential defects on preimplantation development. Of the bulls tested, 9 (14%) negatively affected preimplantation development. Embryo produced with these 9 bulls, had slower progression to the blastocyst stage and increase expression of stress markers, which can be indicative of early embryonic mortality. A project studying the mechanisms by which the SFEarly bulls impact preimplantation development is ongoing.To further explore influences of sire on placentation, a system to derive trophectoderm cell lines from individual embryos produced in vitro was established. A project evaluating SFEarly bulls under this system is ongoing to isolate male influences during early placenta development. For both projects, manuscripts are being prepared for publication. AIM 3: Determine Genetic Mechanisms Underlying Sire Influences on Embryo Survival and Pregnancy Establishment. This aim will test the hypothesis that genetic loci and genomic rearrangements associated with embryo survival and mortality and pregnancy success can be identified using cutting-edge genomic analyses of fertile and subfertile sires identified and phenotyped in Aim 1. Bulls have been sequenced and analysis is being performed to identify genetic and structural variants common in candidate SFEarly and SFLate bulls. A manuscript is being prepared for publication.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Franco, G.A., S.T. Reese, R. Poole, J. Rhinehart, K. Thompson, R. Cooke and K.G. Pohler. (2020) Sire contribution to pregnancy loss in different periods of embryonic and fetal development of beef cows. Theriogenology. 154: 84-91.
  • Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Franco GA, Pereira MHC, Ortega MS, Reese ST, Vasconcelos JLM, Spencer TE, Pohler KG. (2021). Sire effect on pregnancy rate and pregnancy loss in dairy cows: does field fertility associate with sire conception rate (SCR)?. Journal of Dairy Science. JDS.2020-19981.
  • Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Ortega MS, Rizo JA, ONeil EV, Pohler KG, Kerns K, Schmelze A, Green JA, Spencer TE. (2021). Bovine trophectoderm differentiation in vitro: from single cell gene expression to pregnancy associated glycoproteins production. Biology of Reproduction. BIOLRE-2021-0096.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Clark KN, Drum JN, Rizo JA, Ortega MS. (2020) Influence of Sire Conception Rate on Bovine Early Embryo Development. IETS 47th Annual Conference.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Stoecklein KS, Clark KN, Pohler KG, Ortega MS. (2020) Investigating differences in gene expression between in vitro produced bovine embryos and parthenotes. IETS 47th Annual Conference.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: 2020 National Cattlemans Beef Association, Cattlemans College: Sire influence on embryo development.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: 2020 American Society of Animal Science Meetings Endocrinology Symposia: Timing of pregnancy loss in cattle.
  • Type: Other Status: Other Year Published: 2020 Citation: Graduate Seminar Series, Department of Animal Sciences, University of Nebraska (2020). Dissecting paternal influences on pregnancy establishment in the bovine.


Progress 01/01/19 to 12/31/19

Outputs
Target Audience:Target audience include scientists, educators, and producers. Efforts included presentation of seminars to deliver science based knowledge to stakeholders and experiential learning opportunities. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Opportunities for training was primarily via Kelsey Clark, Jason Rizo and Gessica Franco who are pursuing degees in Animal Sciences, and conducting experiments related to the project. How have the results been disseminated to communities of interest?The results have been published in peer-reviewed scientific journals and also presented at regional, national and international scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?For the next reporting period, evaluation of candidate SFEarly bulls for their influences during preimplantation development and placentation is expected to be completed and published. Furthermore, genetic associations between candidate markers/structural variants with the studied phenotypes (preimplantation embryonic development and placentation) will be completed, verified and published.

Impacts
What was accomplished under these goals? AIM 1. Identify sires with increased embryo mortality. This aim will identify subfertile Holstein bulls who sire pregnancies with a higher prevalence of early (< day 32) or late (> day 32) embryo mortality. The approach is to analyze reproduction records of many heifer raising facilities and dairies to identify candidate sires for early (SFEarly) or late (SFLate) embryo mortality and then ensure their phenotype using a controlled fertility field trial. Data from commercial farms was retrieved, and above 40,000 reproduction records comprising both embryo transfer and artificial inseminations from a total of 145 bulls have been analyzed to identify candidate SFEarly and SFLate bulls. From these analyses, a set of 10 high fertility and 40 candidate SF bulls were identified. Initial analysis conclude low correlations with sire conception rate reinforcing the need for these types of analysis.AIM 2. Investigate physiological mechanisms regulating early embryo loss. This aim will test the hypotheses that SFEarly sires have defects in genes controlling embryo development and/or conceptus implantation and/or placentation that underlie increased early embryonic mortality. Candidate SF bulls are being tested to identify potential defects on preimplantation development and trophoblast differentiation in vitro. So far 22 out of 50 bulls have been tested in vitro, and 6 bulls (27%) have been identified to negatively affect preimplantation embryonic development. AIM 3: Determine Genetic Mechanisms Underlying Sire Influences on Embryo Survival and Pregnancy Establishment. This aim will test the hypothesis that genetic loci and genomic rearrangements associated with embryo survival and mortality and pregnancy success can be identified using cutting-edge genomic analyses of fertile and subfertile sires identified and phenotyped in Aim 1. Through the record analysis, bulls identified as candidate SFEarly have been deep-sequenced and genetic analyses are being performed to identified candidate markers and structural variants associated with early embryonic mortality.

Publications

  • Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Ortega MS, Kelleher AM, O'Neil E, Benne J, Cecil R, Spencer TE. NANOG is required to form the epiblast and maintain pluripotency in the bovine embryo [published online ahead of print, 2019 Dec 5]. Mol Reprod Dev. 2019;10.1002/mrd.23304. doi:10.1002/mrd.23304
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Franco, G.A., T.S. Maia, R.F.G. Peres, C.F.G Martins, S.T. Reese, J.L.M. Vasconcelos and K.G. Pohler. 2018. Quantification of PAG genes in semen of high and low fertility sires using droplet digital PCR. International Ruminant Reproduction Symposium. Foz Do Iguaco, Brazil.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Franco, G.A., G. Dalmaso de Melo, S.T. Reese, V.M. Negron-Perez, C.L. Timlin, K. Uh, K. Lee, V.R.G. Mercadante and K.G Pohler. 2019. Conceptus-derived products in circulation during early pregnancy in cattle receiving parthenogenetic vs normal embryos. Brazilian Embryo Transfer Society Annual Meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Franco, G.A., G. Dalmaso de Melo, S.T. Reese, V.M. Negron-Perez, C.L. Timlin, K. Uh, K. Lee, V.R.G. Mercadante and K.G Pohler. 2019. Comparison of developmental capacity of bovine parthenogenetic and biparental IVF embryos and its effects on placental products secretion. Society for the Study of Reproduction Annual Meeting. San Jose, CA.