Location: Agricultural Genetic Resources Preservation Research
2022 Annual Report
Objectives
Objective 1: Build, secure, manage, and facilitate the use of the animal genetic resource collection.
Sub-objective 1A: Operate species committees to advise NAGP on collection development and use.
Sub-objective 1B: Targeted acquisition of animals for breeds already in the collection based upon quantitative or molecular genetic analysis, number of animals and germplasm in the collection.
Sub-objective 1C: Acquire samples from breeds not presently in the collection or with limited numbers of animals and samples and minor species (yak, water buffalo or bison).
Sub-objective 1D: Engagement with other countries through the United Nation’s Food and Agriculture Organization (FAO) Intergovernmental Technical Working Group on Animal Genetic Resources.
Objective 2: Development and implementation of the publicly accessible Animal-GRIN V2 database.
Sub-objective 2A: Redesign and develop public facing webpages, perform necessary software upgrades and increase user friendliness of the genomic component of Animal-GRIN.
Sub-objective 2B: Design GIS interface with Animal-GRIN.
Objective 3: Characterize genetic diversity to guide collection development and increase its utility.
Sub-objective 3A: Use quantitative and/or molecular approaches to evaluate and compare genetic variability within and among livestock populations and the collection. Specifically focusing upon: using Yorkshire and Duroc compare in-situ vs. collection genetic diversity; extend molecular characterization of goat breeds; and initiate molecular characterization of water buffalo.
Sub-objective 3B: Combine genomic and production system parameters into a GIS format to assist in making collection decisions as they relate to production systems and climate change.
a. Evaluate genetic diversity of oysters in relation to environmental factors.
b. Gradients of allele frequency for loci associated with geographic regions.
Objective 4: Develop and refine cryopreservation technologies enabling efficient germplasm collection, evaluation, and utilization by gene banks and stakeholders.
Sub-objective 4A: Establish assays using flow cytometry and CASA to evaluate sperm quality.
Sub-objective 4B: Create an inexpensive device and accompanying methods for vitrification of oocytes in bulk.
Sub-objective 4C: Develop quality control standards and best practices for germplasm repositories.
Approach
Genetic resources underpin the livestock sectors ability to improve productivity and contribute to global food security and economic well-being of rural America. Despite the importance of genetic resources there continues to be a contraction of genetic variability nationally and internationally. Furthermore, genetic resources will likely become more contentious under the Convention on Biological Diversity and its Nagoya Protocol. Developing secure collections of germplasm and tissue from U.S. livestock breeds and associated populations is a mechanism to safeguard and promote US interests. To date substantial amounts of genetic resources and information have been curated. Importantly, large numbers of animals in the collection have been used by industry and researchers for a variety of purposes. However, more work is needed to curate germplasm from livestock populations, understand their genetic diversity, enhance effective mechanisms for cryopreservation, and to make the collection available to a wide array of stakeholders and customers via a robust user friendly information system. Steps to achieve such goals are detailed in this project plan. At the end of this project cycle it is anticipated that the germplasm collection will be more robust, better methods and tools will have been developed for collecting, analyzing and utilizing genetic resources.
Progress Report
This is the final annual report for this research project. With over 1.1 million samples and more than 60,000 animals, the collection of germplasm and tissue the USDA National Animal Germplasm Program (NAGP) collection is a global leader in conserving animal genetic resources. Objective 1 deals with the growth of the germplasm collection and the project’s interaction with various stakeholders. Significant growth in the collection occurred. By all metrics the collection increased during this project plan. Sample and animal number has increased by 180,000 and 9,500, respectively. While the number of breeds and subpopulations within breed increased by 9 and 92, respectively. The Dairy Species Committee (Subobjective 1A) was instrumental in raising awareness about inbreeding of major dairy cattle breeds to the American Dairy Science Association and to artificial insemination companies. This effort culminated in an international meeting on genetic diversity and inbreeding, with 100 attendees from nine countries. As a result, research and industry formed linkages to jointly address this issue. Industry and the research community increased their utilization of the collection to solve a variety of key issues, for example, reintroduction of lost y chromosomes in Holstein and identification of animals carrying lethal mutations (Angus and Brangus). A project scientist served as the U.S. representative to the United Nations Food and Agriculture Organizations Intergovernmental Technical Working Group on Animal Genetic Resources (Subobjective 1D) where U.S. interests were advanced for monitoring and increased understanding of global animal genetic diversity. Additionally, project scientists co-authored a new gene bank manual for the United Nations Food and Agriculture Organizations, which will be formally released in late 2022.
The Animal-Genetic Resources Information Network (Animal-GRIN) is a mission critical element of curating and understanding the animal genetic resources collected. During this project computer programmers maintained functionality of the program as new software was released by software companies (Subobjective 2A). As needed features were identified they were added to the system. This included features to make searching for individual animals easier for public users. With the increasing importance of climate change, it is essential that we know the geographic location where animals in the collection were born. To facilitate this aspect of collection evaluation, the ability to map where specific animals came from was developed and added to the public webpages (Subobjective 2B).
Characterization of genetic resources increases our understanding of the germplasm and tissue collection. During this project cycle several key studies were performed (Objective 3). Both Duroc and Yorkshire collections were compared to the in-situ population of each breed using molecular markers. It was determined that the collection for both breeds mirrored the in-situ populations for genetic variability. For both breeds it was shown that the collection had approximately 98% of the alleles in common with the in-situ populations. Genetic diversity studies for pig and goat breeds, and oysters were performed. The pig study evaluated all major and many minor breeds within the continental U.S. as well as feral pigs in Hawaii and Guam, and three imported Chinese breeds. Goat breeds studied included those from the U.S., South Africa, Costa Rica, Brazil, and Argentina. Both of these studies demonstrated that the U.S. holds substantial genetic variability for these species that U.S. producers can utilize if needed. Oysters were sampled from the Louisiana coast, as divided by the Mississippi River and different coastal features. No subpopulations were found among the varying coastal areas suggesting that large portions of oyster genetic variability can be captured by sampling one or two different locations. For the first time an animal gene bank’s collection of beef cattle was mapped by temperature and heat index, thereby assisting in the identification of geographic areas that are underrepresented in the collection.
Adaptation of flow cytometry assays for field conditions is challenging because the data reflect changes in sperm physiological processes rather than absolute answers. Consequently, attempts to adapt flow cytometric assays to instrumentation, such as the Isperm and Qubit portable fluorometer, did not result in usable products. However, experimentation for the adaptation enabled further refinement and validation of the flow cytometric assays resulting in improved methods for monitoring in vitro capacitation and acrosome reaction processes. The application of the flow cytometric assays will provide NAGP and stakeholders in the commercial semen industry with a means to understand the effects of semen treatments on sperm physiology and potentially screen samples/males for differences in fertilizing potential (Subobjective 4A).
Objective 4. Significant progress was made toward identification and development of assays that can evaluate sperm quality across species (Objective 4A). Our investigation of meaningful sperm motion characteristics resulted in the development of a computer model that can potentially be included in future computer assisted sperm analysis (CASA) systems and enable users to identify inferior/superior samples, thus expanding the current utility of this technology. In addition, the exploration of multiple fluorescent stains to assess quality via flow cytometry permitted us to validate two new methods to analyze sperm fertilizing potential and provided us with information about sperm maturation processes thus enabling an understanding of more subtle indicators of sperm quality and function which we anticipate will be used to distinguish between the fertilizing potential of males. The technologies in these experiments (CASA and flow cytometry) are widely utilized by the commercial semen industry and consequently our findings may be implemented by them, repository management, and other stakeholders.
Currently, vitrification of either single or groups of up to 20 oocytes or embryos may be accomplished using commercially available products or with devices fabricated by laboratories, but these commercial devices can be quite expensive and those developed in laboratories have not been optimized. Consequently, an exploration of new devices for vitrification of large quantities of oocytes and embryos was performed (Objective 4B). The findings demonstrated that when used, the new devices that employed using handles designed with 3D printing and nylon mesh with a variety of pore sizes would result in rates of oocyte survival and embryo development following vitrification and warming that were comparable to those achieved with commercially available products or reported in scientific literature. However, the devices proved difficult to manufacture and handle, and with no improvement in oocyte or embryo survival rates their continued development is not warranted.
When developing and managing a germplasm repository it is critical to monitor the sample quality from collection, through sample preservation, and during utilization to ensure a sample is in its best possible condition when used to meet a purpose (e.g., artificial insemination, genetic analyses). The samples in the NAGP collection are from animals and therefore, there is inherent variation in germplasm quality because of the biological nature of the animal, sampling conditions, species, season, and many other factors. Even so, when all factors are considered, there is an expectation that samples should have reasonable quality and effectiveness if they have been handled correctly. Consequently, quality control procedures and best practices were developed for use by NAGP (Objective 4C) and will also serve as a guide to other gene banks and stakeholders who can reference them when collecting, preserving, and utilizing germplasm. Employing these procedures and practices will ensure that the samples in the NAGP collection, or the collections of the stakeholders that utilize the guidelines, are treated appropriately and safeguarded in perpetuity.
Accomplishments
1. Genetic change in a composite population. For thousands of years, societies have combined various populations of livestock to obtain food security. In this mixing of genetic compositions, the new breed takes on phenotypic characteristics of both breeds and varying genetic composition of each founding population. ARS Scientists in Fort Collins, Colorado, and Miles City, Montana, used a composite population based upon Red Angus (50%), Charolais (25%) and Tarentaise (25%) to demonstrate how the new composite emerged as a unique population and that the intended founder breed proportions were not constant. The instability of composite populations suggests that natural or artificial selection alters populations over time and producers need to be cognizant that the composition of a composite population is not constant and will potentially change animal performance. Specifically, the Tarentaise proportion increased to 57% while Red Angus and Charolais decreased to 38% and 5% respectively. This unmanaged shift in breed composition suggests that the Tarentaise may be better adapted to the cold dry climate found in Montana and thereby has ramifications for breeding cattle under conditions of climate change. The type of genetic change observed in this study supports earlier ARS research with Brangus that are raised in a hot humid climate.
2. Managing genetic diversity and inbreeding. The U.S. dairy breeding industry supplies improved genetic resources to the world; however, they have expressed concern about the rate of inbreeding particularly in Holstein and Jersey. Partnering with the American Dairy Science Association, an ARS scientist in Fort Collins, Colorado, developed an industry wide conference on the question of inbreeding that was attended by 100 people from nine counties. The conference afforded industry and the public sector with an opportunity to discuss and seek common goals that will allow the breeding complex to move forward in addressing this issue. As a result of this meeting, new research partnerships were developed and strategic plans to resolve this issue were initiated.
Review Publications
McManus, C.M., Hermuche, P.M., Paiva, S.R., Guimaraes, R.F., Carvalho, O.A., Blackburn, H.D. 2021. Gene bank collection strategies based upon geographic and environmental indicators for beef breeds in the United States of America. Livestock Science. 254. Article e104766. https://doi.org/10.1016/j.livsci.2021.104766.
Hagedorn, M., Page, C.A., O'Neil, K., Flores, D.M., Tichy, L., Conn, T., Chamberland, V., Lager, C., Zuchowicz, N., Lohr, K., Blackburn, H.D., Vardi, T., Maraver, K. 2021. Assisted gene flow using cryopreserved sperm in critically endangered coral. Proceedings of the National Academy of Sciences (PNAS). 118(38). Article e2110559118. https://doi.org/10.1073/pnas.2110559118.
Purdy, P.H., Graham, J.K., Azevedo, H.C. 2021. Evaluation of boar and bull sperm capacitation and the acrosome reaction by flow cytometry. Animal Reproduction Science. 246. Article e106846. https://doi.org/10.1016/j.anireprosci.2021.106846.
Blackburn, H.D., Torres, L., Liu, Y., Tiersch, T.R. 2022. A framework addressing the temporal aspects of fish sperm motility leading to community-level standardization of assessment. Zebrafish. 19(4):119-130. https://doi.org/10.1089/zeb.2022.0006.
