Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
Animal Sciences
Non Technical Summary
Research initiatives for these projects will focus on improving reproductive efficiency in livestock and humans. Experiments will be designed to improve embryo development and identify paternal contributions to preimplantation embryo development with an overall goal of relieving infertility. These studies are important to improve reproductive efficiency in multi-billion dollar livestock industries while also identifying processes at the cellular level which impact sperm function and decrease fertility. We hypothesize that exposure of post-ejaculatory sperm to environmental stressors and toxicants contributes to loss of embryonic viability and pregnancy establishment. We aim to determine if exposure of sperm to environmental toxicants decreases gamete interaction, fertilization potential and embryo development. We also aim to determine if short-term exposure of ejaculated sperm to common environmental toxicants alters the genome and epigenome of both sperm and embryos fertilized from exposed ejaculates. We expect that sperm exposed to environmental stressors will appear acceptable using conventional semen analyses but will have decreased function including paternal effects on embryo development resulting in idiopathic subfertility. Methodologies employed for these activities will include computer-assisted sperm analyses, CRISPR-Cas9 gene editing and in vitro embryo production derived from sperm exposed to environmental toxicants. Sperm exposure to toxicants followed by fertilization will provide missing information to better understand environmental risk to livestock and humans. Impacts will guide management decisions in livestock to improve reproductive performance. Results from these experiments will also be applicable to human clinical assisted reproductive technology procedures, especially in the case of male-factor infertility.
Animal Health Component
20%
Research Effort Categories
Basic
65%
Applied
20%
Developmental
15%
Goals / Objectives
Understand the biology of gamete development, fertilization and embryogenesis including the underlying cellular and molecular mechanisms.
Refine methods to produce animals by genetic engineering or genome editing for the improvement of livestock production efficiency and development of human biomedical models.
Project Methods
These projects will be conducted using many different semen analyses, sperm biology and embryo production methods. Sperm are typically collected from various animals in accordance with approved semen collection protocols or sourced for a commercial vendor. Semen analyses generally employ the use of Computer Assisted Sperm Assessment, light microscopy and fluorescent imaging to perform various techniques related to observing, quantifying and characterizing events critical for sperm function. Percoll gradient centrifugation is typically used to isolate sperm followed by dilution in appropriate extenders. Often cryopreserved sperm is used.For embryo experiments, oocytes are sourced from a local abattoir or purchased from an independent vendor. Oocytes are collected via follicle aspiration or through mechanical slicing of the ovary. In vitro embryo production is implemented according to our standard laboratory protocol.Gene editing experiments in embryos are conducted in our laboratory through CRISPR-CAS9 mediated delivery either through intracytoplasmic sperm injection or via electroporation. Validation of gene editing is accomplished by immunofluorescence or through Sanger sequencing. PCR amplification is conducted in-house followed by gel electrophoresis. Sequencing is outsourced.Genomic studies conducted with gametes and embryo originate in the laboratory through chemical or toxicant exposure. Downstream analyses are conducted including developmental and fertility indices. DNA/RNA isolation is performed in house along with library preparation. Sequencing and bioinformatics are outsourced to core facilities or in collaboration with an established investigator or colleague.Results from experiments will be analyzed primarily with SAS statistical software and interpreted as significant at P ≤ 0.05. Embryo developmental data will be analyzed in accordance with established laboratory practices. Efforts used to deliver information will include publication of results in peer-reviewed scientific journals, dissemination at local, regional, and international meetings, and through oral and poster delivery to associations of interest. Formal and informal classroom instruction will be provided to graduate and undergraduate students in continuum with these initiatives to include experiential learning of laboratory techniques.Progress to obtain research initiatives will be evaluated through number of peer-reviewed publications (Goal = 5). Measure of success will include successful implementation of projects including graduate degrees awardedthroughout duration, number of personnel trained, number of seminars and presentations delivered. Data collection will include changes to developmental success of bovine embryos, identification of genome sequences susceptible to environmental insult, development of an alternative method to nuclear reprogramming, patenting of semen extenders and implementation of extenders devoid of antibiotics for porcine artificial insemination. Key milestones will include number of publications (n = 1 /year), patenting success and n = 1 alternative methods to contemporary cloning. Impacts will be determined as a change in bovine in vitro embryo production protocol, inclusiveness or adoption of antibiotic-free extender in the porcine industry, inclusiveness or adoption of new semen extenders for bovine and porcine artificial insemination, and identification of new fertility indices for use in human clinical settings.