Location: Warmwater Aquaculture Research Unit
2022 Annual Report
Objectives
1. Develop and adapt existing imaging and photon emitting technologies toward describing the intrauterine environment, and sperm qualities such as motility, and develop strategies to enhance and improve reproductive health and fertility in food animal reproduction systems.
1.A. Examine in vivo uteroplacental hemodynamics following acute maternal infusions with vasoactive supplements.
1.B. Development of in vitro and ex-vivo approaches for cellular and tissue biophotonic imaging using nanoparticles.
1.C. Development of specific molecular-based approaches for in vivo biophotonic imaging.
1.D. Development of alternate biophotonic animal models and the use of digital infrared thermal imaging and near infrared (NIR) spectroscopic approaches to study specific reproductive health, environmental, and/or physiological processes in livestock and poultry.
2. Use novel imaging and related technologies for the tracking of relevant pathogens (disease stressors; e.g. Salmonella, Mycobacterium avian paratuberculosis) in avian and livestock hosts using photon emitting sentinels in the animal system and/or environment (e.g., nanoparticles, energy transfer systems, transformed bacterium) to address bacterial abundance and persistence related to livestock well-being and production performance, and develop mitigation strategies.
2.A. Use of biophotonics imaging strategies to establish potentially unknown infection sites of Mycobacterium avium subsp. Paratuberculosis in a rodent model.
2.B. Development of alternate biophotonic animal models and the use of digital infrared thermal imaging approaches to study various disease states in livestock and poultry.
Approach
There is a critical need for technological innovations that will permit production-based questions to be asked and answered in the context of the living animal. The overall goal of this project is to develop technologies that can perform in situ time-lapse and in vivo bio-imaging of cellular and molecular events and biological processes in livestock and poultry, in real-time. Specifically, this basic and applied research will target the improvement of reproductive efficiency and the mitigation of disease, which are both essential for efficient food and fiber production. Novel technologies will utilize photonic and/or thermal signatures, spectroscopy and/or fluorescence, ultrasonography, and nanotechnology in adaptive research aimed at facilitating physiological assessments related to reproduction and disease monitoring in livestock and poultry. To this end, this project is designed to cover a broad range of research in the organismal, cellular and molecular life sciences aimed at understanding production performance end-points through the development of new life-science research models. With these new models in place, translational research can then be applied in livestock and poultry production-management settings for application to the real-world enviroment.
Progress Report
This is the final report for this project. Refer to project 6066-31000-016-000D, "Improving the Productivity and Quality of Catfish Aquaculture" for additional information.
Mississippi State University researchers have made additional progress in all the planned milestones through the continued efforts of the project's Principal Investigator, an ARS researcher in Stoneville, Mississippi, in concert with existing and new collaborations, and through the support of graduate students and research staff. Significant progress of note has been made specifically in the following areas:
Within Sub-Objective 1A, we have developed and tested a novel fluorescent perfusion technique to examine macroscopic blood vessel density of the placentome. This novel technique has been successfully validated by comparing in vivo Doppler uteroplacental blood flow results with molecular markers of placental blood perfusion and angiogenesis. Additionally, the influence of blood flow classification on genome-wide mRNA expression in the placenta has been evaluated to provide insight into pathways that might be altered due to environmental insults during pregnancy. Interestingly, minimal differences were observed in transcript abundance between different size placentomes in cattle; however, environmental alterations significantly altered transcript abundance. Specifically maternal nutrient restriction increases translational and ribosomal mRNA expression as a compensatory mechanism to increase nutrient partitioning to the developing fetus. In addition, we have made progress in expanding these imaging techniques of blood perfusion in the male, especially bulls. Progress was made in the evaluation of the influence of environment on bovine testis biometrics, thermoregulation, and blood flow in mature and peri-pubertal bulls. Additionally, the influence of blood flow classification on genome-wide mRNA expression in the testis is being evaluated to provide insight into pathways that might be altered due to differential blood flow to the testis.
Within the Sub-Objective 1B, we designed iron-oxide core nanoparticles to permit interaction with X-chromosome-bearing spermatozoa, followed by safe removal from bio-mixtures, under a magnetic field. We co-incubated extended boar semen with various amounts of synthesized magnetic nanoparticles (MNP). After separation, eluted MNP free spermatozoa were kept for sex determination using Polymerase Chain Reaction molecular technique, focusing on sex-bound genes. A stereomicroscope observation clearly showed sperm entrapped with MNP. Further characterization will permit a better visualization.
Within the Sub-Objective 1C, we obtained samples of raw semen that “failed” or “passed” the selection criteria for breeding in a commercial boar stud for seminal plasma purification through serial centrifugations. Thereafter, we extracted exosomes through series of ultracentrifugation and characterized them through western immunoblotting, dynamic light scattering, and high-resolution transmission electron microscopy. Exosomes are subjected to both microRNA and proteome profiling and comparison of both Failed and Passed groups.
Within the Sub-Objective 1D, we tested the Near Infra-red Spectroscopy (NIRS) as a non-invasive method to discriminate between Passed of Failed semen samples for breeding purpose. We obtained samples of raw semen that “failed” or “passed” the selection criteria for breeding in a commercial boar stud for seminal plasma purification. After elimination of sperm cells and other debris through two series of increasing centrifugation forces, purified seminal plasma fractions were collected for NIRS analyses. The study is ongoing.
Within Sub-objective 1D, we applied NIRS to evaluate disease and reproductive health diagnostics. Cattle experiencing respiratory disease (BRD) from bacterial or viral pathogens were examined by NIRS by collecting a suite of biofluids (nasal secretions, saliva, breath condensate, blood). Bovine respiratory syncytial virus (BRSV) is a major contributor to respiratory disease in cattle worldwide. Traditionally, BRSV infection is detected based on non-specific clinical signs, followed by reverse transcriptase-polymerase chain reaction (RT-PCR), the results of which can take days to obtain. BRSV infection is typically diagnosed by evaluation of the clinical signs, followed by time-consuming serological and molecular methods. Near-infrared aquaphotomics evaluation based on biochemical information from biofluids has the potential to support the rapid identification of BRSV infection in the field. Our previous report revealed excellent prediction equations for diagnosing disease based on NIR aquaphotomics in blood plasma. Here we expand the analysis to more the easily obtainable biofluids breath condensate and nasal secretions. Another study evaluated NIR spectra of nasal secretions (NS) from dairy calves challenged with bovine respiratory syncytial virus (BRSV). The near infrared aquaphotomics evaluation of this biofluid unveiled changes between the spectra of samples collected from uninfected and infected states. These chemical differences were discriminated by PCA-LDA using a leave-one-animal-out approach with accuracy, sensitivity, and specificity >88% in the calibration and internal validation. By collecting spectra from nasal secretions, we revealed the potential of NIRS in combination with aquaphotomics and chemometrics for the qualitative detection of this viral infection in-vivo as a first step toward developing a rapid in-field diagnostic tool for BRSV infection. Using blood plasma, NIRS diagnosis of respiratory disease from model systems predicted early-stage respiratory infection ranging from 93-100% accuracy, 93-95% sensitivity and 83-95% specificity for the causative pathogenic element, with variation a function of the biofluid tested.
Within Sub-Objective 2B, we cultured reproductive epithelial cells (vaginal, cervical, and endometrial) to explore the effective intracellular delivery of a synthetic NanoLuc luciferase relaxin mRNA construct. In our future studies, the luc-Relaxin mRNA will be tested to reduce dystocia during parturition in heifers.
Accomplishments
1. Omics of the equine follicular fluid during follicle development. Through cooperative agreements with an ARS researcher in Stoneville, Mississippi, Mississippi State University, Southern Illinois University, and Colorado State University scientists examined developmental changes in ovarian follicle physiology. First, we investigated the proteome of equine follicular fluid during follicle development and over different seasons. Data provide an extensive description and functional analyses of the equine follicular fluid proteome during follicle selection, development, and ovulation. Altogether, the findings provide sets of molecular candidates to serve as potential non-invasive biomarkers of follicle development to boost female fertility.
Review Publications
Mustapha, P.A., Bogoro, S.E., Feugang, J.M. 2022. Biothermoimaging tools for management of climate smart and precision livestock-assisted reproduction. In: Obembe, O.O., Ekundayo, E.O., Okoli, A.S., Gidado, A., Adetunji, C.O., Ibrahim, A.B., Ubi, B.E., editors. Agricultural biotechnology, biodiversity and bioresources conservation and utilization. Boca Raton, FL: CRC Press. p. 315-334. https://doi.org/10.1201/9781003178880.
Kouba, A.J., Langhorne, C.J., Willard, S.T., Vance, C.K., Smith, T. 2021. Spermiation response to exogenous hormone therapy in hibernated and non-hibernated boreal toads (Anaxyrus boreas boreas). Reproduction, Fertility and Development. 34(5):453-460. https://doi.org/10.1071/RD21033.
Contreras-Correa, Z.E., Messman, R.D., Sidelinger, D.R., King, E.H., Sanchez-Rodriguez, H.L., Burnett, D.D., Lemley, C.O. 2021. Melatonin alters bovine uterine artery hemodynamics, vaginal temperatures, and fetal morphometrics during late gestational nutrient restriction in a season-dependent manner. Journal of Animal Science. 99(9):1-14. https://doi.org/10.1093/jas/skab242.
Revord, R.S., Lovell, S.T., Brown, P., Capik, J., Molnar, T.J. 2020. Using genotyping-by-sequencing derived SNPs to examine the genetic structure and identify a core set of Corylus americana germplasm. Tree Genetics and Genomes. https://doi.org/10.1007/s11295-020-01462-y.
Reid, D.S., Burnett, D.D., Contreras-Correa, Z.E., Lemley, C.O. 2022. Differences in bovine placentome blood vessel density and transcriptomics in a mid to late-gestating maternal nutrient restriction model. Placenta. 117:122-130. https://doi.org/10.1016/j.placenta.2021.12.004.
Arregui, L., Kouba, A.J., Germano, J., Barrios, L., Moore, M., Vance, C.K. 2021. Fertilization potential of cold-stored Fowler’s toad (Anaxyrus fowleri) spermatozoa: temporal changes in sperm motility based on temperature and osmolality. Reproduction, Fertility and Development. 34(5):461-469. https://doi.org/10.1071/RD21037.
Chen, L., Santos-Rivera, M., Burger, I., Kouba, A.J., Vance, C.K., Barber, D. 2021. Near-infrared spectroscopy (NIRS) as a method for biological sex discrimination in the endangered Houston toad (Anaxyrus houstonensis). Methods and Protocols. 5(1):4-16. https://doi.org/10.3390/mps5010004.
