Location: Warmwater Aquaculture Research Unit
2021 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
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 researchers in Stoneville, Mississippi, in concert with existing and new collaborations, and through the support of graduate students and research staff. Significant progress of note have 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 is being evaluated to provide insight into pathways that might be altered due to environmental insults during pregnancy. 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 Sub-Objective 1B, we examined the possibility of a newly designed liposome vesicle nanoparticles as a new variant for sperm mediated gene/drug transfer (SMGT). In this novel approach, fluorescently labeled liposome vesicles were formulated to be filled with a naturally fluorescent drug (doxorubicin). Various imaging microscopic imaging techniques were used for assessments. The nanosize and binding capability of the synthesized liposome vesicles were confirmed. The loading of these liposome nanovesicles with doxorubicin and incubation with freshly harvested boar spermatozoa permitted the uptake of doxorubicin within the sperm nucleus. The incubation of doxorubicin-labeled spermatozoa with cell culture monolayers led to intracellular delivery of doxorubicin, with the monolayer of cells’ nuclei exhibiting doxorubicin fluorescence. These observations indicate liposome nanovesicles as possible and viable variants of efficient SMGT during in vitro fertilization. In line with Sub-Objective 1B, the developed liposome nanovesicles have potential to enable in vivo molecular imaging. Within the scope of Sub-Objective 1C, we found that spermatozoa incorporating fluorescent or doxorubicin-loaded liposomes still maintained their motility, which is of great interest for in vivo applications. Indeed, the use of tagged (e.g., membrane markers) liposome nanovesicles loaded with fluorescent compounds for sperm interaction has the potential to permit targeted in vivo molecular imaging, following artificial insemination. We also investigated the RNA transcript profiles of equine ovarian follicular fluids through their microvesicle contents. Follicular fluids were harvested to search for changes and potential biomarkers of follicle development and seasonal variations. Follicular fluids appear as crucial sources of various biomarkers to enable future in vivo interventions in females.
Within Sub-objective 1D, we applied near Infrared Reflectance Spectroscopy (NIRS) to evaluate disease and reproductive health diagnostics. Cattle experiencing respiratory disease (BRD) from either bacterial or viral pathogens were examined by NIRS by collecting a suite of biofluids (nasal secretions, saliva, breath condensate, blood). Chemometric evaluation of changing spectral features associated with infection and disease severity were correlated with traditional modes of disease diagnoses to create prediction equations for improved sensitivity and specificity of pathogenic infection. To this end, NIRS diagnosis of respiratory disease from all sources was > 95% in the model systems and 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. Validation of observed infection through changes in the metabolomics profile was conducted by 1HNMR spectroscopy measuring changes in 52 metabolites observed in both viral and bacterial infections, suggesting the formation of a complex of diagnostic metabolic biomarkers for BRD diagnosis strategies, therapeutic interventions, and management practices. Reproductive cycling in horses is frequently monitored by ultrasound for detection of follicle development and ovulation in conjunction with standard hormone assays on blood serum such as the Radio Immunoassays (RIA) or the Enzyme-Linked-ImunoSorbant Assay (ELISA). Such assays must be conducted separately for each hormone under consideration while NIRS captures multiple facets of information about reproductive cycling in biofluids. We have used NIR-aquaphotomics to distinguish the reproductive profiles for Estrus and Diestrus in cycling mares from serum. PCA analysis described 91% of variance using 7-PCs when analyzed with a 90% classification of Estrus and Diestrus in two different serum treatments. The use of NIR-aquaphotomics thus has potential to identify not only reproductive phase, but also correlate hormone values in serum to spectral profiles, quantitatively. We also found distinct scores plots associated with ovarian persistant corpus lutea (PCL), which, in combination with the simultaneous presence of E2 and P4 makes these spectra distinct. Zearalenone (a mycotoxin with estrogenic effects in pigs, poultry and fish) treated mares have blood levels higher in estrogenicity at ovulation. Dose dependent shifts in NIR spectral peaks can be found in mare serum for estradiol equivalents (E2Eq) and ZON, leading to WAMACS and development of an Aquaphotome for estrogenicity and mycotoxin consumption. Quantitative values of E2Eq were better measured from spectra taken on protein precipitated serum than unprocessed serum, suggesting that smaller molecules and metabolites may be more easily detected in the absence of large serum proteins or if they are bound to serum albumin. Quantitative values of ZON were alternately better measured from spectra taken on unprocessed serum compared to protein precipitated serum-indicating ZON responses may influence serum protein (MW > 30K) composition.
Within Objective 2, progress was made to construct a pHS23_eGFP vector to develop fluorescent Fusobacterium necrophorum for in vivo imaging. Specifically, the anaerobic culture equipment to conduct the study has been established, the vector design supplies have been purchased, and the project workflow has been designed to initiate the project. The development of fluorescent Fusobacterium necrophorum has many applications in livestock microbiome research associated with various infections and dysbiosis. Furthermore, characterization of the bovine vaginal microbiota throughout pregnancy was established during this reporting period and initial studies revealed substantial diversity in the composition of the bovine vaginal microbiota that were not influenced by maternal nutrient restriction during the last third of pregnancy. However, advancing gestational age resulted in compositional changes at the order, family, and genus level. Moreover, supplementing dietary melatonin to dams altered the beta diversity of the bovine vaginal microbiota, specifically melatonin increased the prevalence of aerobic bacteria in the vaginal tract.
Within Objective 2 continued progress has been made into investigating probiotics as alternatives to antibiotics in broiler production. We have worked over the past year with 2 different laboratories to obtain plasmids that carry the Lux ABCDE genes for the expression of bioluminescence. Through those collaborations we are now in possession of 2 probiotic bacteria that express bioluminescence (E. coli Nissle and Bacillus subtilis). We have also become interested in utilizing bioluminescence to evaluate colonization patterns in pathogens that are associated with poultry infections. Therefore, we have also been working on the construction of a bioluminescent strain of Salmonella enteritidis, Salmonella reading, and Avian Pathogenic E. coli (APEC).
Accomplishments
1. Development and validation of a novel techniques to assess blood perfusion. Through cooperative agreement with an ARS researchers in Stoneville, Mississippi, Mississippi State University researchers, examined novel ex vivo approaches of determining blood perfusion in bovine reproductive tissues. Proper fetal nutrition via adequate placental blood flow is critical to maximize bovine offspring production potential while minimizing pregnancy wastage and calf morbidity and mortality. Utilizing ultrasonography and microscopic capillary measurements we validated a novel ex vivo tissue perfusion technique to visualize macroscopic blood vessel density in the bovine placentome. This technique increased knowledge about placental efficiency via local blood perfusion, which is being adopted by other research groups. These results will expand our understanding of environmental insults and stimuli affecting fetal development through the placenta.
2. Nonthermal method for milk pasteurization. Through cooperative agreement with an ARS researcher in Stoneville, Mississippi, Mississippi State University and Southern Illinois University researchers, examined a nonthermal method for milk pasteurization. Camels are the most important animals in the hot arid areas, as they provide transportation and food sources (e.g., meat and milk) to locals. Raw camel milk contains a wide range of pathogenic microorganisms, but the opportunity for its pasteurization is limited. There is a need to optimize the currently available sterilization methods. Hence, we found the beneficial effect of the nonthermal ultraviolet-C light not sufficient to meet the Food and Drug Administration requirements in terms of bacteria pathogens.
Review Publications
Messman, R.D., Contreras-Correa, Z.E., Paz, H.A., Lemley, C.O. 2021. Melatonin-induced changes in the bovine vaginal microbiota during maternal nutrient restriction. Journal of Animal Science. 99(5):1-10. https://doi.org/10.1093/jas/skab098.
Bronson, E., Guy, E.L., Murphy`, K.J., Barrett, K., Kouba, A.J., Poole, V., Kouba (Vance), C.K. 2021. Influence of oviposition-inducing hormone on spawning and mortality in the endangered panamanian golden frog (atelopus zeteki). BMC Zoology. 6(17):1-14. https://doi.org/10.1186/s40850-021-00076-8.
Santos-Rivera, M., Woolums, A., Thoresen, M., Blair, E., Jefferson, V., Meyer, F., Vance, C.K. 2021. Profiling Mannheimia haemolytica infection in dairy calves using near infrared spectroscopy (NIRS) and multivariate analysis (MVA). Nature Scientific Reports. 11. Article 1392. https://doi.org/10.1038/s41598-021-81032-x.
Gillis, A.B., Guy, E.L., Kouba, A.J., Allen, P.J., Marcec-Greaves, R.M., Vance, C.K. 2021. Short-term storage of tiger salamander (Ambystoma tigrinum) spermatozoa: The effect of collection type, temperature and time. PLoS ONE. 16(1):e0245047. https://doi.org/10.1371/journal.pone.0245047.
Dhahir, N., Feugang, J., Witrick, K., Park, S., White, S., Abughazaleh, A. 2020. The effect of different ultraviolet-C light doses on microbial reduction and the components of camel milk. Food Science and Technology International. 27(2):99-111. https://doi.org/10.1177/1082013220935230.
Sanford, C.D., Owen, M.O., Oosthuizen, N., Fontes, P.L., Vonnahme, K.A., Nelson, M., Reyaz, A., Lemley, C.O., Delorenzo, N., Lamb, G.C. 2021. Effects of administering exogenous bovine somatotropin to beef heifers during the first trimester on conceptus development as well as steroid and eicosanoid-metabolizing enzymes. Journal of Animal Science. 99(3):1-7. https://doi.org/10.1093/jas/skab050.
