Location: Warmwater Aquaculture Research Unit
2019 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 scientists have made additional progress in all the planned milestones through the continued efforts of the project's Principal Investigator, an ARS scientist at 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:
Examine in vivo uteroplacental hemodynamics following acute maternal infusions with vasoactive supplements. A novel fluorescent perfusion technique has been established to examine macroscopic blood vessel density of the placentome. These techniques are being validated by sampling placental tissue and comparing in vivo results versus those of molecular markers of placental blood perfusion and angiogenesis. In addition, we have made progress in expanding these imaging techniques of blood perfusion in the male, specially bulls. Interestingly these preliminary results are showing positive therapeutic alterations in male fertility.
Development of in vitro and ex-vivo approaches for cellular and tissue biophotonic imaging using nanoparticles. Fluorescent bio-markers such as quantum-dot (QD) nanoparticles were conjugated to arginine vasotosin (ARV) and gonadotropin releasing hormone (GnRH) for in vivo imaging. These quantum dots were then administered to sedated anurans (frogs) through two different routes (intraperitoneal and intranasal) to determine the effectiveness of hormone delivery to targeted reproductive organs for inducing and controlling reproductive events such as spermiation or breeding behaviors. Frogs were used because they are a good model for birds and fish due to the similarities in reproductive processes, hormone receptors and tissue binding specificity- but without the complexities of feathers and scales that can hinder in-vivo imagining. They are also smaller than birds and more easily handled without the requirement of an aquatic environment for fish. In-vivo imaging tracked the binding of AVT- and GnRH quantum dots over 30 minutes compared to unconjugated QD, and confer that specific binding to target tissues was possible. The densities of the hormone conjugated QDs were much higher in the testes and kidney than for the unconjugated QD, demonstrating receptor binding and initiation of sperm release. Nasal administration of GnRH resulted in spemiation similar to the traditional intraperitoneal treatment, giving a less invasive means to initiate gamete production for breeding. Furthermore, AVT-conjugated QD were found to bind to sperm, indicating the presence of receptors on the sperm head, and suggesting another mode of bio-marking spermatozoa.
Furthermore, the synthesis of specific carbon-based liposome nanoparticles incorporated with fluorescent dyes permitted the evaluation of their binding capacity with boar spermatozoa, without impairing their motility potential. This preliminary study is promising for as it provides an interesting route to enable efficient sperm-mediated gene transfer, for transgenesis and drug delivery, for therapeutic purpose. Indeed, the binding of liposomes to sperm membrane was confirmed and the loaded doxorubicin in the synthesized liposomes subsequently fluoresced within the sperm DNA. Similarly, we found that plain or doxorubicin-loaded liposomes interact with follicle cells following 24 hours microinjection within the antrum of cultured porcine follicles. The results indicate an alternative approach for in situ monitoring and/or treatments of follicle diseases reducing female fertility potentials.
Development of specific molecular-based approaches for in vivo biophotonic imaging and 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. Synthesized liposomes showing efficient binding to reproductive cells in vitro (in sub-objective 1.B.) were used for in vivo monitoring of equine follicle cells. Fluorescent liposomes were microinjected in antral ovarian follicles of living mares, followed by fluorescence measurements of aspirated follicular fluids and biopsied follicle wall fragments after 24 hours post-injection. Fluorescence was mainly observed in the plasma membrane of biopsied follicle cells, especially mural granulosa cells, confirming the liposome binding. These results indicate the possible use of such liposomes, for minimal invasive in vivo labeling to enable further in vivo or ex vivo evaluations. Synthesized liposomes could also serve as cargo for intracellular deliveries (as mentioned in Sub-objective 1.B.) for therapeutic purpose. Additional efforts are geared toward the identification suitable biomarkers (e.g., seasonality and follicle growth) for molecular-based targeting to follow folliculogenesis and improve female fertility. Aspects of bird and fish reproduction can be modeled in a variety of amphibian species that exhibit similar gamete development processes and tolerances to environmental factors such as osmolality, light sensitivity, activation and cryostability. We have used the unique properties of amphibian sperm, which is much more difficult to cryopreserve than most mammalian sperm, to focus on the biophysics of membrane stability and cryotoxicity related to alternative cryodiluents and cryoprotectants. Furthermore, we have tested various staining markers for mapping stability through cryopreservation processes for sperm expressed post exogenous hormone treatment, which is a first for caudates. Traditionally NIR has not been used for water based sampling due to the ‘interference’ of the water peak in chemical identification; however we have taken advantage of the aqueous spectral profile to specifically examine the changes in biological systems. We have evaluated serum and protein precipitated serum from horses using NIR to develop spectral imaging signatures for the reproductive states in cycling mares through estrus and diestrus, and quantified the changes in the spectral profiles with estradiol and progesterone levels measured by traditional ELISA assays. Additionally, we have found that the influence of the mycotoxin zearalenone on serum profiles is also distinguishable using NIR. In both cases we were able to develop multivariate equations using chemometrics to obtain a suite of biochemical information possibly replacing the need for multiple single compound assays.
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. Challenges have been experienced in transformation of our bioluminescent constructs into gram-positive bacteria for poultry disease model development, but these short-comings are being addressed to achieve progress in this aspect of our objectives for the coming year. Studies have revealed in poultry models new methods for tracking bacterial progression in eggs, and these systems will be used to identify sites of colonization by both harmful and probiotic bacteria for further study.
Use of biophotonics imaging strategies to establish potentially unknown infection sites of Mycobacterium avium subsp. Paratuberculosis in a rodent model. Specifically, scientists have developed a highly bioliuminescent Mycobacterium avium subsp. paratuberculosis (MAP) strain and have validated its kinetics and luminescent properties for future in vivo studies; with some preliminary work already accomplished in a chicken embryo model. In parallel, we used genetically modified bacteria for their fluorescence detection to test the efficacy of silver-coated magnetic nanocomposites in real-time evaluation of bacteria growth in both culture systems and livestock meats. These studies revealed the antibacterial effect of the tested nanocomposites, while the usefulness of the real-time imaging appears critical for the development of suitable and rapid methods to investigate bacteria contamination.
Development of alternate biophotonic animal models and the use of digital infrared thermal imaging approaches to study various disease states in livestock and poultry. Research has expanded to include more poultry emphasis through applications of digital infrared thermal imaging in broiler production environments and have revealed, through serial imaging over the past year in production houses, potential deficiencies in housing insulation and construction that may influence environmental controls. In addition, the use of thermal imaging in production broiler poultry houses have revealed structural changes that may need to be made to improve environmental control systems. Experimental types of insulation are being monitored in breeder houses that have begun to reveal “hot spots” of inconsistency within the insulated structure. These may directly impact the efficiency of environmental controls as well as the inputs and costs associated with optimal boiler/breeder housing, and research is ongoing to define these anomalies discovered through this research.
Accomplishments
1. Real-time in vivo imaging of the intrauterine environment in livestock. Real-time screening of the intrauterine environment is critical to maximize livestock production potential while minimizing pregnancy wastage and calf morbidity and mortality. Through cooperative agreement with ARS scientist in Stoneville, Mississippi, Mississippi State University scientists successfully used Doppler ultrasonography to screen novel therapeutic supplements to increase uteroplacental blood flow in sheep and cattle. This rapid screening technique will allow scientists to target efficient and economically feasible therapeutic supplements. Specifically, a therapeutic supplement, melatonin, has been shown to increase uterine blood flow during pregnancy and increase weaning weights of calves born to melatonin supplemented dams by 13% compared to controls. These results will benefit scientists examining fetal programming and beef cattle producers focused on cow-calf operations.
2. New method for enhancing fertility in food animal reproduction systems. The newly synthesized carbon-based liposome nanoparticles exhibited efficient binding to both male and female reproductive cells. The specific properties of such liposomes will be beneficial as an approach for efficient transgenic animal production, through sperm-mediated gene transfer and therapeutic achievements, through in situ drug delivery. The search for critical developmental biomarkers (e.g., sperm or follicular fluid proteins) will allow for specific molecular and cellular targeting to enable selective improvements (e.g. sperm nanopurification using magnetic nanoparticles conjugated with specific biomarkers). Through cooperative agreement with ARS scientist in Stoneville, Mississippi, Mississippi State University scientists have synthesized conjugated quantum dot nanoparticles and applied in-vivo for imaging of hormone receptor binding in reproductive tissues and can be used a as bio-markers in sperm cells. These results specifically benefit the scientific community by expanding knowledge that will further permit applied in vivo studies targeting livestock fertility.
3. Real-time monitoring of antimicrobial properties of nanoparticles against foodborne pathogens. Foodborne pathogens increasingly exhibit drug resistance against antibiotics, and despite the broad-spectrum of developed antibiotics during the last decades, bacteria still show multidrug-resistance. The advance in nanotechnology and combination with microbiology have promising applications in the inhibition of bacterial growth. Here we tested the antimicrobial properties of silver magnetic nanoparticles (Ag-MNP) against various foodborne pathogens (E. Coli, Salmonella Typhimurium, and Salmonella Anatum) that were rendered bioluminescent for real-time monitoring of bacterial growth inhibition and survival. Molecular changes were also evaluated. The results indicated the antimicrobial effects of Ag-MNP that was likely exerted through protein changes affecting critical bacterial functions. These proteins can serve as novel biomarkers for further targeting of bacterial contamination with potential impacts in multiple facets of livestock production.
Review Publications
Dutra, G., Ishak, G., Pechan, P., Pechan, T., Peterson, D., Jacobs, J., Willard, S., Ryan, P., Gastal, E., Feugang, J. 2019. Seasonal variation in equine follicular fluid proteome. Reproductive Biology and Endocrinology. 17(1):29. https://doi.org/10.1186/s12958-019-0473-z.
Julien, A., Kouba, A., Kabelik, D., Feugang, J., Willard, S., Kouba, C. 2019. Nasal administration of gonadotropin releasing hormone (GnRH) elicits sperm production in Fowler’s toads (Anaxyrus fowleri). Zoology. 4(3):1-10. https://doi.org/10.1186/s40850-019-0040-2.
Yang, Z., Md Shamimul, H., Htoo, J., Burnett, D., Feugang, J., Crenshaw, M., Liao, S. 2019. Effects of dietary supplementation of l-methionine vs. dl-methionine on performance, plasma concentrations of free amino acids and other metabolites, and myogenesis gene expression in young growing pigs. Translational Animal Science. 3(1):329-339. https://doi.org/10.1093/tas/txy109.
Feugang, J., Rhoads, C., Mustapha, P., Parrish, J., Willard, S., Ryan, P. 2019. Treatment of boar sperm with nanoparticles for improved fertility. Theriogenology. 137:75-81. https://doi.org/10.1016/j.theriogenology.2019.05.040.
Ishack, G., Dutra, G., Gastal, G., Gastal, M., Cavinder, C., Feugang, J., Gastal, E. 2018. In vivo antral follicle wall biopsy: a new research technique to study ovarian function at the cellular and molecular levels. Reproductive Biology and Endocrinology. 16(71):1-12. https://doi.org/10.1186/s12958-018-0380-8.
Feugang, J., Eggert, M., Park, S., Mustapha, P., Steadman, C., Arnold, R., Ryan, P., Willard, S. 2018. Newly designed liposome nanoparticles for drug delivery into boar spermatozoa. Journal Of Reproduction, Fertility And Development. 31(1):227. https://doi.org/10.1071/RDv31n1Ab203.
Castaneda, C., McDaniel, C., Abdelhamed, H., Karsi, A., Kiess, A. 2019. Evaluating bacterial colonization of a developing broiler embryo after in ovo injection with a bioluminescent bacteria. Poultry Science. 98(7):2997-3006. https://doi.org/10.3382/ps/pez053.
McCarty, K., Owen, M., Hart, C., Thompson, R., Burnett, D., King, E., Hopper, R., Lemley, C. 2018. Effect of chronic melatonin supplementation during mid to late gestation on maternal uterine artery blood flow and subsequent development of male offspring in beef cattle. Journal of Animal Science. 96:5100-5111.
Lemley, C., Hart, C., Lemire, R., King, E., Hopper, R., Park, S., Rude, B., Burnett, D. 2018. Maternal nutrient restriction alters uterine artery hemodynamics and placentome vascular density in Bos indicus and Bos taurus. Journal of Animal Science. 96:4823-4834.
Gruhot, T., Park, S., Mustapha, P., Liao, S., Mote, B., Feugang, J. 2018. Dietary L-arginine supplementation affects boar seminal plasma proteome. Journal of Reproduction and Development. 31(1):203-204. https://doi.org/10.1071/RDv31n1Ab157.
Durfey, C., Swistek, S., Liao, S., Crenshaw, M., Clemente, H., Thirumalai, R., Steadman, C., Ryan, P., Willard, S., Feugang, J. 2019. Nanotechnology-based approach for safer enrichment of semen with best spermatozoa. Journal of Animal Science and Biotechnology. 10:14. https://doi.org/10.1186/s40104-018-0307-4.
Gastal, G., Aguiar, F., Ishak, G., Cavinder, C., Willard, S., Ryan, P., Feugang, J., Gastal, E. 2018. Effect of cryopreservation techniques on proliferation and apoptosis of cultured equine ovarian tissue. Theriogenology. 126:88-94. https://doi.org/10.1016/j.theriogenology.2018.11.034.
Park, S., White, S., Steadman, C., Cavinder, C., Willard, S., Ryan, P., Feugang, J. 2018. Real-time bioluminescence analysis of Escherichia coli O157:H7 survival on livestock meats stored fresh, cold, or frozen. Journal of Food Protection. 81(11):1906-1912. https://doi.org/10.4315/0362-028X.JFP-18-207.
Julien, A., Kouba, A., Kabelik, D., Feugang, J., Willard, S., Vance, C. 2019. Nasal administration of gonadotropin releasing hormone (GnRH) elicits sperm production in Fowler’s toads (Anaxyrus fowleri). BMC Biology. 4(3):2-10.
