Location: Warmwater Aquaculture Research Unit
2020 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 researcher 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:
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. In addition, within a subordinate project, we have made progress in expanding these imaging techniques of blood perfusion in the male, specially bulls. Progress was made in the evaluation of the influence of chronic melatonin supplementation on bovine testis biometrics, thermoregulation, and blood flow in mature and peri-pubertal bulls. Melatonin had limited effects on testis biometrics and thermoregulation but did increase blood flow at the end of a 120-day melatonin supplementation period. 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, 1) a novel label-free and high-sensitivity microscopy technique, the spatial light interference microscopy (SLIM), was tested for high-throughput nanoscale imaging of unlabeled and paraformaldehyde-fixed spermatozoa on slides. Comparisons with the Atomic Force Microscope (AFM) imaging enabled a maximal characterization of sperm morphometrics. Both SLIM and AFM were validated with correlation analyses of different compartments of (boar and stallion) spermatozoa and dry mass. Positive (boar) and negative (stallion) correlations of the sperm head and length and width of the acrosome existed between both techniques. The SLIM appeared as a useful technique for high-throughput and rapid sperm evaluation for high confidence selection of samples during assisted reproductive technologies (e.g., in vitro fertilization); 2) the effective delivery of a synthetic NanoLuc luciferase mRNA and protein synthesis in the sheep cervicovaginal mucosa was explored in a multi-institution effort. Hand pressure was used to spray the luc-mRNA on cleaned vaginal lumen of sedated sheep. Results indicated successful intracellular delivery and synthesis of a functional protein enabling bioluminescence imaging (Lindsay et al., 2020). In the future, luc-mRNA will be used as a tool to visualize the efficiency of sperm-mediated gene transfer (SMGT) during in vitro fertilization.
Within Sub-Objective 1C, investigations included the: 1) proteome profiles of porcine follicular fluid harvested during follicle development (Small, Medium, and Large). Follicular fluids (FF) obtained from non-atretic follicles revealed comparable protein concentrations but different protein numbers (1627, 1699, and 1756 proteins, respectively). Proteins were shared across follicle sizes (~247) or were specifics to each follicle size (60-63%). Functional enrichment analyses revealed numerous protein candidates of follicle growth, oocyte maturation and fertilization. Identified markers will serve for bioconjugation to fluorescent nanoparticles to enable fertility prediction through non-invasive and real-time bioimaging; 2) effects of insulin and thyroxine alone, or in combination, during in vitro culture of caprine preantral follicles enclosed in ovarian tissue. Expression levels of apoptotic-related genes and receptors of insulin and thyroid hormones were evaluated, and the reactive oxygen species and estradiol levels. Findings indicated beneficial effects on follicle survival and development; therefore, setting the stage for future work to improving in vitro culture of preantral to/and antral follicles; and 3) the existence of protein markers for real-time monitoring of follicle health and growth. The presence of mural granulosa cells, theca interna, and theca externa layers was validated on harvested equine ovarian follicle wall biopsy (FWB) and performed histological and molecular characterizations. The quality of FWB was validated through various biomarkers of follicle development and endocrinology. The FWB technique appeared as an excellent tool for real-time in situ study of the follicular fluid composition, folliculogenesis and oogenesis.
Within Sub-Objective 1D, we conducted Near Infrared Reflectance (NIR) Spectroscopy in alternative models of amphibian species (caudates) by considering their chemosignaling communication during courtship through pheromone exposure and expression. Biochemical analysis of pheromone content, a hallmark of reproduction in many species, is verified by NMR and HPLC. Characteristic NIR spectral-chemical profiles were collected in-vivo from caudate chemosignaling glands and reproductive skin regions. NIR spectral profiles were able to map signatures of pheromones most prominently from the postcloacal gland which induces reproductive behaviors. Furthermore, NIR spectra revealed distinct sexually dimorphic chemical signatures in these otherwise monomorphic species, demonstrating potential in-vivo gender discrimination - with application to poultry science in embryo gender selection. 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, such as those associated with bovine respiratory disease (BRD) in cattle. Diagnosis of BRD traditionally relies on visual assessment, which can be untimely, insensitive, and nonspecific leading to imprecise treatment and further spread of the disease in the herd creating the need for a rapid an accurate diagnosis method. In this study, Near Infrared Spectroscopy (NIRS) was applied to identify and discriminate the spectrum from blood plasma that profiles responses of dairy calves to M. haemolytica infection using multivariate analysis (MVA). Samples were obtained by collecting blood from five Holstein steers over four days prior to, and 23 days after, intrabronchial challenge with M. haemolytica. Discriminant Analysis (PCA-LDA) classified plasma from baseline and infected periods with an accuracy, sensitivity and specificity =90% for the calibration and internal validation processes. The external validation displayed =75% for the same quality parameters. These results showed that physiological and biochemical changes occurring in the blood plasma of dairy calves during M. haemolytica infection make possible the creation of a NIRS profile and establish a basis for further development of NIRS as a BRD diagnosis and monitoring tool of this infection over time.
Within Objective 2, we: 1) examined the silver-coated magnetic (iron oxide) forming magnetic nanoparticles (Ag-MNP) as an alternative to multidrug resistance. The antibacterial effect of the synthesized Ag-MNP was demonstrated using various foodborne pathogenic bacteria (E. coli and Salmonellas). The Ag-MNP nanoparticles and interactions with bioluminescence-transformed bacteria was further characterized through various sophisticated imaging techniques, 2) used E. coli and Salmonella that were transformed for bioluminescence emission to optimize ultrasound sterilization of animal products. This work, initiated and conducted at both Mississippi State University and Southern Illinois University, indicated the effectiveness of the proposed pasteurization technique on raw camel milk.
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 at estrus and 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 circulating concentrations of estradiol or progesterone.
During the summer of 2019, the possibility of utilizing Bacillus as a probiotic became of interest to our laboratory due to their ease of use, which has increased their popularity in the poultry industry. As spore formers, Bacillus can be easily supplemented into chicken feed due to their ability to withstand the high pelleting temperatures. Therefore, our main objective has been to construct a bioluminescent Bacillus probiotic that can be easily followed to determine their exact site of colonization. Therefore within Objective 2, progress is being made to work on the transformation of the B. subtilis serotypes to include the bioluminescence plasmids. To achieve this, 2 methods were identified: 1. Utilizing the Bacillus Biobrick box previously mentioned and transforming the bacteria using the chemical procedure as well as the electroporation method. 2. The transformation of Gram-positive bacteria is a complicated procedure and therefore we have identified the use of CRISPR-CAS9 as a good method for efficient bacterial transformation.
Accomplishments
1. Development and validation of a novel approach for in vivo biopsy of follicle wall. Through cooperative agreement with an ARS researcher in Stoneville, Mississippi, Mississippi State University and Southern Illinois University researchers examined a novel in vivo biopsy approach of the follicular wall. The continuous growth of ovarian follicle is influenced by various intrinsic and extrinsic factors related to the animals. In vivo investigations of these factors using the same individuals are important to elucidate the associated physiological changes. The previously developed in vivo follicle wall biopsies (FWB) technique was validated as a new and viable approach to obtain reliable fragments for the study of ovarian function. Both cellular (e.g., histology) and molecular (e.g., gene expression and protein detection) techniques were performed on FWB of the same living mare to follow important stages of follicle development and ovulation. This technique does not jeopardize ovarian function and may be applied in different species, including humans.
2. Magnetic nanoparticle conjugate and foodborne pathogens. Through cooperative agreement with an ARS researcher in Stoneville, Mississippi, Mississippi State University researchers examined magnetic nanoparticle conjugates. Nanotechnology has a huge impact in the advancement of biomedicine, while the cytotoxicity of nanoparticles remains of concern in human and animal bodies. Iron-oxide core coated with silver ions (Ag-MNP) were designed to permit interaction with foodborne pathogenic bacteria and to allow safe removal from bio-mixtures, under a magnetic field. The synthesized Ag-MNP nanocomposites were characterized with various imaging techniques and their incubation with bacteria showed efficient bactericide effects. The synthesized Ag-MNP appeared as potential alternative antimicrobial agents to treat foodborne pathogens with antibiotic resistance avoidance (Park et al., 2019).
3. Nonthermal method for milk pasteurization. Through a cooperative agreement with ARS an 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, the nonthermal ultrasound processing was an effective approach to reduce microbial contamination of raw camel milk. Amongst the investigated milk components (e.g., fatty acid, lipid peroxides, protein fractions), only volatile compounds showed few changes that may affect the sensory quality of raw camel milk (Dhahir et al., 2020). The present work offers new horizon for further valorization of camel milk (e.g., longer storage and commercialization).
Review Publications
Guy, E., Gillis, A., Kouba, A., Barber, D., Poole, V., Vance, C. 2020. Sperm collection and cryopreservation for threatened newt species. Cryobiology. 94:1-9.
Vance, C., King, E., Bowers, S., Ryan, P., Walters, K., Shappell, N.W. 2019. Reproductive performance of mares fed dietary zearalenone. Frontiers in Veterinary Science. 6:423.
Owen, M., Northrop, E., Rich, J., Perry, G., Messman, R., Dinh, T., Mccarty, K., Yang, J., Wan, D., Lemley, C. 2019. Oxylipin concentrations in bovine corpora lutea during maternal recognition of pregnancy. Theriogenology. 142:384-389.
Lemley, C., Bowers, K., Yankey, K., Tu, M., Hart, C., Steadman, C., Mccarty, K., Owen, M. 2020. Investigating ovine placentome blood perfusion using power flow Doppler ultrasonography. Small Ruminant Research. 184:106051.
Guy, E., Martin, M., Kouba, A., Cole, J., Vance, C. 2019. Evaluation of different temporal periods between hormone-induced ovulation attempts in the female Fowler’s toad Anaxyrus fowleri. Conservation Physiology. 8(1):1-8.
Messman, R., Contreras-Correa, Z., Paz, H., Perry, G., Lemley, C. 2020. Vaginal bacterial community composition and concentrations of estradiol at the time of artificial insemination in Brangus heifers. Journal of Animal Science. 98(6):1-9.
Rubessa, M., Feugang, J., Kandel, M., Schreiber, S., Hessee, J., Salermo, F., Meyers, S., Chu, I., Popescu, G., Wheeler, M. 2020. High-throughput sperm assay using label-free microscopy: morphometric comparison between different sperm structures of boar and stallion spermatozoa. Animal Reproduction Science. https://doi.org/10.1016/j.anireprosci.2020.106509.
Shamimul, H., Feugang, J., Liao, S. 2019. A nutrigenomics approach using RNA sequencing technology to study nutrient–gene interactions in agricultural animals. Current Developments in Nutrition. 3:18.
Dhahir, N., Feugang, J., Witrick, K., Park, S., Abughazaleh, A. 2019. Impact of ultrasound processing on some milk-borne microorganisms and the components of camel milk. Emirates Journal of Food and Agriculture. 3:18.
Ryan, P., Willard, S., Feugang, J. 2020. Comparative analysis of porcine follicular fluid proteomes of small and large ovarian follicles. Biology. 9(5):101.
Ishack, G., Dutra, G., Gastal, G., Elcombe, M., Gastal, M., Park, S., Feugang, J., Gastal, E. 2019. Deficiency in proliferative, angiogenic, and LH receptors in the follicle wall: implications of season toward the anovulatory condition. Domestic Animal Endocrinology. 70:106382.
Lindsay, K., Vanover, D., Thoresen, M., King, H., Xiao, P., Peres, B., Arainga, M., Park, S., Tiwari, P., Peck, H., Blanchard, E., Feugang, J., Olivier, A., Zurla, C., Villinger, F., Woolums, A., Santangelo, P. 2020. Aerosol delivery of synthetic mRNA to vaginal mucosa leads to durable expression of broadly neutralizing antibodies against HIV. Molecular Therapy. 28(3):805-819.
Feugang, J., Santos-Rivera, M., Grant, A., Park, S., Devos-Burnett, D., Lemley, C., Vance, C. 2019. Discriminating intrauterine growth-restricted piglets through near-infrared spectroscopy of amniotic fluids. Journal of Reproduction and Fertility. 32(2):230.
Ishak, G., Dutra, G., Gastal, G., Gastal, M., Feugang, J., Gastal, E. 2019. Transition to the ovulatory season in mares: An investigation of antral follicle receptor gene expression in vivo. Molecular Reproduction and Development. 86(12):1832-1845.
Liao, S., Willard, S., Ryan, P., Feugang, J. 2019. Proteome changes of porcine follicular fluid during follicle development. Journal of Animal Science and Biotechnology. 10:94.
