Performing Department
College of Agriculture, Food and Natural Resources
Non Technical Summary
Heat stress reduces sperm quality and fertility, thus efficiency of reproduction and production of food animals. There is a lack of knowledge in molecular and cellular level effects of heat stress on buck sperm and biomolecular markers to evaluate sperm quality and predict male fertility. These gaps in the knowledge base are important problems because they are preventing advances in basic and applied food animal production science. The overall goal of the proposed innovative research is aimed at ascertaining and alleviating the impacts of heat stress on buck sperm fertility to build capacity for reproductive biology research in food animal production. Specific research objectives are to determine the effects of heat stress on sperm, and to ascertain the effects of heat stress on seminal plasma that are mitigated by melatonin. By unraveling the underlying mechanisms using comprehensive animal experimental setup at PVAMU's International Goat Research Center and analyses through functional genomics, we will provide valuable insights into both mitigating the negative effects of heat stress on buck sperm and discovering potent biomarkers associated with heat stress-induced fertility decline. Project outcomes will have practical implications in smart agriculture for improving reproductive outcomes in agriculture, ensuring the long-term viability and productivity of livestock systems in the face of climate change. The proposed project is an integral part of a R&D effort in animal systems for the research arm of the land-grant program and is also in line with the need area i. "Studies and Experimentation in Food, Agricultural, Natural Resources, and Human Sciences", and with the priority area of "Sustainable Agriculture" of the CBG program
Animal Health Component
100%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
The purpose of this proposal is to establish capacity on innovative research aimed at developing robust livestock resilient to climate change. This capacity building grant support will enable a novel and pioneering research project on the effects of heat stress on buck sperm, with a focus on the role of extracellular vesicles (EVs) within seminal plasma in fertility. Heat stress is an urgent concern in both livestock animals and humans because of climate change, as it can lead to reduced fertility and reproductive problems (Boni, 2019; Dovolou et al., 2023). Better understanding of how heat stress impacts sperm function and fertility is fundamental for reproduction and artificial insemination in livestock and human reproductive biology. Stress has detrimental effects on sperm quality and fertility, based on research from our lab and those of others (Hitit et al., 2020; Rosyada et al., 2022). Elevated temperatures cause oxidative stress in sperm, DNA damage, and disruption of many biological processes (Houston et al., 2018; Capela et al., 2022). Recent research indicates that EVs (Barranco et al., 2023), which are small membrane-bound vesicles released by cells, and seminal plasma have a role in enabling intercellular communication and affecting sperm function.However, there are significant gaps in the knowledgebase concerning the specific molecular mechanisms underlying the effects of heat stress on buck sperm cells, EVs, and seminal plasma. More research is required to determine the molecular and cellular underpinnings of heat stress-related changes in the composition and function of EVs as well as their interactions generated from buck sperm and seminal plasma. Our long-term goal is to advance our understanding of the impact of heat stress on male fertility and discover novel approaches for mitigating its adverse effects and establish collaborations with our colleagues at other HBCUs. This CBG grant will train students with innovative research on evaluating the changes in EVs and seminal plasma associated with heat stress in buck sperm. Our central hypothesis is that melatonin improves the impaired sperm function and reduced fertility induced by heat stress. We will use a multidisciplinary approach to accomplish this, utilizing techniques encompassing computational biology, molecular biology, including proteomics, and reproductive biology. We will subject bucks to acute and chronic heat stress using a unique model of scrotal insulation with melatonin and without melatonin treatment and collect semen samples before and after heat exposure. We will analyze to characterize the changes in sperm parameters, EV composition, cargo, and function, as well as seminal plasma proteome alterations, analyze the data and interpret the results. We will gain a mechanistic understanding of how heat stress affects the communication between sperm, EVs, and seminal plasma, and how these alterations contribute to compromised sperm function and fertility outcomes. The new knowledge will not only advance our understanding of the effects of heat stress on male fertility but also pave the way for the development of novel diagnostic markers and therapeutic interventions targeting EVs and seminal plasma to mitigate the adverse effects of heat stress on fertility. The proposed research addresses critical knowledge gaps in the field of male fertility under heat stress conditions to develop climate smart agriculture and resilient goats. By investigating the interplay between buck sperm, EVs, and seminal plasma, we aim to unravel the molecular mechanisms underlying the impact of heat stress on fertility. Empowering students in this research will provide them with valuable hands-on experience and training the next generation of researchers in the field of reproductive biology and fertility for sustainable food security.We will pursue the following Specific Research Objectives: 1. Determine the effects of heat stress on sperm. The working hypothesis is that heat stress disrupts buck sperm fertility, and that melatonin alleviates the negative effects. 2. Ascertain the effects of heat stress on seminal plasma. Our working hypothesis is that heat stress induces alterations in the composition, cargo, and functional properties of extracellular vesicles (EVs) derived from seminal plasma, leading to impaired sperm function and reduced fertility. The rationale and significance of the proposed research are that by building capacity and pursuing transformative and translational research, we will produce new knowledge on the effects of heat stress on buck sperm and extracellular vesicles (EVs) within seminal plasma in fertility and develop biotechnological approaches to improve livestock fertility.
Project Methods
The study will use eight mature bucks, each having satisfactory semen quality and normal fertility data. These animals will provide the biological samples necessary for all experimental evaluations. The bucks will be divided into two main groups. One group will receive melatonin implants, while the other will serve as the control group. Each group will be further subdivided to undergo either acute or chronic heat stress conditions. For the melatonin-treated group, 18 mg of melatonin will be implanted in each buck's ear. The acute and chronic heat stress will be simulated via scrotal insulation using diapers that cover the entire scrotum, applied for specified durations to evaluate the physiological impacts of heat stress. Semen samples will be collected bi-weekly using an artificial vagina over the course of the study to analyze the effects of melatonin and heat stress on sperm quality and function over time.Sperm concentration will be quantified using a Nucleocounter SP-100, which provides precise measurements crucial for evaluating the impacts of treatments on sperm production. Morphology will be assessed through dilution with buffered formal saline and microscopic examination to identify morphological abnormalities. This technology will be used to assess detailed motility patterns and kinetic parameters of sperm, providing insights into the functional impacts of heat stress and melatonin treatment. Sperm viability will be assessed using a combination of SYBR-14 and propidium iodide stains, analyzed through flow cytometry to determine the proportion of live versus dead sperm. Oxidative stress levels will be quantified using specific fluorescent dyes, helping to elucidate the cellular damage caused by heat stress.EVs will be isolated from seminal plasma using ultracentrifugation. Their size distribution and concentration will be determined through nanoparticle tracking analysis and dynamic light scattering, providing insights into the cargo and potential signaling roles of these vesicles under stress conditions. Both sperm and EV samples will undergo proteomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This will identify and quantify proteins, allowing for the exploration of molecular pathways affected by heat stress and potentially uncovering new biomarkers of fertility.Heat stress will be induced through scrotal insulation, carefully controlled and monitored to replicate acute and chronic conditions. This method affects the testicular temperature, directly impacting sperm health and production. Temperature and humidity will be continuously recorded using a data logger, ensuring precise correlation with physiological changes observed in the bucks.Data will be analyzed using statistical software like SAS and R. This will include analysis of variance (ANOVA) for comparing treatment effects and other statistical tests to validate the findings. Advanced bioinformatics tools will be employed to analyze proteomic data, helping to map the interactions and pathways altered by heat stress, contributing to a deeper understanding of the underlying mechanisms.