Performing Department
Department of Animal and Dairy Sciences
Non Technical Summary
Heat stress poses a significant global challenge to sustainable livestock production, leading to detrimental impacts on animal production and welfare. Dairy cows are particularly sensitive to heat stress due to elevated internal heat loads caused by high milk production and suffer from reduced appetite, impaired rumen fermentation functions, and gut microbiota dysbiosis. The central hypothesis for the proposed research is that the ruminal microbial communities of dairy cattle possess immense neuroactive potential in modulating heat stress responses through the brain-rumen axis. Our research objectives are to gain a high-resolution rumen microbiome characterization of lactating cows under heat-stressed and heat-stress-relieved conditions, uncover heat stress-related neuroactive substances derived by rumen microbiota and host, and explore the uniqueness in the brain-rumen-microbiome crosstalk between heat-tolerant Jersey cows and heat-susceptible Holstein cows. We expect to identify heat-stress-associated rumen microbes in dairy cattle as well as understand their neuroactive potential and relationships with hosts' heat-tolerance properties and heat-stress responses. The findings from this project are expected to illustrate: the connections between rumen and brain of ruminants, how this systematic communication responds to environmental stress, and differences in this crosstalk between stress-susceptible and stress-tolerant animals. These outcomes will direct future development of rumen-microbiota-brain axis-guided strategies including targeted breeding and precise diet management to optimize microbial communities, and the development of probiotics such as microbe-derived neuroactive additives to alleviate heat stress responses and improve animal welfare.
Animal Health Component
100%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Goals / Objectives
Our long-term goal is to develop practical strategies targeting the rumen-microbiota-brain axis to alleviate animal stress and improve animal welfare. The central hypothesis for this project is that there is a rumen-microbiota-brain axis that modulates heat stress responses. Specifically, the rumination behaviors and microbial communityare influencedwhen the hypothalamus senses high temperature and sends the signal to the rumen via the nervous system. The shifted rumen environment and microbiota may transmit information to the central nervous system (CNS) and enteric nervous system (ENS) through host and bacteria-produced neurotransmitters that activate neurotransmitter receptors and regulate stress responses.The specific aim of this study isto understand the heat stress-related crosstalk between rumen microbiota, enteric nervous system, and central nervous system in lactating cows.Based on the literature review and our previous studies, we hypothesize that colonization of specific bacterial species in the rumen of Holstein lactating cowsis influencedby heat stress (Obj. 1). The shifted rumen microbiota will affect host and bacteria-derived neurotransmitters and interact with downstream neurotransmitter pathways in CNS and ENS (Obj. 2). The crosstalk between rumen and nervous system via microbiota and neuroactive substances is different between heat-tolerant Jersey cows and heat-susceptible Holstein cows, partly explaining the advantage of Jersey cows in dealing with heat stress (Obj. 3). The objectives are as follows:Objective 1: Identify specific heat stress-associated ruminal microbial species and genes in Holstein lactating cows using the high-resolution long-read sequencing technologyObjective 2: Detect the influence of heat stress on neuroactive substances and neurotransmitter pathways in the rumen and nervous system of Holstein lactating cowsObjective 3: Investigate the uniqueness of the rumen-microbiota-brain axis in heat-tolerant Jersey cows compared to heat-susceptible Holstein cows
Project Methods
Experimental designThe animal experiment will be conducted at the Bearden Dairy Research Center, Mississippi State University (MSU). There will be a two-week animal experimental period in early springof 2025 and mid-summer of 2024, respectively, which are considered as natural thermoneutral and heat-stressed conditions. Twelve multiparous early to middle lactation Holstein (n=12) and Jersey (n=12) cows will be recruited from MSU Holstein and Jersey milking herds. The 24 cows will be housed together in the same barn with the freestall management system. After one-week adaptation, there will be a 2-week sampling period.For summer experiment, the 24 Holstein cows will be divided into heat-stressed (n=12) and heat stress-relieved groups with parity, milk day, and estimated milk yield balanced between groups. Holstein heat-stressed and Jersey groups will be housed on the same side of the barn, while Holstein heat stress-relieved group will be housed on the opposite side. After one-week adaptation, only the Holstein heat stress-relieved group will be treated with fan a sprinkler cooling system for the following 16 days. The automatic sprinkler system will be set on at 74 °F controlled by an Edstrom C-440S controller divided into two zones per barn. Each zone has 18 nozzles with a 0.75 gallon per hour output. The frequency will be set for 3 min on and 5 min off throughout the day. The animals will be offered ad libitum access to water and total mixed rations (TMR) to meet the NRC nutrient requirements throughout the experimental period. On day 16 of the summer experimental period, four cows with similar parity and milk yield at day 0 from each group will be slaughtered in the MSU Meat Science Laboratory for tissue collection.Environmental conditionThe temperature (T, °C) and relative humidity (RH, %) at 0700h, 1200h, and 1700h will be recorded everyday throughout the experimental periods to calculate the temperature-humidity index (THI): THI = 0.8 × T + (RH/100) × (T - 14.4) + 46.4 (Llamas-Luceño et al., 2020).Animal behavior and milk productionRespiratory rate and rumination frequency will be visually observed three times a day for 2-h periods at 0630-0830h, 1130-1330h, and 1630-1830h on day 0, 3, 7, 10, 14 in the two sampling periods, respectively, as described in a previous study (Becker et al., 2021). Rectal temperatures will be recorded manually using a digital thermometer after the measurement of rumination frequency (Li et al., 2020). Cows will be milked twice daily (0500 h and 1700 h). Automated milk yields will be recorded for every cow at each milking through the experimental periods. The daily feed intake per group will be recorded.Sample collectionOn days 0, 7, and 14 of each experimental period and day 16 of the summer experimental period, mixed rumen digesta samples (50 mL) will be collected from each cow via an oral rumen tube and a hand vacuum pump and blood samples (10 mL) will be collected into EDTA-containing evacuated tubes from the tail vein before morning feeding, respectively. Blood samples will be centrifuged for 20 min at 1,200 × g and 4°C to obtain the blood plasma. On day 16 of the summer sampling period, the head and neck of each slaughtered cow will be evacuated together to collect hypothalamus (1 cm × 1 cm) and two nodose ganglia samples (2.5 cm in length). The digestive tract of each slaughtered cow will be removed to collect the ruminal tissue samples from the ventral sac of the rumen (2 cm × 1 cm) and celiac ganglion sample (1 cm × 1 cm). All the samples will be stored at -80 °C for downstream analysis.Rumen microbiota composition and functional analysisMicrobial genomic DNA will be extracted from the rumen digesta sample using the QIAamp PowerFecal Pro DNA Kit. To investigate rumen microbiota composition, Oxford Nanopore full-length 16S rRNA gene amplicon sequencing using MinION Mk1B sequencer will be conducted to gain high taxonomic resolution. The raw sequencing data will be base-called by Guppy and filtered by Chopper. The taxonomic classification and microbial alpha and beta diversity will be analyzed by Emuand NanoRTax. Rumen microbial functional profile will be characterized by shotgun metagenomic sequencing for pooled and representative rumen digesta samples from each group at each sampling point using the Oxford Nanopore platform. The base-called metagenomic data will be assembled by metaFlye. Taxonomic assignment of contig will be performed with the BlobTools pipeline. Gene annotation will be conducted by Prokka and KEGG mapper. The abundance and microbial host of genes involved in neurotransmitter synthesis will be particularly focused.Short-chain fatty acids measurementThe rumen digesta samples will be thawed and centrifuged at 20,000×g at 4 °C for 20 min, and the supernatant will be filtered through a 0.45 μm syringe filter. The SCFAs, including acetic, propionic, and butyric acid will be analyzed by gas chromatography.Neurotransmitter concentration measurementSamples of rumen digesta, rumen tissue, blood plasma, hypothalamus, and ganglia will be shipped to Vanderbilt University Neurochemistry Core (Nashville, Tennessee) supported by the Vanderbilt Brain Institute and the Vanderbilt Kennedy Center for neurotransmitter concentration analysis. Briefly, the chief inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and major excitatory neurotransmitter glutamate will be measured using LC/MS, while emotion closely related neurotransmitters serotonin and dopamine will be measured by HPLC-electrochemical detection.Transcriptome analysisTotal RNA will be extracted using a TRIzol-Based RNAiso Plus kit. The RNA will be sent to MSU Institute for Genomics, Biocomputing & Biotechnology (IGBB) for transcriptome analysis. Briefly, the RNA library will be constructed using the TruSeq RNA Library Prep Kit v2 and sequenced on an Illumina HiSeq 2500 platform. The raw sequencing data will be filtered using Trimmomatic software. The remaining reads will be aligned to the Bos taurus genome (ARS-UCD1.2) using HISAT v2-2.1.0 software. The number of fragments per thousand exons (FPKM) will be used to analyze the differentially expressed genes (DEGs) between groups using the voom package installed in R. The functions of DEGs will be analyzed using goatools and KOBAS v3.0 to invest GO functional enrichment and Kyoto Encyclopedia of Genes and Genes (KEGG) pathway enrichment. The neurotransmitter receptor, short-chain fatty acids receptors, and stress-related genes and their related pathways will be particularly focused on.Statistical analysisStatistical analyses will be primarily conducted using RStudio (version: 2023.06.1+524). The normal distribution of variables will be assessed using the Shapiro-Wilk's test. Student's t-test or one-way analysis of variance test will be used to compare animal behavior, SCFAs, neurotransmitters concentration, and microbiota alpha-diversity index between Holstein cows under thermoneutral, heat-stressed, and heat stress-relieved conditions, or between Jersey and Holstein cows under thermoneutral, heat-stressed conditions at different sampling points. Permutational multivariate analysis of variance (PERMANOVA) test will be conducted to compare microbiota beta-diversity between groups. Linear discriminant analysis Effect Size (LEfSe) will be used to determine the bacterial species or genes that are differently present or expressed between groups. Pearson correlation will be analyzed to examine associations between the relative abundance of bacterial species, the concentration of neurotransmitters, and transcription levels of host genes. The influence of other factors, such as parity, age, milk day, milk yield, and body weight, will be evaluated using the linear regression model.