Progress 05/01/23 to 04/30/24
Outputs
Target Audience:Scientists and researchers in academia who are interested in the transcriptomic and epigenetic basis of male reproductive performance. Changes/Problems:We did not encounter any major problems, and no major changes are planned. What opportunities for training and professional development has the project provided?Two graduate students were supported at 0.5 FTE each by this project. During this project year, they had the opportunity to learn catfish breeding, sperm cryo-preservation and in vitro fertilization techniques, hybrid catfish hatching and fry development, genomic DNA extractions, total RNA extractions, next-generation sequencing library preparation, quality control, and sequencing. The students acquired a comprehensive understanding of aquatic farming practices and the technological innovations driving the industry forward. The hands-on experience on aquaculture husbandry and aquaculture biotechnology is pivotal to prepare them as well-trained next-generation workforce for US agriculture sector. Their real-world skills and genomic literacy will help enhance sustainability and food security in the near future. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?As originally scheduled, we will finish the RNA-seq and DNA methylome sequencing in the cryo-preservation study, and start the data analysis during the next reporting period. We will evaluate and analyze the short-term storage phenotypic data and pilot the multi-omics sequencing. We plan to work on three manuscripts entitled 1) Effect of short-term storage on sperm quality and microbiome dynamics in blue catfish; 2) Paternal impacts on industry-relevant offspring performance traits in blue catfish, Ictalurid furcatus; and 3) Effect of age on spermatogenesis, reproductive hormones, testicular gene expression, and sperm quality in blue catfish.
Impacts
What was accomplished under these goals?
We made good progress on all three objectives as planned in this proposal towards fulfilling the project's goals. Broodstock management (Objective 1-3): Mature blue catfish males were obtained from Jubilee Farms in Indianola, MS, USA. Mature channel catfish females were obtained from Harvest Select in Inverness MS, USA. Fish were transported to the Auburn University E.W. Shell Fisheries Center in Auburn, Alabama, USA in Spring 2023. At Auburn, fish were held in 0.02 to 0.04 ha excavated ponds and fed every other day until satiation with a 32% protein diet until one month before spawning, when feed was switched to a 36% protein diet. At the beginning of the catfish spawning season blue catfish males (n = 44) were randomly collected by seining and then euthanized following AU-IACUC protocols. From each male, blood samples were collected for sex steroid analysis. The total body weight, fork length, head width, head circumference, and hump diameter were recorded. Lastly, testes were collected for sperm samples. Testes were removed from the abdominal cavity and placed in 50 mL centrifuge tubes with Hank's Balanced Salt Solution (HBSS) to prevent sperm activation. Thereafter, testes were separated from the peritoneum and blood vessels and blotted on Kimwipes®to remove blood/adherent tissues before being macerated through a fine mesh strainer (200 μm) into sterile centrifuge tubes. Samples from each male were processed separately. A testes sample was collected from each male for molecular analyses. Sperm quality analyses (Objective 1): Sperm density was then determined using a Neubauer hemocytometer. For each male, an aliquot of sperm was used for cryopreservation, while another aliquot was used to quantify sperm quality traits before cryostorage. For sperm cryopreservation, sperm samples were diluted to 1.0 × 109cells/mL using HBSS. Methanal (10%) was added as the cryoprotectant. The diluted sperm were pipetted into 0.5 mL straws before being placed in a controlled-rate freezer. Straws were frozen at a rate of − 5oC/min until they reached -80oC and immediately transferred into liquid nitrogen. Sperm swimming kinematics, includingvelocity, motility, progressive VCL, and progressive motility, were assessed at 10, 20, and 30 s post-activation. Lipid peroxidation and cell viability were also measured.Three technical replicate activations were conducted per male. Maternal spawning induction and egg collection (Objective 1): Females were selected based on secondary sexual characteristics (i.e., swollen abdomen and urogenital pore). Gravid females were then held in mesh spawning bags that were suspended in fiberglass raceways with a flow rate of ~80 L/min. Gravid females were administered two intraperitoneal injections of luteinizing hormone-releasing hormone analog, totaling 100 μg/kg. Ovulating females (n = 3) were anesthetized to reduce stress. The urogenital pore was wiped dry to avoid contamination from urine, blood, or feces. Eggs were hand-stripped by applying gentle pressure on the abdomen toward the vent. Stripped eggs were deposited into metal spawning pans lined with Crisco®All-Vegetable Shortening. The total egg mass of each female was weighed to the nearest ±0.1 g. Egg density was estimated by weighing and counting 1 g of eggs with two replicate counts. Artificial fertilization (Objective 1): Cryopreserved sperm was thawed at 40ºC using a 5 L water bath for 20 s before it was transferred into 1.5 mL microcentrifuge tubes. Males 1 to 15 were used to fertilize Female 1, Males 16 to 30 were used to fertilize Female 2, and Males 30 to 44 were used to fertilize Female 3, creating 44 hybrid catfish families. All eggs were fertilized using a sperm-to-egg ratio of 5.0 × 104sperm per egg and allowed to sit for a 2 min gamete contact time. Eggs were allowed to incubate with the sperm and Fuller's Earth solution for ~5 min with continuous manual stirring to ensure sufficient gamete contact. The fertilized eggs were then transferred into incubation aquaria. Embryo and fry husbandry conditions (Objective 1): Embryos and fry were housed in recirculating aquaculture systems. Individual aquaria had a water flow rate of ~7.5 L/min and were maintained at 27.0 ± 0.8?C. Each aquaria had 3 to 4 mesh basketsfor incubating embryos. For hatched fry, three 5.7 L white tanks were used in each aquarium. To minimize fungal infections, dead and unfertilized embryos were removed daily until hatch, with little disturbance, using a transfer pipette. These eggs were identified by their enlarged size and/or opaque/white coloration. Dead eggs that adhered to healthy embryos were left undisturbed. Fry kept for experimentation were stocked into 5.7 L white tanks at 50 fry per tank with 2-3 replicate tanks for each male-female combination (3 females × 44 males × 2-3 replicates = 131 tanks). Three 5.7 L tanks were housed in each aquarium. Fry mortalities were checked daily, and feeding began just before signs of "swim-up". Fry were fed Purina AquaMax Fry Powderthree times per day to satiation. Fry were sampled at 0, 20, and 40 days post-hatch. Here pictures of 12 larvae per aquaria for body morphometrics and then preserved in liquid nitrogen for molecular analyses. Genomic DNA and total RNA extraction (Objective 2-3): Four fresh-frozen sperm samples and four cryo-preserved sperm samples with two replicates were used for DNA and RNA extraction. The samples were immediately transferred from liquid nitrogen to a water bath and thawed at 40 °C for 20 s to ensure a consistent processing time. The PowerLyzer24 instrument was used to disrupt and homogenize the samples. Genomic DNA and total RNA were extracted with AllPrep DNA/RNA/miRNA kit following the manufacturer's instructions. The concentration of DNA/RNA yield was measured by a Qubit 3.0 Fluorometer. RNA-seq library preparation, Sequencing and data analysis (Objective 2): RNA-seq library for each sample was constructed using NEBNextUltraII RNA Library Prep Kitwith 1 µg of total RNA input. The concentration of each library was quantified by Qubit 3.0 Fluorometer and the size distribution was evaluated by D1000 ScreenTape assay using TapeStation 4200 System. The libraries were sequenced on Illumina NovoSeq6000 sequencer to generate 2×150 bp paired-end reads at Novogene. The raw reads quality of 16 RNA-seq data was assessed by FastQC. After low-quality bases and adapters were trimmed by Trimmomatic (version 0.39), the RNA-seq data were mapped to the blue catfish reference genome using TopHat aligner. The alignment rate ranges variably from 13.7% to 72.1%, including high rRNA alignment due to the higher rRNA content in sperm samples. After removing the reads aligned to rRNA, the mapping rate for each sample ranges from 9.3% to 69.7%. Bedtools calculated and summarized the gene counts. EM-seq library preparation and Sequencing and data analysis (Objective 3): The library construction for DNA methylome sequencing was performed using NEBNextEnzymatic Methyl-seq kit according to manufacturer's protocol. The gDNA of each sample was sheared to 500 bp by Covaris M220at 75 W for 50 s. The concentration and size distribution were determined by Qubit 3.0 Fluorometer and TapeStation 4200 System, respectively. The libraries were sequenced on Illumina NovoSeq6000 sequencer. To get the alignment rate of EN-seq data, we merged the the EM-seq data, and trimmed the low-quality base and adapters. Then, the methylated and unmethylated blue catfish genome were generated for subsequent alignment. Trimmed assembled and both strands of unassembled reads were mapped to the methylated and unmethylated genomes using bwa-mem2, respectively and the secondary alignments were discarded to calculate the final alignment rate. The average total alignment rate was more than 97% and the average unique alignment rate was more than 87%.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2024
Citation:
Wang H, Su B, Zhang Y, Mei S, Wang J, Johnson A, Dilawar H, Bruce T, Dunham RA, and Wang X* (2024). Transcriptome analysis revealed potential mechanisms of channel catfish growth advantage over blue catfish in tank culture environment. Frontiers in Genetics, in press. doi: 10.3389/fgene.2024.1341555
- Type:
Journal Articles
Status:
Under Review
Year Published:
2024
Citation:
Wang H, Su B, Zhang Y, Mei S, Li S, Xing D, Wang J, Bern L, Johnson A, Al-Armanazi J, Hasin T, Hettiarachchi D, Paladines Parrales A, Dilawar H, Bruce T, Dunham RA, and Wang X* (2024). From heterosis to outbreeding depression: genotype-by-environment interaction shifts hybrid fitness in opposite directions. Genetics, under revision.
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