Progress 05/01/24 to 04/30/25
Outputs
Target Audience:The target audiences of this project period will include: 1) Scientists and researchers in academia who are interested in the transcriptomic and epigenetic basis of male reproductive performance. 2) Catfish farmers, including low-income, disadvantaged producers in rural areas, who are interested in improving hybrid catfish breeding and reproduction. 3) Commercial hybrid catfish hatcheries and fingerling suppliers who are considering cryopreservation and in-vitro fertilization techniques for producing the fry. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the reporting period, two graduate students were supported at 1.0 FTE each through this project. Over the course of the project year, the students received comprehensive training in key methodologies, including catfish sperm cryopreservation, in vitro fertilization, hybrid catfish hatching, and fry development and monitoring. They also gained technical expertise in genomic DNA and total RNA extraction, next-generation sequencing library preparation, quality control, high-throughput sequencing, multi-omics data processing and analysis, and quantitative PCR (qPCR) validation. In addition to laboratory skills, the students acquired a broad understanding of aquaculture husbandry and biotechnological applications within aquaculture systems. This hands-on experience is critical for developing a well-prepared, technically proficient workforce to support the U.S. agriculture sector. Their training in applied genomics and aquaculture biotechnology positions them to contribute meaningfully to the advancement of sustainable aquaculture practices and food security initiatives. How have the results been disseminated to communities of interest?The results of this project were disseminated through oral presentations at the Aquaculture 2025 conference, organized by the World Aquaculture Society and held from March 6 to 10, 2025, in New Orleans, Louisiana. This triennial conference integrates the annual meetings of the World Aquaculture Society (U.S. Chapter), the National Shellfisheries Association, the Fish Culture Section of the American Fisheries Society, and the National Aquaculture Association, making it a premier venue for sharing research with a broad stakeholder audience. Both Co-Project Directors and the two graduate students delivered oral presentations highlighting key project findings. As a result, the outcomes reached a diverse audience, including members of the catfish industry, aquaculture researchers, students, extension personnel, and policy makers, thereby enhancing the visibility and broader impact of the research. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will complete RNA sequencing and DNA methylome profiling of short-term stored sperm samples as outlined in Objective 3. Concurrently, we will continue integrative multi-omics data analysis associated with both Objective 2 and Objective 3. In addition to research activities, we will revise three manuscripts currently under review or revision and prepare and submit an additional manuscript based on our results from the omics data.
Impacts
What was accomplished under these goals?
Our team has made great progress on all three objectives. Parental phenotypes in male blue catfish impact on industry-relevant offspring performance traits using cryopreserved sperm (Objective 1): Sperm samples were collected from 44 males, and sperm kinematics and health indices were assessed post-cryopreservation. Thawed cryopreserved sperm were used to generate 44 families. Hatching success was recorded, and larvae from each family were reared in triplicate (n = 50/tank). Survival, weight, and morphometric data were collected from 0 to 40 days post-hatch (DPH). Larger males generally produced larger offspring at 40 DPH (R² ≥ 0.32, P ≤ 0.03) with higher survival rates (R² ≥ 0.23, P ≤ 0.05). Sperm kinematic traits were positively correlated with hatching success (R² ≥ 0.26, P ≤ 0.05). Significant maternal (VC = 23.2%, P = 0.002) and paternal(maternal) (VC = 32.1%, P < 0.0001) effects were observed for hatching success. Maternal effects remained significant (VC ≥ 20.8%, P < 0.001) across 0, 20, and 40 DPH for most morphometric traits, while paternal effects increased over time (VC ≥ 2.7%, P < 0.029) during early fry development. These findings indicate that sperm kinematic parameters are predictive of hatching success in hybrid catfish. Effect of short-term storage on sperm quality and hybrid hatch rate in blue catfish, Ictalurus furcatus (Objective 1): To evaluate the impact of low-temperature, short-term storage on sperm quality and associated hatch success, eight mature males were selected, and sperm were extracted from dissected testes and stored at 4 degrees Celsius. Assessments were conducted at 0, 24, 48, and 72 hours post-collection, measuring sperm swimming kinematics (including total motility, progressive motility, curvilinear velocity, and progressive curvilinear velocity), viability, oxidative stress, and microbial community composition. Results showed a progressive decline in sperm motility and viability, as well as embryo hatch rate, over the storage period. Concurrently, levels of malondialdehyde (MDA), a biomarker of oxidative stress, increased significantly within the first 24 hours, indicating early oxidative damage during refrigerated storage. These phenotypic data will be integrated with the multi-omics analyses outlined in Objective 3 to identify molecular correlates of sperm quality and offspring performance. Identification of DEGs in cryopreserved sperm samples between high hatch rate and low hatch rate samples (Objective 2): RNAseq was performed in cryopreserved sperm samples from 30 male blue catfish. After the quality control and low-quality bases and adapters removal, the clean reads were aligned to the blue catfish genome (GCA_023701845.2). The read counts were generated for calculating gene expression level. To identify the DEGs related to male reproductive performance, we selected four fish with high hatch rate as high hatch rate (HHR) group and four fish with low hatch rate as low hatch rate (LHR) group based on the hatch success rate of each fish collected in Objective 1. A total of 300 downregulated genes and 3550 upregulated genes were identified in LHR group compared to HHR group. Functional enrichment analysis was performed to understand the functions of these genes. Intriguingly, for KEGG pathway enrichment analysis, the upregulated genes were enriched in mitophagy, RNA degradation, and autophagy pathways, which are essential for maintaining cellular health, preventing damage accumulation, and ensuring the survival of the organism in response to various stressors. The upregulation of these genes implies that the sperm with LHR are likely to have undergone more severe damage during cryopreservation. In addition, for gene ontology (GO) term enrichment analysis, the upregulated genes were enriched in cilium-associated processes, which indicated that the low hatch rate sperm may undertake additional efforts to repair or compensate the damage caused by freezing. Validation of the relationship between cryopreserved sperm gene expression and hatch success of hybrid catfish fry (Objective 2): To understand the relationship between gene expression and hatch success rate in cryopreserved sperm, we calculated the Spearman correlation between the gene expression level of each DEG (LHR compared to HHR) and hatch success rates for all 30 groups of hybrid fry. Using a cutoff of r > 0.5 and log (counts per million) > 2, we got 57 out of 300 downregulated genes and 48 out of 3550 upregulated genes that showed a stronger correlation with hatch success rate. Among these, only 3 downregulated genes and 24 upregulated genes are annotated, including trpm4a, drc3, micos10, kmt2b, zbtb33, etc. Strikingly, the downregulated gene trpm4a showed a correlation of 0.667 with the hatch success rate. Trpm4a plays a critical role in regulating immune cell migration and cytokine production in T cells, upregulation of this gene indicates that the high hatch success group tends to have higher potential to protect cells against cryopreservation. Moreover, drc3 is an essential component of the nexin-dynein regulatory complex during spermatogenesis, playing a crucial role in sperm motility and fertility. The downregulation of drc3 may significantly impair sperm motility, potentially contributing to infertility in the LHR group. In addition, the upregulated genes kmt2b and zbtb33 are both involved in regulating DNA methylation, which can lead to abnormal gene expression in LHR group. To validate our RNA-seq data and confirm the correlation between the gene expression and hatch success rate, quantitative PCR (qPCR) was performed to measure the expression of 4 DEGs with a higher correlation to hatch success. 3 out of 4 DEGs showed consistent results, supporting the reliability of our findings. Development of a machine learning model for accurate prediction of hatch rate from 7 gene expression markers measured in cryopreserved sperm samples (Objective 2): To achieve the accurate prediction of hatch success rate, we employed the random forest regression model, which is a nonlinear model and leverages an ensemble of decision trees to improve predictive performance and reduce overfitting. A total of 12 gene markers with higher correlations to hatch success and 12 physiological parameters were used to train the model and generate the importance score of each feature. After filtering, the top 6 gene markers and the top 2 physiological markers with the highest importance score were selected for hatch success prediction. The Spearman correlation between predictive values and observed hatch success rate reached 0.767, indicating strong predictive accuracy. We have also evaluated models using either the top 7 gene markers or the top 7 physiological parameters for predicting hatch success rate, while the Spearman correlation between predictive and observed values was 0.699 and -0.051, respectively. These results suggest that combining gene markers with physiological parameters has a better predictive performance than using either type of marker alone. Our random forest regression model can serve as a predictive tool for hatch success rate prior to fertilization to optimize sperm selection and improve hatch success rate in the industry. RNA sequencing library preparation, sequencing and data analysis for sperm transcriptome under different short-term storage conditions (Objective 3): Nucleic acid was extracted from 48 sperm samples (8 fish x 3 short-term refrigerated storage durations x two replicates) and 72 larvae samples (8 fish x 3 post-hatch durations x 3 replicates) using Qiagen AllPrep DNA/RNA Kit. Sequencing library preparation is ongoing. EM-seq profiling experiments of sperm DNA methylome under different short-term storage conditions (Objective 3): High-quality DNA samples were extracted, and the sequencing library preparation is ongoing.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
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, 227(4), iyae090.
- Type:
Peer Reviewed Journal Articles
Status:
Published
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, 15, 1341555.
- Type:
Peer Reviewed Journal Articles
Status:
Under Review
Year Published:
2025
Citation:
Liyanage SSN, Bandara KA, Martin KA, Wood KR, Abernathy JW, Beck BH, Bruce TJ, Roy AL, Dunham RA, Wang X, Butts IAE (2025). Effect of short-term storage on sperm quality and microbiome dynamics in blue catfish, Ictalurus furcatus. Aquaculture, in revision.
- Type:
Peer Reviewed Journal Articles
Status:
Under Review
Year Published:
2025
Citation:
Wood KR, Zhang Y, Tackett VM, Martin KA, Liyanage SSN, Roy AL, Beck BH, Abernathy JW, Dunham RA, Wang X, Butts IAE (2025). Parental impacts on industry-relevant offspring performance traits using blue catfish, Ictalurid furcatus, cryopreserved sperm. Theriogenology, under review.
- Type:
Peer Reviewed Journal Articles
Status:
Under Review
Year Published:
2025
Citation:
Wood KR, Litvak MK, Liyanage SSN, Martin KA, Tackett VM, Dunham RA, Roy AL, Faulk C, Beck BH, Abernathy JW, Wang X, Butts IAE (2025). Sperm fatty acid composition as an indicator of quality and cryotolerance in blue catfish, Ictalurid furcatus. Theriogenology, THERIO-S-25-00402, in revision.
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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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