Progress 09/01/23 to 08/31/24
Outputs
Target Audience: The target audience was aquaculture farmers, government scientists, academics, graduate students, undergraduate students, and Extension specialists. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training activities:Students directly involved in this NIFA project (1 PhD and 3 MS) and graduate student helpers (1 PhD and 4 MS) in the Aquatic Reproductive Physiology Lab at Auburn University (PI: Butts) learned fundamental concepts in aquatic reproductive physiology, sperm biology, hatchery science, molecular biology, experimental design, and data analyses. Specifically, they gain hands-on training (one-on-one work mentorship) with a Computer Assisted Sperm Analysis (CASA) system to quantify sperm motility, velocity, and other motion parameters; flow cytometer and florescent microscope to quantify membrane viability and oxidative stress of sperm; molecular tools (i.e., nanodrop, qPCR) to extract RNA for gene expression of testes/sperm; and gamete cryopreservation suite (e.g., controlled-rate freezer, vapor pressure storage freezer) to freeze sperm and store samples in perpetuity. Students also learned how to maintain broodfish in ponds, inject fish, and fertilize eggs using artificial fertilization techniques. An undergrad student also learned aspects of fish husbandry and scientific sampling techniques. Professional development:MS students (×4) are now conducting individual studies for their thesis projects [chair: Butts (PI); committee members: Dunham, Roy (co-PIs)]. Studies include aquatic reproductive biology/physiology, molecular physiology, and cell biology of blue catfish and hybrid catfish (male blue catfish x female channel catfish). How have the results been disseminated to communities of interest?Findings have been presented to scientists, farmers, graduate students, and Extension specialists via peer-reviewed literature, and a national meeting (Aquaculture America). What do you plan to do during the next reporting period to accomplish the goals?Objective 1.0) Determine the minimum quantity of blue catfish sperm that is required to fertilize channel catfish eggs in order to consistently maximize hatching success under hatchery conditions. Objective 1.0 is now completed and published in the peer-reviewed journalAquaculture. This research won theBest Student Conference Abstract,1stPlace Student Oral Presentation (Student Spotlight Award), andThird Place Student Oral Presentation Awardat Aquaculture America. Objective 2.0) Investigate how seasonal and age-related variations impact blue catfish male reproductive performance indices. Blue catfish males have been sampled from 17 August 2021 to 7 February 2024 from ponds at the E.W. Shell Fisheries Center (Auburn University, Auburn, AL). Furthermore, 2 to 9-year-old males (~5 per year class) were sampled in May 2023 and 2 to 10-year-old males (~10 per year class) were sampled in May 2024 from the USDA. We will continue to conduct histology, sex-steroid hormone analyses, and ionic analyses. Sequencing data has been received and we are currently doing bioinformatic analyses. Two papers are currentlyIn Preparationfor this objective. Objective 3.0) Link blue catfish male reproductive performance indices to sperm quality (both fresh and cryopreserved) and offspring performance during early developmental stages to the first feeding stage. Data collection and analyses has been progressing (3 papers published) and other papers are currentlyIn Preparation. Study 1 - Impact of fresh and cryopreserved sperm quality on offspring performance has been published in the peer-reviewed journalAquaculture Study 2 - Molecular biomarkers for sperm velocity has been published in the peer-reviewed journalAquaculture Study 3 - Molecular biomarkers for sperm cryopreservation has been published in the peer-reviewed journalInternational Journal of Molecular Sciences Study 4 - A paper describing the morphometrics and ultrastructure of blue catfish sperm will be submitted in 2025 Study 5 - Effect of short-term storage on sperm quality and microbiome dynamics in blue catfish will be submitted in 2025 Study 6 - A paper investigating paternal impacts on industry-relevant offspring performance traits in blue catfish will be submitted in 2025 Study 7:Identification and differential expression of small non-coding RNAs in testes and extracted testicular sperm of blue catfish will be submitted in 2025. Extension and education activities: We will continue to assist with technology transfer and information dissemination to the U.S. catfish industry and academic community.
Impacts
What was accomplished under these goals?
Impact of project: Catfish farming accounts for ~60% of total U.S. finfish aquaculture production, where nearly 60% of the harvest is composed of female channel catfish × male blue catfish hybrid. Although the hybrid industry has experienced growth, there are challenges associated with reproduction between parent species. Foremost, blue catfish males reach maturity after 4 to 7 years and their sperm cannot be readily extracted by stripping. Consequently, sperm collection for blue catfish is a lethal procedure, requiring removal and maceration of testes. Since milt can only be acquired once per male there is a substantial investment in sperm. In contrast, female channel catfish reach sexual maturity earlier, and their eggs can be readily hand-stripped forin-vitrofertilization. These complications suggest that the male contribution of sperm production and quality is a bottleneck for the hybrid catfish industry. We are using basic/applied research to gain a better understanding of biological and physiological mechanisms underlying reproduction. Objective 1.0)Determine the minimum quantity of blue catfish sperm required to fertilize channel catfish eggs to consistently maximize hatching success. Objectives were to determine the minimum quantity of frozen-thawed sperm required to maximize hatching success. Eggs from 18 females were collected and fertilized using fresh and cryopreserved sperm from 36 males at 10 sperm-to-egg ratios (range from 1.0×104to 9.0×105sperm/egg). Embryos sired with fresh and frozen-thawed sperm were incubated under common conditions. Hatching success using cryopreserved sperm was high but dependent on sperm-to-egg ratio. For instance, at ratios of 1.0×104to 5.0×104sperm per egg, hatch increased from 18.0% ± 8.5 to 43.9% ± 8.2. Adding >5.0×104sperm per egg had no effect on hatching success. Data improve understanding of frozen-thawed sperm quality for blue catfish to improve reproductive sustainability and reduce production costs associated with housing males. Objective 2.0)Investigate how seasonal and age-related variations impact blue catfish.Study 1- Impact of seasonality and age (y 2-5):In 2021, 986 blue catfish males (2 years old) were transferred from a hatchery to Auburn, where they were acclimated in ponds. Fish from 2021 have been sampled (17 times) from 17 August 2021 to 7 February 2024. At each sampling point, we recorded fish body mass and length. Blood was collected for sex steroid analyses and ionic analyses. Fish were then dissected, and testes removed, weighed, and flash-frozen for mRNA sequencing or placed in formalin for histological assessments. Gonadosomatic index (GSI) and Fulton's condition (K) will be calculated. Sex steroid hormone, blood ionic analyses, and histology analyses are ongoing. mRNA sequencing has been completed and bioinformatic analyses is underway.Study 2 - Impact of age (y 2-9): In May 2023 and 2024 (spawning season), blue catfish males from 8 age classes (2 to 10 years old) were collected from USDA. We recorded the total body mass and fork length of each male. Blood was also collected for steroid and ionic analyses. Males were dissected, and testes removed, weighed, and flash-frozen for mRNA sequencing or placed in formalin for histology. GSI and K will be calculated. Testis were also processed, and sperm samples were collected for kinematic analyses using Computer Assisted Sperm Analyses (CASA). Sex steroids, histology, and CASA analyses are ongoing. mRNA sequencing has been completed and bioinformatic analyses are underway.Preliminary results for Study 1/2 show seasonal and age-related variations in key male reproductive indices, which will help us optimize male catfish reproduction. Objective 3.0)Link blue catfish male reproductive performance indices to sperm quality and offspring performance. Study 1: Impact of fresh and cryopreserved sperm quality on offspring andStudy 2: Molecular biomarkers of sperm velocity.Characterized important sperm traits and ranked blue catfish males based on sperm velocity. Of the 43 males characterized, testicular tissue from 4 individuals with low velocity and 4 with high velocity were sequenced for sRNAs and mRNAs.No differentially expressed sRNAs were detected between the two performance groups. However, we identified two differentially expressed mRNA transcripts between low and high velocity groups,aqp3aandly97.3, both upregulated in the high velocity group compared to the low velocity group.These biomarkers of sperm quality can now be applied for more efficient aquaculture. Study 3: Biomarkers for cryopreservation.Investigated expression changes caused by cryopreservation using transcriptome profiles of fresh and cryopreserved sperm.Cryopreservation increased oxidative stress levels and DNA fragmentation and reduced sperm kinematics. Coinciding with these changes, RNA-seq identified 849 upregulated genes after cryopreservation, including members of all five complexes in the mitochondrial electron transport chain. This suggests a boost in oxidative phosphorylation activities, which often lead to excessive production of reactive oxygen species associated with cell death. Interestingly, functional enrichment analyses revealed compensatory changes in gene expression after cryopreservation to offset the detrimental effects of ultra-cold storage.Together, these results provide insight into molecular mechanisms of sperm cryoinjury.Study 4 - Morphometrics and ultrastructure of blue catfish sperm. We did the first complete assessment of sperm morphology using SEM and TEM. Study 5: Sperm microbiome. Sperm from 8 blue catfish males were held in the refrigerator at 4oC. Sperm were then sampled at 0 (fresh), 24-, 48-, and 72-hours post-storage for CASA and microbiome.Sperm VCL decreased over the experimental period. The most common bacterial genera are presented, showing the highest male-to-male variation in microbial diversity in the fresh samples. Shannon index significantly declined from 0 to 24 hours and remained unchanged from 48 to 72 hours. However, the genusPseudomonasshowed higher abundances at 48 and 72-hours post-storage, which coincided with decreases in sperm VCL.Together, these results link residing sperm quality to microbiome structure. Study 6: Paternal effects.In 2023, blue catfish males were seined from ponds. Body metrics were recorded and blood was collected for steroid measurements. Testes were dissected, weighed, and processed for histology, milt extraction, and sperm density. For each male, an aliquot of milt was cryopreserved, while another aliquot was used for evaluation of fresh sperm quality and IVF. Kinematic properties and health of sperm are being analyzed. For IVF trials, sperm from 14-15 unique males fertilized eggs from 3 females to form a series of 44 hybrid families. Hatching success was determined. At peak hatch, and again at 20 and 40 days post-hatch, ~24-36 fry were sampled from each family for body area measurements and deformities. We are now processing data. Data will create innovative tools that predict male sperm quality and offspring performance. Study 7: sRNA in sperm and testes.Objectives were to characterize miRNAs in sperm and testis tissue and examine inter-individual variation in expression of sRNAs between males. Using mature males, we detected 174known mature miRNAs in testes tissue. Additionally, we found 5 novel miRNAs unique to sperm, with the remainder found also in testis tissue, therefore showing low tissue specificity of sRNA between these two tissue types, despite sperm expressingmorepiwi-interacting RNAthan in testis.This study describes sRNA profiles inmale gonadal tissue and gametes, including core miRNAs that should be further explored as sperm biomarkers.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Butts, I.A.E., Montague, H.R., Hess, H., Barzegar-Fallah, S., Martin, K.A., Liyange, S.S.N., Wood, K.R., Wang, X., Bruce, T., Abernathy, J.W., Roy, L.A., Dunham, R.A. 2024. Quantifying male reproductive performance to improve hatchery production of hybrid catfish. Aqua2024. Copenhagen, Denmark
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Martin, K.A., Abernathy, J.W., Barzegar-Fallah, S., Dunham, R.A., Liyange, S., Roy, L.A., Tackett, V.W., Wood, K.R., Butts, I.A.E. 2004. Acute thermal stress impacts sperm quality in blue catfish, Ictalurus furcatus. Aquaculture America 2024. San Antonio, TX
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Butts, I.A.E., Dunham, R, Roy, L., Kjelland, M.E. 2023. Quantifying male reproductive performance to improve hatchery production of hybrid catfish. Annual Animal Reproduction Program Project Director Meeting. Ottawa, Canada
|
Progress 09/01/22 to 08/31/23
Outputs
Target Audience: The target audience was aquaculturefarmers, government scientists, academics, graduate students, undergraduate students, and extension specialists. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Training activities - MS students (×4) in the Aquatic Reproductive Physiology Lab at Auburn University (PI: Butts) learned fundamental concepts in aquatic reproductive biology/physiology, sperm biology, hatchery science, experimental design, and data analyses. Specifically, they gain hands-on training (one-on-one work with a mentor) with a Computer Assisted Sperm Analysis (CASA) system to quantify sperm motility, velocity, and other motion parameters; flow cytometer and florescent microscope to quantify membrane viability and oxidative stress of sperm; molecular tools (i.e., nanodrop, qPCR) to extract RNA for gene expression of testes/sperm; and gamete cryopreservation suite (e.g., controlled-rate freezer, vapor pressure storage freezer) to freeze sperm and store samples in perpetuity (i.e., creating our germplasm repository). Students also learned how to maintain broodfish in ponds, inject fish, and fertilize eggs using artificial fertilization techniques. An undergrad student also learned aspects of fish husbandry and sampling techniques. Professional development - MS students (×4) are now conducting individual studies for their thesis projects [chair: Butts (PI); committee members: Dunham, Roy (co-PIs)], while another MS student defended in May 2023 (won national, college level, and departmental awards for her research). Studies include aquatic reproductive biology/physiology, molecular physiology, and cell biology of catfishes. How have the results been disseminated to communities of interest?Findings have been presented to scientists, farmers, graduate students, and extension specialists via peer-reviewed literature, and a national meeting (Aquaculture America). What do you plan to do during the next reporting period to accomplish the goals?Objective 1.0) Determine the minimum quantity of blue catfish sperm that is required to fertilize channel catfish eggs in order to consistently maximize hatching success under hatchery conditions. Objective 1.0 is now completed and published in the peer-reviewed journalAquaculture. This research won theBest Student Conference Abstract,1stPlace Student Oral Presentation(Student Spotlight Award), andThird Place Student Oral Presentation Awardat Aquaculture America. Objective 2.0) Investigate how seasonal and age-related variations impact blue catfish male reproductive performance indices. Blue catfish males are currently being reared at the E.W. Shell Fisheries Center (Auburn University, Auburn, AL) in aquaculture ponds. Males will continue to be sampled until early 2024. We will continue to conduct histology and sex-steroid hormone analyses. Samples will also be processed for blood ions. Samples for Study 1 [Impact of seasonality and age (yrs. 2-5)] andStudy 2 [Impact of age (yrs. 2-9)] will be shipped in November-December 2023 for RNA sequencing. Bioinformatic analyses will start in early 2024. Data will be submitted for 2 peer-reviewed publications. Objective 3.0) Link blue catfish male reproductive performance indices to sperm quality (both fresh and cryopreserved) and offspring performance during early developmental stages to the first feeding stage. Study 1-Impact of fresh and cryopreserved sperm quality on offspring performancehas been published in the peer-reviewed JournalAquaculture Study 2-Impact of reproductive performance traits on offspring performance: Statistical analyses are underway. This work will be submitted for peer review in Spring 2024. Study 3-Molecular biomarkers for sperm qualityhas been published in the peer-reviewed JournalAquaculture Study 4-Biomarkers for sperm quality and offspring performance: MS students (×2) will continue to process samples in 2023/2024. Thereafter,male reproductive performance indices will be linked to fresh and cryopreserved sperm kinematics (motility, velocity) and offspring performance (embryonic survival, hatch success, and fry morphometry/survival) using polynomial regression and multivariate statistics. Extension and education activities: We will continue toassist with technology transfer and information dissemination to the U.S. catfish industry and academic community.
Impacts
What was accomplished under these goals?
Impact of project: Catfish farming accounts for ~70% of total U.S. freshwater aquaculture production; 68% of the harvest is composed of female channel catfish× male blue catfish hybrid. Although the hybrid industry has experienced growth, there are challenges associated with reproduction between parent species. Foremost,blue catfish males reach maturity after 4 to 7 y and their sperm cannot be readily extracted by stripping. Consequently, sperm collection for blue catfish is a lethal procedure, requiring removal and maceration of testes. Since milt can only be acquired once per male there is a substantial investment in sperm. In contrast, female channel catfish reach sexual maturity earlier, and their eggs can be readily hand-stripped forin-vitrofertilization. These complications suggest that the male contribution of sperm production and quality is a bottleneck for the hybrid catfish industry. We are using basic/applied research to gain a better understanding of biological and physiological mechanisms underlying reproduction. Objective 1.0)Determine the minimum quantity of blue catfish sperm required to fertilize channel catfish eggs to consistently maximize hatching success.(1.1) Major activities completed: Objectives were to determine the minimum quantity of frozen-thawed sperm required to maximize hatching success. Eggs from 18 females were collected and fertilized using fresh and cryopreserved sperm from 36 males at 10 sperm-to-egg ratios (range from 1.0×104to 9.0×105sperm/egg). (1.2) Data collected: Embryos sired with fresh and frozen-thawed sperm were incubated under common conditions. (1.3) Summary of results: Hatching success using cryopreserved sperm was highbut dependent on sperm-to-egg ratio. For instance, at ratios of 1.0×104to 5.0×104sperm per egg, hatch increased from 18.0% ± 8.5 to 43.9% ± 8.2. Adding>5.0×104sperm per egg had no effect on hatching success. (1.4) Key outcomes: Data improve understanding of frozen-thawed sperm quality for blue catfish to improve reproductive sustainability and reduce production costs associated with housing males. Objective 2.0) Investigate how seasonal and age-related variations impact blue catfish Study 1- Impact of seasonality and age (y 2-5): (2.1.1) Major activities completed:In 2021, 986 blue catfish males (2 y) were transferred from a hatchery to Auburn, where they were acclimated in ponds. In 2022, ~400 more males were transferred from a hatchery. (2.1.2) Data collected: Fish from 2021 have been sampled 14 times over 32 months and fish from 2022 have been sampled 11 times over 20 months. At each sampling point, we record fish body mass and length. Blood is collected for sex steroid analyses. Fish are then dissected, and testes removed, weighed, and flash-frozen for mRNA sequencing or placed in formalin for histological assessments. Gonadosomatic index (GSI) and Fulton's condition (K) will be calculated. (2.1.3) Summary statistics and discussion of results and (2.1.4) Key outcomes: Fish care is being done daily. Sex steroid hormone and histology analyses are ongoing. Study 2- Impact of age (y 2-9): (2.2.1) Major activities completed:In May 2023 (spawning season), 35 blue catfish males from 7 age classes (2 to 9 y) were collected from USDA.(2.2.2) Data collected: We recorded total body mass and fork length of each male. Blood also collected for steroid analyses. Males were dissected, and testes removed, weighed, and flash-frozen for mRNA sequencing or placed in formalin for histology. GSI and K will be calculated.Testis was also processed, and sperm samples collected for kinematic analyses using Computer Assisted Sperm Analyses (CASA).(2.2.3) Summary statistics and discussion of results and (2.2.4) Key outcomes: Sex steroids, histology, and CASA analyses are ongoing. Objective 3.0) Link blue catfish male reproductive performance indices to sperm quality and offspring performance Study 1- Impact of sperm quality on offspring. (3.1.1) Major activities completed: Compare sperm kinematics and sperm health metrics before and after cryopreservation and decipher how offspring development is affected when eggs are sired with fresh and frozen-thawed sperm. (3.1.2) Data collected: Fresh and cryopreserved sperm kinematics (n = 36 males) were evaluated by CASA, while health metrics were assessed. Eggs from 3 females were collected and fertilized using fresh and cryopreserved sperm from 9 males. Sired embryoswere then incubated under common environmental conditions and growth performance documented over 40 days. (3.1.3) Summary statistics and discussion of results: Kinematic traits and health metrics declined after cryopreservation. Despite this decrease, hatching success using cryopreserved sperm was high. There were generally no differences in morphology or deformity rates in offspring sired with cryopreserved sperm, compared to a fresh control. (3.1.4) Key outcomes: These data improve understanding of frozen-thawed sperm quality for catfish. Study 2- Impact of reproductive performance traits on offspring. (3.2.1) Major activities completed and(3.2.2) Data collected: Objective was to determine correlations between male (n = 83) reproductive performance traits (body size, testes morphology, sex steroids)and sperm kinematics utilizing fresh and frozen-thawed sperm. (3.2.3) Summary statistics and discussion of results: Univariate and multivariate analyses are underway. So far, preliminary results suggest that blue catfish male reproductive performance traits correlated to fresh and post-thaw sperm kinematic traits when using individual correlations. (3.2.4) Key outcomes or other accomplishments realized: Together, these data will advance our understanding of assisted reproduction to improve reproductive efficiency for hatcheries and enhance living and cryopreserved gamete banks for genetic breeding and production. Study 3-Molecular biomarkers for sperm quality.(3.3.1) Major activities completed and(3.3.2) Data collected: To establish gene expression biomarkers of sperm quality, we characterized sperm traits, ranked blue catfish males based on the velocity of sperm, and sequenced testicular tissue for mRNAs and sRNAs. (3.3.3) Summary statistics and discussion of results: Of the 43 males characterized, testicular tissue from 4 individuals with low-velocity and 4 with high-velocity were chosen for sequencing. No differentially expressed sRNAs were detected between the sperm groups. However, we identified two differentially expressed mRNA transcripts between the low-velocity and high-velocity groups,aqp3aandly97.3, both upregulated in the high-velocity group compared to the low-velocity group. (3.3.4) Key outcomes: These biomarkers of sperm quality in testicular tissue are linked to gamete quality and be applied for efficient catfish aquaculture. Study 4-Biomarkers for sperm quality and offspring performance.(3.4.1) Major activities completed and (3.4.2) Data collected: In 2023,bluecatfish males (n = 44, 4-5 yrs.) were seined from ponds. Body metrics were recorded and blood collected for steroid measurements.Testes were dissected, weighed, and processed for histology, milt extraction, and sperm density. For each male, an aliquot of milt was cryopreserved, while another aliquot was used for evaluation of fresh sperm quality andIVF.Kinematic properties and health of sperm are being analyzed. For IVF trials,sperm from 14-15 unique males fertilized eggs from 3 females to form a series of 44 hybrid families. Hatching success was determined. At peak hatch, and again at 20 and 40 days post-hatch, ~24-36 fry were sampled from each family for body area measurements and deformities. (3.4.3) Summary statistics and discussion of resultsand(3.4.4) Key outcomes: We are now processing data. Data will create innovative tools/models that predict male sperm quality and offspring performancefor hatchery production.?
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Montague, H.R., Hess, H., Tackett, M., Britt, S.L., Lawson, L.L., Aguilar, G., Jenkins, L.E., Roy, L., Holland, L.W., Litvak, M.K., Dunham, R., Butts, I.A.E. 2023. In vitro fertilization with frozen-thawed blue, Ictalurus furcatus, sperm and implications for gene banking. Aquaculture 574, 739611
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
Montague, H.R., Tackett, V.M., Lawson, L.L., Roy, L., Dunham, R., Butts, I.A.E. 2023. Proof of concept: Frozen sperm shows promise for catfish hatchery production. Alabama Fish Farming Center - Fish Farming News 2023(1): 6-7
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Montague, H.R., Hess, H., Tackett, V.M., Britt, S., Lawson, L., Aguilar, G.L., Roy L.A., Dunham, R.A., Litvak, M.K., Butts, I.A.E. 2023. Outcomes of in vitro fertilization with frozen-thawed blue catfish sperm and implications for gene banking. Aquaculture America 2023. New Orleans, LA.
Note: Research completed by MSc student (Won best conference abstract & 1st Place Oral Presentation - Student Spotlight Award & Third Place Oral Presentation Award)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Barzegar-Fallah, S., Montague, H.R., Dunham, R.A., Hess, H., Jenkins, L., Paeniska, M., Litvak, M.K., Tackett, V.M., Gill, H., Butts, I.A.E. 2023. Effects of morphometrics and ultrastructure on swimming kinematics of blue catfish Ictalurus furcatus sperm. Aquaculture America 2023. New Orleans, LA
Note: Research completed by MS student (Won Second Place Poster Presentation Award winner)
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2024
Citation:
Hess, H., Strader, M.E., Montague, H.R., Bruce, T.J., Aguilar, G., S�rensen, S.R., Jenkins, L., Roy, L., Dunham, R., Butts, I.A.E. 2024. Molecular biomarkers for sperm quality in an economically important fish: Blue catfish, Ictalurus furcatus. Aquaculture 579, 740220
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2023
Citation:
Montague, H.R. 2023. Quantifying male reproductive performance of blue catfish (Ictalurus furcatus) to improve hatchery production of hybrid catfish. MS thesis. Auburn University. School of Fisheries, Aquaculture and Aquatic Sciences
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2022
Citation:
Hess, H. 2022. Identification of molecular biomarkers for male reproductive success in blue catfish (Ictalurus furcatus). MS thesis. Auburn University. School of Fisheries, Aquaculture and Aquatic Sciences
|
Progress 09/01/21 to 08/31/22
Outputs
Target Audience: The target audience was farmers (especially those working at hatcheries),government scientists, academics,graduate students, undergraduate students, and extension specialists. Changes/Problems:Unfortunately, in Nov 2022 we detected our second outbreak ofIchthyophthirius multifiliis, which is often termed to as "ich". Fish are still available to completeObjective 2.0.? What opportunities for training and professional development has the project provided?Training activities - MS students (×3) in theAquatic Reproductive Physiology Lab at Auburn University(PI: Butts) learned fundamental concepts in aquatic reproductive biology/physiology, sperm biology, hatchery science, experimental design, and data analyses. Specifically, they gain hands-on training (one-on-one work with a mentor) with a Computer Assisted Sperm Analysis (CASA) system to quantify sperm motility, velocity, and other motion parameters; flow cytometer and florescent microscope to quantify membrane viability and oxidative stress of sperm; molecular tools (i.e., nanodrop, qPCR) to extract RNA for gene expression of testes/sperm; and gamete cryopreservation suite (e.g., controlled-rate freezer, vapor pressure storage freezer) to freeze sperm and store samples in perpetuity (i.e., creating our germplasm repository). Students also learned how to maintain broodfish in ponds, inject fish, and fertilize eggs using artificial fertilization techniques. An undergrad student also learned aspects of fish husbandry and sampling techniques. Professional development -MS students (×2) are now conducting individual studies for their thesis projects [chair: Butts (PI); committee members: Dunham, Roy (co-PIs)], while another MS student defended in May 2022. Studies include aquatic reproductive biology/physiology, molecular physiology, and cell biology of catfishes. How have the results been disseminated to communities of interest?Findings have been presented toscientists, farmers, graduate students, and extension specialists via peer-reviewed literature, a national meeting (Aquaculture America), and university symposia. What do you plan to do during the next reporting period to accomplish the goals?Objective 1.0) Determine the minimum quantity of blue catfish sperm that is required to fertilize channel catfish eggs in order to consistently maximize hatching success under hatchery conditions. Results will be submitted for peer review and presented at Aquaculture America. Objective 2.0) Investigate how seasonal and age-related variations impact blue catfish male reproductive performance indices. Blue catfish males are currently being reared at the E.W. Shell Fisheries Center (Auburn University, Auburn, AL) in aquaculture ponds. Males will continue to be sampled throughout the annual reproductive cycle [pre-spawning (January, March); peak-spawning (May, June); post-spawning (August, October)] to survey a wide range of developmental stages, from immature to maturegonads. This sampling will continue until 2024. We will continue to conduct histology and sex-steroid hormone analyses. Objective 3.0) Link blue catfish male reproductive performance indices to sperm quality (both fresh and cryopreserved) and offspring performance during early developmental stages to the first feeding stage. In May 2023, another cohort of mature blue catfish males (n = ~50) will be sampled for reproductive performance indices, including body metrics; testes morphology;sex steroid hormones; miRNA dynamics (on testes/sperm); and "select"sperm traits (on fresh and cryopreserved sperm), such as density, morphology, activity (motility/velocity), cell health, and DNA damage. MS students(×2)will continue to process the samples 2021 samples. Thereafter, we will use all thesereproductive performance indices to assess if they can accurately forecast offspring performance.miRNAs will also be sequenced to compare gene expression between testes and sperm. Finally,bioinformatic analyses will be conductedto decipher a potential molecular mechanism for sperm quality.
Impacts
What was accomplished under these goals?
Impact of the project: Catfish farming accounts for about 75% of total U.S. finfish aquaculture production, in which the channel catfish female by blue catfish male hybrid constitutes nearly 60% of the harvest.The major bottleneck for hybrid catfish breeding is high-quality sperm production as blue catfish reach sexual maturity in 4 to 7 years, and sperm is collected through a lethal testis removal procedure.The fact that semen can only be stripped once from these males requires a substantial economic investment in sperm. In contrast, female channel catfish reach sexual maturity earlier (after 3 years), and their eggs can be readily hand-stripped for artificial fertilization, allowing the animal to survive and provide eggs during the next spawning season. These paternal complications suggest that sperm production, in terms of quality/quantity, is a major bottleneck for hybrid catfish embryo production,making healthy sperm more valuable than eggs in reproduction.As a result, we are currently using basic and applied research to gain a better understanding of critical biological and physiological mechanisms underlying reproduction. Accomplishments for each specific objective are highlighted below: Objective 1.0)Determine the minimum quantity of blue catfish sperm that is required to fertilize channel catfish eggs to consistently maximize hatching success under hatchery conditions. (1.1)Major activities completed / experimentsconducted- Eggs from 18 females were crossed with the sperm from 2 different males, resulting in a total of 36 parental half-sibling families. Cryopreserved milt from 36 males was thawed at 40ºC for 20 s before it was transferred into 1.5 mL tubes. In 2021, sperm were diluted to obtain five sperm-to-egg ratios: 9.0×104, 1.0×105, 2.3×105, 4.5×105, and 9.0×105, and five sperm-to-egg ratios in the 2022 season: 1.0×104, 2.5×104, 5.0×104, 7.5×104, 1.0×105. Fresh milt was extracted from males less than 12 h before fertilization occurred. All males were tested for sperm quality and stored at 4ºC. A fresh control ratio of 1.0×104sperm per egg was used. The final volume for fresh and cryopreserved sperm was standardized to 1.0 mL using HBSS with 3 replicates for each ratio. Aliquots of 250 ± 10 eggs were weighed according to each female's (n = 19) specific egg density. They were then transferred to 80 mL plastic cups lined with vegetable extract. Sperm solutions for each ratio were pipetted directly onto the eggs and allowed to sit for 2 min. Each cup was then activated with 5 mL of hatchery water. Eggs were allowed to incubate with the sperm in aFuller's Earth solution for 3 to 5 min with continuous manual stirring. Stirring ensured sufficient gamete contact and samples were then transferred into incubation aquaria. Hatch success was determined when there was >50% hatch.(1.2) Data collected: Hatch success was calculated as the total number of hatched larvae divided by the initial number of eggs in each basket (250 ± 10 eggs)multiplied by 100, to get a hatching percentage.(1.3) Summary statistics and discussion of results and (1.4) Key outcomes or other accomplishments realized: Mean hatching success, using cryopreserved sperm at ratios of 1.0×104to 9.0×105sperm per egg were 24.3%±6.6 and 53.4%±5.0, respectively. Sperm to egg ratio significantly influenced hatching success, where the total number of hatched yolk-sac fry increased at ratios ranging from 1.0×104to 5.0×104sperm per egg. Thereafter, adding greater than 5.0×104sperm per egg had no significant effect on hatching success. Results showed no significant difference in hatching success when eggs were sired with either fresh or frozen-thawed sperm. Objective 2.0) Investigate how seasonal and age-related variations impact blue catfish male reproductive performance indices. (2.1) Major activities completed / experiments conducted-In March 2021,986 immature blue catfish males (2 years of age) were transferred from a commercial hatchery to Auburn, where they were acclimated in ponds. In March of 2022, ~400 more immature blue catfish males were transferred from a commercial hatchery.(2.2)Data collected-Fish from 2021 have been sampled 9 times over 18 months and fish from 2022 have been sampled 5 times over 10 months. At each sampling point, we record the total body mass and fork length of each fish. Blood is also collected for sexsteroid hormone analyses.These fish are then dissected, and testes removed, weighed, and flash-frozen in liquid nitrogen for mRNA sequencing or placed in formalin for histological assessments. Gonadosomatic index (GSI) and Fulton's condition factor will be calculated.(2.3) Summary statistics and discussion of resultsand(2.4) Key outcomes or other accomplishments realized- Fish care is being done daily. In addition, sexsteroid hormone and histology analysesare ongoing. Objective 3.0) Link blue catfish male reproductive performance indices to sperm quality (both fresh and cryopreserved) and offspring performance during early developmental stages to the first feeding stage. (3.1) Major activities completed / experiments conducted- In March of 2022,mature blue catfish males were transferred from a commercial hatchery to the E.W. Shell Fisheries Center at Auburn University.(3.2)Data collected-Body metrics were recorded, and blood was collected forsex steroid hormone measurements.Testes were dissected, weighed, and processed for milt extraction, and sperm density quantification using a hemocytometer. Samples were also taken for histological assessment. For each male, an aliquot of milt was then cryopreserved using our controlled-rate freezer and cryogenic storage suite, while another aliquot was used for the evaluation of fresh sperm quality. Sperm quality (both fresh and cryopreserved) was then assessed, which includedkinematic properties,health assessment, and RNA analyses. Finally, embryos were sired with fresh and frozen-thawed sperm and then incubated under common environmental conditions where growth performance was documented over 40 days post-hatch. Using these data, we aimed to (A) compare sperm swimming kinematics and sperm health metrics before and after cryopreservation; (B) investigate expression changes caused by cryopreservation using transcriptome profiles of fresh and cryopreserved sperm; (C) decipher how early offspring development is affected when eggs are sired with fresh and frozen-thawed sperm; (D) compare how secondary sexual characters relate to sperm quantity and quality; (E) link sperm form to function.(3.3) Summary statistics and discussion of resultsand(3.4) Key outcomes or other accomplishments realized- (A) Generally, sperm kinematic traits and health metrics declined after cryopreservation. (B) Sperm quality measurements revealed that cryopreservation significantly increased oxidative stress levels and DNA fragmentation, and reduced sperm kinematic parameters. We identified 849 upregulated genes after cryopreservation, including members of all five complexes in the mitochondrial electron transport chain, suggesting a boost in oxidative phosphorylation activities, which often lead to excessive production of reactive oxygen species (ROS) associated with cell death. Functional enrichment analyses revealed compensatory changes in gene expression after cryopreservation to offset detrimental effects of ultra-cold storage.(C) There were no differences in morphology or rate of deformities in offspring sired with frozen-thawed sperm when compared to a fresh control. (D-E) At present, secondary sexual characteristics are being related to sperm quantity/quality and sperm form link to function.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Wang, H., Montague, H.R., Hess, H.N., Zhang, Y., Aguilar, G.L., Dunham, R.A., Butts, I.A.E*., Wang, X*. 2022. Transcriptome analysis reveals key gene expression changes in blue catfish sperm in response to cryopreservation. International Journal of Molecular Sciences 23, 7618. https://doi.org/10.3390/ijms23147618 (Impact Factor = 6.208) *Shared authorship
- Type:
Other
Status:
Published
Year Published:
2022
Citation:
Montague, H.R*., Hess, H., Tackett, V.M., Britt, S., Lawson, L., Aguilar, G.L., Roy L.A., Dunham, R.A., Litvak, M.K., Butts, I.A.E. 2022. Outcomes of in vitro fertilization with frozen-thawed blue catfish sperm and implications for gene banking. College of Agriculture Graduate Research Poster Showcase, Auburn, AL
*NOTE: Research completed by my MSc student (Won best MSc student poster for Focus Area: Fisheries and Aquaculture and Aquatic Environment
- Type:
Other
Status:
Published
Year Published:
2022
Citation:
Barzegar-Fallah, S., Montague, H.R., Dunham, R.A., Hess, H., Jenkins, L., Paeni?ka, M., Litvak, M.K., Tackett, V.M., Gill, H., Butts, I.A.E. 2022. Effects of morphometrics and ultrastructure on swimming kinematics of blue catfish Ictalurus furcatus sperm. College of Agriculture Graduate Research Poster Showcase, Auburn, AL
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Hess, H., Strader, M.E., Myers, J., He, F., Roy, L.A., Dunham, R.A., Butts, I.A.E. 2022. Identification and differential expression of small non-coding RNAs in testes and sperm of blue catfish, Ictalurus furcatus. Aquaculture 2022. San Diego, CA
- Type:
Other
Status:
Other
Year Published:
2022
Citation:
Seminar for The University of Alabama at Birmingham Biology Department (2022). Title: Creating a fertility clinic for aquatic species in the Southeastern United States.
- Type:
Other
Status:
Other
Year Published:
2022
Citation:
Seminar for Savannah State University Department of Marine and Environmental Sciences (2022). Title: Reproductive physiology and hatchery science for aquatic species in the Southeastern US.
- Type:
Other
Status:
Published
Year Published:
2022
Citation:
Hess, H., Strader, M.E., Myers, J.N., He, F., Roy, L., Su, B., Dunham, R., Butts, I.A.E. 2022. Identification and differential expression of small non-coding RNAs in testes and sperm of blue catfish (Ictalurus furcatus). Auburn Research: Student Symposium 2022. Auburn, AL
|
Progress 09/01/20 to 08/31/21
Outputs
Target Audience:Target audience was scientists, farmers, graduate students, undergraduate students, and extension specialists. Efforts: Ian Butts (PI) gave an overview of his research (title:Aquatic Reproductive Physiology and Hatchery Science) to the College of Agriculture at Auburn University at the Spring Virtual Colloquium. This presentation highlighted this USDA-NIFA funded grant. Changes/Problems:On 24 March 2021, 986 immature blue catfish males (average weight was 0.77 kg, 2 years of age) were transferred from a commercial hatchery to the E.W. Shell Fisheries Center (Auburn University, Auburn, AL). Unfortunately, in April 2021 we detected our first mortalities due to Ichthyophthirius multifiliis, which is often termed to as "ich". At present, we have 270 survivors, which does not give much "leeway" if we experience another major mortality event. As such, we are working with the catfish industry to get additional males at appropriate age classes. What opportunities for training and professional development has the project provided?Training activities - MS students (×2) in the Aquatic Reproductive Physiology Lab at Auburn University (PI: Butts) learnt fundamental concepts in aquatic reproductive biology/physiology, sperm biology, hatchery science, experimental design, and data analyses. Specifically, they gain hands-on training (one-on-one work with a mentor) with a Computer Assisted Sperm Analysis (CASA) system to quantify sperm motility, velocity, and other motion parameters; flow cytometer and florescent microscope to quantify membrane viability and oxidative stress of sperm; molecular tools (i.e., nanodrop, qPCR) to extract RNA for gene expression of testes/sperm; and gamete cryopreservation suite (e.g., controlled-rate freezer, vapor pressure storage freezer) to freeze sperm and store samples in perpetuity (i.e., creating our germplasm repository). Students also learned how to maintain broodfish in ponds, inject fish, and fertilize eggs using artificial fertilization techniques. An undergrad student also leant aspects of fish husbandry and sampling techniques. Professional development - MS students (×2) are now conducting individual studies for their thesis projects [chair: Butts (PI); committee members: Dunham, Roy (co-PIs)]. This includes studies on aquatic reproductive biology/physiology, molecular physiology, and cell biology of catfishes. How have the results been disseminated to communities of interest?In collaboration with Dr. David Cline (Associate Extension Professor in SFAAS) we created a 1-minute YouTube video which overviews our research on sperm physiology and cryobanking. The video can be viewed at: https://www.youtube.com/watch?v=5ZR34k3m_w4 and https://www.youtube.com/watch?v=IKj3MoxOjcI What do you plan to do during the next reporting period to accomplish the goals?Objective 1.0) Determine the minimum quantity of blue catfish sperm that is required to fertilize channel catfish eggs in order to consistently maximize hatching success under hatchery conditions. More artificial fertilization trials will be conducted in May-June 2022. Here, eggs from several females (n = 4-5) will be fertilized with cryopreserved sperm at lower sperm to egg ratios (2.5 × 104:1, 5.0 × 104:1, 7.5 × 104:1, 1.0 × 105:1) and a fresh control. Eggs/embryos will then be transferred to an indoor hatchery with a recirculation aquaculture system (RAS) and held below the water surface in mesh screen baskets incubated within larger aquaria. Dead eggs will be removed daily, and hatch success will be determined upon termination of the experiment and calculated as the total number of hatched fry divided by the initial number of eggs. Data from 2021 and 2022 will be analyzed using mixed-model factorial ANOVA models. Objective 2.0) Investigate how seasonal and age-related variations impact blue catfish male reproductive performance indices. Blue catfish males are currently being reared at the E.W. Shell Fisheries Center (Auburn University, Auburn, AL) in 0.08 ha aquaculture ponds. Males of a given age (2-3 years in 2021/2022) will be sampled throughout the annual reproductive cycle [pre-spawning (January, March); peak-spawning (May, June); post-spawning (August, October)] in an effort to survey a wide range of developmental stages, from immature to mature gonads. Each male will be sampled for body morphometrics, testes morphology using histological techniques, sex steroid hormones using enzyme-linked immunosorbent assays (ELISA), sperm quality (if available) using a Computer Assisted Sperm Analysis (CASA) system, and miRNA dynamics using molecular tools and bioinformatics. This sampling will continue until 2024. Objective 3.0) Link blue catfish male reproductive performance indices to sperm quality (both fresh and cryopreserved) and offspring performance during early developmental stages to the first feeding stage. In May 2021, mature blue catfish males (n = 43) were sampled for reproductive performance indices, including body metrics; testes morphology; sex steroid hormones; miRNA dynamics (on testes/sperm); and "select" sperm traits (on fresh and cryopreserved sperm), such as density, morphology, activity (motility/velocity), cell health, and DNA damage. MS students (×2) are currently processing all samples. Thereafter, we will use these reproductive performance indices to assess if they can accurately forecast the cryopreservation success (i.e., post-thaw activity and fertility) of individual males. miRNAs are also being sequenced from males with good (velocity ranged from 193 to 219 um/s; n = 8 males) and bad (velocity ranged from 138 to 158 um/s; n = 8 males) sperm. In upcoming months, the bioinformatic analyses will be conducted to decipher a potential molecular mechanism for sperm quality.
Impacts
What was accomplished under these goals?
Impact of project: Catfish farming accounts for nearly 70% of total U.S. freshwater aquaculture production, where the channel catfish, Ictalurus punctatus ? × blue catfish, I. furcatus ? hybrid constitutes >50% of the harvest. Current technology to produce hybrid embryos is labor intensive and requires the sacrifice of males for gamete collection and in vitro fertilization. This is costly and time-consuming, as blue catfish do not reach maturity for 4-7 years. Catfish sperm are then often of inadequate quality and quantity and do not necessarily give high fertilization rates and offspring viability. As a result, we are currently using basic and applied research to gain a better understanding of critical biological and physiological mechanisms underlying reproduction, gonadal function, gonadal production, and gamete preservation at the cellular, molecular, genomic/genetic, organ system (i.e., testes), and whole-animal level. Together, this work is allowing us to equip the aquaculture industry with the proper technological innovation and assisted reproductive technologies to fertilize eggs with fresh or frozen-thawed gametes as basis for hatchery and aquaculture production. Accomplishments for each specific objective are highlighted below: Objective 1.0) Determine the minimum quantity of blue catfish sperm that is required to fertilize channel catfish eggs in order to consistently maximize hatching success under hatchery conditions. (1.1) Major activities completed / experiments conducted - In May/June 2021 a series of fertilization trials were initiated using fresh and frozen-thawed sperm. Firstly, testes were dissected from blue catfish males, rinsed with Hanks' Balanced Salt Solution (HBSS), and macerated through a fine mesh strainer into centrifuge tubes. Collected cells were counted using a hemocytometer, diluted to 1.0 × 109 cells/mL, and then assessed for motility/velocity using Computer Assisted Sperm Analyses (CASA) software. Thereafter, the cells were cryopreserved in 0.5 mL cryo-straws using our cryogenic storage suite (i.e., controlled-rate freezer) and state-of-the-art protocols for blue catfish. The cryo-straws were thawed and diluted in Eppendorf tubes with HBSS (final volume per tube standardized to 1 mL) to obtain 5 sperm to egg ratios: 9 × 104:1, 1.0 × 105:1, 2.25 × 105:1, 4.5 × 105:1, and 9.0 × 105:1. Once ovulation was detected, females were anaesthetized. The urogenital pore was wiped dry, and eggs released by squeezing the abdominalarea. Eggs were hand-stripped and weighed. Egg density was estimated by weighing out aliquots of 1 g of eggs and manually counting them. This procedure was replicated, and the mean used to obtain total number of eggs per gram. Aliquots of 250 eggs were then weighed, according to each female's calculated egg density, into ~88.7 mL fertilization beakers. In this design, two unique males were "crossed" with every female (n = 12) for a total of 24 males used for experimentation. Sperm solutions for each ratio were pipetted directly onto the eggs, swirled, and allowed to incubate for 2 minutes. Each beaker was activated with 5 mL of ~28°C hatchery water with 6 g/L Fuller's Earth to reduce clumping of eggs. Eggs/embryos were then transferred to an indoor hatchery with a recirculation aquaculture system (RAS) and held below the water surface in mesh screen baskets (18 × 16 × 14 cm) incubated within larger glass aquaria (60 × 30 × 32 cm). (1.2) Data collected - Dead eggs were identified visually by their white, opaque color or enlarged size and removed daily. Finally, hatch success was determined as the total number of hatched fry divided by the initial number of eggs. (1.3) Summary statistics and discussion of results and (1.4) Key outcomes or other accomplishments realized - No significant differences in hatch success were detected between the 5 sperm to egg ratios (9 × 104:1 to 9.0 × 105:1). Further studies will use cryopreserved sperm at lower sperm to egg ratios. Overall, this will enable the fertilizing capability of males to be measured consistently and each male's sperm, whether stored frozen or in living gene banks, to be used wisely. Objective 2.0) Investigate how seasonal and age-related variations impact blue catfish male reproductive performance indices. (2.1) Major activities completed / experiments conducted - On 24 March 2021, 986 immature blue catfish males (average weight was 0.77 kg, 2 years of age) were transferred from a commercial hatchery (Jubilee Farms, Inc. MS.) to the E.W. Shell Fisheries Center at Auburn University, Auburn, AL, where they were acclimated in 0.08 ha aquaculture ponds. (2.2) Data collected - The experiment started on 9-11 August 2021, where fish (n = 270) were equally distributed into 3 × 0.08 ha ponds. At this time, data were collected from 6 fish (Time 0) for total body mass (average = 0.60 kg) and fork length (average = 39.3 cm). Blood was also collected for sex steroid hormone analyses. These fish were then dissected, and testes removed, weighed, and flash-frozen in liquid nitrogen for miRNA sequencing or placed in formalin for histological assessments. Gonadosomatic index [GSI = testes mass / (total mass - testes mass) × 100] and Fulton's condition factor [K = (total mass/fork length3) × 100] will be calculated. (2.3) Summary statistics and discussion of results and (2.4) Key outcomes or other accomplishments realized - Males of a given age class (2 to 4 years; 2021 to 2023) will now be sampled [for testes morphology, sex steroid hormones, sperm quality, testes miRNA dynamics] throughout the annual reproductive cycle [pre-spawning (January, March); peak-spawning (May, June); post-spawning (August, October)] in an effort to survey a wide range of developmental stages, from immature to mature gonads. Objective 3.0) Link blue catfish male reproductive performance indices to sperm quality (both fresh and cryopreserved) and offspring performance during early developmental stages to the first feeding stage. (3.1) Major activities completed / experiments conducted - In May 2021, 43 mature blue catfish males (average weight was 4.3 kg) were transferred from a commercial hatchery (Jubilee Farms, Inc. MS.) to the E.W. Shell Fisheries Center at Auburn University. These, males were seined from in 0.04 ha aquaculture ponds over 6 days during the reproductive season. (3.2) Data collected - Body metrics were recorded, and blood collected for sex steroid hormone measurements using enzyme-linked immunosorbent assays. Testes were dissected, weighed, and processed for milt extraction, and sperm density quantification using a hemocytometer. Samples were also taken for histological assessment. For each male, an aliquot of milt was then cryopreserved using our controlled-rate freezer and cryogenic storage suite, while another aliquot was used for evaluation of fresh sperm quality. Sperm quality (both fresh and cryopreserved) was then assessed, which included kinematic properties (motility/velocity), sperm morphometry imaging, and sperm health assessment (viability, oxidative stress, mitochondrial activity, DNA fragmentation). Testicular samples were sent to Novogene from males with good (velocity ranged from 193 to 219 um/s; n = 8 males) and bad (velocity ranged from 138 to 158 um/s; n = 8 males) sperm quality. (3.3) Summary statistics and discussion of results and (3.4) Key outcomes or other accomplishments realized - Data is currently being compiled and analyzed. Together, these data will enable us to create innovative models that predict male sperm quality and offspring performance as a basis for hatchery production.
Publications
|
|