Source: AUBURN UNIVERSITY submitted to NRP
REPRODUCTIVE PHYSIOLOGY AND HATCHERY TECHNIQUES FOR AQUATIC SPECIES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1013854
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Nov 29, 2017
Project End Date
Sep 30, 2022
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849
Performing Department
Fisheries & Allied Aquaculture
Non Technical Summary
Over the past several decades, aquaculture production has steadily increased to preserve/restore populations of threatened or endangered species and to meet consumer food demands. For instance, in 2014, global aquaculture production reached nearly 74 million tonnes, with an estimated first-sale value of $160.2 billion USD. In the United States, catfish farming in Mississippi, Arkansas, Alabama, and Texas accounted for nearly 70% of the total freshwater aquaculture production, where the female channel catfish x male blue catfish hybrid constituted the majority (50-75%) of the harvest. Moreover, the oyster industry along the Gulf of Mexico is farming its way to an "oyster revolution". This Hatch Proposal aims to support these expanding aquatic industries, among others. To accomplish this, we will take an interdisciplinary approach (i.e. combining physiology, ecology, behavior) to test hypotheses in reproductive biology and physiology of aquatic species, with the ultimate goal of understanding and controlling processes that impact gamete quality and offspring performance. Within this regard our work revolves around three main research objectives: (1) Applied reproductive physiology: understand and control gametogenesis, gamete physiology, fertilization, and embryonic development for species of commercial importance, and identify factors regulating these processes; (2) Gamete cryopreservation: develop cutting-edge techniques to cryopreserve genetic resources (i.e. create a "germplasm repository") for conservation breeding efforts that may be needed in the future for commercially important or endangered species; and (3) Sexual selection and the evolution of gamete interactions: quantify the relative importance of reproductive traits, sperm competition, and gamete interactions in shaping fertilization dynamics and the evolution of sexual characteristics. Together, this will allow us to equip the aquaculture industry with the proper technological innovation and assisted reproductive technologies (ARTs) to fertilize eggs with fresh or frozen-thawed gametes as basis for hatchery and aquaculture production. Creation of a germplasm repository using advanced cryopreservation techniques will also provide an invaluable means of creating experimental families and preserving genetic diversity in populations that are in imminent danger of extirpation.
Animal Health Component
50%
Research Effort Categories
Basic
30%
Applied
50%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3010810102025%
3010811102015%
3050810102035%
1360810102015%
1360811102010%
Knowledge Area
136 - Conservation of Biological Diversity; 305 - Animal Physiological Processes; 301 - Reproductive Performance of Animals;

Subject Of Investigation
0810 - Finfish; 0811 - Shellfish;

Field Of Science
1020 - Physiology;
Goals / Objectives
I aim to support the development of sustainable aquaculture, fisheries management, and conservation biology. To accomplish this, I take an interdisciplinary approach to test hypotheses in reproductive biology, reproductive physiology, and hatchery science of fishes, with the goal of understanding and ultimately controlling processes that impact gamete quality and early offspring performance. Alabama is particularly rich in marine and freshwater bivalves (i.e. freshwater mussels, clams, oysters). As such, I will also study their reproductive biology in the context of sexual selection and gamete cryopreservation. Overall, the specific questions I address focus on individual and population-level variation, especially during the "critical" early life history stages (i.e. egg to early juveniles), which typically have a high-degree of mortality. Within this regard, my long-term objectives are to (1) understand and control physiological processes that impact gamete quality, fertility, and early offspring performance, and (2) optimize cryopreservation protocols for conserving genes of threatened and economically important species in the Southeastern US. Together, this multi-disciplinary approach will allow me to equip the aquaculture industry with the proper technological innovation and assisted reproductive technologies (ARTs) to fertilize eggs with fresh or frozen-thawed gametes as basis for hatchery and aquaculture production. Creation of a germplasm repository using advanced cryopreservation techniques will also provide an invaluable means of creating experimental families and preserving genetic diversity in populations that are in imminent danger of extirpation.
Project Methods
Our research (~16 independent studies over the next 5 years) will revolve around several main research themes. Below I have outlined the overarching rational and methods for each theme. 1. Sperm Biology and Gamete Interactions - The aim of this work is to test hypotheses, which govern 'optimal' conditions for sperm swimming and fertility using fish/shellfish with different reproductive patterns. Within this context, we will explore the different ways in which ejaculate traits, pre-zygotic selection mechanisms, and competitive and non-competitive environments influence fertilization dynamics and shape the evolution of sexual characteristics. Previous models of gamete biology have focused mostly on aqueous environments that do not simulate natural spawning conditions; i.e. sperm activation media lack ovarian fluid and other egg-derived compounds. However, when these maternally-derived compounds are incorporated during spawning they can change the outcome of a fertilization event. Thus, when testing hypotheses, we will shift from the classic sperm activation medium paradigm (water only) to more natural spawning media that encompass interactions between gametes and their associated fluids. Results obtained will not merely be of theoretical interest, but of practical importance for hatchery production. 2. Applied reproductive physiology - Advances in biotechnology have made aquaculture the fastest growing food producing sector in the world. This includes a wide variety of fishes/bivalves that are now being cultivated to meet food demands. However, because many of these species do not reproduce naturally in captivity, assisted reproductive technology (ART) in terms of induced gamete production and artificial fertilization is frequently required to produce offspring. Therefore, accurate knowledge is needed to develop and implement techniques to manipulate breeders, fertilize eggs, and rear embryos, as basis for hatchery and aquaculture production. In this context, studies will be conducted to assess the impact of nutrition, environment, genetic, and endocrine factors on gamete quality from wild and/or farmed breeders; optimize reproductive success by evaluating the influence of sperm and egg quality on fertility; decipher mechanisms of gamete activation; establish standardized ART and embryo incubation techniques. Success criterion is achievement of large-scale production of viable gametes potentially leading to the production of "high-quality" larvae. 3. Hatchery techniques and technology - Rates of natural mortality are highest during early life and are negatively correlated with rates of growth and body size; thus, subtle differences in mortality can cause large differences in hatchery production. In order to develop new hatchery techniques, we will determine which biophysical factors generate optimal culturing conditions during these critical developmental stages and how they interact with genotype to create phenotypic traits of importance. This is essential for aquatic species as survival during early-life represents a substantial component of variation in lifetime fitness. 4. Gamete cryopreservation - Variation in the ability of sperm to withstand cryogenic freezing and thawing has been reported among individuals within species. This male-to-male variation poses a problem where "bad" sperm donors might be of high genetic value. Research in mammalian species has identified several semen quality indices which can be used to discriminate between "good" vs. "bad" sperm donors. On the contrary, there have been only a handful of studies addressing this aspect in bivalves or fishes (Müller et al. 2008; Butts et al. 2011). It is therefore of utmost importance that we continue to understand the physiological processes associated with cryopreservation of gametes and develop models that predict, prior to freezing, those individuals which are able and/or unable to endure the freezing and thawing process. Together, this will allow us to address these male-to-male short-comings in the cryogenic process so we can i.e. transform a "bad" sperm donor into a "good" sperm donor and sustain a more effective germplasm repository to support selective breeding, aquaculture production, and conservation efforts.

Progress 10/01/20 to 09/30/21

Outputs
Target Audience:Establishment of a fully equipped aquatic reproductive physiology lab [i.e., has computer-assisted sperm analysis (CASA) software; cryopreservation equipment; microscopy and imaging suite; flow cytometry; histology suite; and gene expression tools] and experimental hatchery created research opportunities and "hands-on" instruction for numerous undergraduate students [via my Hatchery Management (FISH 5240) class and Research Fellowship program] and graduate students (3 MSc, 1 PhD). Our work was published in international peer-reviewed journals as well as in Fish Farming News and presented at a national scientific conference. We also communicated our findings to catfish and largemouth bass farmers in the Southeastern United States. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Students (~6) in my lab learned fundamental concepts in aquatic reproductive biology/physiology, sperm biology, hatchery science, experimental design, and data analyses. Specifically, they gain hands-on experiences with: Sperm quality assessment: Sperm motility, velocity, and motion parameters (i.e., linearity, amplitude of lateral head displacement) were quantified using a Computer Assisted Sperm Analyses (CEROS II, Hamilton Thorne Inc.) system equipped with negative phase objectives and high-resolution CCD monochrome progressive scan camera. Muse Cell Analyzer (Millipore Sigma), a robust compact flow cytometer, measured changes in mitochondrial potential, membrane viability, and oxidative stress of sperm. Students measured spermatocrit, cell density, and pH of seminal plasma. Moreover, osmolality was measured using a vapor pressure osmometer (Wescor Inc.), while fluorescent microscopy was used to assess cell health. Gamete cryopreservation: A controlled-rate freezer (Kryo 560, Planer PLC) and 50L cryogenic Dewar with transfer hose froze sperm and enabled my lab to store sperm samples in perpetuity, creating our germplasm repository for later assessment. Artificial fertilization procedures: Students learned how to inject fish and fertilize eggs using artificial fertilization techniques. Gene expression analyses: RNA was extracted from catfish and largemouth bass (sperm, eggs, larvae) and later assessed for gene expression. Early fish rearing: Students learned how to rear embryos and larvae using recirculation aquaculture systems. Fish (i.e., catfish, largemouth bass) were also sampled for growth performance and deformities. How have the results been disseminated to communities of interest?Our research was presented at several Auburn University functions to foster ties with other faculty members. Additionally, we presented our research at a national conference. Catfish farmers have also been informed of our work via extension reports (Fish Fishing News) and phone conversations. What do you plan to do during the next reporting period to accomplish the goals?Below I highlight our data collection and experimental plans for the next reporting period: Catfish - Sperm quality is highly variable among catfish males. This variation can cause major economic losses and is not sustainable, especially when cells from selectively bred individuals need to be discarded.Pinpointing biomarkers for fresh/frozen-thawed sperm quality and effects on offspring performance would have major implications for sustainable production of catfish.Objectives: Develop selection criterion for high-performance males (for IVF and gene banking) by quantifying a series of sperm quality indices (i.e., morphological, physiological, kinematic) between fresh and cryopreserved sperm, and then linking these fresh sperm indices to cryopreservation success.Methods:For each male (n = ~30), an aliquot of milt will be cryopreserved, while another aliquot will be used for evaluation of fresh sperm quality. The following indices will be measured on both fresh and cryopreserved sperm:Densitywill be quantified using a hemocytometer.A complete spermmorphometryassessment will be executed. Specifically,sperm will be pipetted onto a slide and prepared using Hemacolor®staining. Images of sperm will be captured and morphometric analyses of sperm will then be performed using an automated sperm morphology analyzer.Kinematics-Fertility is dependent on the ability of sperm to reach an egg within a short period of time, usually seconds. We predict that males with faster swimming sperm and higher motility are of better quality.Sperm will be pipetted onto a slide and topped with a coverslip. Sperm will be activated with water andvideo recorded at 10-30 s post-activation. Once recorded, sperm traits, includingmotility and velocity,will be analyzed using a Computer Assisted Sperm Analyses system (CASA).Health metrics-Spermviability, oxidative stress, mitochondrial activity, and DNA fragmentation will be assessed using fluorescent probes, flow cytometry, and assay kits.Expected results:1) Sperm quality will vary between fresh and cryopreserved samples, and 2) statistical models will link fresh to cryopreserved sperm quality so we will be able to delineate bad vs. good sperm donors. Eastern oyster- is native to benthic habitats along the Atlantic coast of America from Canada to the Gulf of Mexico, the Caribbean, and the coast of South America. At present, the aquaculture industry for this species is rapidly expanding.In 2022, we will take acomprehensive and integrative approachto assess the immediate and long-term effects of abiotic stressors (i.e., salinity, temperature) on oyster gametogenesis. Methods:Immediately after oyster collection and at monthly intervals (for 4 sampling points), 10 oysters will be collected from each aquatic recirculation system at 10, 20, and 30 ppt. Three transverse sections from the middle region of the gonad will be taken and preserved in fixative for histological analysis. Samples will be dehydrated in increasing concentrations of ethanol, embedded in paraffin, and sectioned with a microtome. Sections will be stained with hematoxylin and eosin. Histological sections will be photographed at 200× magnification and testes and ovarian tissues categorized according to cell types. Area fractions of the different cell types will be estimated by placing a 48-point grid on each image. Progression of gametogenesis will then be assessed for each oyster using a maturity index.During peak spawning season (May-June) mature oysters will be opened with a shucking knife, cup-side down, at the hinge, and sex determined by observation of the gonad samples under a microscope. Each male (n = 20 per salinity) will be thoroughly rinsed with distilled water to flush away any excess seawater. The mantle tissue around the gonad will be dried with paper towels before semen collection by pipette. Collected semen will be stored in 1 mL tubes and placed into a chilling block. Morphometrics of oysters including length, width, and height (±0.01 mm) will be recorded. Osmolality of testicular fluid will be determined in triplicate for each male using a vapor pressure osmometer and pH measured using a benchtop pH meter.The following sperm quality traits will be quantified for the males:Kinematic properties,morphometry,density, and health.

Impacts
What was accomplished under these goals? Wild-caught fish stocks are declining, and as the world population grows, the demand for high-quality protein is increasing. New research shows that fish, crustaceans, mollusks, and other aquatic animals account for ~17% of total animal protein consumed by the global population. At present, global production of all aquatic animals is increasing, with aquaculture or fish farming representing nearly half of the total harvest. Aquaculture is therefore playing a pivotal role in global food security, the environment, and human health. Because the aquaculture sector is rapidly growing and is relatively new compared to the livestock sector, it offers tremendous opportunities for technological innovation to further increase sustainability to meet protein demands. Thus, this Hatch Proposal aims to support this industry, with a focus on hybrid catfish, Eastern oyster, eel, largemouth bass, southern flounder, and other key aquatic species. To accomplish this, we take an interdisciplinary approach (e.g., combine physiology, molecular biology, developmental biology, and behavior) to understand and control processes that impact gamete quality and offspring performance, especially during the "critical" early life history stages (i.e., egg to early juveniles), when mortality is unusually high. Overall, this work will allow us to equip the aquaculture industry with technological innovation and assisted reproductive technologies (ARTs) to fertilize eggs with fresh or frozen-thawed gametes as the basis for hatchery and aquaculture production. Specific objectives: We support the development of aquaculture, fisheries management, and conservation biology. To accomplish this our objectives are to (1) understand and control processes that impact gamete quality, fertility, and early offspring performance and (2) optimize cryopreservation protocols for conserving genes of threatened/economically important species. In this regard, I highlight some of the results for each objective: Objective 1: Project 1: Blue catfish sperm are often of inadequate quality and quantity and do not necessarily give high fertilization rates and offspring viability for aquaculture production. As such,a better understanding of molecular processes will advance our male diagnostic toolbox.Objectives and methods: Weexplored the expression of RNAs in testicular tissue from males with "good" (high sperm velocity) and "bad" (low sperm velocity) sperm to decipher potential biomarkers for male reproductive success. Our bioinformatic analyses showed several genes with differential expression between the good and bad males. This work will be submitted for peer-review in 2022. Additionally, we compared sRNAs in testes vs. sperm. Project 2:Catfish farmers in west Alabama are interested in using existing and available water acres that are not being used for catfish to grow largemouth bass, thus allowing diversification of their production strategies.Objectives and methods: In 2020, an experiment was conducted to determine how temperature (21oC, 24oC, or 27oC ±0.2oC) and bass strain (Florida vs. Northern) impacts hatchery production efficiency. Results have clearly shown that Northern largemouth bass have better growth and survival at24oC or 27oC.We compiled this extensive data set in 2021 (i.e., processed larval photos and gene expression data) and results are now being processed for peer-review in 2022. Objective 2: Project 1: Gamete cryopreservation can cause cellular damage, which may impair offspring.Atpresent, the picture is far from complete for catfish. As such, the industry needs 'proof of concept' for the applicability of cryopreserved sperm for their hatchery management programs. Our work aimed to decipher how offspring performance is affected by cryogenic processes, documenting early offspring ontogeny from eggs fertilized with fresh and cryopreserved sperm. In May/June 2021, fresh and cryopreserved sperm from several males and eggs from several females were used. Eggs/embryos from these treatments were incubated in baskets within aquaria. Dead eggs were counted daily and removed to calculate embryonic survival and hatching success. Thereafter, 15-20 individuals were sampled from the fresh and frozen-thawed sperm treatments during the early stages of ontogeny. Body morphology measurements and the rate of deformities (%) are now being analyzed. We expect that these data will showfarmers that cryopreserved sperm can be used for fertility to synchronize gamete availability, conserve sperm, reduce male broodstock numbers, reduce the risk of disease, and conserve genetic variability; thereby, strengthening overall sustainability. Project 2: Our goal is to develop xenogenesis (method of reproduction in which successive generations differ from each other) resulting in xenogens (organisms comprised of elements typically foreign to its species) to increase efficiency of reproduction for catfish hybridization, and secondarily for other applications, i.e., to increase efficiency of reproduction in difficult to spawn species, like blue catfish. Here, we assessed the impact of using cryopreserved stem cells as donor cells for xenogenic transplantation. Our preliminary data show that frozen-thawed stem cells can be used for future xenogenic applications. Results will be submitted for peer-review in 2022.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Koumpiadis, P., Sganga, D., Politis, SN., Gallego, V., Butts, I.A.E., Asturiano, J., Batjakas, I., Tomkiewicz, J. 2021. Sperm production and quality in European eel (Anguilla anguilla) in relation to hormonal treatment. Reproduction in Domestic Animals 56: 1497-1505
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Abualreesh, M.H,, Myers, J.N., Gurbatow, J., Johnson, A., Xing, D., Wang, J., Li, S., Coogan, M., Vo, K., El Husseini, N., Dunham, R.A., Butts, I.A.E. 2021. Development of an effective cryopreservation protocol for blue catfish oogonia. North American Journal of Aquaculture 83: 336-345
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Hardy, R.S., Zadmajid, V., Butts, I.A.E., Litvak, M.K. 2021. Growth, survivorship, and predator avoidance capability of larval shortnose sturgeon (Acipenser brevirostrum) in response to delayed feeding. PLOS ONE 16(3): e0247768. https://doi. org/10.1371/journal.pone.0247768
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Nichols, Z.G., Rikard, S., Alavi, S.M.H., Walton, W.C., Butts, I.A.E. 2021. Regulation of sperm motility signaling in Eastern oyster spawning naturally in seawater with low salinity. PLOS ONE 16(3): e0243569. https://doi.org/10.1371/journal. pone.0243569
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Politis, S.N., Syropoulou, E., Benini, E., Bertolini, F., S�rensen, S.R., Miest, J.J., Butts, I.A.E., Tomkiewicz, J. 2021. Performance thresholds of hatchery produced European eel larvae reared at different salinity regimes. Aquaculture 539, 736651
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Gazsi, G., Butts, I.A.E., Zadmajid, V., Iv�novics, B., Ruffilli, L., Urb�nyi, B., Csenki, Z., M�ller, T. 2021. Ovarian inseminated sperm impacts spawning success in zebrafish, Danio rerio (Hamilton, 1822) even in the absence of a male stimulus. Theriogenology 172: 315-321
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Nichols, Z.G., Zadmajid, V., Dalal, V.B., Stoeckel, J.A., Wayman, W.R., Butts, I.A.E. 2021. Reproductive aspects of freshwater unionid mussel sperm: Seasonal dynamics, cell quantification, and male-to male variability. Animal Reproduction Science 230, 106768
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Alavi, S.M.H., Hatel, A., Butts, I.A.E., Bondarenko, O., Cosson, J., Babiak. I. 2021. Some recent data on sperm morphology and motility kinematic in Atlantic cod (Gadus morhua L.). Fish Physiology and Biochemistry 47(2): 327-338
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Casey, P., Zadmajid, V1., Butts, I.A.E1., S�rensen, S.R., Litvak, M.K. 2021. Influence of temperature on growth and survival of juvenile winter flounder reared under continuous light. Journal of the World Aquaculture Society 52: 204-215
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Abualreesh, M.H., Myers, J.N., Gurbatow, J., Johnson, A., Xing, D., Wang, J., Li, S., Coogan, M., Vo, K., El Husseini, N., Dunham, R.A., Butts, I.A.E. 2021. Effects of antioxidants and anti-freeze proteins on cryopreservation of blue catfish (Ictalurus furcatus) spermatogonial stem cells. Aquaculture 531, 735966
  • Type: Other Status: Published Year Published: 2021 Citation: Myers, J.N., Dunham, R.A., Abualreesh, M.H., Butts, I.A.E. 2021. Its cold down here in Alabama: Freezing cells for optimizing hybrid catfish hatchery production. Alabama Fish Farming Center - Fish Farming News 2020(1):1-18
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Aguilar, G.L., Sakmar, J., Nicholls, A., Bruce, T.J., Kelly, A.M., Roy, L., Bernal, M.A., Butts, I.A.E. 2021. Temperature effects on larval development of Florida (Micropterus salmoides floridanus) and Northern (Micropterus salmoides salmoides) largemouth bass: Implications for intensive indoor aquaculture and fisheries management. Alabama Chapter of the American Fisheries Society, Ramer, AL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Dunham, R.A., Hettiarachchi, D.U., Alston, V., Bern, L., Mei, S., Su, B., Butts, I.A.E. 2021. Progress in catfish xenogenesis. Aquaculture America 2021. San Antonio, TX
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Hettiarachchi, D.U., Alston, V., Gurbatow, J., Zadmajid, V., Johnson, A., Wang, J., Coogan, M.P., Xing, D., Li, S., Butts, I.A.E., Dunham, R.A. 2021. Effect of seasonality for optimization of stem cell extraction from mature blue catfish (Ictalurus furcatus) to produce alloxenogenic catfish. Aquaculture America 2021. San Antonio, TX
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: James, J., Dahl, S., Teichert-Coddington, D., Kelly, A.M., Creel, J.D., Beck, B.H., Butts, I.A.E., Roy, L.A. 2021. Polyculture of red swamp crayfish and Pacific white shrimp cultured in low salinity water. Annual Meeting of the Alabama Inland Shrimp Producers Association. Alabama Fish Farming Center, Greensboro, Alabama. March 2


Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Establishment of a fully equipped aquatic reproductive physiology lab [i.e. has computer assisted sperm analysis (CASA) software; cryopreservation equipment; microscopy and imaging suite; flow cytometry; and gene expression tools] and experimental hatchery created research opportunities and "hands-on" instruction for numerous undergraduate students [via my Hatchery Management (FISH 5240) class at Auburn University] and graduate students (5 MSc, 2 PhDs). Our work was published in international peer-reviewed journals (e.g.Aquaculture; Theriogenology; and Reviews in Fisheries Science and Aquaculture) as well as in Fish Farming News, and presented at a national scientific conference. We also communicated our findings to catfish farmers in the Southeastern United States to foster ties between Auburn University and the local aquaculture industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Students (~9) in my lab learnt fundamental concepts in aquatic reproductive biology/physiology, sperm biology, hatchery science, experimental design, and data analyses. Specifically, they gain hands-on experiences with: Sperm quality assessment: Sperm motility, velocity, and motion parameters (i.e. linearity, amplitude of lateral head displacement) were quantified using a Computer Assisted Sperm Analyses (CEROS II, Hamilton Thorne Inc.) system equipped with negative phase objectives and high-resolution CCD monochrome progressive scan camera. Muse Cell Analyzer (Millipore Sigma), a robust compact flow cytometer, measured changes in mitochondrial potential, membrane viability, and oxidative stress of sperm. Students measured spermatocrit, cell density, and pH of seminal plasma. Moreover, osmolality was measured using a vapor pressure osmometer (Wescor Inc.). Scanning and transmission electron microscopes were used to explore sperm ultrastructure, while fluorescent microscopy was used to assess cell health (i.e. mitochondrial activity, membrane potential). Gamete cryopreservation: A controlled-rate freezer (Kryo 560, Planer PLC) and 50L cryogenic Dewar with transfer hose froze sperm and enabled my lab to store sperm samples in perpetuity, creating our germplasm repository for later assessment. Artificial fertilization procedures: Students learned how to inject fish and fertilize eggs using artificial fertilization techniques. Gene expression analyses: RNA was extracted from catfish eggs/embryos and sperm and later assessed for gene expression.? How have the results been disseminated to communities of interest?Our research was presented at several Auburn University functions to foster ties with other faculty members. Additionally, we presented our research at a national conference. Catfish farmers have also been informed of our work via extension reports (Fish Fishing News) and phone conversations. See below for details: Institutional Nichols, Z.G., Rikard, S., Alavi, S.M.H., Bradford, A.J., Walton, W.C., Butts, I.A.E. 2020. Physiological mechanisms regulations sperm activation in eastern oyster,Crassostrea virginica. Auburn Research: Virtual Student Symposium. Auburn. Al. Hettiarachchi, D., Alston, V., Gurbatow, J., Vo, K., Zadmajid, V., Coogan, M., Xing, D., El Husseini, N., Johnson, A., Wang, J., Li, S., Dunham, R.A., Butts, I.A.E. 2020. Effects of body size, sex steroids and seasonality for optimization of stem cell extraction from blue catfish (Ictalurus furcatus) to produce alloxenogenic catfish. Auburn Research: Virtual Student Symposium. Auburn. Al. Hettiarachchi, D., Alston, V., Gurbatow, J., Vo, K., Zadmajid, V., Coogan, M., Xing, D., El Husseini, N., Johnson, A., Wang, J., Li, S., Dunham, R.A., Butts, I.A.E. 2020. Effects of body size, sex steroids and seasonality for optimization of stem cell extraction from blue catfish (Ictalurus furcatus) to produce alloxenogenic catfish. AFS/USAS Auburn University Student Research Symposium. National Nichols, Z.Gd., Rikard, S., Alavi, S.M.H., Bradford, A.J., Walton, W.C., Butts, I.A.E. 2020. Physiological mechanisms regulations sperm activation in eastern oyster,Crassostrea virginica.Aquaculture America 2020. Honolulu, Hawaii dResearch completed by my MSc student(Won best conference abstract &1stPlace Oral Presentation (Student Spotlight Award) Nichols, Z.G., Stoeckel, J.A., Wayman, W., Butts, I.A.E. 2020. Quantifying sperm production and morphology for two species of unionid mussels.Aquaculture America 2020. Honolulu, Hawaii Abualreesh, M.H., Gurbatow, J., Hettiarachchi, D., Alston, V., Hallas, V.S., Coogan, M.P., El Husseini, N., Dunham, R.A., Butts, I.A.E., 2020. Effects of cryoprotectants and freezing rates for preserving spermatogonial stem cells of blue catfish.Aquaculture America 2020. Honolulu, Hawaii Butts, I.A.E., Myers, J.N., Chatakondi, N., Dunham, R.A. 2020. Improving hybrid catfish production: Impacts of parental effects and gamete interactions. (Invited speaker). Aquaculture America 2020. Honolulu, Hawaii What do you plan to do during the next reporting period to accomplish the goals?Below I highlight our data collection and experimental plans for the next reporting period: Catfish- We will compare miRNA dynamics between "good" vs. "bad" blue catfish sperm donors (based on computer assisted sperm analyses data) and develop fertilization strategies for hybrid catfish hatchery production using frozen-thawed sperm.Southern flounder- We aim to improve cryopreservation techniques for this species.Largemouth bass- (i) will assess the importance of enrichedArtemia, with emphasis on largemouth bass larval nutritional requirements and (ii) determine whether larvae are receptive to micro-diets and whether there is a time during early life when they show high initial acceptance for ingestion, growth, and survival.

Impacts
What was accomplished under these goals? Wild-caught fish stocks are declining, and as the world population grows, the demand for high-quality protein is increasing. New research shows that fish, crustaceans, mollusks, and other aquatic animals account for about 17% of total animal protein consumed by the global population. At present, global production of all aquatic animals is estimated at ~171 million tons (first-sale value of $232 billion USD), with aquaculture or fish farming representing nearly half (47-53%) of the total harvest. Aquaculture is therefore playing a pivotal role in global food security, the environment, and human health. Because the aquaculture sector is rapidly growing and is relatively new compared to the livestock sector, it offers tremendous opportunities for technological innovation to further increase sustainability to meet protein demands. Thus, this Hatch Proposal aims to support this industry, with focus on hybrid catfish, Eastern oyster, eel, and other key aquatic species. To accomplish this, we take an interdisciplinary approach (e.g., combine physiology, molecular biology, developmental biology, and behavior) to understand and control processes that impact gamete quality and offspring performance, especially during the "critical" early life history stages (i.e., egg to early juveniles), when mortality is unusually high. Specifically, for catfish, we linked blue catfish stem cell production and viability to body size and sex hormones for creation of xenogenic catfish; developed cryopreservation protocols for blue catfish stem cells; and explored miRNA dynamics in blue catfish male gametes. Next for winter flounder and largemouth bass we determined how biophysical parameters (e.g., photoperiod, temperature) impact growth and survival during critical early developmental stages. For European eel, we assessed the impact of maternal dietary levels of fatty acids and dietary regimes on reproductive success and offspring quality; and investigated how "enhanced" feeds with modified amino acid composition, impact sperm performance. Finally, for eastern oyster we studied how salinity, pH, and ionic channels impact sperm swimming behaviors. Overall, this work will allow us to equip the aquaculture industry with technological innovation and assisted reproductive technologies (ARTs) to fertilize eggs with fresh or frozen-thawed gametes as basis for hatchery and aquaculture production. Specific objectives: We support development of aquaculture, fisheries management, and conservation biology. To accomplish this our objectives are to (1) understand and control processes that impact gamete quality, fertility, and early offspring performance and (2) optimize cryopreservation protocols for conserving genes of threatened/economically important species. In this regard, I highlight some of our objectives and results. Objective 1-Blue catfish sperm are often of inadequate quality and quantity and do not necessarily give high fertilization rates and offspring viability for aquaculture production. As such,a better understanding of molecular processes will advance our male diagnostic toolbox.In 2020, we startedexploring the expression of miRNAs in testicular tissue and sperm to decipher potential biomarkers for male reproductive success. This is now being done in conjunction with histology, steroid hormonal profiles, and sperm quality assessments. Our bioinformatic analyses is currently underway and we hope to submit this work for publication in 2021 (pending results). Objective 1-Catfish farmers in west Alabama are interested in using existing and available water acres that are not being used for catfish to grow largemouth bass, thus allowing diversification of their production strategies. In 2020, an experiment was conducted to determine how temperature (21oC, 24oC, or 27oC ±0.5oC) and bass strain (Florida vs. Northern) impact hatchery production efficiency. Preliminary results show that Northern largemouth bass have better growth and survival at24oC or 27oC.Wehope to submit this work for publication in 2021. Objective 2-The goal of our NIFA grant (PI: Dr. Dunham) is to develop xenogenesis (method of reproduction in which successive generations differ from each other) resulting in xenogens (organism comprised of elements typically foreign to its species) to increase efficiency of reproduction for catfish hybridization, and secondarily for other applications, i.e., to increase efficiency of reproduction in difficult to spawn species, like blue catfish. Here, we assessed the impact of using cryopreserved stem cells as donor cells for xenogenic transplantation. In May/June 2020, channel catfish were injected with fresh (control) or cryopreserved stem cells from blue catfish. Our preliminary results show no difference between treatments, meaning cryopreserved cells can be banked and routinely used to create xenogens.We hope to submit this work for publication in 2021. Objective 2 -The ability to store catfish stems cells in perpetuity for future xenogeneic applications requires in-depth knowledge of the mechanisms which govern cell survival during freezing and thawing processes. Here, we have not only developed "cutting edge techniques" to preserve genetic resources, but also defined underlying physiological principles that influence gamete survival during low-temperature storage. Specifically, in 2018/2019 we developed techniques to cryopreserve spermatogonia. Now in 2020, we further improved this cryopreservation protocol by testingeffects of antioxidants (catalase, hypotaurine, and ascorbic acid) and anti-freeze proteins (I and III) on post-thawspermatogonia production and viability. Furthermore, we developed a functional freezing protocol for oogonial stem cells. All this work has now been published or submitted for publication.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Kottmann, J.S., Butts, I.A.E., St�ttrup, J.G., Lund, I., Jacobsen, C.J., Holst, L.K., Tomkiewicz. J. 2020. Effects of essential fatty acids and feeding regimes on egg and offspring quality of European eel: Comparing reproductive success of farm-raised and wild-caught broodstock. Aquaculture, 529, 735581
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Hettiarachchi, D., Alston, V., Gurbatow, J., Vo, K., Zadmajid, V., Coogan, M., Xing, D., El Husseini, N., Johnson, A., Wang, J., Li, S., Dunham, R.A., Butts, I.A.E. 2020. Donor body size and sex steroid hormones impact germ cell production for creation of xenogenic catfish. Aquaculture, 528, 735579
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Casey, P., Butts, I.A.E., Zadmajid, V., S�rensen, S.R., Litvak, M.K. 2020. Prolonged photoperiod regime improves the growth performance of hatchery reared juvenile winter flounder. Aquaculture Engineering, 90, 102089
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Myers, J.N., Bradford, A.J., Hallas, V.S., Lawson, L.L., Pitcher, T.E., Dunham, R.A., Butts, I.A.E. 2020. Channel catfish ovarian fluid differentially enhances blue catfish sperm performance. Theriogenology, 149: 62-71
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Myers, J.N, Senior, A., Zadmajid, V., & S�rensen, S.R., Butts, I.A.E. 2020. Associations between ovarian fluid and sperm performance in marine and freshwater fishes: A meta-analysis. Reviews in Fisheries Science and Aquaculture, 28: 322-339
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Myers, J.N., Dyce, P.W., Chatakondi, N.G., Gorman, S., Quiniou, S., Su, B., Peatman, E., Dunham, R.A., Butts, I.A.E. 2020. Specific maternal gene expression profiles in good vs. bad eggs for hybrid catfish production. Comparative Biochemistry and Physiology: Part A, 243, 110675
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Litvak, M.K., Zadmajid, V., Butts, I.A.E. 2020. Growth and survival of winter flounder larvae reared on different photoperiod regimes from hatch to metamorphosis. Aquaculture Research, 51: 2314-2321
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Myers, J.N., Nichols, Z., Abualreesh, M.H., El Husseini, N., Taylor, Z.G., Coogan, M., Gurbatow, J., Khoi, V., Zadmajid, V., Chatakondi, N.G., Dunham, R.A., Butts, I.A.E., 2020. Impact of sperm density on hatching success for channel catfish (Ictalurus punctatus) ? � blue catfish (I. furcatus) ? embryo production. Aquaculture, 521, 735024 Research partially completed by my Sperm Form and Function in Aquatic Organisms (FISH 7960) class
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Butts, I.A.E., Hilmarsd�ttir, G.S., Zadmajid, V., Kr�ger-Johnsen, M., Politis, S., St�ttrup, J., Jacobsen, C., Gallego, V., Holst, L.K., Asturiano, J.F., Tomkiewicz, J. 2020. Dietary amino acids impact sperm performance traits for a catadromous fish. Aquaculture, 518, 734602
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Myers, J.N., Chatakondi, N.G., Dunham, R.A., Butts, I.A.E. 2020. Genetic architecture of early life history traits for channel catfish, Ictalurus punctatus ? � blue catfish, I. furcatus ? hybrid production. Aquaculture, 514, 734436
  • Type: Other Status: Published Year Published: 2020 Citation: Myers, J.N., Dyce, P.W., Chatakondi, N.G., Gorman, S.A., Quiniou, S.M.A., Su, B., Peatman, E., Dunham, R.A., Butts, I.A.E. Gene expression: The key to success for high-hatch hybrid catfish eggs. Alabama Fish Farming Center - Fish Farming News, 2020(2):13-14
  • Type: Other Status: Published Year Published: 2020 Citation: Dunham, R.A., Su, B., Butts, I.A.E., Lu, C., Gurbatow J. 2020. Transgenesis, gene editing and xenogenesis for controlling reproduction. In: Straus, D. (Compiler), Sterility in Aquaculture  Advances, Performances, Impacts. World Aquaculture, June 2020: 57-61
  • Type: Other Status: Published Year Published: 2020 Citation: Myers, J.N., Lawson, L.L., Pitcher, T.E., Dunham, R.A., Butts, I.A.E. 2020. The fertility games: Effects of male-female gametic interactions on fertility potential for hybrid catfish. Alabama Fish Farming Center - Fish Farming News, 2020(1): 7-8
  • Type: Other Status: Published Year Published: 2020 Citation: Myers, J.N., Chatakondi, N.G., Dunham, R.A., Butts, I.A.E. 2020. Every catfish sperm is sacred. Alabama Fish Farming Center - Fish Farming News, 2020(1): 13-14
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Nichols, Z.G., Rikard, S., Alavi, S.M.H., Bradford, A.J., Walton, W.C., Butts, I.A.E. 2020. Physiological mechanisms regulations sperm activation in eastern oyster, Crassostrea virginica. Aquaculture America 2020. Honolulu, Hawaii Research completed by my MSc student (Won best conference abstract & 1st Place Oral Presentation (Student Spotlight Award)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Nichols, Z.G., Stoeckel, J.A., Wayman, W., Butts, I.A.E. 2020. Quantifying sperm production and morphology for two species of unionid mussels. Aquaculture America 2020. Honolulu, Hawaii
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Abualreesh, M.H., Gurbatow, J., Hettiarachchi, D., Alston, V., Hallas, V.S., Coogan, M.P., El Husseini, N., Dunham, R.A., Butts, I.A.E., 2020. Effects of cryoprotectants and freezing rates for preserving spermatogonial stem cells of blue catfish. Aquaculture America 2020. Honolulu, Hawaii
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Butts, I.A.E., Myers, J.N., Chatakondi, N., Dunham, R.A. 2020. Improving hybrid catfish production: Impacts of parental effects and gamete interactions. (Invited speaker). Aquaculture America 2020. Honolulu, Hawaii


Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Establishment of a fully equipped aquatic reproductive physiology lab [i.e. has computer assisted sperm analysis (CASA) software; cryopreservation equipment; microscopy and imaging suite] and experimental hatchery created research opportunities and "hands-on" instruction for numerous undergraduate students [via my Hatchery Management (FISH 5240) class at Auburn University], and graduate students (4 MSc, 1 PhD, 1 BSc Research Fellow). Our work was published in international peer-reviewed journals (e.g. Aquaculture, Developmental Dynamics, Fish and Shellfish Immunology), as well as in the Fish Farming News, and presented at international scientific conferences, and departmental seminars. We also communicated our findings to catfish farmers in the Southeastern United States to foster ties between Auburn University and the local aquaculture industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Students (~5) in my lab learnt fundamental concepts in aquatic reproductive biology/physiology, sperm biology, hatchery science, experimental design, and data analyses. Specifically, they gain hands-on experiences with: Sperm quality assessment: Sperm motility, velocity, and motion parameters (i.e. linearity, amplitude of lateral head displacement) were quantified using a Computer Assisted Sperm Analyses (CEROS II, Hamilton Thorne Inc.) system equipped with negative phase objectives and high-resolution CCD monochrome progressive scan camera. Muse Cell Analyzer (Millipore Sigma), a robust compact flow cytometer, measured changes in mitochondrial potential, membrane viability, and oxidative stress of sperm. Students measured spermatocrit, cell density, and pH of seminal plasma. Moreover, osmolality was measured using a vapor pressure osmometer (Wescor Inc.). Scanning and transmission electron microscopes were used to explore sperm ultrastructure, while fluorescent microscopy was used to assess cell health (i.e. mitochondrial activity, membrane potential). Gamete cryopreservation: A controlled-rate freezer (Kryo 560, Planer PLC) and 50L cryogenic Dewar with transfer hose froze sperm and enabled my lab to store sperm samples in perpetuity, creating our germplasm repository for later assessment. Artificial fertilization procedures: Students learned how to inject fish and fertilize eggs using artificial fertilization techniques. Gene expression analyses: RNA was extracted from catfish eggs/embryos and later assessed for gene expression. How have the results been disseminated to communities of interest?Our research was presented at several Auburn University functions to foster ties with other faculty members. Additionally, we presented our research to catfish farmers in the Southern USA and at other University institutes, both nationally and internationally. See below for details: Institutional: Myers, J.N., Bradford, J.A., Hallas, V.S., Lawson, L.L., Pitcher, T.E., Dunham, R.A., Butts, I.A.E. 2019. The fertility games: Gamete interactions as mechanisms for sexual selection and intra-specific mate choice in freshwater catfish. Auburn University College of Agriculture (COA) Graduate Student Research Poster Showcase Nichols, Z.G., Rikard, S., Alavi, S.M.H., Walton, W.C., Butts, I.A.E. 2019. Physiological mechanisms regulating sperm motility initiation in eastern oyster, Crassostrea virginica. Auburn University College of Agriculture (COA) Graduate Student Research Poster Showcase Myers, J.N., Chatakondi, N. & Dunham, R.A., Butts, I.A.E., 2019. Genetic architecture of early life history traits for channel catfish, Ictalurus punctatus ? × blue catfish I. furcatus ? hybrid production. Student Research Symposium - AFS/USAS Auburn student subunit, Auburn, AL. Myers, J.N., Nichols, Z.G., Abualreesh, M.H., El Husseini, N., Taylor, Z.G., Coogan, M., Gurbatow, J., Vo, K.M., Zadmajid, V., Dunham, R.A., Butts, I.A.E. 2019. Impact of sperm to egg ratio on hatching success for hybrid channel catfish (Ictalurus punctatus)?× blue catfish (I. furcatus)?embryos. This is Research Student Symposium, Auburn, AL. National: Myers, J.N., Chatakondi, N. & Dunham, R.A., Butts, I.A.E. 2019. Genetic architecture of early life history traits for channel catfish, Ictalurus punctatus ? × blue catfish I. furcatus ? hybrid production. American Fisheries Society and The Wildlife Society Joint Conference. Neva, NV. Myers, J.N., Chatakondi, N. Butts, I.A.E., 2019. Genetic architecture of early life history traits for channel catfish, Ictalurus punctatus ? × blue catfish I. furcatus ? hybrid production. Alabama Chapter of the American Fisheries Society, Spanish Fort, Alabama Gurbatow, J., Abualreesh, M.H., Alston, V., Hettiarachchi, D., Vo, K., Bangs, M., Weng Chen, C.C., Butts, I.A.E., Dunham, R.A. 2019. Xenogenic and stem cell transplantation success in channel catfish Ictalurus punctatus. World Aquaculture Society 2019, New Orleans, Louisiana Butts, I.A.E., Politis, S.N., Sørensen, S.R. Tomkiewicz, J. 2019. Recent progress towards closing the life cycle of European eel, Anguilla anguilla in captivity: physiology of embryos and larvae. (Invited speaker). World Aquaculture Society 2019, New Orleans, Louisiana Butts, I.A.E., Myers, J.N., Chatakondi, N. 2019. Genetic architecture of early life history traits for channel catfish, Ictalurus punctatus ? × blue catfish I. furcatus ? hybrid production (Invited speaker). World Aquaculture Society 2019, New Orleans, Louisiana Dunham, R.A., Alston, V., Hettiarachchi, D., Vo, K., Abualreesh, M.H., Gurbatow, J., Bangs, M., Weng Chen, C.C., Butts, I.A.E. 2019. Effects of donor-recipient developmental stage and stem cell preparation on transplantation and xenogenesis success in catfish. World Aquaculture Society 2019, New Orleans, Louisiana International: Alavi, S.M.H., Hatel, A., Butts, I.A.E., Cosson, J., Babiak. 2019. Sperm morphology and motility signaling in Atlantic cod. The 7th International Workshop on the Biology of Fish Gametes, Rennes, France Butts, I.A.E., Hilmarsdóttir, G.S., Zadmajid, V., Krüger-Johnsen, M., Politis, S., Støttrup, J., Jacobsen, C., Gallego, V., Asturiano, J.F., Tomkiewicz, J. 2019. Dietary amino acids impact sperm performance traits for a catadromous fish. The 7th International Workshop on the Biology of Fish Gametes, Rennes, France Politis, S.N., Syropoulou, E., Benini, E., Sørensen, S.R., Butts, I.A.E., Miest, J.J., Mazurais, D., Servili, D., Zambonino-Infante, J.-L., Tomkiewicz, J. Salinity reduction benefits European eel larvae: An overview. Aquaculture European 2019, Berlin, Germany Kottmann, J., Butts, I.A.E., Jørgensen, M.G.P., Ravn, P., Tomkiewicz, J. Effects of exogenous gonadotropin type (carp vs. salmon pituitary extract) on offspring quality in European eel. Aquaculture European 2019, Berlin, Germany Benini, E., Politis, S.N., Sørensen, S.R., Engrola, S., Butts, I.A.E., Tomkiewicz, J. Use of probiotic, prebiotic, and symbiotic additives for priming the gut of European eel larvae. Aquaculture European 2019, Berlin, Germany What do you plan to do during the next reporting period to accomplish the goals?Below, I highlight our plans for the next reporting period. Hybrid catfish xenogenesis - We will continue our work on catfish xenogenesis (project supported by Agriculture and Food Research Initiative Competitive Grant no. 2018-67015-27614 from the USDA National Institute of Food and Agriculture. Specifically, we will identify the life stage yielding the greatest percentage and quantity of stem cells, identify life stages and techniques for spermatogonial cell implantation giving the greatest transplantation and xenogenesis rate, and continue to develop dependable protocols for freezing/thawing spermatogonia. Bivalve work - The oyster industry along the Gulf of Mexico is farming its way to an "oyster revolution", while freshwater mussel biodiversity is declining at an unprecedented rate. Therefore, we will continue to (i) unravel the physiological mechanisms regulating sperm motility initiation for Eastern oyster and (ii) quantify sperm production and morphology for two unionid freshwater mussels, Ligumia subrostrataandLampsilis straminea, throughout the natural spawning season.

Impacts
What was accomplished under these goals? Wild-caught fish stocks are declining, and as the world population grows, the demand for high-quality protein is increasing. New research shows that fish, crustaceans, molluscs, and other aquatic animals account for about 17% of total animal protein consumed by the global population. At present, global production of all aquatic animals is estimated at 171 million tonnes (first-sale value of $232 billion USD), with aquaculture or fish farming representing nearly half (47-53%) of the total harvest. Aquaculture is therefore playing a pivotal role in global food security, the environment, and human health. Because the aquaculture sector is rapidly growing and is relatively new compared to the livestock sector, it offers tremendous opportunities for technological innovation to further increase sustainability to meet protein demands. Thus, this Hatch Proposal aims to support this industry, with focus on hybrid catfish, Eastern oyster, eel, and other key aquatic species. To accomplish this, we take an interdisciplinary approach (e.g. combine physiology, molecular biology, developmental biology, and behavior) to understand and control processes that impact gamete quality and offspring performance, especially during the "critical" early life history stages (i.e. egg to early juveniles), when mortality is unusually high. Specifically, for catfish, we assessed the impacts of different sperm densities on hatching success in order to optimize resources partitioned from each male. Technical aspects of fertilization were then assessed to decipher the importance of gamete interactions (sperm and ovarian fluid) between males and females during sperm activation. Next, a full-factorial breeding design was conducted between distinct male and female pairings at two different temperatures to quantify maternal and paternal effects for embryonic developmental stages until the first feeding stage. Thereafter, we identified genes that were differentially expressed between high-quality and low-quality egg batches. Next, for Eastern oyster, we examined physiological mechanisms of sperm motility signaling to improve fertility in hatcheries and optimize cryopreservation protocols. Finally, our findings on European eel add evidence that certain amino acids regulate milt biochemistry, and that male ejaculate traits may be promoted by amino acid intake. Overall, this work will allow us to equip the aquaculture industry with technological innovation and assisted reproductive technologies (ARTs) to fertilize eggs with fresh or frozen-thawed gametes as basis for hatchery and aquaculture production. Specific objectives: We support development of aquaculture, fisheries management, and conservation biology. To accomplish this our objectives are to (1) understand and control processes that impact gamete quality, fertility, and early offspring performance and (2) optimize cryopreservation protocols for conserving genes of threatened/economically important species. In this regard, I highlight some of our objectives and results. Objective 1 - Hybrid catfish (gamete interactions): Ovarian fluid has been shown to alter the swimming behavior of fish sperm, making it a determining factor for fertility and a potential mechanism for cryptic female choice. With the expansion of the channel × blue hybrid catfish industry, it is becoming essential to understand the magnitude of these gametic interactions so that the industry can maximize fertility outcomes. Here, we aimed to i) activate blue catfish sperm with/without channel catfish ovarian fluid to determine impacts on sperm performance and ii) assess if sperm behaves differently when activated in the ovarian fluid from individual females. In all cases, ovarian fluid treatments always induced increased sperm longevity, and male × female interactions were highly significant, suggesting that channel catfish ovarian fluid differentially enhances blue catfish sperm performance. This information has important implications for developing biotechnological techniques to preserve gametes and successfully fertilize and rear embryos in an artificial environment. Objective 1 - Hybrid catfish (molecular determinants of egg/embryo quality): High variability in egg quality of spawning catfish females exists due to many factors and can be challenging to assess. In some hatchery species, egg quality and generation of viable embryos is correlated to abundances of specific mRNAs. Channel catfish females show considerable extremes in egg quality, causing inconsistencies in hybrid fry production. Thus, our objectives were to examine mRNAs linked to egg and embryo quality and determine expression profiles between low-hatch and high-hatch eggs batches through development. Here, RNA was extracted from eggs/embryos of nine females and real-time q-pCR was used to quantify relative gene expression. Relative gene expression of eight transcripts were up-regulated in the high-hatch group and peaked at the neurulation stage, indicating the importance these gene products at this threshold to normally progress until hatch. Due to lack of expression during earlier stages, maternally derived mRNAs for these genes do not seem to impact early embryonic development. By pairing these molecular markers with physical indicators of egg quality, it may enhance hybrid catfish aquaculture by eliminating problems associated with poor egg quality. Objective 1 - Eastern oyster (sperm studies): Many oyster farms artificially spawn broodstock to produce seed. Unfortunately, hatcheries have reported poor gamete quality and larval success. In order to improve hatchery production, a better understanding of oyster gamete biology is needed. Eastern oyster males and females release sperm and oocytes into the water column to be fertilized. Upon release, sperm is exposed to the external environment in which sperm motility is initiated. However, physical and chemical properties of the environment can affect sperm motility performances, which is a key determinant for fertilization success. Thus, providing the optimal environmental condition will maximize sperm activity and fertilization success. Therefore, the objectives of our work were to determine the physiological mechanisms regulating sperm motility initiation in this species. Sperm were activated with artificial sea water (ASW) buffered to make a pH gradient from pH 6.5 to 10.5. Sperm were also activated across a range of salinities from 4 to 32 ppt. ASW was tested with 0.5 to 3.5 mM EGTA to find the threshold of extracellular Ca2+ ions needed to initiate sperm motility. Na+, K+, Mg2+, and Ca2+ free ASW and their respective channel blockers were also used to elucidate ionic signaling involved in sperm motility initiation. Our preliminary results show that environmental salinity and pH affect sperm motility initiation and indicate that sperm motility initiation is Ca2+-dependent and requires K+ exchange through the plasma membrane. Overall, this provides insights into physiological mechanisms of sperm motility signaling in bivalves and provides valuable information to improve fertility in hatcheries and optimize cryopreservation protocols. Objective 2 - Blue catfish: The ability to store blue catfish stems cells in perpetuity for future xenogeneic applications requires in-depth knowledge of the mechanisms which govern cell survival during the freezing and thawing processes. Here, we have not only developed "cutting edge techniques" to preserve genetic resources, but also defined underlying physiological principles that influence gamete survival during low-temperature storage. Specifically, in 2018 we tested different cryoprotective agents that reduced cell damage during the freezing and thawing process. Now in 2019, we used these best cryo-agents to optimize cell freezing rates. This was done using a control-rate freezer, where freezing rates of 0.5, 1.0, 5.0, and 10.0oC were tested. Results are currently being analyzed.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Zadmajid, V., Falahipor, E., Ghaderi, E., S�rensen, S.R., Butts, I.A.E. 2019. Outcomes of in vitro fertilization with frozen-thawed sperm: An analysis of post-thaw recovery of sperm, embryogenesis, offspring morphology, and skeletogenesis for a cyprinid fish. Developmental Dynamics, 248: 449-464
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Zadmajid, V., Myers, J.N., S�rensen, S.R., Butts, I.A.E. 2019. Ovarian fluid and its impact on sperm performance in fish: A review. Theriogenology, 132: 144-152
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Miest, J.J., Politis, S.N, Tomkiewicz, J., Butts, I.A.E. 2019. Molecular ontogeny of larval immunity in European eel at variable temperatures. Fish and Shellfish Immunology, 87: 105-119
  • Type: Other Status: Published Year Published: 2019 Citation: Myers, J.N., Dunham, R.A., Chatakondi, N.G., Butts, I.A.E., 2019. Hybrid catfish aquaculture: Dads really do matter. Alabama Fish Farming Center - Fish Farming News, 2019(2): 7-8
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Alavi, S.M.H., Hatel, A., Butts, I.A.E., Cosson, J., Babiak. 2019. Sperm morphology and motility signaling in Atlantic cod. The 7th International Workshop on the Biology of Fish Gametes, Rennes, France
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Myers, J.N., Chatakondi, N. & Dunham, R.A., Butts, I.A.E. 2019. Genetic architecture of early life history traits for channel catfish, Ictalurus punctatus ? � blue catfish I. furcatus ? hybrid production. American Fisheries Society and The Wildlife Society Joint Conference. Neva, NV.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Butts, I.A.E., Hilmarsd�ttir, G.S., Zadmajid, V., Kr�ger-Johnsen, M., Politis, S., St�ttrup, J., Jacobsen, C., Gallego, V., Asturiano, J.F., Tomkiewicz, J. 2019. Dietary amino acids impact sperm performance traits for a catadromous fish. The 7th International Workshop on the Biology of Fish Gametes, Rennes, France
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Myers, J.N., Chatakondi, N. Butts, I.A.E., 2019. Genetic architecture of early life history traits for channel catfish, Ictalurus punctatus ? � blue catfish I. furcatus ? hybrid production. Alabama Chapter of the American Fisheries Society, Spanish Fort, Alabama
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Gurbatow, J., Abualreesh, M.H., Alston, V., Hettiarachchi, D., Vo, K., Bangs, M., Weng Chen, C.C., Butts, I.A.E., Dunham, R.A. 2019. Xenogenic and stem cell transplantation success in channel catfish Ictalurus punctatus. World Aquaculture Society 2019, New Orleans, Louisiana
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Butts, I.A.E., Politis, S.N., S�rensen, S.R. Tomkiewicz, J. 2019. Recent progress towards closing the life cycle of European eel, Anguilla anguilla in captivity: physiology of embryos and larvae. (Invited speaker). World Aquaculture Society 2019, New Orleans, Louisiana
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Zadmajid, V., S�rensen, S.R., Butts, I.A.E. 2019. Morphological and histological atlas of embryonic and early larval development in wild-caught Capoeta damascina. Journal of Morphology, 280: 133-148
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Butts, I.A.E., Myers, J.N., Chatakondi, N. 2019. Genetic architecture of early life history traits for channel catfish, Ictalurus punctatus ? � blue catfish I. furcatus ? hybrid production (Invited speaker). World Aquaculture Society 2019, New Orleans, Louisiana
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Dunham, R.A., Alston, V., Hettiarachchi, D., Vo, K., Abualreesh, M.H., Gurbatow, J., Bangs, M., Weng Chen, C.C., Butts, I.A.E. 2019. Effects of donor-recipient developmental stage and stem cell preparation on transplantation and xenogenesis success in catfish. World Aquaculture Society 2019, New Orleans, Louisiana


Progress 11/29/17 to 09/30/18

Outputs
Target Audience:Establishment of a fully-equipped aquatic reproductive physiology lab and experimental hatchery created research opportunities and "hands-on" instruction for numerous undergraduate students [via my Hatchery Management (FISH 5240) class and Principles of Aquaculture (FISH 5210) class at Auburn University], graduate students (4 MSc, 3 PhDs), and an International Visiting Scientist. Our work was published in international peer-reviewed journals (e.g. Frontiers in Physiology, PLOS ONE) and presented at international scientific conferences, and departmental seminars, both nationally and internationally (i.e. Ocean University of China; Zhejiang Institute of Freshwater Fisheries). We also communicated our findings to catfish farmers in Alabama to foster ties between Auburn University and the local aquaculture industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Students (~5) in my lab learnt fundamental concepts in aquatic reproductive biology/physiology, sperm biology, hatchery science, experimental design, and data analyses. Specifically, they gain hands-on experiences with: Sperm quality assessment: Sperm motility, velocity, and motion parameters (i.e. linearity, amplitude of lateral head displacement) were quantified using a Computer Assisted Sperm Analyses (CEROS II, Hamilton Thorne Inc.) system equipped with negative phase objectives and high-resolution CCD monochrome progressive scan camera. Muse Cell Analyzer (Millipore Sigma), a robust compact flow cytometer, measured changes in mitochondrial potential, membrane viability, and oxidative stress of sperm. Students measured spermatocrit, cell density, and pH of seminal plasma. Moreover, osmolality was measured using a vapor pressure osmometer (Wescor Inc.), while antioxidant capacity was measured using a microplate reader. Scanning and transmission electron microscopes were used to learn about sperm ultrastructure. Gamete cryopreservation: A controlled-rate freezer (Kryo 560, Planer PLC) and 50L cryogenic Dewar with transfer hose froze sperm using state-of-the-art. The MVE 815P-190F-GB liquid nitrogen storage system (CRYO Associates Inc.), enabled my lab to store sperm samples in perpetuity, creating our germplasm repository for later assessment. Artificial fertilization procedures: Students learned how to inject fish and fertilize eggs using artificial fertilization techniques. How have the results been disseminated to communities of interest?Our research was presented at several Auburn University functions to foster ties with other faculty members. Additionally, we presented our research to catfish farmers in the Southern USA and at other University institutes, both nationally and internationally. See below for details: 1. Butts, I.A.E., Alston, V., Hettiarachchi, D., Vo, K., Abualreesh, M., Gurbatow, J., Bangs, M., Weng Chen, C.C., Dunham, R.A. 2018. Effects of donor-recipient developmental stage and stem cell preparation on transplantation and xenogenesis success in catfish. 2018 AAES Faculty Summit and Mixer. Auburn, Alabama 2. Butts, I.A.E., Alston, V., Hettiarachchi, D., Vo, K., Abualreesh, M., Gurbatow, J., Bangs, M., Weng Chen, C.C., Dunham, R.A. 2018. Effects of donor-recipient developmental stage and stem cell preparation on transplantation and xenogenesis success in catfish. 2018 Faculty Research Symposium poster session. Auburn, Alabama 3. Aquatic Reproductive Physiology and Hatchery Science Laboratory. Annual Alabama Catfish Update Meeting. Demopolis, Alabama 4. Seminar for University of Texas at Austin - Marine Science Institute. Title: Creating fertility clinics for aquatic species: Eels, salmon, and catfish 5. Seminar for the Auburn University College of Veterinary Medicine. Title: Creating fertility clinics for aquatic species 6. Seminar for Zhejiang Institute of Freshwater Fisheries, Zhejiang, China. Title: From basic science to applied aquaculture for externally fertilizing fishes 7. Seminar for Ocean University of China, Qingdao, China. Title: Creating fertility clinics for aquatic species: eels, salmon, and catfishes What do you plan to do during the next reporting period to accomplish the goals?Below, I highlight our plans for the next reporting period. Sperm biology and gamete interactions - Rationale: DNA fragmentation and membrane integrity, such as the proportion of intact cell membranes, are likely useful biomarkers for sperm fertility diagnosis. For these biomarkers to be useful diagnostically, they must have threshold values which provide discriminatory power for fertility and embryonic development. Objectives and Methods: We will define relationships between DNA fragmentation and membrane integrity and evaluate how these biomarkers contribute to sperm form/function and embryonic development using catfish as the model organism. Sperm will be extracted from >10 blue catfish males. We will use single cell gel electrophoresis or "Comet assay" in conjunction with fluorescent microscopy to study single-strand or double-strand DNA damage. Sperm membrane integrity and oxidative stress will be quantified using flow cytometry, while CASA will measure sperm form and function; sperm will be activated in water alone, in ovarian fluid, or near an egg. Channel catfish eggs will be fertilized using a known quantity of sperm and ovarian fluid. Fertility and embryonic survival will then be linked to this suite of biomarkers using multivariate selection analyses and statistical modeling. Hybrid catfish xenogenesis - Will continue work our work on catfish xenogenesis (project supported by Agriculture and Food Research Initiative Competitive Grant no. 2018-67015-27614 from the USDA National Institute of Food and Agriculture. Specifically, we will identify the life stage yielding the greatest percentage and quantity of stem cells and continue to develop a dependable protocol for freezing/thawing spermatogonia. European eel - Rationale: Establishment of European eel larval rearing protocols is challenged by lack of insights on biology and ecology of yolk-sac and leptocephalus stages. These bottlenecks may be overcome through systematic, targeted research on factors influencing growth and survival of eel larvae. Objectives and Methods: Will assess biochemical composition of larvae and evaluate potential biomarkers of larval robustness; gain insights into the ontogeny of the larval digestive system and digestive capacity; understand larval nutritional requirements by measuring nutrient metabolism and development of digestive capacity at key developmental stages; and evaluate how pre- and probiotics modulate larval performance. Ovarian fluid meta-analysis - Rationale: Studies have reported that the presence of fish ovarian fluid has an impact on sperm performance traits, but the magnitude of this effect has not yet been quantified within and across taxa. Meta-analysis is a quantitative approach used to systematically assess the results of individual studies to derive an estimate of the overall effect size using statistical methods. The results can be used to make innovative conclusions about effects in which there is variation and/or conflicting reports between individual studies. Objectives and Methods: Here, we will determine statistically if there is a positive effect of ovarian fluid on sperm performance traits for all fish species, and then independently for marine and freshwater species.

Impacts
What was accomplished under these goals? Aquaculture production has increased to meet consumer demands and preserve/restore populations of threatened/endangered species (termed conservation aquaculture). For instance, in 2016, global aquaculture for food fish has surpassed 80 million tonnes, with an estimated value of $231.6 billion USD. In the United States, catfish farming in Mississippi, Arkansas, Alabama, and Texas accounts for nearly 70% of the total freshwater aquaculture production, where the channel catfish female ´ blue catfish male hybrid constitutes the majority (50-75%) of the harvest. Moreover, the oyster industry along the Gulf of Mexico is farming its way to an "oyster revolution", while freshwater mussel biodiversity is declining at an unprecedented rate. This Hatch Proposal aims to support these aquatic industries, among others. To accomplish this, we take an interdisciplinary approach (e.g. combine physiology, developmental biology, behavior) to understand and control processes that impact gamete quality and offspring performance, especially during the "critical" early life history stages (i.e. egg to early juveniles), when mortality is unusually high. Thus far, we have provided evidence that paternity and maternity act independently as well as interactivity to influence early life history (ELH) traits (e.g. embryonic survival, hatching success, larval morphology) for hybrid catfish. Hybrid catfish fertilization procedures were also standardized, where preliminary results suggest that 1.0 × 104:1 sperm to egg is optimal for embryo production. Moreover, initial protocols for extraction and freezing-thawing of blue catfish spermatogonial stem cells were established to increase efficiency of reproduction for catfish hybridization. Variation in morphology and performance (e.g. survival, deformity rate) during ELH and inter-linked expression of targeted genes (e.g. growth, stress, immune genes) were quantified for the endangered European eel, a targeted species for aquaculture and conservation, while another series of studies assessed the impact of endocrine factors on gamete quality from wild and/or farmed breeders to improve hatchery production. Together, this work will allow us to equip the aquaculture industry with technological innovation and assisted reproductive technologies (ARTs) to fertilize eggs with fresh or frozen-thawed gametes as basis for hatchery and aquaculture production. Specific objectives: We support development of aquaculture, fisheries management, and conservation biology. To accomplish this our objectives are to (1) understand and control processes that impact gamete quality, fertility, and early offspring performance and (2) optimize cryopreservation protocols for conserving genes of threatened/economically important species. In this regard, I highlight our accomplishments. Objective 1- Levantine scraper: Studies were conducted on Levantine scraper (species of conservation/economic importance) to (i) investigate the comparative effectiveness of hCG- and Ovaprim-based therapies [(D-Arg6, Pro9Net)-sGnRHa + domperidone injection] on the stimulation of male reproduction; (ii) test how 0.25, 0.5, or 1.0 mL/kg BW injections of Ovaprim affects spawning and gamete quality in females; (iii) and examine anatomical and functional development of early larval stages at a histological level with emphasis on digestive system development and organs and tissues required for foraging. Results showed that Ovaprim stimulates testicular steroidogenesis and milt production with high efficacy for acquisition of fertile sperm in captivity, while 0.5 mL/kg dosage is recommended for assisted reproduction of females. Findings are expected to provide information for improving wild broodstock management and implementation of assisted reproduction therapies for acquisition of high-quality gametes. Objective 1- Hybrid channel catfish: Worked on hybrid channel catfish with USDA in Stoneville MS. Here, we used a genetic breeding design to assess genetic, environmental, and gene by environmental interactions, which allow us to detail the genetic architecture of fitness in the hatchery. Sires were mated in all combinations with a set of dams and 5 sire × 4 dam families were generated. Offspring from each family were split into 2 temperature-controlled environments, based on conditions that mimic early (26.6oC) and late (32.2oC) seasonal environments. Embryonic survival, hatching success, as well as larval morphology and deformities were quantified at various timepoints during early ontogeny. Thus far, we have provided evidence that paternity and maternity act independently as well as interactivity to influence ELH traits (e.g. embryonic survival, hatching success, and larval morphology). In a second study, we standardized fertilization procedures for hybrid catfish. Specifically, we wanted to determine the minimum quantity of blue catfish sperm that is required to fertilize channel catfish eggs in order to maximize hatching under controlled conditions. Peliminary results suggest that 1.0 × 104:1 sperm to egg is optimal for hybrid catfish production. This information has important implications for the long-term sustainable development of hybrid catfish for aquaculture purposes as well as for understanding genetic variation during early life history in fishes. Objective 1- European eel: (i) Explored development of endocrine systems regulating appetite, ingestion, and digestion by targeted gene expression; (ii) molecularly defined the first-feeding window; (iii) examined the potential benefit of green-water during endogenous feeding; (iv) investigated the effect of initiating exogenous feeding on larval biometry, nutritional conditions, and gene expression; (v) investigated how salinity affects larval biometry, deformities, and survival as well as expression profiles of genes related to processes involved in or affected by osmoregulation; (vi) followed expression of thyroid hormone genes and investigated how temperature affects expression of those genes from hatch until first-feeding; (vii) and identified how maternal origin and nutritional profiles impact ELH traits. Together, these results provide much needed information to further develop and establish captive breeding techniques and technologies for this critically endangered fish species. Objective 2 - Hybrid channel catfish: We have not only developed "cutting edge techniques" to preserve genetic resources, but also defined underlying physiological principles that influence gamete survival during low-temperature storage. Specifically, immature testes were collected from blue catfish, washed, cut into small portions) pooled, and then distributed into cryovials containing cryomedia. The cryomedia was then supplemented with cryoprotective agents that reduce cell damage during the freezing and thawing process. These agents are classified as permeating or non-permeating based on their ability to penetrate through cell membranes. Thus, firstly, using a 3 × 3 factorial design, testicular tissues were frozen in cryomedia containing different permeating cryoprotectants (dimethyl sulfoxide, methanol, ethylene glycol) at varying concentrations (1.0, 1.3, 1.6 M). Treatments were equilibrated on ice for 60 min, frozen using a control-rate freezer, and then transferred directly into liquid nitrogen. Testes were later thawed in a water bath, rehydrated, and trypsinized. Post-thaw cell viability was then assessed, and results showed 1 M DMSO was optimal. In the second study, we assessed the effects of non-permeating cryoprotectants (D-lactose, D-(+)-trehalose dehydrate) at varying concentrations (0, 0.1, 0.2 M) in conjunction with fresh hen egg yolk (10% v/v) or bovine serum albumin (1.5% w/v). Samples were thawed and assessed, as above. Results are currently being analyzed. Together, these cryogenic protocols will be instrumental for improving the efficiency for hybrid catfish breeding and production.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Zadmajid, V., & Butts, I.A.E. 2018. Spawning performance, serum sex steroids, and ovarian histology in wild-caught Levantine scraper, Capoeta damascina treated with various doses of Ovaprim⿢ [(D-Arg6, Pro9NEt)-sGnRH + domperidone]. Journal of Animal Science, 96: 5253-5264
  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Politis, S.N., Sørensen, S.R., Mazurais, D., Servili, A., Zambonino-Infante, J-L., Miest, J.J., Clemmesen, C.M., Tomkiewicz, J., & Butts, I.A.E. 2018. Molecular Ontogeny of First-Feeding European Eel Larvae. Frontiers in Physiology, 9:1477. doi: 10.3389/fphys.2018.01477
  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Benini, E., Politis, S.N., Kottmann, J., Butts, I.A.E., Sørensen, S.R., & Tomkiewicz, J. 2018. Effect of parental origin on early life history traits of European eel. Reproduction in Domestic Animals, 53: 1149-1158
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Zadmajid, V., Mirzaee, R., Hoseinpour, H., Vahedi, N., & Butts, I.A.E. 2017. Hormonal induction of ovulation using salmon gonadotropin-releasing hormone analogue (D-Arg6, Pro9-NEt) with dopamine antagonist and its impact on embryonic development of wild-caught Longspine scraper, Capoeta trutta (Heckel, 1843). Animal Reproduction Science, 187: 79-90
  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Politis, S.N., Servili, A., Mazurais, D., Zambonino-Infante, J.-L., Miest, J.J., Tomkiewicz, J., & Butts, I.A.E. 2018. Salinity reduction benefits European eel larvae: Insights at the morphological and molecular level. PLoS ONE 13(6): e0198294. https://doi.org/10.1371/journal.pone.0198294
  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Zadmajid, V., Bashiri, S., Sharafi, N., & Butts, I.A.E. 2018. Effect of hCG and Ovaprim on reproductive characteristics of male Levantine scraper, Capoeta damascina. Theriogenology, 115: 45-56
  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Politis, S.N., Servili, A., Mazurais, D., Zambonino-Infante, J.-L., Miest, J.J., Tomkiewicz, J., & Butts, I.A.E. 2018. Temperature induced variation in gene expression of the thyroid hormone pathway of European eel larvae. General and Comparative Endocrinology, 259: 54-65
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Politis, S.N., Mazurais, D., Servili, A., Zambonino-Infante, J.-L., Miest, J.J., Sørensen, S.R., Tomkiewicz, J., & Butts, I.A.E. 2017. Temperature impacts gene expression and morphological development of European eel, Anguilla anguilla larvae. PLOS ONE, 12(8): e0182726. https://doi.org/10.1371/journal.pone.0182726
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Butts, I.A.E ., Prokopchuk, G., Kaspar, V., Cosson, J., & Pitcher, T.E. 2017. Ovarian fluid impacts flagella beating patterns and biomechanical metrics of spermatozoa between alternative reproductive tactics. Journal of Experimental Biology, 220: 2210-2217 [This paper was highlighted in ⿿Inside JEB⿝. Title: Jack Chinook⿿s sperm beat hooknose⿿s to the ultimate reward by Kathryn Knight. Journal of Experimental Biology (2017) 220: 2129-2131
  • Type: Other Status: Other Year Published: 2018 Citation: Butts, I.A.E., Alston, V., Hettiarachchi, D., Vo, K., Abualreesh, M., Gurbatow, J., Bangs, M., Weng Chen, C.C., & Dunham, R.A. 2018. Effects of donor-recipient developmental stage and stem cell preparation on transplantation and xenogenesis success in catfish. 2018 AAES Faculty Summit and Mixer. Auburn, Alabama
  • Type: Other Status: Other Year Published: 2018 Citation: Butts, I.A.E., Alston, V., Hettiarachchi, D., Vo, K., Abualreesh, M., Gurbatow, J., Bangs, M., Weng Chen, C.C., & Dunham, R.A. 2018. Effects of donor-recipient developmental stage and stem cell preparation on transplantation and xenogenesis success in catfish. 2018 Faculty Research Symposium poster session. Auburn, Alabama
  • Type: Other Status: Other Year Published: 2018 Citation: Butts, I.A.E. 2018. Aquatic Reproductive Physiology and Hatchery Science Laboratory. 2018 Annual Alabama Catfish Update Meeting. Demopolis, Alabama
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Butts, I.A.E., Prokopchuk, G., Kašpar, V., Cosson, J., & Pitcher, T.E. 2017. Ovarian fluid impacts flagella beating and biomechanical metrics of sperm between alternative reproductive tactics. 6th International Workshop on the Biology of Fish Gametes. ?eské Bud?jovice and Vod?any, Czech Republic