Progress 12/01/08 to 09/30/13
Outputs
Target Audience: Target audiences are members of the scientific community; agencies involved with biomedical research, aquaculture, biodiversity, and endangered-species conservation; and oyster aquaculture farmers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The Zebrafish International Resources Center (ZRIC) houses tens of thousands of zebrafish mutant lines which serves the international zebrafish community. My three-week visit to ZIRC allowed completion of collaborative experiments and provided experience in the whole process of zebrafish husbandry -- hatchery, feeding, disease control, and recovery of mutant lines from cryopreserved sperm through in vitro fertilization. As an international resource center, ZIRC has a routine extension program for training staff from different zebrafish laboratories. The sperm freezing protocols developed and improved from this project are part of the training materials. In addition, this project provides opportunities for undergraduate student workers to be trained in practices of zebrafish husbandry, hatchery, feeding, use of circulation systems, and water quality analysis. Professional development included attending webinars for introduction to the aquaculture programs of the U.S. Department of Agriculture, the National Science Foundation, and the Gulf of Mexico Coastal Restoration program of the U.S. Environmental Protection Agency, which provide external funding opportunities. How have the results been disseminated to communities of interest? Publications in peer-reviewed journals, conference abstracts and presentations, and project annual reports. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1) Major activities completed: For zebrafish, protocol and pathway refinement was improved. For Xiphophorus fishes, establishment of sperm repositories was initiated. For oysters, self-fertilization inbred oyster larvae were harvested and confirmed by using protocols for non-lethal sperm collection and cryopreservation established in previous years. Specific objectives fulfilled were: 1) Refinement and improvement of the protocol for sperm cryopreservation in the zebrafish; 2) quality control for sperm samples at each step of the freezing process, including use of flow cytometry for measuring membrane integrity and use of computer-assisted sperm analysis for sperm motility; 3) development of sperm repositories for Xiphophorus fishes with strategies suitable for these fishes; 4) development of non-lethal sperm collection and cryopreservation techniques in oysters, and application of these techniques for self-fertilization inbred production, and 5) development of a preliminary protocol for sperm cryopreservation of tetraploid eastern oysters. 2) Significant results achieved, including major findings, developments, or conclusions: 1) Analysis of fresh zebrafish sperm stored with four different buffer systems -- NaCI, Hanks’ balanced salt solution (HBSS), Ca2+-free HBSS, and glucose at four osmolalities of 150, 300, 600, and 1200 mOsmol/kg -- for up to 72 hr showed that buffer type, osmolality, and storage time were all significant factors (P = 0.000) affecting sperm motility and plasma membrane integrity; interactions among factors also were significant (P = 0.000). Of the four types of solutions tested, glucose, at all of the osmolalities tested, immediately caused significantly lower sperm motility than the other buffers (P ≤ 0.032). NaCl caused differences in sperm motility only at 300 mOsmol/kg (P ≥ 0.159) for up to 2 hr. HBSS and Ca2+-free HBSS, at an osmolality of 300 mOsmol/kg (P ≥ 0.218), showed the best capacity to extend sperm motility and membrane integrity, and no differences were found in motility of sperm suspended in HBSS and Ca-free HBSS at each osmolality and storage time (P ≥ 0.997). 2) A protocol for determination of sperm concentration with flow cytometry was developed. This method of measuring sperm concentration is efficient, accurate and fast, and can simultaneously analyze sperm membrane integrity. This approach can also maximize the use of valuable sperm samples. 3) The eastern oyster Crassostrea virginica can change sex which makes self-fertilization possible if sperm can be cryopreserved. To create self-fertilized inbred lines of oysters by use of non-lethal sperm collection and cryopreservation established in our previous studies, small (~1 yr old) and large (~2-3 yr old) oysters were biopsied for sperm collection. Survival of biopsied oysters after 1 yr was 50% for small oysters, and 17% for large oysters. Oocytes were collected from sex-reversed females and self-fertilized with cryopreserved sperm. Of the 24 cryopreserved samples, 14 individuals had ≤ 1% fertility when crossed with oocytes from unrelated females, indicating that the cryopreserved sperm had reduced fertility. The other 10 individuals had a fertility of 39 ± 25% when crossed with oocytes from unrelated females (non-selfing) but showed a significantly lower success of self-fertilization (12 ± 16 %) (P = 0.008), while aliquots of the same oocytes had a fertilization of 83 ± 11% when fertilized with fresh sperm. Larvae were produced at day 3 in self-fertilized families (12-94% of the fertilized oocytes) and survived to the eyed-larvae stage at days 11-14. Genotyping with 9 microsatellite markers confirmed that the larvae resulted from self fertilization in 4 families. 3) Key outcomes or other accomplishments realized: 1) A non-lethal method for sperm collection from eastern oyster was developed and one manuscript was published; 2) one conference presentation was made; and 3) a manuscript on production of self-fertilized inbred larvae of oysters by use of non-lethal sperm collection and cryopreservation was submitted for review.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Yang H, Supan J, Guo X, Tiersch RT (2013) Non-lethal Sperm Collection and Cryopreservation in the Eastern Oyster Crassostrea virginica. Journal of Shellfish Research, 32, 429-437.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Yang H, Walter RB, Tiersch TR (2013) Strategies for establishment of sperm repositories for Xiphophorus fishes. In: Abstracts of Aquaculture 2013, Nashville, Tennessee.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Results of experiments conducted during 2012 were disseminated through publication in scientific journals and presentations at academic meetings including Aquaculture America, the International Symposium on Genetics in Aquaculture, the American Fisheries Society, and the Gulf Coast Conservation Biology Symposium. In addition, research results were disseminated through annual progress reports to funding agencies. PARTICIPANTS: Huiping Yang (PI), T.R. Tiersch, LSU AgCenter; Dr. DaCosta from Ivory Coast; and collaborators with Louisiana Sea Grant (Dr. J. Supan), Rutgers University (Dr. X. Guo), Zebrafish International Resource Center (Dr. Z. Varga and C. Carmichael), and the Xiphophorus Genetic Stock Center (Dr. R. Walter). TARGET AUDIENCES: Target audiences are members of the scientific community; agencies involved with biomedical research, aquaculture, biodiversity and endangered-species conservation; and aquaculture farmers. PROJECT MODIFICATIONS: No changes were made.
Impacts
In research with biomedical fishes, including zebrafish, medaka and Xiphophorus, flow cytometry was used to refine protocols that were previously established to analyze effects of osmotic pressure, storage time, and concentration of cryoprotectants on sperm quality. The feasibility of establishing sperm repositories for medaka and Xiphophorus fishes also was studied. About 20 genetically modified strains and lines of medaka were processed with refined protocols; sperm viability was evaluated by computer assisted sperm analysis, flow cytometry, and fertilization; and data spreadsheets were developed. Also, three species (with 14 strains) of Xiphophorus fishes were processed for sperm repositories. In research with Fundulus grandis, effects of osmolality and calcium ions on activation of sperm motility and effects of dilution ratio on sperm motility activation and refrigerated storage were investigated. Protocols for cryopreservation of Fundulus grandis sperm were established by evaluating cryoprotectants and cooling rates. Effects of dietary lipids and salinity levels on sperm cryopreservation also were evaluated. Analysis of data from these studies is in progress. Work in shellfish research involved development of protocols for cryopreservation of eastern oyster sperm using automated, high-throughput processing equipment for sample-straw filling and sealing. Protocols developed for cryopreservation of sperm from diploid eastern oysters also were applied to cryopreservation of sperm from tetraploid oysters. Preliminary data were collected and this research will continue as long as tetraploid oysters are available. A non-lethal method of collecting sperm from eastern oyster also was developed. Natural spawning and biopsy after anesthesia or notching were evaluated for this study. Gonad biopsy after notching a total of 20 males produced 3.6E + 08 cells per male and fertilization success with thawed sperm averaged 20 percent. Self-fertilization of eastern oyster was accomplished by integrating protocols for sperm cryopreservation with non-lethal sperm collection, and larvae were produced. Genotype analysis using nine microsatellite markers confirmed that larvae were produced by self-fertilization within five families of eastern oyster.
Publications
- Yang, H., Cuevas-Uribe, R., Savage, M.G., Walter, R.B., Tiersch, T.R., 2012. Sperm Cryopreservation in live-bearing Xiphophorus Fishes: Offspring production from Xiphophorus variatus and strategies for establishment of sperm repositories. Zebrafish 9, 126-134.
- Yang, H., Savage, G.M., Hazlewood, L., Walter, R.B., Tiersch, T.R., 2012. Offspring production with cryopreserved sperm from a live-bearing fish Xiphophorus maculatus and implications with females. Comp Biochem Physiol C Toxicol Pharmacol 155, 55-63.
- Yang, H., Hu, E., Cuevas-Uribe, R., Supan, J., Guo, X., Tiersch, T.R., 2012. High-throughput sperm cryopreservation of eastern oyster Crassostrea virginica. Aquaculture 344, 223-230.
- Tiersch, T.R., Yang, H., 2012. Environmental salinity-induced shifts in sperm motility activation in Fundulus grandis. Aquaculture 324-325, 145-150.
- Yang, H., Guo, X., Tiersch, T.R., 2012. Creation of inbred lines of eastern oyster Crassostrea virginica through self-fertilization using cryopreserved sperm and eggs from the same individuals after sex reversal, Abstracts of the International Symposium on Genetics in Aquaculture XI at Auburn University, Auburn, AL.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Research on sperm cryopreservation of fish and shellfish and results of experiments conducted during 2011 were disseminated through publication in scientific journals and book chapters, and through presentations at academic meetings such as the World Aquaculture Society, American Fisheries Society, and the Gulf Region Conservation Symposium. In addition, the results were reported through annual progress reports to the related funding agencies. PARTICIPANTS: H. Yang (PI), T.R. Tiersch, LSU AgCenter; along with numerous collaborators. TARGET AUDIENCES: Target audiences are members of the scientific community; agencies involved with biodiversity and endangered-species conservation; and producers of oysters and ornamental fishes. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Protocols for sperm cryopreservation of eastern oyster were established by using high-throughput processing automated equipment for sample filling and sealing. Two types of 0.5-ml straws (French straws and CBS straws) were evaluated in this research to meet the processing requirements. With optimized protocols, cryopreserved sperm yielded fertilization percentages of 58% (plus or minus 24%) for French straws, and 54% (plus or minus 21%) for CBS straws (n = 16). The data for this research has been drafted into one manuscript, and it is currently in review by Aquaculture. Protocols developed for cryopreservation of sperm from diploid eastern oysters were applied to cryopreservation of sperm from tetraploid oysters. Preliminary data were collected, and this research will be continued as long as tetraploid oysters are available. In addition, a non-lethal method for sperm collection from eastern oyster was developed. By integrating the protocols for sperm cryopreservation with non-lethal sperm collection, cryopreserved sperm from one-year-old males have been used for fertilizing eggs from the same oyster after sex-reversal (self-fertilization). Inbreeding depression was observed for fertilization and larval survival. Analysis of the data from this research is in progress. A series of experiments was performed with Fundulus grandis for evaluating sperm motility and developing protocols for sperm cryopreservation and in-vitro fertilization. Data analysis is in progress. For biomedical fishes (zebrafish, medaka and Xiphophorus), protocols previously established were refined by using flow cytometry to analyze effects on sperm quality of osmotic pressure, storage time, and concentration of cryoprotectants. Also, the feasibility of establishing sperm repositories for medaka and Xiphophorus fishes was studied. About 20 genetically modified strains, or lines, of medaka were processed by following the strategies proposed; and sperm viability was evaluated by computer assisted sperm analysis, flow cytometry, and fertilization. This research is in progress.
Publications
- Hu E, Yang H, Tiersch TR (2011) High-throughput cryopreservation of spermatozoa of blue catfish (Ictalurus furcatus): Establishment of an approach for commercial-scale processing. Cryobiology 1: 74-82.
- Cuevas-Uribe R, Yang H, Daly J, Savage MG, Walter RB, et al. (2011) Production of F1 Offspring with Vitrified Sperm from a Live-Bearing Fish, the Green Swordtail Xiphophorus hellerii. Zebrafish 8: 167-179.
- Yang H, Tiersch TR (2011) Application of Computer-assisted Sperm Analysis (CASA) to Aquatic Species. In: Tiersch TR, Green C, editors. Cryopreservation in Aquatic Species. II ed. Baton Rouge, LA: World Aquaculture Society.
- Yang H, Tiersch TR (2011) Sperm Cryopreservation in Biomedical Research Fish Models. In: Tiersch TR, Green C, editors. Cryopreservation in Aquatic Species. II ed. Baton Rouge, LA: World Aquaculture Society.
- Yang H, Hu E, Tiersch TR (2011) High-throughput Sperm Cryopreservation for Eastern Oysters. The 32nd Annual Meeting of the American Fisheries Society Louisiana Chapter January 27-28.
- Tiersch TR, Yang H, Hu E (2011) Outlook for development of high-throughput cryopreservation for small-bodied biomedical model fishes. Comp Biochem Physiol C Toxicol Pharmacol 154: 76-81.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Research on sperm cryopreservation of fish and shellfish continued to be performed. Protocols for cryopreservation of eastern oyster sperm, with the potential for high-throughput processing using automated equipment, were established. For eastern oysters, a non-lethal method for sperm collection was developed. Results of experiments conducted during 2010 were disseminated through publication in scientific journals and book chapters, and through presentations at academic meetings such as the World Aquaculture Society, American Fisheries Society, and the Gulf Region Conservation Symposium. In addition, the results were reported through annual progress reports to the related funding agencies. PARTICIPANTS: Participants included Dr. Huiping Yang, Research Assistant Professor, Aquaculture Research Station, Louisiana State University Agricultural Center (Principal Investigator); Dr. T.R. Tiersch, Professor, Aquaculture Research Station, Louisiana State University Agricultural Center; and collaborators with Louisiana Sea Grant (Dr. J. Supan), Rutgers University (Dr. X. Guo), University of Georgia (Dr. R. Winn and M. Norris), Zebrafish International Resource Center (Dr. Z. Varga and C. Carmichael), and the Xiphophorus Genetic Stock Center (Dr. R. Walter). Two undergraduate students (Ereene Tan, Department of Biochemistry, and Jennifer Atilano, School of Renewable and Natural Resources) conducted independent research as part of this project. Graduate students E. Hu and Rafael Uribe also participated in the project. TARGET AUDIENCES: Target audiences are members of the scientific community; agencies involved with biodiversity and endangered-species conservation; and producers of oysters and ornamental fishes. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Protocols for cryopreservation of eastern oyster sperm, with the potential for high-throughput processing using automated equipment, were established. Two types of 0.5-ml straws (French straws and CBS straws) were evaluated in this research to meet the processing requirements. With optimized protocols, cryopreserved sperm yielded fertilization percentages of 58% (plus or minus 24%) for French straws, and 54% (plus or minus 21%) for CBS straws (n = 16). Additional data analysis is in progress and one manuscript is in preparation. Protocols developed for cryopreservation of sperm from diploid eastern oysters were applied to cryopreservation of sperm from tetraploid oysters. Preliminary data were collected, and this research will be continued as long as tetraploid oysters are available. For eastern oysters, a non-lethal method for sperm collection was developed, and a reasonable amount of sperm (1.00-9.00E + 08 sperm) could be obtained from 1-year-old oysters with a mean weight of 98.7 g (plus or minus 22.9 g; n = 20), shell height of 73.8 mm (plus or minus 6.1 mm), and length of 60.4 mm (plus or minus 7.4 mm). We will integrate and extend protocols for sperm cryopreservation with non-lethal sperm collection for use in oyster genetic improvement involving production of polyploid oysters, inbred lines, and preservation of natural and selectively bred genetic resources. This research will be continued in 2011. A series of experiments was performed with zebrafish using flow cytometry to analyze effects on sperm quality of osmotic pressure, storage time, and cryoprotectants. Also, parameters measured with computer-assisted sperm analysis and flow cytometry were collected and screened with fresh sperm and post-thaw sperm from large numbers of individuals to identify correlations between sperm viability after cryopreservation and fertility. These data are being analyzed and manuscripts are in preparation. Protocols for sperm cryopreservation of small-sized model fishes should be expanded for application in different laboratories for different purposes. Four types of sample packaging containers are being evaluated for real-time cooling rates at different sample loading volumes. This research is in process.
Publications
- Savolainen, L.C., Yang, H., and Tiersch, T.R. 2010. Fisheries monitoring and preservation: Computer-assisted sperm analysis (CASA). Abstracts of American Fisheries Society Louisiana Chapter, Baton Rouge, LA.
- Savolainen, L.C., Yang, H., and Tiersch, T.R. 2010. Conservation of small fishes can be facilitated with computer-assisted sperm analysis. Abstracts of Gulf Coast Conservation Biology Symposium, New Orleans, LA.
- Savolainen, L.C., Yang, H., and Tiersch, T.R. 2010. Sperm quality assessment in aquatic species: Computer-assisted sperm analysis (CASA). Abstracts of Aquaculture America 2010, San Diego, CA.
- Hu, E., Yang, H., Baxter, J., and Tiersch, T.R. 2010. Commercial-scale blue catfish sperm cryopreservation for hybrid catfish production. Abstracts of American Fisheries Society Louisiana Chapter, Baton Rouge, LA.
- Hu, E., Yang, H., Baxter, J., and Tiersch, T.R. 2010. High-throughput cryopreservation of fish sperm: Blue catfish protocol development and hatchery application. Abstracts of Aquaculture America 2010, San Diego, CA.
- Carmichael, C., Varga, Z.M., Carter, V., Hagedorn, M., Yang, H., Tiersch, T.R., and Westerfield, M. 2010. High-throughput cryopreservation: The future management tool for line acquisition and turnover of live and cryopreserved lines at the Zebrafish International Resource Center (ZIRC). Abstracts of 2010 International Zebrafish Development and Genetics Conference, Madison, WI.
- Barbosa-Solomieu, V., Hu, E., Yang, H., Tiersch, T.R., and Buchanan, J.T. 2010. Development of a standardized protocol for high-throughput cryopreservation of the sperm of Atlantic salmon Salmo salar. Abstracts of Aquaculture America 2010, San Diego, CA
- Yang, H.P., Norris, M., Winn, R., and Tiersch, T.R. 2010. Evaluation of cryoprotectant and cooling rate for sperm cryopreservation in the euryhaline fish medaka Oryzias latipes. Cryobiology, 61(2):211-219.
- Tan, E., Yang, H., and Tiersch, T.R. 2010. Determination of sperm concentration for small-bodied biomedical model fishes by use of microspectrophotometry. Zebrafish, 7(2):233-240.
- Yang, H. and Tiersch, T.R. 2010. Big problem with little fish: specific considerations for sperm cryopreservation of biomedical research model fishes. Abstracts of Aquaculture America 2010, San Diego, CA.
- Yang, H., and Tiersch, T.R. 2010. Development of protocols for zebrafish sperm cryopreservation in different packaging containers. Abstracts of Aquaculture America2010, San Diego, CA.
- Tiersch, T.R., Yang, H., Westerfield, M., Varga, Z.M., Winn, R., and Hagedorn, M. 2010. High-throughput processing for sperm cryopreservation of small-bodied model fishes. Abstracts of 2010 International Zebrafish Development and Genetics Conference, Madison, WI.
- Tiersch, T.R., Hagedorn, M., Varga, Z., Walter, R., Westerfield, M., Winn, R., and Yang, H. 2010. Workshop and panel discussion: High-throughput cryopreservation of germplasm as an exchange currency for genetic resources. Abstracts of 5th Aquatic Animal Models of Human Disease Conference, Corvallis, OR.
- Tan, E., Yang, H., and Tiersch, T.R. 2010. Determination of sperm concentration for small-bodied fishes by use of microspectrophotometry. Abstracts of Gulf Coast Conservation Biology Symposium, New Orleans, LA.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Results of experiments conducted during 2009 were disseminated through presentations at academic meetings such as the World Aquaculture Society, National Shellfisheries Association, the American Fisheries Society, and Gulf Coast Conservation Biology Symposium, and also through publication in scientific journals and book chapters. In addition, the results were reported through annual progress reports to related funding agencies. A fast, efficient method for measurement of sperm concentration was developed for small-bodied biomedical research model fishes (zebrafish, Xiphophorus, and medaka). A standardized protocol for sperm cryopreservation of medaka was developed by evaluating the toxicity of cryoprotectants and cooling rate. PARTICIPANTS: Participants include: Dr. Huiping Yang, Research Assistant Professor, Aquaculture Research Station, Louisiana State University Agricultural Center (Principal Investigator); Dr. T.R. Tiersch, Professor, Aquaculture Research Station, Louisiana State University Agricultural Center; and collaborators at Louisiana State University (Dr. J. Supan) and Rutgers University (Dr. X. Guo). Two undergraduate students (Ereene Tan, Department of Biochemistry, and Jennifer Atilano, School of Renewable and Natural Resources) conducted independent research as part of this project. TARGET AUDIENCES: Target audiences are members of the scientific community, biodiversity and endangered species conservation agencies, ornamental-fish farmers, and oyster researchers and producers. Audiences also included attendees at meetings of the World Aquaculture Society, the National Shellfisheries Association, the Louisiana chapter of the American Fisheries Society, and the Gulf Coast Conservation Biology Symposium. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A fast, efficient method for measurement of sperm concentration was developed for small-bodied biomedical research model fishes (zebrafish, Xiphophorus, and medaka). Sperm samples were collected by crushing of dissected testis and stripping of live males. Linear correlation was established between sperm concentration counted by hemocytometry and absorbance measured by microspectrophotometry, and equations were generated within the effective absorbance range. The accuracy of these equations was verified by comparison of sample concentrations counted with a hemocytometer and calculated with equations, and no significant differences (P = 0.447) were observed. The precision of techniques was verified by the consistency of measurements of serially diluted aliquots from pooled samples. This method is fast (seconds for one sample), sample-saving (1 microlter sample size), and accurate. A standardized protocol for sperm cryopreservation of medaka was developed by evaluating the toxicity of cryoprotectants and cooling rate. Results showed that methanol and 2-methoxyethanol (ME) were the least toxic to sperm cells compared to dimethyl sulfoxide (Me2SO), N, N,- dimethyl acetamide, and N, N,-dimethyl formamide (DMF) and glycerol. Evaluation of cooling rate showed that post-thaw motility was affected by cryoprotectant, cooling rate, and their interaction. The highest post-thaw motility (50%) was observed at a cooling rate of 10 degree/min with methanol as the cryoprotectant. Fertility testing of thawed sperm cryopreserved with 10% methanol at a rate of 10 degree/min showed average hatching of 70% (from 0 to 100%) which was comparable to that of fresh sperm (86%). Existing protocols for sperm cryopreservation of X. helleri were applied to other Xiphophorus species, which are categorized into three groups: southern swordtails, northern swordtails, and platyfishes. To survey the application of the established protocol for X. helleri (southern swordtail) to other Xiphophorus species, and considering their use for biomedical research, X. couchianus (platyfish), X. maculatus (platyfish) and X.variatus (northern swordtail) were chosen for sperm cryopreservation and artificial insemination. Live young were produced from cryopreserved sperm of all the tested species. Development of protocols for cryopreservation of sperm from diploid and tetraploid eastern oysters is also underway (data analysis in progress).
Publications
- Yang, H., and T.R. Tiersch. 2009. Current status of sperm cryopreservation in biomedical research fish models: Zebrafish, medaka, and Xiphophorus. Comparative Biochemistry and Physiology C, 149, 224-232.
- Yang, H., L. Hazlewood, R.B. Walter and T.R. Tiersch. 2009. Sperm cryopreservation of a live-bearing fish, Xiphophorus couchianus: Male-to-male variation in post-thaw motility and production of F1 hybrid offspring. Comparative Biochemistry and Physiology C, 149, 233-249.
- Yang, H., and T.R. Tiersch. 2009. Sperm motility initiation and duration in a euryhaline fish, medaka Oryzias latipes. Theriogenology 72: 386-392.
- Guo, X., Y. Wang, Z. Xu and H. Yang. 2009. Chromosome set manipulation in shellfish. In: Burnell, G., and G. Allen (eds) New Technologies in Aquaculture: Improving Production Efficiency, Quality and Environmental Management. Woodhead Publishing Limited, 165-194.
- Chen, Y., H. Yang and T.R. Tiersch. 2009. Review of computer-assisted sperm analysis (CASA) in fish and shellfish. Aquaculture America 2009, Seattle, WA, February 15-18.
- Yang, H., and T.R. Tiersch. 2009. Sperm cryopreservation in biomedical research fish models. Aquaculture America 2009, Seattle, WA, February 15-18.
- Hu, E., H. Yang and T.R. Tiersch. 2009. High-throughput cryopreservation in aquatic species: Adapting mammalian technology to fish. Aquaculture America 2009, Seattle, WA, February 15-18.
- Yang, H., and T.R. Tiersch. 2009. Cryopreservation of gametes, embryos, and larvae in shellfish. The 101st National Shellfisheries Association meeting, Savannah, GA, March 22-26.
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