Progress 09/01/19 to 08/31/21
Outputs
Target Audience:The target audiences for the project are state hatcheries, private industry producers of largemouth bass, and the aquaculture industry on a national scale. Largemouth bass is one of the most popular freshwater game fish, which provides significant income to several communities throughout the United States that rely on the income from recreational fishing. Trophy-sized largemouth bass is highly sought after by anglers, and producers are unable to meet the demand for them. Recently, significant demand for largemouth bass as a food fish within ethnic markets in urban areas has been observed. We expect the findings of this project to have an immediate impact on production yields via advantages from mono-sex populations (females) and sterility (triploidy). This project intends to provide evidence that will allow us to confirm the sex-determining mechanism (WZ, ZZ) in largemouth bass by the production of mix-sex gynogens, while simultaneously producing broodstock capable of yielding second-generation mono-sex progenies of females which have been shown to grow larger than males. Producing tetraploid largemouth bass broodstock will additionally allow for triploids to be produced without having to manipulate embryos. The findings of this study could allow for the production of sterile all-female largemouth bass with ease in practical farm conditions where largemouth bass are currently produced. Changes/Problems:There were several challenges and problems that occurred throughout the first year of the project. A major problem that was encountered was the inability to induce the obtained broodstock to spawn within our facility despite using multiple approaches of hormonal injections that have been successfully utilized in other studies with largemouth bass. Being unable to induce these broodstock to spawn prevented us from attempting any shocks to carry out objectives 1 and 2. We had planned to counter this possibility by conducting additional spawning attempts with our collaborator at the Kentucky State University Aquaculture Research Center (KSU), but were unable to travel due to restrictions from both universities because of the COVID-19 pandemic. We had additionally planned to examine different hormonal sex reversal protocols with methyltestosterone on normal diploid largemouth bass to work towards our goal of developing mono-sex progenies, but were similarly impeded due to travel restrictions by our university during the largemouth bass spawning season as a result of COVID-19. In the second year of the project, COVID-19 restrictions were still present but were relaxed enough to enable travel to our collaborators at KSU. The challenges faced in inducing spawning in year 1 led us to focus our work on spawnings conducted at KSU. However, this also presented some new challenges, as KSU did not have the facilities to conduct polyploidy induction with pressure shocks. We also ran into a unique issue with fish produced at KSU where swim bladder inflation was consistently below 10%. Issues with inflation of the swim bladder in largemouth bass are not reported in the literature, which makes this occurrence rather interesting but also limited our abilities to work towards the goals of the project. Since swim bladder non-inflation often resulted in high rates of both mortalities and deformities of surviving individuals. What opportunities for training and professional development has the project provided?One graduate student working on the project established collaborations with producers of largemouth bass within the private industry, specifically Malone & Sons (Lonoke, Arkansas), and Mayer Fish Farm (Bardstown, Kentucky) in year 1. This graduate student also gained experience in largemouth bass handling and had the opportunity to learn several techniques essential to monitor progress in fish reproduction (ovulation, spermiation) using koi carp and zebrafish as surrogate species. The graduate students supported by this project wereable to gain experience in thefollowing procedures: flow cytometric analysis using red blood cells and tissue cell suspensions, techniques in the use of sperm extension for storage, and analysis of sperm quality (duration of motility after activation, character of sperm movement), hormonal induction of ovulation and spermiation,controlled reproduction, assessment and description of embryonic development,characterization of swim bladder inflation and deformities, and methods for induction of polyploidy. One undergraduate student was also involved with the project and assisted with general husbandry and fertilizations conducted at OSUfor one semesters. How have the results been disseminated to communities of interest?The graduate students involved in the project at both KSU and OSU discussed results with other graduate and undergraduate students in the labs. However, due to the pandemic, other methods for dissemination have been restricted during the course of this project. The results will be presented at a relevantaquaculture conference in 2022 (Ohio Aquaculture Association 2022 Annual Meeting & Aquaculture America 2022). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
A total of 21 largemouth bass (LMB) broodstock (2-3 years old, 700g mean weight) were purchased from Mayer Fish Farm (Bardstown, KY) in December 2019. Fish were housed in two 400L flow-through tanks and fed to satiation twice daily. Mortalities were recorded, external morphological characteristics were used to determine sex,and gonads were dissected to determine gonad-somatic index (GSI). Of the 9 mortalities recorded, previously described methods for sexing individuals were found to be inaccurate. GSI of dissected males (n=5) ranged from 0.25-0.50% and did not vary by season, while dissected females (n=4) ranged between 1.58% (January) and 3.7% (March). In March 2020, 8 LMB were separated into pairs andtransferred to individual 400L tanks with spawning mats. The following hormonal injections were given to trigger spawning behavior: 0.5 mg/kg priming dose carp pituitary extract (CPE), followed in 10 hours by 0.5 mg/kg CPE resolving, plus 2,000 IU/kg (male) and 4,000 IU/kg (female) human chorionic gonadotropin or 0.5 ml/kg (male) and 0.75 ml/kg (female) luteinizing hormone-releasing hormone. Fish were monitored for 4 days following resolving dose. Small amounts of sperm and ovarian fluid were identified, but no eggs were released by any pair. Most spawning attempts were halted within 3-5 days due to increased aggression between pairs. In April 2021, 14 female LMB were collected from outdoor concrete ponds at Kentucky State University (KSU) and brought inside to holding tanks. Fish were placed in individual hapas and given hormonal injections. Five females were stripped and eggs were mixed (total egg weight = 164g). The initial temperature of stripped eggs was 18.1°C with a mean storage temp of 8.7 °C and 15.6 °C. Four 24oz lidded Tupperware containers were utilized for egg storage trials with 2 target storage temperatures and 2 containers (replicates) of ~20g of eggs in each temperature. Two control fertilizations were conducted with 10g of eggs with either fresh stripped sperm or macerated testes from the same male at KSU. In addition, 2 sets of 30g of LMB eggs were fertilized with either UV-irradiated yellow perch (YP,Perca flavescens) sperm or LMB sperm to undergo physical shocks. Ninety seconds post-fertilization, eggs were rinsed with 200ppm tannic acid to remove stickiness. Eggs were then triple rinsed with water at 7.5 minutes post-activation (mpa) before being subjected to cold shock (1-3°C). After 176-190mpa, half of the cold shocked eggs from each fertilization were transferred to room temperature (17.8°C) while the rest continued exposure to shock. Cold shock groups were transferred to 18.2°C water at 260-280mpa (259-272min cold shock duration). The group allowed to warm at 3hrs and was further pressure shocked at 8000PSI for 1 minute at 240-250mpa. No surviving embryos were identified following cold shock or cold with pressure shock for either fertilization with UV-irradiated YP or LMB sperm. Stored eggs were transported from KSU to Ohio State University (OSU). Storage duration before fertilization for the 15°C groups was 398-454 minutes and 458-533 minutes for the 10°C group. Once at OSU a series of fertilizations were conducted with eggs at each storage temperature, including 2 control fertilizations with different sperm sources (macerated or stripped sperm). Two attempts to produce tetraploids, meiotic gynogens, two mitotic gynogen groups (shock applied at 70 and 80% of first mitotic cleavage), and haploid control were included. Pressure shock of 8000PSI for 1-min duration was utilized for each polyploidy induction attempt. For production of gynogens, YP sperm was UV-irradiated (8500µJ) and had post-dose motility of 50-60%. Both storage temperatures produced viable fish, however, moderately lower survival to hatching was recorded for the 10°C storage temperature compared to 15°C. Surviving individuals to hatching were identified in controls and tetraploid shocks (60mpa) for both temperatures, but only 15°C meiotic and mitotic gynogens (70% of first mitotic cleavage) were identified. Haploid groups from stored eggs did not survive to hatch, but the haploid group from the KSU fertilization with UV-irradiated YP sperm had survivors. Flow cytometry was conducted on surviving larvae at 5 days post-fertilization. Control groups were diploid (c-value = 0.99±0.01), haploidy (c-value = 0.51±0.00) was confirmed, and all gynogen groups with survivors were confirmed diploids (c-value = 1.01±0.01). Survivors from 15°C presumed tetraploid group were diploid (c-value = 1.00±0.01) and from the 10°C tetraploid group were 2/3 diploid and 1/3 triploid (c-value = 1.50). Results obtained from in vitro egg storage trial determined LMB eggs can be stored for fairly long periods (6.5-9 hours) and retain fertilization ability. Our ability to examine the survival of these progenies was difficult, due to an unusual occurrence of swim bladder non-inflation in a majority of surviving individuals, regardless of treatment or storage conditions. To examine this issue, which has never previously been reported in the literature, an experiment was conducted to examine embryonic development and larval rearing conditions. Freshly hatched larvae were obtained from KSU from natural spawns on 2 mats collected on May 14, 2021. Larvae from each mat were transported to OSU in separate plastic bags filled with oxygen. An additional group of non-fed swim-up larvae from May 6thwere also obtained. Larvae from each of these 3 groups were monitored every 24 hrs using a dissecting microscope to evaluate their development. We formed a hypothesis that LMB is capable of consuming food before inflation of the swim bladder, but that consumption of food before swim bladder inflation may compromise the ability of larvae to successfully inflate. Two experiments were set up in tandem with combined larvae from spawns on May 14. The first experiment was stocked on May 21, 2021. Nine static poly-round (8L) containers were randomly stocked with 120 larvae/container and maintained at 3 ppt salinity. One factor (initial live feed) with 2 levels, either rotifers orArtemianauplii, were applied to 6 poly-rounds, 3 each starting at 8 dpf for two weeks. The 2 additional tanks were also fed with rotifers or artemia but the amount of foodoffered was tripled. The last tank was fed rotifers starting at 7dpf. Results from the static trial revealed a rapid and substantial drop in survival during the first 3 days of the experiment, with approximately a third of the initial individuals dying each of the first 2 days. By the third day, survival had leveled off, at 10% for fish fed artemia and 5 % for rotifer fed treatments. The second experiment was designed in a specialized conical (50L tanks) rearing system with sprayer heads and a continuous supply of algal turbidity and salinity. Sprayer heads broke- up surface tension, which has improved swim bladder inflation in other fish, yellow perch and walleye. Six conical tanks were randomly stocked with 1000 larvae each. The inflow of 3 tanks was supplied via spray head and three with a tube. Feeding began at 9dpf withArtemianauplii fed 6-8 times per day and continued for 20 days. Water quality and mortalities were recorded daily. Survival of individuals in both treatments was similar, and like the static trial, overall survival was low at 8% after 9 days of feeding. Of the surviving individuals in the static trial, approximately 30% of survivors for both live feeding treatments successfully inflated their swim-bladder. In the conical system trial, swim bladder inflation was 0% with a high prevalence of spinal deformities (>80%). Overall, we identified for the first time that swim bladder inflation may be a significant impediment to LMB larval culture, but were unable to determine the specific cause of swim bladder non-inflation.
Publications
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Progress 09/01/19 to 08/31/20
Outputs
Target Audience:The target audiences for the project are state hatcheries, private industry producers of largemouth bass, and the aquaculture industry on the national scale. Largemouth bass are one of the most popular freshwater game fish, which provides significant income to several communities throughout the United States that rely on the income from recreational fishing. Trophy-sized largemouth bass are the most sought after by anglers, and producers are unable to meet the demand for them. Recently, significant demand for largemouth bass as a food-fish within ethnic markets in urban areas has been observed. We expect the findings of this project to have an immediate impact on production yields via advantages from mono-sex populations (females) and sterility (triploidy). This project intends to provide evidence that will allow us to confirm the sex-determining mechanism (WZ, ZZ) in largemouth bass by production of mix-sex gynogens, while simultaneously producing broodstock capable of yielding second generation mono-sex progenies of females which have been shown to grow larger than males. Producing tetraploid largemouth bass broodstock will additionally allow for triploids to be produced without having to manipulate embryos. The findings of this study could allow for the production of sterile all-female largemouth bass with ease in practical farm conditions where largemouth bass are currently produced. Changes/Problems:There were several challenges and problems that occurred throughout the first year of the project. A major problem that was encountered was the inability to induce the obtained broodstock to spawn within our facility despite using multiple approaches of hormonal injections that have been successfully utilized in other studies with largemouth bass. Being unable to induce these broodstock to spawn prevented us from attempting any shocks to carry out objectives 1 and 2. We had planned to counter this possibility by conducting additional spawning attempts with our collaborator at the Kentucky State University Aquaculture Research Center, but were unable to travel due to restrictions from both universities because of the COVID-19 pandemic. We had additionally planned to examine different hormonal sex reversal protocols with methyltestosterone on normal diploid largemouth bass to work towards our goal of developing mono-sex progenies, but were similarly impeded due to travel restrictions by our university during the largemouth bass spawning season as a result of COVID-19. What opportunities for training and professional development has the project provided?The graduate student working on the project established collaborations with producers of largemouth bass within the private industry, specifically Malone & Sons (Lonoke, Arkansas), and Mayer Fish Farm (Bardstown, Kentucky). The graduate student also gained experience in largemouth bass handling and overwintering. Graduate student had opportunity to learn several techniques essential to monitor progress in fish reproduction (ovulation, spermiation) using koi carp and zebrafish as surrogate species. Namely, he was practicing the following procedures: flow cytometric analysis using red blood cells and tissue cell suspensions, techniques in use of sperm extension for storage, and analysis of sperm quality (duration of motility after activation, character of sperm movement), embryonic development, stages of embryogenesis, deformities. One undergraduate student was also involved with the project, and assisted with general husbandry for two semesters. How have the results been disseminated to communities of interest?Graduate student discussed results with other graduate and undergraduate students in the lab. However due to current pandemic restrictions other methods for dissemination have been limited. What do you plan to do during the next reporting period to accomplish the goals?In the following year we will continue to attempt to spawn largemouth bass within facilities at OSU. Broodstock were previously PIT tagged, and a portion were sexed. Growth and appearance of the broodstock will continue to be monitored. Spawns will also be conducted at KSU to initiate trials for induction of meiotic gynogens, triploids, and tetraploids. We will investigate how water temperature effects the optimal timing for applying shocks to inhibit the second meiotic and first mitotic divisions, and whether pressure, cold, or heat shocks will be the most effective in doing so. Sex ratio of gynogenetic progenies will be recorded to make a conclusion on whether largemouth bass are female heterogametic, while simultaneously obtaining multiple family lines of gynogenetic broodstock. Data on growth performance and gonad development will then be collected from the following progenies: mixed-sex diploids, all-female diploids, mixed-sex induced triploids, all-female triploids obtained from crossing tetraploids with diploids.
Impacts
What was accomplished under these goals?
A total of 21 largemouth bass broodstock of two-three years old (approximately 700 g mean weight) were purchased from Mayer Fish Farm (Bardstown, KY) in December 2019. Fish were split into two 450 L flow-through tanks and were provided either pieces of rainbow trout (Oncorhynchus mykiss), yellow perch (Perca flavescens), and koi carp (Cyprinus carpio), or live koi carp and koi x goldfish hybrids (Carassius auratus) to satiation once to twice a day. Mortalities were recorded, and gonads were dissected and weighed to assess how gonado-somatic indices (GSI) varied throughout the year. GSI of males did not vary much throughout the year and ranged from 0.25-0.50% (n = 5). Female GSI had a larger range of variation than males, with the smallest being 1.58% in January and the largest of 3.7% in March (n = 4). We have used several previously described methods in order to sex the fish prior to dissection, such as appearance of genital papilla, body morphology (robustness), or behaviors (territoriality, nesting). Based on the number of individuals (n = 9) classified as either gender (December 2019-June 2020), these criteria were found to not accurately allow for identification of sex. On March 13th, 2020 (8.0 °C), eight of the largemouth bass were transferred to a separate 450L tank which was temperature controlled (11.3 °C). Spawntex spawning mats and dishes filled with gravel and marbles were provided as substrate. We attempted to induce hormonally three different pairs of largemouth from the available stock. On May 9th 2020 (21.0 °C), the largest male and female were transferred to a separate 400L tank and given a priming hormonal injection of 0.5 mg/kg of carp pituitary extract (CPE). The following morning the pair was given resolving doses of 0.5 mg/kg of CPE, in addition to 2,000 and 4,000 IU/kg of human chorionic gonadotropin for the male and female, respectively (according to Glennon et al., 2012). Sexual maturation was monitored for the next four days. Minimal amounts of sperm were obtained and motility was determined to be approximately 30-40%. Ovarian fluid was secreted from the female but no eggs were released. Spawning attempts were halted after 5 days due unsuccessful release of eggs and increased aggression observed between the pair. Another pair was separated on May 11th 2020 (20.2 °C), and was given priming hormonal injection of 0.4 mg CPE/kg body weight. Spawning had to be halted due to excessive aggression between the pair. However, this female was also found to be unresponsive to hormonal injections and was and was returned to the rest of the broodstock. On June 15th 2020 (21.5 °C), two pairs of largemouth bass were individually stocked into separate 450L tanks. Both sexes in each tank were provided 1.25 mg of CPE/ kg. Females and males were additionally given 0.75 and 0.50 ml/kg of luteinizing hormone releasing hormone analogues (LHRHa), respectively. One of the females did release a small number of eggs approximately 50 hours post-injection but regressed after this. These spawning attempts also had to be halted due to aggression between each of the pairs.
Publications
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