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Development of Techniques for Accelerated Growth, Maturation, and Vitellogenesis in Cultured Shortnose Sturgeon (Acipenser brevirostrum)

Final Report
Breviro Caviar Inc
AIMAP 2012-M03

I. EXECUTIVE SUMMARY

Breviro Caviar produces cultured Shortnose sturgeon for caviar and meat products. The company operates two hatchery and grow-out facilities located in Pennfield and Charlo, New Brunswick. The facilities are based on intensive recirculation (Pennfield and Charlo) and flow-through (Charlo outside tanks) water infrastructure.

Several years of sturgeon production and directed research at the Pennfield site has indicated that growth, maturation, and egg development of Shortnose sturgeon may potentially be significantly enhanced by maintaining the fish continuously in elevated water temperature and high oxygen/ low nitrogen environments. The culture infrastructure at the Pennfield and outdoor Charlo sites does not allow economical heating of the water to produce optimal temperature environments and relies on conventional oxygen infusion.

The objective of this project was to develop a new intensive recirculation system at the Charlo Salmonid Enhancement Centre (CSEC) that has the capacity to rear sturgeon at continuously elevated water temperatures and oxygen levels with the result of significant improvements to growth rates, maturation rates, and vitellogenesis cycle time. These parameters are essential to enhance the economics of Shortnose and other sturgeon species raised in Canadian environments.

The project was broken into four phases. The design plans and project preparation (phase 1) was completed in September 2012, with ground broken on July 7, 2012 to start construction of the new facility (phase 2). This was a challenging phase with budget and timeline setbacks, but was completed in February 2013. The transfer of 5,000 Shortnose sturgeon (phase 3) to the Charlo site by truck was completed on schedule in June 2012. However, due to construction delays the fish were put into outside tanks until the facility was ready. Transfers to the new facility began in November and was extremely challenging due to cold weather and ongoing construction, which resulted in 6% mortality. Final transfer of all fish into the new system, following the experimental design layout (i.e.,which year classes would be transferred to which tanks), was completed in April 2013, and fish performance (growth, maturation, and egg development) monitoring and analysis began (phase 4).

Breviro has kept records on performance of its stock across all facilities throughout the project period. The data shows that continuously elevated optimal temperatures of 18-20˚C produce good growth and maturation rates for mature fish across all Breviro year classes. The overall food conversion ratio (FCR) for the Charlo facility was between 1.8 and 1.98 (with some tanks demonstrating FCR's as low as 1.54) and specific growth rates ranged from 70g to 240g per month. Comparison results to Pennfield operations demonstrate that optimal temperatures may not need to be as high as 18-20˚C since Pennfield produced comparable growth and FCR rates over a similar period at temperatures ranging from 12-14.5 ˚C. This same result was also experienced in the seasonal temperature rows in Charlo, which will lead Breviro to continue growth rate testing, feed rations and optimal temperatures. 

Elevated oxygen experiments will be completed in the future due to unforeseen system installation and performance challenges. Vitellogenesis studies are in initial phases with an estimated 5% of the population at stages 1 through 3 of vitellogenesis. Accelerated vitellogenesis is being compared to similar year class and sized sturgeon in Pennfield with early results showing positive signs that elevated temperatures will produce caviar ready fish more rapidly.

Overall the project has been successful in providing Breviro with a cost-effective, heated recirculation facility that provides the basis for achieving optimal Shortnose sturgeon growth. Further commercial experiments will be conducted to validate optimal temperature, complete PurGro oxygen studies, and complete full vitellogenesis cycles in partnership with Dr. Matthew Litvak of Mount Allison University. Taken together the results presented herein and the ongoing studies will allow Breviro to grow its sturgeon on a commercially competitive basis with other international producers.

II. Introduction

Breviro Caviar is the only commercial producer of cultured Shortnose sturgeon caviar and meat products in the world. Currently, Breviro operates two grow-out sites for Shortnose sturgeon: the initial site constructed in 2004 at Pennfield is a large-scale intensive recirculation facility. The site has a 50MT biomass grow-out capacity. Breviro Caviar also operates a flow-through outdoor tank system at Charlo, NB. This site has a production capacity of 22MT. 

Breviro has continually conducted research (frequently in collaboration with university and government) to optimize methods by which to culture and produce Shortnose sturgeon. The company has conducted intensive research projects on a variety of culture topics; caviar and meat production, fish growth, maturation, and egg development which are critical aspects of sturgeon culture.

Evidence from literature review, species studies, and discussions with growers of similar sturgeon species has indicated that maintaining sturgeon in water temperatures of 18 - 22˚C will accelerate growth, reduce time to as compared to sturgeon held in systems influenced only by seasonal temperature profiles.

Breviro has also undertaken small-scale studies to evaluate the impacts of rearing sturgeon in very high oxygen/low nitrogen environment produced by the NB, Canada-based InVentures PurGro2 oxygen infusion technology. Based on the study results, this oxygen infrastructure resulted in a 20% increase in sturgeon growth over that of sturgeon held in water characterized by normal oxygen levels

In 2011 Breviro Caviar partnered with the Charlo Salmonid Enhancement Center (CSEC) to develop another Shortnose sturgeon grow-out facility at Charlo in Northern New Brunswick. A key element to this partnership is to test previous R&D on temperature and oxygenation at the commercial level.

Achieving success would be a breakthrough for culture of the Shortnose species and key to Breviro's ability to be competitive as it exports globally.

This is the primary focus of the Aquaculture Innovation and Market Access Project (AIMAP), and was implemented at the new facility that was constructed at the Charlo site. Breviro Caviar believes that this project can help it gain commercial advantage in global sturgeon and caviar production, and that the benefits of high temperature and oxygen culture environments for Shortnose sturgeon will be a key to successful commercial scale operations.

III. PROJECT OVERVIEW

The overall objective of this project was to construct an intensive recirculation facility at Charlo, NB with the appropriate culture infrastructure to accelerate growth, maturation rate, and vitellogenesis (egg development) of Shortnose Sturgeon. This AIMAP project focused on the latter and looked to compare these biological parameters between 2 recirculation systems with different temperatures and oxygen environments. The applied results would enable Breviro sturgeon to be brought to market size, maturity, and gravid condition at the earliest age possible.

The project was broken down into 4 key phases:

IV. METHODOLOGY AND RESULTS

Phase One: Design Plans, and Project Preparation

Objective:

To design an intensive recirculation facility, select infrastructure suppliers, and develop a construction/installation schedule.

Key Activities Conducted:

Design:
A completed design of the proposed recirculation facility, along with all contractor-ready and as-built engineered drawings of the facility (including the building structure, electrical systems, drain and drainage layout, tank design and construction, and recirculation system layout) were finished in September 2012.

Detailed sets of plans are now available: a construction-ready (as-built) plan and drawings of the new facility, all plans required to complete the building structure, infrastructure for drainage, tanks, recirculation system layout, electrical services, etc., and construction timelines and schedules.

Supplier Selection & Project Preparation:
Qualified contractors, equipment suppliers, and installation specialists were selected by September 2012. The company made an active decision to use Canadian and New Brunswick-based suppliers for all major work and equipment, and was fortunate to have good local expertise in the key building, aquaculture and heating systems. Silk Stevens, and in particular one of its engineers, had been involved in over 50 aquaculture system designs and were thus selected as the primary engineering firm. Valox Ltd, a local aquaculture equipment expert was selected for equipment supply. Prospect Building Contractors from Fredericton completed the building construction. And for the heating systems, Tweedie & Associates from Riverview, NB were selected for their expertise in geothermal systems. Electrical, system installation and plumbing were all completed with local contractors, with DBM Inc providing the majority of work.

A plan for all construction/installation schedules, appropriate to complete the facility, was completed in September/October 2012. All construction activities were supervised to maximize scheduling constraints. As is the case with many large construction projects, contractors did not always adhere to predicted construction schedules and there were delays in the overall completion schedule.

Breviro was successful in obtaining a cost effective building structure that has allowed economic heating and associated maximum system performance. Some schedules of construction/installation were impacted as described under Phase Two and target completion dates for various aspects of the facility were delayed. The primary challenge was the fact that we needed to move sturgeon to the new facility before winter and in particular before the water temperature dropped below 5˚C. Some delays in engineering drawings combined with delays in the building construction caused significant challenges and workarounds in order to move the fish inside. While we achieved the fish movement – a major undertaking – it was at the cost of focus on the project in other areas and that alone delayed the project by about a month. A final list of all contractors, equipment suppliers, and installation specialists who completed the facility is available.

Phase Two: Construction of New Facilities and Systems

Objective:

To construct an intensive recirculation facility and all systems involved in culturing Shortnose sturgeon including the building, drain fields, and installation of tanks and all recirculation filtration equipment.

Key Activities Conducted:

A large, fully functional, intensive recirculation facility for culture of Shortnose sturgeon has been constructed at the CSEC site. All work pertaining to the building erections, tank installation, drain fields (recirculation and effluent dedicated systems), all electrical requirements to service to facility, heating infrastructure, and all recirculation filtration equipment has been completed. All construction and installation activities associated with the completion of the new facility were reviewed and supervised.

Building Construction - Construction of the building began in July/August of 2012 with ground/foundation preparation. The initial basic construction process was comprised of cement pier and support column installation. Once these essential components were in place, the walls and roof structure, with associated insulation, were installed. Building construction occurred over a 4 month period such that the entire structure was complete (ready for occupation by the culture systems) around the end of November 2012.

The new facility consists of one large (250ft x 125ft) building which contains three principal components: the tank field, the recirculation system, and the heating system. Based on the premise of heating water to high temperatures continually (18-22˚C), an insulated building was required. The building was designed to be as air-tight as possible.

The structure is a steel column and side wall/roof type with 6” thick batten style fibreglass insulation covering all internal surfaces of the walls and roof. The floor is sand substrate covered by a 3” layer of crushed rock which facilitates maintenance or repair of any underground piping and provides an under-gravel biological filter for decomposition of waste which falls to the floor. 

The building construction took longer than was originally anticipated (initial assumptions were 2 - 2.5 months for completion) and required nearly 4 months to complete. This was a set-back as any work which was required to be completed inside the building (tank installation, in particular) was severely delayed, pushing back the total facility completion by several months. The original construction and equipment installation schedule called for a functioning system in which to begin the AIMAP trials by November 2012. Delays in construction included re-excavation of internal ground, parallel (as opposed to serial) construction of tanks, concrete, electrical, lighting and plumbing and engineering and design issues during system start-up that forced re-design. As a result it was not until February 2013 (3 - 4 month delay) that the facility became fully operational, and not until April 2013 when the system start-up was far enough along (in particular the bio-filtration systems) to allow Phase 4 to start.

Installation and Commissioning of Equipment - As the building was being constructed, the installation of the systems infrastructure was started inside the building. This included the recirculation system drum filter, effluent treatment, and pump sump concrete pits in the treatment area. This construction was completed in September 2012. Commensurate with this installation was the placement of the concrete pad destined to support the heating system equipment. All of the underground influent and effluent tank field piping was also installed during this period so that the majority of all of this required piping was completed by November 2012.

Tanks: The tank design required a concrete pad for each individual tank. These 32 pads were poured over a period of 4 months from September through December 2012. The pour schedule dictated an installation of 4 tanks bi-weekly so the last tanks were not completed until January 2013. There was a significant delay in construction of the 32 tanks as well, so this delay, in combination with the extended pad preparation, significantly affected the total construction schedule of the facility. Breviro had hoped to have all tanks ready for operation by early November 2012, this was not realized for all of the tanks, and only 8 tanks (one row) were ready for fish in October. An additional 8 tanks (for a total of 16) were ready for sturgeon on or about the 3rd week of November 2012. Because it was imperative to have the sturgeon, which were located in the outside tanks, moved into the facility before water temperatures declined to 3-4˚C significant efforts were directed at completing the 16 tank installations by mid November 2012. All of the sturgeon from the outside tanks were in the new building by November 15, 2012. The remaining 16 tanks were operational by the end of February 2013. Obviously the late completion of tank installation seriously affected the ability to start the system on the originally proposed date (October/November 2012).

Recirculation System: Installation of the recirculation equipment (purchased with AIMAP assistance) began in October-November 2012. Installation of the larger pieces of equipment: drum filters, degassers, biofilters, and pumps (and all associated piping, electronic controls, etc.) was completed by end of December 2012. The completed recirculation systems were operational in terms of successful process flow by mid-January 2013.

The biofilter media required several additional weeks to be colonized with nitrifying bacteria; this process began on March 2013 and the filters became fully functional as of May 2013. All tanks containing sturgeon in the new facility were being serviced by the recirculation systems in February 2013.

The smaller infrastructure (oxygen infusion equipment) was installed and operational in various stages in October 2012 (to provide suitable environments for the fish in the tanks). The PurGro2 ILS units were installed in the recirculation systems in December 2012 and were functional as of February 2013. Optimal performance from the units was not achieved until July 22, 2013 because of issues surrounding flow. It was initially assumed that the gravity-based head pressure provided by the main recirculation systems would be sufficient to provide adequate flow from the units, however it was apparent during system start-up that there was too little head pressure to operate the ILS units properly. This issue was solved (in July) with the installation of a 500gpm (maximum flow) pressure booster pump, which allowed for higher flow at a greater pressure through the units.

Heating Systems: An integral part of the culture environment requirements, the heating system was installed during November-December 2012 and became operational in January 2013. The system relies on the extraction of heat from effluent tank water; this heat is then applied to new (make-up) water which flows to the recirculating systems. The design of the recirculation system allows for the removal of effluent water from 2 tanks only, the remaining 30 tanks run on full recirculation flow return to the treatment system. The effluent pit system incorporates a microscreen drum filter (minimum 60 micron particulate size removal) that catches solids before the water exits to the river and to provide clean water to the heat exchangers which are part of the heating system.

As of March 31, 2013 the heating system had been successful in elevating water temperatures by 4–5˚C (over that of the incoming well water: average temperature of 5–6˚C). As the target temperature for the systems is 18-20˚C, there was a considerable increase in temperature required for heating in the winter. In addition, the heating system took a considerable amount of trial and error and technician learning to initiate, as it required a long-term sustainable solution since it is using geothermal heating, but its start-up was challenging due to heat exchanger efficiency, water flows, and effluent filtration. The heating system extracts and applies heat (it does not heat the water directly) and is purposefully slow at raising temperatures for sturgeon safety. Target temperatures were achieved in May 2013 with the biofilters removing ammonia and nitrites to acceptable levels.

All of the building and tank infrastructure, recirculation, PurGro2 oxygen infusion equipment, and heating systems were in place and fully functional as of March 2013. The lighting over the 32 fish tanks was in place in April 2013. The floor surface (predominantly the water treatment area) was covered by the required crushed rock substrate in May 2013.

The recirculation operations are ultimately contingent on biofilter performance. The full operational status of the biofilter systems is dependent on optimal colonization of the Kaldness media by nitrifying bacteria. Starter cultures of these bacteria were added periodically during February-March 2013 to the filters to accelerate colonization. The standard process of initializing biofilters is not rapid and is contingent on water temperatures of >7-8˚C. General time to full colonization of any biofilter is 2 - 3 months. We expected that biofiltration capacity would be optimal within 2 - 3 weeks of attaining water temperatures of 10-14˚C, but in fact low pH of the water (<6.5) hindered optimal bio-filtration until well in June 2013. However, ammonia removal rates were sufficient to allow feeding of the target food rations in all the experimental tanks, even as water temperatures increased.

The facility was complete in terms of operational readiness in March 2013. Additional increases in biofiltration performance and heating to target water temperatures took until May and June 2013 before reaching target parameters. The recirculation systems are now operating within the predicted parameters to meet the objectives of the accelerated growth, maturation, and egg development project.

An assessment of the financial and construction/installation shows that the total gross project cost was approximately $2.64M as opposed to an original budget of $2.325M for an estimated 13.5% over budget. A combination of factors contributed to the cost over-runs, but were primarily attributed to initial delays in financing and environmental approvals forcing a late-start to the project, which led to winter conditions hindering building completion. The knock-on effect was delays in construction, parallel contractor work (i.e.: plumbing, building, concrete, and electrical happening simultaneously) and cost over-runs as a result of slower completion times due to change-orders or changes to work conditions. Faster initial engineering drawings and resolutions to key issues in parallel (rather than serial) might have resulted in better completion time. System start-up and initiation took longer than expected – primarily due to low water pH, slow and difficult heating system start-up, and resulting bio-filtration challenges. 

Phase Three: Transfer of Shortnose Sturgeon to New Breviro/CSEC Facility

Objective:

To transfer 5,000 Shortnose sturgeon from the ASF (Atlantic Salmon Federation) site in St. Andrews, NB to the completed recirculation facility in Charlo, NB and to place them in the proper tanks to complete the fish performance analysis and monitoring.

Key Activities Conducted:

All sturgeon were successfully transferred from ASF to the Charlo site (~6 hours) with minimal mortality and fish health impacts. All sturgeon which form the basis of the project (and the residual from ASF) were on-site at Charlo by 15 July 2012. We monitored oxygen to ensure it stayed above 7 mg/l, carbon dioxide to ensure it stayed below 18mg/L, total gas pressure, pH to ensure it stayed in the range of 6.4 - 8, and ammonia levels to ensure they stayed below 1.5mg/L. All parameters were within range during all transfers.

The fish were moved in 5m3 fiberglass carriers, 5 per truck load; the standard methodology for commercial-scale transfers of other species (i.e., Atlantic salmon smolts). Fish transfer densities were held to a maximum of 50 - 60kg/m3 (maximum allowable for least stress and negative impacts on individual fish). All of these fish were held outside in 16 large (7.5m diameter) fiberglass tanks until the facility was completed. As tanks in the new facility became available in October/ November 2012, sturgeon from those tanks were transferred to the tanks in the new facility.

Only 6 of the 5000 fish transferred died as result of the initial move; long-term health of the transferred fish was good. No sub-optimal parameters were detected during any transfers.

Subsequent moves of some of this same group of fish (transfer from outside tanks at Charlo to completed tanks in the new facility) resulted in a mortality rate of 6% (N=300) of all fish transferred. This mortality related directly to movement of the fish at sub-optimal temperatures (sturgeon moved at temperatures below 4˚C often experience stress and mortality). Because of the delays in tank installation and the onset of winter conditions, fish had to be moved when water temperatures were 1-2˚C less than the minimal temperatures required for best (lowest mortality) transfer. These transfers occurred in October/November 2012. This was an unplanned mortality event and a direct cost of the project delays.

Phase Four: Fish Performance, Monitoring, and Analysis

Objective:

To determine what effect culture infrastructure and techniques for increased temperatures and oxygen levels have on accelerated growth, egg maturation, and vitellogenesis (egg development) of Shortnose sturgeon.

Key Activities Conducted:

Studies were completed on potential accelerated growth, maturity, and egg development of sturgeon within the two recirculation systems. Some limits (due to time, oxygen installation issues, and a virus outbreak) were imposed on the studies and are explained below.

The original concepts of determining growth, maturation, and egg development rates have not changed from the initial proposal, but the timeline to complete each was extended to July 2013. This end date enabled Breviro to collect the data needed and make commercial decisions to optimize its business and sturgeon growth strategy.

The growth, maturity, and egg development monitoring began in April 2013, with full operational readiness of the tank fields and recirculation systems. From a systems operational perspective, the trials began as soon as the target water temperatures reached ~10˚C. Due to the delays in completion of the facility, Breviro developed a timeline for completion of the trials as follows:

  1. Determination of growth acceleration: April –July 2013
  2. Rapidity of maturation: April – July 2013
  3. Chronology of egg development: April – July 2013

The result of the study is a system design and operations strategy based on an artificially created temperature and potentially an ultra-high oxygen environment (more studies are needed to confirm the oxygen impacts) which allows for the commercial-scale acceleration of sturgeon growth, maturation, and egg development. Growth rates and feed conversion rates during the study period have exceeded Breviro's best results in past studies. This indicates that the commercial advantage being sought (larger fish at harvest, maturing earlier, and developing eggs more quickly) is possible in the new facility.

Methodology:

Construction of the new facility in Charlo (as outlined in phase 2 of this report) resulted in a tank layout visible in the diagram on page 7. The tanks are configured in 4 linear rows of 8 each (A, B, C, and D row); the total water volume of A/B and C/D row respectively is processed by a dedicated recirculation system situated at the row end. Each tank has a volume of 50 cubic meters. Oxygen is introduced through a low head oxygen delivery system combined with hyper-oxygenation and nitrogen stripping via 3 PurGro systems in each of the A/B and C/D rows. Water temperatures at the facility range between 14-15˚C during the coldest winter months to 19-22˚C during the warmest months of summer. The configuration of the facility allows for culture control comparisons to be made between the A/B (heated) and C/D (unheated) rows.

The following year classes (YC) were present at the facility: 2003 (N = 3000, average weight = 2.0kg), 2006 (3000, 2.0kg), 2009 (1000, 1.0kg), 2011 (3000, 0.1kg - **the 2011 YC tanks were subsequently dropped from the study due to a virus outbreak, (see ‘Challenges' section below**). For each year class within each recirculation system, the fish were divided into 3 of the 7.5m tanks each, so that a total of 12 tanks in each system were utilized for the trial. The remaining 4 tanks in each recirculation system contain other year classes currently held at Breviro which were also monitored for the trial, as part of what is expected to be the normal target production of the facility. All groups were stocked at densities which Breviro has determined to be comfortable rearing densities for Shortnose sturgeon; the maximum end densities to be no more than 40kg/m3, initial stocking densities were 12 - 16kg/m3.

The three parameters to be studied were set-up as follows:

  1. Determination of Growth Acceleration:
    • Five tanks from the A/B and C/D rows (10 tanks total) were used as explicit experimental tanks for growth comparisons, but the overall growth within the entire system was also monitored.
    • All of the sturgeon in the tanks in the facility were fed to satiation on a continual basis and the amount of food introduced into the various tanks was recorded in detail.
    • For growth monitoring, a sub-sample group of 100 fish from each tank was weighed once every month.
  2. Rapidity of Maturation:
    • The 2006 and 2009 YC were the subject of monitoring for rate of accelerated maturation.
    • The original protocol for determining maturation rates in the 2006 YC was as follows: 50 fish from each tank were to be biopsied at introduction and again once every 3 months thereafter. The biopsy procedure involves physical removal of a small sample of testes or ovary from each fish and accurate mapping of the onset of puberty is possible based on the structure of these tissues. This protocol was modified due to construction set-backs, time constraints, and virus outbreak as follows: biopsy data from 110 fish (from outside tanks at Charlo) was collected in October 2012 (5 months before the fish were introduced to the experimental tanks).
  3. Chronology of Egg Development:
    • The 2003 YC was already mature and has known stages of egg development; a small number of this group was monitored to determine the potential for rapid progression through vitellogenesis.
    • The original protocol for monitoring potential rapidity of egg development in the 2003 YC was as follows: 50 fish from each tank were to be biopsied at introduction and again once every 3 months thereafter. Each fish would be tagged internally with PIT tags for tracking purposes. This is the same procedure currently used at the Pennfield site to predict caviar production. This protocol was also modified due to the same issues mentioned previously, and also involved the data collected from the 110 fish biopsied in October 2012.

Challenges:

As indicated earlier in this report, there were three study-limiting events that impacted this study:

  1. The PurGro system had installation issues. This meant that not enough time and data was able to be collected to determine the impacts from the PurGro systems. These studies are ongoing.
  2. A virus outbreak occurred in the 2011 year class. It was a serious event with 85% mortality of the 2011 year class. This event limited studies on vitellogenesis and maturation due to our inability to biopsy fish during the virus outbreak. Any incremental fish handling and specifically open biopsy was deemed far too dangerous to the stock to be conducted. As a result biopsies will be conducted in September to determine final vitellogenesis and maturation rate increases for the October 2012 biopsied cohort of fish.
  3. Ultrasound for sex determination and for accelerated egg and maturation rate development was not successful. Extensive work and research was conducted using various machines, but none have proved able to provide the detailed imagery required to properly assess gonad development and egg development. Ultrasound was the back-up and complementary plan to biopsy once the virus outbreak occurred, but was unfortunately unsuccessful.

Results:

Determination of Growth Acceleration

Success Indicators: For growth, an indicator of success would be a 30-40% increase in end weight of fish held in the heated, high oxygen tanks as compared to those fish which were reared in the conventional, seasonally-influenced tanks.

The average weights of fish from 8 tanks within the 2 recirculation systems was compared. Comparing tanks from the A/B rows, which were held for 2 months at water temperatures 8-10˚C above those characteristic of temperatures recorded in the C/D rows, shows that there was no significant differences in the increase in average weight through time for fish within tanks, between rows, or between systems. The highest Specific Growth Rate (SGR) recorded from any sample of fish in any tank during the study period was from the C row system.

Most of the fish which had grown >125g/month in all tanks, in both systems, grew within 6-18% of the highest growth rate recorded during the 2 month period when water temperatures were continuously elevated in the A/B row (18-22˚C) and averaged 6-8˚C less in the C/D row (12-14˚C). There were fish from tanks in both systems that exhibited very high SGR's (average based on sample weights) of >200g/month and fish from tanks in both systems that gained very little weight (<100g/month) in the 2 month period. When comparing fish between tanks based on year class origin, it was also found that older fish (2003) held in warm water of the A/B row did not grow significantly faster than fish of the same year class held at lower temperatures in the C/D row for the same time period. Younger fish (2005-2006 YC) exhibited similar trends in growth; warmer water did not significantly accelerate growth rates for fish of these year classes. As well, older fish did not grow significantly faster than fish originating from more recent year classes when held at elevated or seasonal water temperatures, indicating that age did not necessarily influence fish growth in the different temperature regimes examined. 

We also compared the growth of sturgeon from the 2005-2006 YC held in the Pennfield facility to growth of fish in Charlo. In Pennfield, sturgeon were held in similar recirculation systems to those recently completed in the Charlo site; the fish (from two, 150m3 capacity tanks, N=1341 (A5) and N=1250 (A4), average initial weight of 2.0kg) were measured initially on 15 May and re-measured at the end of July 2013 to determine increase in average weight over the 2.5 month period.

Water temperatures in the 2 tanks ranged from 10.5-14˚C for the period and closely followed the temperature regime which was recorded in the unheated C/D row tanks in Charlo. The sturgeon in the 2 tanks in Pennfield exhibited a specific growth rate of 240g/month in one tank and 180g/month in the other. These SGR's recorded for fish from both tanks closely follow the higher growth rates exhibited by the faster growing fish in both row systems at Charlo. In Pennfield and in Charlo, holding and growing sturgeon in water temperatures several degrees colder than those targeted as potentially accelerating growth (18-22˚C) did not negatively impact growth in terms of SGR. It is possible that a spring-time biological anomaly (i.e.: sturgeon coming into the spring tend to eat and grow at high SGR's) may be contributing to the results.

Food Conversion Ratio (FCR)
We examined growth rates of fish in 10 experimental tanks (5 in the A/B row; 5 in the C/D row). Growth of fish in 8 of the tanks was considered as normal within the historical values observed for cultured Shortnose sturgeon. Fish from two tanks in the D row showed very little growth which could not be reconciled given the uniform growth of fish in all other tanks in the C/D row system. It also couldn't be reconciled against other tanks in the D row which performed in the same range as the tanks below. It is assumed that this growth anomaly was related to sampling error or incorrect transfer of data or possibly that these fish were ill from the virus. The fish from the 2 tanks with anomalous growth are now being re-measured (August 15, 2013) to determine the extent of the discrepancy.

Based on the relatively uniform growth rates of fish in 8 of the 10 tanks, there was no significant difference between the average growth in weight between fish held in warm water and those maintained for the same period in colder water. In terms of the conversion rates for fish in the 8 tanks held in the 2 different environments, there was also no significant difference recorded. Sturgeon monitored in the heated A/B rows converted at an average rate (all 4 tanks) of 2.03; fish from the seasonally-impacted C/D rows converted at an average rate of 1.85 (4 tanks). The expected FCR for Shortnose sturgeon held in water of 12-18˚C is generally 1.4-2.0, so values recorded during the study period are consistent with previous results. In the Charlo recirculation systems holding fish in heated water, at least over 2-3 months, did not result in better food conversion.

Based on literature review and discussions with European growers of other sturgeon species, we had assumed that raising Shortnose sturgeon at continually elevated water temperatures of 18-22˚C would result in greater growth than has been characteristic of sturgeon held in seasonally-influenced temperatures. For Siberian sturgeon (A. baerii) cultured in Europe, grown in recirculation systems with water temperatures maintained at 20-22˚C, the general increase in size for sturgeon of >3kg is suggested as an SGR of 100-200g/month. At both the Pennfield and Charlo sites, growth of fish in many culture tanks has been observed to exceed these values even when the fish are held in much colder water and have smaller initial weights of 2-2.5kg. This is very promising for Shortnose.

Rapidity of Maturation:
Success Indicators: For earlier maturation, success of the project would be indicated by a greater proportion of mature or maturing sturgeon held in the heated, PurGro2 oxygen infused tanks.

The experiments to determine potential for accelerated fish growth conducted to date at Charlo, and the fish growth results recorded in Pennfield, have been of relatively short duration (2.5-3 months). In spite of this feature, there have been significant increases in average size of most fish sampled, and steady and uniform growth at both elevated and seasonal water temperatures has been recorded. The results of the experiments to date suggest that Shortnose sturgeon, unlike some other sturgeon species, may not need to be held at very warm water temperatures to realize optimal growth in culture. Obviously more data collected over an extended time period will further define the role that water temperature play in impacting sturgeon growth. If this can be proven over a longer term, this has positive implications for raising Shortnose in Canadian environmental and water climate regimes and will also save considerably on water heating costs in winter.

Based on the relatively uniform growth rates of fish in 8 of the 10 tanks, there was no significant difference between the average growth in weight between fish held in warm water and those maintained for the same period in colder water. In terms of the conversion rates for fish in the 8 tanks held in the 2 different environments, there was also no significant difference recorded. Sturgeon monitored in the heated A/B rows converted at an average rate (all 4 tanks) of 2.03; fish from the seasonally-impacted C/D rows converted at an average rate of 1.85 (4 tanks). The expected FCR for Shortnose sturgeon held in water of 12-18˚C is generally 1.4-2.0, so values recorded during the study period are consistent with previous results. In the Charlo recirculation systems holding fish in heated water, at least over 2-3 months, did not result in better food conversion.

Chronology of Egg Development
Success Indicators: The accelerated progress through egg development would be indicated by tracking of individual females wherein the eggs periodically examined are farther advanced towards a caviar-ready state than the eggs collected from fish held in the conventional environment.

The results of the 110 fish biopsied in October 2012 were 39 males and 49 mature females (22 fish were unknown in terms of gender based on the inability to find gonad tissue). Of the fish that were determined to be female, 7 exhibited eggs in late stage 3 (2-3 months from caviar-ready state), 14 others had stage 2 eggs (6-8 months from caviar-ready state), 12 had stage 1 eggs (12-18 months from caviar-ready state), and the remaining fish exhibited only granular tissue (germinal epithelium) or eggs of pinhead (those derived from granular tissue) stage (18-24 months from caviar-ready state). We believed that the biopsy results were representative of all of the fish which had been introduced into the 8 experimental tanks at Charlo. Since the fish at Charlo were not introduced into the tanks until April 2013, there was little time in which vitellogenesis rate would have been quantifiable in the 3 month sample/assessment period (May 5 - July 31, 2013).

Additionally, due to issues with potential contamination of uninfected fish from the experimental tanks by those fish which were known to be positive for the virus (2011 year class), we did not complete any scheduled biopsies on any fish at the Charlo site in late July 2013. Based on the historical rate of vitellogenesis observed for Shortnose sturgeon, it is likely that calculating potential increases in the rate will require measurements over at least a 3-4 month period even at higher water temperatures in which fish in the A/B rows have been held. There is now a base line profile of stage of egg development for fish introduced into the experimental tanks; a re-assessment of the profile in late August 2013 will determine if the proportion of fish in the experimental tanks will show accelerated progression in terms of egg development. Histological examinations are indicating positive progression of egg development – particularly in the largest fish.

Routine biopsies and examination of any processed fish and/or mortalities of the fish at Charlo in 2012-2013 indicate that all of the fish which are identifiable as either males or females are mature and capable of continued egg development (females) or sperm production (males). The initial plan for the AIMAP experiments was to determine if the fish held in environments with warm, continuously-maintained water temperatures would mature more rapidly than fish held in colder, seasonally-influenced temperatures. Since nearly all of the fish introduced into the experimental tanks in April 2013 were already mature, defining a rate of maturity based on these fish has been problematic. For males, once mature, there is little or no further development beyond adding a small amount of testicular tissue during the onset of spawning. For females, maturity is defined as the presence of granular tissue (germinal epithelium); there is no further maturation, only egg development.

When the initial plans for examining onset of maturity as related to environment were developed, we assumed that some portion of the fish used in the experiments would potentially be immature; however, it has since been found that fish from the year classes selected for the experiments (2003, 2005, 2006) are indeed mature and are not useful to examine potential accelerated maturation. The 2009 year class fish, which had not yet matured (as of December 2012), were intended to be part of the trials on rate of maturation. Examinations of the pre-gonad structure of 40 of this year class in October-December 2012 indicated that the onset of ovary or testes formation was 15-24 months away. 500 fish from the 2009 year class were held in elevated water temperatures during the 2.5 month sample period in spring 2013.

Due to issues with the virus and potential contamination of the 2009 year class, any biopsies planned for the end of July 2013 were not completed. The viral epidemic has passed at the Charlo facility as of August 2013 and it is now clear that any surviving fish from the 2011 year class which had been infected have recovered and are now immune to the impacts of this pathogen. The level of risk concerning this disease has declined and it is now the intention is to re-evaluate the maturity level of the 2009 year class at the end of August and into September 2013.

System Performance and Water Quality
It is evident that there was a significant difference in water temperatures between the two rows for the period 30 April through 31 July, although water temperatures in both rows were comparable in late July 2013 which was expected given the seasonal increases generally observed at the Charlo site. 

During the 3 month study period the water temperatures in the C/D row averaged 5-7˚C lower than the heated water in the A/B row. This was a significant difference which was assumed to be great enough to show differences in potential growth (accelerated or typical) between fish held in warm water and those held in seasonally-influenced environments. It also had benefit to the future commercial decision of what temperature to hold the entire system at in winter. If the temperatures can be held at 14˚C vs. 18˚C-22˚C then this will result in considerable heating and oxygen savings.

The system water quality parameters recorded during the 3 month sample period are generally comparable to intensive recirculation systems which are used to rear sturgeon. Ignoring the oxygen levels which were produced by the implementation of the Purgro system on row A in late July, the oxygen levels recorded in all experimental tanks were optimal and stable as concerns the requirements to grow sturgeon. There were no days in which oxygen levels in any experimental tanks fell below recommended levels (<80% saturation) for optimal fish growth.

Generally, ammonia (NH3) levels within recirculation systems dictate the amount of food fed to fish. Very high food introduction rates can cause spikes in ammonia levels. Continually (3-4 days at a time) high levels of >1.3-1.5 mg/L are considered as negatively impacting the sturgeon as related to food intake and associated feed conversion rate. The average ammonia levels in both the A/B and C/D rows was 0.85 to 0.90 mg/L, well within acceptable levels for general sturgeon culture. There was no significant differences between the average ammonia levels recorded in the A/B and C/D rows; fish growth comparisons between the 2 row systems was likely not influenced by ammonia levels in the systems.

Continual excessive levels of carbon dioxide (CO2) can be very damaging to cultured sturgeon. Constant CO2 levels of >20-25 mg/L are considered as hindering feeding by the fish and can limit oxygen uptake into the fish blood. The recirculation systems at Charlo have advanced CO2 removal equipment and much of the CO2 produced by the fish in response to feeding is removed during the processing of water through the systems. As water temperatures increased, CO2 levels also increased, which was expected given the large amount of food introduced to each tank. In spite of the warm water temperatures and food introduction, the systems were efficient at removing much of the residual CO2 in the recirculated water so that average values in the tanks were always less than 10-15 mg/L. The CO2 values recorded were nearly identical between the two system rows, in spite of the lower average temperature in the C/D row.

A pH of 6.8-7.5 is the optimal range for most efficient nitrification through the system. River water at the Charlo site, which was the predominate source of new water introduced into the recirculation systems, was characterized by a pH of <6.5 for much of April and May 2013.

Nitrate levels in the systems were also studied during the spring 2013. Nitrite is not impacting to cultured sturgeon unless maintained within the environment at >0.8-1.0 mg/L. The recorded values for nitrite in both the A/B and C/D rows averaged much less than would be considered as negatively impacting sturgeon food conversion and growth.

Considering the water quality parameters measured in the recirculation systems at the Charlo facility, none were considered as negatively impacting the fish reared in the 2 rows kept at different temperature regimes. The objective for the experiments determining growth rates was to have all water quality parameters equal in the two systems (and in all tanks) so that only water temperatures were the dependent variable. This was achieved during the 3 month study period.

Determining the environment which best results in accelerating maturity requires a system design which incorporates known successful sturgeon production methods. Breviro has developed, and now constructed at Charlo, the recirculation systems which meet the specific needs of cultured Shortnose sturgeon. Breviro has now incorporated this technology into the culture systems which can be manipulated to provide high annual temperatures and high oxygen levels. The exact determination of the level of oxygen and temperature has not been finalized through this study and will be part of ongoing R&D at the facility to determine optimal rates that balance cost and accelerated rates of growth, maturity, and egg development. The long-term effects of continual high temperature and oxygen as compared to the seasonal, marginal growing conditions of the winter and spring months which are characteristic of the Pennfield and outdoor Charlo sites is still a critical part of Breviro's ongoing operations and subject to continued work and study.

V. Conclusions and Next Steps

Improvements to the culture infrastructure and operational system parameters for the new building show strong promise towards allowing both caviar and meat to be marketed earlier – perhaps up to 1 year earlier if current study results prove sustainable in growth rates for larger sturgeon from 4kg – 8kg. This has significant economic benefits for the company and will provide it with a commercial competitive improvement and possibly allow it to compete much more effectively against the most prevalent sturgeon species – Siberian (Acipenser baerii). If enhanced maturation rate continues, we should produce larger fish at an earlier age which increases the amount of caviar and meat marketed per annum and reduces the cost of production. Accelerated egg development will allow caviar sales at a much earlier schedule than currently realized at the other Breviro facilities as well as allow Breviro to have a percentage of its fish as 2nd spawners – where caviar yields and egg sizes tend to increase by 10% - 20% over 1st timers.

Recommendations for organizations considering a similar project would be to have complete engineering in advance of project start-up, avoid winter conditions and not to have fish on-site nor in a building as construction is underway. While the project was extremely challenging, on the positive side of perspective, completion of such a project and facility within the timeframes allowed, and with live sturgeon on site throughout, is a major accomplishment.

In addition to completing our studies on the PurGro system and vitellogenesis, recommendations from our Phase 4 study results were:

The results of this project are critical to the enhanced commercial viability and global competitiveness of Breviro Caviar. With the successful completion of the new building and new systems, Breviro is positioned to continue its fish performance analysis and refine its subsequent commercialization and production strategy decisions that form the basis of the company's future business and operating model. 

VI. Communications

The project has been or will be communicated via a variety of strategies: 

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