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Determining Ripeness in White Sturgeon Females to Maximize Yield and Quality of Caviar

ANNUAL PROGRESS REPORT

Part II: Detail

PROJECT TITLE: Environmental and endogenous factors affecting egg quality and caviar yield in farmed sturgeon.

REPORT GIVEN IN YEAR: 2013

REPORTING PERIOD: June 1, 2012 – August 31, 2013

AUTHOR: Serge Doroshov and Joel Van Eenennaam

FUNDING LEVEL:$120,000 (1st year budget) + $120,000 (2nd year budget) + $195,210 (3rd year budget).

PARTICIPANTS: Serge Doroshov*, Bernard May*, and Ermias Kebreab*, University of California, Davis; Barbara Rasco*, Washington State University; Molly Webb* and Chris Guy, Montana State University, USFWS; Terry Patterson*, College of Southern Idaho; Peter Struffenegger, Sterling Caviar, LLC; Ken Beer (Industry Advisor), The Fishery; Linda Lemmon, Blind Canyon Aqua Ranch; Leo Ray, Fish Breeders of Idaho; Fred Conte* (Outreach), University of California, Davis; Jason Mann (Project Monitor), EWOS Canada Ltd.

PROJECT OBJECTIVES: This project aims to improve the yield and quality of caviar in farmed sturgeon. The specific objectives are: (1) conduct feeding trials with adult sturgeon to determine the effect of dietary energy on roe yield in age 7 and age 8 maturing females; (2) conduct a feeding trial with prepubertal sturgeon to determine effects of dietary energy on accumulation of ovarian fat in early ontogeny; (3) determine the effect of farm, diet, and maturity age on chemical and sensory properties of caviar, and evaluate the use of image analysis for measuring ovarian fat content; (4) determine the dietary effect on the crude chemical composition of sturgeon ovaries and proliferation of adipose tissue in prepubertal fish; (5) determine the effect of genotype on roe yield and ovarian adiposity in adults, and on ovarian adiposity in prepubertal sturgeon; (6) determine the energy partitioning in prepubertal sturgeon fed a low and high energy diet; (7) develop an integrated approach for management of farmed sturgeon with high caviar yield and quality as outreach for the project.

ANTICIPATED BENEFITS: The sturgeon industry will know how caviar yield and quality can be affected by low and high energy diets and genetic relatedness. They will see the effect of diets on caviar yield as a percentage of the ovarian weight and as a percentage of body weight, and how these diets may affect caviar sensory evaluations. The genetic component of the study will reveal the effect of relatedness on caviar yield and quality, such as whether the high fat ovary females originate from genetically related families. Based on the outcome of this project, the industry may be able to develop an appropriate dietary regime for caviar production sturgeon, and a broodstock mating plan to reduce ovarian adiposity.

PROGRESS AND PRINCIPAL ACCOMPLISHMENTS:

Objective (1) Feeding trials with production fish to determine the effect of dietary energy on roe yield in maturing females: (1.1) California: Two farms are using the same progeny (2006 year class), and at age 3-4 years and body weight 7-10 kg females were moved into six 9.2 m and six 14.6 m diameter circular tanks at Sterling Caviar LLC and The Fishery, respectively. Three tanks were randomly assigned to a high (HE) and low energy (LE) diet, EWOS at Sterling and Skretting at The Fishery. On December 4, 5, and 6, 2012 all EWOS females, and on January 9, 16, 17, 23, 24, and 30, 2013 all Skretting females were checked for black eggs by making a small abdominal incision and inserting a catheter to obtain a sample of eggs (Table 1). All EWOS and Skretting diet females with black eggs (n= 292 and n=585, respectively) were tagged, to trace them back to their original farm tank, put into a lift net, placed onto a transport tank, and relocated to the coldwater site (8-10°C) until caviar harvest during April, 2013.

The feed trial began during September 2011 and monthly mortalities were recorded by the farm staff. The total mortalities for the three replicate tanks averaged 5.9% and 9.9% for the Skretting LE and HE diets, respectively, and due to disease issues at Sterling, the mortalities were higher; 29.8% and 38.1%, for the EWOS LE and HE, respectively (Table 1). Interestingly, the total mortalities were significantly higher for fish in both HE diets, compared to their respective LE diet, and whether or not this is biologically relevant is not clear, although the overall health benefits of a low fat diet (as in humans) maybe important for sturgeon at this life stage.

Although the average percent of mature females for the LE (14.4%) and HE (18.1%) Skretting diet was significantly different (P<0.05) the mean body size (20-29 kg) was the primary factor in determining the percent maturity (Figure 1). There was a significant relationship between the Fishery replicate tank mean body size at harvest, and the percent maturity for each respective tank (R2 = 0.90). However, the percent maturity for the Sterling HE and LE EWOS tanks were similar (28.5 and 30.7%) and there was no relationship between body size and percent maturity (R2 = 0.01) as the fish had reached an optimal body size for first puberty (> 30 kg). Between April 2nd and April 23rd, 2013 all of the seven year old females were processed for caviar, and all individuals were weighed (+/- 0.05 kg) and fork length measured (+/- 0.5 cm). The ovaries were removed and weighed (+/- 0.05 kg) and after processing, the caviar yield (in-the-tin) recorded (+/- 0.1 g), and the caviar was graded on a scale of 1 to 6, with one being the best. In addition to the above data collection, for a sub-sample of 30 females from each tank, a fin clip was collected for genetic analyses (Objective 5), and after ovary screening the remaining ovarian tissue and fat lobes were dissected apart and individually weighed (+/- 0.1 g) and then a 30g subsample of ovarian tissue bagged for proximate analyses (Objective 4). From these 30 females, a sub-sample of five post-screened egg samples were collected for proximate analyses (Objective 4) and a 50g tin of finished caviar reserved for sensory analyses (Objective 3).

The summary caviar harvest for the EWOS diet females is in Table 2. There were differences between the HE triplicate tanks for a few parameters but no differences between the LE tanks. When comparing between the pooled tank data the HE diet fish were slightly larger (32.2 kg) compared to the LE fish (30.8 kg) but not significantly different. On an average the females gained 6 kg in body weight during the 19 month trial. The HE females had a significantly higher ovary weight and GSI compared to the LE females, but there was no difference in caviar yield (2.4 vs 2.5 kg, respectively). The caviar yield as a percent of the body weight was higher for the LE females (8.0%) compared to the HE (7.6%) but was not significantly different (P=0.062). Although yield when expressed as a percent of the ovary weight, was significantly (P<0.05) higher for the LE females (64.1% vs 58.0%).

The summary caviar harvest for the Skretting diet females is in Table 3. There were differences between the triplicate tanks for both diets, likely due to the differences in mean body size between tanks. When comparing between the pooled tank data the HE diet fish were significantly larger (26.0 vs 24.3 kg), and with a higher condition factor and ovary weight compared to the LE fish. The HE females gained about 10 kg in body weight and the LE diet females gained 8 kg, during the 19 month trial. There was no difference in mean caviar weight or GSI between the two diets, but the caviar yield as a percent of the body weight was significantly higher for the LE females (8.6%) compared to the HE (7.9%), and also when expressed as a percent of the ovary weight (61.3% vs 55.5%).

The caviar grade score was identical for both LE and HE diets, at each farm. But overall, the Skretting diet females were graded lower (3.7 average) compared with the EWOS diet females (2.8 average), likely due to the very different water sources and annual temperature fluctuations between the two farms. The Skretting females are reared on pond water that fluctuates from 10-28°C each year, while the EWOS females are on well water, ranging from 18-22°C.

The partitioning of the fat lobes and the ovarian tissue, after caviar screening revealed that females fed the HE diets had significantly larger lobes of fat compared to females fed the LE diets (Table 4). The weight of the ovarian tissue was similar between the EWOS diets but was different between the Skretting diets, as the smaller mean size females in the LE had a smaller quantity of ovarian tissue (Table 4).

The data for the four diets from Tables 2, 3, and 4 were pooled and the females ovarian adiposity categorized by high, medium, or low. Females with yield of caviar (based on the ovary weight), of over 65% were in the low fat ovary category, females with a yield of less than 50% were high fat ovaries and medium fat ovaries fell in between. Table 5 summarizes the EWOS diet females with the most notable data regarding the percent of females in each category. Only 12% of the LE diet females had high fat ovaries, compared to 25% of the females in the HE diet. Conversely, 52% of the LE diet females had their ovaries categorized as the highly sought after low fat ovaries, compared to only 33% of the females in the HE diet. The remaining data revealed that in both the LE and HE diets the females with the lean ovaries tend to be smaller and leaner, but yield more caviar.

From the ninety females analyzed for the proportion of ovary fat lobes and ovarian tissue, the high fat ovary females had fat lobes making up about 30% of the ovary weight, compared to low fat ovaries with only 7-10% fat lobes (Table 5). Also, the percent of fat lobes in the low fat ovaries of females in the LE diet was lower (7.1%), compared to the low fat ovaries of females on the HE diet (10.4%). The high fat ovaries also had significantly higher losses during screening. The losses were based on the percent of the ovary not accounted for, after adding the % of caviar, fat lobes, and ovarian tissue, based on the total ovary weight. These losses are a combination of broken eggs, fatty ovarian tissue stuck to the screen, and pieces of fat and ovarian tissue rinsed out of the bowl during cleaning. The fatty ovaries take more time and effort to screen and clean. Table 6 summarizes the Skretting diet females and the same trends as described for the EWOS diet were found; the LE diet had about 50% less females with high fat ovaries and about 50% more females with low fat ovaries, females with lean ovaries were smaller and leaner but produced more caviar than the “over-eaters” (larger and more robust individuals), and the low fat ovaries in the LE diet had the lowest percent of fat lobes (8.2%).

(1.2) Idaho: This trial began at Fish Breeders of Idaho Dec, 2011 using the 2001 year class of white sturgeon, however, there are also some older (1990, 1992, and 1994 year class) fish that had been mixed with the 2001 fish over the years. Although only six and seven females have been harvested from the LE and HE diets (Table 7), respectively, the trends are the same as observed in California. The females in both diets had similar size and condition factor, but females had larger ovaries and GSI in the HE diet, but a similar caviar yield to the LE diet females (Table 7). When caviar yield was expressed as a percent of the body weight the females fed the LE diet averaged 5.6%, compared to 4.9% for the HE diet females, and when expressed as a percent of the ovary weight, 60% of the ovary from females fed LE diet ended up as caviar product, compared to 43%, for the ovaries from females fed the HE diet.

Objective (2) Feeding trial with prepubertal sturgeon to determine effects of dietary energy on accumulation of ovarian fat in early ontogeny: The second sampling, at age 2 was completed on 6/3/13 and 6/7/13 (Table 8). We randomly sampled 12-13 females from each tank, to attain a total sample size of 50 fish per diet. From each fish sampled the body weight and fork length was recorded. A fin clip was collected and placed into 95% etoh for genetic analyses (Objective 5), and both gonads were removed, weighed, and sub-sampled for proximate analyses, paraffin histology, and frozen sectioning (Objective 4).

Statistical analyses between the two diets revealed no significant difference in body size at age 2, however the high energy diet fish had significantly higher GSI’s, indicating “fattier” ovaries. These higher GSI’s were also observed at age 1 year (first annual report), although at age one the higher energy diet fish were also slightly larger. There is some size variation among tanks of the LE diet but overall, by age 2 it appears that the lower energy diet has had sufficient calories for good growth, and yet those fish are not accumulating as much ovary fat.

Objective (3) Determine the effect of farm, diet, and maturity age on chemical and sensory properties of caviar, and evaluate the use of image analysis for measuring ovarian fat content: We evaluated a number of methods for analysis, particularly image analysis, to find a system that would be suitable for analysis of these samples. We were able to find methods that will work to correlate digital image and spectral images with chemical assays for total fat. Representative data are presented here, focusing on the April samples since these images were of the best quality. Sturgeon ovary samples were provided that had varying fat content, and were received during February 2012 (n=17), April 2012 (n=23), and May 2012 (n=20) from UC Davis.

Chemical Analysis for Total Fat Determination: The results for ovarian fat content using the chemical analytical method are presented in Table 9, and revealed that the ovarian adiposity (percent yield of caviar based on the ovary weight) categories correlated well with the total fat determination.

Fatty Acid Composition Determination: After lipid extraction, fatty acid composition was determined according to Watts and Brows (2002) with a slight modification. Briefly, 50 µL of ovary lipid extract was mixed with 450 µL hexane. Then, 10 µL of the mixture added to a small screw-cap glass tube. Fatty acids were derivatized to methyl esters by adding 2.5 % H2SO4 in MeOH followed by heating to 80°C for 30 min. After cooling to room temperature, 500 µL hexane and 1.5 ml H2O were added and the mixture shaken vigorously. After centrifugation, two phases were appeared. Then 70 µL of top layer was loaded into a vial using the small inserts. Samples were analyzed by GC using an Agilent 6890 series gas chromatographer equipped with 30m × 0.32mm Omegawax™ 320 capillary column (Supelco™, Sigma Aldrich, St. Louis, MO, USA), helium as the carrier gas at 1.4 ml/min, and a flame ionization detector. To quantify the fatty acids, an internal standard (C17:0-Methyl-Ester, 10 µg for 200 mg of lipids) was added to each sample. In order to determine the quality of the GC, an external standard (Natural Menhaden oil; Matreya LLC, Pleasant Gap, PA, USA) was run by GC.

Fatty Acid Composition of Sturgeon Ovary: The fatty acid composition of sturgeon ovaries from different months are presented in Tables 10-12. Mono-unsaturated fatty acids (MUFA) were higher in high and medium fat ovaries compared with low fat ovaries in all groups from different months. In February samples, n-3/n-6 ratio was significantly higher in the low fat group, compared with medium and high fat ovaries, but no significant differences were observed in ovaries from April and May. In all samples from different months, docosahexaenoic acid (DHA) was significantly higher in low fat ovaries compared to the high fat samples; however, there was no difference between low fat and medium fat ovaries. The ratio of DHA/EPA was higher in low fat ovaries compared with medium and high fat ovaries. Saturated fatty acids were dominated by C16:0, which contributed more than 76-80% of total SAFAs. Oleic acid (C18:1n9) was the most abundant MUFAs in all groups. Among the n-3 fatty acids, C22:6n3 (DHA) was the most abundant fatty acids with 46-54% of total n-3 fatty acids.

Image Analysis of Sturgeon Ovary: Fat content in the ovaries was analyzed using image analysis; the Image J software (National Institute of Health, USA, Version 1.47). The ratio of fat in the ovary was calculated after digital pictures had been converted to the 8 bit images. Sensitivity was determined and images converted to black (non-fat tissue) and white (fatty tissue) using the threshold command. The limitation of this method is that only the visual features of the surface of the ovary are apparent in the digital image, so if the composition of the ovary varies from anterior to posterior region or from the exterior to interior surface, this method will not be suitable unless the ovary is cut into smaller sections and separate images taken. The predicted fat content from image analysis for May samples is presented in Table 13 with select images used for the analysis for low, medium and high fat ovaries shown in Figure 2. The regression coefficients between image analysis and chemical assay for May samples is 87% (Figure 3) which is significantly higher than April samples (79%) (see 1st annual report).

Objective (4) determine the dietary effect on the crude chemical composition of sturgeon ovaries and proliferation of adipose tissue in prepubertal fish: The caviar and ovarian tissue composition (protein, lipid, moisture) was calculated by means of standard proximate analysis. Protein content was determined by thermal oxidation (Leco TruSpec, Leco Corp., St. Joseph, Missouri) following the Association of Official Analytical Chemists method 992.15 (AOAC 1990). Lipid was measured using a petroleum ether extraction (Ankom XT 10; Ankom Technology, Macedan, New York). Tissue moisture was measured by drying a sample for 3 hours at 125°C or until no mass change occurred (Yamato Constant Temperature Oven DKN900, Yamato Scientific America, Inc., Santa Clara, California). Gonads of 2-year-old fish were homogenized using liquid nitrogen and a Waring Commercial Laboratory Blender.

For paraffin histology gonadal tissue was embedded in paraffin, sectioned at 5 µm, and stained by hematoxylin and eosin. Slides are currently being examined under a compound scope (Leica DM 2000, 100x-400x). For frozen section histology, samples were processed at both the Veterinary Diagnostics Laboratory and Rogers Dermatology, Bozeman, MT. Frozen sections were embedded in water-soluble media on a chuck. Temperatures were maintained at -20 to 39°C, and sections were attempted at 5, 7, and 10 µm. The proximate analysis data among tanks within each diet were compared using Kruskal-Wallis rank sum tests followed by pairwise Wilcoxon rank sum tests. Although a tank effect was detected within the same diet at a single farm, the data were pooled for an overall comparison between the HE and LE diets. Mann-Whitney U-tests were used to compare data between HE and LE diets within the same farm, and were also used to compare the data between HE and LE diets in the 2 year-old gonad samples.

There were some statistical differences in moisture, fat and protein content of ovarian tissue observed among the HE Skretting tanks, but there were no differences among the LE tanks (Table 14). Statistical analysis of the pooled data indicates no difference in fat or protein content, but moisture content for the HE diet (56.16 ± 10.56) was lower than that of LE diet (60.32 ± 11.29). Similar results were found with the EWOS diets (Table 15). There were some differences among individual tanks fed the HE diet but no differences among the LE tanks. Analysis of the pooled data indicates the HE EWOS diet resulted in a higher ovarian fat content and a lower percent moisture and protein. Overall, both HE diets resulted in higher amounts of fat deposition within the ovarian stroma (although not statistically greater in the Skretting diet (p=0.098)).

The proximate analyses of the caviar samples was similar among the replicate tanks and between the diets (Tables 16, 17) except for the slightly lower percent moisture and higher protein for the HE EWOS diet, compared to its LE diet. Overall, the caviar was approximately 54% moisture, 10% fat and 27% protein. Although we observed that the GSI was statistically higher for females in the EWOS HE diet at both age one (1st Annual report) and age two (Table 8), there were no differences observed in ovary moisture, fat, or protein between the two diets, except moisture at age one (Table 18).

The histology for the ovary samples is underway, and considerable time has been spent working on frozen section histology techniques. No cryo-sections could be collected as there is too much unsaturated fat present in the gonad tissue resulting in the lack of adequate freezing even at -39°C. We are working with Dermapathology Northwest (Bellevue WA) and researchers on Histonet to develop techniques that would allow for observation of the adipocyte-germ cell interface in sturgeon ovaries embedded in paraffin.

Objective (5) determine the effect of genotype on roe yield and ovarian adiposity in adults, and on ovarian adiposity in prepubertal sturgeon: Fin clips were collected from 100 one year-old white sturgeon on 6/12/12; half were fed a LE EWOS diet and half the HE EWOS diet, since age six months. Fin clips were also collected from the six sires and six dams used to generate the year class. The male parents were: OE5E, 6F6B, 6158, 5C49, 2179, and 5C2E. The female parents were: O610, 2808, 2E4C, 7627, 6C27, and 234A. DNA was extracted from fin clip tissue using a Gentra Puregene Genomic DNA Purification kit. Fish were genotyped at 11 microsatellite loci and genotyping was performed on ABI3730x1 Genetic Analyzers and GeneMapper analysis software. Microsatellite alleles were treated as dominant data and the program Logparent was utilized to assign the most likely sire and dam to each sturgeon. Differences in GSI among groups of offspring from each male and female parent, for each diet were evaluated using a one-way analysis of variance.

The most likely parents of each one year-old sturgeon fed the LE and HE diets are presented in Table 19. Sire 5C2E and dam 234A were not represented in the fish sampled, and there was some variability in the number of families sampled from each tank, ranging from 4 to 9 families in the LE diet tanks, and 6 to 9 families in the HE tanks. The replicate tanks for each diet were pooled and the data summarized in Table 20. The most prominent family in each diet was male OE5E x female O610, which accounted for 52% of the families in the LE diet tanks and 26% of the families in the HE diet. The total number of progeny produced by each sire and dam is summarized in Table 21. The most successful sire and dam, that produced 55% of the one year-old sampled fish, were OE5E and O610, respectively. For the one year-old sturgeon fed the different diets and determination of whether there was a specific sire or dam that had progenies with higher GSI’s was not conclusive, due to the highly variable “n” number of progeny from each sire or dam, and the highly variable GSI between individuals. The only statistically significant ANOVA was among individuals grouped by sire in the HE diet. The recent processing of the two year-old fish, and ongoing analyses, will determine if the most prominent family continues to be represented.

Objective (6) determine the energy partitioning in prepubertal sturgeon fed a low and high energy diet: Monthly sampling began December 2012 using the same tanks of prepubertal fish used in Objective 2, and will continue for one year. Fish from each replicate tank were removed and euthanized until five females were indentified (all males were sent to the processing plant except on even months when one from each diet tank was sampled; to preliminarily establish if there are differences between sexes). Body weight (+/- 1.0g) and fork length (+/- 0.5cm) were recorded, the ovaries removed and weighed (+/- 1.0g) and then the ovaries were pooled from each replicate tank and frozen, for proximate analyses. The carcasses (head removed) were also pooled by tank, stored on wet ice and then ground-up in a large commercial meat grinder, to obtain a sub-sample for proximate analysis. The head was removed from each fish, directly behind the pectoral fins, because the grinder could not break down the skull and pectoral fin ray bones, but one female head, from each replicate tank was collected and frozen (n=4 per diet), and will be used in establishing base line data, after solving the bone grinding issue.

The monthly mean body weight for the sturgeon sampled did not differ between the two diets and the fish are growing at a similar rate (Figure 4). The mean GSI was highly variable between diets and sometimes between months, for the same diet (Figure 5), likely due to the sample size restrictions and the individual variability in fatty adipose tissue proliferation. Some of the LE diet fish have larger ovaries than the average HE fish, and sometimes a HE fish has a much smaller and leaner ovary than the average LE fish. Although we did find a significant difference in mean GSI for the fifty females sampled for genetics at age two (Table 8; HE = 4.15% and LE = 3.38%), the twenty females per diet, at age two for this energy allocation objective, showed the same trend, although not statistically significant, the HE diet females GSI was 3.69% and the LE diet GSI was 3.14% (p=0.303). Although this illustrates the difference in “n” numbers, due to limited funding and limited fish available to remove from farm production, we cannot realistically sample fifty females each month, for each diet, for this objective.

Objective (7) develop an integrated approach for management of farmed sturgeon with high caviar yield and quality as outreach for the project: In preparation for outreach activities, project photography, including graphic materials from the IAC/TC annual reports are being reviewed for incorporation in outreach digital presentations. Discussions with project scientists are being initiated to develop themes and concepts gained from the project that address the outreach objective.

USEFULNESS OF FINDINGS: After an additional year on the LE diet, the sampling of the two year-old prepubertal sturgeon indicates there was still an effect of diet on gonad growth; the LE fed fish had lower GSI’s, although body size was not different from the HE fish, indicating similar caloric intake for fish in both diets. Genetic analyses of the one year-old prepubertal sturgeon revealed that a single sire and single dam were responsible for producing over half of the fish sampled. This finding is important for sturgeon culturists if they want to develop a management strategy to maintain multiple, genetically diverse families. The sampling of age 7 caviar females, after 19 months on the diets, revealed that fish fed the HE diets had lower yields of caviar, both on a body weight and ovary weight basis, and were depositing larger quantities of ovarian fatty adipose tissue lobes. This study also showed that it is possible to predict fat content of sturgeon ovary from digital image and with good quality digital images a correlation of approx. 0.9 is possible. In addition, this work revealed that fatty acid composition is different among the roe according to their fat content. It seems by modulating the diets, it could be possible to decrease the fat in ovary to obtain more roe for caviar processing, and produce high nutritional caviar for human consumption.

WORK PLANNED FOR NEXT YEAR: Objective (1): In California, each tank will be re-sampled during the fall 2013 to identify maturing females at age 7+. Percent mature females in each tank will be determined by count before moving fish to the cold water facility. All females with black eggs will be tagged for tank and farm origin. At the coldwater site (8-10°C) there will be at least one tank (9.1m diameter, 0.85m deep, 55 cubic meters volume) assigned to each diet for each farm (minimum 4 tanks). The diet treatments will continue at the coldwater facility through the winter and spring, until caviar harvest at age 8 (April 2014). At the caviar processing plant, fin clips (for genetic analyses, Objective 5), fish length and weight, ovary weight, screened egg and “in-the-tin” caviar weight, fat lobes and residual ovary weight will be collected from random females (n=30/tank). Samples will be collected for proximate analysis (Objective 4) of the ovary tissue and screened eggs, and sensory evaluation of caviar (Objective 3). In Idaho, each raceway will be checked for mature females each winter, spring, summer and fall, because this farm processes caviar year around. The same caviar processing data described above will be collected from all females. For Objective (2) the sampling of three year old pre-pubertal fish will occur during June 2014. We will continue to collect fin clips, extract DNA, and perform PCR at 11 microsatellite loci, and assign a sire and dam, in prepubertal (age 2 and 3) and caviar females (age 7 and 8), for the heritability studies. We will complete the monthly sampling (Nov 2013) of the prepubertal sturgeon for studies on energy allocation and run proximate analyses on samples collected (Objective 6). And for objective (7) we will continue with workgroup scientists to develop themes and concepts gained from the overall project that address the management of farmed sturgeon for high caviar yield and quality, and work will begin in the construction of digital media presentations using Articulate software.

SUPPORT: University of Washington (Rasco Lab) did get assistance with software applications from others at the university and the value of this training was about $8,000.

YEAR

WRAC-USDA

Funding

OTHER SUPPORT

University Industry Federal Other Total

TOTAL

SUPPORT

2011-12

$120,000

0 4,000 0 0 4,000

$124,000

2012-13

$120,000

0 4,250 0 0 4,250

$124,250

2013-14

$195,210

0 0 0 0 0

$195,210

2014-15

TOTAL

$240,000

0 8,250 0 0 8,250

$443,460

PUBLICATIONS, MANUSCRIPTS, POSTERS OR PAPERS SUBMITTED/PRESENTED:

“Identification of quantitative trait loci associated with caviar yield and ovarian adiposity in white sturgeon (Acipenser transmontanus).” Poster presentation by D. Gille, A. Drauch Schreier, and B. May. Plant and Animal Genome XX, San Diego, CA, January 14-18, 2012.

“Caviar yield of Sacramento and Fraser River stocks of farm raised white sturgeon”. Oral presentation by J. Van Eenennaam, P. Struffenegger, K. Beer, J. Henry, and S. Doroshov. The 7th International Symposium on Sturgeon, Nanaimo, BC, Canada, July 22-25, 2013.

“Using image processing to determine ovarian fat content in farmed white sturgeon Acipenser transmontanus”. Oral presentation by M. Ovissipour, B. Rasco, S. Doroshov, and J. Van Eenennaam, accepted to: Aquaculture America 2014, February 9-12, 2014, Seattle, WA.

“Fatty acid composition of farmed white sturgeon Acipenser transmontanus ovary at different ages.” Oral presentation by M. Ovissipour, B. Rasco, S. Doroshov, and J. Van Eenennaam, accepted to: Aquaculture America 2014, February 9-12, 2014, Seattle, WA.

SUBMITTED BY:___________________________________________________________________

Serge Doroshov & Joel Van EenennaamDate

APPROVED: ___________________________________________________________

Jason Mann, Project MonitorDate

APPENDED TABLES AND FIGURES:

Table 1. Inventory (n) at the start of the feed trial (September 2011) using low (LE) and high energy (HE) Skretting (SK) and EWOS diets with 2006 year-class females, at two California sturgeon farms. Data are total recorded mortalities through December 2012 (≈15 months), calculated mean monthly mortality, and number and percent of mature age 7 females moved to the cold water facility in December 2012. Different letters in columns indicates significant different proportions (Contingency table analyses, P< 0.05) between the pooled data for the specific low and high energy diet.

Diet

Tank

Starting (n)

Total Mortalities

% (n)

Mean Monthly Mortality % (n)

(total/15 mo)

Total Mature Females

% (n)

LE-SK

C2

642

3.7 (24)

0.2 (2)

23.0 (142/618)

LE-SK

C4

628

9.4 (59)

0.6 (4)

10.4 (59/569)

LE-SK

C5

640

4.5 (29)

0.3 (2)

9.8 (60/611)

LE-SK

Pooled

5.9% a

0.4% (3)

14.4% a

Diet

Tank

Starting (n)

Total Mortalities

% (n)


(total/15 mo)


% (n)

HE-SK

A1

659

9.7 (65)

0.6 (4)

13.8 (82/594)

HE-SK

C1

677

8.4 (57)

0.6 (4)

24.0 (149/620)

HE-SK

C3

632

11.6 (73)

0.8 (5)

16.6 (93/559)

HE-SK

Pooled

9.9 % b

0.7% (4)

18.1% b

Diet

Tank

Starting (n)

Total Mortalities

% (n)


(total/15 mo)


% (n)

LE-EWOS

1-1

231

26.8 (62)

1.8 (4)

31.4 (53/169)

LE-EWOS

1-2

249

35.7 (89)

2.4 (6)

36.3 (58/160)

LE-EWOS

2-2

259

27.0 (70)

1.8 (5)

25.4 (48/189)

LE-EWOS

Pooled

29.8% a

2.0% (5)

30.7% a

Diet

Tank

Starting (n)

Total Mortalities

% (n)


(total/15 mo)


% (n)

HE-EWOS

3-3

249

42.2 (105)

2.8 (7)

25.7 (37/144)

HE-EWOS

4-1

248

38.3 (95)

2.6 (6)

31.4 (48/153)

HE-EWOS

4-2

255

33.7 (86)

2.2 (6)

28.4 (48/169)

HE-EWOS

Pooled

38.1% b

2.5% (6)

28.5% a

Figure 1. Percent mature females in each tank (n=6; 3-LE and 3-HE) in relation to the tank mean body weight. Skretting diet (top) and EWOS diet (bottom).

36

Table 2. Caviar harvest data from age seven females fed the low (LE) and high energy (HE) EWOS diets for nineteen months. Total number (n) of females processed is given after the tank ID#. Body (BW) and ovary weight (OW) was used to calculate GSI (ovary weight/body weight x 100), K is Fulton condition factor BW (g)/FL(cm)^3, and caviar yield is given as actual weight and as the percent of the body and ovary weight. Caviar grade was provided by the processing plant and was based on a ranking from 1 to 6, with the rank of 1 being the best. Different sample means (ANOVA, post hoc Tukey, P<0.05) among the tanks are marked by different letters, and differences between the pooled diet data are marked with an asterisk. Data are means ± SD. For reference, the body weight and K are also given for at the start of the trial.

Diet

Tank # (n)

Body Wt (kg)

Condition Factor

(K)

Ovary Wt

(kg)

GSI

(%)

Caviar

Yield

(kg)

Caviar Yield

(% BW)

Caviar

Yield

(% OW)

Caviar

Grade

HE

4-1 (37)

34.8 ± 8.4a

0.92 ± 0.09a

4.8 ± 1.5a

13.7 ± 2.4a

2.7 ± 0.8a

8.0 ± 1.8a

57.9 ± 10.4a

2.6 ± 1.4a

HE

4-2 (40)

31.7 ± 5.0ab

0.90 ± 0.10a

4.0 ± 1.0b

12.6 ± 2.0a

2.4 ± 0.7ab

7.5 ± 1.9a

60.2 ± 12.3a

3.2 ± 1.3a

HE

3-3 (35)

30.1 ± 6.1b

0.90 ± 0.11a

3.9 ± 1.0b

13.1 ± 2.2a

2.2 ± 0.6b

7.2 ± 1.5a

55.7 ± 11.1a

2.7 ± 1.5a

HE Pooled

32.2 ± 6.8

0.91 ± 0.10

4.3 ± 1.2*

13.1 ± 2.2*

2.4 ± 0.7

7.6 ± 1.8

58.0 ± 11.4*

2.8 ± 1.4

Trial Start

24.0 ± 4.7

0.90 ± 0.09

LE

1-1 (42)

31.7 ± 5.7a

0.90 ± 0.09a

4.0 ± 1.1a

12.6 ± 2.1a

2.5 ± 0.7a

7.8 ± 1.8a

62.2 ± 11.5a

2.9 ± 1.5a

LE

2-2 (35)

31.0 ± 6.7a

0.89 ± 0.10a

3.9 ± 1.1a

12.7 ± 2.0a

2.5 ± 0.6a

8.1 ± 1.7a

63.8 ± 9.8a

2.9 ± 1.4a

LE

1-2 (49)

30.0 ± 4.8a

0.91 ± 0.09a

3.7 ± 0.9a

12.3 ± 1.8a

2.5 ± 0.7a

8.1 ± 1.8a

65.8 ± 9.5a

2.7 ± 1.2a

LE Pooled

30.8 ± 5.7

0.90 ± 0.09

3.9 ± 1.0

12.5 ± 1.9

2.5 ± 0.7

8.0 ± 1.8

64.1 ± 10.3

2.8 ± 1.4

Trial Start

24.0 ± 4.7

0.90 ± 0.08

Table 3. Caviar harvest data from age seven females fed the low (LE) and high energy (HE) Skretting diets for nineteen months. Total number (n) of females processed is given after the tank ID#. Body (BW) and ovary weight (OW) was used to calculate GSI (ovary weight/body weight x 100), K is Fulton condition factor BW (g)/FL(cm)^3, and caviar yield is given as actual weight and as the percent of the body and ovary weight. Caviar grade was provided by the processing plant and was based on a ranking from 1 to 6, with the rank of 1 being the best. Different sample means (ANOVA, post hoc Tukey, P<0.05) among the tanks are marked by different letters, and differences between the pooled diet data are marked with an asterisk. Data are means ± SD. For reference, the body weight and K are also given for at the start of the trial.

Diet

Tank # (n)

Body Wt (kg)

Condition Factor

(K)

Ovary Wt

(kg)

GSI

(%)

Caviar

Yield

(kg)

Caviar Yield

(% BW)

Caviar

Yield

(% OW)

Caviar

Grade

HE

C1 (144)

28.0 ± 6.0a

0.93 ± 0.09a

4.2 ± 1.3a

14.9 ± 2.4a

2.3 ± 0.6a

8.4 ± 1.7a

57.3 ± 11.4a

3.6 ± 1.3a

HE

C3 (90)

24.9 ± 5.6b

0.94 ± 0.09a

3.6 ± 1.1b

14.5 ± 2.5a

1.9 ± 0.5b

7.7 ± 1.7b

54.4 ± 12.3a

3.7 ± 1.2a

HE

A1 (81)

23.8 ± 4.8b

0.89 ± 0.10b

3.1 ± 0.9c

13.0 ± 2.7b

1.7 ± 0.6c

7.0 ± 2.4c

53.6 ± 14.9a

3.8 ± 1.6a

HE Pooled

26.0 ± 5.9*

0.92 ± 0.09*

3.8 ± 1.2*

14.3 ± 2.6

2.0 ± 0.7

7.9 ± 2.0*

55.5 ± 12.7*

3.7 ± 1.4

Trial Start

16.3 ± 3.3

0.94 ± 0.07

LE

C2 (135)

26.4 ± 5.6a

0.90 ± 0.09a

4.0 ± 1.2a

14.9 ± 2.6a

2.4 ± 0.6a

9.1 ± 1.7a

62.0 ± 11.0a

3.7 ± 1.2a

LE

C4 (55)

22.9 ± 4.0b

0.89 ± 0.09ab

3.0 ± 0.8b

13.3 ± 2.2b

1.8 ± 0.5b

8.1 ± 1.8b

61.3 ± 10.4a

3.7 ± 1.3a

LE

C5 (56)

20.6 ± 4.2c

0.85 ± 0.08b

2.8 ± 0.8b

13.3 ± 2.4b

1.6 ± 0.5b

7.9 ± 2.0b

59.6 ± 11.4a

3.7 ± 1.3a

LE Pooled

24.3 ± 5.7

0.89 ± 0.09

3.5 ± 1.2

14.2 ± 2.6

2.1 ± 0.6

8.6 ± 1.9

61.3 ± 10.9

3.7 ± 1.2

Trial Start

16.3 ± 4.3

0.95 ± 0.08

Table 4. Body weight at caviar harvest for seven year old females, and weight of the fat lobes and ovarian tissue separated from the ovary, after the eggs had been screened. Data were pooled from triplicate tanks for each diet. The two diets were EWOS low (LE) and high (HE) energy and Skretting LE and HE. Different sample means (t-test, P<0.05) between the LE and HE version of each diet are marked by different letters. Data (n=90) are means ± SD.

Diet

Fat Lobes Weight (g)

Ovarian Tissue Weight (g)

Body Weight (kg)

EWOS-LE

565 ± 473 a

334 ± 99 a

31.0 ± 6.0 a

EWOS-HE

856 ± 600 b

352 ± 116 a

32.8 ± 7.3 a

Skretting-LE

629 ± 462 a

250 ± 65 a

23.9 ± 5.4 a

Skretting-HE

945 ± 694 b

314 ± 119 b

26.6 ± 6.3 b

Table 5. Body (BW), ovary (OW) and caviar weight (CW), condition factor (K), gonadosomatic index (GSI), and caviar yield-CY (as a percent of the female body weight and as a percent of the female ovary weight) for seven year old females grouped as high, medium and low fat ovaries (n), fed a low (LE) and high (HE) fat EWOS diet for 19 months prior to caviar processing. Ovarian fat lobe (OFL) and ovarian tissue (OT) weight was recorded from a sub-sample of 30 females from each tank (90 females from each diet), and both were calculated as a percent of the OW, and then the percent of losses during screening and cleaning was calculated (CY+OFL+OT-100). Different letters in rows, indicate significant differences between the means ± SD (ANOVA, post hoc Tukey Test, P<0.05), and yellow highlighted data indicates significant difference between the two diets.

Age 7, EWOS HE (112)

High Fat Ovaries (28)

Med. Fat Ovaries (47)

Low Fat Ovaries (37)

% of Total (n)

25.0 a

42.0 b

33.0 c

BW (kg)

33.5 ± 7.6 a

33.0 ± 6.7 a

30.3 ± 6.1 a

K

0.92 ± 0.10 a

0.91 ± 0.09 a

0.89 ± 0.10 a

OW (kg)

4.6 ± 1.5 a

4.4 ± 1.1 ab

3.8 ± 1.0 b

GSI (%)

13.5 ± 2.8 a

13.4 ± 2.2 a

12.5 ± 1.5 a

CW (kg)

2.0 ± 0.8 a

2.6 ± 0.6 b

2.6 ± 0.6 b

CY (% BW)

5.8 ± 1.6 a

7.8 ± 1.4 b

8.7 ± 1.0 c

CY (% OW)

42.2 ± 6.0 a

58.1 ± 4.0 b

70.0 ± 3.2 c

For (n=90):

OFL (kg)

OT (kg)

CY (% OW)

OFL (% OW)

OT (% OW)

Losses (%)

(n=22)

1.4 ± 0.7 a

0.4 ± 0.1 a

43.2 ± 6.1 a

28.6 ± 8.6 a

8.8 ± 2.9 a

19.4 ± 8.6 a

(n=37)

0.9 ± 0.4 b

0.4 ± 0.1 a

57.8 ± 4.0 b

19.2 ± 5.1 b

8.1 ± 2.0 a

14.9 ± 4.7 b

(n=31)

0.4 ± 0.2 c

0.3 ± 0.1 a

70.1 ± 3.4 c

10.4 ± 4.2 c

8.3 ± 2.3 a

11.2 ± 3.3 c

Age 7, EWOS LE (126)




% of Total (n)

11.9 a

36.5 b

51.6 c

BW (kg)

32.7 ± 5.2 a

32.5 ± 6.3 a

29.2 ± 4.9 b

K

0.92 ± 0.08 a

0.91 ± 0.09 a

0.89 ± 0.10 a

OW (kg)

4.2 ± 1.1 a

4.1 ± 1.0 a

3.6 ± 0.9 b

GSI (%)

12.8 ± 2.8 a

12.6 ± 2.0 a

12.4 ± 1.9 a

CW (kg)

1.9 ± 0.6 a

2.4 ± 0.6 b

2.6 ± 0.7 b

CY (% BW)

5.7 ± 1.3 a

7.5 ± 1.3 b

8.9 ± 1.5 c

CY (% OW)

43.8 ± 6.0 a

59.6 ± 4.2 b

71.9 ± 3.9 c

For (n=90):

OFL (kg)

OT (kg)

CY (% OW)

OFL (% OW)

OT (% OW)

Losses (%)

(n=11)

1.2 ± 0.5 a

0.4 ± 0.1 a

43.1 ± 6.8 a

28.6 ± 5.5 a

10.2 ± 3.6 a

18.1 ± 7.8 a

(n=33)

0.8 ± 0.4 b

0.4 ±0.1 a

59.7 ± 4.3 b

16.9 ± 6.3 b

8.4 ± 2.1 a

15.0 ± 5.8 a

(n=46)

0.3 ± 0.3 c

0.3 ± 0.1 a

71.9 ± 3.9 c

7.1 ± 4.2 c

8.9 ± 2.5 a

12.1 ± 2.8 b

Table 6. Body (BW), ovary (OW) and caviar weight (CW), condition factor (K), gonadosomatic index (GSI), and caviar yield-CY (as a percent of the female body weight and as a percent of the female ovary weight) for seven year old females grouped as high, medium and low fat ovaries (n), fed a low (LE) and high (HE) fat Skretting (SK) diet for 19 months prior to caviar processing. Ovarian fat lobe (OFL) and ovarian tissue (OT) weight was recorded from a random sub-sample of 30 females from each tank (90 females from each diet), and both were calculated as a percent of the OW, and then the percent of losses during screening and cleaning was calculated (CY+OFL+OT-100). Different letters in rows, indicate significant differences between the means ± SD (ANOVA, post hoc Tukey Test, P<0.05), and yellow highlighted data indicates significant difference between the two diets.

Age 7, SK HE (315)




% of Total (n)

32.1 a

43.5 b

24.4 c

BW (kg)

27.8 ± 5.6 a

26.2 ± 5.8 a

23.4 ± 5.6 b

K

0.94 ± 0.09 a

0.92 ± 0.10 a

0.88 ± 0.08 b

OW (kg)

4.2 ± 1.5 a

3.8 ± 1.1 b

3.1 ± 0.9 c

GSI (%)

15.0 ± 3.4 a

14.3 ± 2.2 a

13.3 ± 1.8 b

CW (kg)

1.7 ± 0.7 a

2.2 ± 0.6 b

2.2 ± 0.6 b

CY (% BW)

6.2 ± 1.8 a

8.3 ± 1.4 b

9.4 ± 1.3 c

CY (% OW)

40.9 ± 8.4 a

57.9 ± 4.2 b

70.6 ± 4.3 c

For (n=90):

OFL (kg)

OT (kg)

CY (% OW)

OFL (% OW)

OT (% OW)

Losses (%)

(n=31)

1.5 ± 0.8 a

0.4 ± 0.1 a

41.1 ± 7.7 a

30.9 ± 9.1 a

8.8 ± 2.8 a

19.2 ± 8.2 a

(n=40)

0.8 ± 0.4 b

0.3 ± 0.1 a

57.8 ± 4.0 b

19.6 ± 6.7 b

7.4 ± 1.5 b

15.2 ± 6.5 a

(n=19)

0.4 ± 0.2 c

0.3 ± 0.1 a

68.9 ± 2.6 c

11.2 ± 4.3 c

8.4 ± 2.2 ab

11.5 ± 2.6 b

Age 7, SK LE (246)




% of Total “n”

17.9 a

39.4 b

42.7 b

BW (kg)

27.2 ± 5.8 a

24.7 ± 5.8 b

22.7 ± 5.0 c

K

0.93 ± 0.09 a

0.89 ± 0.09 b

0.86 ± 0.08 c

OW (kg)

4.2 ± 1.5 a

3.6 ± 1.2 b

3.1 ± 0.8 c

GSI (%)

15.2 ± 3.1 a

14.4 ± 2.6 a

13.5 ± 2.1 b

CW (kg)

1.8 ± 0.7 a

2.1 ± 0.7 ab

2.2 ± 0.6 b

CY (% BW)

6.7 ± 1.6 a

8.4 ± 1.5 b

9.6 ± 1.3 c

CY (% OW)

44.1 ± 5.5 a

58.3 ± 4.0 b

71.9 ± 4.4 c

For (n=90):

OFL(kg)

OT (kg)

CY (% OW)

OFL (% OW)

OT (% OW)

Losses (%)

(n=20)

1.1 ± 0.5 a

0.3 ± 0.1 a

44.0 ± 6.5 a

29.5 ± 7.3 a

7.5 ± 2.0 a

19.0 ± 5.6 a

(n=42)

0.6 ± 0.3 b

0.3 ± 0.07 a

58.2 ± 3.8 b

18.7 ± 5.6 b

8.2 ± 2.0 a

14.9 ± 5.0 b

(n=28)

0.2 ± 0.1 c

0.2 ± 0.06 a

70.8 ± 4.0 c

8.2 ± 3.8 c

7.9 ± 1.9 a

13.1 ± 3.4 b

\

Table 7. Idaho caviar harvest data from females fed the low (LE) and high energy (HE) Skretting diets for approximately seventeen months. For all females processed from each diet (n), body (BW) and ovary weight (OW) was used to calculate GSI (ovary weight/body weight x 100), K is Fulton condition factor BW (g)/FL(cm)^3, and caviar yield is given as actual weight and as the percent of the body and ovary weight. Different sample means (t-test, P<0.05) are marked by an asterisk. Data are means ± SD. For reference, the body weight and K are also given for the start of the trial.

Diet (n)

Body Wt (kg)

Condition Factor

(K)

Ovary Wt

(kg)

GSI

(%)

Caviar Yield

(kg)

Caviar Yield

(% BW)

Caviar

Yield

(% OW)

HE (7)

33.1 ± 9.6

0.77 ± 0.18

4.0 ± 2.0*

11.5 ± 2.7*

1.6 ± 0.6

4.9 ± 1.2*

43.3 ± 11.8*

Trial Start

29.4 ± 9.9

0.72 ± 0.10

LE (6)

30.9 ± 2.4

0.78 ± 0.08

2.8 ± 0.3

9.2 ± 1.1

1.7 ± 0.4

5.6 ± 1.6

60.0 ± 10.6

Trial Start

24.0 ± 7.7

0.68 ± 0.09

Table 8. Sampled (6/3/13 and 6/7/13) age 2 white sturgeon (2011 year class) fed low (LE) and high (HE) energy diets since age six months. Tanks are 6.1m (20 foot) diameter fiberglass circular tanks with a 49.3m3 water capacity. Fish number and density for each tank was at the time of sampling. Metrics are based on 12-13 females randomly sampled from each tank (in parenthesis after tank number). K is Fulton condition factor BW (g)/FL(cm)^3. Different sample means among the tanks of each diet, are marked by different letters (ANOVA, post hoc Tukey Test, P<0.05). Data are means ± SD for individual tanks and also for pooled tanks (n=50), of each diet. Pooled data was significantly different (indicated with an asterisk) for GSI only.

Diet

Tank #

Body Wt (kg)

Fork Length (cm)

Condition Factor

(K)

GSI (%)

Total Tank Fish #

Density (kg/m3)

LE

B-6(12)

3.63 ± 0.84 ab

76.1 ± 5.4 ab

0.81 ± 0.06 a

3.66 ± 1.86 a

941

71

LE

B-7(13)

3.29 ± 0.45 b

74.9 ± 3.6 b

0.78 ±0.05 a

2.75 ± 1.02 a

909

66

LE

B-9(12)

4.37 ± 0.88 a

80.7 ± 4.0 a

0.82 ± 0.06 a

3.68 ± 1.56 a

865

66

LE

B-10(13)

3.95 ± 0.85 ab

77.4 ± 4.7 ab

0.84 ± 0.06 a

3.47 ± 1.77 a

900

71

LE

Pooled

3.80 ± 0.85

77.2 ± 4.8

0.81 ± 0.06

3.38 ± 1.58

Diet

Tank #

Body Wt (kg)

Fork Length (cm)

Condition Factor

(K)

GSI (%)

Total Tank Fish #

Density

(kg/m3)

HE

B-4(12)

3.70 ± 0.61 a

76.6 ± 4.8 a

0.82 ± 0.08 a

4.15 ± 1.73 a

901

71

HE

B-11(13)

3.91 ± 0.68 a

78.7 ± 3.6 a

0.80 ± 0.07 a

4.03 ± 1.54 a

907

72

HE

B-12(12)

3.93 ± 0.88 a

78.0 ± 4.9 a

0.82 ± 0.09 a

4.59 ± 1.49 a

926

75

HE

B-18(13)

3.28 ± 0.57 a

74.3 ± 4.6 a

0.80 ± 0.06 a

3.87 ± 1.44 a

930

74

HE

Pooled

3.70 ± 0.72

76.9 ± 4.7

0.81 ± 0.07

4.15 ± 1.52 *

Table 9: Fat content of sturgeon ovary samples (February and April) modified Bligh and Dyer (1959) method.

Sampling Period

February

April

May

Sample Code

Fat Content (%)

Sample Code

Fat Content (%)

Sample Code

Fat Content (%)

20

21.43±1.72

62

29.41±3.25

88

26.74 ±1.1

18

23.15±1.18

57

26.52±1.43

73

21.30±1.2

30

22.1±0.41

66

25.96±2.1

80

23.25±2.1

17

19.1±0.72

36

21.65±3.4

89

20.71±2.6

22

20.1±1.52

43

24.72±4.07

85

22.92±2.3

29

21.41±1.3

45

19.72±5.10

92

23.54±1.2

25

21.52±3.13

52

25.15±2.55

90

21.77±0.98

2

32.47±2.83

63

21.95±3.22

93

35.45±3.1

8

33.43±1.52

47

29.41±3.66

84

32.08±1.9

28

27.93±1.39

37

28.27±2.81

72

33.98±1.01

3

25.85±2.61

46

29.48±8.07

95

30.41±5.6

11

25.8±3.27

33

27.17±4.36

67

37.45±3.5

31

39.37±2.6

50

29.70±4.01

77

30.12±3.3

19

40.9±0.56

48

29.01±3.12

82

50.14±3.06

16

51.54±3.15

38

23.49±2.88

83

48.48±5.3

23

36.33±1.53

49

47.37±2.58

81

43.38±5.03

21

36.12±0.81

55

44.98±2.63

75

52.44±3.08

-

-

44

46.27±2.46

70

48.11±5.23

-

-

65

43.04±6.5

76

53.94±2.86

-

-

42

43.26±1.52

79

48.09±2.51

-

-

59

38.93±3.58

-

-

-

-

34

36.92±1.48

-

-

-

-

54

36.69±2.38

-

-

Visual assessment of ovarian fat content: Red: Low fat ovary Black: Medium fat ovary

Blue: High fat ovary

Table 10: Fatty acid composition of sturgeon ovary (February samples). Means in the same row with different letters are significantly different (P<0.05).

Low fat ovary (N=7)

Medium fat ovary (N=5)

High fat ovary (N=5)

Fatty acid (%wt)

Mean

SD

Mean

SD

Mean

SD

C14:0

2.17

0.19

1.87

0.22

2.96

0.47

C16:0

18.77

0.47

18.58

0.54

17.77

0.60

C16:1n7

5.61

0.22

5.64

0.52

6.00

0.45

C16:2n4

0.22

0.16

0.17

0.17

0.32

0.05

C16:3n4

7.80

1.19

7.70

1.90

6.34

0.73

C16:4n1

0.23

0.16

0.17

0.17

0.32

0.05

C18:0

3.27

0.24

3.02

0.40

2.56

0.26

C18:1n9

26.85

1.90

33.60

2.14

32.53

2.52

C18:1n7

0.23

0.16

0.17

0.17

0.32

0.05

C18:2n6

5.05

0.14

8.03

1.15

5.56

1.31

C18:2n4

0.22

0.16

0.17

0.17

0.32

0.05

C18:3n4

0.48

0.10

0.25

0.19

0.45

0.07

C18:3n3

0.52

0.04

0.56

0.04

0.91

0.68

C18:4n3

0.48

0.10

0.26

0.21

0.51

0.04

C20:1n9

1.11

0.10

1.16

0.10

1.67

0.15

C20:4n6 (AA)

0.22

0.16

0.17

0.17

0.32

0.05

C20:4n3

2.59

0.32

2.25

0.29

1.89

0.27

C20:5n3 (EPA)

0.58

0.10

0.33

0.13

0.57

0.05

C21:5n3

6.81

0.41

4.35

0.85

5.66

0.73

C22:5n3 (DPA)

2.61

0.25

1.59

0.24

2.21

0.30

C22:6n3 (DHA)

14.12

0.55

9.85

1.51

10.71

1.38

∑SAFA

24.22 a

-

23.47 a

-

23.31 a

-

∑MUFA

33.80 b

-

40.61 a

-

40.54 a

-

∑n-3

27.70 a

-

19.22 b

-

22.48 c

-

∑n-6

5.28 a

-

8.21 b

-

5.89 a

-

n-3/n-6

5.24 a

-

2.34 b

-

3.81 b

-

DHA/EPA

24.33 b

-

29.16 a

-

18.61 c

-

Table 11: Fatty acid composition of sturgeon (April samples). Means in the same row with different letters are significantly different (P<0.05).

Low fat ovary (N=8)



Fatty acid (%wt)

Mean

SD

Mean

SD

Mean

SD

C14:0

1.49

0.23

1.72

0.31

2.11

0.30

C16:0

18.1

0.88

17.56

0.71

18.51

0.64

C16:1n7

5.10

0.40

5.10

0.26

5.87

0.56

C16:2n4

0.17

0.13

0.11

0.12

0.23

0.10

C16:3n4

6.70

0.91

6.62

1.56

5.89

0.72

C16:4n1

0.17

0.13

0.11

0.12

0.23

0.10

C18:0

3.10

0.32

3.10

0.42

2.67

0.27

C18:1n9

35.40

1.15

36.63

1.66

37.10

2.60

C18:1n7

0.17

0.12

0.11

0.12

0.23

0.10

C18:2n6

7.26

0.42

7.17

0.58

7.80

0.51

C18:2n4

0.17

0.12

0.11

0.12

0.23

0.10

C18:3n4

0.34

0.03

0.32

0.11

0.33

0.11

C18:3n3

1.22

0.76

1.07

0.72

0.67

0.43

C18:4n3

0.31

0.17

0.32

0.15

0.42

0.14

C20:1n9

1.34

0.27

1.48

0.29

1.45

0.23

C20:4n6 (AA)

0.17

0.12

0.11

0.12

0.23

0.10

C20:4n3

2.24

0.42

2.15

0.38

1.87

0.30

C20:5n3 (EPA)

0.31

0.10

0.32

0.14

0.36

0.13

C21:5n3

4.34

0.46

4.33

0.61

4.18

0.50

C22:5n3 (DPA)

1.48

0.15

1.66

0.17

1.57

0.26

C22:6n3 (DHA)

10.51 a

1.28

9.89

0.87

7.97

1.10

∑SAFA

22.62 a

-

22.32 a

-

23.31 a

-

∑MUFA

41.92 b

-

43.32 a

-

44.63 a

-

∑n-3

20.43 a

-

19.78 a

-

17.10 b

-

∑n-6

7.44 a

-

7.28 a

-

8.04 a

-

n-3/n-6

2.74 a

-

2.71 a

-

2.12 a

-

DHA/EPA

33.15 a

-

30.29 b

-

21.58 c

-

Table 12: Fatty acid composition for sturgeon ovary (May sample). Means in the same row with different letters are significantly different (P<0.05).

Low fat ovary (N=6)



Fatty acid (%wt)

Mean

SD

Mean

SD

Mean

SD

C14:0

1.57

0.23

1.79

0.25

2.21

0.45

C16:0

18.30

0.77

17.90

0.71

17.77

0.96

C16:1n7

4.77

0.48

4.99

0.11

5.37

0.52

C16:2n4

0.11

0.12

0.14

0.15

0.25

0.13

C16:3n4

7.35

2.28

6.86

1.05

6.03

0.80

C16:4n1

0.11

0.12

0.14

0.15

0.25

0.13

C18:0

3.20

0.23

3.03

0.54

2.38

0.50

C18:1n9

33.74

1.20

34.66

2.17

35.67

1.70

C18:1n7

0.11

0.12

0.14

0.15

0.25

0.13

C18:2n6

7.17

1.15

6.81

0.31

6.89

0.85

C18:2n4

0.11

0.12

0.14

0.15

0.25

0.13

C18:3n4

0.27

0.14

0.30

0.10

0.31

0.02

C18:3n3

1.31

0.66

1.22

0.72

1.83

0.25

C18:4n3

0.34

0.17

0.37

0.10

0.53

0.06

C20:1n9

1.65

0.30

1.92

0.65

2.25

0.48

C20:4n6 (AA)

0.11

0.12

0.14

0.15

0.25

0.13

C20:4n3

2.40

0.32

2.38

0.76

1.67

0.50

C20:5n3 (EPA)

0.26

0.17

0.38

0.06

0.44

0.06

C21:5n3

4.68

0.29

4.83

0.41

5.17

0.68

C22:5n3 (DPA)

1.58

0.27

1.68

0.19

1.70

0.20

C22:6n3 (DHA)

10.82

2.30

10.10

1.96

8.45

2.11

∑SAFA

23.06 a

-

22.74 a

-

22.36 a

-

∑MUFA

40.28 a

-

41.72 b

-

43.57 a

-

∑n-3

21.42 c

-

20.98 a

-

19.80 a

-

∑n-6

7.27 a

-

6.95 a

-

7.14 a

-

n-3/n-6

2.94 a

-

3.02 a

-

2.77 a

-

DHA/EPA

40.45 a

-

26.56 b

-

18.99 c

-

Table 13: Prediction of fat content of sturgeon ovary from image analysis

Sample Code

Observed Value (image analysis)

Predicted Value

88

30.61

31.72

73

40.40

44.59

80

27.12

27.54

89

11.2

12.2

85

48.89

49.81

92

45.72

51.62

90

38.14

22.87

93

47.87

44.56

84

13.26

14.58

72

41.31

38.8

95

49.80

47.03

67

44.29

45.03

77

32.23

25.24

82

8.25

14.18

83

9.35

18.80

81

13.4

11.51

75

12.47

12.80

70

10.85

14.93

76

28.1

29.31

79

27.27

23.23

Red: Low fat ovary

Black: Medium fat ovary

Blue: High fat ovary

A (low fat sample)

B (medium fat sample)

C (high fat sample)

Figure 2: Digital images of ovary before and after processing by Image J software, (a) low fat ovary (#80); (b) medium fat ovary (#93), and high fat ovary (#76).

Figure 3: The relationship between fat content determined by image analysis and chemical method for May samples.

Table 14. Proximate analysis of age-seven white sturgeon ovarian tissue, after egg screening was completed. Females were fed low (LE) and high energy (HE) Skretting diets for nineteen months. Total number (n) of samples analyzed is given after the tank ID#. Different sample means (P<0.05) among the tanks are marked by different letters and differences between the pooled data are marked with an asterisk. Data are means ± SD.

Diet

Tank # (n)

Moisture (%)

Fat (%)

Protein (%)

HE

A-1 (30)

56.63 ± 11.22 ab

30.54 ± 11.64 ab

8.87 ± 2.40 a

HE

C-1 (30)

59.81 ± 8.06 a

29.55 ± 7.85 a

8.05 ± 1.26 a

HE

C-3 (30)

51.99 ± 10.93 b

36.80 ± 11.20 b

6.95 ± 1.43 b

HE Pooled

56.16 ± 10.56*

32.30 ± 10.75

7.96 ± 1.92

LE

C-2 (30)

61.91 ± 13.23 a

27.00 ± 13.67 a

7.81 ± 1.12 a

LE

C-4 (30)

58.61 ± 10.79 a

30.94 ± 11.47 a

8.18 ± 1.60 a

LE

C-5 (30)

60.43 ± 9.70 a

29.02 ± 10.59 a

8.24 ± 1.85 a

LE Pooled

60.32 ± 11.29

28.99 ± 11.96

8.08 ± 1.54

Table 15. Proximate analysis of age-seven white sturgeon ovarian tissue, after egg screening was completed. Females were fed low (LE) and high energy (HE) EWOS diets for nineteen months. Total number (n) of samples analyzed is given after the tank ID#. Different sample means (P<0.05) among the tanks are marked by different letters and differences between the pooled data are marked with an asterisk. Data are means ± SD.

Diet

Tank # (n)

Moisture (%)

Fat (%)

Protein (%)

HE

3-3 (30)

59.65 ± 9.81 a

27.30 ± 8.50 a

7.99 ± 1.76 a

HE

4-1 (30)

60.08 ± 6.83 a

27.15 ± 6.37 a

8.39 ± 1.15 a

HE

4-2 (30)

66.79 ± 8.12 b

21.33 ± 7.94 b

8.26 ± 1.49 a

HE Pooled

62.18 ± 8.88 *

25.26 ± 8.07 *

8.22 ± 1.48 *

LE

1-1 (30)

66.97 ± 8.32 a

19.96 ± 7.98 a

10.19 ± 2.06 a

LE

1-2 (30)

69.54 ± 9.40 a

18.71 ± 9.22 a

9.30 ± 1.05 a

LE

2-2 (30)

67.59 ± 8.28 a

19.73 ± 8.19 a

9.82 ± 1.27 a

LE Pooled

68.03 ± 8.65

19.46 ± 8.40

9.77 ± 1.55

Table 16. Proximate analysis of age-seven white sturgeon caviar samples. Females were fed low (LE) and high energy (HE) Skretting diets for nineteen months. Total number (n) of samples analyzed is given after the tank ID#. Different sample means (P<0.05) among the tanks are marked by different letters and differences between the pooled data are marked with an asterisk. Data are means ± SD.

Diet

Tank # (n)

Moisture (%)

Fat (%)

Protein (%)

HE

A-1 (5)

52.91 ± 0.98 a

10.85 ± 2.18 a

27.62 ± 0.64 a

HE

C-1 (5)

52.73 ± 0.95 a

10.77 ± 2.35 a

27.92 ± 1.92 a

HE

C-3 (5)

53.56 ± 0.65 a

10.01 ± 1.29 a

27.96 ± 0.93 a

HE Pooled

53.07 ± 0.89

10.54 ± 1.89

27.83 ± 1.20

LE

C-2 (5)

53.03 ± 1.14 a

10.06 ± 1.00 a

28.17 ± 1.02 a

LE

C-4 (5)

52.62 ± 1.42 a

9.74 ± 1.88 a

28.67 ± 1.50 a

LE

C-5 (5)

52.32 ± 1.24 a

10.32 ± 1.56 a

28.03 ± 2.37 a

LE Pooled

52.66 ± 1.21

10.04 ± 1.43

28.29 ± 1.62

Table 17. Proximate analysis of age-seven white sturgeon caviar samples. Females were fed low (LE) and high energy (HE) EWOS diets for nineteen months. Total number (n) of samples analyzed is given after the tank ID#. Different sample means (P<0.05) among the tanks are marked by different letters and differences between the pooled data are marked with an asterisk. Data are means ± SD.

Diet

Tank # (n)

Moisture (%)

Fat (%)

Protein (%)

HE

3-3 (5)

52.96 ± 1.73 a

9.76 ± 2.24 a

27.79 ± 1.06 a

HE

4-1 (5)

53.86 ± 0.40 a

11.33 ± 1.58 a

27.31 ± 0.79 a

HE

4-2 (5)

53.70 ± 0.98 a

10.06 ± 1.02 a

26.95 ± 1.82 a

HE Pooled

53.51 ± 1.16 *

10.38 ± 1.71

27.35 ± 1.25 *

LE

1-1 (5)

55.57 ± 1.22 a

9.65 ± 0.76 a

26.75 ± 0.91 a

LE

1-2 (5)

54.57 ± 1.13 a

9.78 ± 2.68 a

26.67 ± 0.91 a

LE

2-2 (5)

56.40 ± 1.33 a

9.38 ± 0.60 a

25.34 ± 1.18 a

LE Pooled

55.51 ± 1.38

9.60 ± 1.53

26.25 ± 1.15

Table 18. Proximate analysis for entire ovaries sampled from age-one (June 2012, n=10 each diet) and age-two (June 2013, n=25 each diet) white sturgeon. Significant differences in sample means (P<0.05) at each age, are indicated with an asterisk.

Age/Diet

Moisture (%)

Fat (%)

Protein (%)

1/HE EWOS

1/LE EWOS

4.86 ± 1.87*

8.54 ± 3.83

90.95 ± 2.47

87.39 ± 5.48

1.12 ± 0.44

1.03 ± 0.45

2/HE EWOS

2/LE EWOS

9.67 ± 2.75

8.92 ± 1.80

84.62 ± 6.11

86.80 ± 2.25

0.98 ± 0.50

0.99 ± 0.38

Table 19. Most likely male and female parent of each one year-old sturgeon fed the EWOS LE and HE diets by tank. The families are described with the male parent listed first.

LE

Tank

Family

n

Proportion of family/tank

HE

Tank

Family

n


A13

OE5E x O610

OE5E x 2E4C

OE5E x 7627

6F6B x O610

6158 x 2080

8

1

1

1

1

0.67

0.08

0.08

0.08

0.08

A5

OE5E x O610

OE5E x 2E4C

6F6B x O610

6158 x 2080

6158 x 2E4C

2179 x O610

5

2

2

1

1

1

0.42

0.17

0.17

0.08

0.08

0.08

A14

OE5E x O610

OE5E x 2E4C

6F6B x O610

5C49 x 2080

OE5E x 6C27

6F6B x 6C27

6158 x 2080

5C49 x 2E4C

2179 x O610

2

2

2

2

1

1

1

1

1

0.15

0.15

0.15

0.15

0.08

0.08

0.08

0.08

0.08

A6

OE5E x O610

6158 x 2E4C

OE5E x 2E4C

6F6B x O610

6158 x 2080

2179 x O610

4

3

2

2

1

1

0.31

0.23

0.15

0.15

0.08

0.08

A15

OE5E x O610

6158 x 2E4C

OE5E x 7627

6F6B x O610

8

2

1

1

0.67

0.17

0.08

0.08

A7

OE5E x O610

6158 x 2080

2179 x O610

OE5E x 2080

OE5E x 2E4C

6158 x 2E4C

2179 x 6C27

2179 x 2E4C

5C49 x 2080

2

2

2

1

1

1

1

1

1

0.17

0.17

0.17

0.08

0.08

0.08

0.08

0.08

0.08

A16

OE5E x O610

OE5E x 2080

6F6B x O610

6158 x 2080

5C49 x 6C27

2179 x 2080

8

1

1

1

1

1

0.62

0.08

0.08

0.08

0.08

0.08

A8

OE5E x 2E4C

6158 x 2080

6158 x 2E4C

OE5E x O610

6F6B x O610

2179 x O610

3

3

3

2

1

1

0.23

0.23

0.23

0.15

0.08

0.08

Table 20. Most likely male and female parent of each one year-old sturgeon fed the EWOS LE and HE diets. The families are described with the male parent listed first.

Diet

Family

n


LE

OE5E x O610

6F6B x O610

OE5E x 2E4C

6158 x 2080

OE5E x 7627

6158 x 2E4C

5C49 x 2080

OE5E x 2080

OE5E x 6C27

6F6b x 6C27

5C49 x 2E4C

5C49 x 6C27

2179 x O610

2179 x 2080

26

5

3

3

2

2

2

1

1

1

1

1

1

1

0.52

0.10

0.06

0.06

0.04

0.04

0.04

0.02

0.02

0.02

0.02

0.02

0.02

0.02

HE

OE5E x O610

OE5E x 2E4C

6158 x 2E4C

6158 x 2080

6F6B x O610

2179 x O610

OE5E x 2080

2179 x 6C27

2179 x 2E4C

5C49 x 2080

13

8

8

7

5

5

1

1

1

1

0.26

0.16

0.16

0.14

0.10

0.10

0.02

0.02

0.02

0.02

Table 21. Identified sire or dam, for each one year-old fish sampled (n=50/diet).

# of Fish in LE Diet

# of Fish in HE Diet

Sire

2179

6158

5C49

6F6B

OE5E

2

5

4

6

33

7

15

1

5

22

Dam

2808

2E4C

6C27

O610

7627

7

6

2

32

3

9

17

1

23

0

Figure 4. Mean body weight and age of white sturgeon sampled for energy partitioning in prepubertal fish fed a low and high energy diet. Bottom right linear regression equation is for low energy and top left, for high energy. Data are means and standard error bars.

Figure 5. Mean gonadosomatic index (GSI) and age of white sturgeon sampled for energy partitioning in prepubertal fish, fed a low and high energy diet. Data are means and standard error bar.

Fourth Year Budget Proposals

INSTITUTION: University of California

PRINCIPAL INVESTIGATOR:

Serge Doroshov

SALARIES:

$16,691

Research Associate (12 mths @ 22%)

$16,691

BENEFITS:

$8,529

Research Associate (@ 51.1%)

$8,529

TRAVEL:

$ 2,780

Workgroup Meeting: Hotel (2 nights x $100), per diem (2 days x $50), Airfare ($400)

$700

Per Diem (2 days)

Scientific Meeting: Airfare ($880), Hotel (4 nights x $125), per diem (5 days x $50), registration ($450)

$2,080

SUPPLIES:

$0

TOTAL:

$28,000

INSTITUTION:

University of California, Davis


Bernie May

SALARIES:

$27,561

Project Scientist (4.5mo @100%)

$20,061

Graduate Research Asst. (3 mo @ 50%)

$7,500

BENEFITS:

$7,259

Graduate Research Asst. (@ 1.3%)

$97

Project Scientist (@ 35.7%)

$7,162

TRAVEL:

$ 850

Work group meeting-Hotel (2 days x 100)

$200

Per Diem (3 days)

$250

Airfare

$400

SUPPLIES:

$2,330

DNA extraction Kits

$700

PCR reagents and consumables

$800

Fragment analysis reagents and consumables

$830

OTHER DIRECT COSTS:

Equipment lease and service contract for Genetic analyzer

$2,000

$2,000

(The $2,000 is this project’s contribution to the annual $15,000 service contract to maintain the ABI 3730XL Genetic Analyzer. This machine analyzes microsatellite loci genetic variation in the individual sturgeon that we will work with in this project and permit us to assign genetic relationships among the individual sturgeon).

TOTAL:

$40,000

INSTITUTION:

Montana State University


Molly Webb/Chris Guy

SALARIES:

$8,400

Undergraduate Student (40 weeks @ 50%)

$8,400

BENEFITS:

$588

Undergraduate Student (@ 7%)

$588

TRAVEL:

$ 0

SUPPLIES:

$7,972

Proximate analyses

$6,972

Histology supplies, slides

$1,000

TOTAL:

$16,960

INSTITUTION:

Washington State University


Barbara Rasco

SALARIES:

$23,814

Graduate Student (9 mths @ 50%)

$23,814

BENEFITS:

$2,002

Graduate Student (@ 8.4%)

$2,002

TRAVEL:

$ 1,084


$300

Per Diem (3 days)

$150

Airfare

$634

SUPPLIES:

$1,100

Lab maintenance supplies/vials

$500

Sensory analysis materials

$600

TOTAL:

$28,000

INSTITUTION:

College of Southern Idaho


Terry Patterson

SALARIES:

$0

BENEFITS:

$0

TRAVEL:

$ 2,000


$300

Per Diem (3 days)

$150

Airfare

$450

Travel to farms (6 trips x 150 miles)

$900

Per Diem

$200

SUPPLIES:

$2,000

Field work (stretchers, nets, saw horses, rain gear, consumables)

$1,200

Sampling (vials, whirlpaks, surgery tools and consumables)

$600

Sample shipping (boxes, dry ice)

$ 200

TOTAL:

$4,000

INSTITUTION:

University of California, Davis


Fred Conte

SALARIES:

$0

BENEFITS:

$0

TRAVEL:

$ 1,300

Scientific Meeting – Hotel (3 days x 100)

$300

Per Diem

$150

Airfare

$500

Registration

$350

SUPPLIES:

$1,740

Camera Supplies/Batteries

Flash Unit/Supplies for Extension Photos

$400

In-state UCD fleet services rental for travel to sturgeon farms, local workgroup and planning meeting (charged as expenses by UCD…not travel)

$1,540

TOTAL:

$3,040

37.45520000000000548.11469999999999933.98980000000000221.30239999999988952.44409999999999953.943730.12229999999998948.09320000000000323.253243.38620000000000250.140248.48409999999999832.08149999999999822.92229999999978726.74609999999984920.70980000000000121.77469999999998923.54050000000000235.45709999999999730.41649999999998930.61900000000003540.40300000000000627.12300000000000111.248.89500000000000345.72400000000001138.14600000000000147.87600000000000513.26700000000000141.31199999999999849.80600000000000444.29300000000001332.2320000000000638.259.350000000000006813.412.4710.85000000000002628.127.271999999999988

Chemical analysis (%)

Image analysis (%)

Low Energy

y = 313.15x - 3677.2R² = 0.9532

098.520595629329762109.88464286169425145.82132902974107122.41728625355229119.60119256578425191.46622451904921148.70484896351937161.05911753795641182.856200332392661819202122232425262055.55000000000022212.17999999999982862.626743206.63176.83893.74228.054599.7High Energy

124.10678551363023131.04250967569737171.36971574255512143.41597760214478185.66292178654217280.26667070939135140.17977930613145151.99631055079203266.2999950713480601819202122232425262454.12662.052757.73068.33295.73750.93530.44048.14367.45

Age in Months

Body Weight (g)

Low Energy10.360813844130406240.245852397080415090.386456473306763190.445228996143971820.373382050590494620.387925377468902270.345537640636345290.276549009079013920.232682292188797961819202122232425264.33.073.753.86999999999999973.93.33.143.143.2800000000000002High Energy0.398832523088731130.319078143265496940.389722907755372380.330770247064694770.403783790447252320.432096673462089130.345477061330115580.428957155020599450.3975004357729764501819202122232425263.44.23.863.84.23.863.693.693.3299999999999987

Age in Months

GSI (%)

Documents

Determining Ripeness in White Sturgeon Females to ...depts.washington.edu/wracuw/research/Project Annual... · Web viewProtein content was determined by thermal oxidation (Leco TruSpec,