1. PROXIMATE COMPOSITION, GROWTH PERFORMANCE AND FILLET QUALITY IN TRANSGENIC FEMALE TRIPLOID ATLANTIC SALMON (Salmo salar) REARED AT THREE TEMPERATURES
E.H. Ignatz*, M.L. Rise, J. Westcott, C.N. Bullerwell,
T. Benfey, T.S. Hori, C.D. Runighan

2. Project Overview This study was designed for analysis of:
Growth Performance
Nutritional Factors
Gene Expression
Study comprised of only transgenic all-female, triploid Atlantic salmon (AquAdvantage® salmon - AAS)
Determining effect of 3 temperatures on AAS:
10.5°C
13.5°C
16.5°C
13.5°C was chosen because that is the standard set for optimal growth for conventional salmon and is also the temperature that AquaBounty uses in its recirc system here in Fortune °C was used as this is the average temperature our salmon are grown at in our Panamanian facility.

3. Study Purpose – Determination of Optimal Rearing Conditions
Growth Performance
AAS reach target size in approximately half the time of conventional salmon (Tibbetts et al. 2013)
Temperature already known to have direct impact on the metabolism of poikilotherms
Temperature effect never measured in AAS
Nutritional Factors
Proximate composition of whole bodies; fatty acid, amino acid and mineral composition of fillets
Nutritional value impacts quality and marketability of product
Gene Expression
Analysing liver samples to measure gene regulation and expression differences between temperature treatments

4. Fish Housing
All fish originated from same commercial batch (1 transgenic neomale parent and 3 non-transgenic female parents)
Each temperature treatment housed in triplicate, identical 1.5 m3 tanks
Each tank started trial with 900 fish (numbers reduced throughout study to maintain proper stocking densities)
All fish come from the same commercial batch (AAS ); consisting of one transgenic (neo)male parent and three non-transgenic female parents.
Each temperature treatment is housed in triplicate, identical 1.5 m3 tanks.
While each tank started the trial with 900 fish, these numbers have been reduced throughout the study to maintain proper densities. At the end point of the trial, each tank only holds 60 salmon.

5. Monitoring and Control
Water Quality
Temperature (Twice daily)
Oxygen (Daily)
Ammonia, Nitrite, Nitrate, Ozone, Carbon Dioxide, Salinity, Turbidity, Alkalinity (Weekly)
Feeding
Accurate measuring of all feed added to automatic feeders
500 g – 1.5 kg stage: counting uneaten pellets to determine accurate FCR values
Photoperiod
24 h light (60W incandescent bulbs)
Density
Scheduled reductions in stocking densities throughout trial
Temperature and Oxygen monitored daily. The rest are monitored weekly in one tank per treatment.
Scale calibrated weekly to maintain accurate feed measurements.

6. Study Design Proximate Composition Fatty Acid & Amino Acid Analysis
Chemical Colour Analysis
Gene Expression Analysis
Sampling:
First Feed, 300g, 500g, 800g, 1.5kg
500g, 800g, 1.5kg
800g, 1.5kg
500g, 800g, 1.5 kg
Sample:
Whole Body
Fillet
Liver
Collaboration with:
Bio Food Tech
Coastal Zones Research Institute
Atlantic Veterinary College
qPCR to help determine the effect temperature plays on hepatic gene expression in relation to growth and metabolism. Exact genes to be examined is still to be determined, but currently considering genes related to muscle growth, fatty acid synthesis, heat stress and satiety (IGF-1, GH, FADSD5, Leptin, HSP90/30/70, Ubiquitin)

7. Averages calculated after every monthly assessment
Averages calculated after every monthly assessment. Prior to pig tagging, a random selection of 100 fish per tank were assessed. After pit tagging (120 fish per tank), every fish in the tank was assessed each month. All fish taking part in the monthly assessments were taken off feed 24 hours in advance and fully anesthetized before weight and length measurements were taken.
Error bars represent plus and minus one standard deviation from the mean weight calculated after each monthly assessment.

8. AAS Growth at 10.5°C Days Post First Feeding Date Fish Assessed
Degree Days Post First Feeding
Weight (g)
Thermal Growth Coefficient 36
26-Jul-16
514
1.3
0.67 66
25-Aug-16
834
4.9
1.08 97
25-Sep-16
1160
18.4
1.36
129
27-Oct-16
1467
39.0
2.21
157
24-Nov-16
1797
67.4
2.28
192
29-Dec-16
2158
120.7
2.44
218
24-Jan-17
2434
181.3
2.60
247
22-Feb-17
2736
248.6
2.08
282
29-Mar-17
3106
349.8
2.04
308
24-Apr-17
3387
456.3
2.41
345
31-May-17
3778
622.8
2.14
373
28-Jun-17
4065
755.6
1.91
401
26-Jul-17
4353
910.4
2.02
435
29-Aug-17
4709
1171.3
2.38
Error bars represent plus and minus one standard deviation from the mean weight calculated after each monthly assessment

9. AAS Growth at 13.5°C Days Post First Feeding Date Fish Assessed
Degree Days Post First Feeding
Weight (g)
Thermal Growth Coefficient 36
26-Jul-16
614
2.19
1.11 66
25-Aug-16
1011
14.1
1.43 97
25-Sep-16
1415
50.1
2.07
136
27-Oct-16
1802
90.1
1.81
157
24-Nov-16
2226
159.6
2.39
192
29-Dec-16
2696
241.6
1.67
213
19-Jan-17
2987
287.9
1.01
247
22-Feb-17
3454
425.5
2.23
282
29-Mar-17
3929
536.5
1.23
310
26-Apr-17
4314
664.8
1.63
345
31-May-17
4792
839.9
1.36
372
27-Jun-17
5161
1068.2
2.00
401
26-Jul-17
5557
1290.1
1.68
422
16-Aug-17
5838
1493.4
1.15
Error bars represent plus and minus one standard deviation from the mean weight calculated after each monthly assessment

10. AAS Growth at 16.5°C Days Post First Feeding Date Fish Assessed
Degree Days Post First Feeding
Weight (g)
Thermal Growth Coefficient 36
26-Jul-16
718
3.4
1.32 66
25-Aug-16
1212
26.9
1.42 97
25-Sep-16
1719
80.0
2.58
136
27-Oct-16
2179
147.1
1.91
157
24-Nov-16
2646
212.6
1.64
192
29-Dec-16
3186
302.6
1.36
219
25-Jan-17
3624
397.6
1.55
248
23-Feb-17
4078
515.9
1.46
283
30-Mar-17
4652
687.1
1.40
310
26-Apr-17
5095
809.8
1.21
345
31-May-17
5677
1130.7
1.80
372
27-Jun-17
6118
1358.8
1.43
401
26-Jul-17
6565
1483.5
0.76
Error bars represent plus and minus one standard deviation from the mean weight calculated after each monthly assessment

11. Averages calculated after every monthly assessment
Averages calculated after every monthly assessment. Prior to pig tagging, a random selection of 100 fish per tank were assessed. After pit tagging (120 fish per tank), every fish in the tank was assessed each month. All fish taking part in the monthly assessments were taken off feed 24 hours in advance and fully anesthetized before weight and length measurements were taken.
Fulton’s Condition Factor = (Weight / L^3) * 100
Error bars represent plus and minus one standard deviation from the mean Fulton’s condition factor calculated after each monthly assessment.

12. b a b a
In conventional salmon, a condition factor above 1.0 is generally deemed acceptable.
Higher condition factor with 16.5C treatment at 500 g, 800 g, 1.5 kg, but was seen as equally high in 10.5C treatment at 800 g stage
Error bars represent plus and minus one standard deviation from the mean condition factor calculated during the month each treatment was sampled for that particular weight target. After 1-Way ANOVA testing, lower case letters denote significance between treatments at the same weight. Asterisk denote significance within the same treatment.

13. Commercial FCR values were calculated until each treatment reached 500 g, after which actual FCRs could be determined. For the 16.5⁰C treatment, this would be starting in February, the 13.5⁰C treatment in March and the 10.5⁰C treatment in May.
Error bars represent plus and minus one standard error from the mean feed conversion ratio calculated after each monthly assessment

14. * b b b ab a ab a a In conventional salmon, FCRs average around 1.2
Error bars represent plus and minus one standard deviation from the mean feed conversion ratio calculated during the month each treatment was sampled for that particular weight target. After 1-Way ANOVA testing, lower case letters denote significance between treatments at the same weight. Asterisk denote significance within the same treatment.

15. VSI and HSI Comparisons* a * a * * a a b b a a a a
Using visceral somatic index as an indicator of how much energy is being devoted towards fat deposition in the abdomen instead of protein building in the fillet. A lower VSI is generally preferable as it is more important to use as aquaculturists so we have more fillet to sell rather than processing by-product. Hepatic somatic index gives insight into the status of energy reserves in the fish. A higher HSI is generally preferable as this indicates a healthy environment.
Error bars represent plus and minus one standard error from the mean somatic index calculated after both sampling events. After 1-Way ANOVA testing, lower case letters denote significance between treatments at the same weight. Asterisk denote significance within the same treatment.

16. SalmoFan™ Colour Analysis
Analysis During 800 g Sampling
Treatment (⁰C)
Values from
Across Fillet
10.5
13.5
16.5
<
Analysis During 1.5 kg Sampling
Treatment (⁰C)
Values from
Across Fillet
10.5
TBD
13.5
16.5
Two trained observers analyzed 4 fillets during each sampling using the SalmoFan colour chart and recorded independent observations. The table provided illustrates the ranges that were found for each treatment during both the 800 g and 1.5 kg samplings. Darker pigmentation was noted to follow along the lateral line, with colour fading moving towards to the edges of the fillet. Distance from light to the bottom of the box is 31.5 in.; 100W Halogen Bulb
The color of conventional farmed salmon fillets sold in the Norwegian market normally range from twenty three to thirty on the salmofan and most common are fillets ranging from twenty five to twenty seven (Acharya 2011)

17. Ongoing Work Proximate Composition & Fillet Analyses Gene Expression
Results expected by end of September 2017
Whole body proximates analysed individually
Amino acids, fatty acids and minerals pooled by tank
Astaxanthin concentration pooled by treatment
Gene Expression
Target genes selected to examine growth and metabolic response to temperature
qPCR on RNA extractions to be performed on liver samples

18. Summary Rearing at 16.5⁰C exhibits a production time savings
AAS held at 16.5⁰C on average have significantly higher condition factors during 500 g – 1.5 kg growth stage
At 10.5⁰C & 13.5⁰C FCRs lowered during 500 g – 1.5 kg growth stage
Fillet composition and colour quality yet to be determined
Hepatic gene expression differences in relation to rearing temperature still to be analyzed

19. Questions?

20. Acknowledgements Co-Supervisors
Dr. Matt Rise (MUN, Department of Ocean Sciences)
Dr. Jillian Westcott (MUN, Marine Institute)
Additional MSc Committee Members
Dr. Tillmann Benfey (UNB, Department of Biology)
Dr. Tiago Hori (CATC, Department of Genomics)
AquaBounty Canada Team
Dr. Laura Braden
Chrissie Bullerwell
Amanda Cudmore
Rich Greene
Dr. Armando Heriazon
Logan Jamieson
Damien MacDonald
Jonathan MacInnis
Lee Pagnutti
Wayne Peters
Sheila Peters
Mauricio Rojas
Dawn Runighan
Jon Veinot

21. References
Tibbetts, S., Wall, C., Barbosa-Solomieu, V., Bryenton, M., Plouffe, D., Buchanan, J., & Lall, S. (2013). Effects of combined ‘all-fish’ growth hormone transgenics and triploidy on growth and nutrient utilization of Atlantic salmon (Salmo salar L.) fed a practical grower diet of known composition. Aquaculture,  ,