PRODUCT CATALOGUE. Salmon eggs 2015/2016 Rainbow trout eggs 2016

Transcription

1 PRODUCT CATALOGUE Salmon eggs 215/216 Rainbow trout eggs 216

2 AquaGen product catalogue Table of contents Table of contents Salmon eggs 215/216 - Start material with the best potential for growth, survival and fillet colour... p. 3 Genetic background for salmon eggs 215/ p. 4 Biosecurity and quality control... p. 5 Delivery time... p. 5 Product overview for salmon eggs 215/ p. 6 Product documentation RED: Strong and even fillet colour... p. 7 CMS: Protection against CMS - better heart health... p. 8 PD: Resistance against PD... p. 9 Information letter: Field documentation of QTL-innOva IPN/PD... p. 1 IPN: Resistance against IPN... p. 14 Information letter: Use of QTL-eggs results in an IPN reduction for the whole of Norway... p. 15 STERILE: Sterile salmon... p. 17 TRACK: DNA tracking of escaped farmed salmon... p. 18 Rainbow trout eggs Start material with the best potential for growth and survival... p. 19 Genetic background for rainbow trout eggs p. 2 Biosecurity and quality control... p. 21 Delivery time... p. 21. Product overview for rainbow trout eggs... p. 22 Product documentation IPN: IPN resistance for rainbow trout... p. 23 2

3 AquaGen salmon eggs 215/216 SALMON EGGS 215/216 Start material with the best potential for growth, survival and fillet colour 3

4 AquaGen salmon eggs 215/216 Genetic Background for Salmon Eggs 215/216 At AquaGen we carry out several selections of broodstock at different stages before the final selection is allowed to produce eggs for the farmers. The selection process occurs both at the family- and individual level. The salmon eggs that AquaGen will deliver are the results of selective breeding for a range of important traits through 11 generations since the 197s. How long the different traits have been selected for, as well as the intensity of the selection is very important for the total progress which today s products represent. At the present time more than 2 traits are recorded from over 6 families. A comprehensive recording program ensures progress in these desirable traits while at the same time minimalizing the risk of unwanted or unexpected side effects. The figure below shows the main traits which are measured, selected and controlled in the selective breeding program (breeding nucleus) set up in relation to the commersial egg producing generation to be phased in. Salmon eggs which will be delivered to our customers hatcheries in the 215/216 season will be produced from the top-ranking families that were first fed in 212 (marked with grey in the chart). Elite material from this generation is transferred to our egg production operations. After a four year production cycle the best individuals are selected as broodstock. In this way the best genetics will be reproduced on a large scale to benefit all fish farmers. Since 29 AquaGen has implemented new modern technology called Marker Assisted Selection. Based on this new technology platform we are able to directly select parents to be used in egg-production that possess specific genetic markers (QTLs) that are highly associated with resistance to specific viral diseases and fillet colour. Previously when all traits were selected using traditional breeding methods, we had to make a compromise when weighting the various traits. Traits which are selected by use of genetic markers will not be detrimental to other traits. This means that we can emphasize the trait growth (traditional selection) to a greater degree while also getting the traits for virus resistance/quality (gene based selection). This double selection, both on family- and individual level, ensures a balanced weighting of robust and effective traits as well as high viral resistance and strong fillet colour in the offspring. Implementation of traits based on traditional- and gene marker selective breeding methods in AquaGen's breeding programme (nucleus) of Atlantic Salmon (Salmo salar ) : 25: First feeding year for juveniles of brood fish The four year classes collected from 41 Norwegian salmon rivers. were merged into one. Generation Class of year (first feeding) GROWTH SEA WATER SEXUAL MATURATION FILLET COLOUR FURUNCULOSIS ISA FILLET FAT SLAUGHTER YIELD Traits SKELETAL DEFORMITIES GROWTH FRESH WATER IPN Production/efficiency traits BODY SHAPE Health/robustness traits Gene marker based breeding method started The top-ranking families first fed in 212 are the base for brood fish stripping in 215/ FILLET TEXTURE PD CMS LICE

5 AquaGen salmon eggs 215/216 Biosecurity and Quality Control Fish health surveillance in AquaGen is carried out through the entire lifecycle of the fish. In the final nine months before stripping the monitoring is intensified. This includes comprehensive autopsy and screening of all broodstock populations in the AquaGen system. Production is monitored by both internal and external fish health personnel, and by government and private entities. AquaGen is certified according to GLOBAL GAP (food safety, environmental protection, fish welfare, and health, safety and welfare for employees), FREEDOM- FOOD (animal welfare), ISO 91:28 (quality leadership) and Code-EFABAR (best practice within animal farm breeding and reproduction). AquaGen s production plants at Hemne and Tingvoll are located in EU-approved ISA-free compartments at the highest security level. ISA has never been detected in these compartments. AquaGen s broodstock populations are routinely screened for IPN virus, ISA virus, PD virus and the BKD bacterium during the entire production cycle of four years. Individual testing of parents at stripping can be carried out in addition if ordered in advance. Relevant health information about brood fish and eggs will be documented in a separate HEALTH CV and included in all egg deliveries. Delivery Time In the egg season of 215/216 AquaGen will deliver eggs from the end of September 215 to the middle of August 216: Categories Delivery Time Early eggs Normal eggs Late eggs Wk (Sep Nov. 15) Wk (Nov Apr. 17) Wk (Apr Aug. 21) In the figure below the differing production timelines are presented for salmon through the fresh-water phase and the salt-water phase, based on early, normal and late eggs. The figure shows how AquaGen by means of managed production of broodstock can contribute to the optimal exploitation of our customer s production facility capacity. Should there be a requirement for unexpected additional eggs, due to operating accidents, diseases or lack of capacity in customers own facilities, AquaGen often has the ability to help out with deliveries in such situations. Production plan for salmon in the FRESH-WATER PHASE Category 1st year 2nd year 3rd year Aug Sep Oct Nov Des Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun -year Early eggs Smolt -/1-year Normal eggs Smolt Smolt During the winter the smolt can also be kept -year Late eggs Smolt in tanks/cages and transfered to sea as big smolt. Production plan for salmon in the SEA-WATER PHASE Category 1st year in sea 2nd year in sea 3rd year in sea 4th year in sea May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar -year Smolt Harvest -/1-year Smolt Smolt Harvest -year Smolt Harvest 5

6 AquaGen salmon eggs 215/216 Product Overview for Salmon Eggs 215/216 Early ordering, before August 1, 215 contributes to better production planning and higher delivery assurance in regard to choice of product type, amount and time of delivery. Depending on the results from genotyping of this season s broodstock, some types of products may have limited availability. Product Description AquaGen Atlantic QTL-innOva The broodstock undergo initially a traditional family-based selection with information from more than 2 measured traits. When the top brood candidates are selected on this level, we are taking samples for DNA analysis to find genetic markers (QTLs*) associated with different traits. IPN is included in every QTL product. Various combinations of QTL traits that can be selected are listed below: IPN/PD/CMS/RED IPN/PD/CMS Provides resistance against IPN, PD and CMS and strong fillet colour. The males are chosen using the genetic markers for IPN, PD, CMS and fillet colour. Provides resistance against IPN, PD and CMS. The males are chosen using the genetic markers for IPN, PD and CMS. Egg types IPN/PD/RED IPN/CMS/RED Provides resistance against IPN and PD and strong fillet colour. The males are chosen using the genetic markers for IPN, PD and fillet colour. Provides resistance against IPN and CMS and strong fillet colour. The males are chosen using the genetic markers for IPN and CMS and fillet colour. IPN/PD IPN/CMS IPN/RED IPN Provides resistance against IPN and PD. The males are chosen using the genetic markers for IPN and PD. Provides resistance against IPN and CMS. The males are chosen using the genetic markers for IPN and CMS. Provides resistance against IPN and strong fillet colour. The males are chosen using the genetic markers for IPN and fillet colour. Provides resistance against IPN. The males are chosen using the genetic markers for IPN. AquaGen Atlantic GREEN After the egg type is selected, one or more treatments of the broodfish/eggs can be chosen: Treatment STERILE TRACK Sterilization of eggs which after pressure treatment on recently fertilized eggs makes the fish sterile. This procedure can be done on all types of eggs listed above. Since this production requires extra planning and greater safety margins than ordinary, the ordering deadline is September 1, 215. DNA tracking of farmed fish where potential escapees can be checked against the parent fish (both male & female) which are used for a specific egg delivery. DNA analysis will confirm or deny the relationship between parents and offspring and track the fish back to the owner. Since this production requires extra planning and greater safety margins than ordinary, the ordering deadline is August 1, 215. ORGANIC Virus testing Organic eggs from broodstock reared at lower stocking densities. Since this production requires extra planning and greater safety margins than ordinary, the order deadline is August 1, 215. Individual testing of female- and male broodstock for IPNV, PDV, ISAV and/or HSMIV at the time of stripping. Broodstock free from virus are used in the egg production only. *QTL = Gene marker (Quantitative Trait Loci). Area on the salmon genome (marker) which is closely connected to a gene which controls a specific characteristic in the individual. 6

7 Product documentation RED Strong and even fillet colour Better and more even pigmentation is an important goal for most farmers. With an increasing degree of processing and product differentiation, a high, stable and predictable pigment level in the fillet becomes increasingly important for both the processing industry and consumer. A strong red colour also means that the fillet contains a high level of astaxanthin, which is documented to have positive health related benefits for both salmon and humans. A precise selection of brood fish using three colour-qtls will ensure a strong and even colour in the fillet. Salmon genes that affect pigmentation A salmons ability to accumulate pigment that is supplied in feed varies between different life stages and seasons. It has also been documented that there is a significant genetic variation and heritability of the trait. Several independent studies have shown that between 35% and 55% of variation in fillet colour can be explained with genetics. With modern breeding technology where selection is based to a greater degree directly on genes or genetic-marker tests, we have far better methods to achieve rapid progress on important traits. Specially developed genotyping tool Through 2 years, more than 15, DNA samples were taken from AquaGen fish with accurate recording of pigment levels for fish. This material has been stored in the Biobank that AquaGen established in association with the breeding companies Geno and Norsvin. In order to search for QTLs significantly connected with fillet colour a newly developed SNP chip, containing a total of 95, genetic markers specially designed for the AquaGen strain was used. Three genetic markers for fillet colour Three significant QTLs were found, all having a moderate to strong effect on fillet colour (Figure 1). By combining the identified QTLs in the selection of brood fish, it was possible to document a substantially increased average level of astaxanthin in the fillet. At the same time variation between fish within the same group was reduced. The effect of the three colour-qtls was also tested in fish of differing size, and results show that the beneficial effect is independent from weight. This provides flexibility in the timing for harvesting (Figure 2). How do the colour-qtls work? Functional studies of the genes associated with the three colour-qtls, show that they increase absorption and reduce breakdown of astaxanthin in the intestine. Astaxanthin is a strong antioxidant that protects cells against oxidative stress and contributes to a strong immune system. Research has shown that astaxanthin has beneficial health effects both for salmon and those that eat it Astaxanthin (mg/kg fillet) Astaxanthin (mg/kg fillet) Q 1-2 Q 3-4 Q 5-6 Q Number of colour-qtls (Q) Q 3-4 Q 5-6 Q Number of colour-qtls (Q) Figure 1. Average fillet colour in relation to the number of colour-qtls, Q, in salmon from three different generations. A total of 6, salmon at around 3.5kgs contributed to the data. Each of the three colour-qtls has 1 or 2 copies of the favourable gene marker, Q. In fish with QTL-innOva RED the number of copies of Q lies between 3 and 6, but is mostly 4 and 5 Q. Products with strong and even fillet colour: AquaGen Atlantic QTL-innOva IPN/PD/CMS/RED AquaGen Atlantic QTL-innOva IPN/PD/RED AquaGen Atlantic QTL-innOva IPN/CMS/RED AquaGen Atlantic QTL-innOva IPN/RED Figure 2. Fillet colour groups of salmon (total 23 fish) of different size from 2g to 7 kg, compared to the number of the favourable gene marker, Q, from three colour-qtls. QTL- innova RED will contain 3 to 6 Q, but mostly 4 and 5 Q. Benefits of QTL-innOva RED: Higher average and reduced variation in fillet colour in batches More effective utilisation of pigment content in feed, especially during periods with strong growth Greater flexibility with timing of harvesting 7

8 Heart damage (Histopathologic score) Product documentation CMS Protection against CMS Better heart health Cardiomyopathy syndrome (CMS), is a heart disease affecting salmon that is caused by Piscine myocarditis virus (PMCV). CMS often occurs late in the seawater phase affecting large fish and has become an increasing problem for the Norwegian aquaculture industry since 21, with 17 cases registered in 214. The use of QTL eggs that are selected for resistance to CMS is documented to provide significantly improved heart health that contributes to a more robust salmon. Development and results of QTL-innOva CMS Based on data and tissue samples from a field outbreak of CMS in 28, there was found to be a very good correlation between virus level in the heart, heart tissue damage and mortality. The fish in the study were genotyped and a strong QTL was found that related to virus level in the heart. Effect of the QTL was subsequently tested and confirmed in both a field outbreak and a controlled disease challenge test. In one field outbreak in 211 around 2% reduced mortality was recorded for fish with the CMS-QTL and an economic analysis showed that as little as 1% CMS related mortality was enough to justify the investment in CMS-QTL. It was in 212 that the offspring of the CMS resistant families from the CMS outbreak in 28 were subjected to an infection challenge with PCMV in the laboratory qq Qq QQ Genotypes for CMS-QTL Figure 1. Effect on heart damage of QTL-innOva CMS after disease challenge with PMCV on salmon of average 2.3kgs. The sum of grading for heart damage by histopathology (± standard error) is conducted on 68 fish for each gene variant. Fish with 1 (qq) or 2 (QQ) copies of the beneficial marker that are included in QTL-innOva CMS, had significantly reduced heart damage compared with the unfavourable gene variant (qq). Heart damage was assessed using histopathology and once again there was significantly reduced damage in fish that had the CMS-QTL (Figure 1). Field experience with QTL-innOva CMS Autumn 214 was the first time that S smolt were transferred of QTL-innOva IPN/PD/CMS and QTL-innOva IPN/PD in two locations that had a history of CMS outbreaks. Until now, CMS has not been recorded in any of the fish groups in these locations. Following 8.5 months in the sea, at location A, good growth was recorded for all fish groups. At location B there is a tendency for better growth of fish type QTL-innOva IPN/PD/ CMS. On both locations there is on average a lower mortality rate for fish of QTL-innOva IPN/PD/CMS (Figure 2). Better heart health gives more robust salmon In QTL-innova CMS the most CMS sensitive individuals (qq) are eliminated from the population. This will result in an increased barrier to the introduction and spreading of PMCV in the population, which will therefore be more robust to stress like lice treatment, transport and storage in stand by cages. Growth rate (TGC3) Cage Growth rate (TGC3) Mortality (%) Figure 2. Growth and mortality after 8.5 months in the sea for salmon that have gene markers for CMS resistance (QTL-innOva IPN/PD/CMS) and salmon without this gene marker (QTL-innOva IPN/PD). Fish populations came from the same hatchery, were transferred in September 214 and went into several separate cages in two locations. There was a diagnosis of HSMI in IPN/PD fish in cage 4 at location B Mortality (%) Products with CMS protection: AquaGen Atlantic QTL-innOva IPN/PD/CMS/RED AquaGen Atlantic QTL-innOva IPN/PD/CMS AquaGen Atlantic QTL-innOva IPN/CMS/RED AquaGen Atlantic QTL-innOva IPN/CMS Benefits of QTL-innOva CMS: Reduced virus levels in the heart, reduced heart damage and lower mortality from CMS outbreak Higher degree of herd immunity by the elimination of the most susceptible individuals Robust fish that withstand transport and handling better in the final phase of production 8

9 Product documentation PD Accumulated mortality (%) 1 Resistance against PD Pancreas Disease (PD), caused by Salmonid alphavirus (SAV), has for many years been one of the most costly diseases affecting salmon farming. In recent time there has been recorded a positive trend of a reduction in the number of PD outbreaks. Based on efficacy data from both laboratory and field trials QTL-innOva IPN/PD will make an important contribution to the continuing fight against PD. Development of QTL-innOva IPN/PD AquaGen has utilised challenge data from both field and laboratory, to identify and document gene markers with effect against PD. Gene markers that have been identified to date are not like the simple and strong IPN-QTL, but used in combination they will give a considerable increase in resistance to PD for fish. Our analysis also showed that among the three strongest QTL s associated with PD protection we find the IPN-QTL. Based on the findings of QTL searches and subsequent documentation from laboratory infection trials (Figure 2), the product QTL-innOva IPN/PD was launched in the market in the egg-season 21/11. Field documentation evaluation of effect In order to evaluate the effect of QTL-innOva IPN/PD in the field, we have, from autumn 211 had a close follow up on a total of 19 sites with nearly 8 million salmon Standard eggs Standard eggs Standard eggs QTL-innOva IPN/PD QTL-innOva IPN/PD QTL-innOva IPN/PD Days post challenge Figure 1. Preliminary efficacy studies of QTL-innOva IPN/PD using waterborne infection. All parallel groups of fry derived from parents selected based on gene markers associated with IPN and PD resistance (grey line), showing significant reduction in mortality compared to standard fry groups without QTL. Products with PD resistance: AquaGen Atlantic QTL-innOva IPN/PD/CMS/RED AquaGen Atlantic QTL-innOva IPN/PD/CMS AquaGen Atlantic QTL-innOva IPN/PD/RED AquaGen Atlantic QTL-innOva IPN/PD All of the sites are situated in areas known historically for a high infection pressure with SAV. Of the 19 follow-up sites, 15 had previously experienced clinical PD outbreaks. Results from field documentation 4 of the 19 sites have been diagnosed with PD, all of them with limited PD specific mortality. In addition the follow up has shown that 11 sites with a history of PD don t have any detection of PD after stocking with QTL-innOva IPN/PD. Mortality has been low across the board at all follow up sites. Records show less than 5% accumulated mortality on 13 of the 19 participating sites that have had fish in the sea for more than 1 months (Figure 2). Both PD and IPN, two of the most costly diseases in the last 1-15 years seem to be on the decline in fish farming. Results so far show that QTL-innOva IPN/PD provides a double effect, by both producing a reduction in an important pre-disposing factor for PD (IPN outbreak), at the same time as increasing the overall ability to resist infection pressure from SAV in the environment. This will contribute significantly to an enhanced virtuous circle with a gradual reduction in the number of PD outbreaks/sav positive populations and a commensurate gradual reduction of the infection pressure in the environment. Losses in whole period (%) Harvested location, PD negative Harvested location, PD positive Not harvested location, PD negative Not harvested location, PD positive Months in sea Figure 2. Recorded mortality/losses and number of months after sea transfer of fish at sites participating in field documentation. Sites are located in the area from Rugsund in Bremanger to Hardanger fjord. Benefits of QTL-innOva IPN/PD: Defence against both PD and IPN Synergistic effect increased IPN defence gives reduced risk for PD Field experience shows that the incidence and mortality of PD is reduced in comparison with previous years that didn t use QTL-innOva IPN/PD 9

10 1/4 Field documentation of QTL-innOva IPN/PD Pancreas Disease (PD), caused by Salmonid alphavirus (SAV), has for many years been one of the most costly diseases affecting Norwegian salmon farming. Producers and the authorities have implemented a number of structural and operational measures with the aim of limiting losses from this disease. In addition different vaccines against PD have been developed as well as different types of special feed To be able to evaluate the effect of these eggs in the field we have closely followed up fish at a total of 19 locations, comprised of 8 locations in autumn 211, 3 locations in spring 212 and 8 locations in autumn 213 (table 1). All of these locations were in the area from Rugsund in Bremanger to Hardanger fjord where historically there has been a high infectious pressure from SAV. PD-free project On behalf of the Norwegian Seafood Federation (FHL) the Norwegian Veterinary Institute carried out a major analysis (211) where a number of different measures were evaluated for the effect they have had to limit the scope and prevalence of PD (PD-free project). The analysis was based on operational, health and harvest data from a total of 22 smolt inputs during the period The two main parameters that showed significantly reduced risk for PD outbreaks and influenced the severity of the outbreaks were (i) Vaccination against PD and (ii) Avoidance of IPN outbreaks during the production cycle. There is reason to believe that the widespread use of PD-vaccination in endemic zones, combined with a significant reduction in the number of outbreaks of IPN has contributed significantly to the positive development of the PD situation, as registered during the most recent period (Figure 1). Development of QTL-innOva-IPN/PD Ever since 25, breeding for increased resistance against PD has been central to our R&D programme at AquaGen. In the period a large scale field trial was carried out, in which 395 families were transferred for PD challenge under natural field conditions. AquaGen has utilised data from both the preliminary field trial and various follow-up laboratory challenge tests, to identify and document the genetic markers that have efficacy against PD. These markers are not as clear and strong as IPN-QTL, but combined, they could significantly increase PD-resistance of fish. Our analysis also showed that among the three strongest QTL s associated with PD protection we find the IPN-QTL. Based on the findings of QTL searches and subsequent documentation from laboratory infection trials (Figure 2), the product QTL -innova IPN/PD was launched in the market in the egg-season 21/11. Egg year class Smolt transfer 21/11 Autumn /11 Spring /12 Autumn 212 Total number of locations Locations with a PD history Number of fish (millions) Status (July 213) Harvested Ongoing harvest About 1 year in sea Total Table 1. Overview of numbers of smolt and locations involved in the field documentation of QTL-innOva IPN/PD. Field documentation set-up The first fish produced from QTL-innOva IPN/PD eggs were transferred to sea in the autumn of 1

11 2/4 Of the 19 locations involved in the field documentation, 15 had previously experienced clinical outbreaks. All of the fish that were in the field trial were also PD vaccinated. Results from field documentation The results from the field documentation are presented in figure 3, 4 and 5. Following there is a brief summary of each smolt transfer. Autumn 211: A total of 8 sites with 3 million fish transferred in autumn of 211 were followed-up from stocking to harvest. Of the 8 sites, 7 had experienced clinical PD outbreaks in the previous intake. PD was detected at 3 of the sites and an average accumulated loss from transfer to harvest for these sites was 13.2%. Average accumulated losses for all 8 sites, from stocking to harvest was 7%. There was no evidence of IPN in any of the sites that were monitored. Spring 212: A total of 3 sites, all with previous PD outbreaks, in total 1.3 million fish were transferred and followed up with respect to PD. Accumulated losses for all 3 sites from stocking to the end of June 213 are 9.8%. To date there hasn t been any diagnosis of IPN or PD on any of the sites (14-15 months post-stocking). Autumn 212: In total, 8 sites with 3.4 million fish stocked and followed up with respect to PD. Of these sites, 5 had PD outbreaks in the previous production cycle. By the end of June 213, 1 of these sites had a diagnosis of PD. Accumulated mortality for this site from intake to the end of June is at 1%. Accumulated losses for all of the 8 transfers up until the end of June is at 1.1% (8-11 months post transfer). By the end of June there hasn t been any diagnosis of IPN or PD at any of the other locations. Number of PD outbreaks Figure 1. Development in the number of PD outbreaks in Norwegian salmon arming in the last 4 years (rolling 12 months interval). In the last 7 months we have seen a clear reduction in the number of cases of PD. Source: Norwegian Veterinary Institute. Aug-9 Oct-9 Dec-9 Feb-1 Apr-1 Jun-1 Aug-1 Oct-1 Dec-1 Feb-11 Apr-11 Jun-11 Aug-11 Oct-11 Dec-11 Feb-12 Apr-12 Jun-12 Aug-12 Oct-12 Dec-12 Feb-13 Apr-13 Jun-13 Accumulated mortality (%) Standard eggs Standard eggs Standard eggs QTL-innOva IPN/PD QTL-innOva IPN/PD QTL-innOva IPN/PD Days post challenge Figure 2. Preliminary efficacy studies of QTL-innOva IPN/PD using waterborne infection. All parallel groups of fry derived from parents selected based on gene markers associated with IPN and PD resistance (grey lines), showing significant reduction in mortality compared to standard fry groups without QTL. 11

12 3/4 Reduction in PD outbreaks Both PD and IPN, two of the most costly diseases of the last 1-15 years are in marked decline in Norwegian aquaculture. We believe that this clear trend of a reduction, in the region of 4% in the number of PD diagnoses in the first half of 213, when compared to the first half of 212 was positively related to a large reduction in IPN that has occurred since 21. Combined with a PD vaccine that has a moderate but significant effect, it has contributed to a virtuous circle with a gradual reduction in the number of affected populations and a commensurate gradual reduction of the infection pressure in the environment. Based on efficacy data from both laboratory and field trials it is reasonable to expect that the situation going forward will improve as of the autumn 213 transfers, which represent the first stocking that will have a significant proportion of fish based on QTL-innOva IPN/PD. Facts To produce eggs that are strong against PD, we must use several markers, each with a moderate effect (under 2%) and combine these with families of salmon that we previously found to be strong against PD in field trials. This compares with production of IPNstrong eggs which uses only one marker with very high effect (responsible for over 8% of variation in survival at IPN challenge). For IPN eggs this means that we have a direct measure of whether the fish is strongly resistant or not (marker + or -). For PD eggs we cannot rely on just one marker. It follows that the selection of fish with the correct attribute will be less precise at the individual level, but will be good at the group level. Losses in whole period (%) Frequency of PD pos/neg (%) % 9 % 8 % 7 % 6 % 5 % 4 % 3 % 2 % 1 % % Harvested location, PD negative Harvested location, PD positive Not harvested location, PD negative Not harvested location, PD positive Months in sea Data from VI-report (211): Evaluation of effects in the PD-free project Number of stockings: 22 1 S-7 A-7 S-8 A-8 S-9 A-11 S-12 A-12 Smolt transfer Data from field trial with QTL-innOva-IPN/PD Number of stockings: PD negative PD positive Figure 3. Recorded mortality/losses and number of months after sea transfer of fish on sites involved in the field documentation trial. Sites are located in the area from Rugsund in Bremanger to Hardanger fjord where there is a historically high infection pressure from SAV. Figure 4. Number and frequency of PD positive and PD negative sites in the field documentation trial. In total 4 out of 19 (21%) sites involved in the follow-up have PD positive status. Corresponding figures from the Veterinary Institute report (211) that included 22 stockings in the period spring 7 spring 9, are 115 out of 22 (57%) PD positive.

13 4/ Number of locations PD HISTORY AT LOCATION PD STATUS WITH QTL-innOva IPN/PD Figure 5. Comparison of historic PD status at sites with PD status after stocking of QTL-innOva IPN/PD. Of the 15 sites that previously had PD on-site, 4 have also diagnosed PD after stocking of QTL-innOva IPN/PD (+,+), but 11 have the status of PD negative after stocking of QTL-innOva IPN/PD (+,-). None of the 4 sites without a history of PD have had a detection of PD after use of QTL-innOva IPN/PD (-,-). 13

14 Product documentation IPN Resistance against IPN Mortality (%) IPN is considered as one of the most serious infectious diseases in most types of intensive aquaculture. Broad geographic coverage and many susceptible species, contribute to a continuous infection pressure from different reservoirs. For such diseases, increased host resistance will be an important contribution in controlling the distribution and extent of the disease. AquaGen introduced QTL-innOva IPN to the market in the autumn of 29. Over the last four egg-seasons a total of 558 million QTL-innOva eggs have been delivered to productions sites in Europe. Field documentation with IPN resistant QTL-fish The first QTL fish that originated from the egg intakes in 29/21 were transferred to sea in the autumn of 21 (S) and spring 211 (S1). From these first generations of QTL-innOva IPN eggs a total of 3.6 million fish at 44 locations were followed up and their performance compared with non QTL fish. The field documentation showed that both S and S1 year class had significantly reduced mortality (Figure 1) and fewer IPN diagnoses ( and 1 against 1 and 7) than S and S1 of non QTL fish to 9 days post seawater transfer. Efficacy testing with highly virulent IPNV field isolate Good challenge models based on a natural waterborne infection are an important prerequisite to both look for potential QTL and for subsequent evaluation of their efficacy and significance in practice. Additional use is made of infection trials as a part of AquaGen s follow up of IPN eggs in the field. 7 locations, 2.3 mill. salmon 12 locations, 11.3 mill. salmon Non QTL 11 locations, 5.6 mill. salmon 14 locations, 11.4 mill. salmon QTL-innOva IPN Figure 1. Total average mortality 9 days after sea transfer of salmon at 44 sites respectively S autumn 21 and S1 spring 211. Products with IPN resistance: All products from the AquaGen Atlantic QTL-innOva -line S S1 Highly virulent IPN virus strains from IPN outbreaks isolated from the field were analysed for genetic modifications (mutations) and are included in challenge tests to determine whether the protective effect of QTL-innOva is being maintained (Figure 2).In all, three genotypes were tested, qq (homozygote, IPN sensitive), qq (heterozygote, IPN resistant) and QQ (homozygote, IPN resistant). Accumulated mortality to completion of the test was 59.2% for qq, 8.% for qq and 2.6% for QQ. That results in a relative percent survival of 86.2 and 95.6% for groups with genotype Qq and QQ. This is in accordance with the expected degree of protection for the commercially available product, QTL-innOva IPN that has a specified protection level of 82%. Results from analyses four years after introduction to the market, don t give any indication that the IPN virus has managed to figure out the new defence mechanism contributed by QTL-innOva. Mortality (%) Control RPS = 86.2 % RPS = 95.6 % qq qq QQ Genotype for IPN resistance Figure 2. Accumulated mortality in IPN challenge test of salmon fry with different genotypes for IPN resistance. A highly virulent field isolate was used for the challenge. Relative percent survival (RPS) was 86.5% for qq, and 95.6% QQ compared with qq as a control. Mortality for each group of genotypes is the average of two parallels. Benefits of QTL-innOva IPN: Fish have defence against IPN for the whole of their life Optimised QTL-analysis provides a high degree of assurance that QTL-eggs are composed of high resistant variants, qq and QQ for IPN Repeated laboratory IPN challenge tests confirm the on-going high level of protection against IPN Field experience has given very positive feedback on performance under commercial conditions 14

15 1/2 Mortality (%) Use of QTL-eggs results in an IPN reduction for the whole of Norway Since the introduction of IPN resistant QTLeggs in 29, there has been seen a reduction in the number of recorded IPN diagnoses for salmon by 5 % during the period Information from both farming companies and national statistics also shows that there has been a considerable reduction in mortality for both 211 and 212 generations of salmon in Norwegian fish farming. In the same period the number of QTL-eggs delivered to Norwegian fish farmers has increased greatly. Field investigations to document performance that began at the time of introduction of QTLeggs underpin our conclusion that both the number and severity of outbreaks has been reduced. Field investigations with IPN resistant QTL-fish The first QTL-fish that had their origin in egg intakes from 29/21 egg generation were transferred to sea as S smolts in the autumn of 21, and as S1 smolts in spring of 211. From this egg generation fish were also transferred to sea that had not been selected with the assistance of the IPN gene marker. A total of 3.6 million fish distributed across 44 locations were followed up so that the relative performance of QTL-fish and non QTLfish could be compared. The results of the field investigation showed that both S and S1 classes of QTL had a lower mortality (1.1 % and 4.6 % compared to 6.4 % and 12.7 %) and fewer IPN diagnoses ( and 3 compared to 1 and 7) than S and S1 of non QTL-fish up to 9 days after sea transfer (figure 1). 7 locations, 2.3 mill. salmon 12 locations, 11.3 mill. salmon Non QTL 11 locations, 5.6 mill. salmon 14 locations, 11.4 mill. salmon QTL-innOva IPN Figure 1. Average mortality of salmon at 44 different locations along the Norwegian coast which totally comprised of 3.6 million fish transferred to sea. The mortality is recorded 9 days after sea transfer for both S autumn 21 and S1- spring 211. S S1 IPN resistance tested with a highly virulent field isolate It has been questioned as to whether the IPN virus would adapt in response to the use of IPN resistant QTL-eggs. As a part of AquaGen following-up on performance of QTL-eggs in the field, highly virulent IPN strains involved in outbreaks have been isolated and analysed for genetic changes (mutations). Part of this follow-up involved a new challenge test that was carried out using a highly virulent IPN virus field isolate by VESO Vikan in the spring of 213. A total of three genotypes were tested, qq (homozygote for the trait IPN sensitive), qq (heterozygote for the trait IPN resistant) and QQ (homozygote for the trait IPN resistant). Accumulated mortality when the challenge terminated was 59.2 % for qq, 8. % for qq and 2.6 % for QQ. This result gives a relative percent survival (RPS) of 86.5 % for qq and 95.6 % for QQ, measured against qq as a control (figure 2). This agrees well with the expected degree of protection for this product (AquaGen declares a RPS of 82 % for QTL-innOva IPN). The results from these analyses, four years after introduction to the market, don t give any indication that the IPN virus has found a way round the defence mechanism posed by the QTL-innOva IPN. Reduction in IPN diagnoses on a national level As the proportion of QTL-eggs used in Norwegian aquaculture has increased, the economic importance of the effect of IPN disease has been greatly reduced. National statistics from the Norwegian Veterinary Institute for fish health in 212 showed that the number of outbreaks for the country had reduced by 5 % from 221 diagnoses in 29 to 11 diagnoses in 212 (figure 3). We believe that this trend will be maintained and show further 15

16 2/2 reductions in by virtue of the fact that 7 to 8 % of all salmon eggs laid down will be of QTL origin. A large reduction in the prevalence of IPN will also make a positive contribution towards the overall health status and resultant fish welfare of farmed fish. IPN outbreaks in freshwater often result in consequential effects in the form of variable or poor smolt quality. Furthermore it has been recently documented that fish populations that have previously experienced an IPN outbreak have a resulting higher risk for developing PD later in the production cycle. Facts A fish is a homozygote when it has two identical variants of a gene on a chromosome pair. This can be described as either qq or QQ. A fish is a heterozygote when it has two different variants of the gene (qq or Qq). Genes can be dominant (denoted by a large letter, Q) or recessive (denoted by a small letter, q). Dominant genes will be expressed in the fish phenotype as long as there is at least one on the chromosomes in a pair (qq, Qq or QQ). Recessive genes must be present on both chromosomes of a pair (qq) to be expressed in the fish. Mortality (%) Control RPS = 86.2 % RPS = 95.6 % qq qq QQ Figure 2. Accumulated mortality from an IPN challenge test of salmon fry with different genotypes for IPN resistance. A highly virulent field isolate of the IPN virus was used for the challenge. Relative percentage survival (RPS) was 86.5 % for qq and 95.6 % for QQ compared with qq as a control. Mortality for each genotype is the average of two parallels. Genotype for IPN resistance No. of IPN diagnoses in salmon No. of salmon eggs (x 1) IPN diagnoses in salmon Total no. of eggs No. of QTL-innOva eggs Figure 3. Number of IPN diagnoses for salmon in fresh and sea water in Norway from 29 to 212 compared to the number of QTLeggs delivered by AquaGen and the total number of eggs sold in the same period. Source: Norwegian Veterinary Institute and Norwegian Seafood Federation. 16

17 Product documentation STERILE Sterile salmon AquaGen first introduced eggs for the production of sterile salmon in the season 212/13. The main reason in Norway for producing these eggs is that sterility eliminates some of the possible negative impacts that escaped farmed salmon could have on wild salmon. Sterile salmon can also reduce other problems such as undesirable early maturation prior to harvest weight. The only available method for producing sterile eggs today is triploidization. Just after fertilization eggs are treated in a pressure chamber, resulting in the fish keeping three sets of chromosomes instead of two (diploid). Egg production AquaGen has installed newly developed equipment that can process large volumes, and that gives eggs a gentle and uniform treatment (Figure 1). The latter is essential for good survival during early development. For the first production in the project mentioned below, the average mortality between egg delivery and first feeding was 3.2% and 2.4%, respectively for triploids and diploids. The new pressure chamber has been verified to produce 1% triploid eggs (Figure 2). Production of triploid salmon AquaGen is currently participating in a project with five fish farming companies, to test the large-scale commercial production of triploid salmon. Growth: Triploid salmon grows at least as well as diploids in freshwater, and up until body weight of 2-3 kg in seawater. From then on growth rate reduces, and in the final phase until harvest weight of 5-6 kg, then approximately 1% less weight should be expected compared to diploid. Experiments with long term exposure at elevated temperatures (19 C) and low oxygen content (< 7% saturation) have shown that triploids are more sensitive to these environmental factors than diploids. Animal welfare: Triploid salmon have a higher incidence of skeletal deformities, but this will reduce to the same level as diploids by increasing the phosphorous content of feed in freshwater and the first three months of the seawater phase (>.9% available phosphorus). A greater incidence of cataracts can be reduced if the food contains sufficient histidine (> 1.8%) Diploid Triploid Number of fish DNA-content in cells (relative values) Figure 2. Testing of yolksac fry that were triploidised using the new pressure chamber. Test method used was flow cytometry, showing as expected that the average cell content of DNA is exactly 1.5 times higher in triploids than in diploids. Figure 1. Egg containers are taken horizontally into the pressure chamber (to the left) for triploidizing. With this automatic equipment eggs that are at a very vulnerable stage are gently handled. Products that can be sterilized: All products from the AquaGen Atlantic QTL-innOva -line Benefits with GREEN STERILE: Escaped sterile salmon cannot reproduce and affect the genetics of wild salmon stocks Reduced quality problems with early maturation Increased flexibility in relation to harvest time 17

18 Product documentation TRACK DNA tracking of escaped farmed salmon The escape of farmed salmon is an unwanted incident that has negative economic consequences for the fish farmer and may lead to genetic impacts on wild fish populations. AquaGen offer to store DNA from broodstock that are used in egg production, to allow tracking back from the offspring to their respective egg deliveries. Each egg batch delivered has unique parents, so should there be a suspect escaped fish it can be checked against the broodfish that were the parents of relevant egg deliveries. For tracking to be carried out in a secure way, it is important that recipients of eggs, smolts, production fish and harvest fish have good control of where the fish groups are at all times in the system. DNA salmons identity Each fish has its own unique DNA which is the best and most secure identification an individual has. With the right tools offspring can be checked against parents to verify relationship. Markers are mapped points on the DNA which indicate variation between different individuals. Each fish gets half of its DNA from each of its parents, so by analysing both offspring and parents for known markers, relationship can be verified. They have developed systems for genotyping from tissue samples. DNA profiles from previously analysed tissue samples from broodfish can be tested for relationship to potentially escaped fish if desired. The analysis uses 1, genetic markers, that will give 1% confidence in the answer to the possibility of a relationship. Procedure for parental assignment 1. AquaGen takes a tissue sample from all broodstock (both female and male) that is used for a specific egg delivery. The tissue samples are sent for genotyping at CIGENE. 2. Tissue samples (preserved in alcohol) from potential escapees are sent to CIGENE where a DNA analysis is carried out of these and all DNA profiles from broodstock used in the relevant egg deliveries. 3. The result of the analysis is first sent to AquaGen who link the analyses to an actual egg delivery, and then forward the results to the farmer. Escaped farmed salmon? DNA Reliable answers on parental assignment CIGENE is the research centre at the Norwegian University of Life Sciences that has high competence within the field of genetics research. FARM STRAIN 1 PARENTS FARMED SALMON FARM STRAIN 2 FARM STRAIN 3 HYBRID STRAIN 1 WILD STRAIN 1 WILD SALMON WILD STRAIN 2 WILD STRAIN 3 EGG DELIVERY HYBRID STRAIN 2 DATE, NUMBER, FARM Figure 1. DNA tracking of parents for each individual egg delivery is performed using tissue samples of all male and female fish taken at the point of fertilisation. Tissue samples are registered electronically so that results from genotyping can be tracked back to the correct egg delivery and fish farm in the future. Products with DNA tracking: All products from the AquaGen Atlantic QTL-innOva -line Figure 2. DNA from a potentially escaped farmed fish can be genotyped and checked against a database of farmed fish parents. If the DNA analysis shows a positive correlation between the salmon and registered parents, specific information on egg delivery can be tracked back to the correct smolt site with 1% confidence. As an alternative, the analysis can also reveal whether salmon belong to different wild strains or hybrid strains of wild and farmed salmon. Benefits with GREEN TRACK: No physical marking of fish that are transferred to sea A secure method of relating broodfish to their progeny DNA tracking performed only at the fish farmers request if the need arises Gives protection against false claims of escapes from fish farms 18

19 AquaGen rainbow trout eggs 216 RAINBOW TROUT EGGS 216 Start material with the best potential for growth and survival 19

20 AquaGen rainbow trout eggs 216 Genetic Background for Rainbow Trout Eggs 216 At AquaGen we carry out several selections of broodstock at different stages before the final selection is allowed to produce eggs for the farmers. The selection process occurs both at the family- and individual level. The Rainbow trout eggs that AquaGen will deliver are the results of selective breeding for a range of important traits through 15 generations. How long the different traits have been selected for, as well as the intensity of the selection is very important for the total progress which today s products represent. At the present time a total of 12 traits are recorded from 2 families. A comprehensive recording program ensures progress in these desirable traits while at the same time minimalizing the risk of unwanted or unexpected side effects. The figure below shows the main traits which are measured, selected and controlled in the selective breeding program (breeding nucleus) set up in relation to the commersial egg producing generation to be phased in. Rainbow trout eggs which are delivered to the hatcheries in the 216 season are coming from the top-ranking families that were first fed in 213 (marked with grey in the chart). Elite material from this generation is transferred to our egg producers. Through a production cycle over three years the best individuals are selected as broodstock. In this way the best genetics will be large scale reproduced and benefit all fish farmers. Since 215 AquaGen has implemented new modern technology called Marker Assisted Selection. Based on this new technology platform we are able to directly select parents to be used in egg-production that possess a specific genetic marker (QTL) that are highly associated with resistance to the viral disease IPN. This double selection, both on family- and individual level, ensures a balanced weight to robust and effective traits as well as high IPN resistance in the offspring. Implementation of traits based on traditional- and gene marker selective breeding methods in AquaGen's breeding programme (nucleus) of Rainbow Trout (Oncorhynchus mykiss ) : 26: First feeding year for juveniles of brood fish The three year collected from Norwegian and Swedish farmers. classes were Originally from the Pacific cost of North-America. merged into one. Generation Class of Year (first feeding) Traits GROWTH SEXUAL MATURATION Production/Efficiency Traits Health/Robustness Traits Gene marker based breeding method started The top-ranking families first fed in 213 are the base for brood fish stripping in 216 SKIN COLOUR BODY SHAPE FILET COLOUR FILET YIELD SKELETAL DEFORMITIES IPN 2

21 AquaGen rainbow trout eggs 216 Biosecurity and Quality Control Fish health surveillance in AquaGen is carried out through the entire life-cycle of the fish. In the final nine months before stripping the monitoring is intensified. This includes comprehensive autopsy and screening of all brood fish populations in the AquaGen system. Production is monitored by both internal and external fishhealth personnel, and by government and private entities. AquaGen is certified according to GLOBAL GAP (food safety, environmental protection, fish welfare, and health, safety and welfare for employees), FREEDOM FOOD (animal welfare), ISO 91:28 (quality leadership) and Code-EFABAR (good practice within animal farm breeding and reproduction). AquaGen s production plants at Hemne and Tingvoll are located in the EU-approved ISA-free compartments at the highest security level. ISA has never been detected in these compartments. AquaGen s broodstock populations are routinely screened for IPN virus and the BKD bacterium during the entire production cycle of three years. Individual testing of parents at stripping can be carried out if ordered in advance. Relevant health information about brood fish and eggs will be documented in a separate HEALTH CV and included in all egg deliveries. Delivery Time In the egg season of 216 AquaGen will deliver eggs from the beginning of January to the beginning of July: Category Early eggs Normal eggs Late eggs Delivery Time week 1-4 (Jan. 4 - Jan. 31) week 5-17 (Feb. 1 - May. 1) week (May. 2 - Jul. 3) In the figure below the differing production timelines are presented for trout through the fresh-water phase and the salt-water phase, based on early, normal, and late eggs. The figure shows how AquaGen by means of managed production of broodstock can contribute to the optimal exploitation of our customer s production facility capacity. Should there be a requirement for unexpected additional eggs, due to operating accidents, diseases or lack of capacity in customers own facilities, AquaGen often has the ability to help out with deliveries in such situations. Production plan for trout in the FRESH-WATER PHASE Category 1st year 2nd year Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Early -year Early eggs Smolt Late -year Normal eggs Smolt 1-year Late eggs Smolt Production plan for trout in the SEA-WATER PHASE Category 1st year in sea 2nd year in sea 3rd year in sea Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Early -year Smolt Harvest Late -year Smolt Harvest 1-year Smolt Harvest 21

22 AquaGen rainbow trout eggs 216 Product Overview for Rainbow Trout Eggs 216 Early ordering, before October 1, 215 contributes to better production planning and higher delivery assurance in regard to choice of product type, amount and time of delivery. Product Description AquaGen Rainbow Ova Eggs Eggs from broodstock which produces a balance between Robust and Effective traits in the offspring. AquaGen Rainbow QTL-innOva IPN Eggs from broodstock which produces a balance between Robust and Effective traits, as well as extra high IPN-resistance in the offspring. The males are chosen using the gene marker (QTL) for IPN. AquaGen Rainbow GREEN Treatment STERILE All female Virus testing Sterilization of eggs which after pressure treatment on recently fertilized eggs makes the fish sterile. Since this production requires extra planning and greater safety margins than ordinary, the ordering deadline is November 1, 214. Milt from verified transformed female broodstock instead of normal male broodstock are used. The offspring will be female fish only. Individual testing of female- and male broodstock for IPNV, PDV and/or ISAV at the time of stripping. Broodstock free from virus are used in the egg production only. Robust: Health-related traits such as resistance to disease, reduced deformities, reduced early sexual maturity. Effective: Production-related traits such as growth, skin color, filet color, harvest amounts, body shape. QTL: Gene marker (Quantitative Trait Loci). Area on the rainbow trout genome (marker) which is closely connected to a gene which controls a specific characteristic in the individual, in this case IPN resistance. 22

23 IPN resistance for rainbow trout The viral disease IPN has traditionally been a problem for rainbow trout in the fresh water phase. The disease may cause major losses, both in increased mortality rates, reduced growth and instances of weakened fish that have survived an IPN outbreak. This means a high degree of uncertainty as to how fish will perform compared to annual production budgets. In 29 AquaGen performed the first selection for IPN resistance in rainbow trout using traditional family based breeding. In parallel with this work, a number of challenge tests were used to identify and document genetic markers that can be used to select broodstock with IPN resistant genes. It was proved that IPN resistance in rainbow trout is to a large extent controlled by one single QTL, just like in salmon. The first eggs selected for this QTL were delivered to fish farmers in January 215. Product documentation IPN significant difference in mortality rates between the different genotypes, where the degree of IPN resistance depends on the frequency of Q (Fig. 1 and 2). In experiment 1 the frequency of the favorable IPN resistant variant Q was relatively low (25%). This has contributed to the building up of a high IPN infection pressure because of the high number of IPN sensitive fish (qq) in the group. In experiment 2 the genotypes were more evenly distributed (48% Q), and had a moderate IPN infection pressure. In both tests the mortality rate was considerably lower in the groups that carried the IPN resistant variant Q, and the difference was especially high when the infection pressure was moderate. These infection experiments document that the QTL for IPN resistance in rainbow trout has a good effect on IPN in laboratory experiments. Effect testing of IPN resistance The effect of the genetic marker has been documented in two separate challenge tests on rainbow trout fry soon after start feeding. In order to eliminate environmental effects between test groups all fish were challenged in the same tank. At the end of the experiments both living and dead fish were genotyped and grouped as respectively qq (homozygote, IPN sensitive), qq (heterozygote) and QQ (homozygote, IPN strong). The results from both experiments show a Great expectations for protection by QTL-selection In the eggs selected for IPN resistance (that only have the genotypes qq and QQ), the frequency of Q will be 6 8%. This level of frequency will provide a positive population effect (a kind of herd immunity ) that means there will be a higher threshold for the IPN virus to overcome to establish itself in the group of fish, and the extent of a potential IPN outbreak will be considerably reduced. Figure 1. Experiment 1, effect testing of IPN resistance in recently start fed rainbow trout fry. In the group, that comprised 1719 fish, the frequency of the favorable IPN resistant variant Q was only 25%. The IPN infection pressure was high resulting in an accumulated mortality rate for the IPN sensitive fish (qq) of 83%. Products with IPN resistance: AquaGen Rainbow QTL-innOva IPN Figure 2. Experiment 2, effect testing of IPN resistance in recently start fed rainbow trout fry. In the group, that comprised 1582 fish, the frequency of the favorable IPN resistant variant Q was at a moderate 48%. The IPN infection pressure was also moderate with an accumulated mortality rate for the IPN sensitive fish (qq) of 23%. Advantages of Rainbow QTL-innOva IPN: IPN protection through the entire life of the fish Greater predictability in production of rainbow trout 23