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HSWRI WSB QA/QC MANUAL DRAFT

PROCEDURES MANUAL FOR QUALITY ASSESSMENT AND CONTROL OF MARINE FINFISH CULTURED FOR

STOCK REPLENISHMENT

Prepared by:

Hubbs- SeaWorld Research Institute

1st EDITION

December 2011


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TABLE OF CONTENTS INTRODUCTION ............................................................................................................................................. 1

Background ............................................................................................................................................... 1

Genetic considerations ............................................................................................................................. 2

Goals and Objectives ................................................................................................................................. 3

PHYSICAL QUALITY FACTORS ASSOCIATED WITH CULTURED MARINE FISH ................................................ 5

Malformations and deformities ................................................................................................................ 5

Other Soft Tissue Quality “Defects” .......................................................................................................... 6

DEVELOPMENT AND MORPHOLOGY OF WHITE SEABASS ............................................................................ 7

QUALITY ASSESSMENT AND CONTROL (QA/QC) POINTS AND PRACTICES – HOW, WHEN AND WHERE ... 11

Quality Assessment (QA) ........................................................................................................................ 11

Quality Control (QC) ................................................................................................................................ 12

QUALITY ATTRIBUTES (see additional detail for each category below**) .................................................. 14

Malformations (primarily hard tissue/bone) .......................................................................................... 14

1. Dorsal Head Indentation ............................................................................................................. 15

2. Head Malformation ..................................................................................................................... 18

3. Cranial Protuberances ................................................................................................................. 20

4. Pug Head ..................................................................................................................................... 22

5. Maxilla ............................................................................................ Error! Bookmark not defined.

6. Lower Jaw Extension ................................................................................................................... 28

7. Lower jaw reduction ................................................................................................................... 31

8. Crossbite ..................................................................................................................................... 33

9. Orbital Malformation .................................................................................................................. 36

10. Malformed Operculum ........................................................................................................... 38

11. Branchiostegal Malformation ................................................................................................. 40

12. Vertebral Axial Deviations ....................................................................................................... 42

13. Vertebral Fusion ...................................................................................................................... 45

14. Malformed Fins ....................................................................................................................... 48

Other “defects” (primarily soft tissue) .................................................................................................... 50

15. Skin .......................................................................................................................................... 50

16. Cloudy Eye ............................................................................................................................... 52


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17. Bubble eye............................................................................................................................... 54

18. Popeye .................................................................................................................................... 56

19. Gills .......................................................................................................................................... 58

20. Fin Erosion ............................................................................................................................... 60

BEST MANAGEMENT PRACTICES ................................................................................................................ 62

THE ROLE OF RESEARCH ............................................................................................................................. 63

GLOSSARY OF DEFINITIONS ........................................................................................................................ 64

Bibliography ................................................................................................................................................ 66

APPENDIX A – DATA FORM FOR QA ............................................................................................................ 68


1

INTRODUCTION

Background

This document provides a detailed description of the Quality Assessment and Control (QA/QC) procedures

applied to white seabass produced at the Leon Raymond Hubbard, Jr. Marine Fish Hatchery in Carlsbad,

California and the network of volunteer-based growout facilities. The emphasis of the QA/QC program is

centered on hatchery operations where malformations tend to originate, and where sorting or culling is

easier to accomplish. The Carlsbad Hatchery is owned and operated by Hubbs-SeaWorld Research Institute

(HSWRI) under contract from the California Department of Fish and Wildlife (CDFW) as part of California’s

Ocean Resources Replenishment and Hatchery Program (OREHP). Since 1984, the CDFW, as part of OREHP,

has contracted for research to evaluate the feasibility of culturing and releasing juvenile marine fish, with the

goal of enhancing depleted wild stocks in southern California. The white seabass (Atractoscion nobilis) was

selected as the first species for experimental population replenishment. The white seabass was chosen

because it is a species of great value to both commercial and sport fishers, and landings of this species have

declined to a fraction of historic levels.

This document is a companion document to the Comprehensive Hatchery Plan (CHP) for the Operation of

the Leon Raymond Hubbard, Jr. Marine Fish Hatchery in Carlsbad California and the Procedures Manual for

Growout and Release of White Seabass (Atractoscion nobilis) as Part of the Ocean Resources Enhancement

and Hatchery Program (OREHP). Both of these companion documents have chapters on fish health, but

neither goes into detail on aspects related to QA/QC. Because a primary objective of the OREHP is to

“Develop and implement hatchery operation methods that provide a supply of healthy and vigorous fish”, a

comprehensive QA/QC program is mandatory and warrants thorough consideration and documentation.

This document specifically focuses on physical quality attributes that can be assessed during an external

examination of the fish, including functionality of the swim bladder. It does NOT include other

important quality attributes such as behavior and physiological function. Deficiencies in physical

attributes may include not only “malformations” of bony tissue but also damage to soft tissue such as

eyes, gills and fins.


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Genetic considerations

Genotypic/phenotypic interactions

Phenotypic malformation or the predisposition toward malformation may be due to genetic mutation. Here, reasons are documented as to why genetic mutations in the white seabass broodstock or de novo mutations in the F1 generation (i.e. cultured juveniles) are not likely to contribute to significant malformation rates in captive-bred progeny.

White seabass broadcast spawn, with females producing an average of 1.7 million eggs during a single spawning event. Energetic investment is placed in the sheer number of gametes produced and released during mass spawning rather than quality. On the surface, this creates a situation that appears ripe for creating high rates of genetic mutation, leading to physical malformation in the progeny. Assuming simple Mendelian inheritance, maximum rates of malformation in the F1 generation may theoretically occur under the following scenarios:

1) 50% frequency a. Malformation is due to mutation at a single locus b. Detrimental allele exhibits complete or incomplete dominance c. Single brood female ord. Detrimental allele is present in that brood female

male contributes or

2) 25% frequency male

a. Malformation is due to mutation at a single locus b. Detrimental allele is recessive c. Detrimental allele must occur in brood female andd. Single brood female and single brood male contribute

male

However, these scenarios likely never occur due to a combination of biology, behavior, and management. The first scenario entails the malformation also be present in the carrier, which should not occur under HSWRI broodstock selection and management protocols, and for both scenarios, single male contribution does not occur under group spawning conditions (see below).

Moreover, these scenarios are far too simplistic. Phenotype (normal or abnormal) is more often the product of multilocus interactions, rather than that of a single gene, and independent assortment and recombination during meiosis shuffles allelic combinations in interacting genes in the resulting germ cells/gametes. Added to that complexity, white seabass exhibit multiple maternity/paternity and repeat spawning. Overall, this creates highly variable genotypic combinations in the offspring within and among broadcast spawning events. For example, true siblings (same mother and father) share only 50% of their DNA on average, and the proportion of true siblings resulting from a typical white seabass spawning event rarely exceeds 20% and is often less than 15%. In fact, broadcast spawning itself may have evolved in part as a means of “bet hedging” to produce fitter, more genetically variable offspring.


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In sum, the chance a malformation resulting from an inherited genetic mutation will reach high frequency is extremely small. Likewise, de novo mutations independent of parental genotype that cause any phenotypic change should occur only sporadically and at extremely low frequency in the offspring. Most, if not all, proportionally high malformations are due to conditions external to the broodstock and their progeny, such as physical forcing (e.g. water quality), disease, or nutrition (e.g. vitamins, fatty acid content in diets), rather than any direct genotype/phenotype link.

Hatchery genetic management

HSWRI employs comprehensive hatchery genetic management, in which the primary focus is conservation of genetic diversity, both in the broodstock with respect to the wild population and in the F1 generation with respect to the broodstock. Two documents detail the current plan, including A Contemporary Plan for Managing White Seabass Broodstock and Production Cohorts for the OREHP, Gruenthal and Drawbridge (in press). The former broodstock genetic management plan, on which the new plan is based in part, is presented in Bartley et al. (1995). As part of HSWRI hatchery genetic management:

1) Potential brood fish are collected from the natural population a. Fish are caught in the wild by hook-and-line in the Evolutionarily Significant or

Management Unit of interest (i.e. Southern California Bight, where replenishment is focused)

b. Captured fish are quarantined for 45 days, PIT tagged, given a health exam, and fin clipped prior to introduction to the brood pool

c. No wild fish with disease or malformations are used for breeding d. No captive-bred progeny are used for breeding

2) A total hatchery population (census size) of 140 – 200 healthy brood fish are maintained, divided evenly among four brood pools

3) Broodstock sex ratio is maintained at 60% female and 40% male to compensate for sex-specific mating patterns in white seabass (fewer females contribute within and among spawns)

4) The broodstock is replaced in total (100%) by new brood fish every four years at ~25% per year a. Resident fish that are compromised (ill, hurt, etc.) are selectively replaced first, followed

by older fish and fish that have been in the breeding population longest b. New fish are introduced as per item 1

5) F1 cohorts are chosen and reared such that parental contribution is equalized among females and among brood pools

6) Stockable juveniles should represent an annual hatchery breeding effective size of ≥84

Goals and Objectives

The primary goal of this QA/QC program is to release fish of the highest physical quality into the

ocean such that post-release survival is not unduly impaired and a hatchery phenotype is not evident.


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Physical attributes with potential effects on post-release survival can be broadly categorized as follows

1) malformed mouthparts that could affect feeding; 2) damaged eyes that can affect feeding and

predator avoidance; and 3) malformed body shape and/or fin damage that could affect mobility (for

feeding and predator avoidance). Phenotypic characteristics would include any body component of the

cultured fish that places that individual outside the natural range of variability found in the wild. Since

the wild phenotype for juvenile white seabass has not been formally characterized, the QA/QC program

is currently guided by best judgment anchored in a firm understanding of fish morphology in general.

This goal will be achieved by completing the following objectives.

1. Develop husbandry methods that routinely minimize defects to 10% or less in each cohort

2. Develop a standardized Quality Assessment (QA) program that:

a. minimizes inter-examiner variability

b. validates the QC program on a continuous basis

c. is coupled with a hatchery worker training program

d. is coupled with a research program aimed at minimizing physical defects

3. Develop a standardized Quality Control (QC) program that:

a. is effective in screening out 95% or greater “defective” individuals

b. is practical and efficient, ideally being implemented in a single pass of screening all

individuals in a group


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PHYSICAL QUALITY FACTORS ASSOCIATED WITH CULTURED MARINE FISH

Malformations and deformities

The larval stage of several species has been a priority of focus in the aquaculture industry, as this life

stage is pivotal in successful development. Skeletal malformations and deformities, in particular, have

been studied most extensively, given these are often readily identified and documented in highest

abundance. A malformation is defined as a deformity in the shape or structure of a part, especially

when congenital. Malformations are often induced during embryonic and early juvenile development as

the structures are formed (FineFish 2011). A deformity, dysmorphism, or dysmorphic feature is a major

difference in the shape of body part or organ compared to the average shape of that part; often referred

to as a change in a structure that is already formed (FineFish 2011).

Nutrition is one of the primary areas affecting bone development in marine species, with vitamin D

maintaining calcium homeostasis and vitamin C essential for collagen synthesis. In European sea bass

larvae, Dicentrarchus labrax, low dietary levels of these vitamins can result in disruption of osteoblast

activity, thus influencing osteogenesis and resulting in skeletal malformation (Mazurais et al, 2008).

Vitamin A plays a role in morphogenetic gene expression and must be at optimal levels at earlier

development (<20 dph), as high dietary levels can result in teratogenic effects. (Mazurais et al, 2009).

Specifically, pug headness, compression of the bones of the anterior head and upper jaw, has been

observed in European sea bass with excess dietary vitamin A (Villeneuve et al, 2006).

Fatty acids have also been examined, with scoliosis and gill cover malformations in European sea bass

being linked to elevated or extreme low levels of docosahexaenoic acid (DHA) and eicosapentaenoic acid

(EPA) in the diet (Villeneuve et al, 2005). Additionally, optimal fatty acid levels of dietary

phosphatidylinostol (PI) have a positive effect on reducing the frequency of malformations observed in

Gilthead seabream larvae, Sparus aurata (Sandel et al, 2010). Mineralization also plays a role with

Helland et al, 2006, describing suboptimal levels of phosphorus and calcium as contributing to jaw and

vertebral malformations in Atlantic salmon, Salmo salar.

Factors other than nutrition, such as water temperature, water currents, and high density larval rearing,

to name a few, have been studied with respect to their effects on malformations and deformities

observed in several species (Divanach et al 1997, Georgakopoulou et al 2007, Izquierdo et al 2010, Roo

et al. 2010). Temperature effects resulting in vertebral fusion have been described in several early


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juvenile freshwater and saltwater species (REFS). Additionally, low developmental temperatures have

shown malformations of the branchiostegal rays in Gilthead seabream and European sea bass larvae

(Georgakopoulou et al, 2007). Lordosis, once thought to be specifically related to poor swim bladder

inflation, has now also been linked to high velocity water currents, resulting in deformity from muscle

overexertion on the haemal vertebrae (Divanach et al, 1997). Cranial malformations and kyphosis

have been described radiographically in intensively reared red porgy, Pagrus pagrus, culture systems

despite similar external appearance to wild populations (Roo et al, 2010).

Experimental documentation of malformation prevalence comparisons between wild and cultured

species is limited. However, wild and cultured sharpsnout seabream juveniles have been shown to have

similar malformations (vertebral, pectoral fin ray and dorsal spine count differences) when present,

though with higher frequencies in cultured populations (Favaloro et al, 2003). Specifically, pectoral fin

anomalies have shown the greatest variability among the wild and cultured sharpsnout seabream, with

higher frequency explained by density, temperature and food factors in cultured environments versus

lower frequencies in wild populations explained by selection pressures (Favaloro et al, 2003). Another

study comparing wild and cultured sharpsnout seabream and pandora has determined that while

cultured fish have higher frequencies of malformations, the prevalence of a particular malformation can

be species-specific in wild populations and age-specific in some cultured populations (Boglione et al,

2003). Nonetheless, this identifies that skeletal anomalies occur in both wild and cultured fish, and that

it is not uncommon to find variants of normal development among wild species.

Clearly, malformations and deformities may present for a multitude of reasons, some of which may be

more specific to one species versus another. Causal factors are often difficult to identify, and may act

concurrently to affect fish quality. External quality assessment is one way in which to identify

abnormalities. However, abnormalities may also present at an undetectable level, in the absence of

diagnostic aids such as radiography, mammography, and clearing and staining techniques.

Other Soft Tissue Quality “Defects”

While skeletal malformations and deformities represent the majority of external tissue inconsistencies

identified within the hatchery setting, soft tissue abnormalities have also been observed. These include

lesions of the skin, eyes, gills, fins, and are most often associated with environmental factors of density,

water quality, parasite infestation, viral and/or bacterial insult.


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Skin lesions may present as minor abrasions, pigment changes or the development of erythema and

ulceration. Ocular lesions identified in white seabass often present as intraocular emphysema and

exophthalmia due to supersaturation (Smiley et al. 2012), but ocular scarring and ulcerations have been

also been observed. Discoloration, fraying, and clubbing of the gills have been identified, second to

diminished water quality affecting osmoregulation, though disease may also play a role in the

development of lesions (Noga 2010). Finally fin lesions of erythema, fraying from cannibalism, and

bacterial necrosis are soft tissue factors affecting quality irrespective of any skeletal malformations

identified during larval development.

In an effort to better assess malformations, deformities, and disease-related sequelae occurring in white

seabass cultured at HSWRI, quality assessment and control guidelines have been developed as an initial

step in identifying areas where additional research is needed and managerial production control points

can be addressed.

DEVELOPMENT AND MORPHOLOGY OF WHITE SEABASS

INFO FROM MOSER, HALL, TARDY

Developmental Osteology (Lisa Hall)

A clearing and staining study was conducted to describe the developmental sequence of appearance of

the skeletal elements in cultured white seabass (Hull, 1998, unpublished report, SDSU). This study

examined the timing of ossification-onset only in the head and pectoral regions from 4 days post-hatch

(dph) through 62 dph.

Below is a red drum skeletal model adapted to demonstrate developmental sequence observed in

cultured white seabass.


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13 dph bone is absent (grey), bone is developing (blue)

35 dph bone is absent (grey), bone is developing (blue), bone is ossified (red)


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51 DPH bone is ossified (red)


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QUALITY ASSESSMENT AND CONTROL (QA/QC) POINTS AND PRACTICES – HOW, WHEN AND WHERE

The steps in the QA/QC process are illustrated in the Figure 1 and described below.

Figure 1. Decision tree for WSB QA/QC program

Quality Assessment (QA)

Quality assessment refers to routine, periodic assessment of fish quality by a highly trained HSWRI staff person using a large enough sample size to represent the population in question. Data is recorded in a

12 dph Incubatorswim bladder assessment

~50 dph J1QA

~75 dph J2QA

~ 85 dphQC followed by QA

~ 90 dph – taggingQC followed by QA

DFG Pre-transportHealth Inspection

<30% not inflated ≥30% not inflated

<20%* ≥20%*

10-20% >20%*

<5%* ≥5%*

Cull all

<10%*

<5%* ≥5%*

Cull all orreassess

Cull all orreassess

Stop, retrain, repeat step 4

Stop, retrain, repeat step 5

1

2

3

4

5

6QA – detailed fish assessment, n=125QC – hand sort every fish and cull malformations* Affected with malformation

Legend


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data form (See Appendix a) and transferred to a database for subsequent analyses, including historical review.

The assessment of malformations in WSB has been attempted on white seabass in the larval stages to a limited degree using microscopy and clearing and staining. For routine QA, aside from assessing swim bladder inflation, working with larval fish is impractical because the larvae are too fragile and it is too time consuming. Based on our experience and current knowledge of bone development in white seabass, initiating the QA process at ~50dph (35mm TL; 0.5g) is appropriate. Identifying malformations in fish of this age still requires magnification using a compound microscope, so it is a relatively time consuming process. At subsequent QA check points, assessments can be made with the naked eye.

The quality assessment begins with a visual examination of the overall shape of the fish looking for any obvious deviations from what would be considered a normal looking white seabass at the size being examined. Abnormalities in the overall body shape are noted at this time and examples would include curvature of the spine, shortened body length, indentations along the dorsal or ventral midline and lesions along the length of the body. Following this initial series of observations, a more comprehensive examination is conducted beginning with the left lateral view of the fish, starting with the head region. The visual examination begins at the tip of the snout and follows the dorsal midline to the insertion of the dorsal fin noting abnormalities in the shape or skew of the head, protrusions along the dorsal ridge, and with the quality of the eye (soft tissue). The examination continues from the dorsal to ventral side along the opercles noting changes in the shape of the opercle or if the gills are exposed. The examination then proceeds along the ventral edge of the opercle proceeding anterior towards the snout noting changes to the shape of the opercle in this region or branchiostegals. Next the mouth is opened extending the maxillaries and the oralmandibular and orbital regions are examined noting abnormalities of the upper and lower jaws and suborbital areas. Next the pectoral, pelvic, anal and caudal fins are examined and abnormalities of the fin base, spines and rays are noted. The fish is then turned over viewed laterally on the right side and the visual examination is repeated. Depending on the size and condition of the fish this visual assessment usually lasts between 20 to 60 seconds.

Quality Control (QC)

Quality control refers to a sorting or culling process by hatchery staff with oversight from a highly trained HSWRI staff person. In QC, the entire cohort is sorted by hand one individual fish at a time, with poor quality fish being sorted out of the population and sometimes euthanized, depending on the overall quality of the fish. The goal of the QC program is to achieve a targeted quality threshold of 95% for fish leaving the hatchery. The 95% of fish leaving the hatchery have either perfect physical quality or minor physical differences, which are interpreted as variations of normal physical attributes previously identified in wild white seabass populations. Because this process is so labor intensive, it is conducted only as dictated by the QA findings immediately prior to tagging or during the tagging process (Table XX).

When conducted prior to tagging, the process QC starts with netting batches of fish from the culture tank into a long narrow trough containing 75 ppm Tricaine (MS-222) in seawater at a depth of about 100


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mm (4 inches). Usually two to four people wearing latex gloves are spaced around the trough and physically inspect, with their naked eyed, each fish for malformations and soft tissue defects. After the fish are lightly anesthetized the examiner removes a single fish from the trough. As they grasp the fish it is positioned in their hand so that the abdomen of the fish is resting in their palm with their fingers curled around the fish and their thumb placed on top of the fish’s head. The examiner looks carefully at the fish’s head, trunk and fins on both sides for obvious malformations (e.g. lower jaw, opercles, spinal). At this time they are also confirming soft tissue defects including eye or skin lesions. If any significant malformations or defects are observed the fish is sorted or euthanized. Asymptomatic fish are returned to the culture tank to recover from anesthesia. With this procedure, three to four examiners can inspect over 1,000 fish per hour.


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QUALITY ATTRIBUTES (see additional detail for each category below**)

Malformations (primarily hard tissue/bone)

Head (including orbital bones, operculum and jaws)


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1. Dorsal Head Indentation

Anatomical Description: Concave indent or curvature of the dorsal aspect of the head. When viewing the fish from a lateral profile, the dorsal midline, from the insertion of the dorsal fin to the snout, has a distinct indentation or notch unlike other individuals of similar size.

Other Nomenclature: microcephaly

Causative Factors: Unknown.

History: Identified during the 2011 WSB culture season; evident in Moser 1983.

Prevalence: Severe grades of this defect are very rare in the hatchery; mild and moderate grades are more common but they have also been observed in the wild.

Impact Factor: We believe the negative effect of this defect on survival is insignificant, supported by observations in among wild fish; Grade 2 and 3 animals are culled to stay within our best guess of the natural phenotypic range

Life Stage at Onset: Juvenile, approximately 60 dph

Species Affected: A. nobilis

References:

Grade1 – Straight line deviating from the normal dorsal curvature/arc, or almost

indistinguishable indentation of the dorsal head.

: 0 – No abnormality.

2 – Moderate concave curvature of a region of the dorsal head. SORT/CULL 3 – Severe concave curvature of a region of the dorsal head. SORT/CULL


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Grade 0 (Normal)

Grade 1

Grade 2 (Sort/Cull)

Grade 3 (Sort/Cull)

Example of variability in the wild

X-rays:


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Clearing and staining diagnostics:

Other species:


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2. Head Malformation

Anatomical Description: The head is skewed to the left or right when viewed from the dorsal profile.

Other Nomenclature: crooked head


History: .

Prevalence: Severe grades of this defect are rare in the hatchery

Impact Factor: We believe the negative effect of this defect on survival and aesthetics is significant, which is why we cull fish with any visible deviation of the head from the central axis.

Life Stage at Onset:


References:

Grade1 – Any noticeable deviation of the head when viewed dorsally. SORT/CULL



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Grade 0 (Normal)

Grade 1 (Sort/Cull)


X-rays:


Other species:


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3. Cranial Protuberances

Anatomical Description: Dorsal projections, horn-like in appearance, either unilateral or bilaterally on the head.

Other Nomenclature: horn head


History:

Prevalence: Severe grades of this defect are rare in the hatchery

Impact Factor: The effect of this defect on survival is unclear but moderate to severe protuberance(s) are unsightly, so all are culled

Life Stage at Onset: Juvenile – this has been detected in WSB as early as 32 dph.

Species Affected: A. nobilis; Salmo solar

References: Kent et al., 1987

Grade1 – When viewed from above, a small dorsal protuberance is noted unilaterally or bilaterally. SORT/CULL

: 0 – No abnormality identified.

2 – Moderate dorsal protuberance noted unilaterally or bilaterally. SORT/CULL 3 – Severe protuberance noted unilaterally or bilaterally. SORT/CULL.


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Grade 0 (Normal)

Grade 1 (Sort/Cull)

Grade 2 (Sort/Cull)

Grade 3 (Sort/Cull)


X-rays:


Other species:


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4. Pug Head

Anatomical Description: Cranio-caudal compression of the head and upper jaw, giving the appearance of a lower jaw protrusion.

Other Nomenclature:

Causative Factors: Excess dietary vitamin A, excess/absence of EPA and DHA fatty acids.

History:

Prevalence: This defect is relatively rare in the hatchery

Impact Factor: The effect of this defect on survival is likely negligible but moderate to severe pug head is unsightly, so they are culled

Life Stage at Onset: This malformation has been observed in WSB as early as 58 dph. Identified in early larval stages in other species.

Species Affected: D. labrax, S. aurata, A. nobilis, M. saxatilis

References: Tandler & Koven, Finefish malformation manual.

Grade1 – Mild shortening or compression of the maxillary region and dorsal head.


2 – Moderate shortening or compression of the maxillary region and dorsal head. SORT/CULL 3 – Severe shortening or compression of the maxillary region and dorsal head. SORT/CULL


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Grade 0 (Normal)

Grade 1

Grade 2 (Sort/Cull)

Grade 3 (Sort/Cull)


X-rays:


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Other species:

D. labrax, Photo ref# Morone saxatilis


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5. Upper Jaw

Anatomical Description: Misshapen or missing/inadequate development of bone and tissue comprising the normal conformation of the upper jaw. Malformations may include fusion (abbreviated as "f"), asymmetry ("a") or missing ("m") components of the maxilla or premaxilla.

Other Nomenclature:


History: .

Prevalence: Mild and moderate grades of this defect are more common in WSB.

Impact Factor: We believe the negative effect of this defect on survival is potentially significant relative to the ability of the fish to feed effectively; therefore fish in this category are culled

Life Stage at Onset:

Species Affected: A. nobilis, O. mykiss, E. coioides, L. lineata

References: Georgakopoulou et al., 2007; Cobcroft et al. 2001.

Grade1 – Minimal fusion of the pre-maxilla to maxilla, shortening of space between the two structures, or curvature of structures that may/may not alter ability to open mouth fully. SORT/CULL

: 0 – No abnormalities noted.

2 –Moderate fusion of the pre-maxilla to maxilla with or without distortion of the maxilla, or moderate curvature of premaxilla or maxilla that alters ability to open mouth fully. SORT/CULL 3– Complete fusion of the pre-maxilla and maxilla, where the pre-maxilla may not be identifiable, and distortion of the maxilla are evident; or curvature of structures causes severe distortion/inability to open mouth. SORT/CULL


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Grade 0 (Normal)

Grade 1 (Sort/Cull)

Grade 2 (Sort/Cull)

Grade 3 (Sort/Cull)

Grade 3 (Sort/Cull)

Grade 3 (Sort/Cull)


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Example of variability in the wild:

X-rays:


Other species:


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6. Lower Jaw

a. Lower Jaw Extension

Anatomical Description: Extension of the lower jaw well beyond the upper jaw, with no obvious deviation to the left or right.

Other Nomenclature:

Causative Factors: Unknown

History: .

Prevalence: Severe grades of this defect are rare in the hatchery; mild and moderate grades are more common but they have also been observed in the wild.

Impact Factor: We believe the negative effect of this defect on survival is relatively minor, supported by observations in among wild fish; Grades 2&3 animals are culled to stay within our best guess of the natural phenotypic range

Life Stage at Onset: Juvenile, larval?

Species Affected: A. nobilis, S. lalandi

References: Georgakopoulou et al., 2007; Cobcroft, et al., 2004

Grade1 – Lower jaw (mandible) extension is hardly noticeable beyond the upper jaw (maxilla) - Mild


2 – Lower jaw extension is noticeable but moderate – Moderate. SORT/CULL 3 – Lower jaw extension is obvious beyond the upper jaw (maxilla) – Severe. SORT/CULL


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Grade 0 (Normal)

Grade 0 (Normal)

Grade 1

Grade 1

Grade 2 (Sort/Cull)

Grade 2 (Sort/Cull)

Grade 3 (Sort/Cull)

Grade 3 (Sort/Cull)


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X-rays:


Other species:


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b. Lower jaw reduction

Anatomical Description: Incomplete development of the lower jaw involving angular/dentary bones.

Other Nomenclature:


History: .

Prevalence: Severe grades of this defect are rare in the hatchery; mild and moderate grades are more common

Impact Factor: The effect of this defect on survival is likely minor but combined with the aesthetic aspect, fish with moderate to severe lower jaw reduction are culled

Life Stage at Onset: Larval

Species Affected: S. aurata, D. labrax, A. nobilis.

References:

Grade1 – Lower jaw shortening is hardly noticeable with respect to the upper jaw – Mild.


2 – Lower jaw shortening is moderate but noticeable with respect to the upper jaw – Moderate. SORT/CULL 3 – Lower jaw shortening is obvious with respect to the upper jaw - Severe. SORT/CULL


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Grade 0 (Normal)

Grade 1

Grade 2 (Sort/Cull)

Grade 3 (Sort/Cull)


X-rays:


Other species:

S. aurata, Photo ref.#


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7. Crossbite

Anatomical Description: Lateral displacement of the lower jaw.

Other Nomenclature:

Causative Factors: Trauma, others unknown.

History: .

Prevalence: This defect is rare in the hatchery; mild and moderate grades have been observed in the wild.

Impact Factor: The effect of this defect on survival is unknown, but combined with the aesthetic aspect and concern for long-term feed impairment due to malalignment, fish with all levels of this malformation are culled.


Species Affected: S. aurata, D. labrax, A. nobilis

References:

Grade1 - Mild lateral displacement of lower jaw - Mild. SORT/CULL

: 0 - No abnormality detected.

2 – Moderate lateral displacement of the lower jaw - Moderate. SORT/CULL 3 – Severe lateral displacement of the lower jaw - Severe. SORT/CULL


34

Grade 0 (Normal)

Grade 0 (Normal)

Grade 1 (Sort/Cull)

Grade 1 (Sort/Cull)

Grade 2 (Sort/Cull)

Grade 2 (Sort/Cull)

Grade 3 (Sort/Cull)

Grade 3 (Sort/Cull)


35


X-rays:


Other species:


36

8. Orbital Malformation

Anatomical Description: Incomplete cartilage, bone, or skin formation at the ventral portion of the periorbital region.

Other Nomenclature: Abbreviated eye tissue (AET)


History: .

Prevalence: This defect has been observed commonly over the last year.

Impact Factor: The potential negative effect of this defect on survival is not known. The aesthetic effect is relatively minor. In the absence of more information, animals observed with the defect are culled.

Life Stage at Onset: Juvenile, larval?


References:

Grade1 – Lack of tissue surrounding the globe, which is hardly noticeable without the use of a dissecting scope. The tissue is seen to thin as it comes in contact at a central location. SORT/CULL

: 0 – No eyes are affected.

2 – Lack of tissue is observed grossly, creating a small gap, in the ventral peri-orbital region of one or both eyes. SORT/CULL 3 – Severe lack of tissue is easily observed in one or both eyes. SORT/CULL


37

Grade 0 (Normal)

Grade 1 (Sort/Cull)

Grade 2 (Sort/Cull)

Grade 3 (Sort/Cull)


X-rays:


Other species:


38

9. Malformed Operculum

Anatomical Description: A. Abbreviated (“ao”) - Incomplete development of the operculum unilaterally or bilaterally, usually with gill exposure evident. Abbreviation has been identified on the ventral (“v”), caudal (“c”) and dorsal (“r”) aspects of the operculum. B. Deformed (“do”) – a. Indentation of tissue between the preopercle to opercle. b. Ventral operculum exhibits an abnormal shape or flaring laterally.

Other Nomenclature:

Causative Factors: Low or lack of dietary EPA and DHA (fatty acids).

History: .

Prevalence: Opercular deformities are moderately common among cultured WSB

Impact Factor: The effect of this defect on survival is likely minor but the aesthetic aspect is considered significant, so fish with moderate to severe opercular malformations are culled

Life Stage at Onset: Early larval

Species Affected: S. aurata, D. labrax, O. mykiss, etc.

References: Galeotti, et al., 2000; Verhaegen, et al., 2007

Grade1– Opercular shortening evident with no gill exposure, with or without flaring identified - Mild.

: 0 – Operculum is normal in appearance and completely covers the gills.

2 – Opercular defect with up to 40% gill exposure or abnormal shape or indentation - Moderate. SORT/CULL 3 – Opercular defect with >40% gill exposure, gill erosion/discoloration noted or operculum not present - Severe. SORT/CULL

Subcategories

: Additional letters (noted above under anatomical description) will be added behind the grading to identify location and type of opercular malformation.


39

Grade 0 (Normal)

Grade 1

Grade 2 (Sort/Cull)

Grade 3 (Sort/Cull)


X-rays:


Other species:


40

10. Branchiostegal Malformation

Anatomical Description: A wave-like appearance of the branchiostegal rays, which may involve flaring, curling inward or outward of the rays as they develop. These are often, but not always, identified in conjunction with a malformed operculum. Subcategories used in data collection include “f” (flare), “i” (curled in), and “o” (curled out).

Causative Factors: Low developmental temperatures (150C) in D. labrax. Other causation unknown.

History: This malformation was first identified at the hatchery in 2012.

Prevalence:

Impact Factor: The potential negative impact of this defect on survival is unknown, though there is a significant negative aesthetic effect.


Species Affected: A. nobilis, D. labrax, S. aurata

References:

Grade

1 – Very mild flare or curling noted, hardly noticeable unless the fish is swimming in water. SORT/CULL

: 0 – Branchiostegal rays are normal in appearance.

2 - Small to moderate flare or curling noted, creating a very small gap where the rays should lie flat. SORT/CULL

3 - A large gap is noted, often exposing the gills, from the flare or curling that is evident. SORT/CULL


41

Grade 0 (Normal)

Grade 0 (Normal)

Grade 1 (Sort/Cull)

Grade 1 (Sort/Cull)

Grade 2 (Sort/Cull)

Grade 2 (Sort/Cull)

Grade 3 (Sort/Cull)

Grade 3 (Sort/Cull)


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11. Vertebral Axial Deviations

Anatomical Description: A. Kyphosis – dorsal or convex curvature of spine. B. Lordosis – Ventral or concave curvature of the spine. C. Scoliosis – S-shaped curvature of the spine, or bilateral bending of the vertebral axis.

Other nomenclature: kyphosis, lordosis, scoliosis

Causative Factors: Non-inflation of swim bladder, high velocity water currents, poor larval nutrition, unfavorable salinity and photoperiod conditions (lordosis); very high or low levels of DHA and EPA, dietary deficiency of vitamin C, tryptophan, parasites, toxins (scoliosis).

History: .

Prevalence: This defect is typically rare in the hatchery, although there have been periods where spinal deformities affected a significant percentage of the population; in addition to juveniles, this is one of the most common malformations observed in brood fish held in captivity

Impact factor: The potential negative effect of this defect on survival, as well as the aesthetic aspect, are considered significant, so fish with noticeable spinal deformities are culled.

Life Stage at Onset: Early larval

Species Affected: A. nobilis, many other species.

References:

Grade1 – Curvature deviations (ventral, dorsal or lateral) hardly noticeable when viewing the lateral line or fish as a whole – may require radiographs for diagnosis.

: 0 – No abnormality detected.

2 – Moderate deviation when viewing the lateral line or the fish as a whole. SORT/CULL 3 – Severe deviations when viewing the fish from any angle. SORT/CULL


43

Grade 0 (Normal)

Grade 1

Grade 2 (Sort/Cull)

Grade 3 (Sort/Cull)


Xrays:

Tissue removal, transmitted light:


44


Other species:


45

12. Vertebral Fusion

Anatomical Description: Fusion and/or shortening of vertebrae. Externally appears as though vertebrae may be missing, resulting in a compressed body like that of a perch.

Other Nomenclature: Stumpy spine


History: .

Prevalence: This defect is typically rare in the hatchery, although there have been periods where vertebral fusion affected a significant percentage of the population.

Impact Factor: The effect of this defect on survival is not clear, and the aesthetic aspects are often very subtle. Regardless, fish known to have fused vertebrae are culled.


Species Affected: Several marine and freshwater species.

References:

Grade1 – Shortening of the tail/spine is not obvious on external exam, but overall, the fish seems abnormal or shorter. Radiographs required to definitively diagnose. *RADIOGRAPHS IF IDENTIFIED IN SEVERAL INDIVIDUALS, CULL ENTIRE GROUP IF FUSION PRESENT. SORT/CULL


2 – Moderate shortening of the peduncle/spine is observed on external exam. SORT/CULL 3 – Severe shortening of the peduncle/spine is observed on external exam. SORT/CULL.


46

Grade 0 (Normal)

Grade 1 (Sort/Cull if confirmed radiographically)

Grade 2 (Sort/Cull)

Grade 3 (Sort/Cull)


47


X-rays:



Other species:


48

13. Malformed Fins

Anatomical Description: Caudal fin elements form abnormally, exhibit fusion and/or erosion.

Other Nomenclature:

Causative Factors: Unknown – possibly water temperature or larval rearing methods, disease.

History: .

Prevalence: Malformed fins can be relatively common among batches of hatchery fish, primarily as a result of “fin rot” caused by bacteria.

Impact Factor: The negative effect of this defect on survival is likely to be significant in severe cases relative to reduced mobility. Because fins regenerate, the malformation may be correctable in certain situations. However, until further long-term survival information is apparent and concern for aesthetic effects, this malformation is cullable.

Life Stage at Onset: early larval, juvenile

Species Affected: Many species.

References:

Grade/CategoryP2 – Pelvic fin malformation. SORT/CULL

: P1 – Pectoral fin malformation. SORT/CULL

A – Anal fin malformation. SORT/CULL C – Caudal fin malformation. SORT/CULL

*All categories are identified infrequently and culled. Malformations of the dorsal fin have not been observed.


49

P1 – Pectoral fin (Sort/Cull)

P2 – Pelvic fin (Sort/Cull)

A – Anal fin (Sort/Cull)

C – Caudal fin (Sort/Cull)

Example of variability in the wild: X-rays: Tissue removal, transmitted light: Clearing and staining diagnostics: Other species:

S. aurata, C, Photo ref#

NORMAL

MALFORMED


50

Other “defects” (primarily soft tissue)

14. Skin

Anatomical Description: This refers to pigment changes, abrasions or open ulcers.

Other Nomenclature:

Causative Factors: Trauma, disease, parasites, etc.

History: .

Prevalence: Skin damage can be relatively common among batches of hatchery fish, primarily as a result of abrasion and/or disease.

Impact Factor: The negative effect of skin defects on survival is likely to be significant in severe cases relative to reduced vitality. Fish with abrasions are allowed to recover or, in severe cases of skin lesions, the fish are culled.

Life Stage at Onset: Any

Species Affected: All species.

References:

Grade1 – Darker or lighter pigmentation compared to normal. High amount of mucus on skin or scale loss identified.

: 0 - Normal coloration and scale numbers

2 – Abrasion present or greater than 40% scale loss 3 – Open, ulcerative lesions. SORT/CULL


51

Grade 0

Grade 1

Grade 2 Grade 3 (Sort/Cull)


X-rays:



Other species:


52

15. Cloudy Eye

Anatomical Description: Opaque discoloration to any layer of one or both eyes.

Other Nomenclature: Perch spine, stumpy spine

Causative Factors: Trauma, chemical burns, disease, developmental abnormalities.

History: .

Prevalence: Cloudy eye can be relatively common among WSB in the hatchery.

Impact Factor: The negative effect of this defect on survival is likely very significant in moderate to severe cases, so fish graded at these levels are culled.


Species Affected: A. nobilis, many species.

References: Smiley

Grade1 – Only one eye is affected with a faint cloudy-white discoloration, less than 10% of the eye demonstrating the lesion.


2 – 10-40% of one eye is affected/discolored, regardless of intensity. SORT/CULL 3– >40% of one eye or both eyes affected, regardless of intensity. SORT/CULL


53

Grade 0 (Normal)

Grade 1

Grade 2 (Sort/Cull)

Grade 3 (Sort/Cull)


54

16. Bubble eye

Anatomical Description: Corneal or intraocular air bubbles observed.

Other Nomenclature: emphysema of the eye

Causative Factors: Supersaturation

History:

Prevalence: Bubble eye can be relatively common among WSB in the hatchery.

Impact Factor: The negative effect of this defect on survival is likely significant in severe cases, so in the absence of corrective measures, fish having this malformation are culled.

Life Stage at Onset: larval, juvenile, adult

Species Affected: A. nobilis, others.

References: Smiley

Grade1 – Gas in cornea, anterior or posterior chambers of one eye, with less than 10% of the globe affected. SORT/CULL


2 – Gas in cornea, anterior or posterior chambers of one eye, with 10% to 50% affected, or both eyes affected at any degree. SORT/CULL 3 – Gas in the cornea, anterior or posterior chambers of one eye, with 50-100% of the eye affected. SORT/CULL


55

Grade 0 (Normal)

Grade 1 (Sort/Cull)

Grade 2 (Sort/Cull)

Grade 3 (Sort/Cull)


56

17. Popeye

Anatomical Description: One or both eyes are noticeably distended and appear to be “popping-out” of the bony sulcus.

Other Nomenclature: Exophthalmos

Causative Factors: Supersaturation, trauma, mass development behind the eye.

History: .

Prevalence: Popeye can be relatively common among WSB in the hatchery.

Impact Factor: The negative effect of this defect on survival is likely significant in severe cases, so fish at all levels are culled since corrective measures are not possible.

Life Stage at Onset: Larval and juvenile

Species Affected: A. nobilis, many others.

References: Smiley

Grade1 – One eye slightly bulges from socket when viewed from the top-down. SORT/CULL


2 – One eye bulges noticeably from socket when viewed from any angle. SORT/CULL 3 – Torn tissues identified of one eye or both eyes affected at any degree. SORT/CULL


57

Grade 0 (Normal)

Grade 1 (Sort/Cull)

Grade 2 (Sort/Cull)

Grade 3 (Sort/Cull)


58

18. Gills

Anatomical Description: Abnormal color deviation, parasites, hyperplasia or fusion of the lamellae.

Other Nomenclature:

Causative Factors: Poor water quality, anemia, parasites, etc.

History: .

Prevalence: Severe grades of this defect are very rare in the hatchery

Impact Factor: The negative effect of this defect on survival is likely significant in severe cases, so fish with a severe gill defect are culled.


Species Affected: All species.

References:

Grade1 – Pale (anemia)

: 0 – Normal coloration and lamellar structure

2 – Clubbed (hyperplasia, thickening of tissue), frayed at tips or nodules present. 3 – Lamellar fusion, heavy parasite load, hemorrhaging or necrotic patches of tissue. SORT/CULL


59

Grade 0 (Normal)

Grade 1

Grade 2

Grade 3 (Sort/Cull)


60

19. Fin Erosion

Anatomical Description: Caudal fin elements exhibit fusion, erosion or hemorrhage.

Other Nomenclature:

Causative Factors: Poor water quality, disease.

History: .

Prevalence: Fin erosion can be relatively common among batches of hatchery fish, primarily as a result of “fin rot” caused by bacteria.

Impact Factor: The negative effect of this defect on survival is likely to be significant in severe cases relative to reduced mobility. Because fins regenerate, the defect may be correctable in minor cases. Therefore, moderate and severe cases are culled.

Life Stage at Onset: early larval, juvenile

Species Affected: Many species.

References:

Grade1 - Discolored, frayed, clubbed with erosion of <30% normal length.

: 0 – No abnormality noted.

2 – Clubbed, frayed with erosion >30% normal length +/- hemorrhage. SORT/CULL 3 - Eroded to base with hemorrhaging. SORT/CULL


61

Grade 0 (Normal)

Grade 1

Grade 2 (Sort/Cull)

Grade 3 (Sort/Cull)


62

BEST MANAGEMENT PRACTICES

• Provide employee training as per the quality assessment/control guidelines. • Edit guidelines annually, as indicated by assessments and observations of fish quality within the

hatchery. • Continue to document new and existing malformations and lesions via data collection and

photographs. • Assess critical control points regularly and identify areas where additional control points may be

necessary. • Consistently achieve a minimum of 95% threshold following the steps in the QA/QC Flow Chart • Perform quality assessments (n=125 fish samples) at 50-55 dph, 75-80 dph. • Perform a detailed health/quality assessment on any crop assessed at greater than 10% culling

rate • Hold fish under good water quality conditions to minimize defects. • Provide fish with the best possible nutrition to minimize defects. • Minimize stress and damage associated with handling and high stocking densities.


63

THE ROLE OF RESEARCH

To apply the best available science to define and identify the etiology, to the extent practical, of malformations, deformities and soft-tissue lesions occurring in cultured white seabass so that their prevalence is reduced or eliminated in this stock replenishment program. These are best achieved by:

1. Maintain vigilance with each cohort as they develop to identify problems arising at their earliest onset.

2. As problems arise, document culture practices leading up to the occurrence to help identify potential patterns or trends resulting in the occurrence.

3. Conduct applied research, to the extent practical, to help understand the etiology of malformations.

4. Maintain working relationships with others involved in finfish malformations (i.e. universities, industry).


64

GLOSSARY OF DEFINITIONS

Caudal - at or near the tail Cranial - on the head area Deformity - a deformity, dysmorphism, or dysmorphic feature is a major difference in the shape of body part or organ compared to the average shape of that part. Often referred to a change in a structure that is already formed. Euthanized - ending of life in a painless manner Haemal arch -a bony arch on the underside of a tail vertebra of a vertebrate. Hyperoxic - elevated concentrations of oxygen. The result of breathing elevated concentrations of oxygen is hyperoxia, an excess of oxygen in body tissues. Hypoxic - reduced concentration of oxygen. is a pathological condition in which the body as a whole (generalized hypoxia) or a region of the body (tissue hypoxia) is deprived of adequate oxygen supply. Kyphosis - axial /\ - shaped deviation seen from a lateral view, a dorso-ventral bending of the spine. Lateral - of or pertaining to the side Lordosis - axial \/ - shaped deviation seen from a lateral view, a dorso-ventral bending of the spine. Malformation - a deformity in the shape or structure of a part, especially when congenital. Often induced during embryonic and early juvenile development as the structures are formed. Morphogenesis - the development of the form or structure of an organism during the life history of the individual Notochord - the longitudinal axial support (skeleton) of the embryos of all chordates, which lies ventral to the nerve cord and dorsal to the alimentary canal. Remnants of the notochord usually remain in the adult between the vertebrae, which come to surround it. Ossification - the process of the synthesis of bone from cartilage. There are two types of ossification—intramembranous and endochondral ossification. Pughead - various degrees of a malformation of the snout in fish where there is a reduction of the snout leaving the lower jaw protruding beyond the upper jaw. Reduction of the front skull and upper jaw bones, anterior – posterior compression of etmoid region and upper jaws, malformation in the maxillaries, pre-maxillaries, parassphenoid and etmoid plate.


65

Scoliosis - axial deviation, S shaped vertebral curvature from a dorsal view, sideways bending of the spine. Specific growth rate (SGR) - SGR (%)=100*(ln. final weight of fish – ln. initial weight of fish)/number of days Stargazer - axial deviation,\/ shaped curvature in the neck Surface skimmer - device for removal of organic film in the water surface Weaning - gradual transfer from live feed to formulated feed (pellets)


66

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Divanach, P., Papandroulakis, N., Anastasiadis, P., Koumoundouros, G., Kentouri, M., 1997. Effect of water currents on the development of skeletal deformities in sea bass (Dicentrarchus labrax L.) with functional swimbladder during postlarval and nursery phase. Aquaculture 156: 145-155.

Favaloro, E. and Mazzola, A. 2003. Meristic variation and skeletal anomalies of wild and reared sharpsnout seabream juveniles (Diplodus puntazzo, Cetti 1777) off coastal Sicily, Mediterranean Sea. Aquaculture Research 34: 575-579.

FineFish Project. “Manual on Control of Malformations in Fish Aquaculture.” Web. 15 Nov. 2011. http://www.aquamedia.org/finefish/manual/manual_en.asp

Hall, Lisa A., unpublished data. Developmental Osteology of the White Sea Bass, Atractosion nobilis (Pisces: Sciaenidae), with the Description of the Chondrocranium, Osteocranium, and Pectoral Girdle Regions.

Galeotti, M., P. Beraldo, S. de Dominis, L. D’Angelo, R. Ballestrazzi, and R. Musetti. 2000. A preliminary histological and ultrastructural study of the opercular anomalies in gilthead sea bream larvae (Sparus aurata). Fish Physiology and Biochemistry 22:151-157.

Georgakopoulou E., A. Angelopoulou, P. Kaspiris, P. Divanach, and G. Koumoundouros. 2007. Temperature effects on cranial deformities in European sea bass, Dicentrarchus labrax (L.). Journal of Applied Ichthyology 23:99-103.

Izquierdo, M.S., Socoroo, J., and Roo, J. 2010. Studies on the appearance of skeletal anomalies in red porgy: effect of culture intensiveness, feeding habits and nutritional quality of live preys. Journal of Applied Ichthyology 26: 320-326.

Kent, M. L., S. R. Wellings, W. T. Yasutake, and R. A. Elston. 1987. Carnial nodules associated with cranial fenestrae in juvenile Atlantic salmon, Slamo salar L. Journal of Fish Disease 10:419-421.


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Mazurais, D., Darias, M.J., Gouillou-Coustans, M.F., LeGall, M.M., Huelvan, C., Desbruyeres, E., Quazuguel, P., Cahu, C., Zambonino-Infante, J.L. (2008). Dietary vitamin mix levels influence the ossification process in European sea bass (Dicentrarchus labrax) larvae. Am. J. Physiol. Regul. Integr. Comp. Physiol. 294: R520-R527.

Mazurais, D., Glynatsi,N., Darias,M.J., Christodoulopoulou,S., Cahu,C.L., Zambonino-infante, J.L., Koumoundouros, G. (2009). Optimal levels of dietary vimtain A for reduced deformity incidence during development of European sea bass larvae (Dicentrarchus labrax) depend on malformation type. Aquaculture 294: 262-270.

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Sandel, E., Nixon, O., Lutzky, S., Ginsbourg, B., Tandler, A., Uni, Z., Koven, W., 2010. The effect of dietary phosphatidylcholine/phosphatidylinositol ratio on malformation in larvae and juvenile gilthead sea bream (Sparus aurata). Aquaculture 304: 42-48.

Smiley, J., Okihiro, M., Drawbridge, M., Kaufmann, R., 2012. Pathology of Ocular Lesions Associated with Gas Supersaturation in White Seabass (Atractoscion nobilis). Journal of Aquatic Animal Health 24(1): 1-10. Verhaegen, Y., D. Adriaens, T. De Wolf, P. Dhert, and P. Sorgeloos. 2007. Deformities in larval gilthead sea bream (Sparus aurata): A qualitative and quantitative analysis using geometric morphometrics. Aquaculture 268:156-168.

Villeneuve, L., Gisbert, E., Moriceau, J., Cahu, C.L., Zambonino-Infante, J.L., 2006. Intake of high levels of vitamin A and polyunsaturated fatty acids during different developmental periods modifies the expression of morphogenesis genes in European sea bas (Dicentrarchus labrax). Brit. J. Nutr. 95: 677-687.

Villeneuve, L., Gisbert, E., Le Delliou, H., Cahu, C.L., Zambonino-Infante, J.L., 2005. Dietary levels of all-trans retinol affect retinoid nuclear receptor expression and skeletal development in European sea bass larvae. Brit. J. Nutr., 93: 791-801.


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APPENDIX A – DATA FORM FOR QA


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