The effect of triploidization of Atlantic cod (Gadus morhua L.) on survival, growth and deformities during early life stages Academic research paper on "Animal and dairy science"

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The effect of triploidization of Atlantic cod (Gadus morhua L.) on survival, growth and deformities during early life stages

Ingegjerd Opstad a* Per Gunnar Fjelldal b, 0rjan Karlsen a, Anders Thorsen c, Tom J. Hansen b, Geir Lasse Tarangerc

a Institute of Marine Research, Austevoll Research Station, NO-5392 Storeb0, Norway b Institute of Marine Research, Matre Research Station, NO-5984 Matredal, Norway c Institute of Marine Research, PO Box 1870, NO-5817 Bergen, Norway

ARTICLE INFO ABSTRACT

This study investigated the performance of triploid Atlantic cod (Gadus morhua L.) produced in an intensive feeding system. Eggs pooled from three females fertilized with sperm from two males were either triploidized by hydrostatic pressure (58,600 kPa, 5 min, 6 °C) 30 min post-fertilization, or were maintained as untreated controls, and then reared under identical standard protocols until day 220 post-hatch. Mortality and growth were monitored from start-feeding while vertebral deformities were assessed by radiology on day 220 post-hatch. The triploid cod had lower survival than the diploid group from start-feeding and during early on-growing. On day 53, there was a significantly higher number of deformed fish among the triploid than the diploid cod. There was also a significantly higher prevalence of fish with one or more deformed vertebrae and prevalence of lordosis in triploids than diploids. The triploid group weighed significantly more than the diploid group before onset of feeding. The results of this study, which is the first that deals with skeletal deformities in triploid farmed Atlantic cod, are in line with previous findings that triploid teleosts are more prone to develop bone deformities. However, the mechanisms underlying this phenomenon are, still unknown.

Published by Elsevier B.V.

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Article history:

Received 29 April 2011

Received in revised form 19 September 2012

Accepted 11 January 2013

Available online 22 January 2013

Keywords: Triploid Diploid Cod

Deformities

Survival

Growth

1. Introduction

The production of farmed Atlantic cod (Gadus morhua) has greatly expanded in the course of the past few years, and cod is now rated as Norway's third most important aquaculture species after Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Increased production has led to growing numbers of escapees (Moe et al., 2007), and several studies have found farmed cod on local spawning grounds (Meager et al., 2009; Svasand, 1998). The stock of Norwegian coastal Atlantic cod consists of genetically distinct local population (Sarvas and Fevolden, 2005). Hybridization between escaped farmed and wild salmon has resulted in genetic introgression and fitness depression in wild populations (Araki et al., 2007; McGinnity et al., 2003). There is therefore some concern that cod escapees will hybridize with local cod, thereby increasing the likelihood of genetic introgression (Bekkevold et al., 2006). A further problem is that, farmed cod spawn in the net pens, releasing fertilized and viable offspring to the environment (Jorstad et al., 2008). Furthermore, early unwanted sexual maturation is a major problem in cod farming, since this process reduces appetite, growth and feed conversion efficiency, and can increase mortality (Karlsen et al., 2006; Kjesbu et al., 2006; Taranger et al., 2006, 2010).

* Corresponding author. Tel.: +47 9124 1705.

E-mail address: ingeggjerd.opstad@imr.no (I. Opstad).

0044-8486/$ - see front matter. Published by Elsevier B.V. http://dx.doi.org/! 0.1016/j.aquaculture.2013.01.015

The use of triploid farmed cod may solve these problems, as they are functionally sterile and their gonadal development is less (Feindel et al., 2011; Peruzzi et al., 2009; Trippel et al., 2008). As in other teleosts that have been studied, hydrostatic pressure on newly fertilized eggs is the best method to induce triploidy in Atlantic cod (Peruzzi et al., 2007; Trippel et al., 2008). Trippel et al. (2008) produced triploid Atlantic cod by hydrostatic pressure and found no sexually mature triploid females, compared to 90% mature fish among diploid females. The same study showed that 12.5% of the triploid males were mature, compared to 55% of the diploid males. Peruzzi et al. (2009) found that mature triploid male Atlantic cod spawned with diploid females, but that the resultant larvae all suffered severe deformities and died before the onset of exogenous feeding. In combination with all-female production, therefore triploid cod could be an efficient means of avoiding genetic introgression with wild populations as well as mitigating production performance problems due to early sexual maturation (Taranger et al., 2010). Substantial research has been done on the production performance of triploids of various teleosts (Piferrer et al., 2009). However, in Atlantic salmon, for example, observations of lower survival and growth rates (Cotter et al., 2002), and a higher prevalence of skeletal deformities (Fjelldal and Hansen, 2010) have limited the use of triploid salmon in commercial faming. The aim of this experiment was therefore to investigate the effect of triploidization induced by hydrostatic pressure on the growth, survival and development of deformities from the egg stage to the juvenile stage in Atlantic cod.

2. Material and methods

The experiment was approved by the Norwegian Animal Research Authority and performed according to current animal welfare regulations.

2.1. Broodstock, gamete collection and production oftriploid eggs

The experiment was carried out at the Institute of Marine Research, Austevoll Research Station. Eggs were obtained by stripping farmed coastal Atlantic cod raised and held at Austevoll. A total of 800 ml eggs from three females were pooled and fertilized with sperm from two males. The fertilized eggs were separated into two groups, one of which was kept as untreated controls, while the other was subjected to a hydrostatic pressure of 58,600 kPa (8500 psi) for 5 min (TRC-APV, Aqua Pressure Vessel, TRC Hydraulics inc., Dieppe, Canada) when they were 180 minute degrees (i.e., 30 min post-fertilization at 6 °C) according to the protocol developed by Trippel et al. (2008).

2.2. Egg incubation

One hundred eggs from each group were checked for fertilization under a binocular microscope. The fertilization rate was 94% in the triploid group and 91% in the diploid group. The fertilized eggs were incubated and hatched in 70 l flow through tanks (van der Meeren et al., 2007) at 5.8 ±0.1 °C. Dead eggs (sinking) were removed and the amount was measured every day. The number of larvae was estimated by tube sampling in the incubators at the transfer to the experimental tanks on day 3 after hatching.

2.3. Startfeeding and weaning

The larvae were transferred to six 500 l tanks at 8 °C, three days after hatching. Approximately 25,000 larvae were transferred per tank with green water (30 ml Algae paste; Nannochloropsis, MicroAlgae AS). The flow rate was 0.6 l min-1 on the first day and then increased to 1 l min-1 on the following day. From day four post-hatch, the larvae were fed rotifers (Brachionus plicatilis) enriched with Rotimac (BioMarine AquaFauna Inc., California, USA). The temperature was gradually increased to 12 °C over a period of 10 days, and kept at 12 °C with a light intensity of400 |j,W cm-2, and a continuous light regime. The flow rate was gradually increased to 8 l min-1 on day 60. The larvae were fed most of their live feed at 11:00 and 15:00 daily, in addition to a continuous supply of live feed (rotifers and algae paste or Artemia) pumped from header tanks. The supply of rotifers was gradually increased as the fish grew; on day 30 post hatch, 60 ml algal paste and 45 million rotifers were supplied to each tank. Algal paste was added only while rotifers were added to the tank. On day 31 the fish were co-fed rotifers and Artemia, enriched with Larviva Multigain (Dana Feed A/S) for three days, and followed by only Artemia until day 45. From day 45, the larvae were weaned onto a commercial dry diet (Aglonorse, 500-700 mm), and co-fed Artemia and dry feed for seven days. They were fed the dry feed by hand twice a day, in addition to automatic feeding every hour. After weaning on day 53, severely deformed fish were counted as dead and removed and numbers reduced to 2250 in each tank. The tanks were cleaned and dead individuals were removed and counted daily from day 54. Salinity was 34 ± 0.5 ppt and oxygen saturation in the outlet water ranged from 96 to 100%.

On day 87 the fish were graded into two size groups per ploidy in order to avoid cannibalism. Each ploidy size group was then transferred to individual outdoor tanks (3 m diameter, 7 m3). The cod were on-grown for 4.5 months to reach a size, were the fish can be radio-graphed and blood-sampled. Subsamples were taken of all tanks on day 220.

2.4. Sampling and measurements

Dry weight was determined on days 3,31 and 53. Larvae were anesthetized with MS222 (Argent Chemical Laboratories Inc., Redmond, WA, USA) washed in distilled water, frozen (- 20 °C), freeze-dried and weighed to the nearest 1 |ag. On days 87 and 220 the fish were anesthetized with Benzocain (Unikem, DK-1503 Kobenhavn) and weighed.

2.5. Validation of triploidization on blood samples

Ploidy was validated on the basis of the red blood cells in triploid fish being larger than in diploids (Peruzzi et al., 2005). Blood was withdrawn from the caudal vasculature of 49 diploid and 96 triploid fish on day 220 post-hatch (Table 1) using heparinized syringes. One drop of blood was placed on a microscopic slide, carefully smeared out using the end of a thin glass and a cover slip added. Within 5 min from of sampling, the microscopic slide was photographed. From each slide three none overlapping pictures (8.733 pixels/^m) were taken at 40x magnification using a 1.4 MP camera (CFW 1308c, http://www. scioncorp.com) mounted on a microscope (Carl Zeiss Axioplan). The images were taken from an area on the slide where the blood cells were not densely packed. From the three images at between 20 and 24 blood cells were analyzed using the open-source image analysis program ImageJ (http://rsb.info.nih.gov/ij) and a custom-made macro. Usually the software randomly detected a large proportion of the blood cells in each image, which then were automatically outlined and measured. Images were then visually checked and obviously incorrect cell outlines and their measurements were deleted. Incorrect measurements were usually caused by cell damage, measurements of cells other than red blood cells, or two red blood cells overlapping each other. In cases with suboptimal image quality (e.g., blood cells packed too densely) automatic measurements were supplemented or replaced by manual measurements in ImageJ. The parameters measured were area, perimeter, circularity (4-n-Area/Perimeter2), and major and minor axis of the best fitting ellipse.

2.6. External deformities

At the end of weaning there was high mortality in the tanks containing triploid fish. Fish with severe deformities were found floating and dying. Therefore all fish were counted on day 53 and checked for severely visible external deformity, removed from the tanks and counted as dead. At that size it is impossible to distinguish between different types of deformities.

Table 1

Size and shape of blood cells for the diploid and triploid groups of cod.

Treatment Variable Area Perimeter Major Minor Roundness

axis axis

Diploid Mean 85.22 38.28 13.25 8.20 0.74

N 49 49 49 49 49

SD 6.94 2.40 0.51 0.46 0.06

Minimum 73.58 34.79 12.37 7.42 0.60

Maximum 100.83 44.21 14.41 9.16 0.81

95% CI lower 83.22 37.59 13.10 8.07 0.72

95% CI upper 87.21 38.97 13.39 8.33 0.76

Triploid Mean 114.05 42.96 15.44 9.42 0.78

N 96 96 96 96 96

SD 6.00 1.35 0.68 0.33 0.02

Minimum 97.71 39.12 13.07 8.60 0.71

Maximum 131.82 45.87 16.90 10.23 0.85

95% CI lower 112.84 42.69 15.30 9.35 0.77

95% CI upper 115.27 43.24 15.58 9.48 0.78

2.7. Radiography and evaluation of vertebral deformities

Radiographs were taken on day 220 using a portable X-ray apparatus (HI-Ray 100, Eickenmeyer Medizintechnik für Tierärzte e.K., Tuttlingen, Germany) and 30x40 cm sheet film (FUJIFILM IX 50, FUJIFILM Corp., Tokyo, Japan). The film was exposed twice at 50 mA s and 72 kV, and developed using a manual developer (Cofar Cemat C56D, Arcore (MI), Italy) with Kodak Professional manual fixer and developer (KODAK S.A., Paris, France). The pictures were digitized by scanning (Epson Expression 10000 XL, Seiko Epson Corp., Nagano-Ken, Japan). The vertebral column of each fish was thoroughly examined (Adobe Photoshop CS2). The number of affected vertebrae and the type of deformity, such as neck curvature and lordosis were determined according to Fjelldal et al. (2009a). Individuals with one or more deformed vertebral bodies were classified as deformed.

2.8. Statistics

Data were analyzed using Statistica (version 8.0, Statsoft, Tulsa, U.S.A.) and Stata (version 11, http://www.stata.com). The significance level was P<0.05. Significant differences in the frequency of fish with one or more deformed vertebrae, and of fish with neck curvatures or lordosis between diploids and triploids were tested by Chi-square tests. The percentages of external deformity and survival were arcsine transformed and analyzed by ANOVA, followed by post hoc comparisons using the Tukey HSD procedure. Dry weights on day 3 were tested by the Mann-Whitney U-test. Blood cell diameters, weight on days 31, 53, 87, were compared using nested ANOVA, where tanks were nested under ploidy, followed by the Tukey HSD post hoc test.

3. Results

3.1. Blood cell sizes

The mean blood cell areas were significantly higher (nested ANOVA, P<0.05) in triploids than diploids (Fig. 1). Triploids had blood cells with an average area of 114 |am2, while those of diploids averaged 85 |jm2 (Table 1). There was a small overlap in mean areas of individuals between the ploidies (Table 1). The maximum value for diploids was 101 |jm2 while the minimum value for triploids was 98 |jm2. For the other size parameters (minor axis, major axis, perimeter) similar trends were found (Table 1).

3.2. Survival

The survival rate of eggs during incubation was high in both groups (triploids: 81.4%, diploids: 71.5%). From startfeeding and through weaning (days 3-53), the survival of diploids (mean 39.3 ± 12.7%) was significantly higher (P<0.001) than that of triploids (mean 22.6±5.4%). During early on-growth (days 53 to 87), survival was still significantly higher (P<0.01) in diploids (mean 94.9±3.7%) than in triploids (mean 69.3 ±7.8 %). After grading the fish (day 87) and until the end of the experiment (day 220), survival was 99% in both the diploid groups and 91 and 96% in the two triploid groups.

3.3. Deformities

Fig. 1. Size frequency histograms of blood cells from triploid and diploid fish. N = 2 (96 triploid fish and 49 diploid fish were investigated).

was no effect of ploidy on the prevalence of neck curvature (Table 2). The predominant location for vertebral deformities was in the region between vertebrae numbers 1 and 4 in diploids, and between vertebrae numbers 1 and 4, and 21 and 33 among the triploids (Fig. 2). Vertebra number 2 was the most often affected in the first region in both ploidies, and number 23 the most often vertebra in the second region in triploids. Vertebra number 2 is within the neck curvature region (Fig. 3A and B), while vertebra number 23 is within the lordosis region (Fig. 3C and D).

3.4. Growth

On day 3 after hatching, before startfeeding, the dry weight of diploids was significantly lower (Mann-Whitney U-test, P<0.001) than that of triploids (Table 3). When the diet was shifted from rotifers to Artemia on day 31, triploids tended to have higher dry weights

At the end of weaning, on day 53, the triploid group included significantly higher numbers of fish with deformities (P = 0.001, Table 3).

3.3.1. Vertebral deformities

Analysis of the radiographs on day 220 showed that there was a significantly higher (Chi-square, P = 0.002) prevalence of triploids with one or more deformed vertebrae. Lordosis was significantly higher in triploids than diploids (Chi-square, P<0.001), while there

Table 2

The prevalence (%) of fish with one or more deformed vertebrae, and of fish with neck curvatures or lordosis among diploid and triploid cod on day 220 post hatch (n = 100; 50 per ploidy).

Radiological deformities (%) Diploid Triploid P-value

Neck curvature 40 56 0.109

Lordosis 2 42 < 0.001

Deformed vertebrae 42 72 0.002

A 1 2 3

than the diploids (nested ANOVA, P = 0.078, Table 3). On day 53, after end of weaning, there was no significant difference in weight. During early on growing, on day 87 (nested ANOVA, P = 0.16) (Table 3), the differences were still not significant. On day 220 the small diploid group weighted 37.7 ± 12.4 g and the large diploid group 40.2 ± 12.2 g and the small triploid groups 28.1 ± 8.5 g and the large triploid group 41.1 ±13.8 g.

4. Discussion

The present study reports the successful production of triploid Atlantic cod by the use of high hydrostatic pressure shortly after fertilization. The size of the red blood cells indicates that the method employed to triploidize cod was effective, and in accordance with the results obtained by Trippel et al. (2008). The method seems to be more efficacious than either heat or cold shock on cod eggs, as described by Peruzzi et al. (2007).

4.1. Survival

The high fertilization rate in both groups and the high survival rates during incubation indicate that the egg groups were of good quality (Kjorsvik et al., 2003). Survival during incubation was slightly higher in the triploids than the diploids, but after hatching and until the fish were graded and moved to larger tanks, the triploid cod suffered higher mortality than the diploids. No comparative data on pressure-treated Atlantic cod has been reported, but triploidization of Atlantic cod using heat shocks significantly reduced survival rates (Peruzzi et al., 2007). Peruzzi et al. (2004) found that triploidization by hydrostatic pressure increased mortality during early development and the larval stages in sea bass. Comparisons of shocked trip-loid and diploid fish have shown that the shock is the main factor causing early mortality (Cherfas et al., 1994), while the ploidy status may affect survival later on (Piferrer et al., 2009).

4.2. Deformities

The proportion of triploid fish classified externally as deformed at the end of weaning was far higher than among the diploids. Lordosis was far more evident in the triploid group, and the prevalence offish with one or more deformed vertebrae was significantly higher in

Fig. 3. Lateral radiographs of Atlantic cod with normal neck (A), curved neck (B), normal vertebral column (C) andlordosis (D) at the termination of the experiment on day 220. Vertebral number is indicated with arrows and numbers on neural arches. Scale bar=1.0 cm.

Fig. 2. Location of vertebral deformities at the termination of the experiment on day 220. Prevalence (%, mean ± standard error) of deformed vertebrae in different parts of the vertebral column. N = 2 per ploidy, which represents the prevalence of deformed vertebrae along the vertebral column in each replicate tank 25 fish were investigated in each tank.

Table 3

Dry weight on days 3, 31, 53, wet weight on day 87, and deformities on day 53. Mean values with standard deviation.

Days post hatch Diploid Triploid P-value

3 Dry weight (mg) 0.089 ±0.016 0.098 ±0.012 <0.001

31 Dry weight (mg) 1.022 ±0.172 1.196±0.235 0.078

53 Dry weight (mg) 15.4 ±6.4 12.6 ±5.9 0.422

53 Deformity (%) 0.1 ±0.1 11.8 ± 1.9 0.001

87 Wet weight (g) 2.1 ±0.7 1.6 ±0.6 0.160

triploids. Although Fjelldal et al. (2009a) provided a detailed description of the radiological pathogenesis of this disorder in cod, the exact region where lordosis develops has not been defined. Our study shows that this region is between vertebrae 21 and 33 in triploid cod. It is likely that the lordosis is induced in vertebra number 23, since this is the most frequently affected vertebra. This vertebra is located in the anterior part of the caudal region of the vertebral column, near the transition zone between the abdominal and caudal region (authors unpublished results). This region may be where the compression force imposed by the lateral muscle during swimming is large. Lordosis in the caudal region of the vertebral column has also been observed in cultured sea bream (Sparus auratus) and sea bass (Dicentrarchus labrax), where it has shown to be induced by high swimming activity (Kihara et al., 2002). In Atlantic salmon, low mineral content and mechanical strength are associated with the development of compressed morphology of the vertebral bodies (Fjelldal et al., 2007). The development of lordosis in cultured cod seems to start with vertebral compression (Fjelldal et al., 2009a). The high prevalence of lordosis in intensively cultured triploid cod may be therefore related to a soft bone structure. Factors that may influence the development of vertebral deformities in gadoids include source of marine protein in the weaning diet (Opstad et al., 2006), vitamin K (Roy and Lall, 2007), dietary levels of phosphorus (B^verfjord et al., 1998; Burke et al., 2010; Fjelldal et al., 2009b; Roy and Lall, 2003; Roy et al., 2002). Furthermore, Kousoulaki et al. (2010) found that dietary P had a strong influence on bone mineralization in cod. Fjelldal and Hansen (2010) studied the pathogenesis of vertebral deformities in triploid Atlantic salmon smolts, and suggested that trip-loids may have different dietary requirements than diploids.

The prevalence of fish with neck curvatures studied by radiography did not differ between diploids and triploids, and was within the range already reported in intensively cultured diploid cod (Fjelldal et al., 2009a; Kousoulaki et al., 2010). Intensively cultured cod are more prone to develop curvature of the cranial region of the vertebral column than extensively cultured (Fjelldal et al., 2009a), but the etiology of this deformity remains uncertain. The type of live feed used during intensive start feeding may affect the occurrence of deformities in cod (Imsland et al., 2006). Whether triploid and diploid cod have different dietary requirements and how the juvenile production regime (extensive vs. intensive production) affects the development of vertebral deformities are questions that remain to be studied. There was no ploidy effect on the proportion of fish with radiological detectable neck deformities.

4.3. Growth

The triploid larval cod had higher dry weight before onset of feeding. During the rotifer feeding period the triploid group tended to weigh more. However, after the diet shift to Artemia, and at the end of the weaning period, the diploid group, tended to be heavier, although not significantly so. The behavior of diploid and triploid fish has been shown to differ, e.g. as unusual swimming and feeding behavior in trip-loid rainbow trout (Myers and Hershberger, 1991; Solar et al., 1984). There is a possibility that the ability of triploid and diploid cod to prey on dry diet/Artemia was different, which might have affected growth during the Artemia feeding period. Mortality in the triploid groups

was higher during early feeding (rotifers, Artemia and weaning/ pregrowing period). Growth data comparing diploid and triploid fish show wide variations. McGeachy et al. (1995) and Friars et al. (2001) found that mixed-sex groups of triploid and diploid Atlantic salmon grew equally well in fresh water, while Cotter et al. (2002) found that diploids were significantly heavier about eight weeks after the commencement of start-feeding. Peruzzi et al. (2004) found slower growth in triploid than in diploid sea bass. The triploid Atlantic cod in our study displayed a higher incidence of vertebrae deformities than the diploid controls, and previous studies have shown that such deformities can negatively affect the growth of Atlantic salmon (Hansen et al., 2010).

5. Conclusions

Triploid cod had lower survival rates and suffered a higher incidence of lordosis than diploid cod. The results of this study, which is the first that deals with skeletal deformities in triploid farmed Atlantic cod, are in line with previous studies showing that triploid teleosts are more prone to develop bone deformities. The mechanisms underlying this phenomenon are unknown. The questions of whether dietary requirements differ in triploid diploid cod, and how juvenile production regime (extensive vs. intensive production) affects the development of vertebral deformities, remain to be studied.

Acknowledgements

This study was funded by the Ministry of Fisheries and Coastal Affairs, Innovation Norway and Grieg Cod Farming. The authors wish to thank the staff of Austevoll Research Station for their skilled technical support.

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