Project
title / Environmental influences on the sex of cultured marine flatfish
/ DEFRA
project code / FC0901

Department for Environment, Food and Rural Affairs CSG 15

Research and Development

Final Project Report

(Not to be used for LINK projects)

Two hard copies of this form should be returned to:
Research Policy and International Division, Final Reports Unit
DEFRA, Area 301
Cromwell House, Dean Stanley Street, London, SW1P 3JH.
An electronic version should be e-mailed to
Project title / Environmental influences on the sex of cultured marine flatfish
DEFRA project code / FC0901
Contractor organisation and location / CEFAS
Weymouth Laboratory
Barrack Road, Weymouth, DT4 8UB
Total DEFRA project costs / £ 454,094
Project start date / 01/04/98 / Project end date / 31/03/02
Executive summary (maximum 2 sides A4)
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CSG 15 (9/01) 3

Project
title / Environmental influences on the sex of cultured marine flatfish
/ DEFRA
project code / FC0901

CSG 15 (9/01) 3

Project
title / Environmental influences on the sex of cultured marine flatfish
/ DEFRA
project code / FC0901

Key Objectives and justification

The overall objective was to determine the causes of observed male predominance amongst stocks of cultured Dover sole. The key objectives for the work programme were as follows:

1.  To determine the period over which sexual differentiation of Dover sole normally occurs;

2.  To assess experimentally the influence of rearing temperature on phenotypic sex;

3.  To assess experimentally the influence of diet on the expression of phenotypic sex;

4.  To assess experimentally the influence of stocking density and associated water quality on phenotypic sex;

5.  To assess the influence of early growth rate on phenotypic sex from the results of the above experiments.

Skewed sex ratios, with male fish predominating, are common in cultured broods of some commercially important fish species. The causes of consistent male predominance cannot be simply explained from genetics and it is presumed that aspects of the rearing environment used in the hatchery are influencing the functional sex of developing fish. Rearing temperature, diet, stocking density or water quality could bring about the effect.

Explaining the phenomenon and then developing techniques to avoid it are important issues for aquaculture as the effect may be considered a problem of fish welfare if rearing conditions are inducing the effect. Additionally the industry would normally wish to preclude high proportions of male fish from their stocks as they usually mature earlier and then grow more slowly than females. If the cause is identified as an environmental effect the results of the work should enable skewed sex ratios to be avoided through changes to rearing conditions. There may also be the opportunity to exploit natural processes to control of sex should it be considered suitable. If there were any sign that the source of the problem lies outside the hatchery, in the natural environment, it would indicate there might be an effect on natural stocks that would also need to be addressed. The information provided to DEFRA should benefit other strategic research programmes on sex control (e.g. FC0903) that may be compromised by uncontrolled influences on sex. It would also allow advice to be given on best practices, contributing to the development of a viable and sustainable aquaculture industry.

Main findings

·  Temperatures normally used for early rearing do not influence sex ratios of hatchery reared Dover sole.

·  Diets during the larval stage and at weaning similarly were shown to have no significant effect on sex ratios, neither were early growth rate and stocking density.

·  The proportion of females in batches of fish examined ranged from less than 5% to more than 15%, but this could not be associated with any experimental treatment.

·  An indication was obtained that soluble small molecular weight substances in the hatchery water might contribute to the effect, but further work is required to confirm this.

Implications for policy and future research

The work has shown that features of standard rearing protocols routinely used for Dover sole husbandry, such as temperature, diet and stocking density do not directly contribute to the phenomenon of male predominance of hatchery reared sole stocks. This lessens the prospect of the procedures needing to be addressed purely from the perspective of fish welfare, but does not lead to a clarification of the source of the problem.

At the moment the most likely explanation is that young sole are susceptible to the influence of dissolved substances present in the hatchery water, and that these substances can affect the differentiation of the developing gonad. Further work is required to explore this and, if the effect is confirmed, to identify the nature and source of the chemicals.

CSG 15 (9/01) 3

Project
title / Environmental influences on the sex of cultured marine flatfish
/ DEFRA
project code / FC0901
Scientific report (maximum 20 sides A4)
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CSG 15 (9/01) 3

Project
title / Environmental influences on the sex of cultured marine flatfish
/ MAFF
project code / FC0901

17

Project
title / Environmental influences on the sex of cultured marine flatfish
/ MAFF
project code / FC0901

Scientific Objectives and milestones

Objectives

Initial 1998-2001

1.  To determine the period over which sexual differentiation of Dover sole normally occurs
Months 1-16

2.  To assess experimentally the influence of rearing temperature on phenotypic sex
Months 2-14

3.  To assess experimentally the influence of stocking density on phenotypic sex
Months 13-25

4.  To assess experimentally water quality effects during early rearing on phenotypic sex
Months 24-36

5.  To assess the influence of early growth rate on phenotypic sex from the results of the above experiments
Months 27-36

6.  To prepare a paper for publication and recommendations for the industry
Months 34-36


Amendment to objectives

7. To assess experimentally the influence of larval diet on phenotypic sex. Months 13-25. ( postponing 3 until following year)


Extension 2001-2002

Evidence of an effect of temperature in unrelated species was published in 1999 and 2000. It was felt that it warranted an additional year's investigation with Dover sole to allow a further experiment on temperature to be undertaken. In addition the work on stocking density was to be followed-up with more emphasis on the contribution of water quality under different stocking regimes. This had been suspended from the original proposal because of the introduction of work on diet in the second year.

  1. To assess experimentally the influence of rearing temperature on phenotypic sex
    Months 1-10
  2. To assess experimentally the influence of stocking density and associated water quality on phenotypic sex
    Months 2-11
  3. To prepare a paper for publication and recommendations for the industry
    Months 9-12
  4. To assess experimentally the influence of weaning diet on phenotypic sex,
    Months 3-12. (additional)

Milestones

Initial 1998-2001

Milestone / Target date / Title
01/01 / 7/1999 / Complete description of phases of gonad development & differentiation in sole
02/01 / 5/1999 / Complete experiment on temperature effects on the expression of phenotypic sex
03/01 / 4/2000 / Complete experiment on density effects on the expression of phenotypic sex
07/01
change / 4/2000 / Complete experiment on the influence of larval diet on the expression of phenotypic sex
04/01 / 3/2001 / Complete experiment on water quality effects on the expression of phenotypic sex
05/01 / 3/2001 / Complete data analysis to assess effects of early growth rate
06/01 / 3/2001 / Complete a publication describing the work and a final report

It was agreed (July 1999) that Milestone 03/01, target date 4/2000 -, be rescheduled to months 24-36. In its place a new milestone 07/01, target date 4/2000 was introduced.

Extension2001-2002

Milestone / Target date / Title
01/01 / 12/2001 / Complete experiment on temperature effects on the expression of phenotypic sex
02/01 / 01/2002 / Complete experiment on density and water quality effects on the expression of phenotypic sex
04/01 (added) / 03/2002 / Complete experiment on the influence of weaning diet on the expression of phenotypic sex
03/01 / 03/2002 / Complete a publication describing the work and a final report

Preamble

Male fish predominate in broods of normal diploid Dover sole reared in the hatchery and the same phenomenon has been recorded in other hatchery-reared marine fish such as Japanese flounder [14] and sea bass [7]. Various explanations for it have been proposed and in several species the rearing temperature during the period when the fish are sexually labile has been associated with determination of phenotypic sex. A specific rearing temperature may lead to a 50:50 sex ratio, but if a temperature is used that is three or four Celsius degrees higher a greater proportion of males develops [11].

It is presumed that if environmental sex determination occurs there will be a short period during late larval or early juvenile development, before the gonad has differentiated, when the fish will be sensitive to environmental effects. Some indication of the timing of this window should be obtained from a knowledge of the stage in development when the fishes' sex is naturally fixed. The first part of the programme of work was therefore to identify when a developing ovary could be first distinguished from a testis in normal, hatchery reared fish. The work would then progress by looking at the influence of the environmental variables suggested to be the most important factors from related work in other species. The variable, the degree of change required to cause an effect and the window of sensitivity all needed to be identified.

Methods and results of the research

The period over which sexual differentiation of Dover sole normally occurs

This aspect of the work was intended to provide information on the normal timing of gonad development and enable the period of differentiation leading to a recognisable testis or ovary to be established. It has been particularly difficult to obtain sufficient samples of ovarian tissue from normally reared fish because of the predominance of males. It has also proven difficult to identify primordial gonad tissue in histological material from late larvae and newly metamorphosed sole.

The difficulty of finding sufficient developing females required an alternative approach to the use of normal hatchery stock to provide material of known age. It had been shown that exogenous steroid hormone in the rearing water could influence phenotypic sex of several species if treated at the correct time. In the same way that skewed sex ratios occur in hatchery-reared sole, they also occur in hatchery-reared Japanese flounder. It has been found however that low doses of 17b-oestradiol in the rearing water restored the 50:50 sex ratio in normal diploid populations of flounder and rendered gynogenetic populations nearly all-female[14]. The interesting aspect of this result is that such a dose appears not to override the genetic sex, but seems to negate the environmental effect. We evaluated whether comparable doses would have a similar effect in sole.

As a preliminary trial, tanks were treated using the same methodology, with 10µg l-1 17b-oestradiol in the rearing water for 2 hours daily over 65 days from day 50 post-hatch, after metamorphosis was complete.

The sex of over 110 fish from each group was assessed from gonad morphology and histology and while 5% of the control group was female, the proportion of females in the treated group was 35%. A few remaining fish in the treated group appeared to have no developed gonad (Table 1).

This treatment confirmed that the fish were sensitive to oestradiol, and that the skewed ratios were not a result of a lack of oestrogen receptors in the tissues. It also demonstrated that phenotypic sex could be influenced by exogenous hormone after day 50 post-hatch.

Treatment / Number of fish in sample / % female / % male
Female / Male / Unclassified / Total
Control / 6 / 107 / 0 / 113 / 5.3 / 94.7
Oestradiol / 41 / 69 / 9 / 119 / 34.5 / 58.0

Table 1. Sex of fish in samples from control and oestradiol treated groups of juvenile Dover sole

A second trial was carried out in the same way the following year to produce more material for histology. The sex of fish sampled between 8 and 10 months of age showed a higher proportion of females in the control groups than in the previous trial (about 15% rather than 5%), but a similar proportion of females (30-40%) in the hormone treatment groups. The replicates differed somewhat and the data are presented separately as well as combined in Table 2.

Treatment / Replicate / Number of fish in sample / % female / % male
Female / Male / Unclassified / Total
Control / 1 / 19 / 84 / 6 / 109 / 17 / 77
2 / 12 / 83 / 5 / 100 / 12 / 83
Combined / 31 / 167 / 11 / 209 / 15 / 80
Oestradiol / 1 / 43 / 54 / 12 / 109 / 39 / 50
2 / 31 / 66 / 2 / 99 / 31 / 67
Combined / 74 / 120 / 14 / 208 / 35 / 58

Table 2. Sex of fish in replicated samples from control and oestradiol treated groups of juvenile Dover sole

Experiment to assess the period of sensiticvity to exogenous oestradiol

To assess whether sensitivity to exogenous oestrogen existed prior to day 50 the work was followed up by exposing larvae to the same concentration of oestradiol for 2 h a day during a five day period beginning at weekly/fortnightly intervals between hatch and day 42 post hatch. For each group there were also control treatments in which the fish were only exposed to the ethanol carrier. Duplicate tanks were used for both hormone and control treatments (Table 3). Although the exposure to the hormone was restricted to five days because of the sensitivity of larvae, those treated early in development still showed higher mortalities in the hormone group compared with the control groups. There was also variation between the replicates. The fish were subsequently reared for about 18 months before the sex was determined. There was no significant difference between treatments and controls and no significant enhancement of the proportion of females in the groups was obtained. The higher mortality in the oestradiol treated groups indicated a sensitivity to the hormone early in development.