Tillmann J. Benfey

The Physiology and Behavior of Triploid Fishes

Induced triploidy is widely accepted as the most effective method for producing sterile fish for aquaculture and fisheries management. Artificially produced triploids generally differ from conspecific diploids in three fundamental ways: they are more heterozygous, they have larger but fewer cells in most tissues and organs, and their gonadal development is disrupted to some extent. Despite these basic biological differences, triploids are similar in most respects to diploids when examined at the whole animal level. The only clear differences relate to the effects of impaired gametogenesis on the reproductive physiology and behavior of triploids, especially in females. Other apparent differences include reduced aggressiveness, occasional specific morphological abnormalities, and inferior performance when reared under suboptimal conditions. The causes of these latter two problems are poorly understood but must be addressed if triploids are to be used more extensively.

Acute stress response in triploid rainbow trout (Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis)

The purpose of this study was to determine whether triploid salmonid fishes differ from diploids in their hematological and physiological responses to an acute handling and confinement stress, induced by netting fish from their tanks and placing them in a bucket. Blood samples were collected from fish prior to the handling stress and at either 30 min (rainbow trout) or 20 and 40 min (brook trout) confinement in the bucket. Plasma cortisol, glucose and chloride levels, hematocrit, hemoglobin concentration, total blood cell concentrations (erythrocytes and leucocytes), and differential leucocyte concentrations and their relative proportions were measured. As expected, resting blood cell concentrations were significantly lower in triploids than in diploids. In all other respects, triploids showed little difference from diploids in either values prior to the stress or in changes in these values induced by the acute stress. Both diploids and triploids showed a marked stress-induced increase in hematocrit and plasma cortisol and glucose, accompanied by a decrease in lymphocyte concentrations. Other values showed minimal or no change resulting from the stress. Triploid salmonid fishes therefore appear to exhibit a typical acute stress response, as has been well described for diploids.

Survival and growth of all-female diploid and triploid rainbow trout (Oncorhynchus mykiss) reared at chronic high temperature

The salmonid aquaculture industry has recently been investigating the benefits and drawbacks of sterile triploids. Although studies have shown that triploids should not be restricted by their altered haematology under optimal conditions, little is known about their performance in sub-optimal environments. This study focused on the performance (in terms of growth and survival) of female triploid rainbow trout (Oncorhynchus mykiss) in comparison to female diploids at chronic high temperature. Triploid and diploid rainbow trout were reared in fibreglass tanks at 21 ± 1 °C for 23 days. At these chronic high temperatures, triploids had a significantly (P < 0.001) higher mortality rate in comparisons to diploids: 68.5% of the triploids (n = 175) died within 3 weeks at 21 ° C, while only 39% of the diploids (n = 167) died in this time period. Diploids had larger fork lengths (by 4.8%), body weights (by 23.9%) and condition factors (by 10.3%) than the triploids by the end of the experiment (P < 0.05 in all cases). The cause of fish mortality in this experiment was likely to be multifaceted, and influenced, and/or directly caused, by the stress of the experimental protocol. However, it is evident from these results that triploid rainbow trout did not survive or grow as well as diploids in chronic high water temperature conditions.

The relationship between sperm density, spermatocrit, sperm motility and fertilization success in Atlantic halibut, Hippoglossus hippoglossus

Abstract The commercialization of Atlantic halibut, Hippoglossus hippoglossus , aquaculture has been hampered by a failure to obtain consistently high fertilization rates. The principal goal of this research was to determine the optimum sperm density for successful fertilization of Atlantic halibut eggs. Sperm densities ranged from 2×10 11 to 6×10 11 spermatozoa/ml at 23% and 99% spermatocrit, respectively, for 36 milt samples collected from 17 males. Regression analysis showed a significant positive linear relationship between sperm density and spermatocrit, supporting the use of spermatocrit as a rapid estimator of sperm density in this species. There was no relationship between sperm density and sperm motility (defined as time elapsed from activation until 8 to 9.4×10 5 spermatozoa/egg, respectively.

Comparisons of cultured triploid and diploid Atlantic salmon(Salmo salar L.)

The use of sterile, triploid salmon for aquaculture would be an effective method of preventing genetic interactions between cultured and wild salmon. The studies presented in this paper compare the performance of six year classes of triploid and diploid salmon in freshwater and seawater stages of commercial production. Freshwater growth was comparable between triploids and diploids. Freshwater survival was also similar between triploids and diploids except that survival was lower in the triploids for the developmental interval between fertilization and first feeding. In sea water, triploids performed better than diploids in terms of growth. However, survival was lower in triploids and they showed a higher incidence of jaw deformities. In summary, the overall yields of triploids was lower than diploids under culture conditions.

Cortisol, glucose and hematocrit changes during acute stress, cohort sampling, and the diel cycle in diploid and triploid brook trout (Salvelinus fontinalis Mitchill).

In this study, groups of diploid (control) and triploid brook trout were subjected to a 5 min acute handling/confinement stress. Blood samples were collected from equal numbers of diploid and triploid fish at regular predetermined intervals, beginning prior to confinement and continuing for up to 3h after confinement. These blood samples were used for the measurement of hematocrit and plasma cortisol and glucose concentrations. The effects of cohort sampling and the diel cycle on these variables were also analysed. Except for minor differences, both groups responded similarly to the acute stress, with short-term elevations in plasma cortisol and hematocrit levels, and a more sustained increase in plasma glucose levels. No significant differences were found between diploid and triploid fish in their reactions to cohort sampling. However, the act of removing an individual from a tank caused significant changes in plasma cortisol levels in cohorts remaining in that tank (p < 0.001). Significant diel effects were observed for both ploidy groups in plasma cortisol (p < 0.05) but not in plasma glucose or hematocrit.

Flow-Cytometric Confirmation of Aneuploidy in Sperm from Triploid Rainbow Trout

Abstract Triploid rainbow trout Salmo gairdneri produce spermatozoa with DNA contents intermediate between haploid and diploid values. This confirms that these spermatozoa are aneuploid and explains why progeny produced from the fertilization of haploid eggs with such sperm are not viable. Triploid males have significantly lower spermatocrit and smaller testes than diploids, but still develop normal secondary sexual characteristics and exhibit postspawning mortality.

The growth and reproductive endocrinology of adult triploid Pacific salmonids

This paper describes the effect of triploidy on growth and reproductive endocrinology in the months leading up to and including spawning in rainbow trout,Salmo gairdneri, and pink salmon,Oncorhynchus gorbuscha. Growth rates were the same for diploid and triploid rainbow trout, but triploid female pink salmon were smaller than maturing diploid females and diploid and triploid males of the same age. Triploid males of both species developed typical secondary sexual characteristics and had normal endocrine profiles, although their cycle appeared to be delayed by about one month. Triploid females remained silvery in appearance and showed no endocrine signs of maturation, even at the level of the pituitary. Thus, although triploids of both sexes are genetically sterile, only the females do not undergo physiological maturation.

Freshwater performance of triploid Atlantic salmon (Salmo salar) in New Brunswick aquaculture

The use of ploidy manipulation to produce sterile salmon has been investigated since the mid-1970s. Today, the techniques for producing triploids are well documented. In this study pressure shock techniques were employed to produce triploid Atlantic salmon on a commercial scale, and performance differences between triploids and diploids were assessed. A new 2.51 pressure vessel was designed and used to produce approximately 36 000 triploid salmon during the 1992 spawning season. This vessel was highly successful in inducing triploidy, as determined by flow cytometry. Performance was based on survival and growth, which were monitored from the egg stage until the time of smolting. There were no significant differences in cumulative survival throughout the experiment. During April, May and June of 1993, triploids were significantly smaller than their diploid counterparts (P < 0.05). However, from July 1993 until February 1994 there were no differences in size. In February 1994 the smolting rate was determined by classifying those fish greater than 13 cm as smolt. The success rates were 86% and 88% for triploids and diploids, respectively.

Hormonal sex reversal of Atlantic halibut (Hippoglossus hippoglossus L.)

Abstract Atlantic halibut (Hippoglossus hippoglossus L.) females grow larger and mature later than males, making monosex (all-female) culture economically advantageous. The objective of this research was to develop methods for the production of monosex populations of halibut through the use of sex steroids. Six hundred postlarval halibut, mean size 30.0 mm fork length (FL), were divided evenly among eight tanks and fed a formulated diet supplemented with either 17α-methyldihydrotestosterone (MDHT; 1 and 5 ppm) or 17β-estradiol (E2; 10 ppm) for 45 days. Fish were then further reared on a standard halibut diet until sex ratios could be determined through histological and macroscopic observations. The MDHT treatments yielded 97–100% males, while the E2 treatment yielded a 70–74% female population. This demonstrates that the direct feminization of halibut can be achieved, and also allows for the evaluation of indirect feminization through the crossing of hormonally masculinized females (“neomales”) to normal females.