Aquacutture and Fisheries Management 1993, 24, 145-150

Effects of LHRHa treatments upon the timing ofovulation and upon egg and offspring quality in Arcticcharr, Salvelinus alpinus (L.)

H. HARALDSSON & T. SVEINSSON Department of Physiology, University ofIceland, Reykjavik, Iceland

S. SKULASON Holar Agricultural College, Molar, Hjaltadalur, Iceland

Abstract. Five groups of Arctic chart, Salvelinus alpinus (L.), were studied, each groupconsisting of 13 females. The fish in four of the groups received a single intramuscular (IM)injection: three groups were injected with LHRHa in various forms and dosages, and one wasinjected with saline (control). The females in the fifth group were implanted with controlled-diffusion implants containing LHRH analogue. Ovulation in two of the treatment groups(30fig/kg D-Ala* LHRH and t50M,g/kg D-Trp* LHRH) occurred earlier, and was bettersynchronized, than in the control group. The treatments did not significantly affect (1) theweight of eggs released from each female; (2) egg size, weight, or chemical composition; (3)mortality rates; (4) hatching times or the length of alevins. The fertilization rate in one of thetreatment groups (I50ti.g/kg D-Trp*") was significantly lower than in the control group.

Introduction

In most stocks of wild fish, spawning is an annual event which is timed to ensure favourableenvironmental conditions for the offspring (Bromage, Randall, Davies & McAndrew 1991).Some species of fish do not spawn in captivity, and in other species spawning is anunpredictable and unsynchronized event. The possibility of inducing and synchronizingspawning can be a considerable aid to aquaculture research (Donaldsson & Hunter 1983), forinstance in connection with cross-breeding and developmental experiments. It can also yieldeconomic benefits, for example by facilitating gamete collection.

Photoperiod is the environmental factor that has the most effect on the timing ofovulation in salmonids (for review see Bromage et al. 1991). The endocrine system mediatesenvironmental effects and directly controls ovulation and spawning. In response to changesin environmental conditions, gonadotropin-releasing hormone (GnRH) is released from thehypothalamus and stimulates the pituitary gland to release gonadotropins (GtH). One of theeffects of GtH is induction of sex steroid secretion from the developing sex organs.

Environmental manipulation is currently us^d to change spawning time in a variety of fishspecies (Bye 1987). A hormonal approach, however, represents the most direct method formanipulating spawning times (Zohar 1989). A variety of hormones of the pituitary-gonadalaxis (GtH) have been used for this purpose. Gonadotropin-releasing hormone and itssuper-active analogues (LHRHa) are less species specific than GtH and are effective in lowerdosages. However, hormone treatments of this kind cannot hope to receive wide commercial

Correspondence: H. Haraldsson, Department of Physiology. University of Iceland, Vatnsm^rarvegur 16, 101Reykjavik, Iceland.

145

146 H. Haraldsson et al.

acceptance unless the quality of eggs and offspring is shown to be similar to that of naturallyspawned eggs and offspring.

In the present project, the effects of four different LHRHa treatments on final maturationand ovulation in Arctic charr, Salvelinus alpinus (L.) and on the quality of eggs and offspring,were studied.

Materials and methods

The experiment was conducted in 1991-1992 at Holar Agricultural College. The fish usedrepresented a mixture of stocks from three rivers in north-west Iceland: Mi6fjar(5ard,Vi'Cidalsa and Vatnsdals^. It was previously known that, in culture, the earliest ovulationin these fish occurs in the middle of October and latest up to 2 months later. A control group,and four treatment groups designated '2' to '5', were established. Each group consisted of 13female charr (each l-5-2-5kg), randomly selected from a larger group. All females weremarked with fioy tags and held together in an outdoor tank with a water temperature of about5°Q.

On 11 October, a few days before the anticipated beginning of the spawning period all ofthe fish received single intramuscular (IM) injections. The females in the control group wereinjected with saline. The females in group 2 were injected with 30̂ JLg/kg of the analoguesdes-Gly'^[D-Ala^]-LHRH Ethylamide (30D-Ala) and those in group 3 with des-Gly'",[D-Trp^]-LHRH Ethylamide (30D-Trp). The females in group 4 were injected with the sameanalogue as those in group 3, but received a larger dose: 150p,g/kg (150D-Trp). Analogueswere obtained from the Sigma Chemical Co., St Louis, MO, USA. Implants containing 75 figof the des-GIy'",[D-Ala'^]-LHRH and coated with a layer of ethyiene-vinyi acetatecopolymer (D-AlaEVAC) (Zohar, Pagelson, Gothilf, Dickhoff, Swanson, Duguay, Gom-botz, Kost & Langer 1990), were implanted under the skin of the females in group 5. Theimplants were kindly provided by Y. Zohar, of the University of Maryland.

For the first 4 weeks, the females were checked every 3 days for ripeness of eggs, andthereafter every 7 days. Each female was stripped shortly after being judged fully ripe(running). The weight of eggs stripped from every female was measured, and a portion (ca.1500 from every female) fertilized (each male used fertilized the eggs from one female in eachtreatment group). The diameter (dial calipers) of 30 eggs and the weight of 15 eggs wasmeasured for each female. Using a microscope on samples consisting of 30 eggs, fertilizationrates were estimated 18-22h after fertilization. The rest of the eggs were frozen for laterchemical analysis. The sibling groups were kept separated until first feeding, with watertemperature maintained at about 4''C. All females were killed about one month afterstripping, and the eggs remaining inside weighed. Shortly after hatching, samples of embryosfrom each family were collected and fixed in formalin. Thirty embryos from each of ninefemales from every group were individually measured for length.

The percentage of protein was measured in frozen eggs in 35 samples by the Kjeldahlmethod (Hach, Bowden, Kopelove & Brayton 1987). The amount of lipids was measured in23 samples by the Folch method (Folch, Lees & Sloane Stanley 1957). Dry weight and ashwere also measured, each in 35 samples. One-way ANOVA or Kruskal-Wallis tests followed byKolmogorow-Smirnov tests were used for statistical analyses.

LHRHa treatments on Arctic charr 147

10 Or

80 •

60 •

£ 402

2 oh

Control30D-Ala "

\

D-AlaEVAC ' / , - ' ' /

if30D-Trp '

/

----

150D-Trp "

.a

• t

10 20 30 40Post Treatment [days]

50 6 0

Figure 1. Cumulative percentages of ovulated female Arctic charr over a 55-day period following LHRHa treatmentsand injection with saline (control). The time between injection and ovulation did not differ significantly among thosetreatments labelled with the same subscripts (a,b) (Kruskal-Wallis [P = 0-003] followed by Kolmogorow-Smirinovtest fa = 0-05]).

Results

Of the four different treatments used in the present study, two of them (30D-AIa and150D-Trp) had significant effects on the length of time between injection and ovulation (Fig.1). The number of females which ovulated over the 55-day experimental period was notsignificantly different between treatment groups. The total weight of eggs stripped from eachfemale did not differ significantly among the groups (Fig. 2A). The difference among groupsin the weight of eggs left behind was not significant (Fig. 2A). Furthermore, the chemicalcomposition of the eggs did not differ significantly between any of the groups (Fig. 2B), nordid egg diameter nor weight (Fig. 3A). However, there were significant differences infertilization rates among the different treatment groups (Fig. 3A). Neither 24-h mortalityrates nor the number of dead embryos after an interval of 3 months differed significantlyamong the five groups (unpublished data). The differences in the average number of daysfrom fertilization to hatching was also not significant (Fig. 3B). ANOVA indicated non-signifi-cant differences in the aievin length among groups (Fig. 3B).

Discussion

The effects of LHRHa have been investigated in a variety of fish species (Lam 1982). Fishoften need to be injected two or three times to obtain the best results, and other chemicalssuch as dopamine antagonists have sometimes been used along with LHRHa (Zohar 1989).In many cases, a dosage of 10p.g/kg seems sufficient to induce spawning (Bromage 1988; Lam1982). The farming of Arctic charr is in its infancy, and little attention has been given toLHRHa treatments in this species. However, Gillet & Breton (1991) found that

148 H. Haraldsson et al.

A.

300

250

- 200

.c"55 150

Z 100I-

50

0

B.

• Eggs stripped from temales• Eggs remaining inside females

- 40

3 0

20

10

Control aOD-Ala 30D-Trp 150D-Trp D-AlaEVAC

• Ash• Lipids• Protein

Control 30D-Aia 30D-Trp 150D-Trp D-AlaEVAC

Figure 2. (A) Total weight of eggs stripped from each female (Kruskal-Wallis, P = 0-74) and the total weight or eggsthat were retained by females one month after stripping (Kruskal-Wallis, P = 0-31). Mean values (bars) and standarderror of mean (error bars) are indicated. (B) Protein, lipids, and ash in eggs, shown as a percentage of wet weight(ANOVA, P > 0-05 for all parameters).

D-ArgSGnRH and a biodegradable sustaitied release of D-Trp*LHRHa readily inducedovulation in Arctic charr, while D-Trp*" (30^.g/kg) given in free form failed to induceovulation.

In the present experiment, the fish were injected only once, to minimize stress. Two of thetreatments, 30D-ala and 150D-Trp, affected the timing of ovulation. More than 75% of thefemales in these two groups ovulated within 16 days of injection, while in the control grouponly 46% did so. The 150D-Trp group received five times more analogue than the 3()D-Alagroup, but the effect on the ovulation time was similar. However, the larger dose reduced

LHRHa treatments on Arctic charr 149

A.

35

30

en 25

S 20

O)LJJ

B.

1 5

1 0

5

0"-

140p

120

ra 100

E 80

X 40

20

O"-

Control91.3%a

30D-Trp8a.5%a,b

D-AlaEVAC86.6%a

30D-Ala74.8%a,b

150D-Trp57,6%b

3 •m

CO

g

-J 0

n 20

Control 30D-Trp D-AlaEVAC

30D-Ala 150D-Trp

o

m3

1 0 o

5 __33

J 0

Figure 3. Diameter and weight of individually measured eggs (ANOVA; P = 0-70 and P = 0-19, respectively). Means(symbols) and standard error of means (error bars) are indicated. Average per cent fertilization rates are a!soindicated. Numbers coded with the same letter (a, b) do not differ significantly (Kruskal-Wallis [P = 002] followedby Kolmogorov-Smirinov test [a = 0-05]). (B) Hatching times in days from fertilization (Kruskal-Wallis. P = 0-64)and embryo lengths at the same age (ANOVA, P = 0-46). Averages (symbols) and standard errorof means (error bars)are indicated.

fertilization success. The smaller dose of D-Trp was rather ineffective, which is consistentwith the findings of Gillet & Breton (1991). Thus, in our study, the D-Ala analogue proved tobe the most effective stimulant to ovulation. The same analogue in pelleted form(D-AlaEVAC) did not affect the ovulation time as markedly as when given in free form. Thediffusion-controlled release of the analogue from the implants may have been too slow at the5°C temperature, applied in the present experiment, to elevate plasma levels sufficiently toaffect GtH release from the pituitary.

150 H. Haraldsson etal

The treatments used had no significant effect on size of eggs and embryos. It has beenshown that the size of eggs and alevins affects their viability and later growth (Wallace &Aasjord 1984). Large eggs appear to produce larger juveniles. Although none of theparameters measured, relating to the eggs and offspring for the 30D-Ala group, weresignificantly different from those for the control group, some trends were noticeable. Forexample, both the diameter and weight of eggs, the number of eggs released, the number offemales which ovulated, fertilization rates and the length of embryos were somewhat lower inthe 30D-Ala group than in the control group, while the number of eggs retained in the bodycavity after stripping, hatch times, and mortality rates were greater. In conclusion, a singleinjection of 30M.g/kg of des-gly'", [D-Ala'^]-LHRH can be recommended as a treatment forincreasing production efficiency and synchronizing broodstock ovulation in Arctic charr,without producing damaging effects.

Acknowledgments

This project was funded by Holar Agricultural College, the Agricultural Productivity Fundof Iceland and the Agricultural Research Institute, Iceland. We are very grateful to all thosewho assisted us in carrying out this project; but special thanks are due to Sveinn KariValdimarsson, whose help with the experiments was invaluable. We also thank Drs JohannAxelsson and Mikael M. Karlsson which provided a thorough review of this manuscript.

This paper is based on a research project for advanced study al the Department ofBiology, University of Iceland.

References

Bromage N. (1988) Propagation and stock improvement. In: Intensive Fish Farming (ed. by J. Shepherd & N.Bromage), pp. 103-150. BSP Professional Books, Oxford.

Bromage N., Randall C , Davies B. & McAndrew B. (1991) The control of reproduction in salmonid fish. IcelandicAgricultural Sciences 6 (in press ).

Bye V.J. (1987) Environmental management of marine fish reproduction in Europe. Proceedings of the ThirdInternational Symposium on Reproductive Physiology of Fish 1987, 289-298.

Donaldson E.M. & Hunter G.A. (1983) Induced final maturation, ovulation and spermiation in cultured fish. In:Fish Physiology (ed. by W.S. Hoar, DJ. Randall & E.M. Donaldson), pp. 351-390. Volume IX, Part B.Academic Press, New York.

Folch J., Lees M. »& Sloane Stanley G.H. (1957) A simple method for the isolation and purification of total lipidesfrom animal tissues, Journal of Biological Chemistry 226, 497-509.

Gillet C. & Breton B. (1991) Research work on Arctic charr in France: Researches on broodstock management.Icelandic Agricultural Sciences 6 (in press) .

Hach C., Bowden B., Kopelove A. & Brayton S. (1987) Method performance. Journal of the Association of OfficialAnalytical Chemists 5, 783-87.

Lam T.J. (1982) Applications of endocrinology lo fish culture. Canadian Journal of Fisheries and Aquatic Sciences39, 111-137.

Sokal R,R. & Rohlf F.J. (1981) Biometry. 2nd ed. W.H. Freemann & Company, New York.Wallace J.C. & Aasjord D. (1984) An investigation of the consequences of egg size for the culture of Arctic charr,

Salvelinus alpinus (L). Journal Fish Biology 24, 427-435.Zohar Y. (1989) Endocrinology and fish farming: aspects in reproduction, growth, and smoltification. Fish

Physiology and Biochemistry 7, 395--W)5.Zohar Y., Pagelson G., Gothilf Y., Dickhoff W.W., Swanson P., Duguay S., Gombotz W , Kost J. & Langer R.

(1990) Controlled release of gonadotropin releasing hormones for the manipulation of spawning in farmed fish.Proceedings of the International Symposium on Controlled Release of Bioactive Materials 17, 8-9.