Directory UMM :Data Elmu:jurnal:A:Aquaculture:Vol194.Issue1-2.2001:

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www.elsevier.nlrlocateraqua-online

The relationship between sperm density,

spermatocrit, sperm motility and fertilization

success in Atlantic halibut,

Hippoglossus hippoglossus

Harald B. Tvedt

a,b,)

_{, Tillmann J. Benfey}

_,

Deborah J. Martin-Robichaud

, Joanne Power

a,b

Department of Biology, UniÕersity of New Brunswick, Fredericton, New Brunswick, Canada E3B 6E1

Fisheries and Oceans Canada, St. Andrews Biological Station, 531 Brandy CoÕe Road, St. Andrews,

New Brunswick, Canada E5B 2L9

Received 21 December 1999; received in revised form 15 August 2000; accepted 12 September 2000

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=1011to 6=1011spermatozoarml 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, support-ing 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 -5% of the spermatozoa maintained forward swimming activity . Implant with a salmon

Ž Ž . .

gonadotropin releasing hormone analogue GnRH a ; Ovaplante was followed by a reduction in sperm density, but this affected neither the relationship between sperm density and spermatocrit, nor between sperm density and sperm motility. There was no effect of sperm density on fertilization success within the range of sperm:egg volume ratios of 1:15.6 to 1:8000, equivalent to

)_{Corresponding author. Fisheries and Oceans Canada, St. Andrews Biological Station, 531 Brandy Cove} Road, St. Andrews, New Brunswick, Canada E5B 2L9. Tel.:q1-506-529-8854; fax:q1-506-529-5862.

Ž .

E-mail address: [email protected] H.B. Tvedt .

Ž .

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Ž .

Keywords: Hippoglossus hippoglossus; Spermatocrit; Sperm density; Motility; Fertilization success; GnRH a

1. Introduction

The Atlantic halibut, Hippoglossus hippoglossus, is one of the most likely alterna-tives to Atlantic salmon, Salmo salar, for cold-water aquaculture in the North Atlantic Ž_{Brown et al., 1995 . Successful commercial culture of this flatfish species has,}. however, been hampered by a variety of problems during early development under culture conditions, prior to weaning and metamorphosis. Among these problems is a failure to obtain consistently high fertilization rates.

The use of high quality gametes is of great importance for ensuring the production of viable larvae, but gamete quality is difficult to assess in a quantitative and meaningful

Ž .

manner Kjørsvik et al., 1990 . Techniques used to assess sperm quality in fish include Ž

monitoring sperm density and motility, and fertilization success Billard, 1978; Mounib, .

1978; Aas et al., 1991; Methven and Crim, 1991 . Egg quality is evaluated by monitoring ovulatory rhythms, blastomere morphology, bouyancy, and fertilization and

Ž .

hatching rates Norberg et al., 1991; Bromage and Roberts, 1995; Shields et al., 1997 . The ratio of spermatozoa to eggs, contact time between gametes, and fertilization method are the other factors that have been evaluated for maximizing fertilization rates Ž_{Suquet et al., 1995; Chereguini et al., 1999 . Sperm:egg volume ratios used in research}.

Ž .

with Atlantic halibut have been in the range of 1:100 Rabben et al., 1986 to 1:1000 Ž_{Mangor-Jensen et al., 1998 , although recently this ratio was reduced to 1:10,000}.

Ž .

without reducing fertilization rate Vermeirssen et al., 2000b .

The standard method for determining sperm density in fish milt is to count spermato-zoa, a time-consuming procedure generally done using a haemocytometer. In the search Ž for faster and more practical ways to estimate sperm density, both centrifugation to

. Ž .

determine spermatocrit and spectrophotometry to determine optical density have been used. A direct relationship between sperm density and spermatocrit has been established

for several salmonid species Bouck and Jacobson, 1976; Baynes and Scott, 1985; Piironen, 1985; Munkittrick and Moccia, 1987; Ciereszko and Dabrowski, 1993; Poole

. Ž

and Dillane, 1998 , whitefish, Coreogonus clupeaformis Ciereszko and Dabrowski,

. Ž .

1993 , yellow perch, Perca flaÕescens Ciereszko and Dabrowski, 1993 and Atlantic

Ž .

cod, Gadus morhua Rakitin et al., 1999 , but not for turbot, Scophthalmus maximus Ž_{Suquet et al., 1992b , the only flatfish species for which this relationship has been}. reported. A direct relationship between sperm density and the optical density of fish milt

Ž .

has, on the other hand, been demonstrated for turbot Suquet et al., 1992a,b , as well as Ž

for salmonids, whitefish, yellow perch and seabass, Dicentrarchus labrax Ciereszko .

and Dabrowski, 1993; Poole and Dillane, 1998; Fauvel et al., 1999 . Since both spermatocrit and optical density are easy to measure, the choice of methods has generally been based on access to equipment.

Ž .

The objectives of this research were 1 to assess the suitability of spermatocrit as a Ž .

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Ž .

motility is influenced by sperm density in this species, and 3 to determine the optimum sperm density for successful fertilization of Atlantic halibut eggs.

2. Materials and methods

Sperm samples were obtained during the 1998 and 1999 spawning seasons, using Ž

wild-caught broodstock held at the St. Andrews Biological Station Fisheries and Oceans .

Canada . The mean weight of the fish was 12"3.3 kg, and they had been held in captivity for 2 to 7 years. Freshly stripped milt was stored on ice in 150-ml plastic

Ž .

containers until used. Any samples that were obviously contaminated e.g., with urine were not kept. A total of 36 usable milt samples were collected from 17 individual males. Seven of these samples were obtained after broodstock had been implanted at the Ž Ž . base of the dorsal fin with a gonadotropin releasing hormone analog GnRH a ;

Ovaplante_{, Syndel International at approximately 15}mgrkg body weight.

Spermatozoan density was determined using a haemocytometer. Milt was first diluted

Ž .

in a 50-ml test tube by adding 10ml of milt to either 30 ml spermatocrit-80% or 40

Ž . Ž

ml spermatocrit)80% of a non-activating solution 150 mM sucrose, 1.7 mM CaCl ,2 .

7 mM MgSO , 86 mM glycine and 30 mM Trisma buffered at pH 8.0 , and then mixed4 on a vortex mixer. Duplicate dilutions were made for each milt sample and triplicate

Ž 3 _.

counts of 50 squares 0.00025 mm rsquare each were made on the haemocytometer for each dilution. The mean of the three counts was calculated for each of the two dilutions, and then the mean of these two values used to calculate the actual sperm density. Samples were left undisturbed on the haemocytometer for a few minutes prior to counting, to allow sperm cells to settle. Counts were conducted using an inverted

Ž .

microscope Hund Wetzlar Wilovert, 320=magnification .

Ž Spermatocrit was determined using milt collected into microhaematocrit tubes 75

mm length, 1.1–1.2 mm inner diameter and centrifuged at 5500=g. An initial experiment was conducted to determine the duration of centrifugation required to achieve stable readings, by measuring spermatocrit at 10-min intervals for up to 1 h of centrifugation. Duration of centrifugation had a significant effect on spermatocrit when

Ž .

considering all time intervals Fs11.1, p-0.001, ns36 , but there was no statisti-Ž . cally significant difference among spermatocrits after centrifugation for 30 min 72% ,

Ž . Ž . Ž . Ž .

40 min 66% , 50 min 64% or 60 min 61% Fs2.3, ps0.11, ns24 . Based on these results, 40-min centrifugation was used for subsequent spermatocrit determina-Ž . tions, as this appeared to be the duration where the curves leveled out Fig. 1 . Spermatocrit for each milt sample was estimated based on the mean value of six tubes, with centrifugation starting within an hour of sample collection.

Sperm motility was determined by diluting 10 ml of milt in 1 ml non-activating Ž . solution, and then 1 ml of this diluted solution was pipetted onto a 1% wrv BSA-coated microscope slide. Activation was achieved by adding 25ml of a

standard-Ž .

ized 33 ppt saltwater solution prepared by adding Instant Oceane _{salt to seawater.} Milt samples were stored on ice and the non-activating and seawater solutions stored at 78C–88C until needed. Once diluted, milt was held on ice in a cooler between preparations of replicate slides. The duration of sperm motility was subjectively

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Ž .

Fig. 1. The influence of centrifugation time at 5500=g on spermatocrit in Atlantic halibut. Each line represents data for milt collected from a different male, as mean values of six replicate samples per male at each time interval. The vertical line denotes the centrifugation time interval used for all subsequent experiments.

evaluated as the time elapsed from activation until -5% of the spermatozoa maintained forward swimming activity. Sperm motility observations were done at room temperature Ž218C–248C , using six replicates per sample, within 2 h of milt collection. One person. conducted all the sperm motility observations, in order to decrease the degree of variation.

Fertilization success was determined at sperm:egg volume ratios of 1:15.6 to 1:8000,

8 5 _Ž

equivalent to 4.6=10 to 9.4=10 spermatozoaregg. Numbers of spermatozoa per egg were calculated using the data in Fig. 2 to convert spermatocrit values to sperm

Fig. 2. The relationship between spermatocrit and sperm density for 36 milt samples collected from 17 male

Ž Ž . Ž .

Atlantic halibut vdenotes samples obtained after GnRH a implantation;Idenotes no GnRH a

implanta-.

tion . For density measurements, each point represents the mean of two dilutions, with three counts per dilution. Plots with no bars indicate SD-1.1=1010.

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densities, combined with egg counts of 5 10 ml samples from different batches of eggs w₄₉₉"38 eggsrsample . Three trials were conducted March 16, March 25 and Aprilx. Ž

27, 1999 using different batches of eggs and milt for each trial. During each trial, triplicate samples from the same batch of eggs and milt were fertilized at each sperm:egg volume ratio. The intermediate ratio of 1:500 was used as a control, using fresh, undiluted milt. In all other cases, spermatocrit was adjusted to 58–60% prior to fertilization in order to obtain a standard sperm density; this was done by adding halibut seminal plasma that had previously been obtained through centrifugation and stored at

y808C for at least 2 days to inactivate any remaining spermatozoa Stoss and .

Donaldson, 1982 . Prior to dilution, spermatocrits of milt samples used for each fertilization trial were 68%, 80% and 93%, respectively.

Eggs were stripped into a 3-l plastic beaker and stored in a cooler on ice. Fertilization experiments were conducted in a cold room at 5.48C–5.98C. After adding the correct

Ž .

volume of milt to 25 ml eggs ca. 1250 eggs measured in a graduated cylinder, 25 ml of Ž .

saltwater 33 ppt was added and the mixture stirred for 20 s. After 10 min, the eggs Ž

were rinsed and transferred to beakers containing 500 ml saltwater UV-treated and .

filtered through 5.0 and 0.35mm cartridge filters for incubation in the dark. At 18–22 h

Ž .

post-fertilization at the 8- and 16-cell stages , samples of eggs were preserved in

Ž .

Stockard’s Solution Bonnet, 1939 for fertilization assessment. Eggs were considered to have been fertilized if cell divisions were visible at 160=magnification. A minimum of 300 eggs was examined from each sample for the calculation of absolute fertilization

Žw x w x.

rate number of eggs with cell divisionsrtotal number of eggs in sample and relative

Žw x w

fertilization rate absolute treatment fertilization raterabsolute control fertilization

x. Žw x. Žw

rate . Egg viability live fertilizedqunfertilized eggs r live fertilizedqunfertilized x.

qdead eggs was calculated for each egg sample. An egg was defined as unfertilized if it was clear or had a well-defined blastodisc; eggs that did not fulfill these criteria were

Ž .

defined as dead. Sperm motility was assessed both for undiluted control samples and for diluted samples at the beginning, middle and end of each fertilization trial. Sperm motility after dilution ranged from 45 to 175 s, and was not affected by the amount of dilution or time of testing within a trial.

Linear regression analysis was used to determine whether spermatocrit could be used to estimate sperm density, and whether there was a relationship between sperm density and sperm motility. Fertilization rate data were analyzed by one-way analysis of

Ž . Ž . Ž . Ž .

variance ANOVA on rank Kruskal-Wallis MINITAB version 11.1 Zar, 1996 .

3. Results

A significant positive relationship was found between spermatocrit and sperm density Ž_{Fig. 2; p}_-_{0.001, r}2_s_{0.89 . Sperm density ranged from 1.95}_. ₌₁₀11 _{to 6.13}₌₁₀11 spermatozoarml at 23% and 99% spermatocrit, respectively. Sperm density and

sperma-Ž .

tocrit decreased when males were implanted with GnRH a , but this did not affect the Ž .

relationship between sperm density and spermatocrit Fig. 2 . The lowest spermatocrit Ž .

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Fig. 3. The relationship between spermatocrit and sperm motility defined as the time elapsed until-5% of

the cells maintained forward swimming activity for 36 milt samples collected from 17 male Atlantic halibut

Žvdenotes samples obtained after GnRH a implantation;Ž . Idenotes no GnRH a implantation . Each pointŽ . .

represents the mean of six replicates. The bars indicate SD.

No relationship was observed between sperm density and sperm motility at the

Ž 2 _.

densities tested Fig. 3; ps0.35, r s0.03 . The mean duration of sperm motility for Ž .

individual samples ranged from 63 to 155 s. GnRH a treatments had no apparent effect

Fig. 4. The relationship between sperm motility defined as the time elapsed from activation until-5% of the

spermatozoa maintained forward swimming activity and the percentage of spermatozoa that were immediately

activated upon addition of water to 36 milt samples collected from 17 male Atlantic halibut v denotes

Ž . Ž . .

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Ž .

Fig. 5. Fertilization rates relative to control for Atlantic halibut eggs fertilized at varying sperm:egg volume

Ž .

ratios. For the control, undiluted milt was used at a 1:500 sprem:egg volume ratio. All test ratios used milt diluted to a spermatocrit of 58–60% with halibut seminal plasma. Values are means of three trials"SD.

Ž .

on the relationship between sperm density and sperm motility Fig. 3 . The duration of sperm motility was not influenced by the percentage of spermatozoa that were

immedi-Ž 2 _.

ately activated upon addition of water to the samples Fig. 4; ps0.76, r s0.003 . The proportion of motile spermatozoa decreased with time from activation.

There was no difference in the fertilization rate among sperm:egg volume ratios

Ž . Ž .

tested Hs4.6, ps0.71, ns18 Fig. 5 The viability of egg batches used for the three fertilization trials were 99%, 99% and 92%, respectively. The actual fertilization rates for the control were 96%, 76% and 73%. Sperm motility estimates prior to fertilization for the trials were 163, 202 and 66 s.

4. Discussion

The highly significant relationship found between spermatocrit and sperm density allows for the use of spermatocrit as a simple and rapid estimator of sperm density in Atlantic halibut. Although such a relationship has previously been reported for several

non-flatfish teleost species Bouck and Jacobson, 1976; Piironen, 1985; Ciereszko and .

Dabrowski, 1993; Rakitin et al., 1999 , an earlier study with turbot failed to find such a

Ž .

relationship Suquet et al., 1992b .

Ž 11 11 _.

The observed sperm densities for halibut 2=10 to 6=10 spermatozoarml are Ž

1–2 orders of magnitude higher than reported for other teleost species Mounib, 1978; Aas et al., 1991; Suquet et al., 1992b; Poole and Dillane, 1998; Fauvel et al., 1999;

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Rakitin et al., 1999 . Atlantic halibut also have high total ejaculate volumes per stripping Ž_{1–60 ml: Methven and Crim, 1991 compared to turbot 0.2–2.2 ml: Suquet et al.,}. Ž

1992a, 1994 . Higher sperm densities in halibut are likely a reflection of their spawning location, fecundity and egg size. Atlantic halibut spawn at depths of 300–700 m ŽDevold, 1938 , in total darkness. Fecundity is very high: a 1.95-m female can produce.

Ž .

up to 7 million eggs per spawning season Haug and Gulliksen, 1988 . Spread among

Ž .

10–15 batches at 3–4 day intervals Norberg and Holm, 1995 , with approximately

Ž .

50,000 eggsrl Mangor-Jensen et al., 1998 , this is equivalent to 140-l eggs per season

Ž .

with 9–14 l eggsrbatch. Although cod have similarly high fecundity Powles, 1958 ,

Ž .

they spawn in shallower water Scott and Scott, 1988 . Halibut also have among the

Ž .

largest eggs of all marine teleosts Russell, 1976 , with an average diameter of 3.0–3.5

Ž . Ž .

mm Haug et al., 1984 compared to 1.2–1.6 mm for cod Scott and Scott, 1988 . This combination of producing large volumes of large eggs in a dark environment may require exceptionally high sperm densities to ensure reproductive success.

Ž .

Spermatocrit values obtained prior to GnRH a implantation were similar to those

Ž .

previously reported for Atlantic halibut Methven and Crim, 1991 . In our study, Ž .

implanting males with GnRH a resulted in reduced spematocrit values but this did not Ž .

affect sperm motility. Increased sperm fluidity following GnRH a implantation has

Ž .

been reported for plaice, Pleuronectes platessa Vermeirssen et al., 1998 , winter

Ž . Ž

flounder, P. americanus Shangguan and Crim, 1999 and Atlantic halibut Martin-. Ž .

Robichaud et al., 2000; Vermeirssen et al., 2000a . GnRH a treatment was found to Ž

improve sperm motility in yellowtail flounder, P. ferrugineus Clearwater and Crim,

. Ž .

1998 , but not in winter flounder Shangguan and Crim, 1999 . However, in a study with

Ž .

Atlantic halibut Vermeirssen et al., 2000a , motility was reported to be significantly higher in treated milt samples compared to controls 40 days after implantation. In our

Ž .

study, samples were collected 2–3 weeks after GnRH a implantation, which may explain why there were no differences among the samples.

Fertilization success was largely independent of sperm density within a range of 9=105 _{to 5}₌₁₀8 _spermatozoa_r_{egg. Thus, although this study failed to find a} minimum sperm:egg volume ratio for ensuring good fertilization success, it did show that high fertilization success can be obtained over a wide range of sperm:egg volume

Ž .

ratios. Vermeirssen et al. 2000b have recently shown that this ratio can be successfully lowered to 104 spermatozoaregg, but this is still higher than the sperm densities

necessary for successful fertilization in other teleost species Gwo et al., 1991; Suquet et .

al., 1995 .

Acknowledgements

This research was funded through an NSERC Strategic Grant, with additional infrastructure support from Fisheries and Oceans Canada. We thank Drs. Michael Reith ŽNRC Institute for Marine Biosciences, Halifax and Allen Curry UNB Department of. Ž

Biology, Fredericton for their input into this study. In addition, we thank Dr. Jim Ž .

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References

Aas, G.H., Refstie, T., Gjerde, B., 1991. Evaluation of milt quality of Atlantic salmon. Aquaculture 95, 125–132.

Baynes, S.M., Scott, A.P., 1985. Seasonal variation in parameters of milt production and plasma concentration

Ž .

of sex steroid in male rainbow trout Salmo gairdneri . Gen. Comp. Endocrinol. 57, 150–160.

Billard, R., 1978. Changes in structure and fertilizing ability of marine and freshwater fish spermatozoa diluted in media of various salinities. Aquaculture 14, 187–198.

Bonnet, D.D., 1939. Mortality of the cod egg in relation to temperature. Biol. Bull. 76, 428–441.

Bouck, G.R., Jacobson, J., 1976. Estimation of salmonid sperm concentration by microhematocrit technique. Trans. Am. Fish. Soc. 105, 534–535.

Bromage, N.R., Roberts, R.J., 1995. Broodstock Management and Egg and Larval Quality. Cambridge Univ. Press, Cambridge.

Ž .

Brown, J., Helm, M., Moir, J., 1995. New-candidate species for aquaculture. In: Boghen, A.D. Ed. , Coldwater Aquaculture in Atlantic Canada. 2nd edn. Can. Inst. Res. Reg. Dev., Moncton, pp. 341–362. Chereguini, O., Garcıa de la Banda, I., Rasines, I., Fernandez, A., 1999. Artificial fertilization in turbot,´

Scophthalmus maximus: different methods and determination of the optimal sperm–egg ratio. Aquacult.

Res. 30, 319–324.

Ciereszko, A., Dabrowski, K., 1993. Estimation of sperm concentration in rainbow trout, whitefish and yellow perch using a spectrophotometric technique. Aquaculture 109, 367–373.

Clearwater, S.J., Crim, L.W., 1998. Gonadotropin releasing hormone-analogue treatment increases sperm motility, seminal plasma pH and sperm production in yellowtail flounder Pleuronectes ferrugineus. Fish Physiol. Biochem. 19, 349–357.

Devold, F., 1938. The North Atlantic halibut and net fishing. Fiskeridir. Skr., Ser. Havunders. 5, 1–47. Fauvel, C., Savoye, O., Dreanno, C., Cosson, J., Suquet, M., 1999. Characteristics of sperm of captive seabass

in relation to its fertilization potential. J. Fish Biol. 54, 356–369.

Gwo, J.-C., Strawn, K., Longnecker, M.T., Arnold, C.R., 1991. Cryopreservation of Atlantic croaker spermatozoa. Aquaculture 94, 355–375.

Haug, T., Gulliksen, B., 1988. Fecundity and oocyte sizes in ovaries of female Atlantic halibut, Hippoglossus

Ž .

hippoglossus L. . Sarsia 73, 259–261.

Haug, T., Kjorsvik, E., Solemdal, P., 1984. Vertical distribution of Atlantic halibut, Hippoglossus

hippoglos-sus, eggs. Can. J. Fish. Aquat. Sci. 41, 798–804.

Kjørsvik, E., Mangor-Jensen, A., Holmefjord, I., 1990. Egg quality in fishes. In: Blaxter, J.H.S., Southward,

Ž .

A.J. Eds. , Adv. Mar. Biol., vol. 26, pp. 71–113.

Mangor-Jensen, A., Harboe, T., Shields, R.J., Naas, K.E., 1998. Atlantic halibut, Hippoglossus hippoglossus L., larvae cultivation literature, including a bibliography. Aquacult. Res. 29, 857–886.

Martin-Robichaud, D.J., Powell, J., Wade, J., 2000. Gonadotropin-releasing hormone affects sperm production

Ž . Ž .

of Atlantic halibut Hippoglossus hippoglossus . Bull. Aquacult. Assoc. Can. In press .

Methven, D.A., Crim, L.W., 1991. Seasonal changes in spermatocrit, plasma sex steroids, and motility of sperm from Atlantic halibut, Hippoglossus hippoglossus. In: Scott, A.P., Sumpter, J.P., Kime, D.E., Rolfe,

Ž .

M.S. Eds. , Proc. Fourth Int. Symp. Reprod. Physiol. Fish, Sheffield. p. 170.

Mounib, M.S., 1978. Cryogenic preservation of fish and mammalian spermatozoa. J. Reprod. Fertil. 53, 13–18.

Ž .

Munkittrick, K.R., Moccia, R.D., 1987. Seasonal changes in the quality of rainbow trout Salmo gairdneri semen: effect of a delay in stripping on spermatocrit, motility, volume and seminal plasma constituents. Aquaculture 64, 147–156.

Norberg, B., Holm, J.C., 1995. Sesonguavhengig produksjon av egg og larver. In: Pittman, K., Kjorrefjord,

Ž .

A.G., Berg, L., Engelsen, R. Eds. , Kveite- fra forskning til nœring. John Grieg, pp. 15–25.

Norberg, B., Valkner, V., Huse, J., Karlsen, I., Lerøy Grung, G., 1991. Ovulatory rhythms and egg viability in

Ž .

the Atlantic halibut Hippoglossus hippoglossus . Aquaculture 97, 365–371.

Piironen, J., 1985. Variation in the properties of milt from the Finnish landlocked salmon Salmo salar m.

sebago Girard during a spawning season. Aquaculture 48, 337–350.

Poole, W.R., Dillane, M.G., 1998. Estimation of sperm concentration of wild and reconditioned brown trout,

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Ž .

Powles, P.M., 1958. Studies of reproduction and feeding of Atlantic cod Gadus callarias L. in the Southwestern Gulf of St. Lawrence. J. Fish. Res. Bd. Can. 15, 1383–1402.

Rabben, H., Nilsen, T.O., Huse, I., Jelmert, A., 1986. Production experiment of halibut fry in large enclosed

Ž .

water columns. Counc. Meet Int. Counc. Explor. Sea F:19 .

Rakitin, A., Ferguson, M.M., Trippel, E.A., 1999. Spermaocrit and spermatozoa density in Atlantic cod

ŽGadus morhua : correlation and variation during the spawning season. Aquaculture 170, 349–358..

Russell, F.S., 1976. The Eggs and Planktonic Stages of British Marine Fishes. Academic Press, London. Scott, W.B., Scott, M.G., 1988. Atlantic fishes of Canada. Can. Bull. Fish. Aquat. Sci. 219, 731 pp. Shangguan, B., Crim, L.W., 1999. Seasonal variations in sperm production and sperm quality in male winter

flounder, Pleuronectes americanus: the effects of hypophysectomy, pituitary replacement therapy, and GnRH-A treatment. Mar. Biol. 134, 19–27.

Shields, R.J., Brown, N.P., Bromage, N.R., 1997. Blastomere morphology as a predictive measure of fish egg viability. Aquaculutre 155, 1–12.

Ž .

Stoss, J., Donaldson, E.M., 1982. Preservation of fish gametes. In: Richter, C.J.J., Goos, H.J.Th. Eds. , Proc. Int. Symp. Reprod. Physiol. Fish, Wageningen, pp. 114–122.

Suquet, M., Omnes, M.H., Normant, Y., Fauvel, C., 1992a. Influence of photoperiod, frequency of stripping

Ž .

and presence of females on sperm output in turbot Scophthalmus maximus . Aquacult. Fish Manage. 23, 217–225.

Suquet, M., Omnes, M.H., Normant, Y., Fauvel, C., 1992b. Assessment of sperm concentration and motility in

Ž .

turbot Scophthalmus maximus . Aquaculture 101, 177–185.

Suquet, M., Billard, R., Cosson, J., Dorange, G., Chauvaud, L., Mugnier, C., Fauvel, C., 1994. Sperm features

Ž .

in turbot Scophthalmus maximus : a comparison with other freshwater and marine species. Aquat. Living Resour. 7, 283–294.

Suquet, M., Billard, R., Cosson, J., Normant, Y., Fauvel, C., 1995. Artificial insemination in turbot

ŽScophthalmus maximus : determination of the optimal sperm to egg ratio and time of gamete contact..

Aquaculture 133, 83–90.

Vermeirssen, E.L.M., Scott, A.P., Mylonas, C.C., Zohar, Y., 1998. Gonadotrophin-releasing hormone agonist stimulates milt fluidity and plasma concentrations of 7,20b-dihydroxylated and 5b-reduced, 3a

-hydroxyl-Ž .

ated C21steroids in male plaice Pleuronectes platessa . Gen. Comp. Endocrinol. 112, 163–177. Vermeirssen, E.L.M., Shields, R.J., Mazorra de Quero, C., Scott, A.P., 2000a. Gonadotrophin-releasing

hormone agonist raises plasma concentrations of progestogens and enhances milt fluidity in male Atlantic

Ž .

halibut Hippoglossus hippoglossus . Fish Physiol. Biochem. 22, 77–87.

Vermeirssen, E.L.M., Mazorra de Quero, C., Shields, R., Norberg, B., Scott, A.P., Kime, D.E., 2000b. The application of GnRHa implants in male Atlantic halibut; effects on steroids, milt hydration, sperm motility

Ž .

and fertility. In: Norberg, B., Kjesbu, O.S., Taranger, G.L., Andersson, E., Stefansson, S.O. Eds. , Proc. Sixth Int. Symp. Reprod. Physiol. Fish, Bergen. pp. 391–401.

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Ž

.

volume of milt to 25 ml eggs ca. 1250 eggs measured in a graduated cylinder, 25 ml of

Ž

.

saltwater 33 ppt was added and the mixture stirred for 20 s. After 10 min, the eggs

Ž

were rinsed and transferred to beakers containing 500 ml saltwater UV-treated and

.

filtered through 5.0 and 0.35

m

m cartridge filters for incubation in the dark. At 18–22 h

Ž

.

post-fertilization at the 8- and 16-cell stages , samples of eggs were preserved in

Ž

.

Stockard’s Solution Bonnet, 1939 for fertilization assessment. Eggs were considered to

have been fertilized if cell divisions were visible at 160

=

magnification. A minimum of

300 eggs was examined from each sample for the calculation of absolute fertilization

Žw

x w

x.

rate

number of eggs with cell divisions

r

total number of eggs in sample and relative

Žw

x w

fertilization rate

absolute treatment fertilization rate

r

absolute control fertilization

x.

Žw

x. Žw

rate . Egg viability

live fertilized

q

unfertilized eggs

r

live fertilized

q

unfertilized

x.

q

dead eggs was calculated for each egg sample. An egg was defined as unfertilized if

it was clear or had a well-defined blastodisc; eggs that did not fulfill these criteria were

Ž

.

defined as dead. Sperm motility was assessed both for undiluted control samples and

for diluted samples at the beginning, middle and end of each fertilization trial. Sperm

motility after dilution ranged from 45 to 175 s, and was not affected by the amount of

dilution or time of testing within a trial.

Linear regression analysis was used to determine whether spermatocrit could be used

to estimate sperm density, and whether there was a relationship between sperm density

and sperm motility. Fertilization rate data were analyzed by one-way analysis of

Ž

.

Ž

. Ž

.

variance ANOVA on rank Kruskal-Wallis

MINITAB version 11.1

Zar, 1996 .

3. Results

A significant positive relationship was found between spermatocrit and sperm density

Ž

_{Fig. 2; p}

_-

_{0.001, r}

_s

_{0.89 . Sperm density ranged from 1.95}

_.

₌

₁₀

_{to 6.13}

₌

₁₀

spermatozoa

r

ml at 23% and 99% spermatocrit, respectively. Sperm density and

sperma-Ž .

tocrit decreased when males were implanted with GnRH a , but this did not affect the

Ž

.

relationship between sperm density and spermatocrit Fig. 2 . The lowest spermatocrit

Ž .

(2)

Fig. 3. The relationship between spermatocrit and sperm motility defined as the time elapsed until-5% of .

the cells maintained forward swimming activity for 36 milt samples collected from 17 male Atlantic halibut Žvdenotes samples obtained after GnRH a implantation;Ž . Idenotes no GnRH a implantation . Each pointŽ . . represents the mean of six replicates. The bars indicate SD.

No relationship was observed between sperm density and sperm motility at the

Ž

_.

densities tested Fig. 3; p

s

0.35, r

s

0.03 . The mean duration of sperm motility for

Ž .

individual samples ranged from 63 to 155 s. GnRH a treatments had no apparent effect

Fig. 4. The relationship between sperm motility defined as the time elapsed from activation until-5% of the .

spermatozoa maintained forward swimming activity and the percentage of spermatozoa that were immediately Ž

activated upon addition of water to 36 milt samples collected from 17 male Atlantic halibut v denotes

Ž . Ž . .

(3)

Ž .

Fig. 5. Fertilization rates relative to control for Atlantic halibut eggs fertilized at varying sperm:egg volume

Ž .

Ž

.

on the relationship between sperm density and sperm motility Fig. 3 . The duration of

sperm motility was not influenced by the percentage of spermatozoa that were

immedi-Ž

_.

ately activated upon addition of water to the samples Fig. 4; p

s

0.76, r

s

0.003 . The

proportion of motile spermatozoa decreased with time from activation.

There was no difference in the fertilization rate among sperm:egg volume ratios

Ž

. Ž

.

tested H

s

4.6, p

s

0.71, n

s

18 Fig. 5 The viability of egg batches used for the

three fertilization trials were 99%, 99% and 92%, respectively. The actual fertilization

rates for the control were 96%, 76% and 73%. Sperm motility estimates prior to

fertilization for the trials were 163, 202 and 66 s.

4. Discussion

The highly significant relationship found between spermatocrit and sperm density

allows for the use of spermatocrit as a simple and rapid estimator of sperm density in

Atlantic halibut. Although such a relationship has previously been reported for several

Ž

non-flatfish teleost species Bouck and Jacobson, 1976; Piironen, 1985; Ciereszko and

.

Dabrowski, 1993; Rakitin et al., 1999 , an earlier study with turbot failed to find such a

Ž

.

relationship Suquet et al., 1992b .

Ž

11 11

_.

The observed sperm densities for halibut 2

=

10 to 6

=

10 spermatozoa

r

ml are

Ž

1–2 orders of magnitude higher than reported for other teleost species Mounib, 1978;

Aas et al., 1991; Suquet et al., 1992b; Poole and Dillane, 1998; Fauvel et al., 1999;

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location, fecundity and egg size. Atlantic halibut spawn at depths of 300–700 m

Ž

Devold, 1938 , in total darkness. Fecundity is very high: a 1.95-m female can produce

.

Ž

.

up to 7 million eggs per spawning season Haug and Gulliksen, 1988 . Spread among

Ž

.

10–15 batches at 3–4 day intervals Norberg and Holm, 1995 , with approximately

Ž

.

50,000 eggs

r

l Mangor-Jensen et al., 1998 , this is equivalent to 140-l eggs per season

Ž

.

with 9–14 l eggs

r

batch. Although cod have similarly high fecundity Powles, 1958 ,

Ž

.

they spawn in shallower water Scott and Scott, 1988 . Halibut also have among the

Ž

.

largest eggs of all marine teleosts Russell, 1976 , with an average diameter of 3.0–3.5

Ž

.

Ž

.

mm Haug et al., 1984 compared to 1.2–1.6 mm for cod Scott and Scott, 1988 . This

combination of producing large volumes of large eggs in a dark environment may

require exceptionally high sperm densities to ensure reproductive success.

Ž .

Spermatocrit values obtained prior to GnRH a implantation were similar to those

Ž

.

previously reported for Atlantic halibut

Methven and Crim, 1991 . In our study,

Ž .

implanting males with GnRH a resulted in reduced spematocrit values but this did not

Ž .

affect sperm motility. Increased sperm fluidity following GnRH a implantation has

Ž

.

been reported for plaice, Pleuronectes platessa Vermeirssen et al., 1998 , winter

Ž

.

Ž

flounder, P. americanus Shangguan and Crim, 1999 and Atlantic halibut

Martin-.

Ž .

Robichaud et al., 2000; Vermeirssen et al., 2000a . GnRH a treatment was found to

Ž

improve sperm motility in yellowtail flounder, P. ferrugineus Clearwater and Crim,

.

Ž

.

1998 , but not in winter flounder Shangguan and Crim, 1999 . However, in a study with

Ž

.

Atlantic halibut Vermeirssen et al., 2000a , motility was reported to be significantly

higher in treated milt samples compared to controls 40 days after implantation. In our

Ž .

study, samples were collected 2–3 weeks after GnRH a implantation, which may

explain why there were no differences among the samples.

Fertilization success was largely independent of sperm density within a range of

9 =

10 _{to 5}

₌

₁₀

_spermatozoa

_r

_{egg. Thus, although this study failed to find a}

minimum sperm:egg volume ratio for ensuring good fertilization success, it did show

that high fertilization success can be obtained over a wide range of sperm:egg volume

Ž

.

ratios. Vermeirssen et al. 2000b have recently shown that this ratio can be successfully

lowered to 10

spermatozoa

r

egg, but this is still higher than the sperm densities

Ž

necessary for successful fertilization in other teleost species Gwo et al., 1991; Suquet et

.

al., 1995 .

Acknowledgements

This research was funded through an NSERC Strategic Grant, with additional

infrastructure support from Fisheries and Oceans Canada. We thank Drs. Michael Reith

Ž

NRC Institute for Marine Biosciences, Halifax and Allen Curry UNB Department of

.

Ž

.

Biology, Fredericton for their input into this study. In addition, we thank Dr. Jim

Ž .

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Chereguini, O., Garcıa de la Banda, I., Rasines, I., Fernandez, A., 1999. Artificial fertilization in turbot,´

Scophthalmus maximus: different methods and determination of the optimal sperm–egg ratio. Aquacult.

Res. 30, 319–324.

Ciereszko, A., Dabrowski, K., 1993. Estimation of sperm concentration in rainbow trout, whitefish and yellow perch using a spectrophotometric technique. Aquaculture 109, 367–373.

in relation to its fertilization potential. J. Fish Biol. 54, 356–369.

Gwo, J.-C., Strawn, K., Longnecker, M.T., Arnold, C.R., 1991. Cryopreservation of Atlantic croaker spermatozoa. Aquaculture 94, 355–375.

Haug, T., Gulliksen, B., 1988. Fecundity and oocyte sizes in ovaries of female Atlantic halibut, Hippoglossus Ž .

hippoglossus L. . Sarsia 73, 259–261.

Haug, T., Kjorsvik, E., Solemdal, P., 1984. Vertical distribution of Atlantic halibut, Hippoglossus

hippoglos-sus, eggs. Can. J. Fish. Aquat. Sci. 41, 798–804.

Kjørsvik, E., Mangor-Jensen, A., Holmefjord, I., 1990. Egg quality in fishes. In: Blaxter, J.H.S., Southward, Ž .

A.J. Eds. , Adv. Mar. Biol., vol. 26, pp. 71–113.

Martin-Robichaud, D.J., Powell, J., Wade, J., 2000. Gonadotropin-releasing hormone affects sperm production

Ž . Ž .

of Atlantic halibut Hippoglossus hippoglossus . Bull. Aquacult. Assoc. Can. In press .

Ž .

M.S. Eds. , Proc. Fourth Int. Symp. Reprod. Physiol. Fish, Sheffield. p. 170.

Mounib, M.S., 1978. Cryogenic preservation of fish and mammalian spermatozoa. J. Reprod. Fertil. 53, 13–18.

Ž .

Norberg, B., Holm, J.C., 1995. Sesonguavhengig produksjon av egg og larver. In: Pittman, K., Kjorrefjord, Ž .

A.G., Berg, L., Engelsen, R. Eds. , Kveite- fra forskning til nœring. John Grieg, pp. 15–25.

Norberg, B., Valkner, V., Huse, J., Karlsen, I., Lerøy Grung, G., 1991. Ovulatory rhythms and egg viability in

Ž .

the Atlantic halibut Hippoglossus hippoglossus . Aquaculture 97, 365–371.

Piironen, J., 1985. Variation in the properties of milt from the Finnish landlocked salmon Salmo salar m. .

sebago Girard during a spawning season. Aquaculture 48, 337–350.

Poole, W.R., Dillane, M.G., 1998. Estimation of sperm concentration of wild and reconditioned brown trout,

(6)

Rakitin, A., Ferguson, M.M., Trippel, E.A., 1999. Spermaocrit and spermatozoa density in Atlantic cod ŽGadus morhua : correlation and variation during the spawning season. Aquaculture 170, 349–358.. Russell, F.S., 1976. The Eggs and Planktonic Stages of British Marine Fishes. Academic Press, London. Scott, W.B., Scott, M.G., 1988. Atlantic fishes of Canada. Can. Bull. Fish. Aquat. Sci. 219, 731 pp. Shangguan, B., Crim, L.W., 1999. Seasonal variations in sperm production and sperm quality in male winter

flounder, Pleuronectes americanus: the effects of hypophysectomy, pituitary replacement therapy, and GnRH-A treatment. Mar. Biol. 134, 19–27.

Shields, R.J., Brown, N.P., Bromage, N.R., 1997. Blastomere morphology as a predictive measure of fish egg viability. Aquaculutre 155, 1–12.

Ž . Stoss, J., Donaldson, E.M., 1982. Preservation of fish gametes. In: Richter, C.J.J., Goos, H.J.Th. Eds. , Proc.

Int. Symp. Reprod. Physiol. Fish, Wageningen, pp. 114–122.

Suquet, M., Omnes, M.H., Normant, Y., Fauvel, C., 1992a. Influence of photoperiod, frequency of stripping

Ž .

and presence of females on sperm output in turbot Scophthalmus maximus . Aquacult. Fish Manage. 23, 217–225.

Suquet, M., Omnes, M.H., Normant, Y., Fauvel, C., 1992b. Assessment of sperm concentration and motility in

Ž .

turbot Scophthalmus maximus . Aquaculture 101, 177–185.

Suquet, M., Billard, R., Cosson, J., Dorange, G., Chauvaud, L., Mugnier, C., Fauvel, C., 1994. Sperm features

Ž .

in turbot Scophthalmus maximus : a comparison with other freshwater and marine species. Aquat. Living Resour. 7, 283–294.

Suquet, M., Billard, R., Cosson, J., Normant, Y., Fauvel, C., 1995. Artificial insemination in turbot ŽScophthalmus maximus : determination of the optimal sperm to egg ratio and time of gamete contact.. Aquaculture 133, 83–90.

Vermeirssen, E.L.M., Scott, A.P., Mylonas, C.C., Zohar, Y., 1998. Gonadotrophin-releasing hormone agonist stimulates milt fluidity and plasma concentrations of 7,20b-dihydroxylated and 5b-reduced, 3a

-hydroxyl-Ž .

ated C21steroids in male plaice Pleuronectes platessa . Gen. Comp. Endocrinol. 112, 163–177. Vermeirssen, E.L.M., Shields, R.J., Mazorra de Quero, C., Scott, A.P., 2000a. Gonadotrophin-releasing

hormone agonist raises plasma concentrations of progestogens and enhances milt fluidity in male Atlantic

Ž .

halibut Hippoglossus hippoglossus . Fish Physiol. Biochem. 22, 77–87.

Ž . and fertility. In: Norberg, B., Kjesbu, O.S., Taranger, G.L., Andersson, E., Stefansson, S.O. Eds. , Proc. Sixth Int. Symp. Reprod. Physiol. Fish, Bergen. pp. 391–401.