Malison, J. A,, and J. A. Held. 1996. Reproductive biology and spawning. Pages 11-18 in R. C. 

Summerfelt, editor. Walleye culture manual. NCRAC Culture Series 101. North Central Regional 

Aquaculture Center Publications Office, Iowa State University, Ames. 

Reproductive Biology 

Spawning 

and 

Jeffrey A. Malison and James A. Held, University of Wisconsin-Madison Aquaculture Program, Department of 

Food Science, 123 Babcock Hall, Madison, WI 53706 

An understanding of the basic mechanisms that control 

reproduction is an important prerequisite for the 

efficient commercial culture of any species. The case 

studies that accompany ths chapter deal primarily with 

techniques to artificially propagate walleye. In order to 

furnish the reader with the underlying rationale for the 

development of these specific techniques, we have 

provided a brief description of the reproductive biology 

of walleye in the first half of this chapter. The second 

half summarizes walleye propagation techniques and 

highlights some of the hfferent spawning, incubation, 

and hatching protocols used by walleye culturists. 

Walleye reproduction 

Sexual maturity in walleye is dependent on both the 

size and age of the fish and, therefore, dependent to 

some extent on ambient water temperatures and food 

availability (Ney 1978). In the wild, males generally 

mature at 2-4 years and at a size greater than 11 in (280 

mm) in total length (TL). Females mature at 3-6 years 

and at a size greater than 14 in (360 mm) in TL (Scott 

and Crossman 1973). With proper control of water 

temperature and photoperiod, captive broodstock can be 

successfully spawned (Nagel 1985; unpublished data). 

Intensive culture can provide improved food availability 

and environmental conditions that are more conducive 

to rapid growth than those generally found in the wild, 

thereby resulting in early maturation (our unpublished 

observations). 

Walleye spawn annually in the spring, when water 

temperatures reach 3844°F (3.6-6.7"C), which is 

shortly after ice breakup in most locations (Becker 

1983). The spawning season may begin as early as 

February in the southern extremes of the walleye's 

range, and end as late as July in the northern extremes 

(Hokanson 1977). Peak spawning activity occurs at 

water temperatures of 42-50°F (5.6-10°C; Becker 

1983), and generally lasts for 1-2 weeks at any one 

location. The level and duration of peak spawning 

activity can be affected by rapid changes in the weather. 

Courtshp behavior has been described by Ellis and 

Giles (1965). In brief, it is characterized by approaches 

and contacts, with no continued relationshp between 

any particular pair of fish. The spawning act culminates 

in a rush to the surface by a female and two flanking 

males, with other males in close pursuit (Priegel 1970), 

and a turning or pushng of the female onto its side 

which indicates spawning has occurred. 

Eggs are broadcast onto suitable substrates, and walleye 

do not build nests or provide any parental protection 

(Becker 1983). Typical spawning sites include gravel 

and rubble shoals (Johnson 1961), gravel bottoms of 

inlet streams and rocky wave-washed shallows (Becker 

1983), or flooded wetland vegetation (Priegel 1970). 

Females can completely spawn in one night (Ellis and 

Giles 1965). O'Donnell (1 938) observed that 200-300 

eggs were released during each spawning act, with the 

acts repeated at 5-min intervals. The number of eggs 

produced and released is related to body size; it 

averages 27,00O/lb (60,000kg; Stickney 1986), and 

ranges from 12,500-55,000/1b (28,000-1 20,OOOkg; 

Smith 1941; Wolfert 1969). 

Like most temperate freshwater fishes, the reproductive 

processes of walleye are controlled by environmental 

cues, and the annual reproductive cycle is geared so that 

NCRAC Culture Series 101 - Walleye Culture Manual 11 
progeny are produced in the spring when conditions are 

most favorable for their survival (Redding and Patiiio 

1993). Seasonal variations in photoperiod and water 

temperature elicit gonadal and hormonal changes that 

result in spawning. The annual cycle for walleye can be 

characterized by changes in gonadosomatic indces 

(GSIs; the ratio of gonad weight to total body weight), 

gonadal histology, and serum levels of steroidogenic 

hormones, particularly testosterone (T) and 11 - 

ketotestosterone (1 1-KT) in males and estradiol-1 7p 

(E,) in females. The following description of the annual 

cycle of walleye (Malison et al. 1994b) pertains to fish 

captured from central Minnesota, and the timing of 

specific stages of gonadal development may vary by 

one month or more in walleye from other latitudes. 

Just after spawning, males exhibit low GSIs and the 

testes are comprised primarily of nongerminal tissue. A 

few scattered lobules of spermatogonia are present at 

the periphery of the testes. Serum levels of T and 11- 

KT are low at this time. By late summer, there is little 

increase in GSIs or androgen levels, but in the majority 

of males an increased number of spermatogonia and a 

few spermatocytes can be observed. By early fall, 

increased spermatogenic activity causes GSIs to 

increase. In mid-fall, approximately one-half of the 

germ cells appear to be mature spermatozoa and GSIs 

continue to increase. Additionally, increases in serum 

levels of T can be measured. By midwinter, almost all 

of the germ cells present are spermatozoa, and some 

milt can be expressed. By early spring, there are few 

additional changes in the physical appearance of the 

testes, but continued increases in the serum levels of T 

as well as elevations in serum 11-KT can be measured. 

Finally, at spawning, sharp decreases in the GSIs and 

both androgen levels occur, as spermatozoa are expelled 

and the gonads return to mostly non-germinal tissue. 

Ovarian development in walleye is group synchronous, 

(Malison et al. 1994b), meaning that two populations of 

oocytes at different developmental stages can be 

hstinguished at some point during the annual reproductive cycle (Nagahama 1983). One of these populations 

consists of a heterogeneous group of small oocytes, and 

the other consists of a group of larger oocytes developing synchronously for the next spawning season. 

Just after spawning, the GSIs of female walleye are low, 

and ovaries are comprised of oogonia and 

nonvitellogenic oocytes in equal numbers. A few 

unspawned vitellogenic oocytes are still present, and 

serum E,levels are low. By late spring, ovaries are 

already filled with a large number of nonvitellogenic 

oocytes, indicating that female walleye have a relatively 

short post-spawning quiescent period. By midsummer, 

yolky vesicles can be observed in most of the oocytes. 

During autumn, with the onset of exogenous vitellogenesis and a correspondmg increase in mean oocyte 

diameters, GSIs rise rapidly and serum E, levels 

increase sharply to their highest levels. Increases in 

oocyte diameters and GSIs continue until late January, 

when the oocytes appear to be near maturity (Malison 

et al. 1994b). Serum E,levels steadily decline throughout winter and spring. 

Shortly before spawning, the oocytes undergo final 

maturation and ovulation. There is some evidence that 

17a, 20P-dihydroxyprogesterone (17-20P) is the steroid 

responsible for the final maturation process in walleye 

(Pankhurst et al. 1986; Barry et al. 1995) which occurs 

just prior to spawning. The stages of final oocyte 

maturation are characterized by changes in the germinal 

vesicle (GV), which breaks down during maturation, 

and the coalescence of the oil droplet (Figure 1, 

opposite page). Maturing oocytes can be classified into 

five dstinct stages: (1) those having a centrally located 

GV with many small oil droplets present; (2) those 

showing migration of the GV, in which the GV is 

located between the center and the periphery, and 

numerous oil droplets are still present; (3) those having 

a GV located at the periphery, and fewer, larger oil 

droplets are present; (4) those in which germinal vesicle 

breakdown (GVBD) has occurred, and the oil droplet 

has coalesced; and (5) oocytes that have ovulated and 

have been expelled from the follicle. 

Spawning occurs shortly after ovulation, and the release 

of the eggs results in rapid decreases in GSIs. Eggs 

must be fertilized within 2 min after contact with water. 

Sperm are active and viable for less than 1 min after 

contacting water. The eggs have an adhesive coating 

that can act to attach them to the substrate over which 

they have been broadcast. Over the next several hours, 

the eggs swell to about twice their original size, a 

process known as “water hardening”, and lose their 

adhesive quality (Becker 1983). The development of 

walleye eggs (Figure 2, page 138) is greatly dependent 

on temperature. Niemuth et al. (1959) observed that 

walleye eggs hatch in 26 d at a water temperature of 

12 NCRAC Culture Series 101- Walleye Culture Manual 
Figure 1. Migration and breakdown of the germinal vesicle during the final maturation of walleye oocytes. A) 

centrally located germinal vesicle (GV), 6) migrating GV, C) peripheral GV, D) germinal vesicle breakdown (GVBD). 

NCRAC Culture Series 101 - Walleye Culture Manual 13 
Figure 2. Development of walleye embryos incubated on a slowly rising temperature regimen (50- 

60°F) that leads to hatch in 11-13 d. A) Approximately three and one-half hours after fertilization the 

first cleavage furrow forms, and one cell becomes 

two.The cell cap is developing in the upper left 

corner of the egg. Most of the remainder of the egg 

consists of yolk and a centrally-located oil droplet. 

B) Four d after fertilization, the developing embryo 

is wrapped around the yolk and oil droplet. The head 

and eyes are in the upper left corner of the photograph. C) A walleye egg seven d after fertilization, 

the head of the developing fish is in the right-hand 

side of the photograph. D) A newly-hatched walleye 

fry. 

14 NCRAC Culture Series 101- Walleye Culture Manual 
40°F (4.4"C), in 21 d at 50-55°F (10-12.8"C), and in 7 

d at 57°F (13.9"C). 

At hatch, walleye fry average 0.3 in (7.6 mm) in TL and 

can range in size from 0.25-0.37 in (6-9.5 mm) in TL 

(Priegel 1970). Generally, the yolk sac is absorbed by 

the time the fish reach 0.37 in (9.5 mm) (Houde 1969). 

According to Forney (1966), fry are photopositive and 

pelagic, and undergo the transition from endogenous to 

exogenous feeding 1-6 d after hatching. Others, 

however, have observed that walleye fry do not have a 

functional mouth until they are at least 3 d old (Robert 

Summerfelt, Iowa State University, personal communication). 

Walleye propagation 

Propagation of walleye begins with obtaining 

broodstock. Although a few hatcheries have captive 

broodstock, most operations depend on the capture of 

wild walleye broodstock. The case studies that accompany this chapter describe various broodstock collection 

methods, such as electroshock, gill nets, and a variety 

of trap nets. Generally, the methods are tailored to the 

spawning site and experience of the collection crew. In 

all cases, however, an emphasis should be placed on 

minimizing the stress on the fish while maximizing the 

quality and quantity of gametes collected. 

The first fish to be caught during the spawning run are 

generally males, and early in the season males produce 

copious amounts of milt. Late in the season, males can 

become difficult to locate, and can contain much 

smaller volumes of milt. This problem can be resolved 

by using one of several sperm extenders and storage 

techniques (see some of the accompanying case 

studies). To insure high fertilization rates, the quality of 

semen should be determined prior to its use. Contaminants such as feces, mucous, and particularly water can 

significantly affect both the duration that milt can be 

successfully stored and its fertilization capacity. Sperm 

viability or motility are usually characterized by 

activating a sample with water and estimating the 

percentage of motile spermatozoa and the duration of 

their activity. Techniques to measure sperm motility are 

described in some of the case studies accompanying 

this chapter. 

Most walleye culturists prefer to work with ripe and 

running females; i.e., females whose eggs have undergone ovulation. Although careful handling is required to 

reduce egg loss, these fish are immehately available for 

spawning, and because they can be quickly returned to 

the wild they are subjected to minimum handling stress. 

Several walleye culturists have described procedures for 

using "green" or unripe females. Tested protocols range 

from simply allowing green females to ovulate in 

captivity, to injecting them with various gonadotropic 

hormones. The identification of the stage of final oocyte 

maturation (described above) can be beneficial when 

working with green females, and by doing so one can 

decide to keep a green female for ripening in captivity 

or to release it for possible recapture. Oocytes can be 

easily collected from green females with a cannula; the 

oocytes are then placed into a clearing solution 

(ethano1:formalin:glacial acetic acid; 6:3: 1, v/v) and 

after 3-5 min observed under a microscope or hand 

lens. Fish having eggs in the early stages of final 

maturation (prior to stage 3) often will not ripen in 

captivity without the use of hormones. At our laboratory, we do not keep females unless the eggs have at 

least reached stage 4. It is important to note, however, 

that our captured broodstock are held in indoor tanks. 

Other holding facilities, such as net pens in a natural 

waterway, may allow less mature fish to ripen. Another 

important factor when holding wild walleye is the 

degree of stress to which the fish are subjected. Repetitive handling and prolonged captivity can result in high 

levels of mortality either during captivity or after 

release. 

Several hormone treatments can induce ovulation in 

green female walleyes. Hormone preparations like 

human chorionic gonadotropin (HCG), des-Gly'O-[DAla4] -LHRH - ethyl ami de (LHRHa) , carp pituitary 

extract, and several maturational steroids have been 

successfully used to induce final oocyte maturation in 

many fish species including walleye (Donaldson and 

Hunter 1983; Goetz 1983; Barry et al. 1995). Although 

such hormone treatments can be effective and are 

generally not harmful to the fish, current FDA regulations severely restrict the use of hormone injections in 

walleye. Another potential problem with using hormones is that poorly timed or improperly dosed 

treatments can be ineffective or induce ovulation prior 

to final oocyte maturation, the latter resulting in nonfertile eggs. Several successful methods of using 

hormones to induce spawning in walleye are hscussed 

in the accompanying case studies. 

NCRAC Culture Series 101 - Walleye Culture Manual 15 
Recent studies conducted by investigators from the 

University of Wisconsin-Ma&son and the University of 

Nebraska-Lincoln (Malison et al. 1994a) have shown 

that environmental manipulations and hormone 

injections can be successfully used to advance spawning in walleye. In one study, over 100 adult walleye 

were captured in the autumn and stocked into ponds at 

the Gavins Point National Fish Hatchery, Yankton, 

South Dakota. In late January, February, and March (as 

much as 2 months before normal spawning) groups of 

these fish were transferred to indoor tanks at the 

Calamus State Fish Hatchery, Bunvell, Nebraska. Water 

temperature was raised from 2-10°C over a 1 week 

interval; photoperiod was held at 12 h light: 12 h dark; 

fish were subjected to one of four injection regimens: 

saline control (d 0 and 2), HCG (150 IUkg on d 0 and 

500 IUkg on d 2), LHRHa (35 IUkg on d 0 and 100 

IUkg on d 2), or HCG at 150 IUkg on d 0 and 17-20P 

at 2 mg/kg on d 2. Over 60% of the females treated 

with HCG or LHRHa spawned 6-8 d after injection. 

Eggs from these fish were fertilized, incubated, and 

hatched; the survival rates of these fry were comparable 

to those produced during the normal spawning season 

(Robert Summerfelt, Iowa State University, personal 

communication). As a result of these studes, the 

production of walleye fry can now be extended for over 

3 months of the year. The availability of walleye fry 

during this interval should increase the efficiency of 

existing intensive fry culture systems (e.g., by allowing 

double- or triple- cropping), aid public and private 

hatcheries in their efforts to produce larger walleyes for 

stoclung, and facilitate research on the intensive culture 

of walleye fry. 

Methods for stripping and fertilizing walleye eggs 

generally follow one of two protocols. One technique is 

the dry method, in which eggs and sperm are mixed 

together before being activated by the ad&tion of water. 

The second technique is the wet method, in which eggs 

or sperm are stripped into water, and the other gametes 

are quickly added. Each technique has its advocates. 

Proponents of the dry method cite better quality control 

and improved hatch rates, whereas those who espouse 

the wet method assert that they have a more efficient 

production-line operation. Regardless of the method 

used, care and attention to detail can make the difference between a 20% and 80% hatch rate. Because of 

such dfferences, egg stripping and fertilization techniques are critical steps in the propagation of walleye. 

As previously mentioned, walleye eggs become highly 

adhesive shortly after they are exposed to water. During 

artificial propagation, eggs will stick to one another if 

left untreated. Such clumped eggs will not roll properly 

in incubation jars, and even small clumps can interfere 

with oxygen distribution, resulting in smothered eggs. 

Large numbers of dead eggs provide an excellent 

substrate for fungus, whch can ultimately lull all the 

eggs in a jar. 

The case studies that accompany this chapter describe a 

variety of methods to alleviate clumping during 

spawning. Suspensions of compounds such as bentonite 

(clay), Fuller’s earth, starch, or pond muck can be 

applied to the eggs; the fine particles coat the eggs and 

reduce egg-to-egg adhesion. Other methods include the 

use of a protease solution, which is effective but quite 

expensive when used on a large scale (Krise et al., 

1986; Krise 1988), or a tannic acid solution (Colesante 

and Youmans 1983). The oldest known method is 

simply to continually stir the eggs for an extended 

period of time (up to 4 h), which remains an effective 

but extremely manpower intensive technique. 

Following treatment to prevent clumping, the eggs are 

allowed to water harden for 2-3 h. During this process, 

eggs are normally held undisturbed in buckets, coolers, 

or fine-meshed nets, and during this time the eggs swell 

to about twice their original size. After water hardening, 

the eggs are loaded into incubators. The most common 

incubators used for walleye eggs are jars supplied with 

an upwelling flow of water. The continuous water flow 

rolls the eggs, it provides oxygen, and removes metabolites. As mentioned previously, the time from fertilization to hatch is highly dependent on temperature and 

can range from 7-26 d or more. During incubation, 

eggs are normally treated with formalin (we use a 

1 : 1000 static bath for 15 min once daily) to inhibit 

fungal growth. Hydrogen peroxide is currently being 

evaluated as a replacement for formalin in this regard. 

Eggs are usually counted by volumetric dqdacement 

after water hardening, with sub-samples of eggs from 

different batches measured by hand or with calibrated 

egg troughs that provide an estimate of the number of 

eggdquart (L). Walleye eggs generally average 

80,000-1 50,000 eggs/qt (85,000-1 60,000 eggsk), and 

egg size varies both within and among walleye populations. Moohe et al. (1989) suggested that larvae from 

small eggs within a population had relatively high 

16 NCRAC Culture Series 101- Walleye Culture Manual 
mortality rates, but that among populations small egg 

size was not a disadvantage. 

One of the most critical times for walleye culture is 

during hatch. A jar of walleye eggs can take from 

several hours up to several days to completely hatch. 

The process can be accelerated to some extent by 

increasing the water temperature by several degrees 

once the eggs begin to hatch. In most facilities fry are 

allowed to swim out of the hatching jars and into a 

centralized collection tank. Care must be taken to 

prevent screens from plugging, and the deterioration of 

water quality that can result from the accumulation of 

egg shells, oil droplets, and dead eggs and fry. Solutions 

to this problem include redundant screens, self-cleaning 

designs (e.g., bubble curtains adjacent to screens), and a 

good measure of attentiveness. 

Acknowledgments 

The information included in this chapter on the 

seasonal endocrine and gonadal changes of walleye is 

the result of a cooperative effort between the University 

of Wisconsin-Madison (UW-Madson), the University 

of Minnesota (UM), the University of NebraskaLincoln (UN-L) and Southern Illinois University (SIU). 

Studies on the induction of out of season spawning 

were conducted by investigators from the UW-Madison 

and the UN-L. We would like to thank Thomas 

Kuczynslu, Terence Barry, Lynne Procarione, and Anita 

Lapp of the UW-Madison, Anne Kapuscinsh, Mark 

Hove, Mark Gross, Jeffrey Mittelmark, and Les Getting 

of the UM, Terrence Kayes and Jess Hansen of the UNL, and Robert Sheehan and Bruce Tetzlaff of SIU for 

their participation. Principal funding for these studies 

came from the North Central Regional Aquaculture 

Center with assistance from the Wisconsin Sea Grant 

College Program. 

Ref ere n ces 

Barry, T.P., J.A. Malison, A.F. Lapp, and L.S. Procarione. 

In press. Effects of selected hormones and male 

cohorts on final oocyte maturation, ovulation, and 

steroid production in walleye (Stizostedion vitreum). 

Aq uacu It u re. 

Becker, G.C. 1983. Fishes of Wisconsin. University of 

Wisconsin Press, Madison. 

Colesante, R.T., and N.B. Youmans. 1983. Water 

hardening walleye eggs with tannic acid in a production hatchery. Progressive Fish-Culturist 45:126-127. 

Donaldson, E.M., and G.A. Hunter. 1983. Induced final 

maturation, ovulation, and spermiation in cultured 

fish. Pages 351-403 in W.S. Hoar, D.J. Randall and 

E.M. Donaldson, editors. Fish physiology, volume 9, 

part B. Behavior and Fertility Control. Academic 

Press, New York. 

Ellis, D.V., and M.A. Giles. 1965. The spawning behavior 

of the walleye, Stizostedion vitreum (Mitchill). 

Transactions of the American Fisheries Society 

103153-58. 

Forney, J.L. 1966. Factors affecting first-year growth of 

walleyes in Oneida Lake, New York. New York Fish 

and Game Journal 13:147-166. 

Goetz, F.W. 1983. Hormonal control of oocyte final 

maturation in ovulation in fishes. Pages 11 7-1 70 in 

W.S. Hoar, D.J. Randall and E.M. Donaldson, editors. 

Fish physiology, volume 9, part B. Behavior and 

Fertility Control. Academic Press, New York. 

Hokanson, K.E.F. 1977. Temperature requirements of 

some percids and adaptations to the seasonal 

temperature cycle. Journal of the Fisheries Research 

Board of Canada 34: 1524-1 550. 

Houde, E.D. 1969. Distribution of larval walleyes and 

yellow perch in a bay of Oneida Lake and its relation 

to water currents and zooplankton. New York Fish 

and Game Journal 16:185-205. 

Johnson, F.H. 1961. Walleye egg survival during 

incubation on several type of bottom in Lake 

Winnibigosh, Minnesota, and connecting waters. 

Transactions of the American Fisheries Society 

90131 2-322. 

Krise, W.F. 1988. Optimum protease exposure time for 

removing adhesiveness of walleye eggs. Progressive 

Fish-Culturist 50:126-127. 

Krise, W.F., L. Bulkowski-Cummings, A.D. Shellman, 

K.A. Kraus, and R.W. Gould. 1986. Increased walleye 

egg hatch and larval survival after protease treatment 

of eggs. Progressive Fish-Culturist 48:95-100. 

Malison, J.A., T.B. Kayes, L.S. Procarione, J.F. Hansen, 

and J.A. Held. 1994a. Induction of out-of-season 

spawning in walleye (Stizostedion vitreum). Page 233 

in Book of Abstracts World Aquaculture '94. World 

Aquaculture Society, Baton Rouge, Louisiana. 

NCRAC Culture Series 101 - Walleye Culture Manual 17 
Malison, J.A., L.S. Procarione, T.P. Barry, A.R. 

Kapuscinski, and T.B. Kayes. 1994b. Endocrine and 

gonadal changes during the annual reproductive 

cycle of the freshwater teleost, Stizostedion vitreum. 

Fish Physiology and Biochemistry 13:473-484. 

Moodie, G.E.E., N.L. Loadman, M.D. Wiegand, and J.A. 

Mathias. 1989. Influence of egg characteristics on 

survival, growth and feeding in larval walleye 

(Stizostedion vitreum). Canadian Journal of Fisheries 

and Aquatic Sciences 46:516-521. 

Nagahama, Y. 1983. The functional morphology of 

teleost gonads. Pages 223-275 in W.S. Hoar, D.J. 

Randall and E.M. Donaldson, editors. Fish physiology, volume 9, part A. Endocrine Tissues and 

Hormones. Academic Press, New York. 

Nagel, T. 1985. Development of a domestic walleye 

broodstock. Progressive Fish-Culturist 47: 121 -1 22. 

Ney, J.J. 1978. A synoptic review of yellow perch and 

walleye biology. Pages 1-1 2 in R.L. Kendall, editor. 

Selected coolwater fishes of North America. American Fisheries Society Special Publication Number 

11, American Fisheries Society, Washington, D.C. 

Niemuth, W., W. Churchill, and T. Wirth. 1959. The 

walleye, its life history, ecology and management. 

Wisconsin Conservation Department Publication 

Number 227, Madison, Wisconsin. 

O’Donnell, D.J. 1938. Natural vs. artificial propagation. 

Wisconsin Conservation Bulletin 3:38-40. 

Pankhurst, N.W., G. Van Der Kraak, and R.E. Peter. 

1986. Effects of human chorionic gonadotropin, DESGLY’O (D-ALA6

) LHRH-ethylamide and pimozide on 

oocyte final maturation, ovulation and levels of 

plasma steroids in the walleye (Stizostedion vitreum). 

Fish Physiology and Biochemistry 1 :45-54. 

Priegel, G.R. 1970. Reproduction and early life history 

of the walleye in the Lake Winnebago region. 

Wisconsin Department of Natural Resources 

Technical Bulletin 45, Madison, Wisconsin. 

Redding, J.M. and R. Patiiio. 1993. Reproductive 

physiology. Pages 503-534 in D.H. Evans, editor. The 

physiology of fishes. CRC Press, Inc., Boca Raton, 

Florida. 

Scott, W.B. and E.J. Crossman. 1973. Freshwater fishes 

of Canada. Fisheries Research Board of Canada 

Bulletin 184, Ottawa, Ontario. 

Smith, C.G. 1941. Egg production of walleyed pike and 

sauger. Progressive Fish-Culturist 54:32-34. 

Stickney, R.R. 1986. Culture of nonsalmonid freshwater 

fishes. CRC Press, Inc., Boca Raton, Florida. 

Wolfert, D.R. 1969. Maturity and fecundity of walleyes 

from the eastern and western basins of Lake Erie. 

Journal of the Fisheries Research Board of Canada 

26: 1877-1 888. 

18 NCRAC Culture Series 101- Walleye Culture Manual 
Bennett, C. D., and J. L. Melby. 1996. Walleye spawning operations at Bonny Reservoir, Colorado. 

Pages 19-20 in R. C. Summerfelt, editor. Walleye culture manual. NCRAC Culture Series 101, 

North Central Regional Aquaculture Center Publications Office, Iowa State University, Ames. 

Walleye Spawning Operations at Bonny 

Reservoir, Colorado 

Charles 5. Bennett and James L. MelbL; Colorado Division of Wildlife, 2126 N. Weber, Colorado Springs, 

Colorado 80907 

Introduction 

Bonny Reservoir is a 1,924-acre (778.6-ha) plains 

reservoir constructed and operated by the United States 

Bureau of Reclamation. The Colorado Division of 

Wildlife and Colorado Division of Parks and Outdoor 

Recreation own the conservation pool and manage fish, 

wildlife, and recreation. It is located on the South Fork 

of the Republican River in Yuma County, 27 mi (43.44 

km) northeast of Burlington, Colorado. 

Walleye spawning at Bonny historically starts on or 

about March 20. Thirty to 45 million walleye eggs are 

collected annually from wild captured broodfish. 

Average hatching rate is 65-75 %. 

noted before removing, because removal techniques are 

different due to average size and body shape differences 

between sexes. Males are usually removed from the 

nets head first, while females are backed out of the net 

to minimize egg loss. Standard net picks are used and 

additional care is given when in the opercular area to 

prevent gill damage. Gravid females that are expelling 

eggs while in the net are held tail up or on their back, 

and the vent is blocked with a finger to prevent further 

egg loss while handling. Brood fish are placed in a 100- 

gal (378.5-L) tank containing lake water and supplied 

with 1-2 L oxygen/min dispensed with airstones. Fresh 

water is supplied as necessary with a 12-v pump. A 

burlap tank cover is used to reduce potential stress from 

bright sunlight. 

A two-slip boat house in a protected cove adjacent to 

the dam is modified for walleye spawning. A floor is 

constructed over one boat slip that contains four, 4 x 4 x 

4 ft x 1 in (1.22 x 1.22 x 1.22 m x 25.4 mm) mesh 

holding nets, a 100-gal (378.5-L) holding tank for 

receiving broodfish, a spawning table, and an oblong 

shallow (50 gal, 189 L) tank for floating egg boxes 

during the egg hardening process. A submersible 

electric pump provides water for all phases of the 

spawning process. Overhead PVC pipes and valves 

deliver water where needed. 

Standard neutral buoyancy 2.5-in (63.5-mm) bar mesh 

gill nets, 6 x 200 ft (1.83 x 60 m) are used to capture 

brood size walleye. Nets are deployed daily at sunset 

parallel to the dam riprap and in sandrock rubble 

shoreline areas. Net placement is in 3-6 ft (0.91-1.83 

m) deep water. 

Gill nets are checked at 0700 h by a boat crew of 3-5 

people. Two crew members pull the nets while 1-2 

personnel remove broodfish. The sex of a netted fish is 

The standard project boat is 18 ft (5.4 m) long, powered 

by a 150 hp outboard engine. The size and power of this 

equipment is deemed necessary due to payload, 

distance from the boathouse (1-1.5 mi, 1.61-2.4 km), 

and potential foul weather con&tions. 

Broodfish are returned to the boathouse when the 

carrying capacity of the tank is reached. The actual 

number of broodfish in any haul varies due to fish size 

and sex ratio, but generally 25-50 fish can be held 

safely in the tank. Fish are transferred to a holding tank 

inside the boathouse. The tank is supplied with recirculating lake water and provided with 1-2 L oxygedmin. 

An overhead counter-weighted submersible pump is 

lowered to empty and refill the boat tank for additional 

trips to the nets. Broodfish are sorted by sex and 

ripeness. Males are placed in a 4 x 4 x 4 ft (1.22 x 1.22 

x 1.22 m) holding net (1-in ,25.4-mm bar mesh) 

suspended through the floor of the boat house. “Green” 

females are held in a similar holding net, They are 

checked each morning and ripe fish are stripped. Fish 

that are still green are marked with a hole punch in one 

NCRAC Culture Series 101 - Walleye Culture Manual 19 
Chapter 2 - Reproductive Biology and Spawning 

lobe of the caudal fin and held back another day. 

Females that have not ripened after two such marks are 

released. Males as needed are placed in a tub with 

oxygen and recirculating water on the spawning table. 

The dry method of spawning is used in Colorado. A 

female and the spawning pan is wiped dry and eggs are 

extruded from the fish into the pan by firm pressure to 

the abdomen. We have found that wearing rubber 

surgical and/or wool gloves and using a closed finger 

rocking motion from the tips of the fingers to the back 

of the hand is an effective method for stripping eggs. 

This technique is thought to be less harmful to the fish. 

Less scale loss and mucus production is observed 

compared with other techniques that have been used. 

However, personnel with small hands may have 

difficulty using this technique. Large females (8-15 lb, 

3.6-6.8 kg) are always handled by the head and tail 

rather than by the tail only. Handling a large fish by the 

tail only can result in skeletal damage. 

Fresh semen from two males is used to fertilize the 

eggs. Extended semen is used when males are in short 

supply. Fertilized eggs are covered with water and 

stirred for 90 s with a feather. Water is then decanted 

from the eggs. This step is done as quickly as possible, 

and can require additional repetitions if blood or fecal 

matter is present. Clumping will occur if too much time 

is spent cleaning the eggs. 

After fertilization and cleaning, a mixture of Fullers 

Earth and water (3-4 cups Fullers earth to 1 gal (3.785 

L) of water) is added to the eggs and stirred for an 

additional 90 s to remove the sticky matrix that causes 

eggs to clump during incubation. The mud solution is 

decanted and the eggs are washed with clean water to 

remove residual mud, blood, and excreta. 

Eggs are then poured into a Saran cloth (32 x 32 mesh/ 

in) covered egg box. Each box is capable of holding 2- 

3 million eggs. The boxes float in a shallow oblong tank 

that is supplied with oxygen and lake water. Minimum 

water hardening time is 1 h. After the eggs are waterhardened, they are &pped from the egg boxes and 

placed into an appropriate sized cooler. Coolers should 

not be filled more than two-thirds full. Additional egg 

swelling can exceed the cooler capacity and oxygen 

depletion can result in substantial losses. Fresh water is 

added to completely fill the containers and lids are 

secured. The coolers are then transported to the Wray, 

CO hatchery for tempering, enumeration, and jar 

incubation in well water, 

Bonny Reservoir has consistently produced 30-45 

million fertilized eggs annually. The average hatching 

rate is 65-75 %. 

20 NCRAC Culture Series 101- Walleye Culture Manual 
Copeland, J. A,, and M. M. Wolgamood. 1996. Walleye spawning in Michigan. Pages 21-23 in R. C. 

Summerfelt, editor. Walleye culture manual. NCRAC Culture Series 101. North Central Regional 

Aquaculture Center Publications Office, Iowa State University, Ames. 

Walleye Spawning in Michigan 

James A. Copeland and Martha M. Wolgamood, Michigan Department of Natural Resources, Fish Health 

Laboratory, 34270 CR 652, Mattawan, MI 49071 

Michigan Department of Natural Resources 

has been utilizing walleye from the Muskegon 

River as a source of walleye eggs for many 

years. Fish migrate upstream and are captured 

by DC electrofishmg below a barrier dam 

(Figure 1). Spawning techniques have evolved 

to the point where we can normally rely on 

SO% development to the eyed egg stage 

(eye-up in 1986 was only about 27%). 

Between 80 and 100 million eggs are taken 

annually. 

Broodfish are selected and captured by 

district field crews when the Muskegon River 

water reaches 36°F (2.2”C). Fish are then 

sorted by sex visually and taken to a springfed pond which is 50°F (10°C). Most females 

are “green” upon arrival, and are all placed in 

54 ft3 (1.53 m’) holding nets. Average weight 

of the females is about 7 lbs (3.2 kg). 

Fish are collected daily from the river and transported 

to the holding nets until 500-600 fish have been 

captured. Females in each net are examined daily to 

check for ripeness and ripe females are spawned daily 

until egg-take goals are met. Experience has shown that 

all fish will be ripe within 2-3 d after capture. The 

various apparatus are illustrated in Figure 2. 

Ripe fish are anesthetized prior to spawning. Spawning 

is done on a small dock at the pond. No protection from 

wind, sun, rain or snow is provided for the eggs during 

stripping. Anesthetized fish are rinsed in fresh water 

and wiped with a towel before spawning. One female is 

stripped into a stainless steel 5 qt (4.75 L) pan (Figure 

3, next page). The number of eggs per female ranges 

160,000 to 200,000. If a female produces > 200,000 

eggs, the eggs are usually split between two pans for 

fertilization. 

Figure 1. Electrofishing as a source for collection of broodfish on 

the Muskegan River, Michigan. 

Prior to 1990, two males would be stripped into a pan 

of eggs and a small amount of water (about a cup) was 

added. The eggs were stirred for 5-10 seconds and then 

allowed to set approximately 2 min before being poured 

into a 5-gal (18.9 L) pail with Fuller’s earth and water. 

Figure 2. Equipment for collecting walleye for eggs. 

NCRAC Culture Series 101 - Walleye Culture Manual 21 
Chapter 2 - Reproductive Biology and Spawning 

Figure 3. Stripping eggs from wild walleye in Michigan. 

If eggs were allowed to set for more than 2 min, they 

would begin to stick together. The Fuller’s earth 

solution was prepared by adding one cup (8 fluid ounce 

capacity) of Fuller’s earth to about 3.5 gal (13.25 L) 

water. 

Beginning in 1990, a new fertilization technique was 

used. Semen from two males is rapidly stripped into a 

1.6 qt (1.5 L) plastic jar full of water. This mixture is 

vigorously shaken, then poured into a pan of freshly 

stripped eggs. The mixture of eggs 

and solution of semen are stirred 

with a feather, then allowed to set 

for 30 sec. If eggs begin to stick 

before 30 sec, they must be poured 

into the Fuller’s earth mixture 

immehately or they will form 

clumps. It is believed that fertilization is almost instantaneous. The 

Fuller’s earth mixture is stirred with 

a feather as the eggs are poured into 

it. This procedure is repeated until 8 

or 10 females have been stripped 

and fertilized, and the entire mass of 

eggs put into a 5-gal (1 8.9 L) pail. 

After the last pan of eggs has been 

added to the Fuller’s earth mixture, 

the pail of eggs is allowed to set for 

a maximum of 5 min. At this time, 

the eggs are poured into a floating 

30 ft3 (0.850m’) holding net (with 

0.0625 in [ 1.59 mm] mesh) to allow 

the eggs to water harden. Eggs from 

up to 100 females are put into each 

net. The net also serves to wash 

excess Fuller’s earth out of the eggs. 

The eggs are packaged for shipment 

1 hr after the final lot of eggs have 

water hardened. Fish that have been 

stripped are held in 54 ft3 (1.53 m3) 

net pens in the spring-fed pond for 

24 h and are then returned to the 

Muskegon River. 

Water-hardened eggs are dipped 

from the net using one of the 

spawning pans. Eggs are put into 12 

x 10 x 9.5 in (305 x 254 x 241.3 

mm) Styrofoam boxes. A box is 

initially filled about half full with water. Eggs are 

gently poured into the box until the water overflows. At 

this point, water is poured off the eggs and this process 

continues until the eggs in the box are about 1 in (25.4 

mm) from the top. The eggs are just covered with water 

for shipment. Each box contains about 16 qts (I 5.1 L) 

of eggs. The trip to the hatchery is approximately 100 

miles (160.9 km) and takes about two h. 

22 NCRAC Culture Series 101- Walleye Culture Manual 
Reproductive Biology and Spawning - Chapter 2 

Upon arrival at the hatchery, the eggs are measured into 

plastic hatching jars. Five qts (4.73 L) of eggs are added 

to a jar, and the volume of eggs in this jar is used as a 

guide for filling the remaining jars. The eggs are 

siphoned into the jars. The jars are then put on the 

hatching battery to incubate the eggs. Each jar is 

supplied with 1.06 qt/min (1 Lhin) of 50°F (10°C) 

spring water. Eggs will not be rolled at this low flow. 

Eggs are treated daily with formalin at concentrations 

of 1500 for 15 min. On day ten, treatments are discontinued, and water flow is increased until the eggs roll 

gently. This allows dead eggs to migrate to the top of 

the egg mass and allows them to be siphoned. It may 

take 3-4 d before most dead eggs will roll to the top. 

During this period, eggs should be monitored for 

fungus (saprolegnia) infestation. If fungus does occur, 

formalin treatment should be repeated. 

In 1986, the average eye-up was 27%. In 1990, 1991, 

and 1992, the mean had risen to 63%, 78%, and 83% 

respectively. 

Eggs will be very near hatch on the 15 d of incubation. 

At this time eggs are transferred to a heated water 62°F 

(16°C) battery. After about 3 d at this temperature, eggs 

will hatch. Hatching fry are dn-ected into rectangular 

9.7 x 13 ft (2.91 x 3.9 m) long troughs where the egg 

shells settle. If tannic acid is used to eliminate egg 

adhesiveness, egg shells will not break down at hatch 

and settle in the troughs. The overflow from ths trough 

leads into a 8-ft (100 ft3, 2.8 m3) circular tank. The egg 

shell settling trough is covered with a black cover and a 

light bulb is placed at the outlet. Fry are attracted to the 

light and are quickly pulled into the overflow. The 

carrying capacity of the circular holding tank is about 

three million fry. Fry are held in these circular tanks for 

2-3 d and are then shpped to rearing ponds or stocked. 

The same Styrofoam containers that were used to 

transport eggs are used to ship fry. Approximately 2 gal 

(8 L) of water and 50,000 fry are put into a plastic bag 

in the Styrofoam container. Fry are measured using 

volumetric displacement in a 500 ml graduated cylinder. The number of fry/ml is determined using a 3-4 ml 

sample of fry in a 10 ml graduated cylinder and 

averaging 3-5 samples from each tank. After adding the 

fry, oxygen is added and the bag is sealed. 

NCRAC Culture Series 101 - Walleye Culture Manual 23 
Chapter 2 - Reproductive Biology and Spawning 

24 NCRAC Culture Series 101- Walleye Culture Manual 
Harvey, J. E., and S. E. Hood. 1996. Collection and spawning of walleye broodfish and incubation 

of fertilized eggs. Pages 25-28 in R. C. Summerfelt, editor. Walleye culture manual. NCRAC 

Culture Series 101. North Central Regional Aquaculture Center Publications Office, Iowa State 

University, Ames. 

Collection and Spawning of Walleye 

Broodfish and Incubation of 

Fertilized Eggs 

James E. Harvey and Sbyrl E. Hood, Pennsylvania Fish and Boat Commission, Linesville Fish Culture 

Station, PO. Box 127, Linesville, Pennsylvania 16424-0127 

c 

Introduction 

walleye is one of the most sought after coolwater 

sport fish in Pennsylvania. To supply the fish needed for 

population enhancement, the Pennsylvania Fish and 

Boat Commission is committed to a large-scale walleye 

spawning activity that supports a &verse walleye 

culture effort. Fertilized walleye eggs are also traded for 

species of fish from other states and the U.S. Fish and 

Wildlife Service that the Pennsylvania Fish and Boat 

Commission needs but cannot produce for its fisheries 

management goals. 

Methods 

Most of the walleye eggs come from a wild population in the 2,500-acre (1,012-ha) Pymatuning Sanctuary 

Lake, which is closed to the public, located near the 

town of Linesville, Pennsylvania (Figure 1). The 

Linesville Fish Culture Station is situated on the shores 

of Pymatuning Sanctuary and has conducted walleye 

spawning operations every spring since the early 1950s. 

Collection of adult walleye begins in March or early 

April as soon as the ice melts. The water temperature at 

that time is 36-38°F (2-3°C). Eight to 10 

Lake Erie New York 

Erie 

Linesville Fish 

Cultural Station 

Cleveland 85 miles 

Pen n s y I va n i a 

Ohio r Pittsburgh 

#- 

Figure 1. Location of the Linesville Fish Culture Station. 

trap nets are set throughout the sanctuary 

by a two-man crew working from a 

Float 

gravel and sand bars, preferably in water 5-7 ft (1.5-2.1 

m) deep. The net opening faces shoreward, with the 

lead extending to the shore. 

Initially, net catches contain a high percentage of males 

and few ripe females. The proportion of females in the 

catch increases as water temperature rises to 45°F (7°C) 

and peak catch usually lasts 7-1 0 d. Males become less 

abundant toward the end of this period. 

Nets are checked daily, and adult walleye are trans- 

/-- i Shoreline - I 

0 

125 ft Leader 

pontoon boat. The Pennsylvania trap net 

(Figure 2) is constructed with knotless 

nylon sewn on 4-ft (1.2-m) square aluminum frames. The net size is 1-in (25.4-mm) 

(bar measure) mesh in the crib and 1.5-in 

(38.1-mm) mesh in the 125-ft (38.1-m) 

lead. The frames in the crib ensure that the 

net will not collapse when it is set in shallow water, and 

also facilitates removal of fish by preventing the net 

from collapsing when it is lifted onto the deck of the 

pontoon boat (Figure 3, next page). Net locations are on 

Anchor 

/ 

Pot I I 

Figure 2. Diagram of the Pennsylvania trap net. 

NCRAC Culture Series 101 - Walleye Culture Manual 25 
Chapter 2 - Reproductive Biology and Spawning 

Figure 3. Checking a trap net. 

ported back to the hatchery in onboard tanks. Fresh 

water is provided to maintain oxygen levels by 

means of a pump and manifold system. At Linesville 

Fish Culture Station, an overhead hoist system lifts 

the tanks off the boat (Figure 4), and positions them 

over the holdmg tanks, and the walleye are released 

into the 3.5-m3 (126-ft3) tanks by mechanically 

lifting an end and pouring out the fish and water. 

Fish arc sorted into the category of males, ripe 

females (flowing eggs), and green females (no 

flowing eggs), then placed in separate tanks. 

Green females are checked daily, and usually ripen 

within 3 d. Warmer well water (5 1 OF, 11 "C) is added 

to speed up the ripening process. The ripe females 

are spawned as soon as sorting is completed. It has 

been learned from experience that it is best not to 

delay spawning of ripe 

females as they will lose a 

Pymatuning Sanctuary yields about 0.5 quart 

(0.47-L) or approximately 70,000 eggs. Egg 

size is related to the size of the female (Table 1). 

The average walleye egg from the Pymatuning 

Sanctuary is 0.078-in (1.98-mm) in diameter or 

140,000 eggs/qt (132,440 eggs&). 

Males and ripe females are placed in separate 

tubs containing 132 ppm FinquelO for several 

min until completely anesthetized and then 

rinsed in clean water so that no anesthetic 

comes in contact with eggs or semen. Eggs are 

expressed into a wet pan by a gentle stroking 

motion from behind the gills to the vent while 

slightly arching the back upwards (Figure 5). 

The eggs from 2 females arc stripped into the 

same pan. Simultaneously, the semen from two 

Figure 4.Tanks with adult walleye being lifted off the boat. 

large number of eggs in the 

holding tanks, especially if 

it is overnight. 

Table 1. Walleye egg size as related to size of female from 

Pymatu n i ng Sanctuary. 

A large female may 

produce as many as 

300,000 eggs. The number 

varies with the size of the 

fish; 25,000-40,000 eggs 

for each pound (55,000- 

88,00O/kg) of fish. On the 

average, each female 

spawned from the 

15.0 381 1 .o 0.5 0.072 1.83 181,300 171,510 

18.5 470 2.2 1.0 0.075 1.91 160,400 151,738 

20.0 508 2.7 1.2 0.082 2.08 122,730 116,103 

23.0 584 4.5 2.0 0.084 2.13 114,172 108,007 

28.0 711 7.0 3.2 0.090 2.29 92,826 87,813 

26 NCRAC Culture Series 101- Walleye Culture Manual 
Reproductive Biology and Spawning - Chapter 2 

Figure 5. Expressing eggs into plastic basin. 

males is added. Semen from 2 males is used as a 

precaution against the possibility of a male having nonmotile sperm. Each male has a sufficient quantity of 

milt to be used more than once if needed. However, 

ample males are usually available and reuse of males is 

seldom necessary except at the end of the run. Walleye 

have rough ctenoid scales, and if many fish are stripped, 

these scales can cause abrasions on the hands. Cotton 

gloves can be worn to prevent 

this from occurring. 

After mixing eggs and semen, 

a small amount of water is 

poured into the pan to activate 

the sperm. The sperm and eggs 

in the pan are then gently 

mixed by a circular motion of 

the hands for 2 min. The 

fertilized eggs are then poured 

into a 5-gal (19-L) plastic 

bucket, % filled with well 

water. No more than 8-qt (7.6- 

L) of eggs are placed in a 

bucket. The eggs and water in 

the bucket are gently stirred 

with a turkey feather for 15-20 

min to keep the eggs from 

clumping. During this time 

period, the water is changed 

frequently to remove excess 

sperm, egg shells, ovarian 

material, and other foreign 

matter. 

After this period of constant 

stirring and water exchange, 

the eggs are poured into a 25 

gal (95-L) oak keg which 

contains a 68-oz (2-L) scoop 

of Fuller’s earth in 2-gal (7.6- 

L) of water. No more than 24 

qt (23-L) of eggs are placed in 

a keg. The Fuller’s earth slurry 

prevents the eggs from 

clumping while they water 

harden. Eggs are stirred every 

5-10 min in order to keep the 

Fuller’s earth in suspension. 

After 45 min, the keg is tilted 

slightly and well water is 

added at the bottom of the keg via a hose. All the 

Fuller’s earth must be flushed away prior to the next 

step. After an hour, the eggs have doubled in size and 

water hardened sufficiently to be placed in incubator 

jars. Before being placed in the jars, the eggs are 

strained through a %-in (3.2-mm) screen to remove any 

clumped eggs or any other material that might interfere 

with water movement in the jars. Walleye eggs may be 

Figure 6. Incubator batteries at the Linesville Fish Culture Station. 

NCRAC Culture Series 101 - Walleye Culture Manual 27 
Chapter 2 - Reproductive Biology and Spawning 

shipped in water, 8 qt (7.6-L) eggs per an equal volume 

of water in sealed plastic bags with pure oxygen added. 

Eggs may also be shipped moist in Heath style boxes, 

4-qt (3.8-L) of eggdtray. 

Four-qt (3.8-L) of eggs are placed in each May-Sloan 

jar in each of four 36-jar incubating batteries (Figure 6). 

Approximately 0.75-gpm (2.8-Lpm) of water enters 

each May-Sloan jar at the bottom through a hose and 

spigot. This flow is regulated to ensure a continuous 

gentle movement of the eggs. Fine screens are placed at 

the top of each jar to prevent eggs from washing away 

with the outflow. Annually, 1,400-2,400 females are 

spawned, and these fish produce 700-1,200-qts (662- 

1135-L) of eggs (98-168 million eggs). 

The water supply for egg incubation is from wells 

located on the grounds at the Linesville Fish Culture 

Station. Water from the Pymatuning Sanctuary Lake, a 

shallow, extremely fertile lake with a very high plankton production has been used, but problems were 

encountered with temperature fluctuations and siltation. 

After a week of incubation, dead eggs become evident. 

Dead eggs are more buoyant than live eggs, they rise to 

the top of the incubator jars, and are siphoned off. Eggs 

are treated daily with a 1500 formalin solution (1,667 

ppm) for 17-min to prevent fungal infestations. 

The incubation period varies with water temperature. 

The incubation period is 21 d with eye-up occurring in 

12-1 5 d, in well water of 5 1 "F (1 1 "C) at the Linesville 

Fish Culture Station. There have been successful 

hatches in 12 d when water temperature was 65°F 

(18°C). Our experience indicates that a lugher hatching 

percent can be achieved, with fewer abnormalities, if 

water temperature is 50-52°F (10-1 1OC). If the 

incubation period is extended by lowering the water 

temperature, the percent of hatch will decrease. 

Once hatching begins, it usually takes 3-7 d for all the 

eggs in a lot to hatch. Most of the eggs hatch during the 

middle of that time period. The retaining screens are 

removed from the incubator jars as soon as the eggs 

begin hatching and the fry are allowed to flow out into 

the battery discharge trough and then into holding 

tanks. Eggs can be force-hatched by interrupting the 

water flow into the incubating jars for about 5-15 min 

and by then restoring flow. Care must be taken when 

force-hatching eggs, because if too many hatch at the 

same time an excessive accumulation of egg shells can 

clog retaining screens in the fry holding tanks. Air from 

a compressor is bubbled at the retaining screen to 

constantly remove organic material. Just prior to hatch, 

egg volume is recorded, and this is used to determine 

percent hatch, which ranges from 70-95%. 

28 NCRAC Culture Series 101- Walleye Culture Manual 
Paddock, D. 1996. Incubation of walleye eggs at Garrison Dam National Fish Hatchery. Pages 29- 

30 in R. C. Summerfelt, editor. Walleye culture manual. NCRAC Culture Series 101. North 

Central Regional Aquaculture Center Publications Office, Iowa State University, Ames. 

Incubation of Walleye Eggs at Garrison 

Dam National Fish Hatchery 

David Paddock, U.S. Fish and Wildlife Service, Garrison Dam Fish Hatchery, PO. Box 530, Riverdale, North 

Dakota 58565 

Introduction 

Garrison Dam National Fish Hatchery is located in 

central North Dakota and has been operated by the U.S. 

Fish and Wildlife Service since 1964. Currently the 

hatchery has three buildings used for the production of 

cold water species and 64, 1.5 acre (0.6 ha) earthen 

ponds used for the production of warm and cool water 

species. The hatchery incubates about 48 million 

walleye eggs a year that are collected from wild stocks, 

primarily from Lake Sakakawea and Devils Lake, North 

Dakota. Survival to the eye stage averages 47%, but 

varies, depending upon the source of the eggs. 

Because not all ponds can be stocked at once due to the 

number of ponds that can be filled or drained at a time, 

it is necessary to stagger the hatching of the eggs to 

coincide with pond filling. Controlling the incubating 

water temperature allows the hatchery to prolong the 

incubation of some walleye eggs, allowing a crop of 

northern pike fingerlings to be raised in a particular 

pond in May, followed by walleye fingerlings in June. 

Prolonging incubation is also used to time hatclvng 

until the weather and water temperatures are favorable 

and zooplankton production is sufficient to support 

the walleye fry. 

Methods 

Before spawning starts, an incubation plan is 

developed to project how many green eggs are 

needed, based on the requests made by fishery 

biologists. This information, along with the number 

of ponds that will be used and the date that they will 

be ready for stocking, determines how the eggs will 

be divided among the incubators. 

Spawning begins in early April. Fertilized eggs are 

transported from the spawning areas to the hatchery 

in coolers. The eggs are tempered if there is more 

than 8°F (1 3.3"C) difference between the water temperature in the incubator and the water temperature 

used to transport the eggs. Each incubation jar has been 

calibrated and marked in 0.5 qt (0.47 L) increments to 

allow for easy inventory. Four qt (3.8 L) of eggs are 

ladled from the cooler into each jar, and the water flow 

is adjusted in each jar until the eggs roll uniformly. 

Because the hatchery's water supply comes from Lake 

Sakakawea, fungus infestations would be a problem 

unless kept under control during incubation by treating 

the eggs with formalin (1,667 ppm for 15 min every 

other day). Because the lake temperature is approximately 35°F (1 -7°C) when spawning starts, some of the 

water is heated by electric boilers to provide warmer 

water for incubation. Each incubator is supplied with 

heated and unheated water and the flow of each is 

controlled to produce the desired water temperature. 

The eggs are hatched in nine incubation units. Each unit 

contains 28-40 incubation jars (Figure 1). Heated water 

is degassed by flow-through a packed column and is 

Figure 1. Side view of incubator 28. 

NCRAC Culture Series 101 - Walleye Culture Manual 29 
Chapter 2 - Reproductive Biology and Spawning 

but an increase in fry mortality has 

been observed. It is not yet known if 

this is due to the low incubation 

temperatures or because the water 

temperatures were not increased 

before the fry started to hatch. A 

comparison of the survival rate of 

these fry in ponds has not been made 

with fish whose incubation period has 

not been prolonged. 

When hatching starts, the water flow 

from the incubator is redirected from 

the drain line into a "catch tank". The 

water flow through the incubator 

directs the fry out of the jars, through 

the troughs, and into the tank. 

Figure 2. View of the tank used to catch the fry and the flood light used to The catch tank has a wire screen that 

congregate the fry. 

mixed with unheated water before it enters the upper 

trough if a cooler temperature is desired. By mixing the 

water in this way, the water temperature of each 

incubator can be different and adjusted to within 0.5"F 

(0.28"C) of a desired temperature. Water temperatures 

are recorded daily for each lot of eggs and adjusted if 

necessary. As the hatching date approaches, the eyed 

eggs are inventoried using the increments on the jars 

and monitored so that the hatching date can be recorded. Past records have been used to generate a 

computer program that determines what temperature the 

eggs should be incubated to obtain hatching at a 

predicted date. 

When an extended incubation period is desired, the 

eggs are incubated at a minimum of 46°F (7.8"C) for 

the first 5 d to allow for initial embryo development. 

The temperature can be lowered after this 5 d period 

until the eggs are close to hatching. A day before 

hatching, the temperature is increased to a minimum 

temperature of 56°F (13.3"C) so the fry will be active 

enough to escape their egg shells. This procedure has 

allowed the incubation period to be extended to 42 d. 

There has not been a noticeable reduction in the percent 

eye up in eggs that were incubated using this procedure, 

is covered with polypropylene filter 

cloth (1,024 openings/in2, 159 

openings/cm*) to prevent the fry from escaping. To help 

keep the screen from becoming clogged with egg shells 

and other debris, a 0.25 in (0.6 cm) perforated rubber 

hose is attached to the bottom, upstream side of the 

screen and connected to an air supply. The agitation 

from the air bubbles keeps the screen relatively free of 

debris. Debris is also siphoned from the bottom of the 

tank periodically using a 0.75 in (1.9 cm) hose when the 

tank has relatively few fry. 

Fry are concentrated into one area of the tank for 

harvest. The tank is covered except for a small area 

which is illuminated by a 150-watt flood light hung 

about 3 in (7.6 cm) above the water. (Figure 2). Fry are 

attracted to the light and congregate in the immediate 

area, which keeps them away from the screen and 

makes their removal easier. 

Fry are removed from the catch tank when they are 1-4 

d old. Fry are captured using a dip net made of dacron 

cloth. Fry are enumerated using the volumetric dsplacement method and placed into buckets. Oxygen is 

supplied to the buckets by small air stones that are 

hooked up to an oxygen bottle. Fry are then quickly 

transported to the ponds and stocked. 

30 NCRAC Culture Series 101- Walleye Culture Manual 
Greiff, E. 1996. Stripping, fertilizing, and incubating walleye eggs with Big Redd incubators. Pages 

31-36 in R. C. Summerfelt, editor. Walleye culture manual. NCRAC Culture Series 101. North 

Central Regional Aquaculture Center Publications Office, Iowa State University, Ames. 

Stripping, Fertilizing, and Incubating 

Walleye Eggs with Big Redd Incubators 

Elizabeth Greiff, St. Croix Tribal Natural Resources Department, PO. Box 287, Hertel, WI 54845 

Introduction 

The St. Croix Chippewa Indans of Wisconsin raise 

and stock fingerling walleyes to maintain populations in 

lakes harvested by Tribal members. Staff of the Tribe’s 

Natural Resources Department have collected, 

spawned, and incubated walleye eggs for five years. 

Wild broodstock are captured with fyke nets during the 

walleye spawning season, the eggs and sperm are 

stripped, and the dry method used for fertilization. 

Fertilized eggs are incubated in Big Redd Incubators 

(Big Redd Incubators, Inc., Frazee, MN). After hatching, the fry are stocked in a natural pond. Harvest 

begins 4- to 5-wk later when the fingerlings are about 2 

in (5 cm) long. 

Spawning wild broodfish 

Spawn collection is often coordinated with the 

Wisconsin Department of Natural 

Resources (WDNR) survey of 

adult walleye populations. The 

WDNR sets fyke nets in lakes in 

mid-April after the ice has thawed. 

The net crew notifies us when the 

peak of spawning approaches. We 

collect the ripe fish after the 

WDNR crew has taken their 

survey data. Because our 3-person 

crew collects the spawn and 

monitors the incubators, we try to 

strip all the spawn we need in 1 d. 

Broodstock are separated by sex, 

then placed in separate 72-gal (272 

L) steel basins in a jon boat. The 

water in the basins is not aerated, 

but is changed frequently. Spawning equipment in the boat includes 

a wooden spawning bench 15 x 36 

x 16 in (38 x 91 x 41 cm) (width x 

length x height) with a circular hole cut in one end of 

the top to secure the 5-qt (5 L) plastic pan used for 

fertilization. The dameter of the hole allows the lip of 

the pan to rest on the bench surface. Two round pans are 

used to hold the spawn, one steel 1.5-gal (5.7 L) bucket 

to clean the fertilized eggs, and one 18-gal (68 L) 

square steel basin for claying the eggs. 

Before collecting the gametes, the small bucket and one 

pan are half filled with lake water. The second pan is 

placed dry in the hole cut for it in the spawning bench. 

Milt from one male is stripped into the dry pan in the 

spawning bench. For stripping both males and females, 

the fish’s head is held between the spawn collector’s 

upper arm and side so that the arm from the elbow 

down remains free (Figure 1). With the other hand, the 

spawn collector grasps the fish just above the tail. The 

fish is held belly-down with its back arched over the 

Figure 1. Stripping eggs from a female walleye. 

NCRAC Culture Series 101 - Walleye Culture Manual 31 
Chapter 2 - Reproductive Biology and Spawning 

basin. Milt or eggs are expressed by pressing the fish's 

abdomen firmly with the free hand beginning forward 

of the vent and working back toward it. Stripping is 

stopped if the milt (or eggs when stripping females) 

does not flow freely or if blood is seen. Care is also 

taken to prevent fish slime, water, or other matter from 

entering the pan. The male is released into the lake after 

stripping once, however, large males may be retained 

and stripped twice if few males are collected. A female 

is then stripped into the pan containing milt. If less than 

2-3 cups (473-710 mL) of eggs are collected, a second 

female is stripped. After collecting the eggs, milt from 

another male is stripped over the eggs. Fertilization 

must be accomplished within about 2 min of stripping. 

The milt and eggs are mixed by placing the fingers of 

one hand firmly against the bottom of the pan and 

stirring rapidly without lifting or touching the sides of 

the pan. After mixing to a homogenous color, water 

from the second pan is added to the spawn and this 

mixture of water, eggs, and milt is poured from pan to 

pan 3-4 times, To minimize egg clumping or sticking to 

the pans, the pans are shaken while the fertilized eggs 

are poured. At this point fertilization is complete. 

The eggs are washed free of mucus and semen by 

pouring them into the small bucket (1.5 gal, 15.7 L) 

previously half filled with water. The eggs are swirled 

to prevent sticking by twisting the bucket. While 

swirling, half the water is decanted and replaced with 

fresh lake water. Rinsing is continued until the water in 

the bucket is clear. 

The rinsed eggs are poured into a clay suspension and 

mixed thoroughly. The clay suspension is prepared in 

the square 18-gal (68 L) basin half filled with lake 

water. A handful of wet bentonite is added and stirred 

into suspension until the water feels slippery. Too much 

clay is better than not enough. The clay sticks to the 

eggs and prevents the eggs from clumping. 

We repeat the procedure until we have stripped all 

available fish or have fertilized 10-20 qt or L of eggs 

from 15-30 female walleye. 

The clay/egg basin is placed in shallow water in shade 

and the eggs are hardened in the clay suspension. The 

suspension is mixed periodcally. The eggs take about 2 

h to harden at 48-50"F (8.9-10OC). Eggs are tested for 

hardness by squeezing them between the thumb and 

forefinger. If they do not break, they are hard. When the 

eggs have hardened, they are poured into a 12 x 4 in 

(30.5 x 10.2 cm) deep window screen box and all the 

clay is rinsed off with lake water. Clean eggs are placed 

into an 18-gal (68-L) plastic ice-chest in a layer no 

more than 4 in (10 cm) deep with most of the balance of 

the ice-chest filled with lake water. It usually takes 

about 8 hr between the first fertilization and arrival at 

the incubators. 

Incubation 

we used two Big Redd Incubators (Figure 2) to hatch 

our walleye eggs. Each incubator can hold 11 qts 

(9.9 L) of walleye eggs. Because the incubators are 

portable, they can be set up where the best water quality 

is found and dismantled for cleaning and storage after 

the eggs have hatched. Since 1990, a temporary 

hatchery has been set up in the facilities of the Tribal 

Construction Company. The water source is a well. In 

Figure 2. Big Redd Incubators loaded with walleye eggs. 

32 NCRAC Culture Series 101- Walleye Culture Manual 
Reproductive Biology and Spawning - Chapter 2 

1990, water samples were taken before the water 

entered the incubator. In 1994, water was sampled 

directly from the incubators prior to egg introduction. 

The different sampling locations account for the 

differences in water quality (Table 1). 

eggs/qt (Table 2 and Figure 4). We measure about 0.95- 

qt (900-ml) of eggs into each tube. 

Once the eggs are in the incubators, we monitor air 

flow, water exchange rate, piezometer head level, 

Table 1. Well water quality of well used for incubating 

walleye eggs in Big Redd Incubators, 1990 and 1994. 

Year Temperature pH Dissolved Carbon Iron 

("C) oxygen dioxide (ppm) 

(PPm) (PPm) 

1990 7.8 8.0 7.0 8.8 

1994 9.8 7.0 6.2 8.8 0.2 

dssolved oxygen, water temperature, 

and pH every 4 h, until the fry are 

removed. Carbon dioxide is measured 

once a day. We do not monitor ammonia 

because traces of formalin used to treat 

the eggs for fungus interfere with the 

performance of the ammonia test lut. 

Differences between Incubator A and B 

(Table 3) occur because each incubator 

operates as a separate unit, with individual water, air intake lines, and 

controls. 

The incubators are connected separately to a water line 

with clear %6 in ID (4.8 mm) plastic tubing, and after 

passage thorough the incubators, water is discharged to 

a floor drain. Compressed air is generated with an AC, 

115-V, 60-Hz air pump that supplies each incubator 

independently. A DC air compressor and a 12-V marine 

deep-cycle battery is available in case of electrical 

failure. 

The basic incubator structure is a 9 x 12 x 30 in (23 cm 

x 30 cm x 76 cm) clear plastic holdmg tank. The tank 

holds 11 clear plastic removable tubes mounted on a 

base plate through which water circulates (Figure 3). 

The tubes are 2.5 x 2.5 x 30 in (6.4 x 6.4 x 76.2 cm). 

Each of the 11 tubes can hold a quart or liter of eggs, 

but we do not use the two interior tubes because the 

eggs cannot be observed from the side of the tube. The 

tank's standpipe, with three water level settings, 

piezometer, and airlift assembly fits into the twelfth 

space. The airlift assembly circulates and oxygenates 

the water with perforated plastic tubing connected to 

the air line at the bottom of the tank. 

Before eggs are placed in the incubators, they are gently 

rolled through a screen box with %2 in (4.0 mm) mesh 

to break up any remaining clumps. At the same time, 

the eggs are gradually brought to the same temperature 

as the incubator water by the addition of warm or cool 

water. The WDNR V-trough method and walleye egg 

count chart, modeled after the Von Bayer method but 

specific for walleye, is used to determine the number of 

Air flow is determined by measuring the head of water 

in the piezometer above the tank's water level. Air flow 

varies with the stage of incubation. Eggs are loaded 

with a piezometer reading of 0.47 in (12 mm), incuFigure 3.Top view of a Big Redd Incubator showing 11 

incubating compartments. 

NCRAC Culture Series 101 - Walleye Culture Manual 33 
Chapter 2 - Reproductive Biology and Spawning 

Table 2. Walleye egg count chart, Wisconsin Department of Natural 

Resources, used by St. Croix Tribal Natural Resource 

Department .a 

No. in No. per No. per No. in No. per No. per 

trough quart liter trough quart liter 

38 

39 

40 

41 

42 

43 

44 

45 

46 

47 

48 

49 

50 

51 

52 

53 

54 

55 

56 

57 

16,960 

18,240 

19,680 

21,744 

23,140 

25,197 

26,901 

28,764 

30,801 

32,268 

34,647 

37,265 

39,161 

41,186 

44,494 

46,899 

49,480 

52,254 

55,239 

58,456 

17,928 

19,281 

20,803 

22,985 

24,461 

26,635 

28,436 

30,406 

32,559 

34,110 

36,625 

39,392 

41,396 

43,537 

47,034 

49,576 

52,304 

55,237 

58,392 

61,793 

58 

59 

60 

61 

62 

63 

64 

65 

66 

67 

68 

69 

70 

72 

74 

76 

78 

80 

82 

84 

61,925 

65,680 

67,670 

71,899 

74,146 

78,927 

84,130 

86,904 

92,826 

95,990 

99, 297 

102,762 

110,190 

11 8,346 

127,333 

137,251 

148,220 

160,400 

173,950 

189,070 

aThe number of hardened eggs in a single row in a 6-in trough. 

bated prior to hatching at 0.98 in (25 mm), and hatched 

out at 1.69 in (43 mm). Fry are held in the tank with a 

piezometer reading of 1.69 in (43 mm). During the 

fungicide treatments, the air flow is adjusted so that the 

piezometer reads 0.98 in (25 mm). Air flow is controlled by a valve in the air supply tube. Each tank also 

has an airstone, controlled by an auxiliary air supply 

valve, to increase dissolved oxygen if necessary. 

The water exchange rate is measured at the tank’s outlet 

and is controlled with two valves per incubator in the 

water supply tube. We use the manufacturer’s water 

flow recommendation of 500 mL/min as a minimum 

and manipulate the water flow primarily to control 

temperature. In the small room where the incubators are 

held, altering room temperature also controls tank 

temperature. The incubators are equipped with 

aquarium heaters but they are fragile, and have broken 

65,460 

69,429 

71,533 

75,993 

78,378 

83,432 

88,932 

91,865 

98,125 

101,469 

104,965 

108,628 

1 16,480 

125,101 

134,601 

45,086 

56,681 

69,556 

83,879 

99,862 

inside the tank. They also 

crowd the tank. Temperature 

is more effectively controlled by manipulating 

water flow and room 

temperature. 

Dissolved oxygen is 

measured with a two-probe 

electric meter. Temperature 

and pH are measured with a 

battery-operated meter in 

each tank. Both dissolved 

oxygen and pH meters 

operate continuously. 

Carbon &oxide is measured 

by titration. 

The hardest water quality 

parameters to maintain 

within desirable limits have 

been dssolved oxygen and 

temperature. Dissolved 

oxygen drops significantly 

during the hatch and when 

fry are in the incubators. 

When DO drops to 5 ppm, 

we use the air compressor to 

increase oxygen levels. Pure 

oxygen could be used for 

aeration, but this has not 

been necessary. Water flow 

and room temperature are adjusted as needed, usually 

several times a day, to maintain the desired water 

temperature. We have never had problems maintaining 

other water quality parameters. 

Two to three d after loading the eggs into the incubators, both tanks are treated for 15 min with 4,500 ppm 

formalin to control fungus. We have used concentrations of 2,200-3,300 pprn in previous years, but they 

were ineffective. After the 15 min treatment, the water 

is drained from the tanks to the lowest standpipe level 

by removing the upper section of the standpipe. The 

tank is flushed for 10 min, with both main and auxiliary 

water valves fully open. After flushing, the standpipe is 

replaced and the tank is refilled. The fungicide treatment is repeated every 48 h until the fry are removed 

from the incubators. Most unfertilized eggs that move 

34 NCRAC Culture Series 101- Walleye Culture Manual 
Reproductive Biology and Spawning - Chapter 2 

220000 1Y = 64,360.8 - 3460.8 * X + 59.9 * X2; R2 = 0.999 -0- 

5 180000 

5 200000 .- 

160000 1 

40000 

20000 

00000 

80000 

60000 

40000 

20000 

0 1 l'l'l~l~l~l~l'l'l'l'l 

35 40 45 50 55 60 65 70 75 80 85 

Number of eggs in 6-inch (152.4 mm) trough (X variable) 

Figure 4. Curvilinear relationship between Von Bayer though counts of 

walleyes and number of eggs/L. A 2nd degree polynomial gives a good 

fit to the relationship. 

to the top of the egg mass can be siphoned off. The 

volume of dead eggs is measured. 

During 1990-1 994, the average operating water 

temperature was 51.3"F (10.7"C). At that temperature, 

the eggs eye-up in 9 d (173.7 TU, where TU= temperature - 32°F x days) and begin to hatch in 13-14 d (251- 

277 TU). We induce complete hatch 3 d after hatching 

begins by pouring 129-145°F (5443°C) tap water into 

the airlift assembly at about 1 qt/min (0.9 Wmin) until 

the tank water temperature has increased by 37°F (3°C). 

The increased temperature is maintained for 20 min by 

the addition of more hot water. After 20 min, the water 

temperature is allowed to return to the normal operating 

range of 50-53°F (10-1 1.7"C). The rapid hatching that 

follows generates foam where aeration agitates the 

water. The incubators are equipped with flexible tubing 

that fits over the airlift assembly to withdraw foam. 

Hatching is complete by day 18. We keep the fry in the 

incubators four more days. The number 

of fry is determined by subtracting the 

volume of siphoned dead eggs from the 

initial volume of eggs placed into each 

tube. 

The average hatch rate is 78%, and has 

ranged from 73-87%. The fertilization 

rate cannot be deduced from the hatch 

rate because eggs lost to fungus are 

included in the volume of dead eggs. 

However, in 1994, when there was 

almost no fungal growth during incubation, the mean hatch rate was 83%. In 

1992, when all fertilized eggs died, 

probably due to temperature shock, the 

WDNR gave us eyed eggs to hatch. The 

average hatch rate, starting with eyed 

eggs, was 96%. Extrapolating from this 

observation suggests that loss to fungus 

infection ranges from 4-5%. 

We do not transport fry in the incubators although Big 

Redd literature says it's possible. We tried it once, 

using a DC air pump, but were unable to maintain air 

or water flow through the tanks. We transport eggs in 

boxed, 10-gal (38 L) plastic bags half filled with water 

that is supersaturated with oxygen. 

Cleaning the Big Redd Incubators is time consuming 

because they have to be completely dxmantled. All 

incubator parts and other equipment that comes into 

contact with eggs or fry must be sterilized with an 

overnight soak in a 20-ppm solution of 70% active 

chlorine. Everything is soaked a second night in 

household rust remover (sodium hydrosulfite and 

bisulfite). The chlorine is removed in a third overnight 

soak of sodium thiosulfate solution four times more 

concentrated than the chlorine bath. Finally, every 

piece is washed with dish soap and water and thorTable 3. Average water quality parameters maintained in Big Redd Incubators 

during spring walleye incubation, 1994. 

Incubator DO (ppm) Temperature ("C) pH C02 (ppm) Flow rate (mumin) 

A 6.6 10.8 7.1 6.6 

B 6.1 10.7 7.0 6.8 

81 6 

81 2 

NCRAC Culture Series 101 - Walleye Culture Manual 35 
Chapter 2 - Reproductive Biology and Spawning 

oughly rinsed. The incubators are stored at room 

temperature in their original boxes. 

Big Redd Incubator operation is most labor intensive 

during and after the hatch because the egg shells must 

be removed manually, and fry that are removed with 

them must be sorted and returned to the tank. If the 

eggshells are not removed, they will impede circulation 

and eventually cause the incubators to overflow. 

Siphoning dead eggs is also time consuming. The 

incubators must be monitored regularly to adjust water 

and air flow and to measure water quality. Although 

they require considerable attention, they can be set up 

anywhere where there is suitable water and a drain. The 

incubators and associated equipment require very little 

space. Big Redd Incubators have been indispensable in 

establishng St. Croix’s walleye culture program. 

36 NCRAC Culture Series 101- Walleye Culture Manual 
Gallinat, M. P. 1996. Stripping walleye eggs collected from speared fish and incubation of eggs 

using Big Redd Incubators. Pages 37-39 in R. C. Summerfelt, editor. Walleye culture manual. 

NCRAC Culture Series 101. North Central Regional Aquaculture Center Publications Office, 

Iowa State University, Ames. 

Stripping Walleye Eggs Collected from 

Speared Fish and Incubation of Eggs using 

Big Redd Incubators 

Michael I? Gallinat, Tribal Fisheries Biologist, Red Cliff Fisheries Department, FO. Box 529, Bayfield, WI 

54814 

Introduction 

Tribally regulated spearfishing usually occurs in 

Northern Wisconsin on treaty ceded lands during midApril through early May when walleyes are in spawning condtion. Fish are speared at night when they have 

congregated in the shallows to spawn, with lights 

shined into the water to reflect the eyes of the walleye. 

Tribal spearfishing harvest accounts for about 4% of 

walleyes taken from all lakes in the treaty ceded 

territory. 

Tribal spearfishng is controversial. The perspective of 

many Wisconsin anglers is that fish are for recreation 

and should be taken only by hook and line angling 

methods, and that it is ethically wrong to harvest 

spawning fish. Tribal harvesters, however, view fish as a 

food source and spearing as a means to maintain a 

tra&tional way of life. In the late 1980s, treaty protesters tried to disrupt the tribal spearfishing season. 

Incidents of harassment included rock throwing, racial 

insults, gun shots, and the use of cement walleye decoys 

to damage spears. 

In 1989, a cooperative effort was established between 

the Cable, Wisconsin area Fish for the Future organization and the Bad River and Red Cliff Tribes to ease 

tensions between Indian and non-Indian user groups. 

Walleye gametes from tribally speared fish were 

collected from inland lakes and fertilized eggs were 

transported to tribal hatcheries for incubation. Once 

incubated, fry were transported back to the Cable area 

to culture ponds tended by the conservation club and 

grown to fingerling size. To maintain genetic integrity, 

gametes were collected from lakes with poor natural 

reproduction and the resultant fish stocked drectly back 

into those lakes or into lakes in the same watershed. A 

similar effort with the Eau Claire Lakes Conservation 

Club began in 1992. 

Methods and results 

There was initial concern that water would enter the 

body cavity through the spear holes and it would harden 

the eggs before they could be mixed with milt. Therefore, initially in these cooperative efforts, gametes were 

collected on the lake, as soon as a female was speared. 

It was soon discovered, however, that there was ample 

time to spawn fish at the boat landing. Most veteran 

tribal spearfishermen had become proficient in spearing 

and speared most of their fish in the head region. Also, 

by stripping fish at the landmg, every ripe fish could be 

spawned. 

Walleye eggs are adhesive and must be mixed with an 

inert substance such as clay or starch to prevent them 

from stichng together. Bentonite clay was obtained 

from a local well dnller and mixed with water to the 

consistency of a thin milkshake. A blender was found to 

provide a better product than either stirring the mixture 

by hand or by shaking it up. The clay mixture was 

stored in gallon (3.8 L) water/milk jugs until used. At 

the boat landing, biological data were collected from 

the speared fish by the Great Lakes Indian Fish and 

Wildlife Commission. After the data were obtained 

from a ripe female, it was spawned using the dry 

method, and the milt of three or more ripe males, was 

used to fertilize her eggs. Availability of ripe females is 

the limiting factor, as males make up approximately 

85% of the speared fish because males tend to congregate in larger numbers and remain on the spawning 

grounds longer. When the weather was bitterly cold or 

heavy snow was falling, eggs were collected using the 

wet method to help protect them from the elements. An 

enamel or stainless steel pan was filled with two to 

three inches (5.1-7.6 cm) of lake water and the eggs 

and milt were expressed into the pan. 

NCRAC Culture Series 101 - Walleye Culture Manual 37 
Chapter 2 - Reproductive Biology and Spawning 

The spawn was stirred after the milt was added to the 

eggs, then the clay-water mixture was immediately 

added to wash the eggs from the pan into a plastic 5 gal 

(18.9 L) bucket filled with approximately 6 in (15.2 cm) 

of lake water. Fresh water was added to the bucket and 

was also used to rinse the pan clean. The fertilized eggs 

were gently stirred in the bucket with a rubber gloved 

hand for a few minutes. Enough water was added to the 

bucket to allow for water absorption by the eggs. The 

fertilized eggs were stirred occasionally throughout the 

nightdmorning, as more eggs were added. Fresh lake 

water was added to the bucket with each returning 

fisherman to allow for absorption. The fertilized eggs 

were transported in the 5-gal (18.9-L) buckets to the 

Red Cliff Tribal Fish Hatchery where the eggs were 

rinsed clean and bits of fish viscera, from spear perforations, and other debris were removed with tweezers. 

The hatchery used for the incubation of the walleye 

eggs was a temporary cold-water facility with a 6 in 

(15.2 cm) well that produced 100 gal/min (378.5 L/ 

min). Water temperature was a constant 47-48°F (8.3- 

8.9"C). Financial restraints precluded heating the water 

for use with standard hatching jars, therefore, Big Redd 

Incubators (Figure 1) were used. This incubator uses 

recirculating water, therefore, it does not require much 

water and it was practical to heat the water to desirable 

walleye incubation temperatures. 

Incubators were set up and operated at least one week 

before the expected start of the tribal spearing season to 

establish the water temperature at the water replacement 

flow rates. These rates were set by flow meters attached 

to the system. Upon arrival, 1 quart (0.95 L) of eggs 

was measured into each of the 11 tubes in each incubator. The incubation tubes in the Big Redd incubators are 

indwidually numbered to identify the source of the 

eggs. The incubator operates with an airlift mechanism. 

A small air compressor is provided by the manufacturer. 

A head of approximately 1 in (2.54 cm) was maintained 

during incubation. About 1 pint (473 mL) of water/ 

minute is recirculated through each incubation tube. 

Water in this system is only used to limit the buildup of 

metabolites and aids in temperature control. 

A 250-watt submersible aquarium heater was used in 

each incubator to maintain and control water temperature. Replacement water flow was regulated to obtain a 

water temperature of 53-55°F (1 1.7-12.8"C) for 

incubation. 

t 

Daily operating procedures involved checking and 

cleaning the screen in the incubator, determining water 

temperature and dissolved oxygen levels, treating the 

eggs with formalin if required, reporting quantities 

(volume) of dead eggs removed, and recording the 

replacement water flow. 

\\ I 

a \\ 

\\ 

-b 

-C 

-d 

- e 

If 

Figure 1. Schematic illustration of Big Redd incubator; a. 

air line, b. adjustable standpipe, c. screened water 

discharge windows, d. water return tube, e. incubation 

tubes, and f. manifold for water distribution to individual 

incubation tubes. 

38 NCRAC Culture Series 101- Walleye Culture Manual 
Reproductive Biology and Spawning - Chapter 2 

Using these methods, we averaged 83 % fertilization 

rates; the rates ranged from 40 to 90%. Variation in 

fertilization was dependent upon the conditions under 

which the gametes were collected. Highest mortality 

occurred during the beginning of the tribal spearfishing 

season when fish were not fully ripe. High mortalities 

also occurred if gametes were collected on extremely 

cold nights. 

To control Saprolegnia, formalin fungicide treatments 

were started 2-3 d after the introduction of eggs and 

were repeated every 48 h. Dead eggs that accumulated 

at the tops of the incubation tubes were removed by 

siphon to help prevent fungal infestations. 

Egg shell collectors were inserted into the incubation 

tubes before hatching commenced. This kept egg shells 

and dead eggs from plugging the outlet screens. 

Flexible pipe was also placed on top of the airlift 

assembly at the beginning of hatching to remove the 

foam that is a by-product of the hatching process. 

Eggs start to hatch in about two weeks. The auxiliary 

air stone was also turned up to produce more oxygen 

during hatching. Three days after initial hatching eggs 

were induced to hatch rapidly by increasing the water 

temperature by 4" or 5°F (2"-3"C) in an 18 min period. 

Attempts were made to stock fry about 3 d after 

hatching in order to prevent cannibalism, Ten percent of 

the fry were immedately returned to the lakes from 

which they came strictly for public relations. The 

remaining fry were placed into fingerling production 

ponds. 

Several problems were encountered with the incubation 

system. Airlocks often developed underneath the 

incubation tubes which resulted in poor movement of 

eggs. Plunging the tubes up and down usually sufficed 

to remove trapped air. Some tubes would not sit firmly 

on the manifold nipple. When this occurred, a heavy 

object, such as a brick, was placed on top of the 

incubator to help hold the incubation tubes down. In the 

event of a power outage, the backup system provided by 

the manufacturer would start a backup air compressor, 

but no fresh water would enter the incubator. If the 

hatchery does not have a backup power supply, to keep 

water flowing, the eggs could be heated to lethal 

temperature. Eggs may also clump at the bottom of the 

incubation tube and decrease circulation throughout the 

tube. A stiff plastic tube or wire with a loop at the end 

was used to break up clumps as they occurred. Hatching 

rates ranged 0-87%; failures were largely the result of 

power outages at the hatchery facility. 

Acknowledgments 

The line drawing (Figure 1) of the Big Redd Inc. 

incubator was redrawn by Rex Heer, Iowa State 

University Media Graphics, from a figure supplied by 

Donald Olson, Bid Redd Incubators, Inc. Bayfield, 

Wisconsin. I am thankful to them for their permission 

to use this illustration. 

NCRAC Culture Series 101 - Walleye Culture Manual 39 
Chapter 2 - Reproductive Biology and Spawning 

40 NCRAC Culture Series 101- Walleye Culture Manual 
Thompson, D. 1996. Stripping, fertilizing, and incubating walleye eggs at a Minnesota hatchery. 

Pages 41-44 in R. C. Summerfelt, editor. Walleye culture manual. NCRAC Culture Series 101 

North Central Regional Aquaculture Center Publications Office, Iowa State University, Ames. 

Stripping, Fertilizing, and Incubating 

Walleye Eggs at a Minnesota Hatchery 

Doug Thompson, Minnesota Department of Natural Resources, 1429 Grant Mcmahon Blvd, EIH Minnesota 

5573 1 

Introduction 

walleye have been cultured in Minnesota since the 

late 1800s. In 1993, about 510 million walleye eggs 

were collected from ten sites and incubated in 11 

hatcheries. Of the 355 million fry that were hatched, 

280 million were stocked in 226 lakes and streams, and 

75 million were stocked in 240 fingerling production 

ponds. The Pike River egg take site and hatchery in 

northeastern Minnesota is one of the largest in Minnesota. This case study describes current techniques used 

to capture broodfish, fertilize eggs, and incubate 

walleye eggs at the Pike River facility 

Pike River site 

The Pike River is located in northeastern Minnesota. It 

is 41 mi (66 km) long, has a drainage area of 192 mi2 

(497 km2), and is a tributary to Lake Vermilion, a 

40,557 acre (16,413 ha) lake with low acid neutralizing 

capacity (total alkalinity 20-35 mg/L). The river has 

one small impoundment and is stained with tannin, so 

that it warms quickly in the spring. In normal years, the 

river becomes ice free about a week before Lake 

Vermilion. A site near the mouth of the Pike River has 

been used as a walleye egg take site from about 1890 to 

1946 and from 1971 to the present time. The river at 

this site is about 100 ft (32 m) wide with a maximum 

depth of 10 ft (3 m). The current at this site varies 

widely with lake stage, spring snowmelt, and rain. A 

hatchery near the egg take site has four stacked 

hatching batteries with a total capacity of 1,000 qt (946 

L) of eggs, or about 122 million eggs. 

Collection of broodstock 

Broodfish capture begins as soon as enough ice has 

left the river channel to allow the trap and associated 

structures to be put in the river. At this time the river 

temperature is about 40°F (4°C) but the lake is still ice 

covered. The average egg take starting date from 1985 

through 1994 was 17 April, and the average ending date 

was 28 April. During the spawning season, virtually the 

entire spawning run of walleye is funneled into a pound 

net trap by lead nets spanning the mouth of the river. An 

average of 3,870 females and 16,926 males arc captured 

annually. Several investigations suggest this run 

comprises about 5% of the Lake Vermilion adult 

walleye population. About 67% of the captured females 

arc stripped; they yield on average about 1,040 qt (984 

L) of eggs. Each stripped female yielded on average 

53,984 eggs; the eggs average 122,442/qt (1 29,383L). 

Trapping 

Six square platforms holding the trap, a work deck, and 

four fish-holdmg cribs, each 18 x 18 ft (5.5 x 5.5 m) 

exterior dimension, are bolted together, floated to one 

side out of the main body of current, and tied in place 

by cables extending to opposite shores (Figure 1). The 

downstream platform near the thalweg holds the fish 

trap. The fish-holdmg cribs are made of 1.5 in (3.8 cm) 

Figure 1. The Pike River site used to collect walleye 

broodstock. Nearly 21,000 walleye are collected annually 

by Minnesota Department of Natural Resources personnel at this site using a large trap net. The facility includes 

a work deck and four fish-holding cribs (18 x 18 ft). 

Workers strip and fertilize eggs on the work deck. 

NCRAC Culture Series 101 - Walleye Culture Manual 41 
Chapter 2 - Reproductive Biology and Spawning 

bar mesh nylon, 6 ft (1.8 m) deep; one crib is used to 

hold males and three are used to hold females. 

Long nylon lead nets, 12 ft (3.7 m) deep with 1.5 in (3.8 

cm) bar mesh, are sewn onto each side of the trap net 

throat; the other ends are attached downstream to steel 

pins on opposite shores. Chain, weighmg 15 lb/ft (22.4 

kg/rn), is sewn onto the bottom line of these lead nets 

with twine, 20 ft (6.1 m) at a time, and lowered into the 

water from a large pontoon boat with a power winch. 

Float logs are tied to the top line of each lead net to 

suspend the nets from the bottom of the river channel to 

the water surface. A nylon mesh funnel extends from 

the lead nets to the interior of the trap, allowing fish to 

enter. 

Fish handling 

Unripe females are held in cribs for 3 d; holdmg 

walleye longer than this results in excessive fin wear 

and reddened skin. Each morning the crib holdmg the 

third-day females is emptied; ripe females are placed in 

18 gal (68 L) tubs and held until stripped, and unripe 

females are released upstream of the trap. Similarly, the 

second-day and first-day females are sorted, with unripe 

females placed in the just-emptied crib, using fish slides 

to facilitate the move. Males from the previous day's 

trap catch (in the male holding crib) are used to provide 

milt during this operation. Lastly, the trap is emptied 

and sorted. Only 19% of females in the trap are ripe, 

while an addtional48% ripen within the 3-d holdmg 

period. Males are held for 1 d, so 2 day's catch of males 

is available during each day's egg stripping, ensuring an 

adequate supply of milt. 

The number of walleye entering the trap follows a bell 

curve over the 10-d run, with numbers doubling each 

day early in the run and halving each day at the end. 

Early in the run, males outnumber females by as much 

as six to one, but late in the run, males outnumber 

females by only two to one. Sizes of both males and 

females are larger early in the run. 

In most years, females first ripen when midday water 

temperatures warm to about 42°F (6°C). The peak of 

the egg take occurs when water temperatures are 46- 

50°F (8-1 0°C); the egg take is essentially over when 

water temperatures reach 52-54°F (1 1-1 2°C). Eggs 

taken during the middle of the spawning run typically 

have survival rates about 5% higher than those obtained 

at the beginning or end of the run (79% vs 74%). 

Hatchability declines for eggs fertilized at temperatures 

warmer than about 54°F (12.2"C). 

There are obvious opportunities for selection to occur 

during this egg take operation: river-run spawners and 

open water spawners; large and small fish; early 

spawners and late spawners. At present, selective 

pressures are not considered during our hatchery 

operations except for restricting the stocking of fry to 

waters within the Hudson Bay watershed or to waters 

with no natural reproduction. 

Stripping and ferti I izing 

Eggs are fertilized using the wet method. About 0.5 qt 

(0.5 L) of river water (pH about 7.0) is added to a flatbottomed, 14 in (35 cm) diameter, 4 in (10 cm) deep 

polyethylene pan. The water is stirred by rotating the 

pan. White pans are used to reduce absorption of heat 

from the sun. After an initial squirt of milt from a male 

into the pan, eggs from 3-6 females are sequentially 

stripped into the pan along with milt from 6-1 2 males 

which are simultaneously stripped into the pan, yielding 

a slurry of eggs, sperm, and water 1.0-1.5 in (2.5-3.5 

cm> deep. Milt is added every 15 s for 1 min after the 

last female is stripped to ensure active sperm are 

present while the micropyles of the eggs are open. 

Stirring continues for another minute prior to the 

ad&tion of filtered pond-bottom muck which is added 

to remove adhesiveness of the eggs and prevent 

clumping in the incubation jars. About 0.5 qt (0.5 L) of 

muck slurry is added to the pan of fertilized eggs and 

the mixture is swirled for approximately 2 min. The 

eggs can then be rinsed immediately or can be left 

immobile for a few minutes before rinsing. Various 

types of bentonite-water suspensions (9% bentonite by 

weight) have also been used as declumping agents. 

Dry fertilization is not used because water drips into 

pans from fish, hands, or rain. Moreover, our attempts 

at using a dry or nearly dry method did not improve 

survival to the eyed-egg stage, 80.0% for dry method 

compared with 84.8% for the wet method. The dry 

method may be useful when there is a shortage of males 

and sperm has to be conserved, when extra time is 

available to ensure dry stripping operations, and when 

slightly lower egg survival rates are acceptable. 

42 NCRAC Culture Series 101- Walleye Culture Manual 
Reproductive Biology and Spawning - Chapter 2 

Water hardening 

After treatment with muck, eggs are poured into a 

cradle that floats in the river, and they are rinsed to 

remove muck. The cradle is 6 x 16 x 30 in (15 x 41 x 76 

cm) (depth x width x length), with the bottom rounded 

towards the ends. The entire bottom is covered with 

brass screen which has 58% open area and 24 openings/ 

in (9.4 openings/cm). 

Eggs are worked to one end of the cradle while rinsing, 

then they are placed in 18 gal (68 L) tubs to waterharden for approximately 4 h. The tubs are filled twothirds full of river water and eggs are added until the 

tub is one-third full of eggs. The eggs will swell until 

the tub is one-half full of eggs. The water is changed 

every hour during this 4 h hardening period. 

Eggs are considered to be fragile during the water 

hardening process, consequently, they are treated gently 

during this period. Several times, however, we have 

shipped freshly fertilized eggs to hatcheries several 

hours away by placing them loose in water into coolers 

or plastic cans. These eggs developed normally and they 

had survival rates similar to eggs taken the same day 

and hardened in the usual manner. 

lncu bat ion 

After water hardening, the tubs are transported a short 

&stance to the hatchery and eggs are placed in 5 qt (4.7 

L) plastic hatching jars. Each day's egg take is incubated separately so survival can be tracked. The jars are 

then placed on hatching batteries. Water flow is 

adjusted in each jar so that the egg mass is buoyed a 

few inches by upwelling and the eggs are gently rolling. 

Each jar requires 0.8-1.0 gal/min (3-3.8 L/min) of 

flow. 

Egg size 

Egg sizes are measured 12-1 8 h after fertilization, 

using a 6 in (15.2 cm) Von Bayer trough. Past measurements have shown no consistent change in egg sizes at 

4, 8, 24, 48, 96, and 168 h post-fertilization. Samples of 

eggs are collected with a 0.5 in (1.3 cm) tube. The tube 

is pushed vertically to the bottom of the jar, the top of 

the tube is covered with a thumb, and the tube withdrawn. Three counts are made for each egg lot and the 

average count is interpolated on a Von Bayer chart to 

determine the number of eggs per quart . Egg sizes are 

larger at the beginning of the spawning run (1 10,190/qt) 

than at the end of the run (127,333/L), probably due to 

the larger size of females at the beginning of the run. 

Wafer supply 

The water source for the hatchery is the Pike River, 

which warms faster than area lakes, often reaching 65°F 

(18°C) during the later stages of egg incubation. Goals 

during incubation are to delay the hatch, to allow a 

gradual warming of incubation water to match the 

temperature of lakes to be stocked when the eggs hatch, 

and to minimize temperature swings during incubation. 

The intake water has sufficient oxygen and contains low 

levels of minerals. Water is pumped into a 10,000 gal 

(37,850 L) holding tank on a knoll above the hatchery 

from which it flows by gravity to the hatchery. An 

adjustable chiller with no recirculation allows hatchery 

water to be cooled as much as 10°F (5.6"C) below 

ambient. 

Temperature units for development and hatching 

Hatching battery temperatures are taken at 4 h intervals. 

Daily Temperature Units (DTU's), expressed as the 

average daily temperature (OF) minus 32"F, are calculated for each egg lot. The number of DTU's at eye-up, 

appearance of first fry, and completion of hatch are 

recorded for each egg lot. Seven years of data shows the 

average time to hatch is 27 d with a range of 22-31 d. 

Incubation begins at about 46°F (8°C) and temperatures 

are gradually increased until hatch occurs at about 57°F 

(14°C). Average DTU accumulations are: 206 to eyeup, 307 to first appearance of fry, and 478 to completion of the hatch. More DTU's are needed when 

incubation temperatures are cool than when they are 

warm. The linear formula for this relationship is: days 

to complete hatch = 23.2 + 0.104 x DTU's (R2 = 0.64). 

Removal of dead eggs 

Dead eggs often develop saprolegnia fungus, usually 

after about 5 d. Dead and fungused eggs are lighter than 

live eggs and they will gradually segregate to the top of 

the egg mass in the hatching jars. Dead and fungused 

eggs are siphoned into a screen box, with 0.125 in (3.2 

mm) apertures, that is floating in a tub of water. 

Over several siphonings, about 10% of the live eggs 

will be drawn out of the jars along with the dead eggs. 

These eggs are saved with the following procedure: 

When several quarts of fungused and live eggs have 

been drawn into the screen box, the box is suspended 

NCRAC Culture Series 101 - Walleye Culture Manual 43 
Chapter 2 - Reproductive Biology and Spawning 

above the water in the tub, and a stream of water from 

the siphon tube is used to break apart the fungus clumps 

and force all eggs through the screen into the tub. Water 

is then poured out of the tub through overflow screens, 

the eggs are returned to the hatching jars and placed on 

the hatching battery, and water is started through them. 

After several minutes the live eggs will remain on the 

bottom and the dead eggs will again separate. Dead 

eggs and fungus “fluff” are siphoned off and discarded. 

One day after embryonic eyes are visible (eye-up) 

virtually all dead eggs have been removed. Eggs are 

then poured into a tub, the jars are brushed clean, and 

eggs are placed into hatching jars at the rate of 2 qt/jar 

(1.9 L/jar) instead of the initial rate of 2.5 qVjar (2.4 L/ 

jar). This lower incubation density is needed because 

the eggs tend to buoy higher in the jar after eye-up. The 

survival rate for each lot is calculated by dvidmg the 

remaining eyed-egg volume by the initial egg volume. 

Virtually all eyed-eggs hatch. 

If the hatchery water warms quickly during incubation, 

gas bubbles may come out of solution, attach to eggs, 

and float them out of the hatching jars. To overcome 

this problem we turn down the flow rates to the jars, 

reduce the rate at which the water is allowed to warm, 

or install agitators or packed-column diffusers. 

Fry holding tanks 

After hatching, fry and egg shells pass to the lowest 

tank on the hatching battery and through a pipe to the 

fry holding tanks. The fry tanks have a total capacity of 

900 gal (3,400 L), but they are inadequate to hold all 

the fry (24 million) from a battery even if water is 

chilled and the flow increased. To handle all fry that 

hatch, fry are occasionally removed for stocking during 

the hatching period. Brass screens, with 0.027 in (0.68 

mm) circular apertures), are placed in front of the 

outlets at each end of the fry tanks and bubblers made 

from 0.5-in (1.3 cm) CPVC plastic plumbing tubes with 

0.09 in (2.4 mm) perforations are placed in front of the 

screens to prevent fry and egg shells from clogging the 

screens. 

Once hatched, fry are removed from the fry tank and 

stocked into lakes or fingerling production ponds within 

48 h to prevent holding mortality. Fry are dpped with a 

net made from fine nylon mesh with 150 p apertures 

and 51 % open area. The net is 2 x 3 ft (0.6 x 0.9 m) and 

is attached to two 1.0 in (2.5 cm) wooden dowel rods. 

When dipped, fry are gently rolled in the net for several 

seconds to let excess water drain. When the stream of 

water from the net becomes dscontinuous and starts to 

form drops, fry are poured into a tub of water and 

weighed with a digital scale. 

Fry enumeration 

we have attempted many methods of measuring fry 

includmg volumetric displacement and weighmg. Most 

such samples yielded about 90,000 fry/lb (1 98,00O/kg, 

or 5 mg each), but weights were highly variable. We 

collect the samples during the midway point of each 

pour from the net because the early portion of each pour 

contains more water than later portions. At present, we 

use a standard rate of 100,000 fry/lb (220,000 frykg, or 

4.5 mg each). 

44 NCRAC Culture Series 101- Walleye Culture Manual 
Brown, G. G. and A. A. Moore. 1996. Comparative storage methods and fertility studies of walleye 

semen. Pages 45-49 in R. C. Summerfelt, editor. Walleye culture manual. NCRAC Culture 

Series 101. North Central Regional Aquaculture Center Publications Office, lowa State University, Ames. 

Comparative Storage Methods and Fertility 

Studies of Walleye Semen 

George G. Brown, Department of Zoology and Genetics, lowa State University, Ames, Iowa 5001 I, and Alan 

A. Moore, Rathbun State Fish Hatchery, lowa Department of Natural Resources, RR 2, Moravia, Iowa 

52571 

Introduction 

The timing and duration of the natural breeding season 

of walleye is limited by environmental and ecological 

conditions. Often, males ripen before females, which 

requires capture and holding of male walleye in the 

hatchery until females ripen, however, this may affect 

reproductive physiology of the male and sperm viability. To circumvent ths problem, it is possible to 

refrigerate or cryopreserve semen for later use. The 

objective of this report is to describe methods for the 

storage of semen and its use in fertilization of walleye 

eggs. Effective use of these techniques can improve the 

reliability of fertilization and facilitate walleye breeding 

programs. 

Methods 

Samples of seminal fluid were obtained from male 

walleyes at the Rathbun Fish Hatchery and Spirit Lake 

Fish Hatchery during April and May, 1991-1994. Males 

were anesthetized with tricaine methanesulfonate 

(FinquelTM) and semen was collected by using semen 

aspiration apparatus (Moore 1996). Semen samples 

were collected in calibrated plastic test tubes, the 

volume of semen measured, then the semen transferred 

to 40 ml, tissue culture flasks (Figure l), that have a 25 

cm3, growth area for transportation from the hatchery to 

Iowa State University. The flasks are durable, reusable, 

Figure 1. Standard tissue culture flasks used for storing 

and transporting walleye semen. 

and excellent for transporting and storing samples. Best 

results were obtained when semen and all containers 

were kept on wet ice (ice allowed to partially melt, 

0°C). 

Analysis of untreated semen involved the measurement 

of volume, pH, sperm concentration, and a dye exclusion test. Sperm concentrations were determined by 

mixing 25 Ils, of semen with 25 ml of a saline solution 

(usually extender 7), and by using a hemacytometer to 

obtain counts. The average count was multiplied by 

50,000 to obtain the concentration of spermatozoa 

(spermatozodml). The dye exclusion test was used to 

determine the concentration of living (viable) spermatozoa. An eosin solution (0.113 g of Eosin B/100 ml of 

extender) was mixed (1:5) with semen and sperm 

counts were made of the semen-eosin solution in a 

hemacytometer. Because only dead spermatozoa 

absorbed the dye, the percentage of viable spermatozoa 

could be determined. 

Sperm motility is used as an indicator of sperm viability 

of fresh and stored semen samples. Motility was 

determined by using the drop method. A drop of semen 

about 1.0 mm in diameter was placed on a coverslip and 

a large drop (about 0.05 ml) of dechlorinated water was 

placed over the smaller drop of semen resulting in a 

rapid dilution of the semen. With high-quality semen, 

an explosive motility which persisted for about 30 s, 

occurred following addition of water. However the 

percentage of samples having sperm motility percentage decreased with age of the samples. 

To evaluate stored samples, a sperm motility index 

(smi) was used to determine the percent of sperm that 

were motile: 6 = explosive and 100% sperm motility; 5 

= 75100%; 4 = about 50% of the sperm were motile; 3 

= 5 to 25%; 2 = ~5%; 1 = 4%; 0 = no motile sperm. In 

all cases, motility was recognized as forward motion of 

NCRAC Culture Series 101 - Walleye Culture Manual 45 
Chapter 2 - Reproductive Biology and Spawning 

spermatozoa, not vibratory motion. Sperm motility was 

determined as soon as possible, usually within 5-15 

sec, after addition of water to the drop of semen. 

Initially, semen samples were stored undiluted (raw), at 

a depth no greater than 0.3 mm (usually 3-5 ml of 

seminal mixture) in the tissue culture flasks. These 

flasks were placed flat in a refrigerated incubator (l’C), 

semen was 8.5. The dye exclusion test averaged 94.1%, 

inhcating that almost all spermatozoa were viable. The 

combination of high sperm count and high viability 

indicated that the fresh semen was of high quality. 

Undiluted semen did not store well, as indicated by a 

rapid drop in the percentage of sperm motility after 3 d 

(Table 2). 

and the lids were loosened to 

provide air exchange. At intervals 

over 28 d, small amounts of semen 

were removed from the flasks to 

determine sperm motility. 

Results and discussion 

The volume of undiluted raw 

semen collected from each male 

averaged 3.6 ml, with a maximum 

of 7.0 ml (Table 1). The sperm 

concentration averaged 38.6 x lo9 

spermatozodml. The pH of the 

Table 1. Analysis of undiluted walleye semen. 

Average Range Samples 

volume (ml) per male 3.6 2.0-7.0 49 

dye exclusion (%)” 94.1 86.2-99.6 12 

sperm concentration (xlO9) 38.6 24.0-59.4 31 

a a measure of sperm viability 

Table 2. Analysis of semen quality after storage at 1°C for 3-28 d: % survival (%S) and the 

average sperm motility index (srni). 

Od 3d 6-7 d 14 d 19-21 d 28 d 

n %S smi %S smi %S smi %S smi %S smi %S smi 

Loosened lids 

undiluted 10 

extended 6 

Tightened lids 

extended 6 

Extendedb 

1 :1 10 

1 :2 44 

Extendersb 

Ext7 21 

Rathbun’s Ext 20 

Extl3 13 

100 

100 

100 

100 

100 

100 

100 

100 

5.2 90 

5.0 100 

5.0 100 

6.0 100 

5.5 98 

5.6 95 

5.6 100 

5.6 100 

5.0 

5.0 

5.0 

5.0 

4.7 

4.5 

5.0 

4.9 

10 

100 

100 

100 

91 

81 

100 

100 

2.0 0 

5.0 83 

5.0 100 

5.0 100 

4.8 70 

4.8 52 

5.0 81 

4.8 92 

5.0 83 4.5 17 1.0 

5.0 83 2.4 0 

4.4 0 

4.9 45 3.5 2 1.0 

4.4 10 2.0 0 

4.8 33 4.3 0 

4.9 62 3.1 7 1.0 

aAverage sperm motility index (srni) in dechlorinated tap water: 6 = explosive, 100% motility; 5 = 75-1 00% motility; 4 = -50% motility; 

3 = 525% motility; 2 = 4% motility; 1 = el% motility; 0 = no motility. 

Samples stored in flasks with loosened (48) and tightened (6) lids. 

46 NCRAC Culture Series 101- Walleye Culture Manual 
Reproductive Biology and Spawning - Chapter 2 

Three extenders were evaluated: extender 7, Rathbun’s 

extender, and extender 13 (Table 3). Extender 7 and the 

Rathbun formula are modifications of an extender 

described by Erdahl and Graham (I 980). Extender I3 is 

a simple saline solution (0.88% NaCI) with 1 % penicillin-streptomycin. Samples were extended and tested in 

a 1: 1 and 1:2 ratio (semen:extender) and with tightened 

and loosened lids (Table 3). Although both ratios of 

semen:extender maintained high-motility samples for 

14 d, we selected the 1 :2 ratio for further studies. No 

differences were determined in sperm motility of semen 

samples stored in flasks with either tightened or 

loosened lids. However, experience of the senior author 

with storage of white bass and striped bass semen 

indicated that storage in containers with loosened lids 

was detrimental. 

All three extenders maintained semen in good condtion 

for 14 d, but a higher percent of samples (81% and 

92%) had a higher smi (4.8 and 4.9, respectively) with 

the Rathbun walleye extender and extender 13 compared with extender 7 (52% and 4.4 srni). In general, we 

found that extending seminal fluid which dilutes the 

sample and reduces the concentration of spermatozoa is 

more important in maintaining the viability of stored 

semen than chemical composition of the extender. 

Semen diluted with extender 13, a saline solution, had 

higher survival and srni than undiluted semen (Table 3). 

Although antibiotics were used in all extenders, they 

may not be necessary because the semen contains 

natural antibiotics. 

Samples of extended semen that were stored 2-14 d 

were used to fertilize freshly collected walleye eggs 

(Tables 4, 5). Eggs were stripped into a dry pan. About 

4 ml sample of seminal fluid from one flask was used to 

fertilize about 60 ml of eggs. Three d post-fertilization, 

aliquots of 25 fertilized eggs were counted. All samples 

were maintained until after hatching. These experiments 

demonstrated that the viability of walleye semen that 

had been diluted with extender and maintained at 1°C 

for two weeks fertilized 75-99% of walleye eggs 

(Tables 4, 5). 

We evaluated two media for cryopreservation of 

spermatozoa. Cryogenic medium A consisted of IO g 

glucosek of Rathbun’s extender, 7.5 mg/ml PRO-FAM, 

4 mg/ml BSA, and 7% 

Table 3. Composition of extenders. Extenders were mixed, pH 

adjusted, then used or frozen. 

Extenders Ext 7 Rath bu n’s Ext 13 

CaC1,.2H20 

NaHC03 

Na2HP04 

KC I 

NaCl 

g I ucose 

pyruvate 

citric acid 

bicinea 

HEPES 

MgCI2.6H20 

ddH20 

KOH(1.27g/lOOml) 

pen-st re pb 

PH 

0.103 g 

0.235 g 

2.558 g 

5.780 g 

6.000 g 

0.220 g 

0.100 g 

2.380 g 

1000 ml 

10 ml 

10 ml 

7.6 

0.117 g 

0.134 g 

0.236 g 

1.872 g 

6.578 g 

10.000 g 

0.100 g 

20 rnl 

1000 ml 

10 ml 

9.0 

8.760 g 

1000 ml 

10 ml 

7.6 

a5.3g/l OOml 

bpenicillin-streptomycin: Sigma, P0906: 5000 units of penicillin, 5 mg streptomycin per ml of 

0.9% NaCl. 

dimethyl sulfoxide (DMSO). 

Cryogenic meQum B consisted 

of 7.5 g glucose and 2.5 g 

sucroseL of Rathbun’s extender, 4 mg/ml BSA, egg yolk 

(1.5 ml yolld100 ml extender), 

and 7% DMSO. Fresh semen 

was mixed 1:2 with cryogenic 

medium in a 5 ml centrifuge 

tube (1°C). The mixture was 

drawn into freezing straws (0.25 

ml), the ends sealed and straws 

placed on a flat piece of dry ice 

for 30 min. Straws with frozen 

semen were stored in the vapor 

area of a liquid nitrogen 

chamber. After about one year 

of storage, the semen in the 

straws was thawed by holding 

under running tap water. The 

ends were cut off and content 

empty onto 2 oz (56.7 g) of 

freshly collected walleye eggs. 

Development was followed until 

hatchng and the fertilization 

rate was measured 3 d posthatch (Table 6). The fertility rate 

NCRAC Culture Series 101 - W a l l e y e C u l t u r e Manual 47 
Chapter 2 - Reproductive Biology and Spawning 

using semen that had been stored one year was 31.3%. 

We think that with some improvement of techniques, 

this percentage could be significantly higher. 

University of Minnesota, Minnesota Sea Grant 

Program 102, St. Paul. 

Ref e re n ces 

Erdahl, D.A., and E.F. Graham. 1980. Cryopreservation 

of spermatozoa of brown, brook, and rainbow trout. 

Moore, A. A. 1996. Use of semen extenders for walleye. 

Pages 51-53 in R. C. Summerfelt, editor. Walleye 

culture manual. NCRAC Culture Series 101. North 

Central Regional Aquaculture Center Publications 

Office, Iowa State University, Ames. 

Table 4. Fertility of walleye semen mixed 1 :2 in extender 7 or 

extender 13 and stored 2-13 d.The control semen was fresh 

and undiluted semen. The percentage of fertilized eggs for 

both extender and control was determined 3 d 

postfertilization. 

No. days Number of Extender Extender Control Extender/ 

stored Trials 7 13 Co nt rola 

2 

4 

4 

5 

6 

6 

7 

8 

8 

9 

10 

10 

11 

13 

1 

1 

2 

1 

1 

2 

1 

1 

2 

1 

1 

2 

1 

1 

99.2 

84.0 

88.0 

59.2 

68.0 

90.4 

77.4 

85.6 

78.4 

97.6 

88.0 

88.0 

97.6 97.6 

93.6 

93.6 

77.6 88.0 

95.2 

95.2 

71.2 93.6 

96.0 

96.0 

76.8 95.2 

97.6 96.0 

100.2 

95.5 

100.0 

100.0 

63.2 

72.6 

88.2 

95.0 

81.3 

76.1 

89.2 

81.7 

80.7 

101.7 

aThe percentage of fertilized eggs from semen stored in extender was divided by percentage of 

fertilized eggs from control semen 

48 NCRAC Culture Series 101- Walleye Culture Manual 
Reproductive Biology and Spawning - Chapter 2 

Table 5. Fertility of semen stored in 1 :2 Rathbun’s extender compared 

with control, undiluted semen. The percentage of fertilized eggs for 

both extender and control was determined 3 d postfertilization. 

(modified Moore 1987). 

Days Number of Rath bun’s Control Extended 

stored Trials Extender Contro la 

10 2 93.6 97.2 96.3 

11 2 92.0 92.8 99.0 

12 2 89.2 90.8 98.2 

13 2 90.8 94.8 95.8 

14 2 89.2 90.4 98.7 

aThe percentage of fertilized eggs from semen stored in extender was divided by percentage of fertilized 

eggs from control semen 

Table 6. Fertility of walleye eggs fertilized with cryopreserved semen and fresh 

(control) semen. 

Cryogenic Number Number Percent hatch of Percent Percent hatch 

females eggs semen con t ro I semen c ryo prese rved 

semen relative 

to controla 

media of of cryo-p reserved hatch of from 

A 1 95 24.0 

B 1 108 13.6 

total 2 203 18.5 

59.2 

59.2 

59.2 

40.5 

22.3 

31.3 

aThe percentage of fertilized eggs from cryopreserved semen was divided by percentage of fertilized eggs from control semen 

NCRAC Culture Series 101 - Walleye Culture Manual 49 
Chapter 2 - Reproductive Biology and Spawning 

50 NCRAC Culture Series 101- Walleye Culture Manual 
Moore, A. 1996. Use of semen extenders for walleye. Pages 51-53 in R. C. Summerfelt, editor. 

Walleye culture manual. NCRAC Culture Series 101. North Central Regional Aquaculture 

Center Publications Office, Iowa State University, Ames. 

Use of Semen Extenders for Walleye 

Alan Moore, Rathbun HatcherN Iowa Department of Natural Resources, Moravia, IA 52571 

Introduction 

The Fisheries Bureau of the Iowa Department of 

Natural Resources (DNR) has two hatcheries dedxated 

to the production of walleye. The hatcheries at Spirit 

the sperm. Semen are activated in water, and they have 

a short period of motility after activation (4 min), 

therefore, during the aspiration process, semen is kept 

Lake and Rathbun hatch about 115 million fry 

and raise 3 10,000, 5-7-in (12.7-1 7.8-cm), 

fingerlings annually. In addition, 500,000, 2 in 

(5.0 cm) walleye are raised at satellite 

facilities. 

Each spring, DNR hatchery personnel collect 

large numbers of wild, spawning size walleye. 

Because males often become ripe several days 

before females, males may be held in the 

hatcheries for two to three weeks. This period 

of captivity may lead to thick, viscous semen, 

reduced fertility, and mortality. Since 1987, the 

stripping of male walleye prior to female 

readiness and the preservation and cold storage 

of semen has enhanced hatchery efficiency, 

reduced male handling loss, and increased egg 

fertility. Preserved (extended) semen has been 

used successfully at both the Spirit Lake and 

Rathbun hatcheries. This paper will discuss the 

methods used at Rathbun and introduce the 

basics of walleye semen preservation. 

General semen collection and storage 

methods 

walleye broodstock are collected by gill net, 

transported to the Rathbun Hatchery, sorted by 

sex, and held in raceways. Prior to stripping, 

males are anesthetized in water with 200-300 

ppm carbon dioxide, rinsed in fresh water, 

wiped dry, and held ventral side up. Semen is 

forced from the fish with gentle external 

pressure along the gonad area and semen is 

collected using a water-powered sink aspirator 

(Figures 1 and 2). Only enough suction is used 

to gently move the semen through the collection tubes because too strong a suction will kill 

free from contamination by water or urine. 

Figure 1. Water powered sink aspirator used to collect walleye 

Figure 2. Stripping semen from a walleye with gentle external 

pressure. 

NCRAC Culture Series 101 - Walleye Culture Manual 51 
Chapter 2 - Reproductive Biology and Spawning 

an ice-water bath. One 9-ml flask of 

extended semen is used for each 

group of eggs to be fertilized; about 

one quart (0.9 L) of eggs is maximum volume. The extended semen is 

poured onto the dry eggs, mixed 

gently with a feather or finger, and 

lake water added to activate sperm 

cells. Use of extended semen during 

spawning has increased egg fertility 

by 5%. 

Walleye semen may also be frozen 

for long-term storage and used to 

preserve genetic lines. To freeze 

walleye semen, the extender formula 

given in Table 1 is modified by 

reducing the amount of glucose to 

15.0 g, and addmg 5.0 g. mannitol, 

4 mg/ml bovine serum albumin, 

7.5 mg/ml protein, and 7% dimethyl 

Figure 3. Collecting semen which flows from the aspirator directly 

into a test tube that is cradled in an ice water bath. 

Semen is aspirated into 10-ml vials that are held in an 

ice-water bath (Figure 3) at a temperature of 34-39°F 

(1 .O-4.O0C). Immehate cooling of the semen prevents 

premature sperm activation and prolongs cell life. For 

refrigerated storage, 3.0 ml of semen is placed in a 

25-cm2 tissue culture flask, followed by 6.0 ml of 

extender (Figure 4) (Table 1, 

opposite page). The mixture of 

semen and extender is gently 

swirled. To prevent sperm activation, the extender is always added 

to the semen. Total semen-extender 

volume is 9.0 ml. To allow for gas 

exchange, the maximum storage 

depth per flask is 2.0-3.0 mm, and 

flask lids are screwed on but left 

loosened. Flasks are stored in a 

refrigerator at 33-36°F (0.6- 

2.2"C). To prevent the semen and 

extender from separating, flasks 

are gently swirled on a daily basis. 

Semen may be stored in this 

manner for up to 14 d with little 

loss of fertility; however, beyond 

14 d, results may be variable. 

sulfoxide to the mixture. 

Semen is collected in the same manner as in the 

refrigerated semen process, and mixed with the modfied extender at a 1:2 ratio of semen to extender. The 

semen-extender mixture is aspirated into 0.25-ml 

During the stripping of female 

walleye, semen flasks are removed 

from the refrigerator and placed in Figure 4. Semen is measured into tissue culture flasks and extender added. 

52 NCRAC Culture Series 101- Walleye Culture Manual 
Reproductive Biology and Spawning - Chapter 2 

plastic straws, sealed with a 

cryo-sealer, placed on dry ice 

for 30 min, and stored in liquid 

nitrogen. 

To use the cryopreserved 

semen, the straws are placed in 

70°F (21.1 "C) extender, then 

mixed with eggs immediately 

upon thawing. Approximately 

5 straws are used per 2 fl oz 

(59.1 rnl) egg volume. Fertility 

rates ranging 48-83% have 

been achieved using 

cryopreserved sperm. 

Table 1. Composition of the extender added to 

walleye semen used at Rathbun and Spirit Lake 

Fish Hatcheries. 

Constituent Amounta 

Distilled water 1,920 ml 

Calcium chloride dihydrate (CaC1 2.2H20) 0.234 g 

Magnesium chloride (MgC1 p6H20) 0.267 g 

Sodium phosphate dibasic (Na2HP04) 0.472 g 

Sodium chloride (NaC1) 13.155 g 

Potassium chloride (KC1) 3.744 g 

Glucose 20.000 g 

Citric acid monohydrate 0.200 g 

HOCCOOH (CH2COOH)2mH20 

Sodium hydroxide (NaOH) solutionb 40 ml 

Bicine solutionC 40 ml 

a Total volume equals 2000 ml. 

NaOH solutions is 1.27 g NaOH/100 ml water: 

Bicine (N,N-bis[2-hydroxyethl]glycine) solution is 5.3 g bicine/lOO ml water. 

NCRAC Culture Series 101 - Walleye Culture Manual 53 
Chapter 2 - Reproductive Biology and Spawning 

54 NCRAC Culture Series 101- Walleye Culture Manual 
Koupal, K. D. 1996. Field utilization of extended semen. Pages 55-58 in R. C. Summerfelt, editor. 

Walleye culture manual. NCRAC Culture Series 101. North Central Regional Aquaculture 

Center Publications Office, Iowa State University, Ames. 

Field Utilization of Extended Semen 

Keith D. Koupal, Colorado Division of Wildlife, 6060 Broadway, Denver, CU 80216 

Introduction 

In 1949, the Colorado Division of Wildlife (CDOW) 

introduced walleye into artificial lakes of Colorado on 

the eastern side of the Rocky Mountains. They are now 

a popular sport fish with Colorado anglers. Because 

natural recruitment has been inadequate to sustain a 

sport fishery, the CDOW developed a large-scale 

program of stochng artificially propagated walleyes 

into Colorado’s eastern man-made impoundments. 

Traditionally, Bonny Reservoir and, more recently, 

Pueblo and Horsetooth Reservoirs have been the major 

sites for walleye spawning operations by the CDOW. 

Broodstock collection on these reservoirs has provided 

between 50 and 200 million eggs annually for use by 

the CDOW. In the mid-1 980s, however, adequate fry 

production was impaired because insufficient numbers 

of ripe males were captured. The shortage of male 

walleye seems to be the consequence of selective 

angling mortality for males whch are more vulnerable 

to anglers’ catch than females. 

To increase the availability of male walleye, CDOW 

personnel established a walleye population in Marston 

Reservoir, which is controlled by the Denver Water 

Department and is not open to public angling. This 

walleye population provides a greater proportion of 

male fish. Milt from Marston Reservoir walleye is 

collected, preserved with semen-extender, then shipped 

to spawning operations that require additional semen. 

Field operations 

Spawn collection site 

The initial step of the spawning operation is to set up a 

site on the shoreline for spawn-taking. The ideal site 

has a permanent building located adjacent to the 

shoreline that can be used annually to obtain milt, eggs, 

or both. A source of dechlorinated running water would 

also be ideal, but it is not a necessity. Because a 

permanent shoreline site is not always available, a wall 

tent is helpful. It is extremely important, however, to 

conduct walleye stripping operations in some form of 

shelter, because precipitation and sunlight can compromise the results. The spawn-takmg area will also 

require at least two sturdy tables, one to keep the 

spawning equipment out of the mud, and the other to 

strip males. It is important to have a table that is a 

comfortable height (adjustable height preferred), tubs 

that can hold at least 15 gal (56.8 L) of water, paper 

towels to wipe off fish and equipment, rubber gloves, a 

supply of 10 mL syringes, an oxygen bottle and hose 

hook-up, microscope with slides to check sperm 

motility, and a portable electrical generator. 

Broodstock collection methods 

Trahtionally, CDOW biologists captured walleye with 

gill and trap nets, however, we have found that night 

electrofishing is more efficient. Personnel first spotlight 

groups of spawning walleyes along rip-rap and shallow 

rocky shorelines, then electrofish these areas. 

Electrofishing spawning aggregation can provide up to 

50 male walleye per hour of electrofishng. Although 

there was initially some concern that electrofishing 

would reduce the viability of the gametes, a difference 

in the hatching percentage of eggs fertilized with semen 

of walleye collected with electrofishng and passive 

gear has not taken place. 

Semen collection 

Semen is collected and extended at the spawn taking 

area. At Marston Reservoir, which is closed to public 

fishmg, male walleye are anesthetized with 100 mg/L of 

Finquel, but on public waters, a dfferent anesthetic is 

needed because Finquel requires a withdrawal period of 

21-days before treated fish can be released. When using 

Finquel to anesthetize fish, we wait until fish are 

motionless, then they are removed from the anesthetic 

solution, briefly dipped in fresh water, then wiped with 

paper towels. It is essential that no moisture be present 

on the fish or any of the supplies utilized in this 

operation to avoid activating the sperm before it is 

mixed with the eggs. Milt is extracted from the walleye 

into a small plastic cup that rests in a larger cup 

containing an ice bath, which keeps the milt temperature between 33 and 35°F (0-1°C). The composition of 

the extender that we use is the same as that described 

NCRAC Culture Series 101 - Walleye Culture Manual 55 
Chapter 2 - Reproductive Biology and Spawning 

by Moore (1987, 1996). It is maintained at a temperature close to that of the milt. 

It is important to prevent contaminating milt in the 

collecting cup. The longer it remains in the open the 

more chance it has of becoming contaminated. Large 

quantities of milt in the collecting cup are at greater risk 

of contamination. A guideline for determining the 

amount of milt collected is that male walleye on 

average produce 3 mL, 1.25 mL, and 0.5 mL on their 

first, second, and third daily strippings, respectively. 

These numbers can be used to judge the amount of 

semen that has been collected within the cup until 

personnel are able to use visual estimates. 

To prepare a 12 semen:extender ratio, we place 8 mL 

of milt in a plastic sandwich container (Figure 1) and 

add 16 mL of extender with a syringe. We collect 

sufficient milt to fill at least two of the plastic sandwich 

containers, then we use a dry syringe to remove the 

amount needed to make the desired semen:extender 

ratio. We store extended semen in 24 mL quantities 

because this volume produces a shallow film in the 

container and allows for good oxygen transfer. After air 

is blown into the container, the container is quickly 

sealed, labeled with the date and semen:extender ratio 

then placed in a cooler filled with cubed ice for storage. 

The stored semen is effective for fertilization for 10 d if 

it is oxygenated daily with pure oxygen, sheltered from 

all moisture, and stored at temperatures 3346°F (0.6- 

7.7"C). Even when stored at 46"F, which 

may seem hgh, extended semen has 

produced satisfactory results. 

Sperm motility of all extended semen 

should be checked with a microscope 

(400X) before use. This can be done by 

placing a thin film of extended milt on a 

slide and observing sperm motility 

(movement) when water is added. Batches 

of extended semen that are considered to 

be good will have nearly 100% motile 

sperm cells, and motility will last at least 

30 sec. 

fertilized by fresh semen, as far as water hardening, 

storage, and hatching are concerned. 

Problems 

Sperm activation by accidental contamination with 

water is of major concern. Water contamination occurs 

from wet gloves that were utilized to &p fish in fresh 

water after Finquel bath, filling the larger ice bath cup 

too full and pushing water over the edge of the cup 

holding the milt when extracting the milt for preservation, and placing tubs used for anesthetizing and 

recovery where water from splashing fish may contaminate the milt. Finally, after extended semen has been 

successfully preserved in plastic sandwich containers 

the container must be securely fastened to prevent 

contamination from melting ice. 

Another concern is fecal material contaminating 

extended semen. It is common for fecal material to be 

expelled when stripping semen, especially during the 

second and third strippings when additional pressure is 

needed to expel semen. Fecal material reduces the 

quality of extended semen and should be removed 

immediately with a dry spoon. Blood in semen is also a 

common sighting, however, blood is not considered 

detrimental to semen quality and need not be removed. 

We think that extended semen must be oxygenated 

daily (Figure 1) to ensure that sperm cells will have 

Four mL of extended milt are used to 

fertilize 1 L of eggs by the dry method 

(Figure 2). Eggs fertilized with extended 

semen are treated similarly to those 

Figure 1. Addition of pure oxygen to a plastic sandwich container 

containing extended semen (1 part semen: 2 parts extender). 

56 NCRAC Culture Series 101- Walleye Culture Manual 
Reproductive Biology and Spawning - Chapter 2 

Figure 2. A syringe with 4 rnl of extended semen is used to fertilize 

1 L of eggs by the dry method. 

adequate oxygen to sustain life, even in their cold, 

Extended semen must be checked for 

motility before it is utilized. Many field 

personnel forget the importance of 

insuring the quality of extended milt they 

are using. All containers must be checked 

to avoid contamination of extended 

semen. 

Hand1 i ng procedures 

Undoubtedly, capture and handling of 

walleye utilized in spawning operations 

will be stressful to the fish. Typically, 

males are stripped for three consecutive 

days and then released. To evaluate postspawning mortality of walleye, we held a 

group in a culture pond for 45 d after 

spawning. We observed 22% mortality in 

this group. Since that observation, we 

have taken steps to decrease mortality 

from spawn-taking operations. If ample males are 

collected, it is suggested that they be released after only 

two strippings. The third stripping provides minimal 

amounts of milt which is often more &lute and contaminated with fecal material. If practical, males should 

be stripped on alternate days. This practice allows the 

almost dormant state. It is the opinion of some personne1 that the initial oxygenation should be sufficient to 

maintain quality sperm cells, and they think that 

subsequent oxygenation is not only unnecessary, but 

that the handling of the extended milt may be stressful 

and increase the chances of contamination bv water. 

Daily oxygenation may not be necessary, but until 

further research is conducted, it is suggested that 

containers be reoxygenated daily. 

A condensate will often be present on the lid of the 

plastic sandwich containers holQng the semen, but this 

condensate has not stimulated sperm motility when it 

mixes with the extended semen. However, it is probably 

advisable to be careful to avoid dripping the condensation into the semen container. 

Accidental freezing of stored semen has been observed 

in field operations. This may reduce semen viability. 

Preserved semen should be kept between 33 and 46°F 

(14°C). We found that crystallization starts in the 1:2 

semen:extender ratio at 26.4"F (-3.1OC) and solidfication can be accomplished at 23°F (-50°C). Semen that 

has undergone partial freezing or solidification of the 

extended semen should be used only as a last resort. 

Extended semen that has been partially frozen or 

solidified results in less than optimum motility when 

viewed under the microscope. A propane heater placed 

within the structure where extended semen is kept may 

prevent freezing from occurring on colder nights. 

males to rejuvenate milt, and they will provide more 

milt on alternate days than when they are stripped in 

two consecutive days. 

Spawning operations that collect adequate numbers of 

males can use the first stripping for fresh milt fertilization and milt from the second stripping for preparation 

of extended semen. Even with adequate numbers of 

males, extended semen is a valuable back-up supply. 

When insufficient numbers of males are available to use 

only fresh semen, then extended semen at 1:2, 1:4, or 

1:6 ratios should be used. 

Advantages and disadvantages of extended 

semen 

The use of extended semen requires adhtional labor, it 

is time consuming, and stored semen can become 

contaminated with water or feces. However, experiments conducted at Marston Reservoir during the 1993 

and 1994 spawning seasons proved the value of 

extended semen. Over two years: 63.7% of eggs (9.2 

million) fertilized with fresh semen hatched; 59.8% of 

eggs (2.2 million) fertilized with 1 :2 semen:extender 

NCRAC Culture Series 101 - Walleye Culture Manual 57 
Chapter 2 - Reproductive Biology and Spawning 

hatched; 60.7% of eggs (2.4 million) fertilized with 1:4 

semen:extender hatched; and 66.9% of eggs (2.8 

million) fertilized with 1 : 6 semen:extender hatched. 

These results show that extended semen is just as 

effective as fresh semen. 

In Colorado, we do not plan to replace fresh semen in 

spawning operations entirely with extended semen. We 

consider use of extended semen an alternative that field 

biologists can use effectively in special circumstances. 

When these opportunities arise, however, extended 

semen becomes especially valuable. Although a higher 

hatching percentage was obtained with fresh semen in 

the first two-thrds of the spawning operation, in the last 

one-third of the season, gamete quality decreases and 

thus lower hatching percentages occur. However, 

hatching success of eggs fertilized with extended semen 

has been less variable and extended semen results in 

higher hatchmg percentages in the last one-third of the 

spawning season. Thus, the practical advantage of 

extended semen is the capability to preserve gametes 

collected earlier in the spawning season for use when 

fresh semen quality decreases or is less available. 

Extended semen can be transported from one spawning 

site to another. It is far easier to transport extended 

semen than live fish. Transported males often become 

stressed and fail to produce sufficient amounts of milt. 

Extended semen is especially advantageous for making 

hybrid crosses. Hybrid walleye are prepared using 

sauger semen and walleye eggs. Extended semen eases 

the transportation process of male gametes and allows 

for crosses of geographically isolated populations. Also, 

extended semen can be used for mating different 

genetic stocks. 

Colorado Division of Wildlife has utilized extended 

walleye semen since 1988 and found it to be comparable to the results obtained by fresh semen. This 

experience has allowed field personnel to obtain 

confidence in the extended semen product. Extended 

semen is a reliable source of milt as long as the appropriate steps are taken during its preparation, storage, 

and use. 

References 

Moore, A. 1987. Short-term storage and cryopreservation of walleye semen. Progressive FishCulturist 49: 40-45. 

Moore, A. 1996. Use of semen extenders for walleye. 

Pages 51-53 in R.C. Summerfelt, editor. Walleye 

culture manual. NCRAC Culture Series 101. North 

Central Regional Aquaculture Center Publications 

Office, Iowa State University, Ames, Iowa. 

58 NCRAC Culture Series 101- Walleye Culture Manual 
Heidinger, R. C., B. L. Tetzlaff, and R. C. Brooks. 1996. Hormone induced spawning of walleye. 

Pages 59-62 in R. C. Summerfelt, editor. Walleye culture manual. NCRAC Culture Series 101 

North Central Regional Aquaculture Center Publications Office, Iowa State University, Ames. 

Hormone Induced Spawning of Walleye 

Roy C. Heidinger, Bruce L. Tetzlaff and Ronald C. Brooks, Fisheries Research Laboratorx Southern Illinois 

Universitx Carbondale, IL 6290 1-65 7 1 

Introduction 

It is often dfficult to capture walleye in “running-ripe” 

condition because that stage of spawning is of short 

duration. Walleye culturists in Illinois often collect hard 

(green) broodfish and attempt to bring them into 

spawning condition in the hatchery. This case study 

describes how hormone injections can be used to 

accelerate the ripening process. Several hfferent types 

of hormone preparations have been used to successfully 

advance oocyte maturation and/or induce ovulation in 

walleye. The hormones include human chorionic 

gonadotropin (HCG), carp pituitary extract, luteinizing 

hormone (LH), luteinizing hormone-releasing hormone 

(LH-RH), and pimozide. The use of such hormones had 

variable success, with low hatchability of eggs often 

occurring. Use of hormones in fisheries is not approved 

by the U.S. Food and Drug Administration (FDA). The 

work reported here was conducted under an investigational new animal drug (INAD) permit issued by FDA. 

FDA is currently monitoring a pharmaceutical company 

funded study on the safety of HCG and a study on the 

efficacy of HCG. 

During the maturation phase of the ova, a germinal 

vesicle migrates from the center to the periphery of the 

eggs and breaks down prior to ovulation (GVBD). For 

walleye, this process may begin during February and 

require up to 2.5 months to complete. When females are 

injected with appropriate maturating hormones, such as 

HCG, the germinal vesicle migration and subsequent 

GVBD may take 96 h or less. 

Objectives 

The objective of this study was to determine the dose 

of HCG that needs to be interperitoneally injected into 

female walleye that have eggs at various stages of 

maturation to maximize both the number of females 

that ovulate and the hatching success. We also wanted 

to minimize the length of time wild, walleye broodfish 

were held in the hatchery. Hatch rates of the HCG 

injected females were compared with females that were 

“running ripe” at the capture site and those that ovulated in the hauling tank on their way to the hatchery. In 

addition, time to ovulation and percentage of injected 

females that ovulated were compared with non-injected, 

hard females that were allowed to ripen in hatchery 

raceways. 

Materials and methods 

The study was conducted at Jake Wolf Memorial Fish 

Hatchery, Manito, Illinois. Walleye broodfish were 

collected from Heideke Lake, Lake Shelbyville, and 

Kaskaslua River above Lake Shelbyville during April of 

1987, 1988, and 1989. Fish were collected when water 

temperatures ranged from 42-52°F (5.6-1 1.1OC). 

Female walleye were initially classified into three 

groups: 

Group 1. Fish that were “running-ripe” at the capture 

Group 2. Fish that ovulated in route to the hatchery 

site; 

and were stripped immedately upon reaching the 

hatchery; 

Group 3. Fish that had firm ovaries (hard condition). 

Hard females were catheterized with a 0.12-in (3-mm) 

O.D. glass tube to collect a sample of ova for determination of developmental stage. Subsequently, the 

females were injected with various doses of HCG. The 

egg samples were placed in a modification of 

Stockard’s preservation solution until they could be 

examined for stages of development. Egg stages were 

classified by degree of oil globule coalescence, egg 

membrane opacity, and position of the germinal vesicle 

(GV). Eggs from injected females (group 3) were thus 

classified into seven stages of maturity: 

Stage 3.0. Many small oil droplets; membrane 

Stage 3.5. Some oil droplet coalescence; membrane 

opaque and granular; GV centered. 

opaque and granular; GV off center. 

NCRAC Culture Series 101 - Walleye Culture Manual 59 
~ 

Chapter 2 - Reproductive Biology and Spawning 

Stage 3.75. Oil droplets fewer and larger; some 

membrane clearing; GV migrating and approaching periphery. 

Stage 4.0. Single, large oil globule with few, smaller 

droplets; oocyte membrane translucent; most eggs 

with GV peripheral. 

Stage 4.5. Single, large oil globule; few smaller 

droplets on periphery of large globule; oocyte 

membrane slightly granular; GV break down 

(GVBD) beginning. 

Stage 4.75. Oil globule similar to stage 4.5; egg 

membrane not completely transparent; GVBD 

complete. 

Stage 5.0. Oil globule usually completely coalesced; 

oocyte membrane transparent; GVBD and 

ovulation complete. 

Walleye eggs staged at 3.0-3.75 were immature, and 

eggs staged at 4.0-5.0 were nearly mature. 

Group-3 (hard) females were intra-peritoneally injected 

with HCG at doses of either 0, 50, 150, 250, or 500 IU/ 

lb (0, 110, 330, 550, or 1100 IUkg) of female weight 

during 1987-89 (Table 1). At the time of injection, each 

fish was marked with an individually numbered tag for 

later identification. After hormone injections, the fish 

were placed in concrete raceways with water temperature of about 54°F (12.2”C) and checked twice daily for 

ovulation. In 1987, walleye received only one injection 

of HCG of 150 IU/lb (330 IUkg). In 1988 and 1989, 

fish which had not ovulated 48 h after the first injection 

were injected a second time. In 1988, initial injections 

were at either 50 or 500 IU/lb (1 10 or 1100 IUkg) and 

second injections, if required, were at 500 IU/lb (1 100 

IUkg). In 1989, females were given initial doses of 

HCG at either 50,250 or 500 IU/lb (1 10,550, or 1100 

IUkg), and second doses were at the level of the first. 

Some group-3 (hard) females were allowed to ripen 

without injection in the raceways. 

Ovulation was determined to have occurred when eggs 

would freely flow from the vents with gentle, posterior 

stroking of the fish’s abdomen. Standard “dry method” 

was used to fertilize the eggs. Refrigerated sperm, 

which had been mixed with extender solution at a ratio 

of 1 ml sperm to 2 ml extender (Moore 1987), was used 

during the experiments. Each batch of extended semen 

was examined for motility prior to its use. In a previous 

experiment, we ascertained that using 0-9 d old 

refrigerated, extended semen solution resulted in mean 

hatch rates that were similar to rates when fresh sperm 

was used. We also compared the amount of semen 

solution used per 100 ml of eggs with the hatch rate. 

There were no differences in mean hatch rates between 

0.25, 0.5, 1 .O, 1.5 or 2.0 ml of solutiodl00 ml of eggs. 

For this experiment, we used 0.5 ml of 1:2 sperm 

solution/100 ml of eggs. 

Following fertilization, eggs were mixed in a bentonite 

solution to remove adhesiveness, water hardened for 

about 1 h, and incubated in 16.8-oz (0.5 L) glass 

cylinders that simulated the McDonald hatching jars 

that are commonly used by hatchery personnel. In a 

previous study, we determined that egg viabilities could 

be microscopically quantified accurately at either 6 h, 3 

d, or at 8 d after fertilization. In this study, egg viability 

was quantified 3-4 d after fertilization. 

Results 

During the three-year study, HCG dose, egg maturation stage at time of initial injection, and handling of 

broodfish were monitored to examine their effects on 

egg maturation and viability. There were no significant 

differences among mean egg viabilities, time-toovulation, or percent ovulation among injection dose 

treatment groups, whether the walleye required only the 

initial injection or an additional injection after 48 h 

(Table 1). Therefore, data from various injection dose 

trials were combined in the following analysis. 

Walleye “running ripe” (group l), and spawned at 

collection sites or immehately after arriving at the 

hatchery (group 2), had higher mean egg viabilities than 

hard females (group 3) injected with HCG or held in the 

hatchery raceways without injection (Table 1). Egg 

viability of “running-ripe” females that were stripped at 

walleye collection sites was 76%, and 66% for the 

females that were transported to the hatchery prior to 

stripping. Mean egg viabilities of injected and noninjected females (group 3) were lower than “running 

ripe” fish, but egg viability of females injected with 

HCG was 37% compared with 45% of hard fish held 

without injection at the hatchery. 

Although egg viability was not affected by the HCG 

dose levels of 50-500 IU/lb (1 10-1 100 IUkg), degree 

of egg ripeness at the time of injection did have an 

effect on egg viability. Eggs of non-ripe walleye given 

HCG injections to promote ovulation were staged as 

immature and mature. When the walleye were initially 

60 NCRAC Culture Series 101- Walleye Culture Manual 
Reproductive Biology and Spawning - Chapter 2 

Table 1. Comparison of ovulation percentage, days to ovulation, and egg viabilities of 

walleye females injected with various doses of human chorionic gonadotropin or 

uninjected. Numbers in parentheses represent number of females in treatment. 

Initial Second 

HCG HCG Ovulation Days to ovulation 

(I U/I b) (I U/I b) Ima Nmb Ima Nmb 

injection i n ject io n (% 

Injected 

50 50 

(35) (22) 

150 

(23) 

500 500 

(55) (35) 

Mean 

Non-l njected 

Running ripe 

at site 

Running ripe 

at hatchery 

Hard 

93 96 4.0 

(1 04) (53) (97) 

NA 

NA 

23 

(44) 

Mean hatch 

("/I 

Ima Nmb 

27.0 

(18) 

(7) 

(1 7) 

(1 5) 

(34) 

34.4 

29.6 

31.6 

35.8 

31.4 

(91) 

a Walleye with eggs at immature stages (3.0-3.75) at the time of initial HCG injections. 

b Walleye with eggs at nearly mature stages (4.0-5.0) at the time of initial HCG injections. 

injected, eggs staged as nearly mature had a mean hatch 

rate of 48.0%, while eggs staged as immature had a 

mean hatch rate of 3 1.4%. 

Injections of HCG (irrespective of dosage) significantly 

increased the percentage of fish which ovulated and, 

depending on egg stages, reduced the period of time 

until ovulation. During the three year period, a total of 

157 female walleye were given HCG injections of 50, 

150,250, or 500 IU/lb (110,330,550, or 1100 IUkg). 

Including fish which were given a second injection, 

94% of the females injected with HCG ovulated within 

5 d (Table 1). In contrast, only 23% of 44 females taken 

to the hatchery and held without injections ovulated 

within 7 d. Walleye with immature eggs required a 

mean of 4.0 d to ovulate after injection, and fish with 

nearly mature eggs at initial injection required only a 

mean of 2.4 d to ovulate. 

NCRAC Culture Series 101 - Walleye Culture Manual 61 
Chapter 2 - Reproductive Biology and Spawning 

Conclusion 

Use of HCG is effective for inducing ovulation of 

walleye captured with unripe eggs. HCG injections effectively shortened the time to ovulation 

for walleye that were not “running-ripe’’ upon 

arrival at the hatchery and induced a higher 

percentage of walleye to egg ovulation than fish 

held without injections. 

Egg hatch percentages were similar for injected 

and non-injected walleye that required a holding 

period prior to egg ovulation, however holding 

stress on female fish may adversely affect 

viability of their eggs. 

Acknowledgments 

Funding for this project was provided from the Federal 

Aid in Sport Fish Restoration Act (F-60-R) and 

sponsored by the Fisheries Division of the Illinois 

Department of Conservation. The authors, not the 

granting agency, are responsible for the content of this 

paper. 

References 

Moore, A. A. 1987. Short-term storage and cryopreservation of walleye semen. Progressive FishCultu rist 49:40-43. 

62 NCRAC Culture Series 101- Walleye Culture Manual