Prepared in cooperation with the Kootenai Tribe of Idaho

Effects of Incubation Substrates on Hatch Timing and Success 

of White Sturgeon (Acipenser transmontanus) Embryos

U.S. Department of the Interior

U.S. Geological Survey

Scientific Investigations Report 2013–5180
Cover: Photographs showing white sturgeon embryos attached to incubation 

substrates prior to placement into water-filled incubators. Photographs show three 

substrates from left to right—clean river rocks, riparian vegetation, and waterlogged 

wood. Photographs taken by Eric Kofoot, U.S. Geological Survey, 2007.
Effects of Incubation Substrates on Hatch 

Timing and Success of White Sturgeon 

(Acipenser transmontanus) Embryos 

By Michael J. Parsley and Eric Kofoot

Prepared in cooperation with the Kootenai Tribe of Idaho

Scientific Investigations Report 2013–5180

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

SALLY JEWELL, Secretary

U.S. Geological Survey

Suzette M. Kimball, Acting Director 

U.S. Geological Survey, Reston, Virginia: 2013

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Suggested citation:

Parsley, M.J., and Kofoot, Eric, 2013, Effects of incubation substrates on hatch timing and success of White Sturgeon 

(Acipenser transmontanus) embryos: U.S. Geological Survey Scientific Investigations Report 2013–5180, 16 p., 

http://dx.doi.org/10.3133/sir20135180.

ISSN 2328-0328 (online)
iii

Contents

Abstract ..........................................................................................................................................................1

Introduction.....................................................................................................................................................1

Methods...........................................................................................................................................................2

Incubation Substrates..........................................................................................................................3

Seeding the Incubation Substrates ...................................................................................................6

Incubation, Hatch, and Analysis of Data ..........................................................................................7

Effect of Substrate on Hatch Timing and Success...................................................................................8

Effect of Substrate on Free-Embryo Length at Hatch............................................................................12

Summary........................................................................................................................................................14

Acknowledgments ......................................................................................................................................14

References Cited..........................................................................................................................................14

Glossary.........................................................................................................................................................16

Figures

1. Photograph showing arrangement of incubators in the 6×3 block design........................2

2. Photograph showing an incubator with developing embryos attached to a rock

typical of a substrate covered with periphyton and algae....................................................3

3. Photograph showing developing embryos attached to clean river rocks typical of

the substrate in many white sturgeon spawning areas.........................................................4

4. Photograph showing examples of the riparian vegetation used as substrates.................5

5. Photograph showing an incubator with white sturgeon embryos on a sand substrate ..6

6. Graph showing water temperatures recorded hourly in the three headboxes that

supplied water to the incubation tanks.....................................................................................7

7. Photograph showing an incubator with sturgeon embryos that had floated off the

sand substrate and become lodged in the notch of the water-level control weir ............8

8. Boxplot showing total lengths of free-swimming hatched embryos from different

incubation substrates ................................................................................................................12
iv

Conversion Factors

SI to Inch/Pound

Multiply By To obtain

Length

millimeter (mm) 0.03937 inch (in.)

millimeter (mm) 1,000 micrometer (mm)

centimeter (cm) 0.3937 inch (in.)

kilometer (km) 0.6214 mile (mi)

Volume

liter (L) 0.2642 gallon (gal)

milliliter (mL) 0.0002642 gallon (gal) 

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows:

°F=(1.8×°C)+32.

Tables

1. Incubation and hatch data associated with each incubator. ...............................................9

2. Two-way analysis of variance results for incubation substrate effects on

incubation time to first hatch. ...................................................................................................9

3. Tukey multiple comparisons of means of time to first hatch for each substrate...............9

4. Two-way analysis of variance results for incubation substrate effects on

incubation time to achieve 50-percent hatch. .....................................................................10

5. Tukey multiple comparisons of means of time to achieve 50-percent hatch from

each substrate ............................................................................................................................10

6. Two-way analysis of variance results for incubation substrate effects on

proportion of hatched embryos. .............................................................................................11

7. Tukey multiple comparisons of means of the proportion of hatched embryos from

each substrate ............................................................................................................................11

8. Number of free-swimming embryos measured for total length from each incubation

substrate. ...................................................................................................................................13

9. Two-way analysis of variance results for incubation substrate effects on total

length of free-swimming hatched embryos. ........................................................................13

10. Tukey multiple comparisons of means of total lengths of newly hatched embryos

from each substrate ...................................................................................................................13
Effects of Incubation Substrates on Hatch 

Timing and Success of White Sturgeon 

(Acipenser transmontanus) Embryos 

By Michael J. Parsley and Eric Kofoot

Abstract 

The Kootenai River white sturgeon (Acipenser 

transmontanus) was listed as endangered under the 

Endangered Species Act in 1994 because several decades of 

failed spawning had put the population at risk of extinction. 

Natural spawning is known to occur at several locations in the 

Kootenai River, Idaho, but there is little natural recruitment. 

Microhabitat where embryo incubation occurs is known to be 

an important factor in white sturgeon reproductive success. 

This study was conducted to address questions regarding 

the suitability of different substrates as egg attachment and 

incubation sites for these fish. A comparative laboratory study 

using six types of incubation substrates—clean river rocks, 

periphyton- and algae-covered rocks, waterlogged wood, 

sand, riparian vegetation, and clean glass plates—tested the 

hypothesis that survival to hatch of white sturgeon eggs differs 

among incubation substrates. The results showed that sand 

was unsuitable as an incubation substrate, as the adhesive 

embryos were easily dislodged. Periphyton- and algae-covered 

rocks had the lowest hatch success, and all other substrates 

had similar hatch success. 

Introduction

The Kootenai River white sturgeon (Acipenser 

transmontanus) was listed as endangered under the 

Endangered Species Act in 1994 (U.S. Fish and Wildlife 

Service, 1994) because of the gradual decline in adult fish that 

began around 1960 (Duke and others, 1999; Beamesderfer 

and others, 2009). The decline was caused by the failure of 

spawning to produce enough young sturgeon to sustain the 

population. Spawning by white sturgeon is known to occur at 

several locations over a 20-km reach in the Kootenai River, 

Idaho (Paragamian and others, 2001); however, production of 

free-swimming embryos and feeding larvae from the spawning 

areas is negligible. The failed spawning has been attributed to 

many factors including the overall loss of spawning habitat, 

altered river flows owing to hydropower and flood‐risk 

mitigation (Anders and others, 2002; Paragamian and 

Wakkinen, 2011), and egg suffocation from accretion of fine 

sediments (Kock and others, 2006).

Microhabitat where egg incubation and embryonic 

development occur may be an important factor in white 

sturgeon reproductive success. White sturgeon are broadcast 

spawners and eggs are dispersed over the riverbed as they 

sink. The fertilized eggs are about 3.5 mm in diameter (Deng 

and others, 2002) and are somewhat heavier than water. The 

eggs have a jelly coat that becomes adhesive about 5 minutes 

(range 2−13 minutes; Markov, 1978) after contact with 

water. This jelly coat anchors the eggs to the substrate during 

natural spawning (Conte and others, 1988). Coutant (2004) 

describes what is known about the adhesion of sturgeon eggs 

and the role of river hydraulics on egg dispersal and quality 

of substrates for incubation sites. Clean, smooth surfaces 

generally are expected to provide better incubation sites 

than overly rough, “oily,” and “fluffy” surfaces, which can 

result from periphyton or algae growth on the substrates. 

River hydraulics and hydrological cycles can clean substrates 

through scour and repeated cycles of exposure and inundation, 

but anthropogenic changes have moderated flows and 

water-surface elevation changes in many areas, which may 

result in “dirtier” substrates in sturgeon-egg incubation areas.

Successful sturgeon spawning and egg incubation 

habitat generally is composed of boulders, cobbles, gravels, 

or combinations of these materials. Sand currently (2013) 

dominates the riverbed at most known spawning areas 

for white sturgeon in the Kootenai River as a result of the 

decrease in peak flows (Barton, 2003). Sand is not considered 

a good substrate for spawning and subsequent incubation of 

sturgeon eggs (Parsley and Beckman, 1994; Kock and others, 

2006), although spawning by white sturgeon does occur in 

areas where sand is present in the Kootenai River (Paragamian 

and others, 2001) and in the Fraser River (Perrin and others, 

2003). Coutant (2004) suggested that low-relief overflow 

zones adjacent to river channels that become inundated 
2 Effects of Incubation Substrates on Hatch Timing and Success of White Sturgeon (Acipenser transmontanus) Embryos

with high flows during the sturgeon spawning season offer 

various clean substrates that sturgeon eggs can adhere. These 

substrates would include stones of all sizes and vegetation that 

would provide a meshwork of clean surfaces for attachment of 

eggs in clean, well-aerated water (Coutant, 2004).

The recovery plan for white sturgeon in the Kootenai 

River outlined a three-part strategy including implementing 

measures to restore natural recruitment, the use of 

conservation aquaculture to prevent extinction, and continued 

monitoring and evaluation of population trends (U.S. Fish and 

Wildlife Service, 1999). Efforts to increase success of natural 

spawning include habitat restoration projects to improve 

physical habitat for spawning and egg incubation. Planning for 

the restoration of habitat naturally led to questions regarding 

the suitability of different substrates as egg-attachment and 

incubation sites for these fish. The goal of this study was to 

evaluate different materials as egg-incubation media in white 

sturgeon spawning areas. Several hypotheses were tested to 

meet this goal:

• Survival to hatch of white sturgeon embryos did not 

differ among incubation substrates. 

• Incubation period did not differ among incubation 

substrates.

• Incubation substrate had no effect on total lengths of 

newly hatched white sturgeon. 

Methods

In this comparative study, six incubation substrates were 

tested—clean river rocks, periphyton- and algae-covered 

rocks, waterlogged wood, sand, riparian vegetation, and clean 

glass plate. All test substrates, with the exception of the clean 

glass plates, are present at some level in known spawning 

areas in the Kootenai River, Idaho. The study was conducted 

at the Kootenai Tribal Sturgeon Hatchery near Bonners Ferry, 

Idaho, and all routine broodstock capture and spawning 

operations were handled by hatchery personnel. Eggs and 

sperm were obtained from wild Kootenai River white 

sturgeon captured by angling. Females were held in captivity 

and injected with luteinizing hormone (LHRH-A) to induce 

ovulation. Sperm was immediately expressed from ripe males 

and held in refrigerated containers for later use. All fish were 

released back into the river after gametes were obtained.

The experiment followed a randomized block design and 

each substrate type (n = 6) constituted a treatment. Incubators 

were arranged in three blocks of six incubators. Each of the 

six incubators within a block was randomly assigned to hold a 

specific substrate type (fig. 1). 

Incubators were constructed from glass aquariums 

(40.6 cm long×20.3 cm wide×25.4 cm deep) filled with 

water to a minimum depth of 18.5 cm. A constant flow of 

Figure 1. Arrangement of incubators in the 6 ×3 block design. The three headboxes supplied water to the 

incubators. Headboxes received water that contained a mix of unheated and heated Kootenai River water. 

Photograph taken by Eric Kofoot, U.S. Geological Survey, 2007.

headbox 1

headbox 2

headbox 3

Incubators
Methods 3

water to the incubators was provided using three 113.4-L 

headboxes. The headboxes received water from a common 

line containing a mix of filtered heated and unheated Kootenai 

River water. Each headbox supplied water to a block of six 

incubators. Water temperatures in each of the three headboxes 

were recorded by autonomous temperature loggers (HOBO®

Water Pro, Onset Computer Corporation) each hour for the 

duration of the experiment. Water flow from the headbox 

to the individual incubators was regulated by gate valves. 

Water entered at the head of the incubator through 12.7-mm, 

polyvinyl chloride tubing with a 13-mm diameter diffuser 

placed near the bottom of the incubator. The distal end of the 

incubator was ported with a 1.9-cm-diameter, screen-covered 

bulkhead fitting for water outflow. Incubators were fitted with 

an 18.5-cm-high Cipoletti style overflow weir positioned 

30 cm from the head of the incubator dividing the incubator 

into two chambers, the larger chamber held the substrates. The 

smaller chamber was designed to retain the free-swimming 

embryos when they passed over the weir. Equal flow of water 

through the incubators was achieved by monitoring water 

elevation through the notches in the overflow weirs.

Incubation Substrates

The periphyton- and algae-covered rocks used in the 

experiments were collected from near the mouth of Myrtle 

Creek (river kilometer [rkm] 234.0), a tributary to the 

Kootenai River. Rocks selected were relatively uniform in size 

and covered with moderate amounts of periphyton and algae 

growth (fig. 2). A single rock was used in an incubator. 

Figure 2. An incubator with developing embryos attached to a rock typical of a substrate covered with 

periphyton and algae. Photograph taken by Eric Kofoot, U.S. Geological Survey, 2007.
4 Effects of Incubation Substrates on Hatch Timing and Success of White Sturgeon (Acipenser transmontanus) Embryos

The clean river rocks used were collected from the Moyie 

River (rkm 258.1), another tributary to the Kootenai River. 

These rocks were brushed and rinsed to remove any surface 

growth, and then allowed to dry in the sun prior to the start 

of the experiment. The clean river rocks were selected for 

uniform size, smoothness, and flatness to provide adequate 

surface area for egg attachment (fig. 3). Two clean rocks were 

used in an incubator. 

Stems from four species of riparian vegetation were 

collected from along the banks of the Kootenai River—reed 

canarygrass (Phalaris arundinacea), Pacific willow (Salix 

lucida), black cottonwood (Populus trichocarpa), and redosier 

dogwood (Cornus sericea) (fig. 4). Three bunches of stems, 

containing equal numbers of stems from each of the four 

species, were created; one for each block. 

Figure 3. Developing embryos attached to clean river rocks typical of the substrate in many white 

sturgeon spawning areas. This photograph was taken shortly after the embryos were fertilized and 

before the clean river rocks were placed in the incubators. At that time, the substrates and embryos 

were under a misting system to keep them wet. Labels were used to ensure the proper placement of 

these substrates into a randomly selected incubator within a block (block 1 in this case). Photograph 

taken by Eric Kofoot, U.S. Geological Survey, 2007.
Methods 5

Figure 4. Examples of the riparian vegetation used as substrates. A. Reed canarygrass (Phalaris 

arundinacea), B. Pacific willow (Salix lucida), C. Redosier dogwood (Cornus sericea), and D. Black 

cottonwood (Populus trichocarpa). Photograph taken by Eric Kofoot, U.S. Geological Survey, 2007.

A. B.

D.

C.

A 30.6-centimeter ruler provides a scale for reference.

The sand substrate was composed of a 2-cm layer of 

sifted general-purpose construction sand (600 μm–1 mm 

particle size) that covered the bottom of the incubator in 

each block.

Non-floating, waterlogged limbs from trees were 

collected from along the Kootenai River to serve as the 

waterlogged wood substrate. These limbs were about 

10.5 cm in diameter, and were cut into 25-cm lengths and 

split lengthwise to fit into the incubators. A single piece of 

waterlogged wood was used in an incubator. 

Clean glass plates were cut from window-pane glass 

(17.5×26.5 cm). A single plate was placed in horizontal 

orientation and supported 3 cm off the bottom of the incubator 

in each block. 
6 Effects of Incubation Substrates on Hatch Timing and Success of White Sturgeon (Acipenser transmontanus) Embryos

Seeding the Incubation Substrates

About 10,000 eggs were obtained from a single Kootenai 

River white sturgeon female on June 1, 2007, at 2000 hours. 

The eggs were placed in a 3.8-L stainless steel mixing bowl. 

The milt from two Kootenai River white sturgeon males was 

pooled and then mixed into 1,500 mL of water to activate the 

sperm. The activated sperm was then added to the eggs and 

gently stirred with a primary wing feather from a large bird 

for about 1 minute to allow fertilization to occur. Because 

of the rapidness of the onset of adhesion and the need to 

distribute the fertilized eggs (now embryos; Balon, 1975a), 

among 18 incubators, the embryos were then divided into 

three separate bowls and mixed for another 1–2 minutes 

until the jelly coat started to form and the eggs began to 

become adhesive. A subsample of embryos was then quickly 

collected with a common kitchen baster that then was used to 

gently distribute the embryos onto the individual substrates. 

Because the embryos were quickly developing their adhesive 

envelopes, there was no time for a precise method to equalize 

the number of embryos placed onto substrates that were 

distributed among the 18 incubators. This resulted in variation 

in the number of embryos among incubators.

Substrates generally were seeded with embryos outside 

of the incubator and moved to the incubators after a 20-minute 

waiting period for adhesion to fully form. All substrates except 

for the riparian vegetation and sand were seeded with embryos 

under a commercial off-the-shelf patio misting system. 

Rock, wood, and glass substrates were wetted and placed in 

plastic trays (80 cm long×60 cm wide×7 cm deep) that were 

beneath the misting system. The misters kept the substrates 

and embryos moist during the initial stages of adhesion to the 

substrates. After 20 minutes had passed to allow embryos to 

fully adhere to the substrates, the individual substrates then 

were placed in the assigned incubator and water flow was 

initiated. 

Each of the three riparian vegetation bunches was 

submersed in a separate 18.9-L bucket filled with water. The 

embryos then were distributed across the water surface and 

allowed to settle into the stems and leaves. After 30 minutes, 

each bunch with attached embryos was removed from the 

bucket and placed into the appropriate incubator. 

The incubators containing sand had been previously filled 

with water to a depth of 7 cm. The fertilized embryos were 

seeded over the water surface to settle onto the sand (fig. 5). 

Water flow was initiated after 30 minutes. 

water inlet pipe

Figure 5. An incubator with white sturgeon embryos on a sand substrate. Embryos had just been placed 

in the incubator and the water was not flowing. Photograph taken by Eric Kofoot, U.S. Geological Survey, 

2007.
Methods 7

Incubation, Hatch, and Analysis of Data

A visual inspection of the incubators was made each 

morning and evening and periodically throughout the day. 

Free-swimming embryos were removed from the incubators 

immediately after being observed. The time (in minutes) 

was noted and total lengths were measured (to the nearest 

0.01 mm) under an illuminated magnifying lamp with a digital 

caliper (Mitutoyo model CD-6-CS). Damaged or deformed 

embryos were enumerated but not measured to provide a total 

number of hatched embryos from each incubator. 

The experiment was terminated between 1900 and 

2100 hours on June 12, 2007. At that time, all unhatched 

embryos and the substrates that they were attached to were 

removed from the incubators and preserved in 10-percent 

buffered formalin. In the laboratory, the preserved embryos 

were rinsed, separated from the substrate materials, and 

transferred to 70-percent ethanol. The unhatched embryos 

then were counted under a dissecting microscope. Clusters 

of embryos that were bound together by fungus were gently 

separated to determine how many embryos were in the 

cluster. The total number of unhatched embryos remaining 

May 31 June 2 June 4 June 6 June 8 June 10 June 12 June 14

12.0

12.5

13.0

13.5

14.0

14.5

15.0

15.5

2007

Water temperature, in degrees Celsius

EXPLANATION

1

2

3

Headbox

Figure 6. Water temperatures recorded hourly in the three headboxes 

that supplied water to the incubation tanks. The graph shows that water 

temperature varied over time but there was little difference among headboxes 

that supplied the incubation chambers. The decrease in temperature on June 5 

was caused by a decrease in hot water delivered to this experiment because of 

the need for additional hot water in the Kootenai Tribal Sturgeon Hatchery.

in each incubator was determined from counts made by two 

individuals. When counts differed, additional counts were 

made until a consensus was reached. 

The water temperature data loggers were downloaded and 

review of the hourly temperature s revealed some variation 

in water temperature during the experiment but no difference 

in water temperature among the headboxes (fig. 6). A drop 

in water temperature of approximately 2.5°C within the 

experiment occurred on June 5 when the hatchery facility 

demands for heated water reduced heated water supply to 

our experiment. 

Several metrics were used to evaluate the effects of 

substrate type on incubation of white sturgeon embryos—

the proportion of hatched embryos, incubation time to first 

hatch, incubation time to 50-percent hatch, and total length of 

free-swimming embryos. The initial number of embryos that 

were placed into each incubator was determined by adding the 

number of hatched free-swimming embryos to the number of 

unhatched embryos counted in the laboratory. The effect of 

substrate type on these metrics was assessed using a two-way 

analysis of variance. 
8 Effects of Incubation Substrates on Hatch Timing and Success of White Sturgeon (Acipenser transmontanus) Embryos

Effect of Substrate on Hatch 

Timing and Success

All substrates with embryos attached had 

been placed into the incubators by 2200 hours 

on June 1, 2007. Approximately 9 hours 

later, many bubbles had formed on surfaces 

throughout the incubators and on many of 

the embryos. Bubbles that had formed on the 

embryos on the sand substrates had increased 

the buoyancy of the embryos enough to lift 

them from the substrate. Most of the embryos 

had floated to the surface and were aggregated 

at the notch in the water-level control weir 

(fig. 7). Given that the embryos on the sand 

substrate in all three incubators were no longer 

attached to the sand, the decision was made 

to exclude this substrate type from further 

analysis. All incubators then were inspected 

and, despite the presence of bubbles in all 

incubators, no embryos had detached from the 

other substrates.

Hatch timing and success was first 

observed in an incubator with a clean river 

rock substrate at 0905 hours on June 9, 2007, 

approximately 7.5 days (179.1 hours) after 

the eggs were fertilized (hours to first hatch; 

table 1). Hatch began in six other incubators 

within hours. Hatch was first observed in six 

more incubators the following day, and hatch 

was observed in the last two incubators on 

June 11. Substrate had an effect on incubation 

time to first hatch at α=0.1 but not at α=0.05 

(table 2). A multiple comparison test (Tukey 

HSD) showed that incubation time to first hatch 

was shorter on clean river rock substrate than 

on periphyton- and algae-covered rock substrate 

and waterlogged wood substrate (table 3). 

Substrate also influenced incubation time to 

50-percent hatch at α=0.001 (table 4) and the 

multiple comparison test revealed significant 

differences between most substrates (table 5).

Hatch success varied among incubation substrates 

and between blocks for each substrate type (table 1). Hatch 

success ranged from a low of 2.7 percent in an incubator 

with a periphyton- and algae-covered rock substrate, to a 

high of 60.7 percent in an incubator with a clean glass plate 

substrate. Substrate had a significant effect on incubation 

success (table 6), and the multiple comparison test showed 

that differences in mean proportion of embryos that hatched 

were lowest for embryos attached to the periphyton- and 

algae-covered rock substrate (table 7).

water-level control 

weir with notch

Figure 7. An incubator with sturgeon embryos that had floated off the sand 

substrate and become lodged in the notch of the water-level control weir. Other 

clusters of embryos can be seen floating at the water surface. The water inlet 

pipe with diffuser is in the back of the chamber. Photograph by Eric Kofoot, U.S. 

Geological Survey, 2007.
Effect of Substrate on Hatch Timing and Success 9

Table 1. Incubation and hatch data associated with each incubator. 

[Eggs were fertilized at 2000 hours on June 1, 2007. Abbreviation: mm, millimeter]

Substrate type

Incubator 

No.

(block)

Number of 

embryos in 

incubator

Date and time 

hatch was 

first noted

Hours to

first hatch

Hours to 

50 percent 

hatch

Total 

length 

(mm)

Number of 

embryos 

hatched

Percentage

of hatch

Clean river rocks 1 (1) 167 June 9 17:00 184.5 228.2 11.66 62 37.1

7 (2) 177 June 9 13:54 185.9 213.2 11.64 45 25.4

13 (3) 176 June 9 12:30 181.1 208.0 11.37 39 22.2

Periphyton- and algae-covered rocks 3 (1) 134 June 11 07:50 227.8 228.8 11.64 11 8.2

9 (2) 89 June 10 08:35 204.6 213.0 11.09 15 16.9

15 (3) 150 June 10 11:40 207.7 214.8 11.81 4 2.7

Waterlogged wood 6 (1) 104 June 10 08:05 204.1 253.0 12.04 20 19.2

12 (2) 141 June 10 10:10 206.2 236.8 11.76 80 56.7

18 (3) 197 June 11 11:37 231.6 259.4 11.61 38 19.3

Sand 4 (1) 304 – – – – – –

10 (2) 363 – – – – – –

16 (3) 349 – – – – – –

Riparian vegetation 5 (1) 417 June 9 17:00 189.0 253.1 11.97 77 18.5

11 (2) 198 June 9 17:40 189.7 237.3 11.90 84 42.4

17 (3) 198 June 10 11:00 207.0 237.8 11.89 49 24.8

Clean glass plates 2 (1) 120 June 9 16:45 188.8 254.0 12.33 57 47.5

8 (2) 272 June 10 09:45 205.8 235.9 12.13 165 60.7

14 (3) 108 June 9 16:55 188.9 231.8 11.72 34 31.5

Table 2. Two-way analysis of variance results for incubation 

substrate effects on incubation time to first hatch. 

[Response: Incubation time to incubation time to first hatch]

Df Sum sq Mean sq F value Pr (>F)

Substrate 4 2,063.72 515.93 3.6656 0.05572

Group 2 71.55 35.77 0.2542 0.78159

Residuals 8 1,126.00 140.75

Table 3. Tukey multiple comparisons of means of time to first hatch for each substrate.

[Difference is the difference between the means of time for each substrate pair comparison. Lower and Upper are the confidence intervals. 

p adjusted is the test statistic]

Substrate

Tukey multiple comparison test results

Difference Lower Upper p adjusted

Clean glass plates – Clean river rock 10.6667 17.8860 39.2194 0.8018

Periphyton- and algae-covered rock – Clean river rock 29.5333 0.9806 58.0860 0.0871

Riparian vegetation – Clean river rock 11.4000 17.1527 39.9527 0.7640

Waterlogged wood – Clean river rock 30.1333 1.5806 58.6860 0.0800

Periphyton- and algae-covered rock – Clean glass plates 18.8667 -9.6860 47.4194 0.3670

Riparian vegetation – Clean glass plates 0.7333 27.8194 29.2860 1.0000

Waterlogged wood – Clean glass plates 19.4667 -9.0860 48.0194 0.3410

Riparian vegetation – Periphyton- and algae-covered rock -18.1333 46.6860 10.4194 0.4005

Waterlogged wood – Periphyton- and algae-covered rock 0.6000 27.9527 29.1527 1.0000

Waterlogged wood – Riparian vegetation 18.7333 -9.8194 47.2860 0.3729
10 Effects of Incubation Substrates on Hatch Timing and Success of White Sturgeon (Acipenser transmontanus) Embryos

Table 4. Two-way analysis of variance results for incubation 

substrate effects on incubation time to achieve 50-percent hatch. 

[Response: Incubation time to 50-percent hatch]

Df Sum sq Mean sq F value Pr(>F)

Substrate 4 2,710.51 677.63 15.7512 0.0007

Group 2 735.63 367.81 8.5497 0.0103

Residuals 8 344.17 43.02

Table 5. Tukey multiple comparisons of means of time to achieve 50-percent hatch from each substrate.

[Difference is the difference between the means of time for each substrate pair comparison. Lower and Upper are the confidence intervals. 

p adjusted is the test statistic]

Substrate

Tukey multiple comparison test results

Difference Lower Upper p adjusted

Clean glass plates–Clean river rock 24.1000 8.2175 39.9825 0.0131

Periphyton- and algae-covered rock – Clean river rock 3.0000 -12.8825 18.8825 0.9779

Riparian vegetation–Clean river rock 26.2667 10.3841 42.1492 0.0079

Waterlogged wood–Clean river rock 33.2667 17.3841 49.1492 0.0018

Periphyton- and algae-covered rock –Clean glass plates -21.1000 -36.9825 -5.2175 0.0269

Riparian vegetation–Clean glass plates 2.1667 13.7159 18.0492 0.9934

Waterlogged wood–Clean glass plates 9.1667 -6.7159 25.0492 0.4829

Riparian vegetation–Periphyton- and algae-covered rock 23.2667 7.3841 39.1492 0.0159

Waterlogged wood–Periphyton- and algae-covered rock 30.2667 14.3841 46.1492 0.0033

Waterlogged wood–Riparian vegetation 7.0000 -8.8825 22.8825 0.6993
Effect of Substrate on Hatch Timing and Success 11

Table 6. Two-way analysis of variance results for incubation 

substrate effects on proportion of hatched embryos. 

[Response: Proportion of hatched embryos]

Df Sum sq Mean sq F value Pr(>F)

Substrate 4 2,118.54 529.63 5.2690 0.02235

Group 2 1,091.70 545.85 5.4303 0.03237

Residuals 8 804.16 100.53

Table 7. Tukey multiple comparisons of means of the proportion of hatched embryos from each substrate.

[Difference is the difference between the means of time for each substrate pair comparison. Lower and Upper are the confidence intervals. 

p adjusted is the test statistic]

Substrate

Tukey multiple comparison test results

Difference Lower Upper p adjusted

Clean glass plates – Clean river rock 18.3333 -5.7961 42.4628 0.2567

Periphyton- and algae-covered rock – Clean river rock -18.9667 -43.0962 5.1628 0.2325

Riparian vegetation – Clean river rock 0.3000 -23.8295 24.4295 1.0000

Waterlogged wood – Clean river rock 3.5000 -20.6295 27.6295 0.9917

Periphyton- and algae-covered rock – Clean glass plates -37.3000 -61.4295 -13.1705 0.0117

Riparian vegetation – Clean glass plates -18.0333 -42.1628 6.0962 0.2689

Waterlogged wood – Clean glass plates -14.8333 -38.9628 9.2962 0.4284

Riparian vegetation – Periphyton- and algae-covered rock 19.2667 -4.8628 43.3962 0.2218

Waterlogged wood – Periphyton- and algae-covered rock 22.4667 -1.6628 46.5962 0.1319

Waterlogged wood – Riparian vegetation 3.2000 -20.9295 27.3295 0.9940
12 Effects of Incubation Substrates on Hatch Timing and Success of White Sturgeon (Acipenser transmontanus) Embryos

Effect of Substrate on Free-Embryo 

Length at Hatch

Total lengths were obtained from 612 newly hatched 

embryos (table 8). Total lengths ranged from 8.81 to 13.44 mm 

(fig. 8) and incubation substrate had an effect on total length 

at hatch at α=0.1 but not at α=0.05 (table 9). The multiple 

comparison test failed to identify a significant difference 

(α=0.1) in total length at hatch between any two incubation 

substrates (table 10) perhaps because of the small number 

of free-swimming embryos available to measure from the 

incubators containing the periphyton- and algae-covered 

rocksubstrate.

Clean river 

rock

Clean glass

plate

Periphyton- and 

algae-covered

rocks

Riparian 

vegetation

Waterlogged

wood

9

8

10

11

12

13

14

Substrate

Total length, in millimeters

n=129 n=226 n=20 n=127 n=110

n=110

EXPLANATION

Number of values

Outlier

Outlier

75

90th percentile

10th percentile

th percentile

50th percentile

(median)

25th percentile

Interquartile

range 

Figure 8. Total lengths of free-swimming hatched embryos from different incubation substrates.
Effect of Substrate on Free-Embryo Length at Hatch 13

Table 8. Number of free-swimming embryos measured for total length from each incubation 

substrate. 

Block

Number of free-swimming embyros measured for total length

Clean river

rock

Periphyton- and 

algae-covered 

rock

Waterlogged 

wood

Riparian 

vegetation

Clean glass 

plates

1 53 4 19 40 45

2 39 13 76 60 156

3 37 3 15 27 25

Totals 129 20 110 127 226

Table 9. Two-way analysis of variance results for incubation 

substrate effects on total length of free-swimming hatched embryos. 

[Response: Total length of free-swimming embryos]

Df Sum sq Mean sq F value Pr(>F)

Substrate 4 0.66233 0.165582 3.0002 0.08703

Group 2 0.18751 0.093756 1.6988 0.24273

Residuals 8 0.44152 0.055190

Table 10. Tukey multiple comparisons of means of total lengths of newly hatched embryos from each substrate.

[Difference is the difference between the means of time for each substrate pair comparison. Lower and Upper are the confidence intervals. 

p adjusted is the test statistic]

Substrate Tukey multiple comparison test results

Difference Lower Upper p adjusted

Clean glass plates – Clean river rock 0.5039 -0.1588 1.1665 0.1545

Periphyton- and algae-covered rock – Clean river rock -0.0459 -0.7086 0.6168 0.9991

Riparian vegetation – Clean river rock 0.3635 -0.2992 1.0262 0.3901

Waterlogged wood – Clean river rock 0.2467 -0.4160 0.9094 0.7063

Periphyton- and algae-covered rock – Clean glass plates -0.5497 -1.2124 0.1129 0.1118

Riparian vegetation – Clean glass plates -0.1404 -0.8030 0.5223 0.9429

Waterlogged wood – Clean glass plates -0.2572 -0.9198 0.4055 0.6768

Riparian vegetation – Periphyton- and algae-covered rock 0.4094 -0.2533 1.0721 0.2930

Waterlogged wood – Periphyton- and algae-covered rock 0.2926 -0.3701 0.9553 0.5756

Waterlogged wood – Riparian vegetation -0.1168 -0.7795 0.5459 0.9696
14 Effects of Incubation Substrates on Hatch Timing and Success of White Sturgeon (Acipenser transmontanus) Embryos

Summary

Benthic fish embryos are exposed to numerous 

environmental hazards (Peterka and Kent, 1976). These 

hazards are a major ecological factor determining the 

reproductive success in fishes (Balon, 1975b, 1984), including 

sturgeons. Incubation substrates that result in the earliest hatch 

and greatest overall hatch success have the greatest potential 

to sustain populations. This study showed that clean river 

rocks generally were more suitable than other tested substrates 

expected to be found in the Kootenai River. However, other 

substrates, such as waterlogged wood and riparian vegetation 

also performed well. Sand, which currently (2013) dominates 

the riverbed in some known spawning areas in the highly 

altered Kootenai River, was unsuitable as a substrate for 

adhesive eggs because the developing embryos were easily 

mobilized from the substrate. In these experiments, the 

formation of small air bubbles showed the ease with which 

embryos deposited on sands could be dislodged. Water 

currents in rivers, even at the low velocities typical of some 

Kootenai River spawning areas, would likely mobilize eggs 

that were deposited on sands. It was beyond the scope of this 

study to determine the fate of dislodged embryos.

Incubating white sturgeon embryos are susceptible 

to death from suffocation by sediment cover (Kock and 

others, 2006), predation (Miller and Beckman, 1996), and 

parasitism by spreading aquatic fungi (Scott and O’Bier, 

1962; Conte and others, 1988). Fungus growth on developing 

embryos can quickly spread to adjacent embryos and cause 

high mortality. Although fungal infection was not quantified 

in these experiments, fungus growth was more prevalent 

on the periphyton- and algae-covered rocks than on other 

substrates, and was believed to be the primary cause of the 

reduced hatch success of embryos seeded on this substrate. 

Operation of Kootenai River dams for flood risk management 

and hydropower benefits during the white sturgeon spawning 

season have largely eliminated spring scouring flows that 

typically clean rocks of overlying growth.

This laboratory study suggests that several substrate types 

may be suitable for spawning habitat restoration—clean river 

rock, waterlogged wood, and riparian vegetation. Waterlogged 

wood is present in the Kootenai River and may currently 

(2013) be serving as an embryo incubation substrate in some 

known spawning sites of white sturgeon. The inclusion of 

waterlogged wood in Kootenai River sturgeon spawning 

habitat restoration efforts in areas dominated by sand or 

periphyton- and algae-covered rocks could improve incubation 

success. Coutant (2004) suggested that submerged aquatic 

and riparian vegetation may serve as important incubation 

sites for white sturgeon embryos; however, white sturgeon 

embryos have not yet been observed incubating on stems and 

leaves of vegetation, although few field studies have looked 

specifically for white sturgeon embryos in shallow riparian 

habitats (vander Leeuw and others, 2006). Field studies that 

have collected incubating white sturgeon embryos in other 

river systems reported that catches were greatest over rocks 

(Parsley and others, 1993; McCabe and Tracy, 1994; Schaffter, 

1997; Perrin and others, 2003) and gravels (Schaffter, 1997). 

However, it is apparent from this study that substrates for 

adhesion and incubation of white sturgeon embryos must 

remain relatively clean to reduce the spread of embryo-killing 

fungus. Achieving clean substrates may require restoration 

strategies that maximize scouring flows prior to or during the 

spawning period for white sturgeon.

Acknowledgments 

The Kootenai Tribe of Idaho graciously allowed us to use 

their sturgeon hatchery facility to conduct the experiments. 

Kootenai Tribal Sturgeon Hatchery staff–including John 

(Jack) Siple, Eric Wagner, and Chris Lewandowski–captured 

the brood stock and spawned the eggs that were used in this 

experiment. Bjorn van der Leeuw and Tim Blubaugh of the 

U.S. Geological Survey assisted with the experiment and 

with data entry. Funding for this project was provided by the 

Bonneville Power Administration through the Kootenai Tribe 

of Idaho.

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er.usgs.gov/publication/sir20065225.)
16 Effects of Incubation Substrates on Hatch Timing and Success of White Sturgeon (Acipenser transmontanus) Embryos

Egg An unfertilized gamete from a female white sturgeon.

Embryo A fertilized gamete.

Free-swimming embryo An embryo that has hatched and is feeding endogenously off the yolk sac.

Incubator A modified aquarium in which embryos on various substrates were placed.

Glossary
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For more information concerning the research in this report, contact the 

Director, Western Fisheries Research Center

U.S. Geological Survey

6505 NE 65th Street

Seattle, Washington 98115

http://wfrc.usgs.gov/
Parsley and Kofoot—Effects of Incubation Substrates on Hatch Timing and Success of White Sturgeon (Acipenser transmontanus) Embryos—Scientific Investigations Report 2013–5180

ISSN 2328-0328 (online)

http://dx.doi.org/10.3133/sir20135180