www.elsevier.nlrlocateraqua-online
Single pair mating indicates maternal effects on
embryo survival in rainbow trout, Oncorhynchus
mykiss
James J. Nagler
a,), James E. Parsons
b,1, J.G. Cloud
a aCenter for ReproductiÕe Biology, Department of Biological Sciences, UniÕersity of Idaho, Moscow, ID
83844-3051, USA
b
Pisces InÕestments, Hagerman, ID 83332, USA
Accepted 13 September 1999
Abstract
The maternal and paternal influences on early embryo survival in rainbow trout are not established. The purpose of this study was to determine whether variability in the survival of rainbow trout embryos could be attributed to either the female or male parent. Gametes from individual female and male rainbow trout were used in single pair matings to produce families
Ž
whose survival was followed from fertilization to the time of swim-up i.e., ;7 weeks .
post-fertilization . Survival was assessed at 0.5, 9, 19, 33, and 48 days post-fertilization, corresponding to second cleavage, embryonic keel formation, retinal pigmentation, hatch, and Ž . swim-up, respectively. The variability of survival at all times was significantly P-0.01 influenced by the female parent, whereas the influence of the male parent was negligible ŽP)0.05 . Therefore, in rainbow trout embryo survival can be equated with the quality of the.
Ž .
egg. To predict survival at swim-up i.e., after 48 days it was found that embryonic keel formation, measured 9 days after fertilization, was the earliest time at which a highly significant
Ž .
positive correlation rs0.889, P-0.001 was demonstrated.q2000 Elsevier Science B.V. All
rights reserved.
Keywords: Gametes; Salmonids; Embryo; Development; Maternal effect
)Corresponding author. Tel.:q1-208-885-4382; fax:q1-208-885-7905; e-mail: [email protected]
1
Present address: Troutlodge, PO Box 1290, Sumner, WA 98390.
0044-8486r00r$ - see front matterq2000 Elsevier Science B.V. All rights reserved. Ž .
1. Introduction
The viability of fish embryos and larvae is important for both aquaculturists and fisheries biologists, as early embryo survival of freshwater and marine fishes can vary
Ž . Ž .
dramatically Bromage et al., 1992; Nagler et al., 1999 . Numerous biotic e.g., genetic
Ž .
andror abiotic factors e.g., temperature, oxygen concentration may contribute to this
variability, one important factor undoubtedly being the gametes that combine to produce the zygote.
Ž
Embryo viability is often considered as being synonymous with egg quality Blaxter,
.
1988; Kjørsvik et al., 1990; Bromage et al., 1994 . This implies that the maternal contribution is significantly more important than the paternal, but until the paternal contribution is adequately assessed the idea that fish embryo viability is equivalent to
Ž .
egg quality is not tenable Brooks et al., 1997 . In salmonids, studies have shown that
Ž . Ž
both the female parent Springate et al., 1984 or the male parent Aas et al., 1991; Gile
.
and Ferguson, 1995; Babiak et al., 1998 can independently affect embryo development
Ž .
or survival e.g., fertilization, retinal pigmentation, or the number of embryos hatching . However, these studies were not designed to compare the contribution of each sex equally on an individual basis at numerous early developmental timepoints. A clear appreciation of which sex is contributing significantly to embryo survival in previous studies has always been obscured by pooling of the gametes from several individuals of one sex. It is known that the numbers of progeny resulting from fertilization of eggs by
Ž
sperm of given males contributing to a sperm pool can differ considerably Gharrett and
.
Shirley, 1985; Danzmann and Ferguson, 1988; Withler, 1988; Gile and Ferguson, 1990 . This is presumably due to qualitative and quantitative sperm differences amongst males and the resulting competition for fertilizing eggs. An experimental design that separated female effects from male effects would permit an assessment of whether one sex had a greater impact than the other on embryo viability. Such information would be important for focusing research on the parent that has the greatest impact on the embryo.
The question posed in the present study was whether variability in the survival of rainbow trout embryos was under stronger maternal or paternal influence. Gametes from individual female and male rainbow trout were used to produce families whose survival was followed to the time of swim-up. The variability in embryo survival was strongly
Ž .
associated with the female parent i.e., the egg , while the influence of the male parent was negligible.
2. Methods and materials
2.1. Gametes
Ž .
Rainbow trout Oncorhynchus mykiss gametes were obtained from three-year-old
Ž .
broodstock first-time spawners maintained at Pisces Investments, Magic Springs Farm,
Ž . Ž
Hagerman, ID Experiment 1 or Mt. Lassen Trout Farms, Red Bluff, CA Experiment
.
2 . Female and male broodstock were checked twicerweek for ovulated eggs or running
48C, and used within 24 h of collection. The motility of the sperm in each semen sample was assessed before it was used to fertilize eggs. This was done by diluting a semen
Ž . Ž . Ž
aliquot )1:1000 with 0.5% saline buffered to pH 8.9 with Tris–glycine Benau and
.
Terner, 1980; Christen et al., 1987 on a glass microscope slide and determining the proportion of motile sperm in the field of a light microscope at 100=magnification. All
Ž .
males used had at least 50% of the sperm motile ranges50–80% .
2.2. Experimental protocol
Ž
A standard procedure for in vitro fertilization of trout gametes was followed Nilsson
.
and Cloud, 1992 : groups of about 300 eggs from individual females were combined
Ž .
with 200ml aliquots of semen i.e., an excess from individual males in plastic beakers.
The eggs from each pair combination were placed separately in 6=6=6 cm plastic
boxes with Nitex screen bottoms, that were installed in a vertical fish egg incubator
ŽHeath, Tacoma, WA . Normal husbandry practices were followed, with eggs being.
checked daily, dead eggs removed, and numbers recorded.
2.2.1. Experiment 1
Ž .
In this experiment performed at Pisces Investments; water temperatures14"0.58C
a 4=4 factorial design was used in which gametes from four females and four males
were crossed to generate 16 families. At 18 days post-fertilization the appearance of retinal pigmentation in all embryos remaining in each family was determined. Survival
was calculated as the number of embryos with obvious retinal pigmentationrtotal
number of eggs incubated.
2.2.2. Experiment 2
Ž .
In this experiment performed at University of Idaho; water temperatures10"0.58C
a 3=3 factorial design was used in which gametes from three females and three males
were used to produce nine families. This design was repeated with six additional parent fish to produce two separate trials yielding a total of 18 different families. Survival was measured at five times post-fertilization: 0.5, 9, 19, 33, and 48 days. At 0.5 day evidence of the second cleavage division was used to assess survival. Second cleavage was
Ž .
determined by removing 10 eggs, fixing them in methanol:acetic acid:water 1:1:1
ŽSpringate et al., 1984 , and examining them under a stereoscopic microscope for the.
Ž . Ž
presence of a cleavage embryo i.e., four-cell embryo . Embryonic keel formation i.e.,
.
embryonic shield; Ballard, 1973 was assessed in 10 eggs removed and fixed as above, after 9 days of incubation. After 19 days of incubation, the appearance of retinal pigmentation was used to assess embryo survival as described above in Experiment 1. Hatching success was calculated after 33 days of incubation as the number of embryos
that hatchedrtotal number of eggs incubated. Swim-up success was evaluated after 48
days of incubation by placing the yolk-sac embryos in a small aquarium and observing the number that entered the water column versus those that remained on the bottom.
Swim-up success was assessed as the total number swimming uprtotal number of eggs
Table 1
Statistical summary of 4=4 factorial cross between gametes from individual female and male rainbow trout
Ž .
Timepoint survival measure Mean"SEM Range Source df F Pr)F
Ž .
18 days retinal pigment 70"3% 55–87% Female 3 13.19 0.0012
Male 3 0.49 0.06964
2.3. Statistical analysis
Data were calculated as percentages and are reported as a mean"the standard error
Ž .
of the mean SEM . An arc-sine transformation was used to normalize the data for
Ž
statistical analysis. ANOVA using a General Linear Model was used on SAS SAS
.
Institute, Carey, NC to analyze each endpoint independently. Correlation coefficients for the relationship between survival at second cleavage, embryonic keel formation, retinal pigmentation, and hatch versus swim-up, were calculated as Pearson Product Moment correlations. The level of statistical significance was set at P-0.05.
3. Results
In Experiment 1, there was a statistically significant maternal effect on the proportion of embryos with retinal pigmentation, while the effect of the male parent was
non-sig-Ž .
nificant Table 1 . In Experiment 2, there were statistically significant maternal effects
Ž .
on embryo survival at all five times measured Table 2 , but there were no significant
Ž .
paternal effects. A significant difference between the two trials Table 2 was noted at
Ž .
four of the five times 9, 19, 33 and 48 days survival was assessed.
Table 2
Statistical summary of replicated 3=3 factorial cross between gametes from individual female and male rainbow trout
Ž .
Timepoint survival measure Mean"SEM Range Source df F Pr)F
Ž .
0.5 day second cleavage 78"4% 40–100% Trial 1 1.59 0.2431
Female 4 7.06 0.0098
Male 4 1.94 0.1975
Ž .
9 days embryonic keel formation 61"8% 0–100% Trial 1 20.20 0.0020
Female 4 12.10 0.0018
Male 4 1.46 0.2996
Ž .
19 days retinal pigment 56"8% 0–87% Trial 1 771.88 0.0001
Female 4 225.81 0.0001
Male 4 0.29 0.8740
Ž .
33 days hatch 51"8% 0–83% Trial 1 392.36 0.0001
Female 4 88.76 0.0001
Male 4 0.38 0.8188
Ž .
48 days swim-up 49"8% 0–81% Trial 1 481.08 0.0001
Female 4 98.99 0.0001
To provide an indication of the predictive ability of survival measurements taken
Ž .
early in embryonic development, relative to later i.e., swim-up after 48 days the data
Ž .
collected in Experiment 2 ns18 families were further analyzed. Correlation
coeffi-Ž .
A single pair mating scheme was used to compare maternal and paternal influences on the survival of rainbow trout embryos. Variability in embryo viability up to swim-up
Ži.e., ;7 weeks post-fertilization was due to the female gamete, there being a highly.
significant effect of the egg on development and survival, in both experiments, at all five
Ž
timepoints studied. Therefore, once a rainbow trout egg is fertilized i.e., penetrated by a
.
sperm , embryo survival and development can be equated with egg quality.
The contribution of the male to the developing embryo, in the form of paternal DNA, had minimal impact on embryo survival up to the time of swim up. This might imply
Ž .
that the genetic component of the fertilized egg i.e., maternal and paternal DNA was not as important as other constituents, such as maternal mRNA and yolk content for early development and survival. Maternal mRNAs are synthesized and stored within the
Ž
cytoplasm of the developing oocyte in oviparous animals for review, see Davidson,
.
1986 and are known to be important during early embryonic development before the embryonic genome is utilized. The period over which maternal mRNA is functional in
Ž .
fishes is not known, but based on information drawn from amphibians Davidson, 1986 it could be expected to extend to the blastula stage. In the current study, most mortality occurred between 0.5 and 9 days, so early embryogenic events such as the translation of embryonic proteins from maternal mRNAs may be crucial to survival.
Additionally, yolk proteins, lipids, vitamins, and minerals incorporated into the
Ž
oocyte during ovarian development could influence embryo viability Brooks et al.,
.
1997 . These organic and inorganic constituents are the sole nutrient supply for the embryo until it begins to feed exogenously. Oocyte composition has been extensively
Ž .
investigated for reviews see, Mommsen and Walsh, 1988; Weigand, 1996 , but the
Ž .
relationship to embryo survival has been little studied. Olin and von der Decken 1990 reported that the rate of the disappearance of two yolk proteins during early
develop-Ž .
ment was indicative of embryo viability in Atlantic salmon Salmo salar . However, these proteins have not been identified andror definitively linked to embryo survival. Until a better understanding of the utilization of the nutrient stores in fish eggs during embryogenesis is attained no conclusions relative to embryo viability can be made.
Ž .
The earliest time at which swim-up success i.e., after 48 days could be confidently predicted was after 9 days of incubation. At this time a highly significant correlation
Žrs0.889 between the proportion of embryos with embryonic keel formation and.
Ž .
Ž .
correlate strongly with swim-up rs0.389 . The conflicting results may be a
conse-Ž
quence of either differences in the sample sizes used for correlation analyses ns10
.
families versus 18 families in this study or differences in numbers of eggs sampled for
Ž .
cleavage assessment ns20–30 versus 10 in this study .
To conclude, survival from fertilization to swim up in the rainbow trout is directly related to the female gamete, the egg, as the maternal influence on early development and survival appears much more significant than the paternal contribution.
Acknowledgements
The authors would like to acknowledge the technical assistance of Mr. Devin Bolz, Ms. Kara Kral, and Ms. Ann Pool for the fish husbandry, and thank Dr. Kirk Steinhorst for the statistical analysis.
References
Aas, G.H., Refstie, T., Gjerde, B., 1991. Evaluation of milt quality of Atlantic salmon. Aquaculture 95, 125–132.
Babiak, I., Glogowski, J., Luczynski, M., Goryczko, K., Dobosz, S., Kuzminski, H., 1998. The effect of individual male potency on fertilization ability of fresh and cryopreserved milt of rainbow trout,
Ž .
Oncorhynchus mykiss Walbaum . Aquat. Res. 29, 337–340.
Ballard, W.W., 1973. Normal embryonic stages for salmonid fishes, based on Salmo gairdneri Richardson and
Ž .
SalÕelinus fontinalis Mitchell . J. Exp. Zool. 184, 7–26.
Benau, D., Terner, C., 1980. Initiation, prolongation, and reactivation of the motility of salmonid spermatozoa. Gamete Res. 3, 247–257.
Ž .
Blaxter, J.H.S., 1988. Pattern and variety in development. In: Hoar, W.S., Randall, D.J. Eds. , Fish Physiology, Vol. XI, The Physiology of Developing Fish: Part A. Eggs and Larvae. Academic Press, San Diego, pp. 1–58.
Bromage, N., Jones, J., Randall, C., Thrush, M., Davies, B., Springate, J., Duston, J., Barker, G., 1992. Broodstock management, fecundity, egg quality and timing of egg production in the rainbow trout
ŽOncorhynchus mykiss . Aquaculture 100, 141–166..
Bromage, N., Bruce, M., Basavaraja, N., Rana, K., Shields, R., Young, C., Dye, J., Smith, P., Gillespie, M., Gamble, J., 1994. Egg quality determinants in finfish: the role of overripening with special reference to the timing of stripping in the Atlantic halibut Hippoglossus hippoglossus. J. World Aquacult. Soc. 25, 13–21. Brooks, S., Tyler, C.R., Sumpter, J.P., 1997. Egg quality in fish: what makes a good egg?. Rev. Fish Biol.
Fish. 7, 387–416.
Christen, R., Gatti, J.L., Billard, R., 1987. Trout sperm motility, the transient movement of trout sperm is related to changes in the content of ATP following the activation of flagellar movement. Eur. J. Biochem. 166, 667–671.
Danzmann, R.G., Ferguson, M.M., 1988. Temperature-dependent genotypic selection and embryonic survival of rainbow trout. Biochem. Genet. 26, 69–81.
Davidson, E.H., 1986. Gene Activity in Early Development. Academic Press, Orlando, FL.
Gharrett, A.J., Shirley, S.M., 1985. A genetic examination of spawning methodology in a salmon hatchery. Aquaculture 47, 245–256.
Gile, S.R., Ferguson, M.M., 1990. Crossing methodology and genotypic diversity in a hatchery strain of
Ž .
rainbow trout Oncorhynchus mykiss . Can. J. Fish. Aquat. Sci. 47, 719–724.
Gile, S.R., Ferguson, M.M., 1995. Factors affecting male potency in pooled gamete crosses of rainbow trout,
Kjørsvik, E., Mangor-Jensen, A., Holmefjord, I., 1990. Egg quality in fishes. Adv. Mar. Biol. 26, 71–113.
Ž .
Mommsen, T.P., Walsh, P.J., 1988. Vitellogenesis and oocyte assembly. In: Hoar, W.S., Randall, D.J. Eds. , Fish Physiology, Vol. XI, The Physiology of Developing Fish: Part A. Eggs and Larvae. Academic Press, San Diego, pp. 348–395.
Nagler, J.J., Adams, B.A., Cyr, D.G., 1999. Egg production, fertility and hatch success of American plaice
Ž .
held in captivity. Trans. Am. Fish. Soc. in press .
Nilsson, E.E., Cloud, J.G., 1992. Rainbow trout chimeras produced by injection of blastomeres into recipient blastula. Proc. Natl. Acad. Sci. U.S.A. 89, 9425–9428.
Ž .
Olin, T., von der Decken, A., 1990. Yolk proteins in salmon Salmo salar oocytes, eyed eggs, and alevins differing in viability. Can. J. Zool. 68, 895–900.
Springate, J.R.C., Bromage, N.R., Elliott, J.A.K., Hudson, D.L., 1984. The timing of ovulation and stripping and their effects on the rates of fertilization and survival to eying, hatch and swim-up in the rainbow trout
ŽSalmo gairdneri R. . Aquaculture 43, 313–322..
Weigand, M.D., 1996. Composition, accumulation and utilization of yolk lipids in teleost fish. Rev. Fish Biol. Fish. 6, 259–286.
Ž .
