Research database

Project information
Project title
Influence of ocean acidification and temperature on sperm motility, fertilization and embryonic development in Atlantic cod
Year
2011/2012
Project leader
Helge Tveiten, Nofima
Participants
  • Helge Tveiten (project leader), Nofima, P.B. 6122, N-9291 Tromsø
  • Marianne Frantzen, Akvaplan-niva, Fram Centre, N-9296 Tromsø
  • Geir Rudolfsen, Norwegian Radiation Protection Authority, Fram Centre, N-9296 Tromsø
Flagship

Ocean acidification, Theme: Effect of ocean acidification on species and ecosystems

Summary of Results
In order to investigate the effect of temperature and pH on Atlantic cod sperm characteristics
and fertilization ability, two experiments (exp. I and exp. II) were conducted during the natural
spawning period in April 2011. In both experiments sperm from five males and eggs from one
female were utilized. All gametes were stripped from broodfish of the National Cod Breeding
Programme at the same day as the experiments were conducted.
 
Sperm characteristics:
Sperm characteristics were investigated using Computer Assisted Sperm Analysis
(CASA) according to the established cod protocol (Rudolfsen et al., 2005). Sperm was activated in seawater at different temperatures (4 °C and 9 °C) and pH (8.1 [control],
7.7 and 7.4) and recorded at different time intervals (0, 10 and 20 minutes post activation).
The relationships between sperm quality over time (0, 10 and 20 min post activation) and
stressors (temp and pH) were analyzed in mixed effects models with sperm velocity and
motility as the dependent variable, individual as within subject and random factors, and pH,
temperature and time since activation as the fixed factor. We ran three different models
where in the full model all factors could interact, and compared this with two reduced
models, one with all two way interaction or additive effects only. We Followed Baayen
et al. 2008 and used the 'anova' function in the lme4 package to compare the quality
of the fit between models. Model fit and the significance of including the interactions
were tested using AIC (Akaike's Information Criterion) and log-likelihood ratio statistics
(LLR λ2) (Bates 2005). Model fitting and estimates were obtained with linear mixed-effects (lmer) package lme4 in R (version 2.11.0, R Development Core Team, 2007) using
maximum likelihood estimates (ML). Significant of coefficients are gauged by checking
the summary for whether the absolute value of the t-statistic exceeds 2.
Finally, the model fit was verified using visual examination of normal probability plots
and residual plots.
 
Fertilization ability:
 
Eggs (1000 eggs per replicates, three replicates per treatment) were fertilized in seawater at different temperatures (4°C and c. 9°C) and pH (8.1 [control], 7.7 and 7.4) using a protocol under conditions where fertilization is sperm-density dependent (sperm to egg ratio = 5 x 105: 1, gamete contact time < 30 min.; own unpublished results; Butts et al., 2009). In exp. I fertilization was undertaken immediately after sperm activation and fertilized eggs where incubated under pH and temp conditions identical to the fertilization conditions. In exp. II the number of incubators was doubled and eggs were fertilized with sperm either activated at fertilization or 10 minutes pre-fertilization. In this experiment fertilized eggs were incubated at temp conditions identical to fertilization conditions but at ambient pH (pH 8.1).
Fertilization rate was monitored, and fertilized eggs were investigated with respect to early cell
cleavage according to Tveiten et al. (2001).
 
Results
Sperm characteristics:
Parameters included in this report are mean curvilinear velocity (VCL, μm s–) and percentage of motile cells (MOT) of exp. I and II combined (n=10 males per treatment) (see table I and fig.1 in attachment).
The results reviled no effect of pH on sperm VCL or MOT. However, increased temp (9°C)
induces a significantly stronger temporal decrease in both VCL and MOT compared to lower
temp (4°C).
Fertilization ability:
Unexpected and surprising results were obtained regarding the fertilization ability in both
exp. I and exp. II where the eggs in general, independent of pH, fail to fertilize at 4 ºC (ambient temperature; control). Image analyses of the eggs fertilized at 9 ºC
did not revealed any effect of pH on percentage fertilized eggs in any of the experiment.
However, in exp. I there was a tendency of higher prevalence of activated eggs that had failed
to initiate any cell cleavage, and eggs with abnormal cell cleavage at pH 7.7 and 7.4 compared
to pH 8.1 (see fig. 2 and 3 in the attachment). The fertilization success were in general lower in exp. I than exp. II indicating a higher egg quality in the latter exp. The tendency of increased abnormal egg development at reduced pH in exp I may indicate that future ocean acidification scenario can influence the recruitment potential of populations suffering a suboptimal physiological condition caused by unidentified stressors. This hypothesis needs further investigation. Also, the failure of eggs to fertilize at 4 ºC needs to be further investigated.
 
In order to investigate the effect of temperature and pH on Atlantic cod sperm characteristics
and fertilization ability, two experiments (exp. I and exp. II) were conducted during the natural
spawning period in April 2011. In both experiments sperm from five males and eggs from one
female were utilized. All gametes were stripped from broodfish of the National Cod Breeding
Programme at the same day as the experiments were conducted.
 
Sperm characteristics:
Sperm characteristics were investigated using Computer Assisted Sperm Analysis
(CASA) according to the established cod protocol (Rudolfsen et al., 2005). Sperm was activated in seawater at different temperatures (4 °C and 9 °C) and pH (8.1 [control],
7.7 and 7.4) and recorded at different time intervals (0, 10 and 20 minutes post activation).
The relationships between sperm quality over time (0, 10 and 20 min post activation) and
stressors (temp and pH) were analyzed in mixed effects models with sperm velocity and
motility as the dependent variable, individual as within subject and random factors, and pH,
temperature and time since activation as the fixed factor. We ran three different models
where in the full model all factors could interact, and compared this with two reduced
models, one with all two way interaction or additive effects only. We Followed Baayen
et al. 2008 and used the 'anova' function in the lme4 package to compare the quality
of the fit between models. Model fit and the significance of including the interactions
were tested using AIC (Akaike's Information Criterion) and log-likelihood ratio statistics
(LLR λ2) (Bates 2005). Model fitting and estimates were obtained with linear mixed-effects (lmer) package lme4 in R (version 2.11.0, R Development Core Team, 2007) using
maximum likelihood estimates (ML). Significant of coefficients are gauged by checking
the summary for whether the absolute value of the t-statistic exceeds 2.
Finally, the model fit was verified using visual examination of normal probability plots
and residual plots.
 
Fertilization ability:
 
Eggs (1000 eggs per replicates, three replicates per treatment) were fertilized in seawater at different temperatures (4°C and c. 9°C) and pH (8.1 [control], 7.7 and 7.4) using a protocol under conditions where fertilization is sperm-density dependent (sperm to egg ratio = 5 x 105: 1, gamete contact time < 30 min.; own unpublished results; Butts et al., 2009). In exp. I fertilization was undertaken immediately after sperm activation and fertilized eggs where incubated under pH and temp conditions identical to the fertilization conditions. In exp. II the number of incubators was doubled and eggs were fertilized with sperm either activated at fertilization or 10 minutes pre-fertilization. In this experiment fertilized eggs were incubated at temp conditions identical to fertilization conditions but at ambient pH (pH 8.1).
Fertilization rate was monitored, and fertilized eggs were investigated with respect to early cell
cleavage according to Tveiten et al. (2001).
 
Results
Sperm characteristics:
Parameters included in this report are mean curvilinear velocity (VCL, μm s–) and percentage of motile cells (MOT) of exp. I and II combined (n=10 males per treatment) (see table I and fig.1 in attachment).
The results reviled no effect of pH on sperm VCL or MOT. However, increased temp (9°C)
induces a significantly stronger temporal decrease in both VCL and MOT compared to lower
temp (4°C).
Fertilization ability:
Unexpected and surprising results were obtained regarding the fertilization ability in both
exp. I and exp. II where the eggs in general, independent of pH, fail to fertilize at 4 ºC (ambient temperature; control). Image analyses of the eggs fertilized at 9 ºC
did not revealed any effect of pH on percentage fertilized eggs in any of the experiment.
However, in exp. I there was a tendency of higher prevalence of activated eggs that had failed
to initiate any cell cleavage, and eggs with abnormal cell cleavage at pH 7.7 and 7.4 compared
to pH 8.1 (see fig. 2 and 3 in the attachment). The fertilization success were in general lower in exp. I than exp. II indicating a higher egg quality in the latter exp. The tendency of increased abnormal egg development at reduced pH in exp I may indicate that future ocean acidification scenario can influence the recruitment potential of populations suffering a suboptimal physiological condition caused by unidentified stressors. This hypothesis needs further investigation. Also, the failure of eggs to fertilize at 4 ºC needs to be further investigated.
 
Published Results/Planned Publications

The results from the project will be submitted as a "short communication" to a relevant peer review journal (e.g. Marine Biology)

Communicated Results

-

Interdisciplinary Cooperation

-

Budget in accordance to results
The funding from the Fram Centre have made it possible to start up some activities
which are part of a bigger research project that has not yet succeeded to receive the
necessary funding. The results from the Fram Centre project are important for
emphasizing the objectives and strengthen the feasibility for getting the big project
funded. Further, throughout the Fram Centre project we have been able to identify
needs for optimization of the experimental exposure systems and contacts have been
established to ensure appropriate water chemistry analysis in future experiments.
The funding from the Fram Centre have made it possible to start up some activities
which are part of a bigger research project that has not yet succeeded to receive the
necessary funding. The results from the Fram Centre project are important for
emphasizing the objectives and strengthen the feasibility for getting the big project
funded. Further, throughout the Fram Centre project we have been able to identify
needs for optimization of the experimental exposure systems and contacts have been
established to ensure appropriate water chemistry analysis in future experiments.
If Yes

Yes, the results from the project are highly relevant for optimization of fertilization protocols utilized by the cod aquaculture industry (juvenile producers).

Conclusions
The result of no fertilization at 4 °C is highly interesting and need further
investigation. The experiments conducted in this project are only snapshots of the
parameters that may be affected regarding reproduction by temp and pH and further
experiments need to also consider effect on the reproductive development prior to
spawning (effects on the broodfish) as well as effects on egg quality, larvae and
juveniles. Also, effects of temp and pH in combination with other stressors (i.e.
environmental pollutants) are of highly relevance.
The result of no fertilization at 4 °C is highly interesting and need further
investigation. The experiments conducted in this project are only snapshots of the
parameters that may be affected regarding reproduction by temp and pH and further
experiments need to also consider effect on the reproductive development prior to
spawning (effects on the broodfish) as well as effects on egg quality, larvae and
juveniles. Also, effects of temp and pH in combination with other stressors (i.e.
environmental pollutants) are of highly relevance.