Experiment on hatching the eggs of the Simp magnificus

[timebomb]

I'll make you a bet that increased CO2 in hatch water will force hatch quicker and more surely than oxygen tablets (who knows what those really add). I have never used the latter, so could be surprised.

I was intrigued by what Wright said in this thread and particularly, the bet he wanted Tyrone to take up (quoted above).

So I conducted a little experiment. I got a bag of Simpsonichthys magnificus "Itacarambi B7" eggs from Au S L the other day. The eggs were collected on the 5th of December last year and Au said they should be ready for wetting in 2 months. Yesterday was about 10 weeks so I decided it was time to hatch the eggs. Here's a pic of the container Au uses to incubate the eggs:


I checked and most of the eggs were already well eyed-up. There's a difference between "eyed-up" and "well eyed-up". In the case of the latter, besides the eyes, there's also body . I can't see that clearly though but that's what my maid said. Something about the Filipinas, they sure have sharp eyes. "Sir, got body", that's what she said.


Anyway, I asked my maid to pick out those eggs which were well eyed-up and she found plenty. Au didn't know how many eggs were there in the container but he was sure there were at least 50. Well, if you are reading this, Au, there were something like more than 200 eggs. Thanks, Buddy.


To conduct the experiment, I used 3 different containers to hatch the eggs. For the first one which I'm going to call the "Oxygen tray", I used a low plastic container and filled it up with water taken from my main tank to about 1 cm of water.


I also chopped up an oxygen stone and sprinkled it all over the container. I then put in 40 eggs


After that, I attached an air-stone to the tray and connected it to an air pump.


For the second hatching tray which I'm going to call my CO2 tray, I use a plastic tub and filled it with water taken from the same tank to a height of about 8 cm. 40 well eyed-up eggs went into this container.


I then detached one of the tubing from my CO2 splitter and and place one end of it into the container. The idea is to feed CO2 into the water.


I didn't use a diffuser or a reactor as I didn't think it was necessary. As long as there's a constant stream of CO2 bubbling out from the end of the tube, there should be more CO2 in the tub. Although most of the bubble will rise to the surface and evaporate into thin air, some of it must surely be absorbed by the water.


For the 3rd tray which I'm going to call my "normal tray", I use a plastic container filled with water to a depth of about 3 cm. I put in a generous portion of Java Moss and squeezed in a drop or 2 of Liquidfry No 1. This is how I usually hatch Killifish eggs. I asked the maid to put in all the good eggs she can find and she found 53.


Here's a pic of the 3 containers lying side by side:


Well, that was yesterday at about 5 pm. It's about 5 pm now (24 hours later) and I'm sure you are keen to know the results. Place your bets, folks, while I go and do a headcount

Loh K L

[stormhawk]

Of all the methods I've used, only one has given me a good hatchrate.

Oxygen tablets + Cool water + large surface area container + low water level, about 1 to 2 cm above the peat surface

Seems to simulate the cool raining effect that triggers them to hatch in the wild.

In this case the oxygen tablets that I only trust are JBL's Oxyletten pellets. I would be much interested to know the results from the "Oxygen Stone" you used.

Bubbling into the container serves only to confuse and tire the fry. I did that once and ended up with at least 15 dead fry. When wetting the eggs you should always have some peat around the eggs. The fry seem to require the presence of the peat to hatch out normally. I don't know why this is so, only the fry will know.

[turaco]

I thought the 'traditional' way is CO2 to force hatch non-annual while O2 tablet for annual? & I thought peat is a must in annual hatching? I don't thing you'll get many free swimming fry but will still be keen to know your result though.

[TyroneGenade]

Hello,

Unfortunately I'm not a betting person (unless the outcome is 100% predictable).

However, if you got here you will read that the purpose of the O2 tab is to reduce the number of belly sliders not to force hatch the eggs. Annual eggs hatch when they want to. Force hatching annual eggs normally ends in tears as eggs forced to hatch are under developed and prematurely forced out of diapause III. "Normal" belly-sliders that hatch spontaneously belly-slide because they have exhausted their energy reserves and had to metabolize their own bodies.

Look at a healthy fry and you will see the remnants of a yolk sac attached to the belly. An unhealthy fry will have a concave belly and no yolk sac. It will also display a bent back.

When it comes to hatching, there are a cluster of glands on the head that are stimulated to release an enzyme that breaks down the chorion. These glands are stimulated to produce the enzyme by an increase in respiration, i.e. increase in CO2 concentration. (We in biochemistry define respiration as the use of of O2 not the increase in CO2 but I'm not going to split hairs today...) This CO2 can come from the environment (force hatch) or from the fry whose metabolism receives a kick-start by wetting and an O2 influx.

Now, were it not for:

After that, I attached an air-stone to the tray and connected it to an air pump.

which would drive off the excess O2 needed to stimulate the fry I would guess that both methods 1 and two yield of hatched fry but with less belly-sliders in the O2 tray. This would be hard to say for only 1 experiment per class. The O2 vs CO2 trays would need to be repeated 3x.

I am however, going place 2 Zimbabwe Dollars on the Java-moss tray yielding the best success because:
1) the liquifry will will stimulate the microbes so that more CO2 is produced and
2) the Java moss will increase the O2 levels and release tannins etc... that will reduce belly-sliding.

What I would like to see in further experimentation is the following combinations:
1) O2 tab
2) peat extract
3) O2 tab + peat extract
and see which gives the best result.

Regards

[timebomb]

Well, fellas, here are the results (after 24 hours of wetting) ;

Oxygen tray with 40 eggs - 10 hatched


CO2 tray with 40 eggs - 26 hatched


Java Moss tray with 53 eggs - 0 hatched

The CO2 tray being the most effective one, I thought it would be a good idea to feed CO2 into the Java Moss tray too. So that's what I did.


I came home this afternoon (about 20 hours later) and found 2 fry in the Java Moss tray. But one was already dead. I'm not sure if there's more CO2 in the tray because I also saw many tiny bubbles around the Java Moss. It was pearling.

I checked on all the fry and found quite a few already dead. Those alive seemed to be belly-sliders but I can't be sure. The experiment doesn't indicate much of course and the results are far from exhaustive but it seems to suggest that more CO2 is the way to go for hatching annual eggs. I'm not sure if the lack of peat causes belly-sliding but that would be my next experiment.

Tyrone, if tannins can prevent belly-sliding, would it be the same if I use water from a tank that is stained brown by a piece of new driftwood? It so happens I'm soaking the driftwood I bought from Malaysia and the water is all brown.

Loh K L

[TyroneGenade]

Tyrone, if tannins can prevent belly-sliding, would it be the same if I use water from a tank that is stained brown by a piece of new driftwood? It so happens I'm soaking the driftwood I bought from Malaysia and the water is all brown.

Beats me... The dismal success of the Java moss tray has pretty much sunk my titanic reasoning. So much for that crazy hypothesis...

The CO2 fry, what is the proportion of belly-sliders to normal fry compared to the O2 tray?

The "tannins" are not just tannins but a mix of various organic compounds. Some of these have anti-oxidant properties, some are anti-bacterial and many may have hormonal activity. Some may posess all three properties. You can try your driftwood tannin mix and see what happens.

Returning to magnificus experimentation. A nice way to figure out what is happening is to take a large number of eggs of the same age and wet a portion of the eggs every month once you begin to see eggs eye-up.

For instance, you obtain 200 eggs. Put them on peat so you can monitor their development. When you see an eyed-up egg take it out and put it in another container on peat. When you have say about 20 of about the same age wet them and see what happens. How many hatch?

The eggs that don't hatch, put them back on peat but make a note as which they are. When another bunch eggs eye-up take them out as before but let them sit eyed-up for a few more weeks. Lets say, 2 weeks.

In the mean time wet the remainder of the of eggs that didn't hatch 1st time. Of the remaining eggs what % hatched this time? Wet these 2 weeks after the 1st wetting. Always use the same wetting technique (we get away with an O2 tab and peat extract here in the lab).

The idea is to see when the eggs are actually ready to hatch. I don't buy the idea of force hatching annual eggs. They are designed to stay in the egg till they are ready to get out. If the magnificus are not hatching it means they are still in DIII and are not ready to come out. Why? Do they need to wait longer? or perhaps because of the high incubation temperatures they have a very short window in which to hatch between eying-up and withering away?

From the experiment I described, it would be interesting to see if the viablity of the fry and the propensity of the eggs to hatch when wet deminishes as the DIII embryo ages.

I get the impression that the reason for the staggered development of these fish's eggs is to safe guard against all the eggs eying-up at the wrong time and so damning the unhatched fry to wither and die in the egg.

This is exactly the problem with N. symoensi and sp. Mansa/Kasanka.

Loh, based on current exchange rates I owe you 0.0005992 SGD for the 2 Zim$ I bet.

Regards

[timebomb]

Loh, based on current exchange rates I owe you 0.0005992 SGD for the 2 Zim$ I bet.

Well, it's still money. Remind me to collect when we meet up

Tyrone, the experiment isn't meant to force-hatch the eggs. Those I wetted were already well eyed-up and theorically, should hatch when they're soaked in water. I don't know if I can carry out the steps which you suggested. It seems like a lot of work for one person, more so when I can't see the eggs clearly.

I'm sorry I can't tell you the ratio of belly-sliders in the oxygen and CO2 trays. I did something stupid, you see. When I took out the fry from the 2 trays, I put them together into a new tray. They're all mixed up now and it's impossible to identify which fry is from which tray. However, I did noticed when I was taking out the fry with a turkey baster, that those in the CO2 tray looked more lively. They were swimming stronger than those in the O2 tray.

My maid said there are at least another hundred eggs in the peat Au gave me. But mostly, they aren't ready for wetting. I'll wait another 2 weeks or so before carrying out the other experiment, the one you suggested earlier about using different combinations of peat extract and O2. But considering that more CO2 gives a better hatch rate, wouldn't it be better to use different combinations of peat extract and CO2?

Loh K L

[TyroneGenade]

considering that more CO2 gives a better hatch rate, wouldn't it be better to use different combinations of peat extract and CO2?

Perhaps. The big problem is that there is no control (i.e. water with just eggs or water with eggs in peat).

How many eggs would of hatched had you just put in a predefined number of eggs in the peat they were incubated as per usual? Then we could compare if adding CO2 or O2 has a real effect.

The best way to proceed would be (perhaps):
1) X eggs in peat + water (control group)
2) X eggs in peat + O2 tab + water
3) X eggs in peat + CO2 + water
4) X eggs in peat + peat extract
5) X eggs in peat + O2 tab + peat extract
6) X eggs in peat + CO2 + peat extract

Then we can more accurately assess if O2 tabs, CO2, peat extract and combinations there off yield any better result than simply wetting the peat.

The important thing would be use eggs that are all in the same state: i.e. all recently eyed-up.

The problem is that you would need to hatch at least another 40 eggs per experiment else the intrinsic experimental error would be too high (10 eggs, error = 10% or 1 fish out of 10!, 20 eggs -> 5%, 40 eggs -> 2.5%) and a difference may get lost in the noise.

Keep well

[TyroneGenade]

Hello,

Last night I wet 2 lots of N. furzeri eggs. One was of 11 eggs and the other 20.

The setup, large shallow dish with snap fast lid. The eggs were taken off peat and placed into the dish. To this peat extract was added (I just put peat into a bucket, add warm water, wait and then store the water in bottles) and 1 oxygen tablet. No CO2 or peat added at all.

The results:
Tub 1: 11/11 eggs hatched, tub 2: 19/20 eggs hatched (I had to manually free the 20th fry who was too weak to break out of the egg). Alas, these eggs were old and many of the fry are not looking too good BUT, this is just to let you know that adding CO2 is not needed to hatch eggs if they are good ready to go.

Regards

[timebomb]

this is just to let you know that adding CO2 is not needed to hatch eggs if they are good ready to go.

I'm not so sure about that, Tyrone. Yours are Nothobranchius eggs whereas my experiment is on eggs of the Simpsonichthys. Over here, many of us don't have any problems with Notho eggs but Simp eggs are often hard to hatch. In fact, when we first started, Ronnie and I never succeeded with any eggs of the Simp until we learnt about oxygen tablets. I'm just experimenting to see if CO2 gives better results.

Loh K L

[selena]

Got a bag of Simpsonichthys magnificus "Itacarambi B7" eggs from Au S L last Thursday. The eggs were collected in Dec'04 and incubated for 5 wks. Did a partial wetting for the eyed-up eggs and in 2 batches last night, and managed to collect about 40 frys. Did not find any belly-slider.

Did the same procedure on the Austrolebias nigripinnis Villa Soriano's eggs after a incubation period of 2 months. Count 12 eggs but have 22 frys

Method - (KL said "Find out for Au and follow his method, he is the most successful with magnifus.") Aged water, Jiffy plant peat (could not find coco peat) Japanese O2 tablet (since I bought the wrong type, just dump in too) and JBL O2 tab. Leave breakup O2 tab in hatching tray for 10 minutes and popped in the eyed-up eggs. Wait for frys to swim up and spoon into hatching tray.

Simple . Next headache - will they survive under my care.


Question- Are O2 tablet really necessary? With the slow rate of bubbling and only ten minutes, is there signifiant oxygen to defused into the peat water to enhance hatching.

Au,
Thank you for the guidance and eggs.


Selena

[stormhawk]

Hi Selena, glad to hear the good news and welcome to the world of the SAAs. The next time we have an SAA order come up do join in. The more people the merrier.

Here's my method, which should be similar, if not the same as Au's.

1) I empty the peat containing the eggs into a shallow square plastic tray.
2) Rinse out the plastic bag / container of the remaining bits of peat using cold water straight from the fridge. Pour it out into the tray.
3) Fill the tray with cold water to a level thats about 2 cm or slightly above the peat layer.
3) Then I break up one JBL O2 tablet into four pieces and just dump it into the tray.
4) 1 to 2 hours later the first few fry will appear, followed by their siblings over a period of 8 hours or so.

Below is my reasoning for doing so:

I usually do this at night because the temperature is cooler and darkness somehow gives the fry less things to be frightened of.

I use cold water because it simulates the cold of rainwater and also because cold water seems to contain more oxygen as compared to warm water. As the temperature gradually changes in the hatching tray over time, the fry slowly acclimatise to our room temperature as they hatch out. I don't use aged water though.

I fill up the tray to such a shallow depth because the deeper the water level the more deaths I will experience. This is from personal observation. Since young hatchling SAAs need to inhale in air to inflate their swim bladder the first time round, they usually require a shallow depth of water to allow better oxygen diffusion. Shallow depth also means an easier time scooping out fry since they can't run around much.

I use JBL O2 tablets because when used with cold water, I get no belly sliders. I once used the air tube to bubble in air to agitate the water a little bit, thinking that it may help in hatching out the fry or perhaps increase the oxygen content. I ended up with alot of dead fry so please don't repeat my mistake. The tablets allow a supersaturation of the water with oxygen necessary to facilitate the hatching of the fry.
_________________________

In short, those JBL O2 tablets are HIGHLY necessary when it comes to hatching out SAAs. In conjunction with the use of cold water, there is a significant increase in the viability of the hatchling fry.

Ronnie has also had a good result using my method except that he uses ice cubes to cool a container of water before using that water to wet the peat. Since I usually have a bottle or two in the fridge for drinking, I use the same water to wet the peat.

[CM Media]

Question- Are O2 tablet really necessary? With the slow rate of bubbling and only ten minutes, is there signifiant oxygen to defused into the peat water to enhance hatching.

Hi Selena,

I feel happy for you to be able to hatch the 2 species without any problem.

When I first started with Simp. magnificus which was more than 2 years ago, I was hit badly with most of the fry being bellysliders.

I happened to know someone from France who is quite successful with the Simp. magnificus. He was kind enough to guide me through and told me that by increasing the amount of oxygen dissolve in the water will overcome this problem.

So my buddy and I started to use various methods to increase the oxygen level in the hatching tray. After several experiments, I finally settle down with JBL oxygen tablets which dissolve easily in water as compare to those from Japan which is used for planted tanks and do not dissolve in water.

As in layman term, with the increase of O2 in the hatching tray, the fry will not have to swim to the water surface to fill their swim bladder. If you pay closer attention, you'll realise that SAA fry are much bigger in size and thus they require more strength to get to the upper level of the water. This will usually stress them out. So by increasing the amount of O2 in the hatching tray, the fry won't have to struggle all the way up.

Selena, when you start to harvest enough eggs from the Simp. magnificus, try to hatch the eggs without the O2 tablets and see what happens. There will bound to have bellysliders.

[hobbit6003]

As in layman term, with the increase of O2 in the hatching tray, the fry will not have to swim to the water surface to fill their swim bladder. If you pay closer attention, you'll realise that SAA fry are much bigger in size and thus they require more strength to get to the upper level of the water. This will usually stress them out. So by increasing the amount of O2 in the hatching tray, the fry won't have to struggle all the way up.

Hmm...thought Tyrone told me in the other thread that killies do not have a direct oral/gastrointestinal route access to the swim bladder, and so do not need to surface for air to fill their swim bladder this way?

Cheers,

Kenny

[CM Media]

Hmm...thought Tyrone told me in the other thread that killies do not have a direct oral/gastrointestinal route access to the swim bladder, and so do not need to surface for air to fill their swim bladder this way?

Hi Kenny,

As far as I know, the fry do not graps the air from the surface of cause! My line of thinking is that the dissolve O2 in the water somehow will be able to get to the bladder of the fish...Maybe by way of difussion?

[hobbit6003]

Hi Au,

Yes, oxygen will be carried from the bloodstream to the swimbladder.

You mentioned increasing dissolved oxygen concentration in the water so that frys needn't have to struggle all the way up, so I thought you were implying that they need to surface for air.

Tyrone convinced me that this is not the case,as the air in the swim bladder contains higher percentage of oxygen than any other gas, which would not be the case if it was a direct intake of air from the atmosphere. Some fishes do that.

Cheers,

Kenny

[selena]

Hi Au,

Did another wetting last night. This time I gather two batches of dead frys.
Left the oxygen tab a little longer than before and forgot to skim the oxygen whitish residue floating on the surface. Found only 4 dead fry.

Question - Would the film on the surface cause some difficulties in air exchange? Found no living frys except 4 dead fry.

After putting in a Jap tab, I was able to havest some live fry. Immediately after they could swim, I pop them into the growup container of Java Moss.
By then it was 3am, there were still about 10 frys in the tray. Thought I try your method of leaving them in the peat overnight so I took a nap. Woke up 2 hours to find only 2 live fry and the rest lying in the watery grave.

Question - Those that I spooned up into the growup containers, I checked again in the morning are still alive. Why did those left in the hatching tray died? (Au, is there something you are not telling me, some secret ingredent you add into the water?)

Selena, when you start to harvest enough eggs from the Simp. magnificus, try to hatch the eggs without the O2 tablets and see what happens. There will bound to have bellysliders

.

Au, you are talking about something of distant future Let me try and see if the fry live beyond this week. Thanks again Au.


Selena

[CM Media]

Hi Au,

Did another wetting last night. This time I gather two batches of dead frys.
Left the oxygen tab a little longer than before and forgot to skim the oxygen whitish residue floating on the surface. Found only 4 dead fry.

Question - Would the film on the surface cause some difficulties in air exchange? Found no living frys except 4 dead fry.

First of all, let me ask you afew questions.

1. How big is the hatching container and how deep is the water?
2. What type of containers are you using for hatching?
3. How many O2 tablets you use? (Reason I ask is too much O2 tablets will kill the fry as I've mentioned to you when I pass the eggs to you.) When the water turn cloudy, it's signs of over dosing of O2 for your info..

[selena]

Hi Au,

1. How big is the hatching container and how deep is the water?
2. What type of containers are you using for hatching?

Disposable rectangular container ( I think Nasi Lemak container ) 4" X 6" x2" with water not more than 2 cm above peat.

3. How many O2 tablets you use? (Reason I ask is too much O2 tablets will kill the fry as I've mentioned to you when I pass the eggs to you.) When the water turn cloudy, it's signs of over dosing of O2 for your info..

One JBL break into 4 pieces but only different from previous night was I left it dissolved longer in the peat. Then after I discovered the 4 dead fry, quickly remove the dead, skimmed the debris off the surface and put in some breakup (less than half a tab) Jap oxygen tab. If I have not forgotten, water did not turn cloudy.

Au, my decision for next wetting will be: oxygen tab for ten minutes, popped not more than 10 eggs, quickly get them out as soon as they start swimming.

-------------------------------------------------------------------------------
Jianyang,

Lost my confidence after last night. Will participate in SAA order maybe 5 years later when I have more experience .After this failure I am going to withdrew into some dark hole and nursed my wound.Luckily KL not in S'pore.


Selena

[stormhawk]

Selena, don't lose confidence. My first attempt at hatching out the magnificus ended in disaster too but that didn't stop me from trying others. Right now I have some young flammeus fry that hatched out from a bag of peat that a friend from overseas sent me.

They're doing quite well since the small tank they're in contains lots of those tiny animals in the water.

I should be trying daphnia for them in the next few days. They seem too small to be able to ingest daphnia for now.