Nothobranchius Killifish Breeding

[Schuyler]

I tried out the water incubation method with some N Guentheri eggs and after two weeks it's pretty obvious that it's working. The embryos look almost as developed as the Eggersi ones in the last post.

This time I put together a little video. I wish the camera quality on the microscope was better but it's clear enough to be able to see what's going on in there

 

[Grin Ghost]

Very nice video! What kind of microscope are you using? Thank you very much for sharing!

Edited June 6, 2023 by Grin Ghost

[Schuyler]

On 4/7/2023 at 4:36 AM, Simon Buys said:

Very nice video! What kind of microscope are you using? I don't think a higher quality camera would give you much more value for money. We use very expensive microscopes at the lab and see pretty much the same things. Eye development, pigmentation, heart and bloodflow are all very clearly visible. Maybe if you want to see dividing cell stages more clearly you could eventualy upgrade but i don't think it would be very usefull except for that it is cool to see. Thank you very much for sharing!

Thanks! It's a cheap handheld microscope my dad got off of Amazon and let me borrow. 

I have a light from one of my kid's toys that I use under the eggs. My main thing is that it's hard to control because there isn't an obvious up vs down. Then the stand doesn't really work either.

It does get closer up views of stuff though and that's the important thing.

[Schuyler]

The success with the water incubation made me curious if there was anything special about the water vs just the temp.

Looking through the bags of eggs that I have, there was an interesting pattern. The eggs collected in December were starting to eye up, the bags of eggs from January and early February weren't, but then newer bags from the middle of February were eyed up.

February was when I started making a concerted effort to keep them warmer than just room temp. Egg that reached diapause II before that point went into diapause while ones that hit that point after they started being incubated just developed directly.

If you don't like waiting, well first off if recommend a different genus if fish... But if you don't like waiting longer than you need to, then I would definitely recommend getting a little seed warning mat or some way of Incubating the eggs.

[Schuyler]

It's time to wet the eggs. I added roughly an inch of dechlorinated water to the eggs in the container. Checking back a few hours later and there are a few tiny fish swimming around. There are also subs very wiggly eggs.

Once free they will rest for a little while as they attempt to fill there swim bladder. If this fails they will become belly sliders and will likely need to be culled.

I picked a few eggs out and put them under the microscope again:

I didn't get video of them moment they actually break out but I did get some nice footage of them working on it. I'll keep checking on them and hopefully I'll get lucky with timing.

Edited April 14, 2023 by Schuyler

[Schuyler]

I think I've collected them all with a grand total of 46 confirmed fry. There may have been a few more that were collected but I forgot to mark down but I tried to verify that I actually caught it in the turkey baster because they'll occasionally swim back out of fall in a drip.

I've transferred them to a grow out tray that is a slightly modified version of @Lowells Fish Lab's design.

Something else I noticed was that they'll get the egg shell stuck on their head. I managed to remove it from a few by sucking then up with a pipette.

If you zoom in on the red circle you can barely see that the fry has its head stuck. The green circle has an unhatched but ready egg.

Here are some fry in the grow out tray.

[Schuyler]

Not much to say other than I hope to do a write up about hatching and fry care soon like I did with the other steps earlier.

Oh and the count just went up by six to 47. Though there are a decent number of belly sliders so I expect that number to drop quite a bit. My goal is to beat my 50% survival rate of my first batch but the absolute minimum would be ~20% survival rate (10) to get the BAP points

[Schuyler]

Pulled the last three fry from the coco coir today. So now the total is up to 50.

This podcast released a dirt spawning killifish episode and I actually got mentioned (not by name) because I emailed them about the study that links the age of maturity to longevity. That was a pretty cool surprise.

I found one of the fry that had an egg stuck on it's head and got a picture of it under the microscope

[Schuyler]

 

Hatching eggs

Before you hatch make sure that you have good cultures of small fry food. They may be big enough to eat San Francisco Bay brine shrimp (which are smaller than the Salt Lake like Aquarium Coop carries) but you risk the smaller fish not getting food and starving. The smaller fry tend to be female so this can cause skewed sex ratios.

When you are ready to hatch, fill two small clear containers (like a plastic shoebox or tupperware) with about an inch of room temperature dechlorinated water, add the dirt to one, swirl it around some to help it sink, add some vinegar eels for any fry that hatch before you find them, and wait. Fry could start hatching as soon as an hour but others could take a day or two. After they initially hatch they will rest on the bottom and fill their swim bladder. The large shallow container helps increase surface area so that there is enough oxygen in the water for them to do this. I tend to “wet” the dirt in the afternoon then come back to look for fry in the morning.

The fry will be tiny and hard to see. I’ve found that the easiest thing to do is place the container on a white sheet of paper (the color helps contrast with the fry), shine a bright light from the side, and slowly scan across the top. It can also help to slowly run a pipette, finger, chopstick, etc through the dirt. This gets them to move which makes them much easier to see.

As you find fry, use a pipette, turkey baster, or large spoon to suck/scoop them up and move them to the container without the dirt. This second container will be used for growing them out for the first little while.

After about three days, strain the dirt out in a fine mesh net, squeeze out the water, and put it back in the bag. You can then store that for a few more weeks and try again. The eggs come out of diapause (suspended animation between development phases) at different times so some of them may not have been ready to hatch.

Occasionally there are eggs that are ready to hatch but just need a bit of encouragement. There are three things that can be used to encourage them: heat, agitation, and CO2. One lab protocol I read suggested placing them in a small tube with a tight fitting lid, using a straw to blow bubbles in the tube (to add CO2), putting the lid on, and putting the tube in your pocket for a few hours while you work on other things.

Some will actually get the egg shell stuck on their head. I’ve been able to help remove them by sucking them up with a pipette and squirting them out a few times.

You may notice that some fry spend the majority of their time on the bottom and visibly struggle to get up off of the ground. These are referred to as belly sliders and tend to have a lower survival rate. There are lots of ideas about what causes this. The main one I've seen in a few places (including a lab protocol) is that their incubation time was either too short making them under developed or too long leavening then weakened and low on energy.

Growing out fry

Feed the fry vinegar eels, microworms, or powdered fry food 2-3 times a day for the first week or so, until you can get them onto baby brine shrimp. The small container helps make sure the fry can find the food.

You’ll also want to frequently clean up any uneaten food off the bottom. Some people add a snail or small shrimp to help pick up uneaten food before it fouls up the water. This also consolidates leftover food into nice packages that are easy to clean up.

Keeping the water clean will help the fry grow faster and avoid disease. Floating plants can help reduce nitrates in the water and make the fry feel safe.

Sometimes fry will grow at different rates. If the difference becomes big enough then you may need to separate them by size. That way the smaller fry don't get eaten by the larger ones.

[Schuyler]

Today I did a more thorough cleaning and check on the fry. I pulled out the plants and sucked up the poop with a turkey baster. 

There was a good amount of poop but the fry didn't look like they had growth all that much until this day and happy chonker swam by. Look at the size difference

The big one is just right if the center and there is a small one in the top left.

They seemed to like the places where the coir was on bottom. I'm guessing it's helps then camouflage and feel safe.

There were also a ton of seed shrimp, cyclops, and other tiny critters from when I left the tank to run by itself with some moss and stuff. I think those are adding extra food 

[Grin Ghost]

On 4/24/2023 at 5:54 AM, Schuyler said:

 

When you are ready to hatch, fill two small clear containers (like a plastic shoebox or tupperware) with about an inch of room temperature dechlorinated water, add the dirt to one, swirl it around some to help it sink, add some vinegar eels for any fry that hatch before you find them, and wait. 

You could also try to use colder water from the fridge instead of water at room temperature. After 24h hours transfer the eggs that didn't hatch into a new container with colder water. U can repeat this for 3 days as you mentioned in the post. This, together with water aggitation, simulates 3 days of rain which triggers the hatching of the eggs that won't hatch in the first day.

The containers should be in a warm environment to try this. This way the water warms up quick enough for the fry that do hatch more quickly.

[Schuyler]

Today I wet another batch and I'll likely hatch another batch immediately after this. I'm doing a bit of an experiment. I divided the soil in half (by weight) and then added 350 mL of coke water to each container (there was a mark on the container so it seemed like a good way of making sure they had the same amount of water). I placed them side by side but added an airline with a slow bubble to see if it makes any noticable difference.

[Schuyler]

Maybe I'm paranoid but I think I may have wanted to wait a little longer to hatch these.

For the experiment, the tub with the air hatched first and has 4-6 fry while the other one only has 1-2. The tub with air also seems to stay roughly 1°F cooler.

That said, they all look like belly sliders. Hopefully they will develope out of it.

I did a cleaning for the older batch and now that I'm looking at newly hatched fry again I can see the difference but most of them aren't growing all that fast. I probably need to separate out the bigger ones to their own container. This is a good picture to demonstrate the size difference in the old batch.

 

[Schuyler]

The fry seen to be growing well with fat little bellies:

The bigger ones are in their own little container so they don't out compete the others

[Schuyler]

The larger ones ones are looking more adult like. From what I can tell, one is definitely male and one is probably female.

Male:

Female:

They are now eating microworms, BBS, daphnia, and I'm trying to introduce flake

[Schuyler]

I was getting ready to do some cleanup when I like in the bucket and noticed a fry!

That poor little thing has been in there at least four days in the garage floor with no heat, no aeration, poopy water, and only leftover vinegar eels for food. Just shows you how hardy these little fish are.

Then I went to clean the grow out tray and noticed the size discrepancy...

Here it is after cleaning but better replacing the plants:

Look at that big guy there compared to his little siblings!

I also did a little excitement based on things I've read about belly sliders. Seems like one major theory is that you get them when they are under or over developed.

So I decided to wet four batches of eggs at once. Each was harvested at least a week apart.

Unfortunately, two only really hatched about 3-4 fry. But that middle one is just teaming with them!

(Hard to get a good visual on that... But there's at least four visible there near the bottom)

So there's not much that can be learned about if I agree with the idea of not. It does seem reasonable and it shows up in lab protocols. They probably know what they're talking about.

[Schuyler]

On 5/2/2023 at 8:27 PM, Schuyler said:

 

 

On 5/13/2023 at 11:18 PM, Schuyler said:

 

Check-out that uplift tube in the top left. Recently a batch of snails hatched and the tiny snails climbed up and picked that area clean. It was honestly a but shocking to see.

Another shocking find was this little fish below the tray:

Somehow a female got out of the grow out tray and into the tank below. Either it jumped through a tiny gap, it dripped out of the baster while cleaning/moving, or is a stowaway from the süsswassertang. Either way it's managed to survive with a full grown male in the same small area which is impressive.

[Schuyler]

I moved the more developed fry out of the grow out tray and into the main tank.

The male is coloring up nicely:

Here's a slightly better picture of the female:

[Schuyler]

I fed them pretty heavily with some live daphnia from my tub outside. Needless to say, I think he likes it:

The good news is daphnia has a lot of fiber so he shouldn't get bloat.

Something else exciting that happened: I had an article pop up on Google from the professor who shared his protocol!

https://neuroscience.stanford.edu/news/study-aging-researchers-give-killifish-crispr-treatment

[Schuyler]

His belly is gone already.

I also moved the rest of the fry in this batch down to the main tank and it out the tray. In all there weren't many that made it.

I suspect there was some fry that were eaten and a lot of belly sliders that weren't very healthy to start off with.

I'm moving the next batch into the fry tray and in going to make a few adjustments: more plants to give smaller fry hiding places, pulling larger fry before they can eat the smaller ones, and checking on them more frequently.

Speaking of the fry tray, a shot out to @Lowells Fish Lab. He just put out a video of an updated version of it that look pretty great. 

https://youtu.be/16aFdagCLGk

[Schuyler]

Sad day...

I went to clean up the fry tray and after pulling the floating plants off I noticed that the fry from the recent multiple batch excitement were all gone. Not even a trace. It could have been disease, too hot, not enough food, shock from transferring... No way to tell now. Should have been checking on them more often.

Next time I'll not put as many floaters and make sure to actually look at them daily rather than just feeding.

I still have the leftover coir from the experiment, hopefully that still has some eggs left that will hatch next time. If not then it's all up to the remaining fry from the last batch to keep the line going.

[Grin Ghost]

On 5/25/2023 at 3:14 AM, Schuyler said:

Sad day...

I went to clean up the fry tray and after pulling the floating plants off I noticed that the fry from the recent multiple batch excitement were all gone. Not even a trace. It could have been disease, too hot, not enough food, shock from transferring... No way to tell now. Should have been checking on them more often.

Next time I'll not put as many floaters and make sure to actually look at them daily rather than just feeding.

I still have the leftover coir from the experiment, hopefully that still has some eggs left that will hatch next time. If not then it's all up to the remaining fry from the last batch to keep the line going.

That's sad to hear, hopefully the rest will do fine. Good luck!

[Schuyler]

I gave them some extra attention today to try and get an idea of how many there are hiding in the Süßwassertang. There were at least two belly sliders (probably female), the two males that are always out and about, and one non-belly sliding female. There still hope!

I wish noticed that Eggersi makes seem to extend their gills downward when sparring. Showing I haven't noticed with the Guentheri. I couldn't get a picture of it because they would get distracted by the phone but he's an updated picture of the main male.

Edited May 26, 2023 by Schuyler

[Schuyler]

This was just recommended to me by Google:

https://neuroscience.stanford.edu/news/study-aging-researchers-give-killifish-crispr-treatment

One of the professors on that was one one who answered some of my questions and said that there is an idea that the first fish to mature have shorter lifespans but it hasn't been rigorously tested.

That was really cool to see pop up.

[Schuyler]

Starting another N Guentheri batch that came from the first batch I hatched.

Completing the lifecycle!