2018 Incubation Experiment

Markw84 said:

@Tom

I just had the first hatch in the clutch I incubated in vermiculite/peat mix. This is a younger female I mentioned before that has have very low fertility rates. I put these eggs without any rinsing into a container of 50/50 vermiculite / peat mixed completely and then an equal weight of water added and mixed. This produced a slightly wetter mix than I normally had used as the peat was already damp when i got it from the bag. I buried the eggs just over 1/2 way into this mix.

The baby took the longest I have ever had a sulcata take for a first pip out of a clutch - 96 days when normally I got 86 days. The yolk sac was substantially smaller than previous hatchlings and was the biggest hatchling I've ever gotten out of this female - 37 grams. The eggshell was quite thin and crumbled in my hand as I took some out to add to the brood box when I took the hatchling out.

I believe both the acidity / humic matter of the peat, along with the better water potential of the media are key things I am watching as this progresses.

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Did you measure the pH of your vermiculite/peat mix at the completion of incubation? Suspect it will be similar to pH of straight peat.
Thanks

CarolM said:

I will see if I can get a test kit from the garden centre near me. If it is not too expensive I will get it and test my soil where I have seen the torts laying eggs.

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I haven't managed to get a soil tester yet. I must google and see if there is a DIY way if testing the soil here in my garden.

They are finally hatching! 90 days would have been 6/18, so they are a few days later than normal for some reason. The first pip was this morning and it was one from the peat side. This afternoon, I discovered a pip on the "regular" side. How strange that the eggs all sit for months and then begin hatching within a few hours of each other, even in different shoe boxes.




I will get weights after rinsing, but before soaking, and again after soaking. Peat group will be marked so I can track growth for a few weeks.

4 currently pipping from each box now.

This is so interesting! Thanks for the pics!

Tom said:

4 currently pipping from each box now.

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Awesome. And can you see if the shells in the peat are thinner than the shells in the other box? From your pic in the above thread the shell looks quite thin.

CarolM said:

Awesome. And can you see if the shells in the peat are thinner than the shells in the other box? From your pic in the above thread the shell looks quite thin.

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Not yet. I like to wait until they come out of their shell on their own before I mess with them. They usually pip, tear open the egg, and then sit in it for a day or two. Once they leave the egg, I then rinse them and their egg shell and put them into brooder boxes. At that point, I'll have more a of a feel for any differences or similarities in the egg shell thickness.

Tom said:

Not yet. I like to wait until they come out of their shell on their own before I mess with them. They usually pip, tear open the egg, and then sit in it for a day or two. Once they leave the egg, I then rinse them and their egg shell and put them into brooder boxes. At that point, I'll have more a of a feel for any differences or similarities in the egg shell thickness.

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Oh right. I forgot about that. Me being impatient again. [emoji17]

This thread is great- I absolutely love seeing some good scientific process happen in the tortoise hobby world. To often I feel like excellent reptile breeders operate on "feel," which is amazing- but means that it is hard to teach other people what and how they do things. Threads like these are what has got me addicted to this forum (which I recently found). With my background in research and reptile breeding, I have a great appreciation for how much work projects like these take- so thank you to all of the forum members who take the time for these projects and desiminate the information to us all.

-Wade

PS- about the defication and urination in nests. I wanted to propose that the fecal material might be getting "squeezed" out by the egg as it comes out of the cloaca. The eggs really stretch things out as they are being deposited, so any fecal material that is near the cloaca might just be forced out. As for urine- I have heard people propose that urination is a means of softening the nesting location, in order to make digging a nest easier for the female. The moisture could also help a nesting chamber hold its shape better.

First baby out of its egg was on the peat side and weigh in at 38 grams after a quick rinse, but before soaking. 39 grams after its first soak.

As an aside to another recent thread, the first hatchling came out of a cracked/dimpled egg. I don't know why some people have so much trouble with cracked eggs. Most of mine hatch just fine. One of them went bad, but the other are all hatching.

I don't know how to scientifically quantify the thickness and "brittleness" of the remaining egg shell, but its thin and doesn't have much structural strength any more. I don't have to correct type of caliper to measure the thickness, but I will compare how it "feels" to the non-peat egg shells when the time comes.

Tom said:

First baby out of its egg was on the peat side and weigh in at 38 grams after a quick rinse, but before soaking. 39 grams after its first soak.

As an aside to another recent thread, the first hatchling came out of a cracked/dimpled egg. I don't know why some people have so much trouble with cracked eggs. Most of mine hatch just fine. One of them went bad, but the other are all hatching.

I don't know how to scientifically quantify the thickness and "brittleness" of the remaining egg shell, but its thin and doesn't have much structural strength any more. I don't have to correct type of caliper to measure the thickness, but I will compare how it "feels" to the non-peat egg shells when the time comes.

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I think the relative feel of the shells side by side would be a good indication for what we are looking for. Also relative size of the yolk sac upon emerging. 38 g is a pretty good size for the hatchling, so relative weights also a difference to look for.

I am of the mindset currently that the organic matter, along with water potential of the media are key ingredients to test in this type experiment. The water potential of the media would be key in facilitating calcium absorption/chelation in a more acidic environment.

The incubation medium seemed drier in these two boxes to me. Summer rains adding moisture to the nest chamber may not only help trigger hatching, but also facilitate development at the end stages of incubation as the larger, more developed embryos would have the highest demand for nutrients/minerals in their final stages.

Got some numbers going now. Here is the original photo of the eggs. If you look closely you can see some that are dimpled and cracked in the back row of each box. The one in the peat box on far right rear rotted and had to be chucked about month ago. All of the other ones hatched. Here is a pic of one of the dimpled ones after the baby hatched out of it. It is the one from the back row of the peat box, 2nd from the right. You can see the dimple in the original pic.

Here is the vermiculite side hatching. In my rush to get to work on time today, I neglected to take a pic of the peat side…


All of the babies on the peat side have hatched or are still in the process, except for the one egg that rotted. On the vermiculite side 3 eggs have not pipped yet. I just candled them and it looks like two should still hatch and one is clear inside. I don't draw much conclusion from this yet, but it will be interesting to see if we end up with three of the vermiculite not hatching compare to one from the peat side.

The hatch weights are pretty standard for Miss Daisy's babies. No surprises there. I guess what is surprising is that there really isn't much difference between the two groups. Here are the weight of the ones that have hatched so far:
Vermiculite Side:
34
35
38
42
37
35
38
38
34
40

Peat Side:
38
39
36
39
38
36
36
37
32
39
34
37

There are more still coming out of their eggs, but the largest ones were on the vermiculite side, and the smallest was on the peat side. On average though, the peat side ones seem slightly larger over all, but not by much at all.

The eggshells from each group feel about the same. They are all very thin and brittle and its difficult to rinse them off with out the "shell" part crumbling off of the flexible inner membrane part. This is also normal for Daisy's eggs after hatching. It does not appear to me that the peat made anything different happen in regard to egg shell thickness or strength during the incubation process.

Here are the final tallies:
The vermiculite side hatched 13 of 16 eggs. I'll give the last three another week or two and then consider opening them to see what went on in there.
Peat side hatched 15 of 16. The only one that didn't hatch was the one that rotted and got stinky.

Here are the hatch weight numbers. All numbers are after being rinsed, but before soaking:


I added them up and averaged them. Vermiculite babies had an average weight of 36.8 grams. Peat babies had an average weight of 36.6 grams. This is close enough for me to call them the same. Seems like the peat babies were a little closer to the same weights in general and the vermiculite babies had more highs and lows, but not by much.

I will chart their weights for the next few weeks and see if any differences appear, but at this stage, I'm ready to say that the peat didn't make any difference. The difference in the number of eggs that hatched is numerically significant, but its pretty normal for 2-4 eggs in a clutch of 30+ to not hatch. Interesting that 3 of the 4 that didn't hatch in this batch were on the vermiculite side, but there is not conclusive evidence here about why that is. I think the experiment bears repeating a few more times. I think my sulcatas are done laying for the year now that it is consistently hot every day. Any one else want to run this experiment and see what they get? I'll do it again next year. And next year I'll try a clutch with half the eggs on vermiculite and half on long fibered peat moss too.

In the mean time, anyone want to buy a sulcata baby to support science? I've got 28 more than I need. $50 each, and volume discounts available...

We have a late hatcher to the party. A 4th of july baby! 36 grams.

@Tom I have some hatching now from a clutch I put in 50/50 vermiculite/peat moss and covered just over 1/2 way up. I kept the mix fairly moist as I am interested in keeping the water potential higher in this type experiment as I believe it the the water availability that allow for the better chelation and utilitzation of calcium. Especially towards the end of the incubation cycle. These babies all were in the same range of weight as you have here. However the parts of the eggshell that were in contact with the medium was amazingly thinner than the top that was exposed to air. The lower 1/2 of the eggs seemed almost like the hard shell was almost gone and only the membrane left by the time the baby hatched. Although I have not measured the water potential of the medium, I did keep it noticeably wetter than yours felt to me.

I think I need to get a meter to measure moisture content and try to quantify this more. In talking with Dave and going through some research papers - it does seem the water potential of the nest is a key ingredient. Perhaps the beginning of the rains we always think triggers hatching and facilitates digging out, is also accelerating calcium availability to the developing embryo its last two weeks or so. That would be when calcium demand would be the highest for the tortoise as well.

So the humic matter PLUS increased moisture may be needed. There is also still the increased CO2 levels increasing acidity that could also be coming into play in the latter stages as much larger embryos are creating more CO2?? In your box of 24 vs more empty box, both these things could have come into play as the much greater percentage of egg volume would have put more water and CO2 into that closed box.

One more late hatch:


This means each side hatched 15 out of 16, and 30 out of 32 for the whole clutch, for a 94% hatch rate which is typical for Daisy's clutches. The last egg on the right in this pic isn't going to hatch. Nothing in it upon candling.

I'll get a weight on this little one once he breaks out of his shell.

Markw84 said:

Agree. Lots we don't know.

My thoughts are not as much about acidity but the chelating effects of humic substances. Certainly the insides of a tortoise are organic material. How about the mucus deposited with tortoise eggs whlie laying? Or the observation that most nests I've dug up contain some fecal matter the mother seems to deposit in the chamber? All could contribute to chelation and calcium mobilization as well as establishing microbes for gut flora when the hatchlings emerge. How much we change that balance when we artificially incubate is worth investigating.

The thinning of the eggshell also would seem to aid in respriation / gas exchange in the egg and as an embryo grows, that need would increase. The growth uses calcium and we have a relationship between oxygen need and eggshell thinning. The more the calcium is used, the thinner the eggshell should be and the larger the embryo and its gas exchange needs.

In his talk at the TTPG and experiments, Dave also investigated the water chemistry and how it effects the intitial "setting" of the embryo to begin development. That brought into play Ph and total alkalinity. That also seemed to offer a strong relationship to split scutes. But that is a whole different line of investigation. That of course is more apparently of concern with aquatics, and in his case, Amazonian chelonians. But his experiements were on N American aquatics and quite remarkable.

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Each tub had a 1 : 1 water to substrate ratio by weight. It may have looked dry on top, but it was damp underneath. Any more water than what was in there and the eggs are likely to start splitting open, based on past mistakes of mine. Plus the humidity in the incubator is typically in the 80%'s.

My shoe boxes allow a lot of air circulation. So much so that even in my humid brooder, the egg shell membranes, paper towels and leaves all dry out with the lids on. This is why I don't make more or bigger vent holes on the side of the boxes.

I intend to repeat this experiment next year when they start laying again, and I hope you and others will repeat it. I'd like to see it done about 6 times for us to really start seeing a pattern and drawing some firm conclusions. Next year I will also try it with long fibered sphagnum moss too.

If y'all will take notice, this is also another dimpled egg that is hatching. You can see the dimple in the last two pics I've posted on this thread.