# 2018 Incubation Experiment



## Tom (Mar 27, 2018)

At last year's TTPG conference one of the speakers talked about better hatch rates coupled with bigger and healthier babies when he incubated his aquatic turtle eggs on some sort of acidic substrate instead of something inert like vermiculite. He used peat moss and explained that the acids in the decomposing moss dissolved the calcium in the egg shell making it more bio-avaialble to the baby, allowing more oxygen into the egg during the end stages of incubation, and making it easier to hatch since the egg shell walls were thinner.

@Markw84 and I both had the same ideas about it, and we wondered if the same thing would apply to tortoise eggs. Mark brought this up in another thread a few days ago and it reminded me that I wanted to try it. In addition to Mark, I think this experiment will be of interest to @Sterant @HermanniChris @kingsley and @Will too.

My 60 pound female sulcata, Daisy, lays pretty large clutches. Frequently more than 30 and her record last year was 42 in one clutch. She's also a favorite of my male, Tuck, and she has high fertility and high hatch rates. This clutch was laid on 3/18/18 and consisted of 32 eggs. 4 eggs were dimpled, all others were perfect. Good shell formation and consistent large size. As I dug the eggs up, I put them in order in the large egg flat. Once the egg boxes were set up, I put every other egg in each box so that there would be a relatively even distribution of eggs from the top, middle, and bottom of the nest in each egg box. 16 eggs per box. One box will be incubated in my usual style on fine vermiculite with a 1 : 1 water to vermiculite ratio by weight. Eggs are sunk about 2/3 to 3/4 into the vermiculite. The other box has about half the normal amount of vermiculite to maintain the normal moisture ratio and the other half is lightly dampened "organic" peat moss with no additives of any kind. The eggs in this box are in contact with the peat only and not touching the vermiculite on bottom.











The eggs will be placed on top of each other on the same shelf in my large circulating air incubator.






The eggs are already showing significant chalking, and we will have answers around mid June.

Comments and questions are welcome.


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## Bambam1989 (Mar 27, 2018)

I am excited to see the results. Though I am not a breeder I still find it fascinating!


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## Tom (Mar 27, 2018)

Bambam1989 said:


> I am excited to see the results. Though I am not a breeder I still find it fascinating!


You've seen the results of my usual style. Can you imagine bigger, healthier and even more vigorous???


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## Bambam1989 (Mar 27, 2018)

Oh my that really would be something! Im pretty confident that mine will exceed the 400gram mark before the end of this month and I thought that was some pretty vigorous growth..
I love the idea of this working for other types of torts too.


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## G-stars (Mar 27, 2018)

Are you planning on keeping these hatchlings for a couple months to see the outcome?


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## Tom (Mar 27, 2018)

G-stars said:


> Are you planning on keeping these hatchlings for a couple months to see the outcome?


Yes. I usually keep hatchlings for two months. I like to get them started well, eating a wide variety of foods and I want to see some good growth before I send them to their new homes. In the case of this experiment, I might even hold on to some of them a bit longer to really give things a chance to either equalize after hatching, or demonstrate an even greater difference over a longer term.


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## Tom (Mar 28, 2018)

Bambam1989 said:


> Oh my that really would be something! Im pretty confident that mine will exceed the 400gram mark before the end of this month and I thought that was some pretty vigorous growth..
> I love the idea of this working for other types of torts too.


Amazing how well things can go when it is done right. When I started raising sulcatas and other species in this monsoon season style, which is what they hatch into in the wild, one of the things that was used as an argument against what I was doing was that it was "un-natural". Looking back, I find it interesting how wrong they were, and how monumentally un-natural their dry methods really were.


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## Sterant (Mar 28, 2018)

Glad you are doing this - also glad you have enough eggs TO do this.


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## Yvonne G (Mar 28, 2018)

When I had the Manouria here I had a bad gnat problem with my eggs. The gnat maggots even ate through the egg shells. So I tried different mediums, peat moss being one. All the eggs in that batch rotted.


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## Sterant (Mar 28, 2018)

Yvonne G said:


> When I had the Manouria here I had a bad gnat problem with my eggs. The gnat maggots even ate through the egg shells. So I tried different mediums, peat moss being one. All the eggs in that batch rotted.


Yeah - I guess that's the balance. The acid has to be just right to partially dissolve the egg over the incubation period, but not before.


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## Markw84 (Mar 28, 2018)

Really like the experiment and that you have a proven good female to give you a nice sampling of "identical" eggs for each method.

I have a clutch of sulcata eggs from a young female that has had low fertility with last year's eggs. I have gone this way with her first clutch this year. I did mix 50:50 peat moss vermiculite completely instead of just peat moss alone on top. Your test will be much more telling as you have a known producer for the eggs. My clutch is from Feb 9 and do look really good so far.


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## Markw84 (Mar 28, 2018)

Sterant said:


> Yeah - I guess that's the balance. The acid has to be just right to partially dissolve the egg over the incubation period, but not before.



The theory is not as much about acidity but the observation that humic substances (Organic like peat moss) actually chelates calcium. This bonds an amino acid with the calcium for motility and is a form the calcium can take that some evidence shows is much more useable by the body. For example, calcium supplements are avaialbe in different forms - calcium citrate, chelated calcium, etc.


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## Sterant (Mar 28, 2018)

Markw84 said:


> The theory is not as much about acidity but the observation that humic substances (Organic like peat moss) actually chelates calcium. This bonds an amino acid with the calcium for motility and is a form the calcium can take that some evidence shows is much more useable by the body. For example, calcium supplements are avaialbe in different forms - calcium citrate, chelated calcium, etc.


Ok Got it. Does this have the physical effect of breaking down the structure of the egg as well?


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## Tom (Mar 28, 2018)

Sterant said:


> Ok Got it. Does this have the physical effect of breaking down the structure of the egg as well?


According to the presenter, yes.

And given what we just learned from @HermanniChris and his sanding of the egg shell, I would love to go to South Africa where Chersina occur and test the soil where they lay for acidity. I wonder if we could get @CarolM or @JeannineD to help us with that. We have soil test kits here in the states. I wonder if they have them available in the RSA?


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## Markw84 (Mar 28, 2018)

Sterant said:


> Ok Got it. Does this have the physical effect of breaking down the structure of the egg as well?



The calcium in the eggshell is a source of calcium for the developing embryo. There is a limited amount of calcium the yolk can carry. Less than a fully developed hatchling requires. The observations and studies have shown that as the calcium is utilized, the eggshell does thin substantially, which in at least some species, aids in the ability of the hatchling to break through the egg. Measuring average weights of hatchling, and weights of the eggshell after hatching, have shown a substantial difference in hatchling size and corresponding decrease in eggshell mass. The eggshells are much thinner and lighter. The environment we put an incubating egg in seems to be able to either augment calcium uptake, or retard it.


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## Kapidolo Farms (Mar 28, 2018)

Kelly Hull @tortadise and my incubation set up for Manouria eggs would suggest this is a huge difference. The talk was regarding south American aquatic species that live in areas where plant acids are very abundant everywhere. A stronger already look-ed at aspect might be any indication as to where red foots and yellow foots may favor for egg laying within the Amazon basin.

I don't know if there is a more common location for sulcata to lay eggs than other places within the larger habitat where they live. North American tortoise species will lay at or in the burrow mouth for climate control of the eggs, and though I did not look I'd have to imagine some one somewhere has evaluated the soil's physical conditions as well as the climate of the burrow.

Many field works in Australia have many parameters observed and recorded, but they are all aquatic side necks.


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## Tom (Mar 28, 2018)

Will said:


> Kelly Hull @tortadise and my incubation set up for Manouria eggs would suggest this is a huge difference. The talk was regarding south American aquatic species that live in areas where plant acids are very abundant everywhere. A stronger already look-ed at aspect might be any indication as to where red foots and yellow foots may favor for egg laying within the Amazon basin.
> 
> I don't know if there is a more common location for sulcata to lay eggs than other places within the larger habitat where they live. North American tortoise species will lay at or in the burrow mouth for climate control of the eggs, and though I did not look I'd have to imagine some one somewhere has evaluated the soil's physical conditions as well as the climate of the burrow.
> 
> Many field works in Australia have many parameters observed and recorded, but they are all aquatic side necks.


What are you guys doing for your Manouria? What incubation media? What are the results? Tell me more!!!

I'm using this type of peat moss because it is what I could find. I intend to duplicate this experiment with the next clutch using long fibered sphagnum moss. Any thoughts? Any tips for how to improve the quality of the experiment?


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## Sterant (Mar 28, 2018)

Tom said:


> According to the presenter, yes.
> 
> And given what we just learned from @HermanniChris and his sanding of the egg shell, I would love to go to South Africa where Chersina occur and test the soil where they lay for acidity. I wonder if we could get @CarolM or @JeannineD to help us with that. We have soil test kits here in the states. I wonder if they have them available in the RSA?


The research paper that lead to Chris sanding the eggs stated that when Chersina retain eggs, which develop internally, then are oviposited within a couple weeks of hatching - the shells are much thinner. It was suggested that the shells of retained eggs were shelled thinner to allow for oxygen diffusion inside the body of the mother where oxygen was less available. Perhaps the deposited eggs are thinner because the calcium was used during development, and not because of an oxygen requirement, or at least not ONLY an oxygen requirement. Is the internal chemistry of Chersina acidic?


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## Tom (Mar 28, 2018)

Sterant said:


> The research paper that lead to Chris sanding the eggs stated that when Chersina retain eggs, which develop internally, then are oviposited within a couple weeks of hatching - the shells are much thinner. It was suggested that the shells of retained eggs were shelled thinner to allow for oxygen diffusion inside the body of the mother where oxygen was less available. Perhaps the deposited eggs are thinner because the calcium was used during development, and not because of an oxygen requirement, or at least not ONLY an oxygen requirement. Is the internal chemistry of Chersina acidic?



I know that the human urinary tract is supposed to have an acidic pH. When it doesn't, problems ensue. I have no idea what the pH of the oviduct in a Chersina would be, but I'd be curious to find out. I suspect there are mechanisms at work here that we don't know about.


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## Markw84 (Mar 28, 2018)

Tom said:


> I know that the human urinary tract is supposed to have an acidic pH. When it doesn't, problems ensue. I have no idea what the pH of the oviduct in a Chersina would be, but I'd be curious to find out. I suspect there are mechanisms at work here that we don't know about.


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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## Tom (Mar 28, 2018)

Markw84 said:


> 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.



In line with the quoted paragraph, I've been having poor hatch rates for my SA leopards. The breeder I bought them from also reported poor hatch rates with artificial incubation, and he let them incubate naturally in the ground. He reports "incubation" times of up to 18 months, because the eggs must undergo diapause and can't begin to develop until the summer heat kicks in after a winter underground. This year, I intend to leave all the eggs in the ground and cover each nest with an open mesh cage so that predators can't get to the eggs, and other females can't dig them up or disturb them to deposit their own eggs. Next spring, I will dig them all up and artificially incubate them. Best of both worlds. I'll leave a nest or two in the ground just out of curiosity.


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## Kapidolo Farms (Mar 28, 2018)

Tom said:


> What are you guys doing for your Manouria? What incubation media? What are the results? Tell me more!!!
> 
> I'm using this type of peat moss because it is what I could find. I intend to duplicate this experiment with the next clutch using long fibered sphagnum moss. Any thoughts? Any tips for how to improve the quality of the experiment?



Both Kelly and myself following his practice to some extent have posted what we do here on TFO. My post is https://tortoiseforum.org/threads/live-naked-people.126107/page-17#post-1461695 as a guide to most Mep posts, and others in the thread "live Naked People". Kelly can chime in as it suits him.


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## CarolM (Mar 28, 2018)

Tom said:


> According to the presenter, yes.
> 
> And given what we just learned from @HermanniChris and his sanding of the egg shell, I would love to go to South Africa where Chersina occur and test the soil where they lay for acidity. I wonder if we could get @CarolM or @JeannineD to help us with that. We have soil test kits here in the states. I wonder if they have them available in the RSA?


I could look for you guys. Just give me a brand name or two and i will take a look for you.


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## Tom (Mar 28, 2018)

CarolM said:


> I could look for you guys. Just give me a brand name or two and i will take a look for you.


At nurseries and garden centers they have soil test kits for people to test their soil parameters. We also have a service where samples can be sent in to a lab for testing for a small fee. Do they do that there? I would imagine the farmers would all know about this. Know any farmers?


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## Kapidolo Farms (Mar 28, 2018)

_Live Naked People_ post 240
"One experiment that my sister consulted on (she works with medical isotopes for use in human health therapies and assured me I would not glow if I did this), but the vets at the Philly Zoo would not sign off on, was to incubate eggs side by side of pancake tortoises, one set that had a night time drop and another that did not. The incubators would be in larger enclosures with gas concentration monitors. The oxygen would be radio isotope labeled, and at some point the some eggs would be sacrificed to see if the night time drop eggs had a different accumulation of oxygen isotope after the same number of 'incubator days'. Also would night time drop eggs incubate at a different average number of days, and what would be the robustness of those neonates. "My" idea is that the change in temperature makes the eggs respire, more than at a stable temperature. Respiration is the result of metabolic processes in the egg. It would potentially do two things along two paths. Just pumping the air in and out might accelerate development, and it might also give rest to some processes. This as a thought experiment may be even more complex than narrated, but at least two factors could explain any differences in the observable parameters I hoped to look for. Days to emergence, and robustness. At the time we were generating many pancake tortoises with a high hatch rate."

Chris did two things that are variables in the egg that hatched. 1) He 'made' the egg respire by fluctuating the temp, and 2) he reduced the layer of egg shell making passive respiration work better. No telling which or if the combination had a synergistic effect.

Some of the successful Pyxis breeders use the night time temp swing as well. Eggs are seeds in a fashion and many seeds need to be 'scarified' to grow. The seeds that need abrasive scarification are often those that are in an arid environment (extra thick seed coat prevents desiccation. They are scarified by the gut of something that eats them, or by being tumbled with sand in a seasonal water way.

I found Chersina eggs during the morning that still had mucus slime on them in water ways in RSA. Wow.


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## CarolM (Mar 28, 2018)

Tom said:


> At nurseries and garden centers they have soil test kits for people to test their soil parameters. We also have a service where samples can be sent in to a lab for testing for a small fee. Do they do that there? I would imagine the farmers would all know about this. Know any farmers?


My brother farms grass I will ask him.


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## CarolM (Mar 29, 2018)

Tom said:


> At nurseries and garden centers they have soil test kits for people to test their soil parameters. We also have a service where samples can be sent in to a lab for testing for a small fee. Do they do that there? I would imagine the farmers would all know about this. Know any farmers?


Okay, My brother farms grass in the Artlantis area and there are quite a few angulate tortoises there. I asked him about the acidity level of the soil in Atlantis and he repsonse was " It is naturally between 3.5 and 4" I hope that means something to you guys.


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## CarolM (Mar 29, 2018)

CarolM said:


> Okay, My brother farms grass in the Artlantis area and there are quite a few angulate tortoises there. I asked him about the acidity level of the soil in Atlantis and he repsonse was " It is naturally between 3.5 and 4" I hope that means something to you guys.


Artlantis is ATLANTIS.


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## Sterant (Mar 29, 2018)

CarolM said:


> Okay, My brother farms grass in the Artlantis area and there are quite a few angulate tortoises there. I asked him about the acidity level of the soil in Atlantis and he repsonse was " It is naturally between 3.5 and 4" I hope that means something to you guys.


Wow! That's pretty acidic. 

"A *pH* less than 7 is *acidic*. A *pH* greater than 7 is basic. The *pH* scale is logarithmic and as a result, each whole *pH* value below 7 is ten times more *acidic* than the next higher value. *For* example, *pH 4* is ten times more *acidic* than *pH* 5 and 100 times (10 times 10) more *acidic* than *pH* 6"


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## CarolM (Mar 29, 2018)

Sterant said:


> Wow! That's pretty acidic.
> 
> "A *pH* less than 7 is *acidic*. A *pH* greater than 7 is basic. The *pH* scale is logarithmic and as a result, each whole *pH* value below 7 is ten times more *acidic* than the next higher value. *For* example, *pH 4* is ten times more *acidic* than *pH* 5 and 100 times (10 times 10) more *acidic* than *pH* 6"


Do you think it is wrong?


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## Sterant (Mar 29, 2018)

CarolM said:


> Do you think it is wrong?


Not at all. I have no opinion on it - I know nothing about soil chemistry - but it would seem your brother does! Just interesting input into our shell chelation conversation. Thanks for getting this!


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## CarolM (Mar 29, 2018)

Sterant said:


> Not at all. I have no opinion on it - I know nothing about soil chemistry - but it would seem your brother does! Just interesting input into our shell chelation conversation. Thanks for getting this!


No problem.


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## Sterant (Mar 29, 2018)

CarolM said:


> No problem.


I am going to test the sand/soil mix I have the bowsprits on as I also incubate in that medium. From what I read, sandy soils are similarly acidic to your brothers.


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## CarolM (Mar 29, 2018)

Sterant said:


> I am going to test the sand/soil mix I have the bowsprits on as I also incubate in that medium. From what I read, sandy soils are similarly acidic to your brothers.


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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## Anyfoot (Mar 29, 2018)

Interesting thread @Tom. I shall sit back and enjoy read to come. 

I wonder how acidic their urine is. Some of mine urinate when digging a nest.


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## Markw84 (Mar 29, 2018)

Anyfoot said:


> Interesting thread @Tom. I shall sit back and enjoy read to come.
> 
> I wonder how acidic their urine is. Some of mine urinate when digging a nest.



All of my tortoise do that.


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## Tom (Mar 29, 2018)

Anyfoot said:


> I wonder how acidic their urine is. Some of mine urinate when digging a nest.


Good question. Mine do too. All species, every time. This one seemed to be making a mud nest:


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## Anyfoot (Mar 29, 2018)

It may well be all mine urinate whilst digging and I’ve just not observed closely enough. 

Makes you wonder what impact we are having on the eggs when we remove them from the nesting site. 
Mine also sniff the potential nesting sites, what are the smelling? Temperature and/or soil qualities, vitamins,minerals or acidity levels for example, And mine spend hours checking the bottom of the nest with their feet before laying, if this is for checking the temperature then the sniffing is more likely for soil quality. 
Then some of us wash the eggs before placing into the incubator. 
I wonder what the ph level is of the surface of a freshly laid egg? 

Excuse my ignorance, what is the slime called on s freshly laid egg? Mucus!!!!


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## Anyfoot (Mar 29, 2018)

Just found this female laying.


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## Tom (Apr 11, 2018)

Nothing new to report about the clutch that is the subject of this experiment, but I have something interesting from a previous clutch from this same male and female. Tuck and Daisy.

Daisy lays large clutches. Normal is 30-32 and last year she laid 42 in one nest. That was a record for me. 36 was her highest before that. In any case, she laid 32 eggs in the ground on 1-8-18. This was her first clutch of the year. I dug them up and in random order placed them onto their egg flat, which I then carried into the reptile room, where I placed the eggs into their pre-prepaired shoe boxes with moistened vermiculite. Only 24 eggs will fit in one shoe box, so when I see Daisy laying, I always prepare two shoe boxes. Again, in random order, I placed 24 eggs in one box and the remaining 8 in the other. Same vermiculite from the same bag mixed with the same water, in the same ratio, in the same brand and type of shoe box, mixed within minutes of each other. The boxes went into the incubator at the same time and sat side by side on the same shelf. Everything identical.

The box with 8 pipped and began hatching about 5-6 days ago. All 8 hatched and everything seemed normal. Normal egg shell thickness, normal weights of 32-34 grams on the hatchlings. Nothing happening yet in the other box though… Until about two days ago, when the box with the other 24 eggs pipped and began hatching:



Again all looked normal, and I wondered why they were hatching several days apart. I remove hatchlings from the egg boxes as soon as they leave their egg under their own power. I don't like to mess with them while they are still sitting in their eggs. Some bust the top off the egg, but then remain sitting in the egg for a day or two to absorb their yolk sac. Another thing I do is rinse their egg and put it in the brooder box with them after hatching. I picked up the first hatchling to be rinsed and soaked, and it just felt heavy and solid. That is subjective. Lately I've been handling a lot of smaller leopard and star hatchlings, so maybe my perspective is a little "off". I got out the scale and this baby was 39 grams. Hmm… I pulled a few more out of the box and they were all 37-39 grams after rinsing. Why were they heavier, and why did they hatch 3-4 days later than their clutch mates in the other box next door? Next surprise, and the detail that is pertinent to this thread: Their egg shells were wafer thin. They just crumbled in my hand when I tried to rinse them. I often peel off the excess rubbery inner layer of the egg and put just the calcified "shell" in their brooder boxes with them. That is what I did about 3-4 days ago with their clutch mates. But these eggs wouldn't even hold their shape when I tried to peel the inner membrane off. It is clear that the calcification of the eggs in the box with 24 had been greatly reduced over the three month incubation period when compared to the box with only 8 eggs, whose shells remained more "normal". The hard "shell" portion had become so thin in the box with the 24 eggs, that it just crumbled in my fingers. It wouldn't even hold a shape.

Why? How? Where did the calcium go? Into the babies as it appears? The only variable I can see is the number of babies in each box. Did the crowded box of 24 create more carbon dioxide and generate carbonic acid inside the shoe box? This can sometimes happen in under-aerated fish tanks, or fish tanks with a lot of decaying organic matter, and it makes the water more acidic. A major problem for a salt water tank. I'd love to hear any ideas or explanation. Why did the larger batch hatch later? Why were their egg shells so much thinner? Why were they bigger than their clutch mates?

Random bunch of names with a lot of breeding experience here:
@Markw84 
@Sterant 
@HermanniChris 
@kingsley 
@zovick 
@Will 
@Anyfoot 
@Yvonne G

Forgive me if I've left anyone out, and please contribute.

Please feel free to alert any other member to this conversation.


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## Anyfoot (Apr 11, 2018)

Interesting. 

Seems to me that the 8 eggs hatched at an earlier stage for some reason and we’re less developed. The 24 hatchlings hatched later and were more developed, they absorbed more calcium from the eggshell to aid more growth whilst in the egg. 

Did you notice if the eggsacks were smaller on the 24 eggs? 

They must use protein from the yolk and calcium from the eggshell to develop within the egg so you would imagine the eggsacks to be smaller on the 24, the extra usage of eggshell for calcium would also explain the thinner wall of the eggs. 

Where is the heat source in your incubator reletive to the 2 shoe boxes for these 32 eggs?

Have you ever sprayed near the eggs? 

Was there lids on the shoe boxes to contain carbon dioxide?


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## Anyfoot (Apr 12, 2018)

Just a side thought. When I have eggs that don’t develop I always open them and they are always very thick walled. When an egg blows it’s shell is always very thick walled. Even if the eggs are 8 months old they are still thick walled. This means the outside elements most likely don’t have an effect on the egg walls thinning. I’m assuming that also carbon dioxide doesn’t thin the wall, because for an egg to blow must take such a build up of carbon dioxide(gases) to blow a thick wall. 
On this assumption the only way an egg can thin after being layed is through embryo development.


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## Sterant (Apr 12, 2018)

@Tom - Are these shoe boxes covered? Do you open them during the incubation period? If so, how often?


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## Markw84 (Apr 12, 2018)

@Tom This is great! Bigger hatchlings with better calcium utilization resulting in the thinner eggshells. That's what we want to see with your test group with peat added! But why now here??

Have you incubated 24 in one shoebox before? It seems that's the only difference we are talking about here. So the better development and great usage of calcium from the eggshell is aided by having the 24 in one box. The variables I see...

- Eggs touching each other. I have planned on doing an experiment myself where I stack eggs together more like they would be in a nest where they are all in contact with each other instead of spaced out evenly on vermiculite and not touching.

- Larger volume of organic matter (left on the eggs) per the amount of space and vermiculite. The mucous from the female laying and any dirt stuck to the egg is 3 times more in the group of 24 vs. the group of 8 with the same amont of vermiculite in each box. So we do have 3 times the organic (humic) matter in that box.

- 3 times the number of developing eggs with the corresponding water exchange through the egg going on in one box. Would have been interesting to weigh the vermiculite left in both boxes after the hatchlings were removed to see if there was a substantial difference in water weight.

- 3 times the number of developing eggs that are respiring and adding 3 times the amount of respired gasses going on in one box. I know you cover your boxes and only have a few small holes towards the top. There could be (as you suggest) a substantial difference in carbon dioxide build up.

Have you ever stacked 24 eggs in one box like this before? What happened to those clutches?


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## Tom (Apr 12, 2018)

Anyfoot said:


> Interesting.
> 
> Seems to me that the 8 eggs hatched at an earlier stage for some reason and we’re less developed. The 24 hatchlings hatched later and were more developed, they absorbed more calcium from the eggshell to aid more growth whilst in the egg.
> 
> ...


Yolk sacs on each group were variable in size and typical of what I normally get.

The incubator is a large stand up freezer. I removed the compressor and other "guts". I have a 72 watt RHP and a computer fan mounted on the ceiling above the eggs and on the floor under the eggs. The fans create a constant circular air movement. I have tubs of water on the bottom shelf for humidity, and the thermostat is a DBS-1000 by Helix controls. It is a digital proportional unit.



I don't spray water near the eggs, but I will dribble a little water into the vermiculite away from the eggs once or twice during incubation if it "feels" dry. Whenever I see my first pip, I add water to the media to increase moisture and dampness to both simulate the wetness from the rain that they would hatch into, and because I find it helps them extricate them selves form the egg when things are more damp.

I do keep the lids on all the boxes and there are four 1/8th inch holes drilled on the sides of each box, near the top.


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## Tom (Apr 12, 2018)

Sterant said:


> @Tom - Are these shoe boxes covered? Do you open them during the incubation period? If so, how often?


Yes. I use the lids. All the boxes have a total of four 1/8th inch holes drilled on the sides of each box, near the top. I open them 3 or 4 times during the course of the 90 day incubation, and only for a few seconds to inspect eggs and smell for any rotten ones.


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## Tom (Apr 12, 2018)

Markw84 said:


> @Tom This is great! Bigger hatchlings with better calcium utilization resulting in the thinner eggshells. That's what we want to see with your test group with peat added! But why now here??
> 
> Have you incubated 24 in one shoebox before? It seems that's the only difference we are talking about here. So the better development and great usage of calcium from the eggshell is aided by having the 24 in one box. The variables I see...
> 
> ...


I regularly incubate 24 eggs in each box. I can't recall this phenomenon happening before, but if it did happen in years past, I might not have noticed it since this whole "absorbing the egg shells" thing is new info to me. If it happen in the past, I might have assumed the female laid some thin walled eggs and needed more calcium in the diet. I vaguely recall having that thought in my early years of sulcata breeding, and I was regularly getting 35-40 gram hatchlings back then too. Maybe this has been under my nose the whole time, but my ignorance prevented me from seeing it?

I've never stacked the eggs, but the eggs are definitely touching when I crowd 24 of them into one box. The 8 eggs were clumped together on one side in the same fashion as the 24 in two rows of 4 eggs. I like to have the eggs touching since they touch in the nest when mom lays them and because I've read evidence than when one hatches, the others cue off of that.

Your humic material theory might have some merit. I would add that the amount of dirt on each egg is, on average, about the same for both groups, but clearly there would be 3 times more of it with 3 times more eggs. The eggs that are the subject of the experiment featured in this thread are divided half and half. We have the added variable now of the peat, but still interesting…

I add water to the boxes a few times randomly and very unscientifically during incubation by "feel", so I've ruined any chance of gaining insight by weighing the vermiculite after incubation. There would be a lot of variables involved in this too. How many times was the lid taken off and for how long? What was ambient temp and humidity each time the lid was off? Does one lid fit or even sit a little tighter than the other? Etc…

I can't answer _why_ this happened, but it did happen and I can measure the results easily enough. Much food for thought here...


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## Sterant (Apr 12, 2018)

Anyfoot said:


> Have you ever sprayed near the eggs?



What was your thought on spraying the eggs?


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## Anyfoot (Apr 12, 2018)

Sterant said:


> What was your thought on spraying the eggs?


I was just thinking that if Tom was spraying at all inside the incubator that some may have been sprayed more than others. If that would make a difference anyway, I don’t know.

Do they absorb water through the egg shell? 

Spraying a dud egg that is gased up will increase chances of exploding, I’ve never had a good egg explode yet when I’ve sprayed it.

I’m not advising spraying eggs at all, I’m just curious about things and like to mess about and have sprayed eggs purposely. 

Curiosity kills the cat. Good job I’m not a cat.


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## Sterant (Apr 12, 2018)

Anyfoot said:


> I was just thinking that if Tom was spraying at all inside the incubator that some may have been sprayed more than others. If that would make a difference anyway, I don’t know.
> 
> Do they absorb water through the egg shell?
> 
> ...


OK - Just curious what you were thinking there.....I spray my eggs twice a week with a very fine mist from a distilled water container I keep in the incubator. I have heard of concerns that spraying eggs could crack them, but I've never experienced that.


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## Anyfoot (Apr 12, 2018)

It would be interesting to see if there is any difference in growth between the 8 and 24 moving forward. 
How long will you be keeping these for @Tom.


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## Tom (Apr 12, 2018)

Anyfoot said:


> It would be interesting to see if there is any difference in growth between the 8 and 24 moving forward.
> How long will you be keeping these for @Tom.


I usually only keep sulcatas 4-6 weeks before selling.


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## Pearly (Apr 13, 2018)

Tom said:


> I regularly incubate 24 eggs in each box. I can't recall this phenomenon happening before, but if it did happen in years past, I might not have noticed it since this whole "absorbing the egg shells" thing is new info to me. If it happen in the past, I might have assumed the female laid some thin walled eggs and needed more calcium in the diet. I vaguely recall having that thought in my early years of sulcata breeding, and I was regularly getting 35-40 gram hatchlings back then too. Maybe this has been under my nose the whole time, but my ignorance prevented me from seeing it?
> 
> I've never stacked the eggs, but the eggs are definitely touching when I crowd 24 of them into one box. The 8 eggs were clumped together on one side in the same fashion as the 24 in two rows of 4 eggs. I like to have the eggs touching since they touch in the nest when mom lays them and because I've read evidence than when one hatches, the others cue off of that.
> 
> ...



Another great thread! Really enjoying reading it! Thanks!


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## Markw84 (Apr 13, 2018)

@Tom I thought it would be interesting to get Dave's (TTPG talk on water submersion and water chemistry effects on eggs) thoughts on this so I forwarded him the link to this thread. He responded this morning. Easier to just copy and paste his response. I think everything will make sense even though a lot of it is with the background of a lot of my previous discussions with him.

(the Packards refers to a research paper he and I have discussed quite a bit that looked at what the water potential of an incubation media does to hatchling vitality. The "redheads" refers to the Podocnemus erythrocephala he is working with.)

_"Hi Mark,

I enjoyed the thread! The results from the incubation where the only known difference is the number of eggs sounds very similar to what the Packards found by varying moisture levels for painted turtle and snapping turtle eggs, though 1:1 water:vermiculite was considered wet. Eggs that were kept in moister conditions seemed to transfer more calcium from shell to turtle. The dryer eggs hatched 5-6 days sooner. The authors didn’t phrase it this way, but it seemed like the eggs would hatch if they reached a point where the calcium bioavailability couldn’t keep up with developmental requirements, so dryer eggs hatched sooner.

So, “something” in the box with 24 eggs increased the calcium bioavailability. Some possibilities:
1) Moisture: If we think of “moisture exchanges” going in and out of the egg with the same water, look at how much moisture entered the “system” from the eggs themselves. There may have been more moisture at T=0 due to that in the box of 24.
2) More CO2 from egg respiration: This idea has merit. When we were having problems with baby matamatas in rock hard eggs that couldn’t escape, my dad tried restricting air exchanges to the incubator, so CO2 could build up and form carbonic acid, but he didn’t do a controlled experiment. He went from vermiculite and larger ventilation holes to peat moss with more restricted ventilation holes. The eggs with peat moss and smaller holes hatched on their own. We never figured out the contribution of CO2, but I was worried about potential anoxia, so I’ve focused more on using different incubation media.

For many years I’ve thought about pH effects on shell dissolution. The chelation ideas have come more recently. I had a turtle with metastatic mineralization of soft tissues including the corneas. The Vet prescribed EDTA (chelating agent) drops for symptomatic relief by dissolving the calcium crystals from the corneas. Separately, with the red heads coming from blackwater rivers, I’ve explored the attributes of humic substances in blackwater to assess if they are beneficial/required for animals from this environment. The humic substances are chelating agents and they also serve as a pH buffer. They can release protons to solution, but each proton on the big molecule has a different pKa (equilibrium at a slightly different pH) - giving the buffering effect. They buffer the Rio Negro to about 4 pH. So, finding a pH of 4 in a sandy soil with peat is very reasonable. It was the EDTA experience that guided me to the idea that chelating agents can be used to dissolve calcium crystals.

Have to run, but I have more thoughts on other parts of the thread.

Another quick thought - 
Oscillating temps and eggs “breathing”: I think consumption of O2 and release of CO2 vary with temperature, but I don’t think of the oscillating temps facilitating exchange of these gasses.

On the other hand I have viewed the oscillating temps as impacting the direction and rate of water transport across the shell."_


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## bouaboua (Apr 13, 2018)

Very interesting and educational ! ! !


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## tortoiseplanet (Apr 13, 2018)

Found this online:

“The pH of the urine of healthy tortoises fed on vegetarian diet is usually alkaline and the urine pH in anorexic tortoises (and in tortoises with high protein intake) is often acidic”

I think urine deposits in nests are rather only for humidity (according to this source):
http://c.ymcdn.com/sites/members.arav.org/resource/resmgr/Files/Proceedings_2000/2000_35.pdf


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## Markw84 (Apr 14, 2018)

tortoiseplanet said:


> Found this online:
> 
> “The pH of the urine of healthy tortoises fed on vegetarian diet is usually alkaline and the urine pH in anorexic tortoises (and in tortoises with high protein intake) is often acidic”
> 
> ...



Thank you for adding this to the discussion. Interesting paper that I have gone over before and is in my files. My issue is that it is old - all the references are at least 20 years old. It is only Testudo species and the diet was kept to strictly vegetarian for samples. It also does not quantify the Alkaline range found vs. Acidity found in others. Since it can go either way it seems it may be 7.1 vs. 6.8?? So, most likely fairly neutral. The paper itself states with protein in the diet, it does turn acidic. Normally a tortoise of the types discussed here are opportunistically taking protein whenever they see it. What if a female with developing eggs purposefully seeks out more protein in the diet? The same as she does for calcium sources.

Additionally, all my tortoise nests I've dug up, contained some fecal matter the female deposits in the bottom of the nest - addings substantial humic matter. The respiration of the eggs packed in tight proximity in a closed nest also would seem to increase CO2 levels. All leads to a very possible acidic effect at work.


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## Markw84 (May 20, 2018)

@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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## Tom (May 20, 2018)

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.
> 
> ...


96 days is a long time, and 37 grams is a big hatchling. Great observations here.

My eggs are due in the next week or so, and candling reveals that most should hatch. There are a few clear ones, but most look good.


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## Yvonne G (May 20, 2018)

Tom said:


> Nothing new to report about the clutch that is the subject of this experiment, but I have something interesting from a previous clutch from this same male and female. Tuck and Daisy.
> 
> Daisy lays large clutches. Normal is 30-32 and last year she laid 42 in one nest. That was a record for me. 36 was her highest before that. In any case, she laid 32 eggs in the ground on 1-8-18. This was her first clutch of the year. I dug them up and in random order placed them onto their egg flat, which I then carried into the reptile room, where I placed the eggs into their pre-prepaired shoe boxes with moistened vermiculite. Only 24 eggs will fit in one shoe box, so when I see Daisy laying, I always prepare two shoe boxes. Again, in random order, I placed 24 eggs in one box and the remaining 8 in the other. Same vermiculite from the same bag mixed with the same water, in the same ratio, in the same brand and type of shoe box, mixed within minutes of each other. The boxes went into the incubator at the same time and sat side by side on the same shelf. Everything identical.
> 
> ...


I don't know the whys, but what jumped out at me was that the "full term" babies used the calcium from the shell right before hatching. I don't know how to explain it. But I'm thinking that the shells were the same thickness in both batches at the time of the "premature" hatching, and then the other batch started using more of the calcium. Or rather, they use the calcium at the very end of the incubation???????

I had a group of Manouria eggs in a 5 gallon bucket, in leaf litter, just like they would have been in a nest. Never knew if this was a good way to hatch them or not because the gnat maggots age them.


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## Olddog (May 20, 2018)

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.
> 
> ...



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


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## CarolM (May 20, 2018)

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.


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.


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## Tom (Jun 22, 2018)

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.


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## Tom (Jun 22, 2018)

4 currently pipping from each box now.


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## teresaf (Jun 22, 2018)

This is so interesting! Thanks for the pics!


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## CarolM (Jun 23, 2018)

Tom said:


> 4 currently pipping from each box now.


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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## Tom (Jun 23, 2018)

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.


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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## CarolM (Jun 23, 2018)

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.


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


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## wccmog10 (Jun 24, 2018)

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.


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## Tom (Jun 24, 2018)

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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## Markw84 (Jun 24, 2018)

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.


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.


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## Tom (Jun 26, 2018)

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.


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## Tom (Jun 26, 2018)

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.


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## Tom (Jun 27, 2018)

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...


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## Tom (Jul 5, 2018)

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


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## Markw84 (Jul 5, 2018)

@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.


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## Tom (Jul 7, 2018)

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.


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## Tom (Jul 7, 2018)

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.
> 
> ...



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.


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## Tom (Jul 7, 2018)

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.


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