2018 Incubation Experiment

[Anyfoot]

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?

 

[Anyfoot]

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.

 

[Sterant]

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

 

[Markw84]

@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?

 

[Tom]

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?

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.

 

[Tom]

@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.

 

[Tom]

@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?

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...

 

[Sterant]

Have you ever sprayed near the eggs?

What was your thought on spraying the eggs?

 

[Anyfoot]

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.

 

[Sterant]

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.

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.

 

[Anyfoot]

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.

 

[Tom]

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.

 

[Pearly]

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...

Another great thread! Really enjoying reading it! Thanks!

 

[Markw84]

@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."

 

[bouaboua]

Very interesting and educational ! ! !

 

[tortoiseplanet]

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

 

[Markw84]

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

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.

 

[Markw84]

@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.

 

[Tom]

@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.

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.

 

[Yvonne G]

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:
View attachment 235792

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.

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.