Temperature Sex Determination VIDEO- how this may relate to the Burmese Star TSD issues

Markw84

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Today I was forwarded this video on a study of a discovered effect of epigentic influence and TSD in Red Eared Sliders. I found it very interesting and of great value.

Here is the link to that short, 4 minute video:


Here is my response after watching it:

Now this is very interesting. Thank you for sharing this! This is great stuff for those of us working on TSD and especially when diapause is involved.

I immediately thought of the issues we have with TSD in G platynota and the way most babies artificially incubated turn out male despite high "female expected" temperatures. In studying diapause and TSD, I have had several conversations with my sister (one of the world's leading authorities on epigenetics) about possible epigenetic effects starting the cascade of events setting gender. I had always proposed that the way we diapause is affecting this. The Behler Center had good success in determining a pivot temperature and reliable sex outcomes of their G platynota. I noted that they used a diapause technique of just putting the eggs on a shelf in a room and let it fluctuate from high 60°s to high 70°s then incubate. This produced a definite and reliable pivot point for them. However, most breeders that were willing to share info, use a cooler diapause where they cool eggs from anywhere from 60° to 65° then incubate. Despite incubation at temps above the pivot point Behler Center found, most still turn out to be male.

In studying ground temps and weather data from Myanmar, I came to the conclusion that no natural nest in Myanmar would ever reach temperatures much below 70° in their winter. So I took the chance to change this trend and diapause my platynota eggs with warmer, fluctuating temps and not use the steady low temperature cooler method. I then incubated at temps most use of about 89°-90°. Of the holdback from my early 2019 clutches out of 7 I held back, 1 is male, 1 is yet undetermined and 6 are female.

Here we have remarks that the calcium channels as well as the proteins involved for epigenetic effects are both affected by temps. With the lower temps of diapause, I'll bet the calcium is not being utilized as noted in the higher temps for red ears since the diapause is so low. The same may be true for the protein STAT3 which would not able to be phosphorylated at these lower temps in diapause. As incubation begins the lack of this protein - pSTAT3, and possibly the lower calcium channel levels, would turn off the Kdm6b expression. Although not developing, the diapaused egg could certainly be establishing a chemical balance the later incubating temperatures cannot turn around, or turn around quick enough - and we have males developing, despite "female" incubation temperatures.

Mark
 

gummybearpoop

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Thank you for always sharing Mark. There have been/are selfish breeders out there who will withhold information for the betterment of themselves for various reasons (ego, money, etc.) Instead you are always willing to share valuable information for the betterment of the species.

I can’t think of any other breeder of Burmese Stars that provides detailed history and care sheets like you do when anyone purchases babies from you.

Keep up the great work!
 

turtlesteve

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Very interesting, thanks!

I have done quite a bit of reading and analyzing research on this topic too. This isn’t the first time I remember reading that calcium was involved the process (not sure if it was this group or someone else though). And I recall, in some of the earlier research on TSD, that it was proven that sex could be selected chemically by allowing the appropriate hormone to be absorbed through the egg during the critical period for TSD in the second ‘trimester.’ I do not recall of anyone that tried to expose developing eggs to soluble calcium, to look for an effect on sex ratio, but it seems like something that might be worth trying in a controlled experiment (presumably, more calcium would bias things toward female).

I am further surprised that the possibility of chemically hijacking TSD has not been used as a conservation tool.

With regards to the apparent unreliability of TSD in some cases, I must also consider the simple hypothesis - that egg temperatures are not being measured accurately in all cases. Incubators I have tested have had a temperature error of 2-4 degrees F, and always to the high side (e.g displayed reading hotter than the egg temperature). So, given that nobody seems to be using calibrated thermometers to double check everything, I tend to be skeptical.

I also suspect that in species that lay in fall or winter and undergo diapause, the proper incubation profile is one of increasing temperatures, not constant temperature. Specifically, eggs should be kept slightly cooler for the first trimester of development, and then heated to the desired TSD temperature at the onset of the ‘critical’ period early in the 2nd trimester. This is attempting to mimic a situation where eggs are timed to develop in spring and hatch in summer. This also avoids the “split scute” problem (e.g. female temps believed to cause a higher risk of defects) since the shell and major organs form earlier, before the TSD period, when incubation temperatures are lower.

Unfortunately, I don’t yet breed enough tortoises to test any of this myself.

Steve
 

Markw84

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Very interesting, thanks!

I have done quite a bit of reading and analyzing research on this topic too. This isn’t the first time I remember reading that calcium was involved the process (not sure if it was this group or someone else though). And I recall, in some of the earlier research on TSD, that it was proven that sex could be selected chemically by allowing the appropriate hormone to be absorbed through the egg during the critical period for TSD in the second ‘trimester.’ I do not recall of anyone that tried to expose developing eggs to soluble calcium, to look for an effect on sex ratio, but it seems like something that might be worth trying in a controlled experiment (presumably, more calcium would bias things toward female).

I am further surprised that the possibility of chemically hijacking TSD has not been used as a conservation tool.

With regards to the apparent unreliability of TSD in some cases, I must also consider the simple hypothesis - that egg temperatures are not being measured accurately in all cases. Incubators I have tested have had a temperature error of 2-4 degrees F, and always to the high side (e.g displayed reading hotter than the egg temperature). So, given that nobody seems to be using calibrated thermometers to double check everything, I tend to be skeptical.

I also suspect that in species that lay in fall or winter and undergo diapause, the proper incubation profile is one of increasing temperatures, not constant temperature. Specifically, eggs should be kept slightly cooler for the first trimester of development, and then heated to the desired TSD temperature at the onset of the ‘critical’ period early in the 2nd trimester. This is attempting to mimic a situation where eggs are timed to develop in spring and hatch in summer. This also avoids the “split scute” problem (e.g. female temps believed to cause a higher risk of defects) since the shell and major organs form earlier, before the TSD period, when incubation temperatures are lower.

Unfortunately, I don’t yet breed enough tortoises to test any of this myself.

Steve
Lots of interesting thoughts, Steve. Thank you!

A note on split scutes: Scute delineation occurs at the exact same time in embryo development as the "sex setting" is popularly thought to occur - stage 17 of the embryo development. It has also pretty conclusively been shown that the presence of flouride and/or very soft water will produce an abundance of split scutes at least in some aquatic turtles experimented with...
 

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It has also pretty conclusively been shown that the presence of flouride and/or very soft water will produce an abundance of split scutes at least in some aquatic turtles experimented with...
This whole thing is fascinating, but this last part in particular is. I'm using well water to moisten my incubation media. Zero fluoride or chloramines, and extremely hard and alkaline. I'm also using moderate temperatures for incubation ranging from 86-88. Most clutches see no split scutes, but occasionally I'll get a few. Usually and consistently 3, if any, from a typical sulcata clutch, as many as 30-40% in the SA leopards, and none in any other species. I've tried to link it to high spikes in temperature, but that doesn't seem to be the case.
 

Markw84

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This whole thing is fascinating, but this last part in particular is. I'm using well water to moisten my incubation media. Zero fluoride or chloramines, and extremely hard and alkaline. I'm also using moderate temperatures for incubation ranging from 86-88. Most clutches see no split scutes, but occasionally I'll get a few. Usually and consistently 3, if any, from a typical sulcata clutch, as many as 30-40% in the SA leopards, and none in any other species. I've tried to link it to high spikes in temperature, but that doesn't seem to be the case.
Tom - when do you sleep? 4:18 AM!
I've had 2 split scutes out of about 40 sulcatas and none out of 90 Burmese. I stopped letting any tap water touch the eggs. I've use the rescue solution to wash eggs and moisten incubation medium this past year. I also have been using fluctuating temps from -88°-91° = high enough to create split scutes with the traditional wisdom. I also mix peat moss in with vermiculite for incubation medium increasing acidity. I think alkalinity is a possible issue. It also can inhibit as much calcium use from the egg.

I seems funny that the platynota seem almost impervious to split scutes, while pardalis are very prone. Sulcatas sometimes. Yet all three are fairly closely related genetically.
 

Tom

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Tom - when do you sleep? 4:18 AM!
I've had 2 split scutes out of about 40 sulcatas and none out of 90 Burmese. I stopped letting any tap water touch the eggs. I've use the rescue solution to wash eggs and moisten incubation medium this past year. I also have been using fluctuating temps from -88°-91° = high enough to create split scutes with the traditional wisdom. I also mix peat moss in with vermiculite for incubation medium increasing acidity. I think alkalinity is a possible issue. It also can inhibit as much calcium use from the egg.

I seems funny that the platynota seem almost impervious to split scutes, while pardalis are very prone. Sulcatas sometimes. Yet all three are fairly closely related genetically.
Sleep? Who has time for sleep? I've got stuff to do!

All of the above sounds brilliant and I will follow your lead and report the results.

I don't know what "rescue solution" is? Since m well water is fluoride and chemical free, I think I will continue to use that, but with the addition of the peat.

Per our previous discussions, I've also been allowing the egg temps to fluctuate more, and my last batch of Gpp had zero scute anomalies.

Two of my adult platynota came with scute anomalies from the breeder back in 2013. Both are female, and I bought two others from that same breeder that turned out to be male. All four were "incubated for female".
 

Markw84

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Sleep? Who has time for sleep? I've got stuff to do!

All of the above sounds brilliant and I will follow your lead and report the results.

I don't know what "rescue solution" is? Since m well water is fluoride and chemical free, I think I will continue to use that, but with the addition of the peat.

Per our previous discussions, I've also been allowing the egg temps to fluctuate more, and my last batch of Gpp had zero scute anomalies.

Two of my adult platynota came with scute anomalies from the breeder back in 2013. Both are female, and I bought two others from that same breeder that turned out to be male. All four were "incubated for female".
One of the original platynota I got for my adult group had a scute abnormality as well. Turned out to be male. But none I have hatched have had any scute abnormalities. Did any of your platynota?

The Rescue Solution is from Dave Drajeske's talk at TTPG a few years ago on Submersion of aquatic turtle eggs. His "Rescue Dip" is: distilled water with calcium chloride - 300 mg/L and baking soda - 600 mg/L
 

Tom

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One of the original platynota I got for my adult group had a scute abnormality as well. Turned out to be male. But none I have hatched have had any scute abnormalities. Did any of your platynota?

The Rescue Solution is from Dave Drajeske's talk at TTPG a few years ago on Submersion of aquatic turtle eggs. His "Rescue Dip" is: distilled water with calcium chloride - 300 mg/L and baking soda - 600 mg/L
No split scute playtnota for me either.
 

turtlesteve

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Lots of interesting thoughts, Steve. Thank you!

A note on split scutes: Scute delineation occurs at the exact same time in embryo development as the "sex setting" is popularly thought to occur - stage 17 of the embryo development. It has also pretty conclusively been shown that the presence of flouride and/or very soft water will produce an abundance of split scutes at least in some aquatic turtles experimented with...
You are correct. I went back and reviewed the papers involved (studies on red eared sliders) for more detail, and I may be interpreting this incorrectly. I think for some reason, I am assuming that mutations affecting scute pattern would be occurring significantly before scute delineation is visible - but there is not necessarily any justification for this assumption.

I guess this all leaves four hypothesized causes for scute malformations, that I've read in various places:

1. Incubation temperature, based on observations that split scute females are more common than males.
2. Constant incubation temperatures, presumed due to reduced gas/oxygen flow across the egg shell.
3. It's largely genetic
4. (Now) soft water or fluoride.

I'd like to see the evidence you're referencing, as it's the first I've heard it. Generally hard water contains soluble calcium, and fluoride strongly binds (and reduces availability of) calcium - is this the hypothesized mechanism of action?

I have a (female) platynota with a split scute. Produced by Andy at ATC 6 or 7 years ago, if memory serves. I also raised 4 Pyxis arachnoides, now adults. Two had split scutes, and turned out female. The two with normal scute patterns were male. I've certainly seen split scute males, but less commonly than females.

Steve
 
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