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Easy sex - an interesting bit of snake biotechnology

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5rings.chondroforum

Easy sex - an interesting bit of snake biotechnology

Lead

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Posts: 4

(07/01/09 12:26:52)

My wife and I are molecular biologists by training and we are total science geeks. We started wondering about snake genetics and began hatching some plans. So far we have done a little of the preliminary work but it looks promising so far. Here is where we are......

1. We think, emphasis on the "think", that we will be able to positively sex neonates with a fragment of their initial shed. That means no waiting for the babies to get old enough to probe. It also means no risk "sexing" on snakes of all ages. We have yet to do our double blind studies but we are certainly pointed in the right direction. We hope to have the details worked out in the next few months.

2. Next up are specific genes, such as albinism. This is a little more complicated and at this point we are still collecting journal articles and sequences. Some traits are probably "do-able", others will likely remain a mystery as gene sequences are not yet known. I expect this will take years rather than months.

The idea came about when shopping for high dollar animals with unknown sex. We knew there had to be a better way. Keep your fingers crossed.

VonSamhain

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Posts: 57

(07/01/09 12:50:29)

lets hope it works, that would change a lot for this community.

http://www.iherp.com/VonSamhain2

Nick Metherd

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Posts: 185

(07/01/09 13:43:15)

MVF Post Breeder

I have always loved the research that went into as well as the trial and error of proving or disproving a new hypothesis. A few ideas of mine that I have yet to prove but am totally open to share are these:

A snake of a chondro can be determined by the length of the tail at any age. I have observed that in neonate, yearling, and adult animals that two animals of equal length and weight will have different lengths of tails if one is a male and the other is a female. This has only been observed in the few animals I have kept and I don't have access to a large collection to get more specifics on this. My original thought was to measure neonate tails but that runs a high risk of kinks similar to probing. I was also going to try and count the number of scales from the ventral opening to the tip of the tail to see if there is a number difference but have been bitten in yearling several times trying to attempt this so I would only imagine trying this by a breeder in a neonate of only a few months old.

If anyone with neonates currently would like to try this, you are more than welcome to. I would guess that by either measuring the distance from opening to tip on like sized neonates or scale count on any size animal would be able to be used to determine sex.

MegF42

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Posts: 2448

(07/01/09 14:35:18)

Forum super duper Ultra Chondro!

How do you count scales without removing the baby from the perch. Again, forcing the tail straight to count would t me pose a risk of kinds as well. It will be really interesting to see if sex can be determined using the sheds.

2.2~Cornsnakes
3.4.2~Green Tree Pythons
4.2.1~Amazon tree boas
1.0~California Kingsnake
1.1~Snail Eaters
1.0~Eyelash Viper
1.1~False Water Cobras
Assorted: Horses, dogs, and cats
www.Franclycac.com

tylersquare

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Posts: 41

(07/01/09 15:39:01)

is there a reason the first shed would reveal the sex but not later sheds?

greg stephens2

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Posts: 1957

(07/01/09 16:12:07)

Forum Ultra Chondro!

If you guys need snakes to include in a study I have sex known animals and will have unsexed neonates hatching soon.
I typically hold back many of the neos so that could be helpful in verifying the process.
If you would be interested please shoot me an email.

Greg Stephens
gstephens1@zoominternet.net
Arboreal Specialties

Nick Metherd

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Posts: 186

(07/01/09 22:05:05)

MVF Post Breeder

Sorry I meant counting scales off of the shed and this could be done at any age. It is the measuring of the tail that would use the first shed and couldn't really use any other shed. Probably wouldn't work since sheds can be stretched and size varries in hatchlings. Greg I might shoot you an email in the next few days to see if we can come up with some ideas about counting scales.

I believe the author of this thread is probably doing some DNA testing on the initial shed. I had a friend in Houston that did some work on how to extract DNA using a snake shed from African house snakes. You could use this testing to determine if the Y chromosome is present. You could also use this testing to determine if an animal was het for albino if you knew which marker to look for. Easiest method that I could think of for this is to get sheds from Damon of known albino and het. for albino animals, identify all of the markers they have in common, and then eliminate markers by testing known animals that are not het. by using animals with long documented pedigrees. May not take years, but definitely some time and patience.

Good luck with all of your tests and experiments.

Last Edited By: Nick Metherd 07/01/09 22:38:10. Edited 3 times.

Tim Morris1

sheds

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Posts: 233

(07/02/09 04:58:22)

Forum Chondro Junkie

Having made almost exclusive use of sheds in molecular work I'm certain that any shed subsequent to the first can be used for determining the sex of an animal - the DNA is all the same. Finding the genes responsible for other traits such as albinism however will take considerable work (and money). Typically this type of work includes mapping the genome then working out what genes are responsible for what- this step can take a long time though it can (and has been) be done.

Tim

CarlosEcharte007

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Posts: 2099

(07/02/09 05:35:55)

Forum super Ultra Chondro!

Keep up the good work, this will change history!

Carlos Echarte
Coneroks1@aol.com

5rings.chondroforum

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Posts: 5

(07/02/09 06:43:03)

Yes, we are using DNA from shed scales. The sex chromosomes of snakes are visually pretty difficult to distinguish, unlike a human. Karyotypes are nearly useless in snakes. Therefore we had to find a gene sequence specific to male or female snakes. We think we have found one. Time will tell. Snake genetics are quite different than humans and we have a quite a bit yet to learn.

Yes, we can theoretically sex any snake, at any age, if the protocol works like we think it will. It only takes a handful of cells or scales. I may ask for volunteers once we have done our double blinds with our collection.

Some sequences for genes associated with albinism are well known in other animals. We have yet to determine if these sequences can be used to identify albinism in snakes. If it works out, there will be no more 50% het for albino. They will be proven to carry the gene or not. No guesswork. Some breeders might not like that idea but consumers should love it.

We have several intrinsic "business" issues to contend with in regards to our services. You might give us some valuable input here.

1) What do we charge for the service? We have time and money invested. We aren't looking to get rich, but we'd like to make ends meet.

2) How can we make sure you don't send us a contaminated sample? One or two cells from another snake can void the results.

3) How do we make sure that you are applying our results to the snake in question? Our reputation is on the line and since you would have direct contact with the buyer it would be easy to point an errant finger toward us. The protocol itself would be virtually 100% accurate, but as we saw in the O.J case, all of the things that happen before and after the protocol can cause problems.

We are still in preliminary stages so I wouldn't count your chickens just yet, but it does look promising. We certainly may encounter more difficulties than we think. We are busy people with kids and snakes to feed. We might also get frustrated and give it up at any moment. I would ask that you not spread this information to other forums. I am writing this because we need to know if it is worthwhile to continue spending our own money on something that may not be used. I would also like to keep this a bit of a secret for business reasons. I would hate to iron out the protocol and then find somebody else taking the credit for it. If you have any positive input I would like to here it. I'll keep you posted.

Steve

http://www.serpentinelogic.com

Christopher Feenan

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Posts: 613

(07/02/09 10:43:43)

MVFonator

Are you sure that you are planning double blind trials ? How ? I mean, does it mean something different with snakes than it does with humans ?

tylersquare

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Posts: 42

(07/02/09 12:56:03)

double blind usually means that both the tester and testee are unaware of the treatment which they are being given (which could be a placebo type treatment). People usually just use that phrase to say that all possible bias has been eliminated from the testing process.

i'm a little curious as to how you're going to find markers for albinism without spending a LOT of money though. theres no albino reptiles with genomes sequenced that i could find

Christopher Feenan

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Posts: 614

(07/02/09 13:37:47)

MVFonator

Yeah, I know what double blind means, and no, it doesn't mean all possible bias has been removed.

It means something simple, and specific. No, people "don't just say that ". If variables are being controlled, including investigator bias, then those controls are described. Researchers don't describe their method as double blind when it is not, unless they are trying to pretend that they are expert in something when they are not.

My BS alarm is going off.

Nick Metherd

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Posts: 187

(07/02/09 13:50:07)

MVF Post Breeder

Here is my opinion on the answers to your questions.
1. Cost- it probably wouldn't be cheap. This would be something used on high end animals since most interested in buying a 300.00 animal isn't going to spend that amount for the test. What is the cost of electrophoresis these days? 10 yrs ago when I was in college it was about 300.00 a pop. Or on the flip side without telling us your costs, what is a generic range that you were looking to price this at?

2 + 3. The best you could do is ask for a complete shed, rinse it in a non-damaging solution and then use a piece of that shed. You can only guarantee your results, not the reputability of the dealer.

To answer your question Tyler, to the best of my knowledge and again it's been ten yrs and I'm not in a science field now, you would not actually have to map the entire genome in order to discover which marker is responsible for albinism or any other trait that is only controlled by one allele. You don't have to know what the allele does necessarily, just that it doesn't have anything to do with albinism.

-Take Damon's two animals and map out the markers using sheds
-Circle all of the markers that they have in common (and there will be a lot since they are siblings)
-Compare the markers to Merauke lines that have been in the US for generations. Since the animals come from the same region (in theory), they would share more markers that you would not find in other CB animal line like the Trooper Blues. Continue this until you have eliminated all of the markers except one.

Again those are rough terms and I might be way off so if someone would like to correct me, I am open to it. That is what I remember though.

And as for the double blind study, I can only think of studies that can be done that are blind on one end. Double blind usually refers to both the test subject and the researcher don't know who is taking what medicine for example. Then blood is taken and analyzed after the results of any negative side affects have been recorded to find out who took the meds and who didn't. With this the test subject is inanimate (the shed) so it cannot affect the outcome. As long as the researcher doesn't know where the shed came from, all tests will be true in the results.

One more question to throw out there and I'm sorry for the long post- I thought I read somewhere that in reptiles, the XY is backwards in that the females carry the Y chromosome equivalent? Is that true? Wouldn't that make things easier to just look for that in the tests?

Last Edited By: Nick Metherd 07/02/09 13:59:57. Edited 1 times.

geckos70

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Posts: 1381

(07/02/09 13:51:04)

Forum Ultra Chondro!

Interesting idea on genetic sex determination. Good luck on optimizing your protocol. However, I would not worry about this being talked about on other forums because this forum has more than it's fair share of science geeks from multiple disciplines. I do have a couple of questions: Where are you currently working on this project if you don't mind me asking? I noticed in your profile that it said you are in Tennessee. Are you at a University and if so how did you get an IACUC (Institutional Animal Care and Use Committee) to approve the use of your personal animals?

Thanks and good luck
Michael

"A government big enough to give you everything you want, is big enough to take away everything you have" Thomas Jefferson

Rico Walder

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Posts: 791

(07/03/09 04:39:03)

MVFonator

Interesting concept and one that's been thought of by others as well. Good luck working the details out. If you need sheds I have a stockpile of individually isolated & ID'd sheds. Of course there are a few more snakes here to get more if necessary.

Rico Walder
Signal Herpetoculture
www.signalherp.com
Chondro Coalition
www.chondrocoalition.com

5rings.chondroforum

Now guess why I dont post on forums.

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Posts: 6

(07/04/09 17:46:07)

I rarely post on forums because some people have no lives of their own and spend all of your free time critiquing everyone else. Some of you obviously got distracted and missed the salient point to my post. Did you find any dangling participles? I can't stand those. Perhaps I misspoke when I said "double-blind". It certainly was not the point to my post, but a couple of you seemed to think it was the only issue of import. This version of double blind, is that I am supplying sheds from snakes without knowledge of what snakes they came from. We have help and we have access to snakes that are of known sex. I will give them to my wife only identified by a number. Only females are supposed to have the gene in question. In simple terms, if we get results it should indicate female. We will have to go back and correlate numbers with snakes after the results. With me not knowing the sex as I collect the sheds and my wife not knowing the sex of the animals I counted that as "double-blind", maybe I counted wrong. Let's call it a 1.5 blind study. If the snakes could talk, they might willingly take part and give us a proper double blind. Perhaps not.

We are fervent protectors of animals. We are very aware that NIH has animal protocols that must be followed when working under an NIH grant. We are working with our own money and time. IACUC is used for the protection of animals in the confines of an research institution. We are collecting animal waste and not even touching the animal. I would suggest that probing animals that we are selling in commercial fashion would be more likely to draw the attention of IACUC. It is certainly more dangerous and invasive to the animal. I am not sure we would qualify as an institution either. I should likely spend some time in one though.

As I understand snake genomes, and I know precious little, their sex is determined by chromosomes Z and W. The ZW being female, whereas in humans the "heterozygous" condition is male. (I put that word in quotes so you semantic eagles don't don't bombard me with Mendelian vernacular). Reptiles chromosomes don't seem to be very much like human chromosomes from a visual perspective. Yet, DNA is DNA and some genes are often highly conserved between species. Again, we are still reading as much and as often as we can. I've got a lot to learn and very little free time. We are doing our best to shorten the curve and welcome any help that can be offered.

I do appreciate the posivite and constructive input. I am listening to what you are saying and have plenty of concerns myself. The cost for the protocol itself isn't huge but equipment and time can get a little pricey. We would like to make it affordable to everyone. Like I said earlier, we are just doing this on a lark. We don't have a grand business plan. Nobody is planning on quitting their jobs and living loca vida. We are just a couple of science nerds trying to solve an intersting problem. I suspect that we will have to let demand inversely dictate price. We have to pay for equipment but if we only do 10 tests a month they will be pricey as a function of our cash output. If we did 50, then it might be considerably more affordable. We will simply spread the cost over the number of tests we did. Without sticking my neck out it will most likely be less than $50. I hope. I can't imagine the average collector would want to absorb anything more costly than that.

The albinism question is a tough and complicated issue. There are several types of albinism and we do not yet know if they are conserved sequences. The sequence for human tyrosinase is well known and a couple of mutations seem to lead to forms of albinism in people. One form is a single point mutation that has been found by directly sequencing the region in question. I don't think that is a do-able proposition for us or snake owners. I read one paper that indicated one mutation allowed the DNA to be cut by a rare cutter. That might be a clue. Anyways, we obviously have our work cut out for us. Nobody said it would be easy.

As I said before, we dont know if this thing will fly. We have been encouraged by what we have found and read. It may be a total and resolute failure. If I were a betting man, I would bet against us. It is quite likely that better minds than ours have addressed this issue before. Who knows, we might get dumb-lucky! We simply wanted to know if it would be something you would use if it were available. We might also need your help in the next few months.

I am going off to drink myself double-blind.

Steve

http://www.serpentinelogic.com

Christopher Feenan

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Posts: 615

(07/05/09 06:10:03)

MVFonator

Steve - I am glad that you are interested in solving a vey difficult genetic puzzle - Good luck !

My question to you was not "because I have no life ", and it wasn't a critique. It was the sort of straightforward question that practising scientists have to answer. An appropriate question, given your inappropriate claim.

Attacking me for asking it isn't professional - leave emotion out of it, and be prepared to defend your claims. No, your study isn't 1.5 blind either.

The "salient point to your post" was simple. You hope to someday do something that is very difficult.

You should publish your results if you fall short so that others can start where you left off, or learn from your mistakes.

tylersquare

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Posts: 43

(07/07/09 22:48:31)

Christopher Feenan wrote:
Yeah, I know what double blind means, and no, it doesn't mean all possible bias has been removed.

when I say all possible bias, I mean the bias which is possible to remove. And yes, it does. look up any definition you want... the sole reason for using a double blind method is to eliminate the human bias. It doesn't apply to this research simply because there is no control type group to be had, but removing bias is still a good thing regardless if the term blind applies to it.

one thing that would help remove bias for this experiment would be to have someone who is unaware of your snakes' sexes take the samples and number them, stir em up, run your tests.

5 rings, sorry if you thought that I was taking the double blind part of this as being the most important, I was simply trying to help someone out

tylersquare

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Posts: 44

(07/07/09 23:11:41)

Nick Metherd wrote:
To answer your question Tyler, to the best of my knowledge and again it's been ten yrs and I'm not in a science field now, you would not actually have to map the entire genome in order to discover which marker is responsible for albinism or any other trait that is only controlled by one allele. You don't have to know what the allele does necessarily, just that it doesn't have anything to do with albinism.

In order to know if alleles are present, you have to be able to test for them, and in order to test for them, you have to know what you're testing for. Microsatellites could be used too, but those can only be identified if you know where the gene is, or can identify a pattern among many individuals.

There are other ways, but they don't work very well very often. By plucking cells responsible for pigment production and making cDNA from the mRNA present in the cell, you can get (among other things) the exon sequences of the genes being used for pigment production. Do this with normal and albino snakes, and eventually some type of pettern may be seen. once you have a known exon sequence of a gene which either causes or contributes to the albinism phenotype, you can test animals by doing a southern blot or sequencing the area of their genome which may or may not hold said allele. the problem is, DNA works as a huge network, and albinism could very easily be controlled by multiple genes working a specific way.

What may be even more of a business venture would be to figure out genes responsible for pigment, breeding your snakes, and knocking out the gene with a morpholino. Getting to the embryo for injection at the one cell stage would be quite the hurdle, however.

Last Edited By: tylersquare 07/08/09 19:04:05. Edited 1 times.

bushman

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Posts: 1

(07/08/09 10:37:30)

I wish you all the best from down under. A similar project is underway in Melbourne, although there have been set-backs with contamination of samples.
Please let us know if we can help in any way and keep us updated on your progress. Perhaps a collaborative effort would increase chances of success.

Last Edited By: bushman 07/08/09 10:47:19. Edited 2 times.

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Easy sex - an interesting bit of snake biotechnology

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